UNIVERSIDAD COMPLUTENSE DE MADRID    

FACULTAD DE FARMACIA 
Departamento de Parasitología 

 

 
 
 

 

CARACTERIZACIÓN PARASITOLÓGICA E 
INMUNOLÓGICA DE LA LEISHMANIASIS VISCERAL EN 

EL ESTADO DE AMHARA, ETIOPÍA 
 

 MEMORIA PARA OPTAR AL GRADO DE DOCTOR 
PRESENTADA POR 

Endalamaw Gadisa Belachew 
 
 

Bajo la dirección de la doctora 
 

Israel Cruz Mata 
Francisco Javier Moreno Nuncio 

 
 

 Madrid, 2012 
 
 
 
 

©Endalamaw Gadisa Belachew, 2012 



 

 

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Universidad Complutense de Madrid 

Facultad de Farmacia  

Departamento de Parasitología 

 

 

 

Caracterización parasitológica e inmunológica de la 

leishmaniasis visceral en el Estado de Amhara, Etiopía.   

 

Tesis doctoral  

 

 

 

 

Endalamaw Gadisa Belachew 

Madrid 2012 

 



 

 

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El presente trabajo de investigación ha sido realizado en el Centro Nacional de 

Microbiología - Instituto de Salud Carlos III (Majadahonda, España) y en el 

Armauer Hansen Research Institute (Addis Ababa, Etiopia) bajo la dirección de 

los Drs. Israel Cruz Mata y Fco. Javier Moreno Nuncio  



 

 

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AGRADECIMIENTOS 

Me gustaría aprovechar esta oportunidad para expresar mi más profunda 

gratitud a todas las personas, que de una u otra forma, han colaborado en la 

elaboración de esta tesis.  

En primer lugar, quiero dar las gracias de todo corazón a mis directores, Dr. 

Israel Cruz y Dr. Javier Moreno, por hacerme ver los errores y orientarme hacia 

adelante a pesar de  todos los altibajos. Esta tesis no hubiera posible sin vosotros, os 

estoy muy agradecido.  

A la Dra. Carmen Cañavate, por acogerme en su laboratorio y por su 

colaboración para sacar adelante este trabajo. Carmen, llevaré tus palabras en mi 

corazón “tienes que reflexionar Endalamaw”. Querida Carmen, te estoy 

agradecidísimo.   

Al Dr. Jorge Alvar, que me brindó esta oportunidad desde el principio, y 

generosamente me ha ayudado en realizarla. Apreciado Jorge tu generosidad es 

ejemplar, ha sido una bendición.   

A la Dra. Estefanía Custodio y al Dr. Luis Sordo, fue una gran oportunidad 

conoceros. Vuestros conocimientos multidisciplinarios me han ayudado para llevar a 

cabo el proyecto, especialmente en el manejo de datos, muchísimas gracias amigos.  

  Al Dr. Abraham Aseffa por su ayuda continua, por su cariño y porque sus 

consejos fueron mi bastón. Querido Abraham, te estoy agradecidísimo.  

A los amigos de Majadahonda, del grupo de Leishmania/Parasitología: Amigos, 

no voy a nombraros a todos, sois todos muy simpáticos y tenéis una mente abierta. 

Mucho os  agradezco sobre todo por aceptarme como soy y tratarme cariñosamente. 

Una vez más, muchísimas gracias a todos vosotros.  

A mis amigos de Armauer Hansen Research Institute por sus ayudas material, 

intelectual y moral. Amigos sois especiales y muchísimas gracias.  

A todos los miembros del laboratorio regional de Amhara, por crear un 

ambiente agradable durante mi estancia en el laboratorio. Muchísimas gracias Amigos.  

A todos los niños de Libo Kemkem y Fogera que han participado en la 

realización de esta tesis. Quiero daros las gracias desde lo más profundo de mi 

corazón: Chiquillos os debo mucho.  



 

 

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A todos encuestadores quien trabajado conmigo y Zelalem Abraham: por su 

amistad durante el trabajo de terreno, debo dar mi agradecimiento especial a Zelalem. 

Queridos amigos, vuestra contribución es insustituible, os estoy muy agradecido.  

A mis padres y hermanos quien de una u otro forma están en esta tesis. 

Queridos, hemos compartido tiempos inolvidables dúrate la realización de esta tesis; 

en feliz o tristeza; Gracias queridos.   

Por último, quiero agradecer de una manera muy especial a mi mujer, por su 

apoyo, cariño, ánimo y consejos. Querida mía, sin ti esta Tesis no hubiera sido posible, 

muchísimas gracias.        

Y por encima de todo, agradezco a Dios y Santa María porque están conmigo 

dando luz a cada paso de mi vida. Gracias Dios, Amen 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

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RESUMEN 

 La leishmaniasis visceral (LV) o kala-azar es una enfermedad infecciosa que 

resulta fatal sin diagnóstico y tratamiento adecuado. Está causada por parásitos 

pertenecientes al complejo Leishmania donovani y es transmitida por dípteros 

hematófagos, concretamente hembras de diversas especies del género Phlebotomus 

en el Viejo Mundo y del género Lutzomyia en el Nuevo Mondo. Se estima que la 

incidencia mundial de LV es de 500 000 casos, de los cuales el 90% ocurre en 

diferentes países que se encuentran en tres grandes focos: el subcontinente indio 

(Bangladesh, India y Nepal), y el cuerno de África (Sudan y Etiopía), donde se 

considera que la transmisión es principalmente antroponótica, y América (Brasil), 

donde se considera una zoonosis, con el perro como principal reservorio. Cada año 

mueren entre 50 000 y 60 000 personas debido a la LV, cuya tasa de mortalidad, entre 

las enfermedades parasitarias, sólo es superada por la malaria. En los últimos años, 

se ha documentado la (re)emergencia de la LV en numerosos países. El número de 

casos en las zonas endémicas ha aumentado y además están apareciendo  nuevos 

focos en regiones donde previamente no se había detectado la enfermedad.  Entre los 

factores responsables destacan: los cambios medioambientales, la inmunodepresión, 

el movimiento en masa de población no inmune a áreas endémicas y la resistencia a 

los antimoniales pentavalentes.  

 En Etiopía se calcula que la  incidencia de LV es 4 500 a 5 000 casos. Este 

país ostenta el mayor porcentaje de coinfección Leishmania/VIH; entre los años 1998-

2007 la proporción de casos de LV asociados a infección por VIH aumentó del 18,5 al 

al 41%. Recientemente, el norte del país ha sufrido un brote de LV que costó la vida 

de centenares de personas. El 90% de los afectados tenía entre 8 y 45 años de edad y 

más del 20% sufrían malnutrición. Estos hechos demuestran que la LV está 

convirtiéndose en una preocupación creciente para la salud pública de Etiopía, siendo 

prioritario un mayor conocimiento de su epidemiología en este país para poder diseñar 

un programa de vigilancia y control adecuado. 

 En ausencia de inmunodepresión, sólo un pequeño porcentaje de los 

infectados desarrolla la enfermedad, mientras que la mayoría permanecen 

asintomáticos. Dado que los portadores asintomáticos podrían actuar como 

reservorios, el conocer la prevalencia de infección en este grupo contribuiría a una 

mejor comprensión de la dinámica de la transmisión, y por tanto sería de gran utilidad 

a la hora de desarrollar estrategias de control.  



 

 

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 Actualmente se considera necesario el diseño de un programa de control eficaz 

de la LV en Etiopía, que utilice herramientas que permitan determinar adecuadamente 

la exposición al parasito (para poder estimar la prevalencia e incidencia), y que 

identifique factores de riesgo asociados a infección y enfermedad. En este último caso 

merecen especial atención aquellos que afectan a la respuesta inmune. 

 Para contribuir a esta iniciativa UBS-Optimus Foundation financió el proyecto 

Visceral leishmaniasis and malnutrition in Amhara State, Ethiopia, que contempla la 

caracterización nutricional, inmunológica y parasitaria de la población infantil del 

Estado de Amhara, área en la que se registró el brote de LV arriba mencionado. Y es 

dentro del marco de este proyecto en el que se plantea el presente trabajo, cuyos 

objetivos, asociados a distintos artículos científicos, se detallan a continuación:  

 1- Evaluación de herramientas diagnósticas para la detección de la infección 

 asintomática por Leishmania (Artículo 1).  

 2- Determinar la prevalencia post brote de LV activa e infección asintomática 

 para establecer si aún existe transmisión activa (Artículo 2). 

 3- Identificar factores de riesgo asociados a la infección (Artículo 3). 

 4- Estudiar la influencia de la malnutrición en la inmunidad adaptiva y su 

 posible asociación a la susceptibilidad a infección y/o enfermedad (Artículo 4).  

 

 La población de estudio de este trabajo ha estado formada por los niños de 4 a 

15 años de edad de los distritos de Libo Kemkem y Fogera (Estado de Amhara, 

Etiopía). Los datos presentados se han obtenido a través de un estudio por 

conglomerados multi-etapas y del registro del centro de tratamiento de LV de Addis 

Zemen (Estado de Amhara). Los datos sociodemográficos, antropométricos, clínicos y 

las muestras biológicas utilizadas en el estudio fueron obtenidos por profesionales de 

la salud debidamente entrenados para realizar este.   

 La población de estudio utilizada para evaluar la utilidad de las herramientas 

diagnósticas en la detección de infección asintomática, se seleccionó de sub-distritos 

con una alta prevalencia (≥ 8 casos) después del brote de LV ocurrido en 2004-2005, 

según el registro del centro de tratamiento de LV de Addis Zemen (datos de 2008) 

(Población 1). Se evaluaron dos aproximaciones serológicas (test de aglutinación 

directa-DAT, y tira inmuno-cromatográfica rápida basada en el antígeno recombinante 

rK39-rK39-ICT) y una determinación de la respuesta de hipersensibilidad retardada 



 

 

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(test de la leishmanina-LST). Para ello se encuestaron 605 niños sin LV previa y se 

detectó infección asintomática en 61 de ellos (10.1%).  Se encontraron, además, 

durante este estudio, tres casos activos de LV. Por tanto se pudo confirmar que existe 

transmisión activa de Leishmania en los sub-distritos estudiados. Además se 

comprobó que el uso combinado de DAT y LST es la mejor opción para detectar la 

infección asintomática. 

 Para determinar la prevalencia en el área de estudio se muestrearon todos los 

sub-distritos en los que había habido, de acuerdo al registro del centro de tratamiento 

de LV de Addis Zemen, al menos un caso de LV durante el brote (Población 2). De un 

total de 386 niños encuestados, sólo se encontró un caso activo de LV. Mientras que 

la prevalencia de infección asintomática fue del 1.02%. Se observó que la prevalencia 

aumentaba con la edad y que, además, era mayor entre el sexo masculino. La baja 

prevalencia observada (tanto de casos activos como de infección asintomática) indica 

que las condiciones que originaron el brote ya no persisten, y que actualmente se da 

una situación de baja transmisión. No obstante, la ausencia de datos sobre las causas 

que originaron el brote hace necesario mantener un sistema de vigilancia para poder 

predecir un nuevo brote o incremento de los casos.   

 La identificación de factores de riesgo asociados con la infección se realizó 

estudiando la Población 1. Se analizaron los datos sociodemográficos y nutricionales 

de la población infantil estudiada con respecto a la infección asintomática por 

Leishmania. Se realizaron análisis multi-variante y uni-variante. Los factores mostraron 

una asociación positiva con la infección: mayor edad, sexo masculino, malnutrición 

aguda, el hábito de dormir fuera de la casa, el cuidado de ganado, número creciente 

de miembros en la familia, historia previa de  LV en algún miembro de la familia, casa 

con techo de paja y casa con grietas en las paredes. Como factores protectores se 

encontraron: posesión de un mayor número de cabezas de ganado y gallinas por 

familia. En conclusión, estos resultados indican que factores modificables como las 

condiciones de la vivienda, la malnutrición y los hábitos personales influyen en la 

transmisión de LV, subrayando la necesidad de educación y movilización social para 

mitigar el problema. Por otra parte, la protección asociada al aumento del número de 

cabezas de ganado que posee una familia podría estar indicando bien un mayor grado 

de riqueza (con una influencia positiva en la salud y otros elementos físicos del hogar), 

o un comportamiento zoofílico del vector, de manera que el ganado hiciese de barrera 

a la transmisión; no obstante cualquiera de estas explicaciones neceistaría de un 

estudio más detallado. 



 

 

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 Para estudiar el impacto de la malnutrición sobre la inmunidad se realizó un 

análisis de parámetros hematológicos, poblaciones linfocitarias de sangre periférica, 

niveles séricos de PGE2 y de citoquinas, así como los niveles de estas últimas en el 

sobrenadante de cultivos de células mononucleadas de sangre periférica (PBMC) 

estimuladas con fitohemaglutinina (PHA) en niños de la población de estudio 

(Población X). Se observó que la malnutrición severa en el sexo masculino se asocia a 

un valor de hematocrito (HCT), niveles de hemoglobina (HGB) y leucocitos circulantes 

(WBC) más bajos que en los no malnutridos. Sin embargo, no se observaron 

diferencias para estos parámetros dentro del sexo femenino. Independientemente del 

sexo, los individuos con malnutrición severa mostraron una disminución en el número 

absoluto de leucocitos circulantes y de las sub-poblaciones de linfocitos T (CD4+ y 

CD8+) y B en comparación con los individuos no malnutridos. El análisis de 

sobrenadantes de cultivo de PBMC estimuladas con PHA mostró que los no 

malnutridos tenían una mayor concentración de IL-10, IL-2 e IFN-γ en comparación 

con los que presentaban malnutrición severa. Por el contrario, los sueros de individuos 

no malnutridos presentaron títulos más bajos de PGE2 en comparación con los 

malnutridos. Al integrar el factor infección (determinado por DAT) se observó que la 

capacidad de las PBMC de producir IL-10 e IFN-γ de manera constitutiva era mayor en 

los no malnutridos que en los malnutridos, independientemente del estatus de 

infección.  Lo que indica una mejor condición inmunológica en los individuos no 

malnutridos.  

 

 

 

 

 

 

 

 

 

 



 

 

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INTRODUCCIÓN   

 La leishmaniasis representa un complejo de enfermedades con una importante 

diversidad clínica y epidemiológica. Está causada por diversas especies de protozoos 

del género Leishmania que son transmitidos a través de la picadura de hembras de 

flebótomo, pertenecientes al género Phlebotomus en el Viejo Mundo y al género 

Lutzomyia en el Nuevo Mundo. Las diferentes formas clínicas dependen del resultado 

de la interacción entre la especie infectante y la respuesta inmune del huésped; y 

varían desde distintas manifestaciones tegumentarias (leishmaniasis cutánea, 

cutáneo-difusa o mucosa) a la leishmaniasis visceral, una afección sistémica que es 

mortal sin tratamiento. La leishmaniasis se encuentra en el grupo de las Enfermedades 

Olvidadas, debido a los limitados recursos invertidos en su diagnóstico, tratamiento y 

control, y a su fuerte relación con la pobreza. (WHO, 2010; Bern et al., 2008).      

 

I- EPIDEMIOLOGÍA DE LA LEISHMANIASIS 

 

 La epidemiología de la leishmaniasis depende de la especie de Leishmania, las 

características ecológicas de las áreas de transmisión, incluyendo la biología del 

vector, y la exposición actual y pasada de la población humana al parásito. La 

leishmaniasis es endémica en 98 países o territorios, con más de 350 millones de 

personas en riesgo, y una prevalencia (probablemente muy subestimada) de 12 

millones. De acuerdo a los datos publicados la incidencia se estima en 2 millones de 

casos nuevos al año (0.5 millones debidos a la leishmaniasis visceral, y 1.5 millones 

debidos a las diferentes formas tegumentarias). Genera una pérdida de 2 357 000 

años de vida ajustados por discapacidad, lo que sitúa a la leishmaniasis cuarta en 

cuanto a morbilidad dentro de las enfermedades tropicales y novena en un análisis 

global de las enfermedades infecciosas. La leishmaniasis visceral (LV) causa unas 50 

000 muertes al año, una tasa superada, entre las enfermedades parasitarias, sólo por 

la malaria (Desjeux, 2004; WHO, 2010).     

 La leishmaniasis presenta una amplia distribución, existiendo transmisión a 

humanos en 5 continentes. No obstante, la mayor carga de enfermedad se localiza en 

unos focos concretos. El 90% de los casos de LV ocurre en Bangladesh, Brasil, 

Etiopía, India, Nepal y Sudán; mientras que en Afganistán, Argelia, Arabia Saudí, 

Brasil, Irán, Perú y Sudán se da el 90% de los casos de leishmaniasis tegumentaria. 

Por otra parte, la distribución es dinámica, de manera que varias áreas endémicas 

muestran una amplia fluctuación en la incidencia a lo largo del tiempo, lo que en 



 

 

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ocasiones se atribuye a eventos específicos como el desplazamiento de poblaciones y 

factores climáticos. Cambios ambientales, climáticos y socioeconómicos podrían 

expandir el rango geográfico de la transmisión de la leishmaniasis (Alvar, et al., 2006; 

Chappuis et al., 2007).  

 Un fenómeno de creciente preocupación es la co-infección Leishmania/VIH, 

que intensifica la carga de enfermedad, causando formas más severas y más difíciles 

de manejar. Si bien en Europa la incidencia de esta co-infección disminuyó desde 

finales de los 90, en otras partes del mundo está aumentando lentamente, según la 

pandemia de VIH se expande a las áreas rurales endémicas de leishmaniasis. 

Particularmente, en el norte de Etiopía la tasa de VIH en enfermos de leishmaniasis 

visceral incrementó desde el 19% en los años 1998-9 al 34% en 2006-7. En Brasil, 

India, Nepal y Sudán la prevalencia aún se mantiene por debajo del 10%, pero se 

espera que aumente (WHO, 2010). 

 

I.1- Ciclo biológico 
 

La leishmaniasis está presente en ecosistemas extremadamente diversos, con 

adaptaciones específicas para cada especie de vector. Además, Leishmania es capaz 

de infectar una amplia variedad de mamíferos. Desde el punto de vista de la fuente de 

infección en humanos, la leishmaniasis puede clasificarse en dos amplias categorías: 

i) leishmaniasis zoonótica, en la que el reservorio de infección lo constituyen animales 

salvajes o domésticos; y ii) leishmaniasis antroponótica, en la que el reservorio es el 

hombre. Así, la existencia de la leishmaniasis queda determinada por una serie de 

condiciones epidemiológicas que permiten el contacto del parásito con el vector y con 

los huéspedes vertebrados, llevándose a cabo el desarrollo completo y continuo del 

ciclo biológico de Leishmania (Figura 1) (Marzinovsky y Schurenkowa, 1924; Adler, 

1940). 

  Leishmania (Leishman, 1903; Donovan, 1903; Wright, 1903) es un parásito 

digénico y dimórfico, que realiza parte de de su ciclo vital en el tubo digestivo del 

flebótomo, donde se encuentra en la forma promastigote (1.5-3 µm x 10-20 µm), 

presentando un flagelo anterior, y en el huésped vertebrado, parasitando las células 

fagocíticas del sistema retículo-endotelial en la forma amastigote (2.5 x 6.8 µm), con 

un flagelo residual. El modo en que se multiplican ambas formas del parásito es por 

fisión binaria (Bryceson, 1996). 

 

 

 



 

 

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Figura 1. Ciclo biológico de Leishmania (www.who.int/tdr/diseases/leish/default.htm) 

 

 

Leishmania es transmitida al huésped mamífero mediante la picadura de una 

hembra de flebótomo infectada. Las hembras de flebótomos vectores necesitan ingerir 

sangre para que se produzca el desarrollo completo de sus huevos. La preferencia de 

los vectores por los diferentes vertebrados varía de acuerdo a la especie y a la 

disponibilidad de huéspedes. Durante el proceso de alimentación sobre el huésped, el 

flebótomo introduce, además de los parásitos, su saliva. Esta, junto con los 

proteofosfoglicanos del parásito se cree que ejercen un papel importante en el 

establecimiento del parásito en la piel (WHO, 2010). Cuando la hembra del flebótomo 

toma sangre de un mamífero infectado, que presenta parasitemia, en el caso de la 

leishmaniasis visceral, o de la propia úlcera en las cutáneas, esta puede ingerir 

macrófagos infectados con amastigotes de Leishmania. En el interior del aparato 

digestivo del vector, los amastigotes se transforman en promastigotes; estos se 

multiplican por fisión binaria y van migrando hacia la porción anterior del aparato 

digestivo, donde se transformarán en promastigotes metacíclicos, que es la forma 

infectiva que transmitirá el vector (Molyneux y Killick-Kendrick, 1987).  

 Al inocular los parásitos en la dermis del huésped, aquellos que sobreviven a la 

acción del complemento se adhieren rápidamente a las células residentes o a las 

Leishmaniasis 
Visceral 

Leishmaniasis 
Cutánea 



 

 

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reclutadas pertenecientes al linaje de monocitos/macrófagos (Moll et al., 1995). La 

adhesión está mediada por receptores de membrana en las células diana que se unen 

a factores del complemento adheridos al parásito; posteriormente este penetra al 

interior celular mediante fagocitosis. Leishmania queda entonces englobada en un 

fagolisosoma que evoluciona a lo largo de la ruta fagocítica hasta convertirse en una 

vacuola parasitófora, con unas características finales entre endosoma tardío y 

lisosoma (Amer y Swanson, 2002; Waller y McConville, 2002). Una vez en la vacuola, 

y tras 2 a 5 días, los promastigotes se transforman en amastigotes (Antoine et al., 

1998; Handman, 1999). Los amastigotes se dividen en el interior de la vacuola 

parasitófora hasta que la célula no puede acumular más y se rompe liberándolos al 

exterior, donde son captados por otras células competentes.  

 En el caso de las especies o cepas viscerotrópicas, una vez la infección se ha 

establecido, los parásitos pueden persistir en el huésped durante toda su vida en las 

células del sistema retículo-endotelial, en médula ósea o bazo, bien en forma activa, 

causando enfermedad, o acantonados cursando una infección subclínica, 

asintomática, con la posibilidad de reactivarse y causar patología si consiguen escapar 

al control del sistema inmune del huésped. Sin embargo, las especies o cepas 

dermotrópicas generalmente se mantienen localizadas junto al punto de inoculación, 

causando la enfermedad cutánea.  Cualquier dispersión de las especies dermotrópicas 

suele ser tardía y sólo hacia porciones de piel adyacentes (produciendo lesiones 

satélites), o hacia el tejido o nódulos linfáticos próximos. Algunas especies del 

subgénero Viannia (como L. braziliensis) migran a la mucosa oro-faríngea donde 

pueden permanecer en estado latente durante años hasta reactivarse y causar 

lesiones mucosas destructivas (Alvar, 2001). 

 

II- LEISHMANIASIS VISCERAL 

La Leishmaniasis Visceral (LV), también llamada Kala-azar (KA), es endémica 

en 65 países. Actualmente se reconocen dos especies de Leishmania asociadas a 

esta enfermedad: Leishmania donovani y Leishmania infantum. La primera se asocia a 

una transmisión antroponótica y se distribuye en los principales focos del sub-

continente indio y el este de África, si bien hay datos que indican que en determinadas 

áreas de este último foco la transmisión podría ser antropozoonótica. L. infantum se 

asocia a una transmisión zoonótica, con el perro como principal reservorio, y se 

distribuye principalmente por toda la cuenca mediterránea, algunas regiones de 

Oriente Medio y Asia, y por Sudamérica (WHO, 2010) (Figura 2). 



 

 

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Figura 2. Distribución geográfica de la LV (WHO, 2010). 

 

II.1- (Re-)emergencia de la leishmaniasis visceral 

 La LV es una enfermedad dinámica y las circunstancias asociadas a su 

transmisión varían de un modo continuo en función de los cambios asociados al 

ambiente, y del comportamiento humano. De acuerdo con la OMS los programas de 

control de la leishmaniasis activos son escasos y la mortalidad y morbilidad asociadas 

a la leishmaniasis muestran una preocupante tendencia al alza en todo el Mundo 

(WHO, 2010). 

 En los últimos años, se ha documentado la emergencia de la LV en diversas 

áreas en las que no se había descrito antes la presencia de la enfermedad, como en la 

ciudad de Posadas, Argentina (Salomón et al., 2008), el estado de Amhara, noroeste 

de Etiopía (Alvar et al., 2007), o Bután (Bhattacharya et al., 2010). Así como la re-

emergencia en focos que se consideraban controlados en Uzbekistán y Tajikistán 

(Alam et al., 2009). Y la rampante propagación de la LV urbana en Brasil (Maia-

Elkhoury et al., 2008). 

 Entre los factores de riesgo asociados a la re-emergencia de la LV se 

encuentran aquellos relacionados con el ambiente, que aumentan la exposición al 

vector y/o estrechan el contacto con los reservorios, pero también están aquellos que, 



 

 

16

en el huésped, facilitan la evolución de la infección a la enfermedad, y la propagación 

en la comunidad.  

 

 II.1.1- Factores ambientales 

 La leishmaniasis se caracteriza por una distribución en microfocos, este tipo de 

distribución se debe a condiciones micro-ecológicas que afectan al vector, el parásito y 

al reservorio. En función de la eco-epidemiología de cada foco en particular. Las 

alteraciones del ambiente, de origen natural o por acción humana, pueden generar un 

aumento o disminución de la incidencia de la enfermedad. Entre los cambios 

ambientales que afectan la incidencia se encuentran la urbanización, la domesticación 

de los ciclos de transmisión y la incursión de asentamientos humanos en entornos 

forestales (WHO, 2010). 

 

 II.1.2- Cambio climático 

 La distribución de la leishmaniasis presenta una asociación muy fuerte con el 

clima, estando muy afectada por factores como la lluvia, la temperatura atmosférica y 

la humedad. Estos factores tienen efecto en la distribución, supervivencia y tamaño de 

población de vectores y reservorios. Además, grandes efectos climáticos, como las 

sequías o inundaciones pueden provocar movimientos masivos de población hacia 

áreas endémicas, incrementando el número de individuos susceptibles (WHO, 2010). 

 

 II.1.3- Movimientos de población 

 Los movimientos de población, por causas ambientales o provocadas por la 

acción del hombre, están detrás del origen de algunas epidemias de LV; bien al poner 

a una población en contacto con un ciclo de transmisión, o al introducir un número 

importante de personas infectadas en un área no endémica en la que existe la 

posibilidad de transmisión vectorial. Un ejemplo dramático fue la epidemia de LV en la 

provincia de Western Upper Nile, en el sur de Sudán, asociada a la movilización de 

población a un área de transmisión zoonótica, provocada por la guerra civil que sufrió 

el país (1984-1994), y generando una epidemia que causó la muerte de 100 000 

personas (WHO, 2010; Zijlstra y El-Hassan, 2001).    

  



 

 

17

 II.1.4- Inmunodepresión 

 La respuesta inmune innata juega un papel crucial en la resistencia del 

huésped a la infección por Leishmania. Esta respuesta actuaría tanto en el control de 

la multiplicación del parásito en la fase inicial de la infección, como en la generación de 

una cascada inmunoreguladora dirigida a ejercer una respuesta específica contra el 

parásito (Peruhype-Magalhães et al., 2005). En el caso de la LV, hay dos fenómenos 

que, al alterar esta respuesta inmune, están especialmente relacionados con un mayor 

riesgo de desarrollo de la enfermedad: la co-infección con VIH y la malnutrición.   

 El desarrollo de la pandemia de VIH/SIDA durante las últimas décadas ha 

modificado el espectro de la LV, tanto a nivel clínico como epidemiológico. Este 

fenómeno surge como consecuencia del solapamiento de ambas infecciones, que 

adquiere mayor importancia en los últimos años como consecuencia de la ruralización 

del SIDA y la urbanización de la leishmaniasis. En los pacientes co-infectados los dos 

patógenos ejercen un efecto sinérgico, lo que tiene implicaciones muy importantes en 

su expresión y propagación. El tiempo de desarrollo de SIDA se ve acelerado y la 

leishmaniasis puede no tener una presentación clásica. Estos pacientes presentarán 

una pobre respuesta a la terapia y una elevada tasa de recaídas; así como una 

elevada parasitemia y una serie de manifestaciones atípicas que dificultarán y 

retrasarán el diagnóstico. Así, los enfermos co-infectados engrosarían el número de 

reservorios humanos en áreas donde la transmisión es antroponótica. Pero además, 

las mismas características podrían ayudar a crear nuevos focos de transmisión 

antroponótica en áreas donde la LV es zoonótica. Quizás un ejemplo de la magnitud 

de este problema lo constituye el sur de Europa, que fue el paradigma de la co-

infección Leishmania/VIH, encabezando la lista de casos reportados, durante la 

primera década de la pandemia de VIH/SIDA, y donde se observó que la prevalencia 

de LV en enfermos de SIDA era entre 100 y 2000 veces mayor que en individuos 

inmunocompetentes u otros grupos de inmunodeprimidos no VIH+ (Desjeux y Alvar, 

2003; Molina et al., 2003; Cruz et al., 2006-a; Alvar et al., 2008).  

 Existen fuertes evidencias que indican un vínculo entre la malnutrición y un 

déficit de las respuestas immunes innata y adaptativa, de manera que en casos de 

deficiencia nutricional la LV presentaría formas más severas y letales. Particularmente 

en niños menores de 5 años la malnutrición juega un papel significativo en la evolución 

clínica de la LV. Si bien las bases que explican la asociación entre malnutrición y LV 

no están claras, se sabe que la malnutrición genera inmunodepresión y que esta es un 

factor de riesgo para el desarrollo de la enfermedad. La lactancia materna se asocia 



 

 

18

con una mayor posibilidad de permanecer asintomático tras la infección, mientras que 

un peso bajo al nacer se asocia con una mayor posibilidad de desarrollar la 

enfermedad tras la infección. Del mismo modo, los niños que presentan bajas medidas 

antropométricas tienen mayor riesgo de desarrollar LV que los sanos (Maciel et al., 

2008; Malafaia, 2009). A nivel epidemiológico se ha descrito el papel de la malnutrición 

en el desarrollo de epidemias de LV en el este de África; un claro ejemplo es el 

descrito por Marlet et al. (2003), que describen un brote de LV que afectó a 904 

individuos entre mayo del año 2000 y agosto del 2001 en los distritos de Wajir y 

Mandera en una región fronteriza entre Kenia, Somalia y Etiopía. 

 

  II.1.5- Fallo terapéutico y resistencia a fármacos 

 La quimioterapia es crítica tanto para el paciente como para reducir el número 

de reservorios en áreas donde la transmisión es antroponótica. De manera que la 

monitorización del acceso al tratamiento en las diversas áreas endémicas es una pieza 

clave a la hora de desarrollar un programa de control, esta estrategia debe incluir 

también el control de la calidad del fármaco. Un acceso no controlado al mismo puede 

llevar a un uso incorrecto, tratamiento con dosis sub-óptimas, fallo terapéutico y, a 

largo plazo, el desarrollo de resistencias. Un fenómeno que ha merecido especial 

atención ha sido el desarrollo de resistencia al tratamiento con antimoniales 

pentavalentes (SbV). Estos han sido la primera opción para tratar la LV durante las 

últimas siete décadas, sin embargo en determinadas áreas de India y Nepal, el fallo 

terapéutico asociado a los SbV llega al 60%; lo que implica un mayor número de 

individuos infectados y capaces de actuar como reservorios. Afortunadamente, en los 

últimos 10 años se ha progresado bastante en la terapéutica de la leishmanaisis, y se 

cuenta con nuevas opciones como las formulaciones lipídicas de Anfotericina B, la 

miltefosina y la paromomicina. No obstante continúa siendo necesario fortalecer la 

farmacovigilancia de los tratamientos antileishmania, y monitorizar la aparición de 

resistencia a fármacos (Dujardin, 2006; WHO, 2010; Den Boer et al., 2011). 

 

II.2- Presentación clínica 

 La mayoría de las infecciones cursan de manera asintomática, si bien el 

seguimiento a largo plazo indica que una pequeña proporción de estos individuos 

termina desarrollando una LV clínica.  



 

 

19

 En aquellos en los que se desarrolla la enfermedad tras la infección, el periodo 

de incubación puede variar de unos pocos días a un año, y el desarrollo generalmente 

es gradual. Los síntomas más comunes son fiebre, malestar, pérdida de peso y 

anorexia. Y los signos más frecuentes son esplenomegalia, en ocasiones asociada a 

hepatomegalia, caquexia y palidez de las membranas mucosas. El oscurecimiento de 

la piel es un signo encontrado típicamente en India (kala-azar en Hindi significa fiebre 

negra). A medida que la enfermedad progresa aparecen signos de malnutrición, 

siendo frecuente la aparición de infecciones concomitantes. Estos síntomas persistirán 

durante semanas o meses. Sin tratamiento, la mayoría de los casos pueden ser 

fatales, siendo las co-infecciones bacterianas, hemorragias masivas o anemia 

(causada por un estado inflamatorio persistente) las principales causas de muerte 

(Dujardin, 2006; Chappuis et al., 2007; WHO, 2010).  

 Las manifestaciones clínicas de la LV pueden ser diferentes en función de si 

esta es endémica, esporádica o epidémica (Cascio et al, 2002; WHO, 2010):  

 i) La LV endémica, en general, presenta un curso relativamente crónico. 

Cuando el agente causal es L. infantum, como en el sur de Europa, el norte de África, 

Asia central y oriental, y América, el grupo de edad más afectado son niños entre uno 

y diez años de edad. No obstante, en los últimos años la pandemia de SIDA y el 

aumento del uso de inmunosupresores han provocado que la mitad de los casos de LV 

de Europa ocurran en adultos. Cuando el agente causal es L. donovani, como en el 

este de África e India, la mayor incidencia se da en niños y adultos jóvenes.  

 ii) La LV esporádica suele ocurrir en población no autóctona de cualquier edad 

que entra en un foco endémico. Estos casos suelen ser agudos, y la enfermedad 

progresa rápidamente. Estos pacientes son más propensos a desarrollar formas 

complicadas de la enfermedad, como anemia hemolítica severa, daño renal y 

hemorragia mucosa. 

 iii) La LV epidémica ocurre generalmente en áreas de transmisión 

antroponótica, en este caso todos los grupos de edad son susceptibles, excepto 

aquellos que adquirieron inmunidad durante una epidemia previa. Se observan formas 

agudas de la enfermedad, y la tasa de mortalidad es generalmente elevada. 

 Se considera que determinados factores de riesgo pueden facilitar la 

progresión de la enfermedad tras la infección, como son la inmunodepresión (donde 

juegan un papel importante, en el contexto de la LV, la malnutrición y la infección por 



 

 

20

VIH), la susceptibilidad genética y  la existencia de otras infecciones concomitantes 

(Dujardin et al., 2006; WHO, 2010).  

 

II.3- Respuesta inmune y patogénesis  

 El desarrollo de las diferentes formas clínicas de la leishmaniasis está 

determinado por la respuesta inmune del huésped y la especie de Leishmania 

implicada en la infección.  En la mayor parte de los casos, la presencia del parásito 

desencadena una respuesta compleja por parte del huésped, en cuya fase inicial 

activa la respuesta inmune innata con la participación de células NK y producción de 

IL-12 que conduce a la respuesta inmune adaptativa gobernada principalmente por 

células colaboradoras de tipo 1 (Th1), en la que participan células dendríticas 

presentadoras de antígenos y células efectoras T CD4+ y CD8+ que secretan 

citoquinas pro-inflamatorias como la IL-12, IFN-γ y TNF-α, que a su vez activan los 

mecanismos leishmanicidas de los macrófagos infectados como son la producción de 

óxido nítrico y reactivos intermediarios del oxígeno (Murray et al., 2005).  Esta 

respuesta genera una inmunidad celular sistémica y específica contra el parásito que 

permite controlar su multiplicación y diseminación, siendo capaz de mantener la 

infección subclínica y de proteger al individuo frente a futuras infecciones por 

Leishmania.  

 En otros casos, el parásito es capaz de evadir la respuesta inmune específica 

del huésped, afectando la capacidad presentadora de las células dendríticas, que evita 

el desarrollo de una respuesta Th1 específica, y también la capacidad leishmanicida 

de los macrófagos (MacMahon-Pratt  y Alexander, 2004), lo que resulta en una 

inmunosupresión específica frente a Leishmania que lleva a la multiplicación y 

diseminación  del parásito por diferentes órganos y tejidos y a la aparición de los 

síntomas clínicos característicos de la leishmaniasis visceral. La infección por L. 

donovani o L. infantum genera hiperplasia reticuloendotelial, afectando a bazo, higado, 

mucosa del intestino delgado, medula ósea, ganglios linfáticos y otros tejidos linfoides. 

Pudiéndose observar atrofia de estos órganos. Esto causa una reducción de la vida 

media de leucocitos y eritrocitos, provocando granulocitopenia y anemia. Puede 

alterarse la función hepática, disminuyendo el tiempo de producción de protrombina; 

esto, junto con la trombocitopenia puede generar hemorragia severa. También se 

puede observar hipoalbuminemia, que se asocia a la aparición de edema. Son también 

comunes la hipergammaglobulinemia y la activación policlonal de células B. En la fase 

avanzada la aparición de enfermedades concomitantes es frecuente, especialmente 



 

 

21

neumonía, disentería y tuberculosis, que son la causa común de muerte en estos 

enfermos (WHO, 2010). Solo la quimioterapia efectiva es capaz de reducir la carga 

parasitaría en estos pacientes, lo que a su vez permite el desarrollo de una respuesta 

inmune tipo Th1 que coopera con el tratamiento en la recuperación completa del 

paciente con LV  y es capaz de mantenerlo inmune frente a futuras infecciones. 

  

  

 II.3.1-Leishmaniasis dérmica post kala-azar 

 La leishmaniasis dérmica post-kala-azar (PKDL) es una complicación de la LV; 

se caracteriza por un sarpullido macular, maculopapular y/o nodular en pacientes que 

se han recuperado (o se están recuperando) de un episodio de LV. El sarpullido 

generalmente comienza alrededor de la boca, desde donde se propaga a otras partes 

del cuerpo en función de la severidad. Se observa principalmente in Sudán e India, 

ocurriendo tras el tratamiento de LV en el 50% y 5-10% de los casos respectivamente. 

El intervalo entre el episodio de LV y el PKDL varía de 0 a 6 meses en Sudán y de 2 a 

3 años en India. Probablemente los enfermos de PKDL juegan un papel importante 

como reservorios entre epidemias de LV. El mecanismo exacto por el que se 

desarrolla el PKDL se desconoce, sin bien existe una creciente evidencia de que la 

patogénesis está mediada por la respuesta inmune (Zijlstra et al., 2003).  
 

 

 

      Figura 3. Esplenomegalia en paciente de  

        LV (A). Formas nodulares de PKDL  

        facial (B). (Autor: Endalamaw Gadisa) 

         

 

 

 

II.4- Control de la leishmaniasis visceral 

 La transmisión de la leishmaniasis se mantiene  en un complejo sistema 

biológico que implica huésped humano, parásito, vector y en algunas ocasiones un 

reservorio animal. Por tanto, cualquier estrategia de control requiere de un adecuado 

conocimiento del ciclo biológico en cada contexto epidemiológico. Se requerirá un 

A  B 



 

 

22

conocimiento exhaustivo de la dinámica de la enfermedad, y deberán combinarse 

distintas aproximaciones para romper la cadena de transmisión: diagnóstico, 

tratamiento y profilaxis, control del vector y del reservorio (si procede). 

 

  II.4.1- Diagnóstico 

 Las manifestaciones clínicas de la leishmaniasis no son específicas y pueden 

ser compartidas con las de otras infecciones sistémicas. Además, en función del área 

geográfica, habrá que prestar especial anterior al diagnóstico diferencial con otras 

patologías como la malaria o la esquistosomiasis. Por tanto, en la medida de lo 

posible, se precisa la confirmación mediante alguna prueba de laboratorio. 

 Diagnóstico parasitológico 

 La demostración del parásito tras crecimiento en cultivo, o mediante 

observación microscópica, de aspirados de médula ósea, bazo o ganglio linfático es la 

prueba confirmatoria de la infección y aun constituye el método gold standard. La 

observación microscópica de aspirados de bazo presenta una gran sensibilidad (93-

99%), seguido de la de médula ósea (52-85%). El estudio del aspirado de ganglio 

linfático presenta una sensibilidad menor (52-65%), si bien en el este de África se ha 

reportado un buen rendimiento (Siddig et al., 1988; Zijlstra et al., 1992; Sundar y Rai, 

2002; WHO, 2010). 

 Una alternativa que permite mejorar la sensibilidad en la detección del parásito 

es la reacción en cadena de la polimerasa (PCR). Además, la especificidad de la PCR 

puede adaptarse a necesidades particulares al utilizar como diana regiones 

hipervariables, variables o conservadas, y/o combinarse con otras técnicas de biología 

molecular, como la secuenciación, hibridación con sondas o digestión con 

endonucleasas de restricción. De este modo es posible caracterizar de manera rápida 

el parásito presente en la muestra en el nivel taxonómico de complejo, especie o 

incluso aislado individual, solventando los inconvenientes de las técnicas clásicas de 

identificación y caracterización (Reithinger y Dujardin, 2007). No obstante, una de las 

mayores aportaciones de la PCR al diagnóstico de la LV es su elevada sensibilidad en 

muestras de sangre periférica (70-100%), permitiendo realizar el diagnóstico a partir 

de muestras biológicas obtenidas mediante procedimientos menos invasivos (Antinori 

et al., 2007).  Lamentablemente, su uso aún s emantiene restringido a hospitales de 

referencia y a centros de investigación (WHO, 2010). 



 

 

23

 Diagnóstico inmunológico 

El diagnóstico inmunológico, ya sea a través de la evaluación de la respuesta 

humoral o celular, tiene gran interés en el caso de la LV. Pues es utilizado tanto en el 

diagnóstico de la enfermedad, como en la detección de infección asintomática o 

infecciones pasadas, lo que resulta de gran utilidad en estudios epidemiológicos. 

Los métodos serológicos aprovechan la elevada producción de IgG anti-

Leishmania durante la fase activa de la enfermedad. Se utilizan varios formatos, desde 

enzimoinmunoensayos y tests de aglutinación a inmuno-blots e inmunofluorescencia 

directa, considerándose esta última el método de referencia en el diagnóstico 

serológico de la LV. No obstante, no conviene olvidar que estos métodos no son 

capaces de distinguir entre infecciones activas o pasadas y que, en función del 

antígeno utilizado, podría obtenerse reacciones cruzadas con otros agentes 

infecciosos o enfermedades autoinmunes. Por otra parte, estos métodos pueden 

perder hasta un 50% de sensibilidad en el caso de enfermos coinfectados con el VIH 

(Cruz et al., 2006-a; Cruz et al., 2006-b). Existen dos métodos serológicos, el test de 

aglutinación directa (DAT) y una tira inmunocromatográfica rápida (rK39-ICT), que han 

sido desarrollados para poder ser aplicados sobre el terreno. El primero utiliza 

promastigotes completos y permite determinar el título de anticuerpos; el segundo 

utiliza el antígeno recombinante rK39 y es cualitativo. Ambos son baratos y fáciles de 

usar y han mostrado una sensibilidad (93-94%) y especificidad (95-97%) adecuadas 

en diversas áreas endémicas (Chappuis et al., 2006).  

La respuesta inmune mediada por células T juega un papel crucial en el control 

de la infección por Leishmania (Murray et al., 1989). En infecciones asintomáticas o 

tras un tratamiento exitoso se desarrolla una respuesta de linfoproliferación específica 

que puede valorarse in vivo a través de la prueba de la leishmanina o test de 

Montenegro y ex vivo mediante un ensayo de linfoproliferación en placa.  El test de 

Montenegro se encarga de medir una respuesta de hipersensibilidad retardada tras la 

inoculación intradérmica de una solución de promastigotes de Leishmania formolados 

(Weigle et al., 1991). Los ensayos de linfoproliferación, al determinar la capacidad de 

proliferación linfocitaria frente a antígeno de Leishmania, proporcionan (al igual que el 

test de Montenegro) información sobre la capacidad del sistema inmune de montar 

una respuesta celular específica y protectora frente al parásito (Sacks et al., 1987). 

Los ensayos basados en la determinación de la respuesta celular frente a Leishmania 

no presentan utilidad a la hora de diagnosticar un episodio de LV, pero sí aportan 



 

 

24

mucha información sobre la tasa de contacto con el parásito en población de área 

endémica (Alvar et al., 2007; Gidwani et al., 2009). 

  

II.4.2- Tratamiento 

El tratamiento debe suministrarse sólo a aquellos casos confirmados, y en 

ocasiones se aportará un suplemento nutricional o de rehidratación. De manera ideal, 

el tratamiento debe curar al paciente, reducir el riesgo de recaídas y de aparición de 

PKDL y reducir la transmisión. Durante las últimas siete décadas, los antimoniales 

pentavalentes han sido el tratamiento de primera opción para la LV, estando en la 

segunda línea la pentamidina y la anfotericina B deoxicolato. Afortunadamente, en los 

últimos diez años han aparecido nuevas opciones como las formulaciones lipídicas de 

anfotericina B, la miltefosina o la paromomicina. Actualmente se recomienda la 

combinación de fármacos, ya que ofrece varias ventajas: i) acorta el periodo de 

tratamiento, aumentando la adherencia al mismo, ii) se reduce la dosis, y por tanto 

disminuyen los efectos  tóxicos y el precio, y iii) disminuye la posibilidad de aparición 

de cepas resistentes, aumentando así la vida útil de los fármacos disponibles (WHO, 

2010). 

 

II.4.3- Control del reservorio 

En áreas donde la transmisión es antroponótica, las piezas clave en el control 

del reservorio serán un programa de detección activa de casos, vigilancia y la 

disponibilidad de un tratamiento eficaz. En áreas en que la LV es zoonótica (siendo el 

perro principal reservorio), es preciso determinar la distribución y frecuencia de la 

infección. Es importante considerar que no todos los perros infectados manifiestan 

síntomas de la enfermedad; por tanto, deben emplearse herramientas validadas en 

cada contexto epidemiológico a la hora de determinar la tasa de infección en los 

reservorios. Idealmente, los perros infectados deberían ser eliminados; si bien esta 

medida no ha resultado del todo eficaz en áreas donde se ha empleado durante largo 

tiempo, como en Brasil. Alternativamente, se debe potenciar el uso de insecticidas y/o 

repelentes, bien en forma de lociones o de collar (WHO, 2010).   

 

 



 

 

25

II.4.4- Control vectorial 

Un control vectorial efectivo tendrá un gran impacto en la transmisión del 

parásito, particularmente si se realiza en el hábitat doméstico y peridoméstico. Es de 

vital importancia conocer la ecoepidemiología de la LV en el área de intervención, así 

como la especie de vector (o vectores) implicada, su hábitat, rango de vuelo, 

preferencias alimentarias, lugares  de reposo y refugio, así como su estacionalidad. Se 

puede proceder al control químico mediante el uso de insecticidas residuales de 

aplicación intradomiciliaria o el de telas mosquiteras impregnadas en insecticida 

(WHO, 2010).     

 

III- LEISHMANIASIS VISCERAL EN ETIOPÍA 

 Se desconoce la carga total de LV en Etiopía, pero se estima una incidencia de 

aproximadamente 4 000 casos clínicos anuales (Alvar et al., 2008). También se 

considera emergente, pues se está propagando hacia áreas no endémicas dentro del 

país. Diferentes estudios epidemiológicos, realizados de forma esporádica, han 

identificado hasta 40 focos (Fig. 4). En la actualidad hay transmisión de  LV en tres 

diferentes zonas ecológicas: en las tierras bajas del noroeste y suroeste, a menos de 1 

500 metros sobre el nivel del mar, y en las tierras altas del la región centro-norte, a 

más de 1 800 metros sobre el nivel del mar. 

 El gran foco de LV de Humera y Metema se encuentra al noroeste del país, en 

las tierras bajas en la frontera con Sudán. Es el principal foco de LV de Etiopia, y 

acumula el 60% de los casos. Estas regiones son semiáridas, y el vector 

(Phlebotomus orientalis) se encuentra asociado a los suelos arcillosos y a los bosques 

de acacia. Desde  principios de 1990 ha habido pequeños brotes en este foco debido a 

nuevos proyectos agrícolas a gran escala y a la ruta comercial desde Port-Sudan a 

Addis Abeba, que atraviesa los grandes focos de LV del sur de Sudan (Mengesha y 

Abuhoy, 1978; Maru 1979). Este foco tiene la mayor tasa de confección 

Leishmania/VIH, hasta un 40% de los pacientes con LV está infectado por el VIH 

(Alvar et al., 2008).  

El 20% de los casos del país ocurre en la región suroeste, que engloba la 

sabana del suroeste de Etiopia, el área del lago Abaya, la meseta del río Omo, el área 

de Aba Roba y el valle de los ríos Segen y  Woito (Ayele y Ali, 1984). En esta región 

los vectores implicados son P. martini y P. celiae que se encuentran en asociación con 

los nidos de termita (Macrotermes termite) (Gebre-Michael y Lane, 1996). En esta 



 

 

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zona, en las regiones de Afder y Liban, ha habido brotes de LV que han afectado 

regiones fronterizas de Kenia y Somalia (Marlet et al., 2003). 

 

 

Figura 4. Distribución de la leishmaniasis en el Este de África 

 

 El foco más reciente se encuentra al nordeste del país, en los distritos de Libo 

Kemkem y Fogera (estado de Amhara), que se encuentran a 1800-2000 metros sobre 

el nivel del mar. En esta región, las actividades agrícolas han reducido la vegetación 

natural a grupos acacias dispersas por todo el territorio. Durante la estación de lluvias 

la mayoría del área queda inundada, durante la estación seca el suelo queda surcado 

por profundas grietas. Esta área se consideraba libre de LV hasta 2005, cuando 

Médicos sin Frontera-Grecia (MSF-G) reportó un brote en el distrito de Libo Kemkem 

(Alvar et al., 2007).  



 

 

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IV- BIBLIOGRAFÍA 

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Babaniyi O, Gudeta N, Cañavate C, Bern C (2007). Kala-azar outbreak in Libo 

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Cascio A, Corbellino M (2007). Clinical use of polymerase chain reaction performed on 

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(2010). Occurrence & significance of kala-azar in Bhutan. Indian Journal of Medical 

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diagnostic performance of the direct agglutination test and rK39 dipstick for visceral 

leishmaniasis. British Medical Journal; 333: 723. 

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Comparison of new diagnostic tools for management of pediatric Mediterranean 

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access. Clinical Microbiology and Infection; 17: 1471-7.  

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Annals of Tropical Medicine and Parasitology; 97(S1): S3–S15.   

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(Diptera: Phlebotominae) as vectors of visceral leishmaniasis in the Aba Roba focus, 

southern Ethiopia. Medical Veterinary Entomology; 10: 53-62. 

- Gidwani K, Rai M, Chakravarty J, Boelaert M, Sundar S (2009). Evaluation of 

leishmanin skin test in Indian visceral leishmaniasis. American Journal of Tropical 

Medicine and Hygiene; 80: 566-7. 

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India. British Medical Journal; 1: 1252-4. 

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(2008). Visceral leishmaniasis in Brazil: trends and challenges. Cadernos de Saúde 

Pública, Rio de Janeiro; 24: 2941-7. 

- Maciel B L L, Lacerda H G, Queiroz J W, Galvão J, Pontes N N, Dimenstein R, 

McGowan S E, Pedrosa L F C, Jerônimo S M B (2008). Association of nutritional status 

with the response to infection with Leishmania chagasi. American Journal of Tropical 

Medicine and Hygiene; 79: 591-8. 

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leishmaniasis: a review. Parasite Immunology; 31: 587-96. 

- Marlet M V L, Sang D K, Ritmeijer K, Muga R O, Onsongo J, Davidson R N (2003). 

Emergence or re-emergence of visceral leishmaniasis in areas of Somalia, 

northeastern Kenya, and south-eastern Ethiopia in 2000-01. Transactions of the Royal 

Society of Tropical Medicine and Hygiene; 97: 515-8.  

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leishmaniasis in hospitalized patients in Northwestern Ethiopia. American Journal of 

Tropical Medicine and Hygiene; 28: 15-8. 

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Humera region of Ethiopia. Tropical Geographical Medicine; 30: 199-206. 

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mice harbor persistent parasites and mediate an antigen-specific T-cell immune 

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leishmaniasis. Lancet; 366: 1561. 

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Teixeira-Carvalho A, Figueiredo R M, Guimarães-Carvalho S F, Ferrari T C, Correa-

Oliveira R (2005). Immune response in human visceral leishmaniasis: analysis of the 

correlation between innate immunity cytokine profile and disease outcome. 

Scandinavian Journal of Immunology; 62: 487-95. 

- Reithinger R, Dujardin J C (2007). Molecular diagnosis of leishmaniasis: current 

status and future applications. Journal of Clinical Microbiology; 45: 21-5. 

- Sacks D L, Lal S L, Shrivastava S N, Blackwell J, Neva F A (1987). An analysis of T 

cell responsiveness in Indian kala-azar. Journal of Immunology; 138: 908-13. 

- Salomón OD, Sinagra A, Nevot MC, Barberian G, Paulin P, Estevez JO, Riarte A, 

Estevez J (2008). First visceral leishmaniasis focus in Argentina. Memórias do Instituto 

Oswaldo Cruz, Rio de Janeiro; 103: 109-11.  

- Siddig M, Ghalib H, Shillington D C, Petersen E A (1988). Visceral leishmaniasis in 

the Sudan: comparative parasitological methods of diagnosis. Transactions of the 

Royal Society of Tropical Medicine and Hygiene; 82: 66-8. 



 

 

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- Sundar S, Rai M (2002). Laboratory Diagnosis of Visceral Leishmaniasis. Clinical and 

Diagnostic Laboratory Immunology; 9: 951-8. 

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morphology and function Leishmania parasites. International Journal of Parasitology;  

32: 1435-45. 

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skin test standardization and evaluation of safety, dose, storage, longevity of reaction 

and sensitization. American Journal of Tropical Medicine and Hygiene; 44: 260-71. 

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kala-azar dermal leishmaniasis. Lancet Infectious Diseases; 3: 87-98. 

 
 
 
 
 
 
 
 
 
 



 

 

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V- EL PROYECTO Visceral Leishmaniasis and Malnutrition in Amhara State, 

Ethiopia DE LA FUNDACIÓN UBS-Optimus  
 

 El brote de LV en los distritos Libo Kemkem y Fogera  resultó en la muerte de 

centenares de personas y puso en riesgo a más de 420 000 habitantes, por ello la 

OMS aconsejó su estudio y control. En respuesta a ello, el Centro Colaborador de la 

OMS para Leishmaniasis del Centro Nacional de Microbiología y el Centro Nacional de 

Medicina Tropical (ambos pertenecientes al Instituto de Salud Carlos III), y en 

colaboración con el Armauer Hansen Research Institute de Etiopía propusieron a la 

Fundación UBS-Optimus un proyecto para estudiar la leishmaniasis visceral desde un 

punto de vista nutricional inmunológico y parasitológico. Teniendo en cuenta que se 

trata de una región con una elevada tasas de malnutrición, y que la relación 

malnutrición-leishmaniasis visceral ha quedado ya demostrada; este proyecto pretende 

establecer el papel de la malnutrición en la epidemiología de la leishmaniasis visceral 

en este nuevo foco e identificar los factores socio-económicos están asociados a ella. 

De este modo se plantearán nuevas estrategias a la hora de prevenir la enfermedad. 

 Es en el marco de este proyecto donde se desarrolla el trabajo de la presente 

Tesis Doctoral.  

 Para mostrar mejor las características físicas del entorno, las condiciones de la 

población y las circunstancias en las que se ha realizado el trabajo, se ha incluido una 

galería de imágenes (ANEXO I) con fotos realizadas a lo largo del estudio que 

documentan sus diferentes fases y que pueden ayudar a comprender mejor el trabajo 

realizado. 

 



 

 

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VI- OBJETIVOS 

1- Evaluación de herramientas diagnósticas para la detección de la infección 

asintomática por Leishmania (Artículo 1).  

2- Determinar la prevalencia post brote de LV activa e infección asintomática para 

establecer si aún existe transmisión activa (Artículo 2). 

3- Identificar factores de riesgo asociados a la infección (Artículo 3). 

4- Estudiar la influencia de la malnutrición en la inmunidad adaptiva y su posible 

asociación a la susceptibilidad a infección y/o enfermedad (Artículo 4). 

 

 



 

 

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VII- ARTÍCULOS CIENTÍFICOS 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



For Peer Review

 
 
 

 
 

 
 

Usefulness of rK39-immunocromatrographic test, direct 
agglutination test, and leishmanin skin test to detect 

asymptomatic Leishmania infection in children from a new 

visceral leishmaniasis focus in Amhara State (Ethiopia) 
 
 

Journal: American Journal of Tropical Medicine & Hygiene 

Manuscript ID: AJTMH-11-0196 

Manuscript Type: Original Research Paper 

Date Submitted by the 
Author: 

04-Apr-2011 

Complete List of Authors: Gadisa, Endalamaw; AHRI/ALERT, Armauer Hansen Research 
Institute 
Custodio, Estefanía; Instituto de Salud Carlos III, Centro Nacional de 
Medicina Tropical 
Cañavate, Carmen; Centro Nacional de Microbiología, Instituto de 
Salud Carlos III, WHO Collaborating Centre for Leishmaniasis, 
Servicio de Parasitología 
Sordo, Luis; Instituto de Salud Carlos III, Centro Nacional de 
Epidemiología 
Abebe, Zelalem; Amhara Regional State Research Laboratory, Health 
Centre 
Nieto, Javier; Centro Nacional de Microbiología, Instituto de Salud 
Carlos III, WHO Collaborating Centre for Leishmaniasis, Servicio de 
Parasitología 
Chicharro, Carmen; Centro Nacional de Microbiología, Instituto de 
Salud Carlos III, WHO Collaborating Centre for Leishmaniasis, 
Servicio de Parasitología 
Aseffa, Abraham; AHRI/ALERT, Armauer Hansen Research Institute 
Yamuah, Lawrence; AHRI/ALERT, Armauer Hansen Research Institute 
Engers, Howard; AHRI/ALERT, Armauer Hansen Research Institute 
Moreno, Javier; Centro Nacional de Microbiología, Instituto de Salud 
Carlos III, WHO Collaborating Centre for Leishmaniasis, Servicio de 
Parasitología 
Cruz, Israel; Centro Nacional de Microbiología, Instituto de Salud 
Carlos III, WHO Collaborating Centre for Leishmaniasis, Servicio de 
Parasitología 

Key Words: 
Leishmaniasis, Parasitology, Protozoan Infections, Surveillance, 
Vector-borne diseases, Infectious Diseases, Epidemiology, 
Diagnostics 

  

American Journal of Tropical Medicine & Hygiene



For Peer Review

 

 

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LRH: GADISA AND OTHERS 

RRH: ASYMPTOMATIC LEISHMANIA INFECTION IN ETHIOPIA 

Usefulness of rK39-immunochromatographic test, direct agglutination test, and leishmanin skin 

test to detect asymptomatic Leishmania infection in children from a new visceral leishmaniasis 

focus in Amhara State (Ethiopia) 

Endalamaw Gadisa, Estefanía Custodio, Carmen Cañavate, Luis Sordo, Zelalem Abebe,    

Javier Nieto, Carmen Chicharro, Abraham Aseffa, Lawrence Yamuah, Howard Engers, Javier 

Moreno, Israel Cruz* 

Armauer Hansen Research Institute, Addis Ababa, Ethiopia; Centro Nacional de Medicina 

Tropical, Instituto de Salud Carlos III, Madrid, Spain; WHO Collaborating Center for 

Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de 

Salud Carlos III, Madrid, Spain; Centro Nacional de Epidemiología, Instituto de Salud Carlos 

III, Madrid, Spain; Amhara Regional State Laboratory, Bahir Dar, Ethiopia 

*Corresponding author: Israel Cruz, WHO Collaborating Center for Leishmaniasis, Servicio de 

Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. 

Majadahonda-Pozuelo Km2, 28220, Majadahonda, Madrid, Spain, Telephone: 34-918223623, 

Fax: 34-915097034, E-mail: cruzi@isciii.es 

 

ABSTRACT 

In areas where visceral leishmaniasis is anthroponotic, asymptomatic infected patients may 

play a role in transmission. Additionally, the number of asymptomatic patients in an endemic 

area will also provide information on transmission dynamics. Libo Kemkem and Fogera 

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districts (Amhara Region, Ethiopia) are now considered newly established visceral 

leishmaniasis endemic areas. In selected villages from these districts we have conducted a 

study to assess the usefulness of different approaches to estimate asymptomatic infection rate. 

Out of 639 participants rK39 immunochromatographic test was able to detect asymptomatic 

infection in 2% (13/639), direct agglutination test in 7.7% (49/639), and leishmanin skin test in 

5.8% (37/623); the combined use of serological methods and leishmanin skin test allowed 

detecting asymptomatic infection in 12.8% (82/639). We conclude that the best option to detect 

asymptomatic infection is the combined use of both direct agglutination test and leishmanin 

skin test. 

INTRODUCTION 

 Visceral leishmaniasis (VL) is a vector borne disease caused by members of the 

Leishmania (Leishmania) donovani complex, is endemic in 65 countries and, among them, 

Bangladesh, India, Nepal, Brazil, Sudan, and Ethiopia account for approximately 90% of the 

cases. The estimated annual incidence is 500 000 clinical cases with 59 000 associated 

deaths.1,2 Poor populations are particularly affected by VL, which is considered as one of the 

´most neglected diseases´.3 In addition, VL is currently spreading and (re-)emerging in different 

areas of the world with increasing public health concern.4  

 In full-blown disease, VL is fatal if left untreated; and even with treatment the case 

fatality rate ranges from 4 to 10%.5,6 In stable endemic areas clinical disease appears only in a 

fraction of those infected, while another fraction will not develop the disease and remain 

asymptomatic.7 The prevalence of asymptomatic infection is quite different between and within 

different endemic countries, and the number of asymptomatic usually exceeds the number of 

symptomatic infections, though this ratio can vary from 0.4 : 1 to 50 : 1.2 

 Visualization of parasite amastigotes by microscopic examination of bone marrow, 

spleen or lymph node aspirates has been the Gold Standard method of VL diagnosis for many 

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years. However, as this procedure is based on an invasive sampling, its use for asymptomatic 

infection surveillance is not justified. Even more, in poor remote endemic areas the expertise 

and facilities required for these procedures may not be within reach. Thus procedures based on 

less invasive sampling, such as serology or leishmanin skin test (LST) seem to be more suitable 

for this purpose.  

 Although the detection of anti-Leishmania antibodies does not discriminate between 

current or past infection, serological methods have been used to assess asymptomatic infection 

in different VL endemic areas. These have been based either on the direct agglutination test 

(DAT), rK39-immunochromatographic test (rK39-ICT), enzyme-linked immunosorbent assay 

(ELISA), indirect immunofluorescent antibody test (IFAT) or Western blot (WB).8,9,10,11,12 The 

LST is a useful method to detect cell-mediated immunity against Leishmania, this test becomes 

positive after subclinical infection and, in this case, persists for much longer than anti-

Leishmania antibodies; LST turns also positive within weeks-months after successful therapy 

against VL, indicating a healing or protective response.13,14 This makes LST a valuable tool to 

detect exposure to Leishmania parasites in epidemiological surveys, and its usefulness to detect 

asymptomatic infection has been shown by different authors in different endemic areas.11,15,16 

In general LST would detect a higher proportion of asymptomatic infection than serology. 

However (given that serology and LST are based on different types of  immune responses), in 

the absence of a Gold Standard, the combination of these two approaches would give a more 

realistic picture of the asymptomatic infection rate in a given endemic area.17,18 

 In areas where VL transmission is anthroponotic asymptomatic individuals may have a 

role as reservoirs, and even in areas where VL is zoonotic it is speculated that these individuals 

could also contribute to transmission.7,19 Thus the assessment of the prevalence and distribution 

of asymptomatic cases would contribute to a better understanding of VL transmission, helping 

in control efforts. 

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 In Ethiopia, VL is an endemic disease of increasing public health concern. It is 

estimated that thirty percent of the VL patients in Ethiopia are malnourished, while HIV co-

infection affects 40% in the northwest of the country; both conditions are known to facilitate 

the spread of VL.20,21 In addition to the classical foci in the northwest along the border with 

Sudan (Humera and Metema) and those in the south (Lake Abaya region, Omo river, and Aba 

Roba plains), the disease has recently spread to previously non-endemic areas, such as Libo 

Kemkem and Fogera districts (Amhara State) where a VL outbreak occurred in 2004--2005.15 

Thus VL is currently a priority in the public health agenda of the Amhara State Health Bureau; 

and there is a need to generate epidemiological data on VL in Amhara State. To support VL 

control, facilities for treatment, mobile teams for surveillance, community mobilization, and 

active case detection strategies have been established. In order to contribute to this initiative the 

UBS-Optimus Foundation granted the project entitled Visceral leishmaniasis and malnutrition 

in Amhara State, Ethiopia, which among its specific objectives aims to characterize nutritional, 

immunological, and parasitological aspects in the child population from this area; and it is in 

the frame of this project that we have explored the usefulness of rK39-ICT, DAT, and LST to 

detect asymptomatic Leishmania infection in children from different sub-districts of Libo 

Kemkem and Fogera. Furthermore, the detection of asymptomatic infection in children will 

give information about the status of VL transmission in this highland focus; additionally this 

will contribute to assess the magnitude of asymptomatic infection, which can help in early 

detection and treatment, thus contributing to decrease transmission as well as disease morbidity 

and mortality. The information obtained with this kind of studies can also be interesting in a 

scenario where HIV spreads to Leishmania endemic areas, helping to foresee new VL cases 

among asymptomatic individuals as a consequence of a reactivation of previous Leishmania 

infections after acquired HIV.22     

MATERIALS AND METHODS 

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 Study site, the study was conducted during May--July 2009 in the districts (weredas) of 

Libo Kemkem and Fogera (Amhara State, Ethiopia) (Figures 1 and 2). These are adjacent 

districts most affected by the outbreak of VL occurred in 2004-2005.15 According to year 2009 

census, the population was 198 374 (male 100 951 and female 97 423) and 226 595 (male 115 

693 and female 110 902) for Libo Kemkem and Fogera respectively.  The districts are located 

in a black cotton clay soil flat plain (1800--2000 meters above sea level). Human activities 

related to intensive cultivation of teff, maize, beans, oilseeds, rice and cotton, have reduced the 

natural vegetation to scattered clumps of acacia trees. Most of the area is flooded during the 

rainy season (July--September) and dried up during the dry season (November--May), resulting 

in deep cracks in the soil surface, which could turn into breeding sites for the putative vector 

Phlebotomus orientalis.23,24  

 Study population, population sampling was carried out by multi-staged cluster survey. 

Primary sampling units were sub-districts (kebeles) with high incidence of VL according to the 

2008 register of the Addis Zemen VL Treatment Center: Agita, Bura, and Yifag from Libo 

Kemkem district and Dibasifatra and Rib Gebriel from Fogera district. Secondary sampling 

units were randomly selected villages (gotts) in each of the selected sub-districts. Third 

sampling units were randomly selected households in each of the villages. All children with a 

reported age between 4 and 15 yr living in the household at the time of the survey were eligible 

for the study, as long as they were asymptomatic and had no past history of VL.  

 Data collection, information on age, gender, residence, and clinical assessment was 

obtained for all participants by trained medical personnel (nurses and health officers) using 

pretested questionnaires and protocols.  

 Asymptomatic case definition, asymptomatic individuals were defined by a positive 

result in rK39-ICT, DAT or LST, and the absence of VL signs and symptoms (fever for > 2 

weeks, in combination with either enlargement of spleen and/or liver, or weight loss).     

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 Sample collection and storage, peripheral blood was collected in Na2-EDTA tubes 

(SIGMA, UK) and immediately one drop was used for rK39-ICT and two drops were spotted 

on Whatman 3MM filter paper (Whatman International Ltd., England), filter papers were left to 

air dry and placed individually in sealed plastic bags. The plastic bags containing filter papers 

were kept in a chilled ice-box and sent on the same day to the Amhara State Regional 

Laboratory, where they were stored at 4 ºC for further DAT analysis.  

 Ethical considerations, the study was approved by the ethical review boards of 

Instituto de Salud Carlos III, Armauer Hansen Research Institute, and the Ethiopian National 

Ethical Review Committee. Support letters were obtained from the Amhara State and the 

different districts´ Health Bureaus. Parents/guardians gave written informed consent prior to the 

enrolment of their children in the study, and for children above 11 yr of age verbal assents were 

also obtained in addition to the consent of their parents /guardians. 

 Detection of anti-Leishmania antibodies, rK39-ICT (Kalazar Detect® Rapid Test, 

InBios International Inc., USA) was performed using one drop of blood and 3 drops of chasing 

buffer following the manufacturers’ instructions. DAT with freeze-dried antigen (ITMA-

DAT/VL, Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium) was initially 

performed with the screening method according to the manufacturer’s protocol. Blood samples 

giving a titer ≥ 1:3200 were considered positive.  

 Leishmanin skin test, LST was performed using L. major antigen (Leishmanin batch 

123-2; Pasteur Institute, Iran). One hundred µL of the antigen were intradermally inoculated on 

the volar surface of the forearm with a 1 mL sterile syringe and disposable needle. The test was 

read 48 hr later by the ballpoint pen method. An induration with an average of two 

perpendiculars ≥ 5 mm was considered as positive. 

 Data analysis, infection prevalence was calculated using rK39-ICT, DAT, and LST 

results. The differences in infection prevalence between age group, sex, and location were 

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compared by the Fisher’s exact and χ 2 tests. A p value < 0.05 was considered statistically 

significant. Data analysis was performed using SPSS version 16.0 (SPSS Inc., Chicago, Illinois, 

USA,) and STATA version 10 (Stata Corp., College Station, TX, USA).  

RESULTS 

 A total of 639 asymptomatic participants were included in the study: 331 were boys 

(51.8%) and 308 girls (48.2%), being the boy : girl ratio close to 1 : 1 in the two districts 

studied.  

The mean age of the participants was 8.9 yr (SD: 3.2), without differences between both sexes. 

They were grouped according to their age in 3 different groups: group 1 (< 5 yr) consisted in 

78/639 individuals (12.2%), group 2 (5--9 yr) in 310/639 (48.5%), and group 3 (10--15 yr) in 

251/639 (39.3%). 

 Anti-Leishmania antibodies were detected in 13 out of 639 children (2.0%) by rK39-

ICT and in 49/639 (7.7%) by DAT. Sixteen out of the 639 children (2.5%) initially tested by 

LST were lost for reading. This test returned a positive result in 37 out of 623 (5.9%).  

 Globally, 57 out of 639 children (8.9%) were found to be seropositive (rK39-ICT and/or 

DAT positive), and 82 out of 639 children (12.8%) were considered as infected (positive by 

rK39-ICT and/or DAT and/or LST). For the group of 623 children tested by the three methods 

we observed that 44/56 seropositive children had a negative LST result. While 25 out of 37 

LST positive children were seronegative. A detailed description of the performance of these 

three methods in the group of 623 children is given in Table 1.  

 The mean age in those with asymptomatic infection was 10.0 yr (SD: 2.9). Analysis by 

age group revealed a positive association between asymptomatic infection and age, children 

between 10--15 yr being the group with a higher asymptomatic infection rate (18.7%; p = 

0.005). This finding was common to all test employed.  

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  A strong association was also found with regard to gender, with infection in boys being 

higher than in girls (17.2% vs 8.1%; p = 0.001), differences in asymptomatic infection rate by 

age group and gender is provided in Table 2.  

The highest prevalence of asymptomatic infection was found in the selected villages from Bura 

(27.9%) and the lowest in those from Agita (3.6%). 

 A detailed description of the results obtained by the three methods employed by gender, 

age and location is given in Table 3. 

DISCUSSION 

 The present work reveals the presence of asymptomatic Leishmania infection among 4-

15 yr old children from the districts of Libo Kemkem and Fogera, in the new VL focus of 

Amhara State, Northwestern Ethiopia. The observed overall asymptomatic infection rate was 

12.8% (82/639); determined by the combination of serological methods, which detected 57/639 

seropositive individuals (8.9%), and LST, detecting 37/623 (5.9%) positive individuals. As 

proposed initially the combination of serology and LST allowed a wider detection of 

asymptomatic infection. 

 The discordances observed between serology and LST were expected. Both LST and 

serology have been used to assess exposure to Leishmania, irrespective of disease presentation, 

and are frequently used in epidemiological studies in Leishmania endemic areas.9,25,26 

Nevertheless, comparison of LST positivity and seroprevalence rates is complicated due to the 

different type of immune response detected by each test. LST measures a delayed type 

hipersensitivity reaction to Leishmania and relies on an in vivo cellular immune response to 

Leishmania antigens, while seropositivity is the result of a significant level of Leishmania-

specific antibodies in the peripheral blood, which is based on a humoral immune response. The 

two differentiated groups of LST-positive/seronegative and LST-negative/seropositive children 

observed in our study are in agreement with the lack of association between LST and serology. 

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LST positivity appears later after infection and seems to be a sign of protective immunity 

against VL, while seropositivity is considered a marker of more recent infection and has been 

related to disease progression.27,28 However, a recent sero-epidemiologic study in Bihar (India) 

observed low disease conversion rate in asymptomatic DAT positive individuals. 29  

 Different studies in VL endemic areas have shown that LST detects a higher 

Leishmania infection rate in asymptomatic individuals than serological approaches. 8,11,26,30,31,32 

In contrast our study shows that the infection rate obtained with serology is higher than that 

obtained with LST, 8.9% vs 5.9% using DAT and rK39-ICT and 7.6% vs 5.9% using DAT 

alone. Given that VL has recently been reported in our study area this finding can be associated 

to the longer time needed for the development of a LST positive response compared to sero-

conversion. If we consider that LST positive conversion is the result of a repeated exposure to 

natural infection this would also explain why LST positivity is higher in the older age group.  

 We have observed a high discordance between DAT and rK39-ICT in our work (Table 

1). Eight individuals with a positive rK39-ICT result were negative by DAT, while 54 DAT 

positive individuals presented a negative rK39-ICT result; and only 4 individuals presented a 

positive result by both serological methods. Although Ritmeijer and others reported a lower 

sensitivity of rK39-ICT in Sudan, a meta-analysis on the performance of DAT and rK39-ICT 

for active VL diagnosis concluded that both tests have a similar level of sensitivity.33,34 

Additionally a recent study in Libo Kemkem evaluating the performance of DAT and two 

different rK39-ICT brands indicated that either approach is suitable for VL diagnosis in this 

area of Ethiopia.35 However our study population is asymptomatic and higher positivity rates 

have been reported for DAT vs rK39-ICT when these methods are used to assess asymptomatic 

infection in other VL endemic areas.7 Given that performance of a serologic test can depend on 

the stage of the disease/infection, the lower performance of rK39-ICT can be explained by its 

ability to detect antibody response against only a single antigen (rK39), while DAT relies on 

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the detection of antibodies against a wide range of Leishmania antigens (the whole freeze-dried 

promastigote).36 Another explanation can be based on the nature of the tests. As proposed by 

Ter Horst and others, antibodies detected by rK39-ICT could be less able to react in a rapid 

reaction than in the overnight incubation used for DAT.37  

 Although DAT has shown better performance to detect asymptomatic infection in our 

study population, the presence of 8 individuals with a positive rK39-ICT result but negative for 

DAT merits attention. A possible explanation for this could be the different volume of blood 

tested by each method. While in our study rK39-ICT was performed on one drop of blood, 

DAT was performed on a 5 mm disk punched out from a spot of two drops of blood on a filter 

paper, which can be considered a lower amount of blood. Additionally, it was observed by 

Zijlstra and others that an rK39-based test (though in ELISA format) could detect 

asymptomatic infection earlier than DAT.38 

 The increase of asymptomatic infection rate with age was consistent with an endemic 

focus of VL, with a marked increase for the age group of 5--9 yr onwards. In addition, the 

presence of asymptomatic infection in individuals aged less than 5 yr is also consistent with the 

presence of active transmission, in spite of the low VL incidence situation reached after the 

outbreak.6  

 The present study indicates the appropriateness of combining serology (particularly 

DAT) and LST to obtain a consistent picture of the asymptomatic infection rate in a VL 

endemic area. This work also indicates that after the 2004--2005 VL outbreak active 

transmission is still happening in the villages studied, and also that L. donovani transmission 

can potentially be established in Ethiopian highlands (1800--2000 meters above sea level) 

which are commonly considered free of VL. 

Acknowledgments 

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 We would like to thank: the study participants for volunteering, the data collectors for 

the field work, the AHRI/ALERT and the Fundación Española para la Cooperación 

Internacional, Salud y Política Social for logistic and technical support, the Amhara State 

Regional Laboratory for allowing us to use their laboratory facility and for creating conducive 

environment during the field work. We would also like to thank Dr. Jorge Alvar 

(WHO/CDS/NTD/IDM) and the American Journal of Tropical Medicine and Hygiene for their 

permit to adapt the figures published on Alvar and others [Am. J. Trop. Med. Hyg., 77(2), 2007, 

pp. 275–282] to obtain figures 1 and 2 in this manuscript. 

Financial support 

 We also gratefully acknowledge the financial support by the UBS-Optimus Foundation 

(Switzerland), through the project Visceral leishmaniasis and malnutrition in Amhara State, 

Ethiopia, and the Spanish Ministry of Science and Innovation and the Instituto de Salud Carlos 

III through the Network of Tropical Diseases Research (RICET RD06/0021/0009 and 

RD06/0021/0000).   

Authors´ addresses 

 Endalamaw Gadisa, Armauer Hansen Research Institute, POB 1005, Jimma Road, 

ALERT Compound, Addis Ababa, Ethiopia, Telephone: 251-113211375, Fax: 251-113211563, 

E-mail: endalamawgadisa@yahoo.com. Estefanía Custodio, Centro Nacional de Medicina 

Tropical, Instituto de Salud Carlos III, Sinesio Delgado 6, 28029, Madrid, Spain, Telephone: 

34-918222282, Fax: 34-913877756, E-mail: ecustodio@isciii.es. Carmen Cañavate, WHO 

Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de 

Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220, 

Majadahonda, Madrid, Spain, Telephone: 34-918223623, Fax: 34-915097034, E-mail: 

ccanave@isciii.es. Luis Sordo, Centro Nacional de Epidemiología, Instituto de Salud Carlos 

III, Monforte de Lemos 5, 28029, Madrid, Spain, Telephone: 34-918222699, Fax: 34-

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913877815, E-mail: lsordo@isciii.es. Zelalem Abebe, Amhara Regional State Research 

Laboratory, POB 531, Bahir Dar, Ethiopia, E-mail: gebriyehailu@gmail.com. Javier Nieto, 

WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de 

Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220, 

Majadahonda, Madrid, Spain, Telephone: 34-918223623, Fax: 34-915097034, E-mail: 

fjnieto@isciii.es. Carmen Chicharro, WHO Collaborating Center for Leishmaniasis, Servicio 

de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. 

Majadahonda-Pozuelo Km2, 28220, Majadahonda, Madrid, Spain, Telephone: 34-918223623, 

Fax: 34-915097034, E-mail: cchichar@isciii.es. Abraham Aseffa, Armauer Hansen Research 

Institute, POB 1005, Jimma Road, ALERT Compound, Addis Ababa, Ethiopia, Telephone: 

251-113211375, Fax: 251-113211563, E-mail: aseffaa@gmail.com. Lawrence Yamuah, 

Armauer Hansen Research Institute, POB 1005, Jimma Road, ALERT Compound, Addis 

Ababa, Ethiopia, Telephone: 251-911608706, E-mail: yamuahlk@ahrialert.org. Howard 

Engers, Armauer Hansen Research Institute, POB 1005, Jimma Road, ALERT Compound, 

Addis Ababa, Ethiopia, Telephone: 251-113211375, Fax: 251-113211563, E-mail: 

engersh@ahrialert.org. Javier Moreno, WHO Collaborating Center for Leishmaniasis, Servicio 

de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. 

Majadahonda-Pozuelo Km2, 28220, Majadahonda, Madrid, Spain, Telephone: 34-918223623, 

Fax: 34-915097034, E-mail: javier.moreno@isciii.es. Israel Cruz, WHO Collaborating Center 

for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de 

Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220, Majadahonda, Madrid, Spain, 

Telephone: 34-918223623, Fax: 34-915097034, E-mail: cruzi@isciii.es.  

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first 10 years. Clin Microbiol Rev 10: 298--312.  

23. Elnaiem DA, Connor SJ, Thomson MC, Hassan MM, Hassan HK, Aboud MA, Ashford 

RW, 1998. Environmental determinants of the distribution of Phlebotomus orientalis in 

Sudan. Ann Trop Med Parasitol 92: 877--887. 

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24. Gebre-Michael T, Balkew M, Alamirew T, Gudeta N, Reta M, 2007. Preliminary 

entomological observations in a highland area of Amhara region, northern Ethiopia, 

with epidemic visceral leishmaniasis. Ann Trop Med Parasitol 101: 367--370. 

25. El-Safi SH, Bucheton B, Kheir MM, Musa HA, El-Obaid M, Hammad A, Dessein A, 

2002. Epidemiologyof visceral leishmaniasis in Atbara River area, eastern Sudan: the 

outbreak of Barbar el Fugara village (1996-1997). Microbes Infect 4: 1439--47.   

26. Hailu A, Gramiccia M, Kager PA, 2009. Visceral leishmaniasis in Aba Roba, south-

western Ethiopia: prevalence and incidence of active and subclinical infections. Ann 

Trop Med Parasitol 103: 659--70  

27. Mahmoodi M, Khamesipour A, Dowlati Y, Rafati S, Momeni A, Emamjomeh M, 

Hejazi H, Modabber F, 2003. Immune response measured in human volunteers 

vaccinated with autoclaved Leishmania major vaccine mixed with low dose of BCG. 

Clin Exp Immunol 134: 303--308.   

28. Sinha PK, Bimal S, Pandey K, Singh SK, Ranjan A, Kumar N, Lal CS, Barman SB, 

Verma RB, Jeyakumar A, Das P, Bhattacharya M, Sur D, Bhattacharya SK, 2008. A 

community-based, comparative evaluation of direct agglutination and rK39 strip tests in 

the early detection of subclinical Leishmania donovani infection. Ann Trop Med 

Parasitol 102: 119--125.  

29. Gidwani K, Kumar R, Rai M, Sundar S, 2009. Longitudinal seroepidemiologic study of 

visceral leishmaniasis in hyperendemic regions of Bihar, India. Am J Trop Med Hyg 80: 

345--346. 

30. Manson-Bahr PEC, 1961. Immunity in kala-azar. Trans R Soc Trop Med Hyg 55: 550--

555. 

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31. Evans TG, Teixeira MJ, McAuliffe IT, Vasconcelos I, Vasconcelos AW, Sousa AA, 

Lima JW, Pearson RD, 1992. Epidemiology of visceral leishmaniasis in Northeast 

Brazil. J Infect Dis  166: 1124--1132.   

32. Riera C, Fisa R, Udina M, Gállego M, Portús M, 2004. Detection of Leishmania 

infantum cryptic infection in asymptomatic blood donors living in an endemic area 

(Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop 

Med Hyg 98: 102--110. 

33. Ritmeijer K, Melaku Y, Mueller M, Kipngetich S, O'keeffe C, Davidson RN, 2006. 

Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral 

leishmaniasis. Am J Trop Med Hyg 74: 76--80. 

34. Chappuis F, Rijal S, Soto A, Menten J, Boelaert M, 2006. A meta-analysis of the  

diagnostic performance of the direct agglutination test and rK39 dipstick for visceral  

leishmaniasis. BMJ 333: 723. 

35. Cañavate C, Herrero M, Nieto J, Cruz I, Chicharro C, Aparicio P, Mulugeta A, Argaw 

D,  Blackstock AJ, Alvar J, Bern C, 2011. Evaluation of two rK39 dipstick tests, direct 

agglutination test, and indirect fluorescent antibody test for diagnosis of visceral 

leishmaniasis in a new epidemic site in highland Ethiopia. Am J Trop Med Hyg 84: 102-

-106. 

36. Boelaert M, Rijal S, Regmi S, Singh R, Karki B, Jacquet D, Chappuis F, Campino L, 

Desjeux P, Le Ray D, Koirala S, Van der Stuyft P, 2004. A comparative study of the  

effectiveness of diagnostic tests for visceral leishmaniasis. Am J Trop Med Hyg 70: 72--

77.   

37. Ter Horst R, Tefera T, Assefa G, Ebrahim AZ, Davidson RN, Ritmeijer K, 2009. Field  

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evaluation of rK39 test and direct agglutination test for diagnosis of visceral 

leishmaniasis in a population with high prevalence of human immunodeficiency virus in 

Ethiopia. Am J Trop Med Hyg 80: 929--934. 

38. Zijlstra EE, Daifalla NS, Kager PA, Khalil EA, El-Hassan AM, Reed SG, Ghalib HW,  

1998. rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donovani  

infection. Clin Diag Lab Immunol 5: 717--720.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 1: Location of the study area 

Figure 2: Location of the sub-districts on which the study was performed (grey background) 

 

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Table 1: Detailed description of the performance of rK39-ICT, DAT and LST in the group of 

623 children 

 

rK39-ICT DAT LST n/N (%) 

Negative Negative Negative 542/623 (87.0) 

Negative Negative Positive 25/623 (4.0%) 

Negative Positive Negative 37/623 (6.0%) 

Positive Negative Negative 5/623 (0.8%) 

Positive Positive Negative 2/623 (0.3%) 

Positive Negative Positive 3/623 (0.5%) 

Negative Positive Positive 7/623 (1.1%) 

Positive Positive Positive 2/623 (0.3%) 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Table 2: Rate of asymptomatic infection by gender and age (N = number of children tested by 

variable group; n = number of infected children per variable group; % = percentage of infected 

children by variable group) 

 

 Rate of asymptomatic infection   

Age group (yr) Boys [n/N (%)] Girls [n/N (%)] Both sexes [n/N (%)] 

< 5 1/34 (2.9) 2/44 (4.5) 3/78 (3.8) 

5--9 21/147 (14.3) 11/163 (6.7) 32/310 (10.3) 

10--15 35/150 (23.3) 12/101 (11.8) 47/251(18.7) 

Total 57/331 (17.2) 25/308 (8.1) 82/639 (12.8) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Table 3:  Asymptomatic infection rates detected by the three different tests and their 

combination displayed by gender, age and location (N = number of children tested per variable 

group; n = number of positive children by each test per variable group; % = percentage of 

positive children by each test per variable group; *Figures for LST are related to a 623 children 

population)  

  TESTS    

VARIABLES rK39-ICT 

n (%) 

DAT 

n (%) 

LST* 

n (%) 

Seropositive 

n (%) 

Infected 

n (%) 

WHOLE POPULATION 

(N = 639) 

 

13 (2.0) 

 

49 (7.7) 

 

37 (5.9) 

 

57 (8.9) 

 

82 (12.8) 

GENDER      

Boys (N = 331) 9 (2.7) 33 (9.9) 27 (8.3) 40 (12.1) 57 (17.2) 

Girls (N = 308) 4 (1.3) 16 (5.2) 10 (3.3) 17 (5.5) 25 (8.1) 

Age group (yr)      

< 5 (N = 78) 1 (1.3) 3 (3.8) 1 (1.3) 3 (3.8) 3 (3.8) 

5--9 (N = 310) 5 (1.6) 23 (7.4) 8 (2.6) 26 (8.4) 32 (10.3) 

10--15 (N = 251) 7 (2.8) 23 (9.1) 28 (11.4) 28 (11.1) 47 (18.7) 

SUBDISTRICT      

Agita (N = 139) 1 (0.7) 5 (3.6) 0 (0.0) 5 (3.6) 5 (3.6) 

Bura (N = 147) 5 (3.4) 22 (14.9) 24 (16.4) 24 (16.3) 41 (27.9) 

Dibasifatra (N = 140) 6 (4.3) 4 (2.8) 6 (4.4) 9 (6.4) 13 (9.3) 

Rib Gebriel (N = 144) 1 (0.7) 13 (9.0) 7 (4.9) 14 (9.7) 18 (12.5) 

Yifag (N = 69) 0 (0.0) 5 (7.2) 0 (0) 5 (7.2) 5 (7.2) 

 

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ETHIOPIA

UGANDA
KENYA

SUDAN

SOMALIA

Blue Nile

W
h

it
e

 N
il

e
Humera

Metema

Libo

Lake

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Belessa

300 km

ERITREA

DJIBOUTI

FOGERA 

DISTRICT

Mender 
Mariam

Micheldebr

Asta
Mariam

Martadios

Bilb
Wuha

Libo Giorgis

Ameno

Ajmeda
Bir

Kute

Deleta

Tehara

Mante Woger

Birra Abo
Estifanos Shemo

Godgua
Dit

AGITA

Tara Gedam

Addis
Zemen

Angot 

BURA
Shina

Bandiko

Ginaze
Yekog
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YIFAG

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Abakiros

M
eneguzer

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LIBO

DISTRICT

LAKE

TANA

DIBASIFATRAWoreta

Figure 1

Figure 2

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Low prevalence of Leishmania infection in post-epidemic 

areas of Libo Kemkem, Ethiopia 
 
 

Journal: American Journal of Tropical Medicine & Hygiene 

Manuscript ID: Draft 

Manuscript Type: Short Report 

Date Submitted by the 
Author: 

n/a 

Complete List of Authors: Sordo, Luis; Instituto de Salud Carlos III, Centro Nacional de 
Epidemiología. CIBER en Epidemiología y Salud Pública 
Gadisa, Endalamaw; Armauer Hansen Research Institute, ALERT 
Compound 
Custodio, Estefanía; Instituto de Salud Carlos III, Centro Nacional 
de Medicina Tropical 
Cruz, Israel; Instituto de Salud Carlos III, Centro Nacional de 
Microbiología, Servicio de Parasitología 
Simón, Fernando; Instituto de Salud Carlos III, Centro Nacional de 
Epidemiología. CIBER en Epidemiología y Salud Pública 
Abebe, Zelalem; Amhara Regional State Research Laboratory, 
Amhara Regional State Research Laboratory 
Moreno, Javier; Instituto de Salud Carlos III, Centro Nacional de 
Microbiología, Servicio de Parasitología 
Aseffa, Abraham; Armauer Hansen Research Institute, ALERT 
Compound 
Tsegaye, Hailu; Armauer Hansen Research Institute, ALERT 
Compound 
Nieto, Javier; Instituto de Salud Carlos III, Centro Nacional de 
Microbiología, Servicio de Parasitología 
Chicharro, Carmen; Instituto de Salud Carlos III, Centro Nacional 
de Microbiología, Servicio de Parasitología 
Cañavate, Carmen; Instituto de Salud Carlos III, Centro Nacional 
de Microbiología, Servicio de Parasitología 

Key Words: 
Leishmaniasis, Emerging Diseases, Epidemiology, Infectious 
Diseases, Protozoan Infections, Parasitology 

  
 
 

American Journal of Tropical Medicine & Hygiene



For Peer Review

 

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LRH: SORDO ET AL. 

RRH: PREVALENCE OF LEISHMANIA INFECTION IN LIBO KEMKEM 

 

Low prevalence of Leishmania infection in post-epidemic areas of Libo Kemkem, Ethiopia 

 

Luis Sordo, Endalamaw Gadisa, Estefanía Custodio, Israel Cruz, Fernando Simón, Zelalem 

Abebe, Javier Moreno, Abraham Aseffa, Hailu Tsegaye, Javier Nieto, Carmen Chicharro, 

Carmen Cañavate* 

 

Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, Spain; CIBER en 

Epidemiología y Salud Pública (CIBERESP); Armauer Hansen Research Institute, Addis 

Ababa, Ethiopia; Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, 

Madrid, Spain; WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, 

Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain; Amhara 

Regional State Laboratory, Bahir Dar, Ethiopia 

 

*Corresponding author: Carmen Cañavate, WHO Collaborating Center for Leishmaniasis, 

Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 

Ctra. Majadahonda-Pozuelo km 2, 28220 Majadahonda, Madrid, Spain. Telephone: +34 

918223623, Fax: +34 915097034, E-mail: ccanave@isciii.es 

 

ABSTRACT 

In Libo Kemkem (a district of the Amhara region, Ethiopia), no cases of kala-azar had 

ever been reported until 2005, when an outbreak occurred. Over one third of those affected 

were children under 15 years of age. The aim of the present study was to determine the 

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prevalence of Leishmania infection in children aged 4--15 years. A cross-sectional survey 

was conducted in 2009. Sampling was performed in a multi-staged cluster survey. A total of 

386 children were included in the study. The overall prevalence of Leishmania infection 

(DAT- and/or rK39-ICT- and/or LST-positive subjects) in this population was then estimated. 

Only one case of active disease was encountered. The overall prevalence of infection was 

1.02% (95% CI: 0--4.54), being higher among boys and in children older than 12 years. The 

results suggest that the conditions responsible for the outbreak no longer reign.  However, 

active surveillance remains necessary. 

 

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East Africa has suffered a marked increase in the number of cases of visceral 

leishmaniasis (VL) or kala-azar over the last two decades, probably due to a combination of 

demographic and climatic changes.1,2 

The areas traditionally regarded as endemic for VL in Ethiopia lie in the north-west 

(bordering Sudan) and south of the country.3,4 Libo Kemkem district (wereda) is located in 

the highlands of the Amhara region in northwestern Ethiopia at an altitude of 1800--2000 m. 

No cases of kala-azar had ever been declared in this area until 2005. Earlier, in 2004, the 

Amhara Regional Health Bureau reported a 5-fold increase in crude mortality rates in the 

Libo Kemkem district, attributing this to an outbreak of drug-resistant malaria. However, in 

May 2005 an outbreak of kala-azar was determined to be the culprit.5 The epidemiological 

background - a few cases over a 1-year period followed by an explosive increase - was 

consistent with the rapid emergence of the disease in a population with little pre-existing 

immunity.5 By December 2007, 2543 patients with kala-azar had been treated by Médecins 

sans Frontières.6 More than one third were children under 15 years of age, for whom a 

fatality rate of over 3% was reported.6 The rapid spread of the disease between 2004 to 2007 

suggested that transmission would not be easy to control.5 Before the Libo Kemkem outbreak 

there was no epidemiological surveillance system for leishmaniasis in Ethiopia, making it 

difficult to determine whether the epidemic between 2004--2007 was an outbreak due to a 

recent introduction of the parasite or, as suggested by Herrero et al.,6 the parasite was 

endemic to the area but in low numbers.  

The aim of the present study was to determine the prevalence of Leishmania infection 

in children aged 4--15 years from Libo Kemkem, four years after the outbreak. 

A cross-sectional survey was conducted between May and July 2009, as part of the 

project Visceral Leishmaniasis and Malnutrition in Amhara State, Ethiopia, funded by the 

UBS-Optimus Foundation. Sampling was undertaken as part of a multi-staged cluster survey. 

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The Primary sampling units were randomly selected sub-districts (kebeles) of Libo Kemkem 

that, according to the records of Médecins sans Frontières-Greece held at the Addis Zemen 

Health Centre, had reported at least one case of VL during the 2004--2007 epidemic. These 

were selected taking into account their size according to a recent census.7 The secondary 

sampling units were randomly selected villages (gotts)  in each of the selected sub-districts. 

The tertiary sampling units were households randomly selected from an updated census for 

each village. All children between 4 and 15 years of age residing in these household were 

tested. A total of 386 children were included in the study. 

Ethical clearance was obtained from the review boards of the Instituto de Salud Carlos 

III, the Armauer Hansen Research Institute, and the Ethiopian National Ethical Review 

Committee. Parents/guardians gave written, informed consent prior to the enrolment of their 

children in the study. For children over 11 years of age, verbal assent was obtained in 

addition to the consent of their parents or guardians.  

Each participant was clinically assessed by health professionals for any complaint of 

fever lasting longer than two weeks, weight loss, and the presence of splenomegaly and 

lymphadenopathy, in order to determine the presence of any active infection. All children 

were tested using the leishmanin skin test (LST), the rK39-immunochromatographic test 

(ICT) (Kalazar Detect® Rapid Test, InBios International Inc., Seattle, WA), and the direct 

agglutination test (DAT) (ITMA-DAT/VL, Institute of Tropical Medicine, Antwerp, 

Belgium).  Sociodemographic data were recorded using pre-tested questionnaires. The rK39-

ICT test was performed immediately after blood sampling, according to the manufacturer’s 

instructions. The DAT test was performed on blood-impregnated filter paper using freeze-

dried antigen. The screening method followed the manufacturer’s protocol; titres of ≥1:3200 

were deemed positive. Leishmanin skin testing was performed using L. major antigen 

(Leishmanin batch 123-2; Pasteur Institute, Tehran, Iran), as previously described.8,9 

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 The overall prevalence of Leishmania infection (DAT- and/or rK39-ICT- and/or LST- 

positive) in the population was then calculated. Prevalence rates were expressed in 

percentages with 95% confidence intervals (95% CI). Stata v.10.1 software was used to 

perform all statistical analyses. Data were weighted according to selection probabilities and 

analysed using the Stata v.10.1 complex samples procedures, which takes into account 

sample clustering. 

One hundred and ninety nine of the 386 children (50.9%) were girls. The mean age of 

the participants was 8.9 years (SD 3.03); 44.76% of the children were under 8 years of age, 

and 33.08% between 8 and 11 years. 

Only one case of active VL was found, which returned positive results for both rK39-

ICT and DAT. Nine children were DAT-positive only; four of them had suffered kala-azar 

previously. However, five children that previously had VL showed negative results for both 

rK39-ICT and DAT. None of the children returned a positive LST result. 

The overall prevalence of infection (DAT and/or rk39 positive) was 1.02% (95% CI: 0-

-4.54).  The prevalence among boys was higher (1.78%; 95% CI: 0--7.98, vs. 0.3%; 95% CI: 

0--1.31 in girls). The greatest prevalence was recorded in children older than 12 years 

(2.56%; 95% CI: 0--10.54), followed by those between 8 and 11 (0.82%; 95% CI: 0--3.53); 

the under 8 years subgroup showed the lowest prevalence (0.49%; 95% CI: 0--2.59). 

However, these prevalence values were not statistically different to one another (Table 1).  

To the best of our knowledge, these are the first Leishmania infection prevalence data 

for Libo Kemkem since the 2004--2007 epidemic. Higher prevalence rates for similar 

populations have, however, been reported for other regions of Ethiopia.10,11 

The permanence of Leishmanin skin test reactivity is thought to depend on latent 

infection and the continuous exposure to biting, Leishmania-carrying sand flies;10,17 a 

decrease of these vectors might explain the absence of positive results in the present study 

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population.  This would also explain the reduction in the number of VL cases reported in this 

area.6 Some authors report a natural conversion rate from positive to negative of 14.8% to 

9.3% in other settings in Ethiopia.12 Nevertheless, the absence of positive results is 

noteworthy, especially in comparison with previous results in the same age range (23.6%),5 

though in the latter study rapid assessment via convenience sampling was undertaken. 

The present study also used DAT and rK39 ICT detection of infection, methods that 

reveal the presence of anti-Leishmania antibodies appearing early after infection. The 

combined use of these methods, which has performed well in VL diagnosis in this area,13 

yielded just 1% prevalence. It should be remembered that this result is only for 4--15 year-old 

and not for the whole population. However, although the prevalence of Leishmania infection 

increases with age,10 the presence of only one active case in the study population and the low 

prevalence even among the older children, suggests a low prevalence for the general 

population.  

 The present results appear to indicate that the conditions that provoked the kala-azar 

epidemic in the study region no longer reign. The parasite may have been introduced by 

migrant agricultural labourers who, returning to their villages after completing seasonal work 

on the border of Sudan,14 acted as a reservoir of the causal parasite – a hypothesis put forward 

at the time of the epidemic. However, the available evidence indicates that the affected 

population was not made up simply of migrant workers, and there is no evidence that any 

such migration has ever ceased.  This suggests that, as well as an increase in the size of the 

human reservoir, some change in the vector population must have occurred. Libo Kemkem 

lies at an altitude of 1800--2000 m, beyond that at which phlebotomes are normally found.  

However, as described by other authors,15 changes in the temperature or relative humidity 

could have encouraged their increased presence.  In other scenarios such changes have been 

attributed to global warming.15,16 It should be noted, however, that the response to the 

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outbreak included the establishment of centres where specific treatment could be received. 

The clearance of patients’ infections would have led to a reduction in the seroprevalence rate. 

Until 2004, no case of leishmaniasis had ever been reported in Libo Kemkem. The very 

low prevalence in the study population may suggest that the district may be experiencing a 

pre-epidemic status similar to that seen prior to the outbreak. Efforts to identify areas of high 

prevalence and then focus control efforts in these places might be wiser than blanket control 

of the entire district. However, the doubts surrounding the reasons for the outbreak means 

vigilance with respect to the impact of possible climate changes should be considered; such 

changes might encourage new outbreaks. 

Acknowledgements 

The authors thank the study participants for volunteering, the data collectors for their 

field work efforts, the AHRI/ALERT and the Fundación Española para la Cooperación 

Internacional, Salud y Política Social for logistic and technical support, and the Amhara 

State Regional Laboratory for allowing us to use their laboratory facilities and for creating a 

conducive environment during field work. 

Financial support 

We also gratefully acknowledge the financial support of the UBS-Optimus Foundation 

(Switzerland).  Support was also provided by provided via the Visceral Leishmaniasis and 

Malnutrition in Amhara State, Ethiopia project, and the Instituto de Salud Carlos III via the 

Tropical Diseases Research Network (RICET RD06/0021/0009 and RD06/0021/0000). 

Authors´ addresses 

Luis Sordo, Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Monforte 

de Lemos 5, 28029 Madrid, Spain, CIBER en Epidemiología y Salud Pública (CIBERESP), 

Telephone: 34-918222699, Fax: 34-913877815, E-mail: lsordo@isciii.es. Endalamaw 

Gadisa, Armauer Hansen Research Institute, POB 1005, Jimma Road, ALERT Compound, 

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Addis Ababa, Ethiopia, Telephone: 251-113211375, Fax: 251-113211563, E-mail: 

endalamawgadisa@yahoo.com. Estefanía Custodio, Centro Nacional de Medicina Tropical, 

Instituto de Salud Carlos III, Sinesio Delgado 6, 28029 Madrid, Spain, Telephone: 34-

918222282, Fax: 34-913877756, E-mail: ecustodio@isciii.es. Israel Cruz, WHO 

Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de 

Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220 

Majadahonda, Madrid, Spain, Telephone: 34-918223623, Fax: 34-915097034, E-mail: 

cruzi@isciii.es.  Fernando Simón, Centro Nacional de Epidemiología, Instituto de Salud 

Carlos III, Monforte de Lemos 5, 28029 Madrid, Spain, CIBER en Epidemiología y Salud 

Pública (CIBERESP), Telephone: 34-918222033, Fax: 34-913877815, E-mail: 

fsimon@isciii.es. Zelalem Abebe, Amhara Regional State Research Laboratory, POB 531, 

Bahir Dar, Ethiopia, E-mail: gebriyehailu@gmail.com. Javier Moreno, WHO Collaborating 

Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, 

Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220 Majadahonda, Madrid, 

Spain, Telephone: 34-918223623, Fax: 34-915097034, E-mail: javier.moreno@isciii.es. 

Abraham Aseffa, Armauer Hansen Research Institute, POB 1005, Jimma Road, ALERT 

Compound, Addis Ababa, Ethiopia, Telephone: 251-113211375, Fax: 251-113211563, E-

mail: aseffaa@gmail.com. Hailu Tsegaye,  Armauer Hansen Research Institute, POB 1005, 

Jimma Road, ALERT Compound, Addis Ababa, Ethiopia, Telephone: 251-113211375, Fax: 

251-113211563, E-mail: tsegsha2@gmail.com. Javier Nieto, WHO Collaborating Center for 

Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de 

Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220 Majadahonda, Madrid, Spain, 

Telephone: 34-918223623, Fax: 34-915097034, E-mail: fjnieto@isciii.es. Carmen Chicharro, 

WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de 

Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo Km2, 28220 

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Majadahonda, Madrid, Spain, Telephone: 34-918223623, Fax: 34-915097034, E-mail: 

cchichar@isciii.es. Carmen Cañavate, WHO Collaborating Center for Leishmaniasis, 

Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 

Ctra. Majadahonda-Pozuelo Km2, 28220 Majadahonda, Madrid, Spain, Telephone: 34-

918223623, Fax: 34-915097034, E-mail: ccanave@isciii.es. 

REFERENCES 

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leishmaniasis in a new epidemic site in Amhara Region, Ethiopia. Am J Trop Med 

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15. Maroli M, Rossi L, Baldelli R, Capelli G, Ferroglio E, Genchi C, Gramiccia M, 

Mortarino M, Pietrobelli M, Gradoni L, 2008. The northward spread of leishmaniasis 

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Guerrero I, Cubero E, Molina R, 2010. Seasonal trends and spatial relations between 

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17. Weigle KA, Valderrama L, Arias AL, Santrich C, Saravia NG, 1991. Leishmanin skin 

test standardization and evaluation of safety, dose, storage, longevity of reaction and 

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Table 1: Prevalence of Leishmania infection 

 

 
Sample 

Prevalence of Leishmania infection (rK39- and/or 
DAT-positive) 

  Na %b Na %b  95% CIb OR (95% CI) p 

Overall 386  10 1.02 0-4.54   

Sex        
Girls 199 50.9 2 0.3 0-1.31 Ref.  

Boys 187 49.1 8 1.78 0-7.98 5.94 (0.38-93.84) 0.291c 

Age        
<8 years 169 44.76 2 0.49 0-2.59 Ref.  

8-11 years 132 33.08 3 0.82 0-3.53 1.675 (0.1-29.12)  

>11 years 85 22.16 5 2.56 0-10.54 5.228 (0.43-63.4) 0.39c 
 
aUnweighted 
bWeighted 
cFisher 
 

 

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PLoS Neglected Tropical Diseases
 

Factors Associated with Leishmania Asymptomatic Infection in Highland Northern
Ethiopia

--Manuscript Draft--
 

Manuscript Number:

Full Title: Factors Associated with Leishmania Asymptomatic Infection in Highland Northern
Ethiopia

Short Title: Leishmania Asymptomatic Infection Correlates

Article Type: Research Article

Keywords: Visceral leishmaniasis, Ethiopia, Leishmania asymptomatic infection, Amhara state

Corresponding Author: Estefania Custodio, Ph.D.
Instituto de Salud Carlos III
Madrid,  SPAIN

Corresponding Author Secondary
Information:

Corresponding Author's Institution: Instituto de Salud Carlos III

Corresponding Author's Secondary
Institution:

First Author: Estefania Custodio, Ph.D.

First Author Secondary Information:

All Authors: Estefania Custodio, Ph.D.

Endalamaw Gadisa

Luis Sordo

Israel Cruz

Javier Moreno

Javier Nieto

Carmen Chicharro

Abraham Aseffa

Zelalem Abebe

Haiulu Tsegaye

Carmen Cañavate

All Authors Secondary Information:

Abstract: Background: In northern Ethiopia the prevalence of visceral leishmaniasis is steadily
rising posing an increasing public health concern. In order to develop effective control
strategies on the transmission of the disease it is important to generate knowledge on
the epidemiological determinants of the infection.
Methodology/Principal Findings: We conducted a cross-sectional survey using a multi
staged stratified cluster sampling on high incidence sub-districts of Amhara state,
Ethiopia. The survey included a socio-demographic, health and dietary questionnaire
and anthropometric measurements. We performed RK39-ICT and DAT serological
tests in order to detect anti-Leishmania antibodies and carried out Leishmanin Skin
Test (LST) using L. major antigen. Logistic regression models were used. Of the 605
children surveyed 61 children were positive to infection (10.1%). The individual
variables that showed a positive association with infection were increasing age, being
male and sleeping outside [odds ratios (95% CI): 1.12 (1.02, 1.23), 2.06 (1.10, 3.82)
and 2.10 (1.15, 3.85) respectively] and in relation to the household: increasing number
of people living in the house, past history of VL in the family, living in a straw roofed

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house and if the family owned sheep [OR (95% CI): 1.26 (1.06, 1.50), 2.66 (1.44, 4.92),
2.35 (1.24, 4.45) and 3.25 (1.52, 6.98) respectively]. The presence of dogs in the
house [OR (95% CI): 0.44 (0.23, 0.85] showed an inverse association.
Conclusions/Significance: Behavioural patterns like sleeping outside are determinant in
the transmission of the infection in this area. Protective measures should be
implemented against these identified risk activities. Results also suggest a
geographical clustering of the infection and a transmission within the homestead,
human to human, but more studies are needed on the behaviour of the vector to clarify
possible entomological interventions related to housing conditions. The role of
domestic animals in transmission needs to be studied further before giving any
recommendation.

Suggested Reviewers: Jorge  Alvar
World Health Organization
alvarj@who.int

Philippe Desjeux
One World Health Organization
pdesjeux@oneworldhealth.org

Caryn Bern
Center for Disease Control and Prevention, Atlanta
cxb9@cdc.gov

Lashitew Gedamu
Calgary University
lgedamu@ucalgary.ca

Opposed Reviewers:

Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation



 

To the PLOS Neglected Tropical Diseases Editors: 
 
 

It is my pleasure to send you enclosed a paper entitled “Factors 
associated with Leishmania asymptomatic infection in highland northern 
Ethiopia” for consideration as an article in the PLOS Neglected Tropical 
Diseases. 

 
Visceral leishmaniasis prevalence is steadily rising in northern Ethiopia, 

posing a public health challenge in the region. In two highland (1800-2000 mts 
above sea level) provinces of Amhara state an outbreak occurred in 2005 that 
has ever since transited into an endemic focus with sustained transmission. 

 
 The role of asymptomatic infected individuals in the control and 

prevention of the disease is important, as they can and act as reservoirs for new 
infection or become ill if immunosuppression occurs. Factors associated with 
infection can differ from disease correlates, and may also change in relation to 
the vector behaviour in the local environment. 

 
We find important to disseminate the results of our study not only 

towards the reinforcement of the program control developed by the Amhara 
Regional Health Bureau, but also in order to contribute to the scientific 
community with the epidemiological determinants of Leishmania asymptomatic 
infection in an environment that, to the best of our knowledge, has not been 
described before. We certainly consider the PLOS Neglected Tropical Diseases 
the best vehicle for it.  
 

All authors have contributed to the conception and design of the work, 
the acquisition of data, or the analysis of the data in a manner substantial 
enough to take public responsibility for it. All authors believe the manuscript 
represents valid work and have reviewed the final version of the manuscript and 
approve it for publication. None of the authors had any conflict of interest.                                                                                  

 
Finally, the material included in the manuscript is our original work and 

has not been published before, nor has been submitted for publication 
elsewhere.  

 
Looking forward to hearing from you, 
 
 
Yours sincerely, 
 
 
Estefanía Custodio 

 

Cover Letter



 1 

Title: Factors associated with Leishmania asymptomatic infection in 1 

highland northern Ethiopia 2 

 3 

Short Title: Leishmania asymptomatic infection correlates 4 

 5 

Authors: Estefanía Custodio1, Endalamaw Gadisa2, Luis Sordo3,4 , Israel Cruz5, 6 

Javier Moreno5, Javier Nieto5, Carmen Chicharro5, Abraham Aseffa2, Zelalem 7 

Abebe6, Hailu Tsegaye2 and Carmen Cañavate5 8 

1. Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, 9 

Madrid, Spain 10 

2. Armauer Hansen Research Institute, Addis Ababa, Ethiopia 11 

3. Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, 12 

Spain 13 

4. CIBER en Epidemiología y Salud Pública (CIBERESP), Spain  14 

5. WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, 15 

Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, 16 

Spain 17 

6. Amhara Regional State Laboratory, Bahir Dar, Ethiopia 18 

19 

AsymInfecNorthEthio_ECustodio
Click here to download Manuscript: AsymInfRiskFact_ECustodio.doc 

http://www.editorialmanager.com/pntd/download.aspx?id=57824&guid=c8183611-61f3-47b9-ae2a-ed092d8c0149&scheme=1


 2 

ABSTRACT 20 

 21 

Background: In northern Ethiopia the prevalence of visceral leishmaniasis is 22 

steadily rising posing an increasing public health concern. In order to develop 23 

effective control strategies on the transmission of the disease it is important to 24 

generate knowledge on the epidemiological determinants of the infection. 25 

Methodology/Principal Findings: We conducted a cross-sectional survey 26 

using a multi staged stratified cluster sampling on high incidence sub-districts of 27 

Amhara state, Ethiopia. The survey included a socio-demographic, health and 28 

dietary questionnaire and anthropometric measurements. We performed RK39-29 

ICT and DAT serological tests in order to detect anti-Leishmania antibodies and 30 

carried out Leishmanin Skin Test (LST) using L. major antigen. Logistic 31 

regression models were used. Of the 605 children surveyed 61 children were 32 

positive to infection (10.1%). The individual variables that showed a positive 33 

association with infection were increasing age, being male and sleeping outside 34 

[odds ratios (95% CI): 1.12 (1.02, 1.23), 2.06 (1.10, 3.82) and 2.10 (1.15, 3.85) 35 

respectively] and in relation to the household: increasing number of people 36 

living in the house, past history of VL in the family, living in a straw roofed house 37 

and if the family owned sheep [OR (95% CI): 1.26 (1.06, 1.50), 2.66 (1.44, 38 

4.92), 2.35 (1.24, 4.45) and 3.25 (1.52, 6.98) respectively]. The presence of 39 

dogs in the house [OR (95% CI): 0.44 (0.23, 0.85] showed an inverse 40 

association. 41 

Conclusions/Significance: Behavioural patterns like sleeping outside and 42 

herding the cattle are determinant in the transmission of the infection in this 43 

area. Protective measures should be implemented against these identified risk 44 

activities. Results also suggest a geographical clustering of the infection and a 45 

transmission within the homestead, human to human, but more studies are 46 

needed on the behaviour of the vector to clarify possible entomological 47 

interventions related to housing conditions. The role of domestic animals in 48 

transmission needs to be studied further before giving any recommendation. 49 

 50 

 51 

 52 

 53 
 54 



 3 

   AUTHORS SUMMARY: 55 

 56 

Visceral leishmaniasis is a vector borne disease that can be fatal if left 57 

untreated. Its prevalence is steadily rising in northern Ethiopia posing a public 58 

health challenge in the region. We conducted a study on the factors associated 59 

to asymptomatic infection in Amhara state, where little is known about 60 

Leishmania transmission.  Sleeping outside and herding the cattle were 61 

identified as risk activities, so the implementation of preventive measures 62 

towards them is recommended. Our results also suggested a transmission 63 

within the homestead, human to human, but more entomological studies are 64 

needed in order to clarify the vector’s behaviour in the area. Individuals living in 65 

houses that owned sheep were more likely to be infected and those living in 66 

houses with dogs seemed to be protected. This last result differs from the direct 67 

association between dog’s ownership and the clinical form of the disease found 68 

in a case control study carried out in this same area. These conflicting results 69 

add up to the debate found in the literature regarding the role of domestic 70 

animals in the transmission of Leishmania in different regions of the world. No 71 

specific recommendation should be given until the exact role of the domestic 72 

animal in the transmission cycle is clearly understood. 73 

74 



 4 

INTRODUCTION 75 

 76 

Visceral leishmaniasis (VL) or kala-azar is a neglected vector-borne 77 

parasitic disease that manifests with irregular bouts of fever, substantial weight 78 

loss, weakness, hepatosplenomegaly and pancytopenia, and that is fatal if left 79 

untreated [1]. It has an estimated annual incidence of 500 000 clinical cases 80 

with 50 000 associated deaths and 2 357 000 disability-adjusted life years lost. 81 

It is mainly concentrated in few major foci and the East African L.donovani focus 82 

is the second largest, with the highest incidence in Ethiopia and the Sudan [2]. 83 

 84 

VL is caused by protozoan parasites of the Leishmania donovani species 85 

complex transmitted to human and animal hosts by the bite of phlebotomine 86 

sand flies. It has already been determined that large numbers of individuals in 87 

endemic areas are infected with the parasite but do not develop any signs or 88 

symptoms of the disease. The reported ratio of asymptomatic infections to VL 89 

clinical cases varies widely from 4:1 in Kenya [3] to 50:1 in Spain [4]. This 90 

variation is presumed to reflect differences in parasite virulence and host 91 

population characteristics, and may also depend on the study designs and on 92 

the tests used to define asymptomatic infection [1]. 93 

 94 

The methods more widely used in order to assess asymptomatic 95 

infection in the field are a) serological assays that detect anti-Leishmania 96 

antibodies based either on the direct agglutination test (DAT) or the rK39-97 

immunochromatographic test (rK39-ICT) and b) Leishmanin Skin Test (LST) 98 

that measures cell-mediated immunity against Leishmania [5,6]. 99 

 100 



 5 

It is important to generate knowledge on the factors associated with 101 

asymptomatic infection for the optimal design and implementation of prevention 102 

and control strategies of VL, as asymptomatically infected individuals can 103 

harbor latent parasite and act as reservoirs for new infection or become ill if 104 

immunosuppression occurs [7,8]. 105 

 106 

In northern Ethiopia, the prevalence of VL is steadily rising posing an 107 

increasing public health concern. The region has recently experienced 108 

epidemics in previously unaffected areas [2]. In 2005, a kala-azar outbreak 109 

occurred in the district of Libo Kemkem in Amhara regional state, described by 110 

Alvar et al [9] . A case control study was conducted there in 2007 to evaluate 111 

the risk factors associated with the clinical form of the disease [10]. However, 112 

and as it has been previously stated,  the epidemiological determinants of 113 

clinical VL and sub clinical infection are not necessarily the same [11]. 114 

 115 

Thus, the aim of this study is to describe the determinants of 116 

asymptomatic leishmanial infection among the villages with high incidence of VL 117 

in Libo Kemkem and Fogera in order to provide further information that will help 118 

the Amhara regional health authorities to develop effective strategies to control 119 

the transmission of the disease. 120 

 121 

 122 

MATERIAL AND METHODS 123 

 124 

Study area and population 125 



 6 

 126 

The study was conducted during May-July 2009 in the districts (weredas) 127 

of Libo Kemkem and Fogera (Amhara State, Ethiopia). These are adjacent 128 

districts most affected by the outbreak of VL that occurred in 2005 [9]. In 2009, 129 

the population numbered 198 374 and 226 595 in Libo Kemkem and Fogera, 130 

respectively. The economic status of the population is uniformly low. The 131 

districts are located in a black cotton clay soil flat plain (1800-2000 meters 132 

a.s.l.). Human activities related to intensive cultivation of teff, maize, beans, 133 

oilseeds, rice and cotton, have reduced the natural vegetation to scattered 134 

clumps of acacia trees. Most of the area is flooded during the rainy season 135 

(July-September) and dried up during the dry season (November-May), 136 

resulting in deep cracks in the soil surface, which could turn into breeding sites 137 

for the putative vector Phlebotomus orientalis [12,13]. 138 

 139 

Study design 140 

 141 

Population sampling was carried out by a multi-staged cluster survey. 142 

Primary sampling units were sub-districts (kebeles) with high incidence of VL 143 

according to the 2008 register of the Addis Zemen VL Treatment Center: Bura, 144 

Yifag Akababi and Agita from Libo Kemkem and Sifatra and Rib Gebriel from 145 

Fogera. Secondary sampling units were randomly selected villages (gotts) in 146 

each of the selected sub-districts. Third sampling units were randomly selected 147 

households in each of the villages. All children with reported age between 4 and 148 

15 years living in the household at the time of the survey, and who had not 149 

previously suffered VL, were included in the study, as long as they were 150 



 7 

asymptomatic (absence of VL signs and symptoms: fever for > 2 weeks, in 151 

combination with either enlargement of spleen and/or liver, or weight loss).  152 

 153 

Data collection 154 

 155 

A blood sample was taken from the selected children in order to detect 156 

anti-Leishmania antibodies. The rK39-ICT (Kalazar Detect® Rapid Test, InBios 157 

International Inc., USA) was performed following the manufacturers’ 158 

instructions. DAT with freeze-dried antigen (ITMA-DAT/VL, Prince Leopold 159 

Institute of Tropical Medicine, Antwerp, Belgium) was carried out on blood-160 

impregnated filter paper following the screening procedure according to the 161 

manufacturer’s instructions. Titers ≥1:3200 were considered positive.  162 

 163 

Leishmanin Skin Test was carried out using L. major antigen (Leishmanin 164 

batch 123-2; Pasteur Institute, Iran). The test was read 48 hours later by the 165 

ballpoint pen method. An induration with an average of two perpendiculars ≥5 166 

mm was considered as positive. 167 

 168 

All children were measured and weighted according to standard WHO 169 

procedures [14]. Wasting was defined as Body Mass Index (BMI) for age <-2 Z 170 

scores, and stunting as  Height for Age < -2 Z scores according to the 2006 171 

WHO Growth Standards for children < 5 years and to the 2007 WHO Growth 172 

Reference for children > 5 years respectively [15]. 173 

 174 



 8 

Care providers of the children were interviewed by trained health 175 

professionals using standardized questionnaires that included questions on 176 

demographics, household characteristics, child health, dietary habits and VL 177 

prevention behaviours. The questionnaires used were pretested and translated 178 

into Amharic, the local language. 179 

 180 

 181 

182 



 9 

Data Analysis 183 

 184 

The primary outcome of interest was Leishmania asymptomatic infection 185 

defined as a positive result either in rK39-ICT, DAT or LST. The serological 186 

tests and the LST measure different types of the immune response and are thus 187 

not likely to produce the same results. Therefore we created two secondary 188 

outcomes: a) Seropositive: positive to rK39-ICT and/or DAT irrespective of the 189 

LST result and b) LST Positive: positive to LST irrespective of the serostatus. 190 

 191 

We attempted to estimate the factors associated with asymptomatic 192 

infection and then to isolate the factors associated with the seropositivity and 193 

LST positivity by making independent analysis for the three outcomes described 194 

above. 195 

 196 

Multivariate analysis to examine the socio-economic, behavioural, 197 

nutritional and dietary predictors of asymptomatic infection indicators were 198 

carried out using logistic regression models adjusting for potential confounding 199 

variables that were significant in the univariate analysis (carried out using 200 

bivariate logistic regression). Variables included in the model as numeric have 201 

the p value for trend and the Odds Ratios (OR) and Confidence Intervals (CI) 202 

described in the text and the OR (CI) for categories detailed in the tables. 203 

Multivariate models included all variables for which adjusted estimates are 204 

presented. 205 

 206 

A p value less than 0.05 was considered statistically significant. 207 



 10 

 208 

Data analysis was performed using AnthroPlus v1.02 (WHO, Geneva, 209 

Switzerland), and SPSS version 18.0 (SPSS Inc., Chicago, Illinois, USA). 210 

 211 

 212 

Ethical considerations 213 

 214 

The study was approved by the ethical advisory boards of Instituto de 215 

Salud Carlos III in Spain and the Armauer Hansen Research Institute and the 216 

Ethiopian National Ethical Review Committee in Ethiopia. Support letters were 217 

obtained from the Amhara Regional State and the district Health Bureaus. All 218 

parents/guardians gave written informed consent prior to the enrolment of their 219 

children in the study. Assent was also obtained from children > 11 years of age. 220 

 221 

 222 

RESULTS 223 

 224 

All the gotts selected were rural. Around 90% of the households were 225 

headed by males and in more than 99% of the households the occupation of the 226 

head was related to farming activities (farmer, labourer, cotton worker, etc.). 227 

Ninety eight per cent of the households reported owning land. The mean size of 228 

land owned by a household was 1.6 Ha (range 0.01 – 8 Ha). Only 6.4% of the 229 

households owned more than 3 Ha. More than 95% of the households reported 230 

owning some type of domestic animals, mainly cows (89.8%), chicken (59.2%) 231 



 11 

and sheep (23.6%). Thirty two per cent of the households had radio and only 232 

0.3% had access to electricity. 233 

 234 

A total of 605 children were surveyed (51.1% boys and 48.9% girls) with 235 

a mean age of 8.8 (3.2 SD).  Sixty one children (10.1%) had asymptomatic 236 

infection, of which 38 (6.3%) were seropositive and 33 (5.6%) LST positive. 237 

There was a wide variation in the number of asymptomatically infected children 238 

according to gotts, with the gotts in Bura kebele presenting the highest 239 

frequencies (see Table 1). 240 

 241 

Among the children, 245 (40.7%) were found to be stunted and 130 242 

(21.6%) were wasted. Only 4.6% had consumed animal food source products 243 

the day before the interview.   244 

 245 

Unadjusted analysis of infection  246 

 247 

Table 2 and Table 3 summarize the individual and household 248 

characteristics that showed significant association in the univariate analysis with 249 

any of the outcomes previously described. 250 

 251 

The individual factors that showed a positive association with 252 

asymptomatic infection were: increasing age, male sex, being wasted, sleeping 253 

outside and herding cattle; and the household characteristics: increasing 254 

number of people in the household, having a past history of VL in the family, 255 

living in a household straw roofed house and owning sheep three years before 256 



 12 

and at the time of the survey. On the other hand, owning dogs three years 257 

before and at the time of the survey showed an inverse association with 258 

asymptomatic infection. 259 

 260 

 Sleeping outside at any time of the day was the only individual variable 261 

that showed a direct association with seropositivity besides increasing age and 262 

being male. In terms of household characteristics, those that presented a 263 

positive association were increasing number of people in the household, living 264 

in a family with past history of VL and if the household owned sheep three years 265 

before and at the time of the survey. In the opposite direction, the number of 266 

cattle owned by the family at the time of the survey showed an inverse 267 

association. 268 

 269 

 The individual variables that showed a direct association with LST 270 

positivity were the same as those for asymptomatic infection, except for 271 

wasting.  The use of bed net by a child, although not statistically significant 272 

suggested an inverse relationship (p=0.07). In terms of household conditions, 273 

increasing number of people in the family and straw roof showed a positive 274 

association with a positive LST, and the increasing number of cattle owned by 275 

the family and the presence of dogs three years before and at the time of the 276 

survey showed a negative one. 277 

 278 

Adjusted analysis 279 

 280 



 13 

Table 4 shows the results of the multivariate logistic regression for 281 

asymptomatic infection, seropositivity and LST positivity.  282 

 283 

The individual variables that kept in the model positively associated with 284 

asymptomatic infection after adjustment were: increasing age, being male and 285 

sleeping out at any time [OR (95% CI): 1.12 (1.02, 1.23), 2.06 (1.10, 3.82) and 286 

2.10 (1.15, 3.85) respectively]. In terms of household characteristics: increasing 287 

number of people living in the household, past history of VL in the family, living 288 

in a straw roofed house and if the family owned sheep three years before and at 289 

the time of the survey [OR (95% CI): 1.26 (1.06, 1.50), 2.66 (1.44, 4.92), 2.35 290 

(1.24, 4.45) and 3.25 (1.52, 6.98) respectively]. And with an inverse and 291 

significant association, the presence of dogs in the house three years before 292 

and at the time of the survey [OR (95% CI): 0.44 (0.23, 0.85]. 293 

 294 

Being male and increasing age were the only two individual variables that 295 

kept direct and significant association with seropositivity after adjustment [OR 296 

(95% CI): 2.69 (1.26, 5.77) and 1.12 (1.00, 1.25) respectively]. Living in a family 297 

with past history of VL and if the household owned sheep three years before 298 

and at the time of the survey also showed a positive association  [OR (95% CI): 299 

3.66 (1.84, 7.28) and 2.43 (1.02, 5.78) respectively]. And in the opposite 300 

direction, the number of cattle remained inversely associated with seropositivity 301 

[OR (95% CI): 0.85 (0.74, 0.99)]. 302 

 303 

The individual factors positively and significantly associated with LST 304 

were sleeping outside and herding the cattle [OR (95% CI): 3.14 (1.33, 7.39) 305 



 14 

and 3.80 (1.04, 13.89) respectively]; and in terms of household conditions the 306 

only one positively associated with it was increasing number of people in the 307 

household [OR (95% CI): 1.29 (1.03, 1.63)] and in the opposite direction the 308 

presence of dogs three years before and at the time of the survey [OR (95% 309 

CI): 0.29 (0.13, 0.67)] 310 

 311 

No significant association was found between any of the outcomes 312 

analysed for asymptomatic infection and stunting; sex, age or education of the 313 

head of the household; household electricity, radio or land owning, floor and 314 

walls construction material and condition; number of meals or consumption of 315 

animal source food products; number of bed nets in the household, house 316 

spraying status;  the existence of an animal shed, animal dung or a termite 317 

mound near the house; and number of chicken owned by the household. 318 

 319 

 320 

DISCUSSION 321 

 322 

The prevalence of asymptomatic infection found in our study sample as 323 

well as the factors associated with it differed depending on the outcome variable 324 

used for the analysis.  325 

 326 

The discordances observed between serology and LST have been 327 

discussed elsewhere [16-18]. The last LST screening in the area was 328 

conducted in 2005 as part of the outbreak assessment, and the prevalence of 329 

LST positivity was considerably higher than in our study, 34% for men and 26% 330 

for women [9]. This discrepancy is consistent with the fact that the cited study 331 



 15 

was conducted in a different population age range (0.7 to 60 years old) and at 332 

the peak of the epidemic in an area that has since transited into an endemic 333 

focus with a sustained low transmission, as described recently by Herrero et al 334 

(Herrero, 2009). The strong variation in the prevalence of asymptomatic 335 

infection among clusters highly endemic for VL is congruent with the spatial 336 

clustering observed in other studies of asymptomatic infection [19,20] and of 337 

clinical VL cases [21,22]. Notably, Bura, the kebele where the 2005 outbreak 338 

started, has maintained the highest prevalence ever since [9]. 339 

 340 

The increase of asymptomatic infection rate with age observed in our 341 

study area is consistent with an endemic focus of VL, in spite of the low VL 342 

incidence situation reached after the outbreak [23]. The permanence of LST 343 

reactivity is thought to be a consequence of cumulative past exposure, thus 344 

prevalence typically rises with age [24].  The positive association between 345 

Leishmania infection and older age, as well as with male sex, has also been 346 

related to activities like cattle herding or sleeping outside, that imply an 347 

increased potential exposure to the sand fly vector, and that are culturally 348 

specific to male adolescents and male adults [19,25]. Our results would support 349 

this hypothesis, as cattle herding and sleeping outside were also identified in 350 

our study population as risk factors for asymptomatic infection and had 351 

previously been identified as risk factors for VL in South Ethiopia [26] and North 352 

Ethiopia (in our study area) as well [10]. The greater exposure to sand flies 353 

when herding livestock can be associated with the moving near and/or far away 354 

from homesteads at dusk and down when the sand flies are active [27] and also 355 

with an increased proximity to acacia trees. Resting under acacia trees has 356 



 16 

been identified as a risk factor for VL in our study area [10]. Among our 357 

surveyed population, 82% of the herder children reported resting under acacia 358 

trees while herding. Acacia trees are thought to be diurnal resting sites for 359 

Phlebotomus orientalis,  the described potential vector of the disease in the 360 

area [12,13].  361 

 362 

Poor nutritional status has been associated with a higher risk of 363 

developing visceral leishmaniasis in other studies [28-31] although to the best of 364 

our knowledge, an association with asymptomatic infection has not yet been 365 

described. In our findings wasting appeared as a risk factor for asymptomatic 366 

infection but only in the unadjusted analysis, so we can not conclude there is 367 

association between nutritional status, measured by anthropometry, and 368 

asymptomatic infection in our study population. 369 

 370 

The use of bed net appeared to be protective but did not reach a 371 

significant association with any of the infection outcomes used in the analyses, 372 

which is in agreement with other studies in relation to asymptomatic infection 373 

[18,32]. The protective effect of bed net use against visceral leishmaniasis 374 

remains unclear, with variable results depending on the setting and study 375 

[11,33]. The lack of protective effect found in our study could be associated with 376 

the net condition, nature of utilization and impregnation status, conditions that 377 

were not assessed in our survey.   378 

 379 

A larger family size may appear as a risk factor for asymptomatic 380 

infection based on attraction of sand flies by greater biomass, as it was 381 



 17 

described for the risk of VL in this same area [10]. Other studies have also 382 

shown a positive association with seropositivity  and  previous VL cases contact 383 

[3,20,34], supporting the hypothesis of transmission within the homestead, 384 

human to human. This hypothesis has been further strengthened by the failure 385 

of other studies to relate the household clustering of infection and disease to 386 

characteristics of the house or the surroundings [3,28]. It is important to 387 

highlight that the increased likelihood of asymptomatic infection among children 388 

with a past VL case in the family remained significant only for seropositivity and 389 

not for LST positivity, in concordance with findings of Bern et al in Bangladesh 390 

[32]. In one study conducted in Kenya, it was found that the association 391 

between LST positivity and previous VL cases in the family was significant only 392 

for women and young children, suggesting that women were exposed in and 393 

around the house and males, in addition, exposed elsewhere [35]. We tested 394 

this hypothesis by conducting separate analyses for male and female 395 

populations but results did not vary (data not shown).  396 

 397 

Living in a straw roofed house versus an iron thatched one was the only 398 

house characteristic associated with asymptomatic infection. It could be related 399 

to socioeconomic status or to the potential of straw roofs to provide resting 400 

places for the sand fly that would increase its survival and abundance. Mud-401 

type houses have been identified as risk factors for VL or asymptomatic 402 

infection before and have been associated with better living conditions or with 403 

the vector preference for mud crack walls for breeding and resting 404 

[22,26,36,37]. However, regarding P. orientalis more studies are needed, as the 405 



 18 

few extant studies in the literature point out to an exophagic behaviour of the 406 

vector, ill suited with this hypothesis [38].   407 

  408 

In relation to domestic animals the associations found are conflicting. 409 

Owning sheep increases the risk of asymptomatic infection but the presence of 410 

dogs and an increasing number of cattle owned by the family minimizes it. 411 

 412 

The positive correlation of disease and the presence of sheep has 413 

already been described [19,22], and has been explained by the greater biomass 414 

and the accumulation of animal dung that may be attractive to the sand flies, 415 

drawing the vectors into closer association with humans. 416 

 417 

The role of cattle in the transmission of the disease remains unclear and 418 

has been subject of a recent review [11]. Several studies coincide with our 419 

results in the protective effect of cattle towards VL [21,22,39] and the possible 420 

explanation given is that the number of cattle acts as wealth indicator and/or 421 

that there is a potential vector blood preference for these domestic animals that 422 

will exert a zooprophylactic effect. P. orientalis blood preference for cattle 423 

versus humans has already been shown in other areas of Ethiopia, supporting 424 

this idea [38]. Interestingly, this protective effect was seen in relation to 425 

seropositivity, but not to LST positivity, in concordance with findings in 426 

Bangladesh by Bern et al  [32], where increased cattle density was found to be 427 

protective against VL and DAT positivity but was identified as a risk factor for 428 

LST positivity. 429 

 430 



 19 

The negative correlation between the presence of dogs and 431 

asymptomatic infection is in contrast to the finding of owning dogs as a risk 432 

factor for VL in this same area [10]. The protective role of dogs against 433 

asymptomatic infection had been described in Brazil [34], but other studies have 434 

not found any significant association with either asymptomatic infection or VL 435 

[20,22].  436 

 437 

Our study had a number of limitations. One of them is the cross sectional 438 

nature of the study, which limits the making of causal inferences between the 439 

analysed factors and the infection. Another limitation is that the study subjects 440 

were not tested for HIV, which may alter the results. However, adult HIV 441 

incidence in Amhara state was 0.32 in 2007 [40]  (theoretically lower for children 442 

below 15 years old) so although it is possible that a subset of our cases may 443 

have had HIV it would be a small number and we are confident it would not alter 444 

our general conclusions. 445 

 446 

CONCLUSIONS 447 

 448 

Selected behavioural and housing factors were associated with higher rates 449 

of asymptomatic infection and these can be the focus of interventions. 450 

Protective measures should be implemented against the identified risk activities 451 

such as herding cattle or sleeping outside. More studies are needed on the 452 

behaviour of the P.orientalis in the area in order to clarify possible 453 

entomological interventions related to housing conditions. Given the contrasting 454 

results found in our study and in the literature, and the complex nature of the 455 



 20 

relationship between disease transmission and domestic animals, the exact role 456 

of domestic animals in transmission needs to be studied further before any 457 

intervention is recommend in this regard.  458 

 459 

Acknowledgements 460 

 461 

 The authors thank the study participants for volunteering, the data 462 

collectors for the field work efforts, the AHRI/ALERT and the Fundación 463 

Española para la Cooperación Internacional, Salud y Política Social for logistic 464 

and technical support, and the Amhara Regional State Regional Laboratory for 465 

allowing us to use their laboratory facilities and for creating a conducive 466 

environment during field work.  467 



 21 

 

 

 

Table 1: Leishmania asymptomatic infection, seropositivity and LST positivity prevalence by gott in Amhara state, Ethiopia. 

    Asymptomatic     

    infection* Seropositivity*  LST positivity*± 

Name of cluster/Gott Kebele Wereda N n % n % N n % 

FOGERIE MENDER Agita Libo Kemkem 32 0 0 0 0 32 0 0 

FUAT FUAT Agita Libo Kemkem 34 2 5.9 2 5.9 31 0 0 

GILGEL TERARA Agita Libo Kemkem 32 0 0 0 0 29 0 0 

MELAGUD Agita Libo Kemkem 35 0 0 0 0 35 0 0 

MEDROGE Bura Libo Kemkem 46 11 23.9 10 21.7 46 7 15.2 

MEHAL-EGZIABHERAB Bura Libo Kemkem 31 11 35.5 1 3.2 31 10 32.3 

MENTA-WARKA Bura Libo Kemkem 29 8 27.6 6 20.7 29 2 6.9 

QUARA Bura Libo Kemkem 34 5 14.7 1 2.9 33 4 12.1 

GULTOCH D. Sifatra Fogera 34 4 11.8 3 8.8 34 2 5.9 

LAHADA D. Sifatra Fogera 35 2 5.7 1 2.9 34 1 2.9 

RAS DIBA D. Sifatra Fogera 34 0 0 0 0 33 0 0 

SIFATRA D. Sifatra Fogera 30 4 13.3 3 10 27 1 3.7 

AMAGA Rib Gebriel Fogera 30 3 10 3 10 30 0 0 

DENBOCH Rib Gebriel Fogera 35 2 5.7 1 2.9 35 1 2.9 

GICHOCH Rib Gebriel Fogera 35 2 5.7 2 5.7 32 2 6.2 

GOMBEL Rib Gebriel Fogera 32 4 12.5 2 6.3 32 3 9.4 

ANSHA Yifag Akababi Libo Kemkem 33 2 6.1 2 6.1 33 0 0 

BATA Yifag Akababi Libo Kemkem 34 1 2.9 1 2.9 33 0 0 

Total      605 61 10.1 38 6.3 589 33 5.6 
*As defined in the Materials and Methods section 
LST=Leishmanin Skin Test 
± A total of 589 children had the LST performed. 
 



 22 

 

Table 2: Individual variables associated with Leishmania asymptomatic infection in children. Unadjusted analysis. 
  Asymptomatic infection * Seropositivity* LST positivity*± 
Factor N (%) Positive     Odds ratio§  Positive     Odds ratio§  Positive     Odds ratio§  

  n (%) (95% CI) n (%) (95% CI) n (%) (95% CI) 

  Age        
<5 years 77 (12.7) 2 (2.6) Reference 2 (2.6) Reference 1 (1.3) Reference 

5 to 9 years 295 (48.8) 21 (7.1) 
2.87 (0.66, 

12.53) 15 (5.1) 2.01 (0.45, 8.98) 8 (2.7) 2.16 (0.27, 17.53) 

10 to 15 years 233 (38.5) 38 (16.3) 
7.31 (1.72, 

31.05) 21 (9.0) 3.71 (0.85, 16.22) 
24 

(10.6) 8.87 (1.58, 66.69) 
p for trend   <0.0001  0.03  0.002 

  Sex        
Girl 296 (48.9) 18 (6.1) Reference 18 (6.1) Reference 10 (3.4) Reference 
Boy 309(51.1) 43 (13.9) 2.50 (1.40, 4.41) 43 (13.9) 2.85 (1.36, 5.98) 23 (7.6) 2.30 (1.07, 4.92) 

  p global   0.002  0.006  0.032 

Wasting¶        
No 472 (78.4) 47 (10.0) Reference 29 (6.1) Reference 25 (5.4) Reference 

Yes 130 (21.6) 14 (10.8) 1.10 (0.58, 2.05) 9 (6.9) 1.14 (0.52, 2.46) 8 (6.2) 1.15 (0.51, 2.62) 
p for trend   0.05  0.11  0.14 

Sleeps outside        
No 366 (60.6) 21 (5.7) Reference 16 (4.4) Reference 8 (2.2) Reference 

Yes 238 (39.4) 40 (16.8) 3.32 (1.93, 5.08) 22 (9.2) 2.23 (1.14, 4.34) 
25 

(10.7) 5.21 (2.31, 11.77) 
p global   <0.0001  0.018  <0.0001 

  Herds the cattle        
No 250 (41.3) 12 (4.8) Reference 10 (4.0) Reference 3 (1.2) Reference 

Yes 355 (58.7) 49 (13.8) 3.17 (1.65, 6.10) 28 (7.9) 2.05 (0.98, 4.31) 30 (8.5) 7.54 (2.27, 24.91) 
p global   0.001  0.06  0.001 

  Uses bed net        
No 365 (60.3) 41 (11.2) Reference 21 (5.8) Reference 25 (6.9) Reference 

Yes 240 (39.7) 20 (8.3) 0.72 (0.41, 1.26) 17 (7.1) 1.25 (0.64, 2.42) 8 (3.4) 0.47 (0.21, 1.07) 
p global   0.25  0.51  0.072 

Overall 605             
*As defined in the Materials and Methods section 
LST=Leishmanin Skin Test 
± Only 589 children had the LST performed. 
§OR obtained by logistic regression  
¶Defined as Body Mass Index for Age Z score < -2 SD based on the 2006 WHO Growth Standards for children < 5 y and on the 2007 WHO Growth Reference for children > 5 y  



 23 

 

Table 3: Household characteristics associated with Leishmania asymptomatic infection in children. Unadjusted analysis. 

  Asymptomatic infection*  Seropositivity* LST positivity*± 

Factor N (%) Positive     Odds ratio§  Positive     Odds ratio§  Positive     Odds ratio§  

  n (%) (95% CI) n (%) (95% CI) n (%) (95% CI) 

People in the family        

< 5 people 201 (33.3) 13 (6.5) Reference 10 (5.0) Reference 5 (2.6) Reference 

5-7 people 257 (42.6) 23 (8.9) 1.42 (0.70, 2.88) 14 (5.4) 1.10 (0.48, 17.53) 14 (5.6) 2.24 (0.79, 6.33) 

> 7 people 145 (24.0) 24 (16.6) 2.87 (1.41, 5.85) 14 (9.7) 2.04 (0.88, 4.73) 13 (9.0) 3.71 (1.29, 10.64) 
p for trend   0.003  0.05  0.03 

Past history of kala azar in         
the family        

No 442 (73.2) 34 (7.7) Reference 18 (4.1) Reference 22 (5.1) Reference 
Yes 162 (26.8) 27 (16.7) 2.40 (1.39, 4.12) 20 (12.3) 3.32 (1.71, 6.45) 14 (7.5) 1.37 (0.65, 2.91) 

   0.002  <0.0001  0.4 

House roof material        
Corrugated iron 223 (36.9) 32 (14.3) Reference 19 (8.5) Reference 18 (8.4) Reference 

Straw 382 (63.1) 29(7.6) 2.04 (1.19, 3.47) 19 (5.0) 1.78 (0.92, 3.44) 15 (4.0) 2.19 (1.08, 4.34) 
p global   0.009  0.09  0.03 

Number of cattle owned by         
the household        

No cattle 62 (10.2) 7 (11.3) Reference 6 (9.7) Reference 4 (6.9) Reference 
Less than 10 cattle 502 (83.0) 50 (10.0) 0.87 (0.37, 2.01) 30 (6.0) 0.59 (0.24, 1.48) 27 (5.5) 0.78 (0.26, 2.33) 
More than 10 cattle 41 (6.8) 4 (9.8) 0.85 (0.23, 3.11) 2 (4.9) 0.48 (0.09, 2.49) 2 (4.9) 0.69 (0.12, 3.97) 

p for trend   0.1  0.041  <0.0001 

Household owns sheep        
No 462 (76.4) 38 (8.2) Reference 27 (5.8) Reference 18 (4.0) Reference 

Yes 143 (23.6) 23 (16.1) 2.56 (1.30, 5.04) 11 (7.7) 2.38 (1.04, 5.45) 15 (10.5) 2.36 (0.98, 5.67) 
p global   <0.0001  0.039  0.056 

Household owns dogs        
No 273 (45.1) 35 (12.8) Reference 22 (8.1) Reference 21 (7.8) Reference 

Yes 332 (54.9) 26 (7.8) 0.58 (0.34, 0.98) 16 (4.8) 0.58 (0.29, 1.12) 12 (3.1) 0.46 (0.22, 0.95) 
p global   0.04  0.11  0.035 

Overall 605             
*As defined in the Materials and Methods section 
LST=Leishmanin Skin Test 
± Only 589 children had the LST performed. 
§OR obtained by logistic regression  



 24 

Table 4: Factors associated with Leishmania  asymptomatic infection in children. Adjusted analysis. 

 Asymptomatic*  Seropositivity* LST positivity* 

 infection   

Factor Odds ratio (95% CI) Odds ratio (95% CI) Odds ratio (95% CI) 

Child age    

<5 years Reference Reference Reference 

5 to 9 years 2.61 (0.57, 12.02) 1.99 (0.43, 9.16) 2.03 (0.44, 9.44) 

10 to 15 years 5.62 (1.24, 25.45) 3.57 (0.79, 16.07) 4.20 (0.89, 19.49) 

p for trend 0.014 0.041 0.16 

Child sex    

Girl Reference Reference Reference 

Boy 2.06 (1.10, 3.82) 2.69 (1.26, 5.77) 1.34 (0.59, 3.03) 

  p global 0.02 0.011 0.48 

Child sleeps outside    

No Reference " Reference 

Yes 2.10 (1.15, 3.85)  3.14 (1.33, 7.39) 

p global 0.016  0.009 

Child herds the cattle    

No   Reference 

Yes " " 3.80 (1.04, 13.89) 

p global   0.044 

People in the family    

< 5 people Reference " Reference 

5-7 people 1.54 (0.72, 3.32)  1.45 (0.69, 3.05) 

> 7 people 3.04 (1.32, 7.00)  3.01 (1.37, 6.60) 

p for trend 0.009  0.029 

History of past kala azar in family    

No Reference Reference  

Yes 2.66 (1.44, 4.92) 3.66 (1.84, 7.28) " 

p global 0.002 <0.0001  

House Roof Material    

Corrugated iron Reference   

Straw 2.35 (1.24, 4.45) " " 

p global 0.009   

Number of cattle    

No cattle  Reference  

Less than 10 cattle " 0.38 (0.14, 1.04) " 

More than 10 cattle  0.28 (0.05, 1.57)  

p for trend  0.035  

Household owned sheep 3 years before and 
at the time of the survey    

No Reference Reference  

Yes 3.25 (1.52, 6.98) 2.43 (1.02, 5.78)  

p global 0.002 0.046  

Household owned dogs 3 years before and at 
the time of the survey    

No Reference  Reference 

Yes 0.44 (0.23, 0.85)  0.29 (0.13, 0.67) 

p global 0.014  0.003 
*As defined in the Materials and Methods section 
LST=Leishmanin Skin Test 
± Only 589 children had the LST performed. 
§OR obtained by logistic regresssion  



 25 

 

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1 

 

Effect of childhood protein energy malnutrition on the immunological status of 1 

children from the new Visceral Leishmaniasis focus in Libo Kemkem (Amhara 2 

State, Ethiopia) 3 

Endalamaw Gadisa1, Zelalem Abebe2, Estefanía Custodio3, Tsegaye Hailu1, Luis 4 

Sordo4, Javier Nieto5, Carmen Chicharro5, Abraham Aseffa1, Carmen Cañavate5, 5 

Howard Engers1, Israel Cruz5 and Javier Moreno5 6 

(1) Armauer Hansen Research Institute, Addis Ababa, Ethiopia;  7 

(2) Amhara Regional State Laboratory, Bahir Dar, Ethiopia  8 

(3) Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, Madrid, Spain;  9 

(4) Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, Spain;  10 

(5) WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro 11 

Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain;  12 

 13 

Abstract 14 

Childhood Protein Energy Malnutrition (PEM) is a major public health problem in developing 15 

countries. Epidemiologic studies documented strong association between PEM and risk of child 16 

death and/or severity of infections. However, the mechanisms that govern the relationship 17 

between PEM and infectious diseases are multiple and not well described. The present study 18 

examined the effect of PEM on selected immunological players implicated in the infection by 19 

Leishmania parasites. Our data showed significantly lower total count of white blood cells, 20 

lymphocytes and T cell subpopulations (CD4+ and CD8+) in severely malnourished children. 21 

PBMCs from the severely malnourished children produced significantly low or no cytokine after 22 

24 hours of stimulation with PHA. Moderately malnourished children produced significantly high 23 

level of PGE2 as compared to the non-malnourished children. Non-malnourished children 24 

showed high level of cytokines in the serum, while no detectable levels of any of the cytokine 25 

assayed were found in severely malnourished children and only IL-4 and IL-10 were detected in 26 

the serum of moderately malnourished children. The DAT positive non-malnourished children 27 

produced significantly high level of IFN-γ and IL-10 as compared to the moderately and severely 28 

malnourished children.  The detection of low levels of serum IFN-γ and relatively high levels of 29 

IL-4 and IL-10 in the moderately malnourished children might indicate a Th2 bias in this group 30 



2 

 

probably associated to the high level of PGE2 production. In conclusion, severe malnutrition 31 

affected not only the absolute number of the immune cells in children but also their functional 32 

activity, an adverse effect that might be mediated by overproduction of biochemical factors 33 

induced by malnutrition like PGE2. 34 

  35 

INTRODUCTION 36 

Childhood Protein Energy Malnutrition (PEM) is a major public health problem in developing 37 

countries. In Africa it is estimated that 45% of the children suffer from malnutrition [1]. There is a 38 

strong relationship between malnutrition, infection, and infant mortality. Human epidemiologic 39 

studies have documented that PEM is a primary cause of immunodeficiency and a major 40 

determinant of both progression and severity of infections caused by different pathogens [2-4]. 41 

Children with mild and moderate malnutrition present a reduced cell mediated and humoral 42 

immune function [5] which can make them more susceptible to infections. A recent meta-43 

analysis showed that HIV prevalence is high in malnourished children in sub-Saharan Africa, 44 

and they are at a significantly increased risk of mortality [6]. Zachariah et al, observed that 45 

moderate to severe malnutrition is a risk factor associated with TB [7].  A strong association of 46 

giardiasis and PEM has also been reported [8]. The specific anti-IgG response to Plasmodium 47 

falciparum was found to be significantly lower in severely stunted children as compared to non 48 

stunted [9].  49 

Visceral leishmaniasis (VL) or kala-azar is a (re)emerging neglected tropical disease often 50 

associated with malnutrition. VL is a life-threatening infectious disease caused by protozoan of 51 

the Leishmania (Leishmania) donovani species complex, causing 59,000 deaths per year. Of 52 

the 500,000 annual cases, 90% occur in Bangladesh, Brazil, India, Nepal, Ethiopia and Sudan. 53 

VL provokes in the patients fever, severe cachexia, hepatosplenomegaly, pancytopenia and 54 

hypergammaglobulinaemia [10]. In most endemic areas children of young age are the most 55 

affected and immature immune system and malnutrition were documented as predisposing 56 

factors [11, 12]. A recent study confirms that percentages of VL patients with underweight 57 

ranges from 21.4% in Brazil to 92.6% in Sudan [13]. PEM was also shown to be a determinant 58 

factor for progression to clinical VL [14], and it is associated with more severe form of VL and a 59 

major risk for poor treatment outcomes   [15].  60 



3 

 

The immunodeficiency caused by malnutrition is responsible for the high susceptibility to VL, but 61 

the specific mechanism underlying the interaction between VL and malnutrition are not totally 62 

understood. A protective immune response in human VL is associated with a predominant cell 63 

mediated immunity and a type 1 cytokine profile [16]. Different studies demonstrated a 64 

significant decrease of peripheral blood CD3+ cells, impairment in the development of effector 65 

cell responses and an alteration in the balance of type 1/type 2 cytokine expression in 66 

malnourished children as compared to the non-malnourished once [17, 18]. Moreover, a bias to 67 

T helper 2 (Th2) type responses in malnourished infected children with decreased IFN-γ and an 68 

increased IL-4 and IL-10 production was documented [19]. 69 

Studies of malnutrition in animal models have reported a decrease in the percentage of CD3+ T 70 

lymphocytes in the spleens of moderately and severely malnourished rats [20], and it has also 71 

been shown that the T cells (CD4+ and CD8+) from malnourished mice are quiescent [20, 21]. 72 

Furthermore, Anstead et al. [22] in experimental VL showed that early visceralization of L. 73 

donovani in malnourished BALB/c mice is due to failure of lymph node barrier function, and may 74 

be related to decreased levels of IL-10 and nitric oxide (NO) and excessive production of 75 

prostaglandin E2 (PGE2), an important negative regulator of host immunity.  76 

 77 

Understanding the impact of the immunodeficiency caused by malnutrition in a population at risk 78 

for VL will help in the control effort; preventing the spread to new areas and/or mitigating the 79 

problem in affected communities. Libo Kemkem (Amhara, Ethiopia) is a high land district that 80 

converted to a low transmission area of VL after the outbreak detected in 2004/2005 [23, 24]. A 81 

study assessing the post outbreak situation indicated that children of 3 to 15 age group 82 

accounted for 30% of the cases. Moreover, 18% of the outbreak cases were malnourished [24].  83 

In order to understand the effect of nutritional status on immunity we evaluate hematological 84 

parameters and levels of peripheral blood lymphocyte subsets, as well as the functional 85 

capability of these cells to produce cytokine, in malnourished and non-malnourished children of 86 

4 to 15 years of age living in the new outbreak zone. In addition, we have measured serum 87 

levels of PGE2 in these children to assess the possible role of this factor in immunodeficiency 88 

and susceptibility to Leishmania infection.  89 

 90 

 91 

 92 



4 

 

 93 

 Materials and methods 94 

This study was done as part of an ongoing longitudinal study on childhood malnutrition and risk 95 

factors for asymptomatic VL, in the frame of the UBS-Optimus Foundation granted project 96 

“Visceral leishmaniasis and malnutrition in Amhara State, Ethiopia”.  97 

STUDY SUBJECTS. The study was conducted in children of age 4 to 15 years and was 98 

approved by the ethical review boards of Instituto de Salud Carlos III, Armauer Hansen 99 

Research Institute and the Ethiopian National Ethical Review Committee. Support letters were 100 

obtained from the Amhara State and the different districts’ Health Bureau. Parents /guardians 101 

gave written informed consent prior to the enrolment of their children in the study, and for 102 

children above 11 years of age verbal assents were also obtained in addition to the consent of 103 

their parents/guardians.   104 

The study was carried out in two phases. The first phase was done during May and July 2009, 105 

and included 456 children whose nutritional status, hematological parameters, and peripheral 106 

blood lymphoid subset were analyzed. The second phase was done during May and June 2010, 107 

enrolling 410 children, in whose nutritional, cytokine and serum PGE2 analyses were carried 108 

out. Anthropometric and clinical data were documented using structured questionnaires. Trained 109 

health professionals conducted the physical examination, anthropometric measurements 110 

(height, weight and age), clinical assessments and biological sample collection.  111 

NUTRITIONAL STATUS. All children were measured and weighted according to standard WHO 112 

procedures (WHO Working Group, 1986). Malnutrition was defined according to the Body Mass 113 

Index (BMI) for Age in relation to the 2006 WHO Growth Standards for children ≤  5 years and 114 

to the 2007 WHO Growth Reference for children older than 5 years respectively [25]. Global 115 

malnutrition was defined as BMI for Age Z score (BAZ) < -2 SD, moderate malnutrition as BAZ < 116 

-2 SD and BAZ > = -3 and severe malnutrition as BAZ < -3 SD.  117 

HEMATOLOGICAL ANALYSIS. Blood samples from 394 children of the first phase were 118 

collected in 4 ml Na2-EDTA tubes (SIGMA, UK) for hematological analysis. Analysis was done 119 

with hematology analyzer (Abbot CELL-DYN® 1800, USA) at the Amhara Health Bureau 120 

Regional Laboratory in Bahir-Dar. 121 

CELL STAINING. Percentage of peripheral blood CD4+, CD8+ and CD19+ cells were 122 

determined by cell staining of whole blood samples and flow cytometry analysis. One hundred 123 



5 

 

µL. of blood was incubated with anti-human anti-CD4 FITC conjugated, anti-CD8 PE conjugated 124 

and anti-CD19 FITC-conjugated monoclonal antibodies (Immunotools – Germany), and red 125 

blood cells were lysed using lysis buffer (BD, USA). The stained cells were fixed with 2% 126 

formaldehyde and kept at 4oC. Analysis with four colors FACSCalibur (BD, USA) was done 127 

within one week period of the cell staining date. Absolute number of cell subsets was 128 

determined taken into consideration the percentage of the different cell subsets after gating for 129 

lymphocytes at the FSC-SSC dot plot and the absolute number of peripheral lymphocytes 130 

obtained at the hematological analysis. 131 

CELL PREPARATION AND IN VITRO STIMULATION ASSAY: Blood sample collected in 2 ml 132 

Na2-EDTA tubes (SIGMA, UK) from 88 randomly selected children were used for in vitro 133 

stimulation assay with Phytohemaglutinin (PHA). One mL of whole blood was incubated for 15 134 

minutes at 4 oC after gentle shaking with 1 mL ice cold ACK buffer to lyse red blood cells. After 135 

5 minute centrifugation at 6000 rpm the supernatant was removed and the pellet washed in 2 136 

mL PBS (7.2 pH). The washed pellet was diluted in 1 mL of complete medium (RPMI10 with 137 

10% FCS complemented with antibiotics (100 IU/mL Penicillin and100 µg/mL Streptomycin) and 138 

subsequently 0.5 mL of the suspension of peripheral blood mononuclear cells (PBMC) were 139 

cultured in a flat bottom 24 well-plate with PHA (0.08 pg/µL final concentration). The culture 140 

supernatants were collected after 24 hours and kept frozen at -20 ºC until analysis. 141 

DIRECT AGLUTINATION TEST. Two drops of blood were impregnated on Whatman 3MM filter 142 

paper (Whatman International Ltd., England), left to air dry and placed individually in sealed 143 

plastic bags. The dry blood was used to determine asymptomatic Leishmania infection and/or 144 

exposure to Leishmania. Direct agglutination test (DAT) with freeze dried antigen (ITMA-145 

DAT/VL, Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium) was used according 146 

to the manufacturer’s protocol and titers  ≥1:3200 were considered positive [26, 27]. 147 

CYTOKINE ANALYSIS. The level of cytokines in the serum and culture supernatant were 148 

measured by commercially available kit for flow cytometric detection (Diaclone Research, 149 

Besancon, France) as per the manufactures instruction. 150 

PROSTAGLANDIN E2 ANALYSIS. Venous blood sample was collected in 5 mL plane tubes 151 

(SIGMA, UK) and serum was separated and stored at -20oC.  PGE2 assay was done for 94 152 

randomly selected serum samples from DAT negative children. The ELISA assay was done 153 

using commercially available kit (Parameter™, R&D Systems, Inc, USA) according to the 154 



6 

 

manufacture’s instruction. Cytokine and PGE2 concentrations (picogram per milliliter) were 155 

calculated using a standard curve developed with the standard provided with the kits. 156 

STATISTICAL ANALYSIS. Comparison of mean values of cytokines, PGE2 and hematological 157 

parameters was performed using STATA version 11 (Stata Corp., College Station, TX, USA) 158 

and P values ≤ 0.05 were considered statistically significant. 159 

 160 

RESULTS 161 

Nutritional status 162 

A total of 456 children were enrolled into the first study, of those 227 were boys and 229 were 163 

girls. In terms of nutritional status; 77.2% (352/456) were non malnourished and 22.8% 164 

(104/456) had acute malnutrition. Of the acutely malnourished children 5.0% (23/456) had the 165 

severe form and 17.8% (81/456) had the moderate form. Of the 410 (201 boys and 209 girls) 166 

children enrolled in the second phase; 77.1% (316/410) were non malnourished and 22.9% 167 

(94/410) had acute malnutrition. Of the acutely malnourished, 4.4% (18/410) had the severe 168 

form and 18.5% (76/410) had the moderate form. 169 

Hematological analysis 170 

We managed to get blood from 394 out of 456 children. Table 1 shows the red blood cell (RBC), 171 

Hemoglobin (HGB), hematocrit (HCT), and white blood cells (WBC), lymphocyte and 172 

subpopulations (CD4+, CD8+ and CD19+) of lymphocytes counts by nutritional status and sex. 173 

No difference was seen with sex and within the different nutritional status group of the girls in 174 

mean RBC, HGB and HCT. However, severely malnourished boys had significantly less RBC 175 

and HCT as compared to the normal and moderately malnourished (P<0.05) and their HCT was 176 

lower than the normal reference range. Moreover, their hemoglobin was less than the normal 177 

reference range but it was not significantly (P=0.064) lower as compared to the normal and 178 

moderately malnourished boys. Statistically significant difference was observed between the 179 

different nutritional status groups for total count of WBC, lymphocytes and sub populations of 180 

lymphocytes (P <0.05). There was no statistically significant (P=0.161) difference in CD4/CD8 181 

ratio among the different nutritional status groups. The malnourished children had less CD19+ 182 

cells (P=0.018) as compared to the non-malnourished group (Table 1).  183 



7 

 

 184 

Cytokine production by PHA-stimulated PBMCs 185 

Of the 88 samples used for PHA stimulation 57 were from non-malnourished, 21 from 186 

moderately and 10 from severely malnourished children. In the overnight PHA culture of PBMC, 187 

there was no detectable level of IL-4 and IL-12 in the severely malnourished children. 188 

Statistically significant level (P=0.002) of IL-10, TNF-α and IFN-γ were produced by the PBMC 189 

from the non-malnourished and moderately malnourished children as compared to the severely 190 

malnourished group. Higher level of TNF-α, IL-4, IL-10, and IFN-γ were detected in the 191 

supernatant of the PBMC culture from the non-malnourished children but the difference was not 192 

statistically significant (P>0.05) as compared to moderately malnourished children. There was 193 

no detectable level of IL-12 in the culture supernatant from the moderately malnourished 194 

children (Figure 1).  195 

 196 

Serum levels of Prostaglandin E2 and Cytokines 197 

From the total of 94 DAT negative serum samples assayed for PGE2; 61 were from non-198 

malnourished, 6 were from severely malnourished and 27 were from moderately malnourished 199 

children. The mean PGE2 level was higher in the malnourished children as compared to non-200 

malnourished. Significantly (P=0.007) high level was produced by the moderately malnourished 201 

children compared to the non-malnourished children (Figure 2). 202 

Of the 43 DAT negative children; 29 were non-malnourished, 12 moderately malnourished and 203 

2 severely malnourished while from the 36 DAT positive children; 18 were non-malnourished, 10 204 

moderately malnourished and 8 severely malnourished. We observed difference in the cytokine 205 

production profile in the DAT positive and negative groups. In the DAT negative groups there 206 

was detectable level of all the assayed cytokines (IL-4, IL-10, IFN-γ and TNF-α) in non-207 

malnourished children, but none in the severely malnourished children, and in the moderately 208 

malnourished children low level of IL-4 and IL-10 were detected. In the DAT positive groups 209 

there was significantly high (P=0.00) level of IFN-γ production in the non-malnourished when 210 

compared to malnourished children, higher level of TNF-α, IL-4 and IL-10 were produced by the 211 

non-malnourished children but was not significantly high compared to the moderately 212 



8 

 

malnourished children. Only IL-10 reached at a detectable level in the severely malnourished 213 

children (Fig 3).  214 

  215 

DISCUSSION 216 

The strong association between malnutrition and infections has been established through 217 

epidemiologic studies conducted in several different countries. The severity of malnutrition 218 

determines the risk of death and/or severity of infections [2, 4, 6, 14]. In our study area, 18% of 219 

the visceral leishmaniasis cases were reported to be malnourished and malnutrition was one of 220 

the implicated risk factors for the outbreak [24]. In line with this we assessed the effect of 221 

malnutrition on hematological parameters and cytokine production. 222 

Our data showed significantly lower total count of white blood cells, lymphocytes and sub 223 

populations of lymphocytes (CD4+ and CD8+ cells) in severely malnourished children (P <0.05). 224 

Hematopoietic tissue requires a high nutrient supply, and in consequence bone marrow function 225 

may be altered by nutritional deficiencies that can be reflected in a change of the hematological 226 

parameters (RBC, WBC, etc…). A similar observation was reported in animal model study; 227 

malnourished mice presented anemia with reduced hemoglobin concentration, and total number 228 

of erythrocytes, leucopenia with depletion of polymorphonuclear granulocytes, lymphocytes and 229 

monocytes [29]. Change in the hematopoietic environment (high fibronectin and laminin) and 230 

biochemical evidence of alterations in extracellular matrix proteins in the bone marrow was 231 

observed in malnourished mice [30, 31]. And severe malnutrition during childhood was shown to 232 

affect thymic development, which compromises immunity by a long-term reduction of peripheral 233 

lymphocyte counts [15].  234 

The absence of significant difference in the CD4+/CD8+ ratio in our data indifferent to the report 235 

by Nassar et al 2007 [32], supports the idea that the immunodeficiency associated with the 236 

severe form of malnutrition is not due to increased helper-suppressor T-cells ratio[33]. 237 

Our stimulation data showed that the PBMC from the severely malnourished children produced 238 

significantly low or no cytokine response to PHA. This probably means that the cells from the 239 

severely malnourished children are non-responsive (quiescent). With intracellular staining and 240 

flow cytometric analysis Rodriguez et al 2005 [34] reported significantly lower CD4+ IL-2-241 

positive cells in malnourished children compared to those in well nourished  children. In 242 

addition, they showed that cells from malnourished children had impaired activation capability 243 



9 

 

as compared to the well-nourished, infected children. A reduction in the number of lymphocyte 244 

subpopulation producing a specific cytokine or the dormancy induced by malnutrition could alter 245 

the capacity of cells to produce specific cytokines in response to a stimulus. 246 

 The high level of PGE2 in the serum of the DAT negative moderately malnourished children 247 

observed in our data is in accordance with previous observation. PGE2 production is enhanced 248 

in malnutrition [22, 35]. Also, Anstead et al. [22] demonstrated that mice fed with a diet deficient 249 

in protein, iron and zinc have an altered innate immune response with lymph node cells from 250 

malnourished infected mice producing increased levels of PGE2, decreased levels of IL-10 and 251 

had a lower inducible nitric oxide synthase (iNOS) activity in the spleen and liver. 252 

The serum from both the DAT positive and the DAT negative non-malnourished children 253 

produced a higher level of cytokines than the malnourished. In the DAT negative group, the 254 

severely malnourished children produced no detectable level of any of the cytokines assayed; 255 

the moderately malnourished children produced IL-4 and IL-10, while the non-malnourished 256 

groups produced all the assayed cytokines (Fig2). The bias to the Th1 type in the non-257 

malnourished DAT negative children and Th2 type in the moderately malnourished group is in 258 

agreement with our PGE2 data explained above (Fig 1). Previous in vivo studies have also 259 

demonstrated that PGE2, can affect cytokine production directly during an inflammatory 260 

response; the production of Th1cytokines (IFN-γ and TNF-α) are down regulated [36].    261 

The DAT positive non-malnourished children produced significantly high level of IFN-γ and IL-10 262 

as compared to the moderately and severely malnourished children (Fig 3). Our data is in 263 

agreement with behavior of immune response in L. donovani infection; IFN-γ is secreted at the 264 

very initial stages of the exposure to the parasites as observed in the seroconverted or 265 

subclinically infected individuals in the endemic area [37]. Carvalho et al. [38] observed high 266 

levels of IFN-γ in oligosymptomatic individuals who evolved to spontaneous cure, supporting the 267 

fact that resistance is related to an efficient cellular immune response.  Also Gama et al [39] 268 

described the detection of IL-12 in 85.2%, IFN-γ in 48.1%, IL-10 in 88.9%, and TNF- α in 269 

100.0% subclinically infected children. However the detection of low level of IL-4 in our non-270 

malnourished DAT positive children might indicate that some children might shift the Th2 271 

response and progress to the clinical from of VL. While the detection of the low IFN-γ and 272 

relatively high IL-4 and IL-10 in the moderately malnourished children indicates a Th2 bias in 273 

this group that might be related to the high level of PGE2 production.  274 



11 

 

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136. 394 



Table 1: Comparison of the mean hematological parameters in children of different 

nutritional status groups, Libo Kemkem, Ethiopia.  

Nutritional Status  
(N=number per group) Parameters 

Mean ±SE 
Normal Moderate  Severe 

Reference 
range*1,2 

RBC (106 cells/µL)     

Boys 
4.55±0.42 (134) 4.58±0.08 (42) 4.09±0.21  (12) 3.80-6.20 

Girls 
4.50±0.04 (170) 4.55±0.08 (27) 4.34±0.15 (9) 3.80-5.60 

HGB (g/dL)     

Boys 
12.74 ± 0.14 (134) 12.80±0.21 (42) 11.63±0.61 (12) 12.20-17.70 

Girls 
12.78±0.15 (170) 12.75±0.32 (27) 12.41±0.42 (9) 9.50-15.80 

HCT (%)     

Boys 
37.43±0.34 (134) 37.70±0.72 (42) 34.20±1.83 (12) 35.00-50.80 

Girls 
37.04±0.41 (170) 37.17±0.90 (27) 36.07±1.19 (9) 29.40-45.40 

WBC (103/µL 8.03 (304) 7.01 (69) 5.67 (21) 3.10-9.10 

LYMP (103/µL) 3.63 (304) 2.97 (69) 2.39 (21) 1.20-3.70 

CD4 (103/µL) 1.09 (124) 0.99 (23) 0.64 (10) 0.457-1.628 

CD8 (103/µL) 0.91 (124) 1.05 (23) 0.53 (10) 0.23-1.178 

CD4/CD8 (ratio) 1.38 (124) 1.42 (23) 1.30 (10)  

CD19 (103/µL) 0.34 (124) 0.26 (23) 0.17 (10)  

*1=Establishing Clinical laboratory Reference Intervals in Africa; IAVI (International 
AIDS Vaccine Initiative) and *2= Immunohematological Reference Ranges for Adult 

Ethiopians [28]. 

 

 

 

 



FIGURE LEGENDS  

 

 

Figure 1: Cytokine Concentration (pg/ml) in a supernatant from a 24 hour culture of 

peripheral blood mononuclear cells with PHA, in children from Libo Kemkem, south 

Gondar, Ethiopia. Cytokine measurement was performed for 57 non-malnourished, 21 

moderately malnourished and 10 severely malnourished children.  

 

 

Figure 2. Serum concentration of PGE2 by nutritional status in children from Libo 

Kemkem, South Gondar, Ethiopia: Serum samples from 94 DAT negative children; 61 

from non-malnourished, 6 from severely malnourished and 27 from moderately were 

analyzed. 

 

Figure 3. Serum concentration of cytokines (TNF- α, IL-10, IL-4, and IFN- γ) by 

nutritional status and asymptomatic infection as detected by DAT positivity in children 

from Libo Kemkem, South Gondar, Ethiopia; 43 DAT positive (29 non malnourished, 12 

moderately malnourished and 2 severely malnourished) and 36 DAT positive (18 non 

malnourished, 10 moderately malnourished and 8 severely malnourished) serum 

samples were analyzed.  

 

 

 

 

 

 

 

 

 



FIGURE 1 

P=0.02

0
1,

00
0

2,
00

0
3,

00
0

4,
00

0
C

yt
ok

in
e 

co
nc

en
tr

at
io

n 
(p

g/
m

l)

Non malnorished Severely malnorished Moderately malnorished

TNF-a IL-4 IL-10 INF-g IL-12
Key

Nutritional status

 



FIGURE 2 
 
 

P=0.007

0
5,

00
0

10
,0

00
15

,0
00

P
G

E
2 

(p
g/

m
l)

Non malnorished Severely malnorished Moderately malnorished
Nutritional Status

 
 
 
 



FIGURE 3 
 

P=0.00

0
50

0
1,

00
0

1,
50

0
2,

00
0

Non
 m

aln
or

ish
ed

se
ve

re
ly 

m
aln

or
ish

ed
M

od
er

ate
ly 

m
aln

or
ish

ed

Non
 m

aln
or

ish
ed

Sev
er

ely
 m

aln
or

ish
ed

M
od

er
ate

ly 
m

aln
or

ish
ed

DAT negative DAT positive

TNF-a IL-4 IL-10 INF-g

Key

C
yt

ok
in

e 
co

nc
en

tr
at

io
n 

(p
g/

m
l)

Nutritional status

 



 

 



VIII- DISCUSIÓN  

En Etiopía, la LV está distribuida en todas las tierras bajas con diferentes 

grados de endemicidad. Se estima una incidencia de 4.500 a 5.000 casos 

nuevos por año y la tasa de coinfección Leishmania/VIH está entre el 15% y el 

30% de todos casos de LV (Alvar et al., 2006). En los últimos años la LV se ha 

extendido en Etiopía, se han documentado nuevos brotes en el norte y sur del 

país: Libo Kemkem y Belesa (región de Amhara), Shiraro (región de Tigray) e 

Imey (región de Somalí) (Informe de Ministerio de Sanidad de Etiopía). Se 

piensa que los movimientos de trabajadores temporales a las zonas 

endémicas, la malnutrición asociada a la pobreza y la coinfección 

Leishmania/VIH han contribuido a la expansión de la LV a zonas previamente 

no endémicas (Herrero et al., 2009). Como consecuencia de este incremento la 

LV se ha convertido en una preocupación para la salud pública del país y su 

control se ha convertido en una prioridad. Para plantear un programa de control 

frente a la LV adaptada a las situaciones locales, hay que disponer de datos 

socio-epidemiológicos exhaustivos de buena calidad que permitan monitorizar 

el progreso del programa y la evaluación de su impacto. El entendimiento de la 

influencia de factores de riesgo como la malnutrición, y la disponibilidad de 

herramientas valoradas localmente para establecer las situaciones 

epidemiológicas tendrán un impacto positivo en los esfuerzos realizados para el 

control de la LV.     

            El presente estudio mostró la presencia de infección asintomática por 

Leishmania en los niños de entre 4 y 15 años de edad en los distritos de Libo 

Kemkem y Fogera, en el nuevo foco del región de Amara, noroeste de Etiopía. 

La tasa global de la infección asintomática (positiva por LST y/o serología) en 



los comunidades en que se han documentado mayor número de LV después 

del brote (2008) fue del 10,1% (61/605), el 6,3% fueron individuos seropositivos 

y el 5,6% fueron positivos para LST. La discordancia observada entre LST y 

serología son probablemente debido a la diferencia del tipo de respuesta 

inmune detectada por cada prueba. LST mide la reacción de hipersensibilidad 

retardada frente Leishmania mientras que la seropositividad resulta de la 

respuesta de anticuerpos específicos a Leishmania. La positividad de LST 

aparece más tarde después de la infección, mientras que la seropositividad es 

considerada como un marcador de una infección más reciente. Por la misma 

razón, el LST positivo se produce como resultado de la exposición repetida a la 

infección natural, lo que también justifica porqué el número de individuos 

positivos para LST es mayor en los grupos de mayor edad. La mayor tasa de 

infección detectada por serología (con DAT y rK39-ICT) al comparar con las 

obtenidas con LST puede estar asociado a la necesidad de periodos de tiempo 

más largos para el desarrollo de respuesta positiva en el LST con respecto a la 

seropositividad y a que la LV ha aparecido recientemente en nuestro área de 

estudio. La diferente tasa de infección observada entre las pruebas serológicas 

puede depender de la etapa de la enfermedad/infección, la baja tasa detectada 

por la prueba rK39-ICT puede ser explicada por su capacidad para detectar 

solo los anticuerpos específicos frente al antígeno rK39, mientras que el DAT 

detecta una respuesta de anticuerpos frente a una amplia gama de antígenos 

de Leishmania (el promastigote completo liofilizado) (Boelaert et al., 2004). Otra 

posible explicación se basa en la naturaleza de las pruebas; tal y como 

propusieron ter Horst et al., (2009) los anticuerpos detectados por rK39-ICT 

podrían tener una menor capacidad de reacción en el formato rápido de tira 



inmunocromatográfica que en las incubaciones durante toda la noche usadas 

en el DAT. La observación de que la tasa de infección asintomática aumenta 

con la edad es consistente con un foco endémico de LV, de hecho, el 

incremento de edad es un factor del riesgo para la infección asintomática. La 

presencia de infección asintomática en individuos menores de 5 años de edad 

es consistente con la presencia de una transmisión activa. El mayor número de 

infección asintomática detectado por el uso combinado de las pruebas 

serológicas (particularmente DAT) y LST, indica que este método combinado 

es el procedimiento más apropiado para determinar la tasa de infección 

asintomática en un área endémica de LV.  En efecto, la prevalencia de LV 

asintomática detectada varía según las técnicas y el área endémica por lo que 

se aconseja la combinación de técnicas para determinar la prevalencia más 

cercana a la realidad en ausencia de un método Gold Standard para la 

detección de infección asintomática (de Gouvea Viana et al., 2008). 

La prevalencia global detectada en las áreas donde hubo al menos un caso 

clínico de LV durante el brote de 2004/2005 fue del 1,02%.  Los niños mayores 

de 12 años presentaban la mayor prevalencia (2,56%), seguidos por el grupo 

de niños entre 8 y 11 años de edad (0,82%), y la menor prevalencia fue 

detectada en los menores de 8 años (0,49%). La baja prevalencia detectada en 

nuestro estudio indica que las condiciones que provocaron la epidemia de la LV 

en la región de estudio ya no persisten. Una hipótesis propuesta durante la 

epidemia de LV mantenía que el parásito había sido introducido en la región 

por trabajadores agrícolas, quienes habían regresado infectados a sus pueblos 

después de completar su trabajo estacional en la frontera de Sudán y habían 

actuado como reservorio del parásito (Herrero et al., 2009). Sin embargo, de 



acuerdo con la información disponible, los afectados no fueron sólo estos 

trabajadores agrícolas desplazados, y no hay ninguna evidencia de que tal 

migración haya cesado. Esto nos hace pensar que algún cambio en la 

población del vector debió de provocar el brote, se ha sugerido que cambios en 

la temperatura y humedad relativa pueden aumentar la abundancia del vector 

(Gálvez et al., 2010).  

Por otro lado, también es posible que la respuesta al brote de LV y el 

tratamiento de los enfermos pudiera haber llevado a la reducción de la tasa de 

seroprevalencia. Antes del año 2004 no se había reportado ningún caso de LV 

en Libo Kemkem, y probablemente se está volviendo a la situación pre-

epidémica. No obstante, las dudas sobre las causas del brote y la caída de la 

prevalencia post brote que hemos observado en este estudio resaltan la 

necesidad de vigilar los cambios climáticos para evitar la reemergencia de la 

LV en esta zona.       

            Durante el brote de LV de 2004/2005, la  prevalencia determinada por 

LST fue considerablemente mayor que la observada en nuestro estudio, 34% 

en hombres y 26% en mujeres (Alvar et al., 2007). No obstante, este estudio 

fue hecho para valorar el brote en la población de 0,7 a  60 años de edad, y se 

hizo mediante un muestreo por conveniencia.  

 La fuerte variación en la infección asintomática observada  entre zonas 

altamente endémicas de LV está de acuerdo con la agrupación especial 

observada en otros lugares en estudios de infección asintomática  (Evans et al., 

1992; Singh et al., 2010) y de casos clínicos (Bern et al., 2005; Ryan et al., 

2006).  



            El aumento de la edad constituye un factor de riesgo para la infección 

por Leishmania y se asocia con las actividades específicas de los niños 

mayores o adolescentes que implican un potencial aumento de la exposición a 

la picadura del flebótomo (Singh et al., 2010). Se piensa que esta situación es 

también responsable de la mayor frecuencia de infección entre el sexo 

masculino (Ali y Ashford, 1993). Nuestros resultados respaldan esta hipótesis, 

actividades tales como el hábito de dormir fuera de la casa y el cuidado del 

ganado se han identificado como factores de riesgo para la infección 

asintomática. La mayor exposición a los flebótomos durante el cuidado del 

ganado  se puede asociar con estar en el campo al anochecer y amanecer, 

momentos del día en los que los  flebótomos son más activos (Wijers, 1963) y 

también con una mayor proximidad a las acacias rojas (pudimos comprobar 

que el 82% de los niños que van a cuidar ganados descansaban a la sombra 

de las acacias rojas), que se consideran como sitios de descanso diurno para 

Phlebotomus orientalis, el potencial vector de Leishmania en el área de estudio 

(Elnaiem et al., 1999).  

 En nuestro análisis de factores de riesgo, la malnutrición aguda se 

asocia positivamente a la infección asintomática, pero solo en el análisis 

univariante. Cuando el sexo y la edad fueron introducidos en el modelo la 

malnutrición aguda perdió su significancia, probablemente reflejando la 

interacción entre las tres variables, ya que factores como el sexo masculino y el 

aumento de la edad mostraron una asociación directa y significativa con la 

malnutrición aguda. El riesgo de infección asintomática asociado con un mayor 

número de miembros en la familia podría ser debido a la  atracción de los 

flebótomos por grandes biomasas, que se ha descrito como un riesgo de LV en 



el mismo área (Bashaye et al., 2009). También había  mayor probabilidad de 

infección asintomática entre los niños de una familia con historia previa de 

casos de LV en la familia, aunque esta asociación sólo fue significativa con los 

casos seropositivos y no con los positivos por LST, lo que está de acuerdo con 

las conclusiones de Bern et al. (2007)  en Bangladesh. En relación con la 

seropositividad, otros estudios ya han mostrado la asociación entre este factor 

y el contacto previo con enfermos de LV (Caldas et al., 2002; Evans et al., 

1992; Schaefer et al., 1995), apoyando la hipótesis de  transmisión dentro de la 

familia.  

 El incremento del riesgo de infección asintomática en familias que viven 

en una casa de techo de paja y con grietas en las paredes frente a las que 

viven en casas con chapa ondulada puede estar relacionado con el poder 

económico de la familia y refleja su pobreza/riqueza, además el techo de paja y 

las grietas en los paredes sirven como sitio potencial de reproducción y de 

descanso diurno por los flebótomos, incrementado su supervivencia y 

abundancia. No obstante, con respecto a P. orientalis, se necesitan más 

estudios para confirmar este último punto, ya que los pocos estudios existentes 

en la literatura señalan un comportamiento exofágico del vector, incompatible 

con este hipótesis (Gebre-Michael et al., 2010).  

 Por otro lado, la posesión de un mayor número de cabezas de ganado 

por familia mostró un efecto protector frente a la infección asintomática y 

seropositividad, pero no a la positividad por LST. El efecto protector del ganado 

y gallinas frente a la LV puede explicarse por su papel como indicadores de 

riqueza o también por el efecto zooprofiláctico del ganado (Caldas et al., 2002; 

Schenkel et al., 2006). La preferencia P. orientalis por la sangre del ganado 



frente a la del hombre ya se ha demostrado en otra parte de Etiopía, 

contribuyendo a esta última teoría (Gebre-Michael et al., 2010), pero el efecto 

zooprofilático de las gallinas con respecto a P. orientalis necesita ser resuelta. 

Con respecto a los efectos de la malnutrición sobre el status inmunológico de 

los niños, nuestro estudio mostró un número de leucocitos, linfocitos y células T 

CD4+ y CD8+ significativamente menor en los niños con malnutrición severa (p 

<0,05). La malnutrición severa durante la infancia afecta el desarrollo del timo, 

lo que compromete la inmunidad a  largo plazo y produce una reducción del 

número de linfocitos en sangre periférica. La baja o nula expresión de 

citoquinas observada en las células de los niños con malnutrición severa tras la 

estimulación con PHA está probablemente asociada con la menor actividad de 

estas células. Rodríguez et al.  (2005) reportaron niveles significativamente 

menores de células T CD4+ e IL-2+ en niños malnutridos en comparación con 

los niños bien nutridos. Además, se demostró que las células procedentes de 

los niños malnutridos tienen deficiencia en el poder de activación en 

comparación con las células procedentes de niños bien nutridos.   

 Los mayores niveles de PGE2 observados en el suero de los niños DAT 

negativos y con malnutrición moderada está de acuerdo con observaciones 

previas en las que la producción de PGE2 aumenta con la malnutrición 

(Anstead et al., 2001; Dooper et al., 2002). Además, la predisposición a una 

 respuesta de citoquinas tipo Th1 en los niños no malnutridos y DAT negativos,  

y de  tipo Th2 en los moderadamente malnutridos está también  de acuerdo 

con el dato de PGE2 explicado anteriormente.  

 

 



BIBLIOGRAFÍA 

- Ali A, Ashford RW (1993). Visceral leishmaniasis in Ethiopia. I. Cross-

sectional leishmanin skin test in an endemic locality. Annals of Tropical 

Medicine and Parasitology; 87: 157-61. 

- Alvar J, Yactayo S, Bern C (2006). Leishmaniasis and poverty. TRENDS in 

Parsitology; 22: 552-57. 

- Alvar J, Bashaye S, Argaw D, Cruz I, Aparicio P, Kassa A, Orfanos G, 

Parreño F, Babaniyi O, Gudeta N, Cañavate C, Bern C (2007). Kala-Azar 

outbreak in Libo Kemkem, Ethiopia: Epidemiologic and parasitologic 

assessment. American Journal of Tropical Medicine and Hygiene; 77: 275-82. 

- Anstead G M, Chandrasekar B, Zhao W, Yang J, Perez L E, Melby P C 

(2001). Malnutrition alters the innate immune response and increases early 

visceralization following Leishmania donovani infection. Infection and Immunity; 

69: 4709-18. 

- Bashaye S, Nombela N, Argaw D, Mulugeta A, Herrero M, Nieto J, Chicharro 

C, Cañavate C, Aparicio P, Vélez I D, Alvar J, Bern C (2009). Risk factors for 

visceral leishmaniasis in a new epidemic site in Amhara Region, Ethiopia. 

American Journal of Tropical Medicine and Hygiene; 81: 34-9. 

- Bern C, Hightower A W, Chowdhury R, Ali M, Amann J, Wagatsuma Y, Haque 

R, Kurkjian K, Vaz L E, Begum M, Akter T, Cetre-Sossah C B, Ahluwalia I B, 

Dotson E, Secor W E, Breiman R F, Maguire J H (2005). Risk factors for kala-

azar in Bangladesh. Emerging Infectious Diseases; 11: 655-62. 

- Bern C, Haque R, Chowdhury R, Ali M, Kurkjian K M, Vaz L, Amann J, Wahed 

M A, Wagatsuma Y, Breiman R F, Williamson J, Secor W E, Maguire J H 

(2007). The epidemiology of visceral leishmaniasis and asymptomatic 



leishmanial infection in a highly endemic Bangladeshi village. American Journal 

of Tropical Medicine and Hygiene; 76: 909-14. 

- Boelaert M, Rijal S, Regmi S, Singh R, Karki B, Jacquet D, Chappuis F, 

Campino L, Desjeux P, Le Ray D, Koirala S, Van der Stuyft P (2004). A 

comparative study of the effectiveness of diagnostic tests for visceral 

leishmaniasis. American Journal of Tropical Medicine and Hygiene; 70: 72-7. 

- Caldas A J, Costa J M, Silva A A, Vinhas V, Barral A (2002). Risk factors 

associated with asymptomatic infection by Leishmania chagasi in north-east 

Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene; 96: 

21-8. 

- de Gouvêa Viana L, de Assis T S, Orsini M, da Silva A R, de Souza G F, 

Caligiorne R, da Silva A C, Peruhype-Magalhães V, Marciano A P, Martins-

Filho O A, Rabello A (2008). Combined diagnostic methods identify a 

remarkable proportion of asymptomatic Leishmania (Leishmania) chagasi 

carriers who present modulated cytokine profiles. Transactions of the Royal 

Society of Tropical Medicine and Hygiene; 102: 548-55. 

- Dooper M M, Wassink L, M'Rabet L, Graus Y M (2002). The modulatory 

effects of prostaglandin-E on cytokine production by human peripheral blood 

mononuclear cells are independent of the prostaglandin subtype. Immunology; 

107: 152-9. 

- Elnaiem D A, Hassan H K, Ward R D (1999). Associations of Phlebotomus 

orientalis and other sandflies with vegetation types in the eastern Sudan focus 

of kala-azar. Medical Veterinary Entomology; 13: 198-203. 



- Evans T G, Teixeira M J, McAuliffe I T, Vasconcelos I, Vasconcelos A W, 

Sousa A de A, Lima J W, Pearson R D (1992). Epidemiology of visceral 

leishmaniasis in northeast Brazil. Journal of Infectious Diseases; 166: 1124-32. 

- Gálvez R, Descalzo M A, Miró G, Jiménez M I, Martín O, Dos Santos-Brandao 

F, Guerrero I, Cubero E, Molina R (2010). Seasonal trends and spatial relations 

between environmental/meteorological factors and leishmaniosis sand fly vector 

abundances in Central Spain. Acta Tropica; 115: 95-102. 

- Gebre-Michael T, Balkew M, Berhe N, Hailu A, Mekonnen Y (2010). Further 

studies on the phlebotomine sandflies of the kala-azar endemic lowlands of 

Humera-Metema (north-west Ethiopia) with observations on their natural blood 

meal sources. Parasite and Vectors; 3: 6. 

- Herrero M, Orfanos G, Argaw D, Mulugeta A, Aparicio P, Parreño F, Bernal O, 

Rubens D, Pedraza J, Lima MA, Flevaud L, Palma P P, Bashaye S, Alvar J,  

Bern C (2009). Natural History of a Visceral Leishmaniasis Outbreak in 

Highland Ethiopia. American Journal of Tropical Medicine and Hygiene; 81: 

373–7. 

- Rodríguez L, González C, Flores L, Jiménez-Zamudio L, Graniel J, Ortiz R 

(2005). Assessment by flow cytometry of cytokine production in malnourished 

children. Clinical Diagnostic and Laboratory Immunology; 12: 502-7. 

- Ryan J R, Mbui J, Rashid J R, Wasunna M K, Kirigi G, Magiri C, Kinoti D, 

Ngumbi P M, Martin S K, Odera S O, Hochberg L P, Bautista C T, Chan A S 

(2006). Spatial clustering and epidemiological aspects of visceral leishmaniasis 

in two endemic villages, Baringo District, Kenya. American Journal of Tropical 

Medicine and Hygiene; 74: 308-17. 



- Schaefer K U, Kurtzhals J A, Gachihi G S, Muller A S, Kager P A (1995). A 

prospective sero-epidemiological study of visceral leishmaniasis in Baringo 

District, Rift Valley Province, Kenya. Transactions of the Royal Society of 

Tropical Medicine and Hygiene; 89: 471-5. 

- Schenkel K, Rijal S, Koirala S, Koirala S, Vanlerberghe V, Van der Stuyft P, 

Gramiccia M, Boelaert M (2006). Visceral leishmaniasis in southeastern Nepal: 

a cross-sectional survey on Leishmania donovani infection and its risk factors. 

Tropical Medicine and International Health; 11: 1792-9. 

- Singh S P, Picado A, Boelaert M, Gidwani K, Andersen E W, Ostyn B, 

Meheus F, Rai M, Chappuis F, Davies C, Sundar S (2010). The epidemiology of 

Leishmania donovani infection in high transmission foci in India. Tropical 

Medicine and International Health; 15(S2): 12-20. 

- ter Horst R, Tefera T, Assefa G, Ebrahim A Z, Davidson R N, Ritmeijer K 

(2009). Field evaluation of rK39 test and direct agglutination test for diagnosis of 

visceral leishmaniasis in a population with high prevalence of human 

immunodeficiency virus in Ethiopia. American Journal of Tropical Medicine and 

Hygiene; 80: 929-34. 

- Wijers DJ (1963). Studies on the vector of kala-azar in Kenya. II. 

Epidemiological evidence. Annals of Tropical Medicine and Parasitology; 57: 7-

18. 



 

 



 

 

 

IX- CONCLUSIONES 

 Se han evaluado a nivel local herramientas diagnósticas para la determinación 

de infección asintomática sobre el terreno, aspecto clave a la hora de implementar un 

programa de control en áreas donde la LV es antroponótica. Se ha determinado la 

utilidad de combinar DAT y LST para obtener la mejor medida de la tasa de infección 

asintomática en la comunidad. Esta metodología puede apliacrse igualmente en otras 

áreas endémicas de Etiopía.  

 La prevalencia de LV activa y de infección asintomática en las poblaciones 

estudiadas fue baja, indicando la necesidad de identificar áreas con mayor prevalencia 

y enfocar el esfuerzo de control en ellas. Podemos considerar que la baja prevalencia 

post-brote puede deberse a la rápida instauración de un centro de tratamiento de LV, 

que contribuye a disminuir el número de individuos infectados (reservorios) en áreas 

de transmisión antroponótica. Se debe también considerar una posible disminución de 

la población de vectores a niveles similares a los de antes del brote. Una hipótesis de 

origen del brote es el aumento de la población de vectores en el área debido a 

cambios ambientales. No obstante, esto último no ha podidi probarse al no existir un 

sistema de vigilancia vectorial. 

 Los factores personales (comportamiento), medioambientales y 

socioeconómicos que hemos identificado como asociados a un aumento del riesgo de 

infección asintomática son una aportación indispensable a la hora de plantear un 

programa de control de LV.  

 A través del estudio de factores de riesgo y del estudio de los aspectos 

inmunológicos, hemos comprobado que la malnutrición influye en la infección por 

Leishmania. Por tanto consideramos que es necesario considerar el aspecto 

nutricional, particularmente en los grupos más vulnerables, a la hora de plantear  un 

programa de control y prevención de la LV. 

 

 

 

 

 



 

 



 

 

 

X - ANEXO I 

Galeria de fotos 

   



 

 



ÁREA DE ESTUDIO

El área de estudio es una meseta situada de 1800 a 2000 metros sobre el nivel del mar. Durante la 
estación de lluvias, (junio-septiembre) la mayor parte del área está inundada (1). A partir de 
octubre se empieza a secar y entre diciembre y febrero, la estación seca (2), se forman grietas 
profundas en el suelo que constituyen lugares ideales para el refugio, durante el día, de los 
flebótomos (3) 

1

2

3



 

 



Debido a las actividades agrícolas la cobertura vegetal del área se ha visto reducida y por ello la 
principal fuente de energía para cocinar son los excrementos secos de vaca. Alrededor de 
numerosas viviendas, ya sea de tejado de paja  o de chapa ondulada, pueden encontrarse 
apiladas las heces secas de vacas (4 y 5).  Además, los animales se mantienen cerca (6) o incluso 
dentro de las viviendas (7). 

4 5

6 7



 

 



98

POBLACIÓN DE ESTUDIOS

En este estudio se incluyeron niños y niñas de 4 a 15 años de edad residentes en diferentes 
kebeles (sub-distritos) de Fogera y Libo Kemkem (8, 9 y 10)

10



 

 



COMIDA

La injera es una torta fina hecha de harina de teff fermentada (11), o de una mezcla de harina de 
teff y de otros cereales. Se le suele añadir diferentes tipos de puré de legumbres (shiro), y 
verduras u hortalizas como tomate, patata, zanahoria o pimientos (12). En el shiro se añade aceite 
o a veces manteca, muy ocasionalmente se añade carne. El teff es el principal cultivo de la zona 
(13). 

13

1211



 

 



ENTRENAMIENTO

Antes de realizar el trabajo de campo se organizó un entrenamiento de los enfermeros encargados 
de realizar las encuestas, las medidas antropométricas, la toma de muestras biológicas y los 
diferentes análisis  que se realizaron in situ. Entrenamiento en la prueba serológica DAT  (14) y en 
la toma de medidas antropométricas en el aula del hospital de Bahir Dar (15). Entrenamiento en la 
prueba serológica rK39-ICT (16) y en la intradermorreación de la leishmanina (17).

1716

14

15



 

 



ESTUDIO PILOTO

Se realizó un estudio piloto para optimizar los métodos de colección de los datos y confirmar las 
habilidades prácticas de los enfermeros. Personal experto acompañó al grupo de enfermeros al 
campo para asesorar y evaluar su trabajo práctico a la hora de hacer las medidas antropométricas 
(18) y las pruebas diagnósticas (19). Los problemas y resultados de estudio piloto se pusieron en 
común en una reunión final (20). 

18 19

20



 

 



TRABAJO DE CAMPO

En el momento de realizar el trabajo de campo, el equipo de encuestadores se instalaba en una 
zona de cada uno de los pueblos seleccionados (gotts) a la que acudían los niños para su examen 
(21). El examen físico incluía medida de temperatura (22), peso (23), talla (24) y la toma de sangre 
(25). A la vez se realizaba una encuesta al adulto responsable de los niños (26)

21

22 23



 

 



24 25

26



 

 



27 28

Durante el estudio transversal se realizó a los niños la prueba de la leishmanina. Para ello se 
inoculaba por vía intradérmica, 100 µL de la solución de leishmanina (27), y a las 42 – 72 horas se 
midió la presencia de induración mediante el método del bolígrafo (28). Esta prueba es positiva si 
el diámetro de la induración es mayor de 5 mm (29), en caso contrario se considera negativa (28) . 

29 30



 

 



TRABAJO DE LABORATORIO. 

Las muestras obtenidas durante el trabajo de campo se trasladaban el mismo día al Laboratorio 
Regional de Amhara, en Bahir Dar, para su procesamiento (31). En este laboratorio se realizaba el 
hemograma (32), separación, cultivo  y tinción de linfocitos (33 y 34), la separación del suero y el 
test DAT.  

33 34

31 32



 

 



EVALUACIÓN EXTERNA DEL PROYECTO

En noviembre de 2009 se llevó a cabo una evaluación del proyecto por parte de un experto 
externo, el Dr. Philippe Dexjeux , Institute for One World Health), quien visitó los diferentes centros 
implicados en el proyecto y el área de estudio (35, 36 y 37).

35

36 37



 

 


	Tesis de Endalamaw Gadisa Belachew
	PORTADA
	AGRADECIMIENTOS
	RESUMEN
	INTRODUCCIÓN
	I- EPIDEMIOLOGÍA DE LA LEISHMANIASIS
	II- LEISHMANIASIS VISCERAL
	III- LEISHMANIASIS VISCERAL EN ETIOPÍA
	IV- BIBLIOGRAFÍA
	V- EL PROYECTO
	VI- OBJETIVOS
	VII- ARTÍCULOS CIENTÍFICOS
	VIII- DISCUSIÓN
	IX- CONCLUSIONES
	X - ANEXO I