Exitoso cultivo in vitro de gametocitos de Plasmodium falciparum

Ana Mercedes Rada, Carolina Moreno, Silvia Blair

Grupo Malaria, Universidad de Antioquia, Medellín, Colombia.

Institución en donde se llevó a cabo el trabajo:

Sede de investigación Universitaria, SIU, Universidad de Antioquia.

Recibido: 25/04/08; aceptado:14/08/08

Introducción. Los estadios sexuales de Plasmodium falciparum han sido menos estudiados que los estadios asexuales. Al parecer, esto se debe a la carencia de cultivos estandarizados in vitro y a la dificultad de reconocer sus estadios de desarrollo. Estos hechos no permiten el estudio de aspectos biológicos, aspectos metabólicos, expresión de genes y síntesis de proteínas durante los estadios sexuales, temas de interés en la investigación de nuevos medicamentos antipalúdicos, principalmente los aislados de plantas, y la identificación de un potencial blanco contra Plasmodium.

Objetivos. Establecer un cultivo in vitro de gametocitos, con la identificación de sus cinco estadios de desarrollo, y asegurar su continua producción.

Materiales y métodos. El cultivo in vitro de gametocitos se realizó a partir de la cepa NF54 de P. falciparum en medio RPMI, con determinación de la parasitemia asexual y sexual, adición de glóbulos rojos A-Rh+ sólo el primer día de cultivo y cambio diario del medio con adición de mezcla de gases (90% N2, 5% O2; 5% CO2), asegurándose que el cultivo se mantuviera a 37 °C. Cuando la parasitemia asexual estuvo entre 3% y 5%, se comenzó a agregar el doble de volumen de medio.

Resultados. Se obtuvieron gametocitos en estadios I, II y III a partir del día 11 de cultivo y estadios IV y V a partir del día 14 de cultivo.

Conclusiones. Se estandarizó un cultivo in vitro para estadios sexuales de P. falciparum que puede usarse para futuros estudios de evaluación de compuestos, naturales o sintéticos, que actúen sobre los gametocitos, lo cual podría permitir el desarrollo de nuevas estrategias de control contra el paludismo.

Palabras clave: Plasmodium falciparum, in vitro, paludismo, Anopheles.

Successful in vitro culture of Plasmodium falciparum gametocytes

Introduction. The sexual stages of Plasmodium falciparum have not been studied in as much detail as the asexual stages due to the lack of standardized in vitro cultures as well as difficulties in identifying the sexual development stages of the parasite. These difficulties hamper the studies on biology, metabolism, gene

expression and protein synthesis during sexual stages. Each of these facets are important targets in antimalarial drug research, particularly the identification of potential therapeutic agents against Plasmodium (derived mainly from plants).

Objectives. An  in vitro culture of P. falciparum gametocytes was established to standardize the identification of its five developmental stages and ensure their continuous production.

Materials and methods. The in vitro gametocyte culture was established from the P. falciparum NF54 strain in RPMI culture medium, with assessment of the asexual and sexual parasitaemia. The medium was supplemented with type A Rh+ red blood cells only on the first day of culture. Subsequently, the medium was changed daily, together with addition of gas mixture (90% N2, 5% O2, 5% CO2) and maintenance of the culture temperature at 37 °C. When asexual parasitaemia reached 3 to 5%, the medium was changed by doubling its volume.

Conclusions. We standardized an in vitro culture for sexual stages of P. falciparum that can be used for future studies about evaluation of compounds of synthetic or natural origen against the sexual stage, which may permit to develop new control strategies against malaria.

Key words: Plasmodium falciparum, in vitro, malaria, Anopheles.

Los estadios sexuales de Plasmodium falciparum llamados gametocitos son los responsables de la infección del mosquito vector Anopheles y los estadios asexuales son los responsables de la sinto-matología en el hombre (1). Se conoce que, mediante un proceso complejo de desarrollo del parásito, sólo una pequeña fracción de los parásitos asexuales evoluciona al estadio sexual (2), y se ha observado que las densidades de gametocitos en sangre periférica son considerablemente más bajas que las de los parásitos asexuales (3). Algunos reportes señalan que la proporción entre gametocitos y estadios asexuales de P. falciparum es de 1:10 (4-7) y otros señalan que es de 1:156 (3).

El gametocito, célula especializada, acomoda su vida a diferentes ambientes y pasa por varios estadios en los que se pueden observar no sólo diferencias morfológicas sino también metabólicas; también, cambios en la expresión de ciertos genes y síntesis de diversas proteínas que reflejan una preparación de los gametocitos para los drásticos cambios futuros (8).

Uno de los aspectos más importantes y desconocidos de este estadio sexual es que durante el proceso de maduración atraviesa por cinco estadios morfológicamente diferentes, clasificados como estadios I al V, los cuales expresan proteínas sobre la superficie de los eritrocitos infectados (9). Inicialmente, Field y Shute, en 1956 (10), describieron los cinco estadios de maduración de los gametocitos de P. falciparum. Posteriormente, estos fueron caracterizados por microscopía electrónica, en publicaciones de Cheryl Lobo y Nirbhay Kumar, en 2000 (11), yArthur Talman y colaboradores, en 2004 (8), quienes describieron las características morfológicas de los gametocitos en sus estadios inmaduros I, II, III y IV, y del gametocito maduro en estadio V.

Por el interés que tiene esta fase sexual de Plasmodium, desde hace varios años se han desarrollado técnicas de cultivo para obtener gametocitos en sus diferentes estadios y poder estudiarlos in vitro. La primera descripción de cultivo in vitro de gametocitos de P. falciparum se realizó en 1929 por Row, quien presentó evidencia del crecimiento in vitro de gametocitos de estadios inmaduros (I, II, III y IV), pero no hubo formación de gametocitos maduros (V) (12). A partir de ese momento, se desarrollaron otras técnicas de cultivo in vitro para la obtención de gametocitos.

En 1976, Trager y Jensen (13) reportaron el mantenimiento de un cultivo continuo de P. falciparum en eritrocitos humanos; los parásitos fueron obtenidos de monos Aotus trivirgatus infectados; realizaron diluciones de los parásitos por la adición de eritrocitos humanos con intervalos de tres a cuatro días y cambio diario de medio de cultivo, y obtuvieron así gametocitos imperfectos después de dos meses de mantenimiento. También en 1976, Smalley (14) realizó la técnica de microcultivo que permitió el desarrollo in vitro de gametocitos de P. falciparum morfológicamente maduros.

Posteriormente, aparecieron los cultivos manuales, que con la adición de hipoxantina lograron la producción de gametocitos maduros de P. falciparum (15-17), y los cultivos automatizados, que buscaban el desarrollo y la maduración de gametocitos usando tratamientos con la N-acetil glucosamina para sincronización del cultivo, y se logró la producción en poco tiempo de los cinco estadios sexuales de P. falciparum diferenciables morfológicamente (18,19).

Estas técnicas de cultivo buscaban de una manera simple obtener gametocitos en cantidad suficiente, diferenciables morfológicamente e infectantes, para lograr estudios futuros relacionados con el desarrollo de una vacuna que bloquee la transmisión.

En Colombia, no se ha reportado hasta ahora ningún estudio que utilice el cultivo in vitro de gametocitos de P. falciparum. Por lo tanto, el Grupo Malaria de la Universidad de Antioquia, interesado en la obtención de este estadio del parásito y de su continua producción para la medición de la respuesta del gametocito a nuevas opciones terapéuticas antipalúdicas, como son los compuestos aislados de plantas, estableció un cultivo in vitro de gametocitos en el cual se obtuvieron estadios del I al V, según el protocolo de Bhattacharyya Kanti y Kumar Nirbhay(20).

En este reporte se describe la técnica de cultivo in vitro de gametocitos según el protocolo deBhattacharyya Kanti y Kumar Nirbhay (20), y se muestran imágenes propias de los diferentes estadios, cuya morfología fue analizada por microscopía de luz y estuvo acorde con las características morfológicas de los cinco estadios del gametocito descritas en publicaciones de Cheryl Lobo y Nirbhay Kumar, 2000 (11), y Arthur Talman y colaboradores (8).

Materiales y métodos

Parásitos

Se utilizó la cepa NF54 de P. falciparum (cepa sensible a la cloroquina) para la producción de gametocitos.

Medio de cultivo

El medio de cultivo (1 L) se preparó según protocolo de Trager y Jensen (13),de la siguiente manera: se pesaron 10,4 g de RPMI (SIGMA), 5,94 g de hepes ácido, 2,0 g de dextrosa, 0,5 g de L-glutamina, 0,016 g de sulfato de gentamicina y 0,026 g de hipoxantina; se adicionaron 960 ml de agua desionizada estéril y se mezcló con agitador magnético hasta que todos los reactivos estuvieron completamente disueltos. Se filtró con membrana Millipore® de 0,22 µm y se conservó a 4 °C hasta su uso; este medio se denominó medio incompleto.

Para preparar 50 ml de medio completo, el cual se usó para cambio de medio del cultivo de gametocitos, se tomaron 43,3 ml de medio incompleto y se le adicionaron 5,0 ml de suero humano A-Rh+ al 10%, 43,0 µl de glutatión reducido 1 mg/ml (0,00326 M) y 1,8 ml de NaHCO3 al 5% (0,59 M). Después de la preparación de cada uno de los medios, se tomó una muestra para la prueba de esterilidad en infusión cerebro corazón (BHI), la cual se incubó a 37 °C y se leyó a las 24, 48, y 72 horas; para la utilización de los medios, la prueba de esterilidad debía ser negativa a las 72 horas.

El suero humano se preparó a partir de una mezcla de plasma A-Rh+ (con pruebas biológicas de tamización negativas), en botellas de policarbonato de 1 L que contenían perlas de vidrio que ocupaban, aproximadamente, un cuarto de las botellas; éstas se esterilizaron en formaldehído y posteriormente se les adicionó 1,7 ml de CaCl2 1,5 M estéril por cada 250 ml de plasma; se incubaron durante 6 horas a 37 °C y luego toda la noche a 4 °C. Después del tiempo de incubación, se centrifugaron a 4.500 rpm por 40 minutos a 4 °C, se tomó el suero de las botellas cuidadosamente y se envasó en tubos Falcon® estériles de 15 ml. A continuación se inactivó el suero a 56 °C durante 30 minutos y se almacenó a -20 °C. El suero se descongeló a 37 °C por 20 minutos antes de su uso.

Lavado de glóbulos rojos

Según el protocolo para el manejo de la cepa de P. falciparum in vitro del Instituto Nacional de Salud (21), los glóbulos rojos A-Rh+ de donantes locales de sangre fueron recolectados en tubos secos al vacío de 10 ml. Por cada 10 ml de sangre se adicionó 1,4 ml de solución anticoagulante CPD (14%); para preparar 50 ml de solución anticoagulante CPD (14%) se pesaron 0,164 g de ácido cítrico, 1,317 g de citrato de sodio 2H2O, 0,111 g de fosfato de sodio 1H2O, 1,278 g de dextrosa y mezclar con 50 ml de agua desionizada estéril. Se mezcló suavemente y se almacenó a 4 °C para su preservación durante 30 a 35 días, aproximadamente.

Para el lavado de los glóbulos rojos, se tomó la sangre total que estaba a 4 °C, se pasó a un tubo Falcon® de 15 ml, se centrifugó a 1.500 rpm durante 10 minutos y se descartaron el plasma y la capa de leucocitos. Posteriormente, se adicionó medio de lavado de glóbulos rojos (para preparar 100 ml de medio de lavado: se toman 96,4 ml de medio incompleto y se mezclan con 3,6 ml de NaHCO3 previamente preparado a una concentración de 5% (0,59M) en una proporción 2:1 (medio incompleto: glóbulos rojos); se mezcló suavemente por inversión, se centrifugó a 1.500 rpm durante 10 minutos y con una pipeta Pasteur se descartaron el sobrenadante y, aproximada-mente, 100 µl de la superficie del sedimento de glóbulos rojos. El lavado se repitió tres veces. Los glóbulos rojos lavados se conservaron a 4 °C hasta su uso.

Cultivo de gametocitos

Procedimento de cultivo de gametocitos: la cepa NF54 de P. falciparum se cultivó in vitro para producción de gametocitos, según protocolo de cultivo de Bhattacharyya Kanti y Kumar Nirbhay(20). El procedimiento que se aplicó fue el siguiente: el cultivo in vitro de Plasmodium con un volumen final de 10 ml (hematocrito del 6% de sangre A-Rh+ y una parasitemia de 0,3% de NF54 de P. falciparum), se envasó en botella de cultivo; posteriormente, se adicionó por 20 segundos mezcla de gases (90% N2, 5% O2, 5% CO2) y se colocó a incubar la botella en posición horizontal a 37 °C.

Al cultivo se le adicionaron los glóbulos rojos sólo el primer día y se le cambió el medio diariamente; se eliminó el medio viejo que tenia la botella, se dejó un pequeño volumen de, más o menos, 1 ml de medio por cada 5 ml de cultivo para evitar tomar glóbulos rojos; se tomaron, aproximadamente, 4 µl de glóbulos rojos del fondo de la botella de cultivo con los que se hizo un extendido en una placa portaobjetos, se coloreó con Giemsa y se determinó el crecimiento de la parasitemia asexual y sexual. Después de desechar el medio viejo, se le agregó medio completo nuevo (8 ml) y se adicionó por 20 segundos la mezcla de gases (90% N2, 5% O2; 5% CO2). Durante el cambio de medio se aseguró que el cultivo no estuviera por debajo de 37 °C; para esto se cambió el medio rápidamente, colocando la botella de cultivo sobre una placa de Petri que se puso en una plancha a 37 °C por 5 minutos, previo al cambio de medio. El cultivo fue manipulado siempre bajo condiciones de esterilidad.

Cuando la parasitemia asexual del cultivo estuvo entre 3% y 5%, se comenzó a agregar el doble de volumen de medio completo (16 ml) diariamente.

Según el protocolo de Bhattacharyya Kanti y Nirbhay Kumar (20),alrededor de los días 14 a 18 del cultivo se obtendrían gametocitos maduros (estadio V). Nosotros mantuvimos el cultivo hasta el día 14 porque en un ensayo previo de 17 días se vio que, después del día 15, se presentaron una disminución de glóbulos rojos y alteraciones morfológicas en los gametocitos que no permitían realizar una buena diferenciación de sus estadios.

Determinación de la parasitemia

Se hizo seguimiento diario de las parasitemias en extendidos de sangre periférica coloreados con Giemsa, según el protocolo del Instituto Nacional de Salud (21). Se llevó un registro diario de los conteos, expresados en porcentaje, de glóbulos rojos parasitados por formas asexuales y formas sexuales en 103 glóbulos rojos. Estos conteos se llevaron a cabo en 11 campos de 300 glóbulos rojos cada uno, aproximadamente, y se calculó la parasitemia tomando el número de glóbulos rojos parasitados multiplicado por tres y dividido por 100.

Resultados

Al realizar el análisis morfológico y la clasificación de estadios de P. falciparum en el cultivo, se observaron estadios asexuales y gametocitos morfológicamente diferenciables desde el primer día de seguimiento, los cuales se clasificaron con base en las características descritas (8,11). Esta coexistencia de estadios sexuales y asexuales del parásito posiblemente se debió a que el cultivo se encontraba sin

sincronizar y, de esta manera, pueden aparecer parásitos asexuales que comienzan a diferenciarse en gametocitos y parásitos con predisposición genética para formación de gametocitos. Igualmente, a partir del día 12 del cultivo de gametocitos se observó morfológicamente mayor cantidad de gametocitos compatibles con las características morfológicas de los estadios IV y V descritas por Cheryl Lobo  y Nirbhay Kumar en 2000 (11),y Arthur M. Talman y colaboradores en 2004 (8). Aun en el día 14 en el que se finalizó el mantenimiento del cultivo, se observaron gametocitos V maduros. Además, el porcentaje de glóbulos rojos parasitados con gametocitos fue mayor de 1% a partir del día 4 de cultivo y se mantuvo por encima de este porcentaje hasta el día 14 de su mantenimiento, lo que indica que el presente cultivo permite obtener diariamente producción de gametocitos durante todo el tiempo de sostenimiento del cultivo (cuadro 1).

Cuando se realizó diariamente la relación entre formas asexuales y sexuales de la cepa NF54 de P. falciparum en cultivo, se observó que hubo un mayor porcentaje de glóbulos rojos parasitados con estadios asexuales que de glóbulos rojos parasitados con formas sexuales; la diferencia representó un 2,7% más.

Al efectuar el análisis morfológico de los gametocitos según Arthur M. Talman y colaboradores (8),y Cheryl Lobo y Nirbhay Kumar (11), se observaron en el cultivo de la cepa NF54 de P. falciparum gametocitos desde el estadio I hasta el estadio V. Durante los primeros 11 días del cultivo se observaron en su mayoría gametocitos compatibles con los estadios I, II y III (figura 1, figura 2 y figura 3); los días siguientes se comenzaron a observar en su mayoría gametocitos compatibles con los estadios 4 y 5 (figura 4, figura 5 y figura 6) y el día 14, último día del mantenimiento del cultivo, en su mayoría se observaron gametocitos compatibles con el estadio V maduro, los cuales presentaron características morfológicas que permitieron diferenciarlos en microgametocito (macho) (figura 5) y macrogametocito (hembra) (figura 6).

Discusión

El gametocito ha sido una forma parasitaria poco estudiada, a pesar de cumplir un papel crítico en la continuación del ciclo de vida del parásito que ocasiona la malaria por medio de su transmisión del hombre al mosquito vector Anopheles. Se ha comprobado que la presión de los medicamentos antipalúdicos que no ejercen una acción gametocida puede también aumentar la producción de gametocitos y, de esta manera, intensificar la transmisión (22). Igualmente, estudios in vivo

demuestran que parásitos resistentes producen gametocitos más infectantes para el mosquito (23). Es por esto que los gametocitos constituyen un potencial blanco para combatir la malaria por medio de nuevas alternativas antipalúdicas y, de esta manera, lograr reducir la transmisión. Para esto es necesaria su producción en cantidad suficiente y que permanezcan infectantes para el mosquito para permitir el desarrollo de estudios futuros. En este estudio el protocolo de cultivo in vitro descrito por Bhattacharyya Kanti y Nirbhay Kumar (20), se llevó a cabo en su totalidad con la cepa NF54 de P. falciparum y con él se pudo reproducir el desarrollo de gametocitos de P. falciparum y obtenerlos en sus diversos estadios.

Al analizar el cultivo se observó que, durante su mantenimiento, los gametocitos se encontraron siempre acompañados del estadio asexual. Se han reportado diversas técnicas que utilizan sustancias químicas que eliminan los estadios asexuales, como la mitomicina C (24), la pirimetamina (25) y el sorbitol (26), aunque el uso de estas sustancias generó ciertos inconvenientes como la obtención de un número reducido de gametocitos, no eliminar estadios asexuales resistentes a la pirimetamina y la ausencia de producción de gametocitos funcionales.

Otra técnica desarrollada es el uso de gradientes de Percoll (27), la cual ha sido probada por nosotros, observándose que después de someter el cultivo de gametocitos a diferentes gradientes de Percoll, se concentran los glóbulos rojos parasitados con estadios sexuales y se observa un aumento del porcentaje de estos al compararlo con el porcentaje previo a la purificación. Además de permitir la obtención de un cultivo puro de gametocitos, esta técnica facilita la separación de todos los estadios de gametocitos. Es así como se podrían emplear métodos como éste para obtener únicamente gametocitos en cada uno de sus estadios de desarrollo y estudiar de una manera más amplia su biología.

Una característica importante en el cultivo que se desarrolló fue que los estadios maduros que se comenzaron a obtener a partir del día 11 del cultivo se lograron diferenciar morfológicamente en macrogametocitos (gametocitos hembras) y microgametocitos (gametocitos machos) y, a partir del día 14, se observaron mucho mejor sus características morfológicas, situación que podría permitir el inicio de estudios de capacidad infecciosa con estos estadios de gametocitos maduros de la cepa NF54 de P. falciparum.

Existen algunas diferencias con otros autores en relación con la técnica de cultivo de gametocitos, pero no son significativas. Por ejemplo, J. B. Jensen (28), produjo la maduración morfológica de gametocitos después del día 8 de cultivo y no logró la exflagelación in vitro, por lo cual se menciona que estos gametocitos no son funcionales para estudios futuros. R. S. Phillips y colaboradores (29), utilizaron para el cultivo de gametocitos glóbulos rojos fetales, los cuales suministraron un ambiente favorable para el crecimiento de los gametocitos. En otros artículos se encontró el uso de químicos, como la hipoxantina, que nosotros también adicionamos al cultivo para permitir la maduración de los gametocitos (15).

En Colombia no se ha reportado hasta ahora ningún estudio sobre cultivo in vitro de gametocitos de P. falciparum. Éste se llevó a cabo por el Grupo de Malaria de la Universidad de Antioquia, dada la necesidad de realizar estudios más amplios con los gametocitos, tales como estudios que permitan conocer su proceso de desarrollo (gametocitogénesis), sus mecanismos de citoadherencia, sus procesos metabólicos, la expresión de genes, la inmunidad contra sus diversos estadios, su capacidad infecciosa, sus estrategias de transmisión y evolución, y además,

conocer su respuesta a medicamentos antipalúdicos. Sin embargo, es necesario saber si todas las cepas de P. falciparum tienen la misma posibilidad de producir gametocitos que la cepa NF54 de P. falciparum empleada en este cultivo, la cual fue exitosa en la producción de gametocitos en estadio I, II, III, IV y V.

Esperamos continuar con el estudio de los gametocitos encontrando nuevos blancos para intervenir en el control del paludismo, además de estudiar el patrón de la resistencia a los antipalúdicos, enfocados en nuevos fármacos, que no sólo eliminen los estadios asexuales del parásito sino también a los estadios sexuales, y finalmente, contribuir en el bloqueo de la transmisión del paludismo.

Agradecimientos

A la Universidad de Antioquia y al Grupo Malaria, especialmente a Cecilia Giraldo Castro, bacterióloga de asistencia.

Conflictos de intereses

El manuscrito fue preparado y revisado con la participación de todos los autores, quienes declaramos que no existe ningún conflicto de intereses que ponga en riesgo la validez de nuestros resultados.

Financiación

Este trabajo fue financiado por la Universidad de Antioquia y el Grupo Malaria.

Correspondencia:

Silvia Blair, Grupo Malaria, Universidad de Antioquia, Calle 62 Nº 52-59, torre 1, Sede de Investigación Universitaria. SIU, Medellín, Colombia. Teléfono: (574) 210 6486; fax: (574) 210 6487 sblair@quimbaya.udea.edu.co

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24. Sinden RE, Ponnudurai T, Smits MA, Simm AM, Meuwissen JH. Gametocytogenesis of Plasmodium falciparum in vitro: a simple technique for the routine culture of pure capacitated gametocytes en masse. Parasitology. 1984;88:239-47.

25. Chutmongkonkul M, Maier WA, Seitz HM. A new model for testing gametocytocidal effects of some antimalarial drugs on Plasmodium falciparum in vitro. Ann Trop Med Parasitol. 1992;86:207-15.

26. Saul A, Graves P, Edser L. Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol. Int J Parasitol. 1990;20:1095-7.

27. Kariuki MM, Kiaira JK, Mulaa FK, Mwangi JK, Wasunna MK, Martin SK. Plasmodium falciparum: Purification of the various gametocyte developmental stages from in vitro–cultivated parasites. Am J Trop Med Hyg. 1998;59:505-8.

28. Jensen JB. Observations on gametogenesis in Plasmodium falciparum from continuous culture. J Protozool. 1979;26:129-32.

29. Phillips RS, Wilson RJ, Pasvol G. Differentiation of gametocytes in microcultures of human blood infected with Plasmodium falciparum. J Protozool. 1978;25:394-8.

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Journal of Clinical Microbiology, March 1999, p. 700-705, Vol. 37, No. 30095-1137/99/$04.00+0Copyright © 1999, American Society for Microbiology. All rights reserved.

In Vitro Culture and Drug Sensitivity Assay of Plasmodium falciparum with Nonserum Substitute and Acute-Phase Sera

Pascal Ringwald,* Fleurette Solange Meche, Jean Bickii, and Leonardo K. Basco

Laboratoire de Recherches sur le Paludisme, Laboratoire Associé Francophone 302, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, and Institut Français de Recherche Scientifique pour le Développement en Coopération, Yaoundé, Cameroon

Received 5 June 1998/Returned for modification 9 November 1998/Accepted 2 December 1998

The short-term in vitro growth of Plasmodium falciparum parasites in the asexual erythrocytic stage and the in vitro activities of eight standard antimalarial drugs were assessed and compared by using RPMI 1640 medium supplemented with 10% nonimmune human serum, 10% autologous or homologous acute-phase serum, or 0.5% Albumax I (lipid-enriched bovine serum albumin). In general, parasite growth was maximal with autologous (or homologous) serum, followed by Albumax I and nonimmune serum. The 50% inhibitory concentrations (IC50s) varied widely, depending on the serum or serum substitute. The comparison of IC50s between assays with autologous and nonimmune sera showed that monodesethylamodiaquine, halofantrine, pyrimethamine, and cycloguanil had similar IC50s. Although the IC50s of chloroquine, monodesethylamodiaquine, and dihydroartemisinin were similar with Albumax I and autologous sera, the IC50s of all test compounds obtained with Albumax I differed considerably from the corresponding values obtained with nonimmune serum. Our results suggest that Albumax I and autologous and homologous sera from symptomatic, malaria-infected patients may be useful alternative sources of serum for in vitro culture of P. falciparum isolates in the field. However, autologous sera and Albumax I do not seem to be suitable for the standardization of isotopic in vitro assays for all antimalarial drugs.

    INTRODUCTION

Cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry, molecular biology, immunology, and pharmacology. One of the applications of parasite cultivation is the in vitro drug sensitivity assay, which is a major tool for the screening of potential antimalarial drugs, the monitoring of drug sensitivity, and the detection of cross-resistance patterns against Plasmodium falciparum parasites (5, 25, 34). Although several assays have been developed, the in vitro cultivation technique of the erythrocytic stages of P. falciparum remains essentially the same as that originally described by Trager and Jensen (29). In this standard technique, the following components are required: P. falciparum-infected human erythrocytes, buffered

Top Abstract Introduction Materials and methods Results Discussion References

RPMI 1640 medium, and human serum. The parasitized erythrocytes are incubated in a low-oxygen atmosphere at 37°C.

The culture medium is commercially available at a relatively low cost. The sources of infected blood abound in countries where malaria is endemic. In countries where malaria is not endemic, several reference clones and strains of P. falciparum are available in research laboratories. It has generally been accepted that nonimmune human serum is required for optimal parasite growth. However, the requirement for a regular supply of nonimmune human serum entails difficulties in conducting research in most of the African continent, where malaria transmission occurs at a high level throughout the year. Nonimmune human type AB-positive serum is relatively scarce and expensive in countries where malaria is not endemic. Furthermore, it is recommended that several units of serum from different donors be pooled to reduce batch-to-batch differences in the support of parasite growth (14). Additional problems include blood type compatibility and risks associated with the handling of infectious agents.

Because of these disadvantages, numerous alternative sources of sera and nonserum substitutes have been tested in the past (1, 6, 13, 15, 19, 27, 28, 37). Although some of these substitutes have been successfully used for the continuous cultivation of laboratory-adapted P. falciparum strains and clones, they are generally less effective than human serum and have not been adopted by many research laboratories. In a recent study, Ofulla et al. (21) have identified serum albumin and lipids as the key serum components that are necessary to sustain optimal parasite growth. Further studies have shown that a commercially available lipid-enriched bovine serum albumin, Albumax I (Gibco BRL), can replace human serum for the continuous in vitro cultivation of malaria parasites, leading to its routine use in several laboratories (2, 4, 7, 8, 31).

Another alternative source of serum is malaria parasite-infected patients themselves. It has been thought that semi-immune human serum protects individuals who are continuously exposed to malaria parasites against malarial disease and therefore inhibits parasite growth (14). This assumption has not been proven experimentally through the quantitation and correlation of parasite growth inhibition, the level of acquired immunity, and the presence of antimalarial drugs in sera collected from indigenous populations. In addition, if this hypothesis were true, indigenous adults who have been exposed to the malaria parasite are expected to have acquired immune protection against further attacks of malaria. Contrary to this expectation, in many areas of endemicity in Africa, such as in Yaoundé, Cameroon, symptomatic malarial infection occurs frequently in both adults and children (24). It has also been shown that heat-inactivated serum from healthy, semi-immune African donors can support the growth of laboratory-adapted strains of parasites and fresh isolates and that acute-phase homologous serum may be useful for the continuous in vitro culture of reference strains (2, 20).

A preliminary in vitro study with lipid-enriched bovine serum albumin has reported that serum-free medium can be used instead of nonimmune serum to determine the level of drug activity (22). Autologous and homologous acute-phase sera from malaria parasite-infected

patients and Albumax I have not been evaluated as alternatives for in vitro culture with fresh clinical isolates obtained from indigenous patients. In our present study, we (i)

assessed the growth of clinical isolates of the malaria parasites in two different RPMI 1640 media during a single life cycle or two life cycles with nonimmune type AB-positive serum, Albumax, autologous acute-phase serum, and homologous acute-phase serum with the aim of determining the best medium for short-term culture and in vitro drug assay and (ii) compared the in vitro activities of various antimalarial compounds against fresh clinical isolates of P. falciparum using different sera or serum substitute with the aim of assessing whether these alternative sources can be used to standardize isotopic in vitro assays.

    MATERIALS AND METHODS

Clinical isolates. Thirty fresh clinical isolates were obtained from symptomatic Cameroonian patients residing in Yaoundé before treatment. Eight were children between 5 and 14 years old; 22 were adults ( 15 years old; age range, 19 to 69 years). Our previous studies have shown that populations of patients in this age range in Yaoundé present with similar clinical and laboratory features (24, 26). The following inclusion criteria were set for this study: presence of signs and symptoms of acute uncomplicated malaria, monoinfection with P. falciparum, parasitemia of >0.2%, and no history of recent antimalarial drug intake confirmed by a negative Saker-Solomons urine test (18). Since this study is part of an ongoing clinical study designed to determine the clinical efficacy of first- and second-line drugs, pregnant women and patients with signs and symptoms of severe and complicated falciparum malaria, as defined by the World Health Organization (WHO) (33), were excluded. After informed consent was obtained, 10 ml of venous blood was collected in a tube coated with anticoagulant (EDTA) and in a

tube not coated with anticoagulant. The patients were treated with either amodiaquine or sulfadoxine-pyrimethamine, which are first- and second-line drugs in Cameroon, respectively, and were monitored daily until they were cured. The study was approved by the Cameroonian National Ethics Committee.

Drugs. The following antimalarial drugs were obtained from the indicated sources: chloroquine sulfate, Rhone-Poulenc-Rorer, Antony, France; monodesethylamodiaquine, a biologically active metabolite of amodiaquine, Sapec S. A., Lugano, Switzerland; quinine hydrochloride, Sigma Chemical Co., St. Louis, Mo.; mefloquine hydrochloride, Hoffmann-La Roche, Basel, Switzerland; halofantrine hydrochloride, SmithKline Beecham, Hertfordshire, United Kingdom; dihydroartemisinin, a biologically active metabolite of artemisinin derivatives; Sapec S. A.; pyrimethamine base, Sigma Chemical Co.; and cycloguanil base, a biologically active metabolite of proguanil, Zeneca Pharma, La Defense, France. Stock solutions of chloroquine, monodesethylamodiaquine, and cycloguanil were prepared in sterile distilled water. Stock solutions of quinine, mefloquine, halofantrine, dihydroartemisinin, and pyrimethamine were prepared in methanol. Twofold serial dilutions (fourfold serial dilutions for pyrimethamine and cycloguanil) of the drugs were made in the same solvent used to prepare the stock solutions. The final concentrations ranged from 25 to 1,600 nmol/liter for chloroquine, 50 to 3,200 nmol/liter for quinine, 5 to 320 nmol/liter for

Top Abstract Introduction Materials and methods Results Discussion References

monodesethylamodiaquine, 2.5 to 160 nmol/liter for mefloquine, 0.5 to 32 nmol/liter for halofantrine, 0.25 to 16 nmol/liter for dihydroartemisinin, and 0.09 to 51,200 nmol/liter for pyrimethamine and cycloguanil. Each concentration was distributed in triplicate in 96-well tissue culture plates and air dried.

In vitro assay. Venous blood samples collected in tubes with anticoagulant were washed three times in p-aminobenzoic acid (PABA) and folic acid-free RPMI 1640 medium. Two types of RPMI 1640 medium were used to cultivate the parasites: the standard RPMI 1640 medium containing 1 mg of PABA per liter and 1 mg of folic acid per liter and PABA- and folic acid-free RPMI 1640 medium. Since PABA and folic acid compete with dihydrofolate reductase and dihydropteroate synthase inhibitors (pyrimethamine, cycloguanil, proguanil, and sulfonamides), PABA- and folic acid-free RPMI 1640 medium was used to evaluate the in vitro activities of these drugs (17, 31, 40). Both RPMI 1640 media were supplemented with HEPES (25 mmol/liter), NaHCO3 (25 mmol/liter), and gentamicin (10 µg/ml). Infected erythrocytes were suspended in these culture media at a hematocrit of 1.5% and an initial parasitemia of between 0.2 and 0.6%. The following serum or serum substitute was added to obtain the complete media: 10% (vol/vol) nonimmune type AB-positive serum (obtained from French blood donors who resided in France and who had no previous history of malaria), 10% (vol/vol) human serum from malaria-infected patients, and concentrated Albumax I solution (10% [wt/vol]) diluted to a final concentration of 0.5%. The concentration of Albumax I used in this study was shown

in a previous study (21) to be optimal for parasite growth. If the blood sample had a parasitemia of >0.6%, fresh uninfected, type A-positive erythrocytes were added to adjust the parasitemia to 0.6%. All experiments were performed within 4 h after blood collection.

The growth of 12 parasite isolates in the presence of either autologous or homologous acute-phase sera was compared in the complete standard RPMI 1640 medium. Only freshly obtained clinical isolates were used in our experiments, and the isolates were used without prior adaptation to in vitro culture. Some of the acute-phase sera were stored at 20°C for several months before use. Freshly drawn sera either were used immediately after collection or were stored at 4°C for up to 3 weeks. For these experiments, the parasitemia (range, 0.2 to 3.0%) was not adjusted; one blood sample with a parasitemia of 10%, however, was diluted with fresh uninfected type A-positive erythrocytes from a healthy donor (1:9 [vol/vol]) to obtain an initial parasitemia of 1%. The short-term culture technique was based on the isotopic microtest of Desjardins et al. (5). Two hundred microliters of the suspension of infected erythrocytes was distributed in

triplicate in 96-well tissue culture plates that were either drug-free or precoated with test compounds. The parasites were incubated at 37°C in 5% CO2. [3H]hypoxanthine (specific activity, 16.3 Ci/mmol; 1 µCi/well; Amersham, Buckinghamshire, United Kingdom) was added after the first 18 h of incubation to assess parasite growth. The incorporation of

[3H]hypoxanthine has been established as an accurate and reliable means of determining in vitro parasite growth (3, 5). After an additional 24 h of incubation (additional 48 h for experiments with PABA- and folic acid-free RPMI 1640 medium and drug assays for pyrimethamine and cycloguanil), the plates were frozen to terminate the assays. The plates were thawed to lyse the infected erythrocytes, and the contents of each well were collected on glass-fiber filter papers, washed, and dried with a cell harvester. The filter disks were transferred into scintillation tubes, and 2 ml of scintillation cocktail (Organic Counting

Scintillant; Amersham) was added. The incorporation of [3H]hypoxanthine was quantitated with a liquid scintillation counter (Wallac 1410; Pharmacia, Uppsala, Sweden). For parasite growth assays, the results either were expressed as counts per minute or were normalized to the growth of parasites in their corresponding autologous sera. The 50% inhibitory concentrations (IC50s), defined as the drug concentration corresponding to 50% of the uptake of [3H]hypoxanthine measured in drug-free control wells, were determined by nonlinear regression analysis with Prism software (GraphPad Software, Inc., San Diego, Calif.). The IC50s determined for isolates cultivated in medium supplemented with different sera or serum substitute were expressed as the mean IC50 ratios. IC50 ratios were calculated for the following: Albumax I/nonimmune serum, Albumax I/autologous acute-phase serum, and autologous acute-phase serum/nonimmune serum. If the mean IC50 ratio between media supplemented with different sera or serum substitute was 0.50 to 1.50, the mean IC50s were considered to be equivalent. The threshold IC50s for in vitro resistance to chloroquine, monodesethylamodiaquine, quinine, mefloquine, halofantrine, cycloguanil, and pyrimethamine were estimated to be >100, >60, >800, >30, >6, >50, and >100 nmol/liter,

respectively (25). The level of resistance to dihydroartemisinin is still undetermined.

    RESULTS

Nine fresh clinical isolates were tested for in vitro growth in standard RPMI 1640 medium supplemented with nonimmune or autologous serum or Albumax I. All isolates adapted readily to the in vitro conditions, as shown by an adequate incorporation of tritium-labeled

hypoxanthine (>3,000 cpm) during a 42-h incubation period (Fig. 1). The growth of most clinical isolates was optimal in the presence of autologous serum; the exceptions were two isolates (isolates 18/98 and 22/98) which grew better with Albumax I than with the autologous serum. One isolate (isolate 14/98) developed equally well with nonimmune serum (51,948 ± 2,530 cpm) and autologous serum (51,788 ± 989 cpm). For other isolates, in the presence of the autologous serum, the incorporation of tritium-labeled

hypoxanthine was 1.4 to 5.6 times higher than that obtained with

nonimmune serum. A comparison between nonimmune serum and Albumax I showed that three clinical isolates (isolates 13/98, 14/98, and 19/98) grew better (two- to eightfold better) with the nonimmune serum. For four other isolates, an opposite trend was observed, with higher (two- to sixfold) levels of hypoxanthine incorporation in the presence of Albumax I. Two isolates (isolates 6/98 and 16/98) grew almost equally well with nonimmune serum and Albumax I. Similar results were obtained with the isolates grown in PABA- and folic acid-free RPMI 1640 medium supplemented with nonimmune serum, autologous serum, or Albumax I over a 72-h incubation period.

Top Abstract Introduction Materials and methods Results Discussion References

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FIG. 1.   Comparison of parasite growth over 42 h in standard RPMI 1640 medium supplemented with pooled nonimmune type AB-positive serum (open squares), Albumax I (black circles), or autologous serum (black squares) assessed by the incorporation of [3H]hypoxanthine. Results represent mean counts per minute (n = 1 to 6 triplicate tests). Bars denote standard deviations.

The assessment of parasite growth in the standard RPMI 1640 medium supplemented with different sets of acute-phase sera showed that the parasites generally grew better with autologous or homologous acute-phase sera than with pooled nonimmune sera (data not shown). The homologous sera supported the growth of fresh isolates to a widely different extent, even surpassing the growth with autologous serum for some isolates. Parasite growth was not influenced by an ABO blood type incompatibility or storage of homologous sera at 4°C or 20°C.

The in vitro drug sensitivity patterns were determined for 11 clinical isolates with nonimmune serum, autologous acute-phase serum, and Albumax I. The differences in the IC50s obtained with different sources of serum or serum substitute were relatively small for chloroquine and monodesethylamodiaquine (Table 1). Nonimmune serum gave the lowest IC50 for chloroquine and monodesethylamodiaquine, generally followed by autologous serum and Albumax I. Wide variations in the IC50s of the other test compounds were observed. The IC50s of quinine and mefloquine differed considerably (nonimmune serum < Albumax I < autologous serum), with generally greater than a 2-fold difference between nonimmune serum and Albumax and greater than a 10-fold difference between nonimmune serum and autologous serum for quinine. The differences in the IC50s of mefloquine were less pronounced. The order of the IC50s of halofantrine was as follows: Albumax I < nonimmune serum < autologous serum. The IC50s of dihydroartemisinin, pyrimethamine, and cycloguanil generally varied according to the following order: nonimmune serum   autologous serum < Albumax I. 

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TABLE 1.   Drug sensitivity patterns of P. falciparum isolates obtained with nonimmune or autologous sera or serum substitute (Albumax I)

The IC50 ratios are summarized in Table 2. According to our criterion of an equivalent IC50, defined as an IC50 ratio of between 0.50 and 1.50, the mean IC50s obtained with Albumax I

were not equivalent to any of the IC50s obtained with nonimmune serum. However,

equivalent IC50s were obtained with Albumax I and autologous sera for chloroquine (IC50 ratio, 1.10 ± 0.17) and monodesethylamodiaquine (IC50 ratio, 1.41 ± 0.33). The IC50s of dihydroartemisinin obtained with Albumax I and autologous sera were also similar, but the values were widely dispersed. The mean IC50s of monodesethylamodiaquine were equivalent with nonimmune and autologous sera (IC50 ratio, 1.10 ± 0.17). The IC50s of halofantrine, pyrimethamine, and cycloguanil were also similar, but the IC50

ratios were widely dispersed.

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TABLE 2.   Mean IC50 ratios of antimalarial drugs obtained from nonimmune or autologous acute-phase sera or serum substitute (Albumax I) against fresh clinical isolates of P. falciparum

    DISCUSSION

In previous studies, laboratory-adapted P. falciparum strains and fresh clinical isolates were successfully adapted and maintained in continuous culture with heat-inactivated semi-immune plasma or serum from African donors with no history of malarial infection in the preceding 3 weeks (2, 20). Our results further extend this observation and demonstrate that autologous and homologous acute-phase sera from malaria-infected patients also support the growth of fresh clinical isolates and often do so better than nonimmune sera. Thus, contrary to the unconfirmed assumption that local sera in areas of endemicity are unsuitable for parasite cultivation because they may contain antimalarial drugs and ill-defined immune factors (14), our results show that autologous and homologous sera from symptomatic patients may also be useful for parasite culture in the field. For most isolates, the in vitro growth attained maximal levels when the autologous acute-phase serum was added.

Clinical isolates may be readily adapted to short-term culture if the absence of antimalarial drugs is verified by a simple urine test (18). Our success rate in performing in vitro assays

with nonimmune serum and fresh clinical isolates (>500 primary isolates between 1993 and 1997) exceeds 92% in Yaoundé (23, 25). The in vitro studies conducted by Oduola et al. (20) and Binh et al. (2) with semi-immune and acute-phase sera to maintain cultures of laboratory-adapted strains and clones of P. falciparum support our observation that local sera may be useful for parasite cultivation. Our data suggest that both autologous and homologous acute-phase sera support parasite development for one or two life cycles and that the patients' own acute-phase sera may be the best source of nutrients for the corresponding P. falciparum isolate, at least for short-term cultivation or for the initiation of culture of field isolates.

Top Abstract Introduction Materials and methods Results Discussion References

The use of Albumax I as a serum substitute for a long-term continuous culture has several advantages over the use of human serum. Albumax I costs less than human serum, is compatible with any blood type, and does not have a wide batch-to-batch difference, as is the case with serum. Growth of laboratory-adapted P. falciparum strains was similar in RPMI 1640 medium supplemented with hypoxanthine (0.2 mM) and nonimmune human serum or Albumax (4). Furthermore, Albumax has been used successfully to maintain a continuous culture of several P. falciparum strains in different laboratories (2, 4, 7, 8, 31). However, Albumax may not be suitable for in vitro drug assays since the IC50s of most antimalarial drugs tested in this study were consistently higher with the Albumax-supplemented medium than with the serum-supplemented medium. Using serum-free media containing 5 g of bovine albumin per liter and Cohn fraction V, Ofulla et al. (22) also found that both chloroquine and quinine IC50s are, on average, 1.6 times higher than the values obtained with serum for 14 culture-adapted or fresh isolates. In their study, the ratio of amodiaquine IC50s (serum-free medium versus serum-supplemented medium) was 1.1 for 11 isolates.

The underlying reason for the higher IC50s with Albumax-supplemented media may be due to high levels of protein binding. In vivo, many antimalarial drugs are known to be highly bound to plasma proteins, notably, to albumin, which is the major component of plasma proteins and Albumax I. Quinine (70 to 95% protein binding), mefloquine (>98%), pyrimethamine (87%), and cycloguanil (75%), which had elevated IC50s with Albumax-supplemented medium, are highly protein bound (35). Chloroquine is relatively less protein

bound (50 to 70%), while monodesethylamodiaquine is highly protein bound (>90%). Protein binding alone does not explain the widely different IC50s of some antimalarial drugs since the albumin concentration used in our experiments (for Albumax I, 0.5% [wt/vol], and for serum albumin, 10% [vol/vol]; the normal plasma albumin concentration is 35 to 50 g/liter) is similar in the two media.

Several possible factors may influence in the increase or decrease in the IC50s. First, as suggested by Ofulla et al. (22), the differential IC50s may result, at least in part, from a higher affinity of antimalarial drugs for bovine albumin than for human albumin. Second, chloroquine and amodiaquine (highly hydrophilic, weak bases) undergo marked uptake into infected and uninfected erythrocytes (11, 30), which may explain the similar IC50s obtained in our study. Third, albumin binds to lipophilic compounds, such as halofantrine, by means of hydrophobic binding forces, and plasma lipoproteins, notably, triglyceride, influence the

IC50s of halofantrine (12). Although the protein binding properties of halofantrine are still unknown due to its low solubility in water, these properties of albumin and lipids may explain the increased transport of halofantrine into infected erythrocytes in Albumax-supplemented medium and thus the lowering of the IC50s.

The standard in vitro test developed by WHO uses a mixture (9:1 [vol/vol]) of RPMI 1640 medium and the patient's whole blood to determine the level of parasite growth in the presence of different drug concentrations (38). The results are interpreted by microscopic

examination of thick blood smears. The results of the WHO in vitro test and those of in vitro assays that are based on the incorporation of tritium-labeled hypoxanthine are not comparable (36). The WHO test determines the maximal inhibitory concentration, while

isotopic tests measure the IC50. The former uses acute-phase plasma; the latter test is usually

performed with nonimmune donor serum after washing of the infected erythrocytes. To our knowledge, no study has compared the two in vitro tests, but it is assumed that isotopic tests are more objective and accurate in determining the sensitivity levels (36). Although the standard WHO test uses whole blood, in our study autologous acute-phase sera did not yield consistent drug assay results compared with those obtained with nonimmune serum except for the results for monodesethylamodiaquine and, to a lesser extent, halofantrine, pyrimethamine, and cycloguanil. In most cases, the IC50s obtained with autologous serum were increased more than two times compared with those obtained with nonimmune serum. An opposite trend would have been expected from the observation that the level of albumin in plasma is generally lower in malaria-infected patients than in healthy adults (9, 10), which should lead to a higher concentration of unbound drug available for schizontocidal

action in the autologous serum. The most discordant result was observed with quinine; the IC50s of quinine obtained with autologous serum were more than 10 times greater than the values obtained with nonimmune serum. The most likely explanation lies in the increased circulating concentration of the acute-phase plasma protein -1 acid glycoprotein, which increases the level of protein binding of quinine in malaria parasite-infected patients (10, 16, 32). Furthermore, the level of protein binding of drugs is known to vary widely between patients (39). For these reasons, autologous sera are probably not useful for determination of the drug sensitivity pattern. Whether our observation extends to the WHO standard in vitro test that uses autologous plasma needs to be determined.

Our study demonstrates that autologous and homologous sera from patients with acute uncomplicated falciparum malaria are suitable for cultivation of field isolates and that their use results in a high growth rate compared with that obtained with nonimmune pooled sera and Albumax I during the first and second in vitro erythrocytic cycles. Our study further confirms the usefulness of Albumax I for the short-term cultivation of field isolates.

Although autologous and homologous sera and Albumax I may serve as alternative sources of serum or serum substitute for the in vitro culture of fresh isolates in the field, at present, none of them seems to be suitable for in vitro drug sensitivity assays with the exception of autologous sera for monodesethylamodiaquine and Albumax I for halofantrine. However, if more data from a larger series of studies comparing Albumax I and nonimmune serum are

accumulated, a reliable conversion factor in the form of IC50 ratios can be calculated for

antimalarial drugs for which the IC50 ratios in this study were relatively low (chloroquine, monodesethylamodiaquine, mefloquine, and halofantrine). With such a conversion factor,

threshold resistance values can be adjusted for Albumax I-supplemented medium. Other serum substitutes that provide adequate nutritional needs for the optimal growth of parasites and that do not bind strongly to antimalarial drugs may be necessary for the standardization

of in vitro assays.

    ACKNOWLEDGMENTS

We thank Sister Solange Menard and her nursing and laboratory staff at the Nlongkak Catholic Missionary Dispensary, Yaoundé, Cameroon, for helping us screen malaria parasite-infected patients.

This investigation was supported by a grant from AUPELF-UREF.

    FOOTNOTES* Corresponding author. Mailing address: OCEAC/ORSTOM, B.P. 288, Yaoundé, Cameroon. Phone: (237) 232 232. Fax: (237) 230 061. E-mail: oceac@camnet.cm.

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Top Abstract Introduction Materials and methods Results Discussion References

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Journal of Clinical Microbiology, March 1999, p. 700-705, Vol. 37, No. 30095-1137/99/$04.00+0Copyright © 1999, American Society for Microbiology. All rights reserved.