Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories / Pruebas moleculares ultrasensibles para la detección de Plasmodium: aplicabilidad en los programas de control y eliminación y en los laboratorios de referencia / Testes moleculares ultra-sensíveis para detecção de Plasmodium: aplicabilidade em programas de controlo e eliminação e laboratórios de referencia

ABSTRACT Objective. To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods. We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results. The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman's test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions. The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

RESUMEN Objetivo. Evaluar herramientas moleculares para detectar bajos niveles de parasitemia y las cinco especies de Plasmodium que infectan a los seres humanos, a fin de emplearlas en los programas de control y eliminación y en los laboratorios de referencia. Métodos. Se evaluaron 145 muestras de sangre de pacientes positivos por reacción en cadena de la polimerasa anidada (nPCR), de individuos asintomáticos y de muestras del Programa Mundial de Malaria de la Organización Mundial de la Salud/Servicio Nacional de Evaluación Externa de Calidad del Reino Unido. Las muestras se analizaron con el kit de PCR RealStar® Malaria 1.0 (alt-Gen; altona Diagnostics), específico para cada género, y con el kit de PCR RealStar® Malaria Screen & Type (alt-S&T; altona Diagnostics). Se compararon los resultados de las pruebas moleculares con los de la PCR cuantitativa (qPCR), la nPCR y el frotis de gota gruesa. Resultados. Los niveles de parasitemia oscilaron entre 1 y 518 000 parásitos/µl, según la especie. En comparación con la nPCR, la prueba alt-S&T tuvo una sensibilidad del 100%, excepto para la identificación de P. falciparum, para el cual la sensibilidad fue del 93,94%. Todas las muestras positivas por alt-Gen lo fueron también por nPCR. Al comparar alt-Gen con la qPCR, la sensibilidad fue del 100% para P. vivax, P. malariae y P. falciparum. Para todas las especies de Plasmodium, la correlación entre los valores del umbral de ciclo de alt-S&T y alt-Gen en comparación con la qPCR fue significativa (P < 0,0001, prueba de Spearman), con r = 0,8621 para alt-S&T y r = 0,9371 para alt-Gen. Cuando se consideraron todas las especies de Plasmodium hubo una correlación negativa entre el nivel de parasitemia y los valores de umbral de ciclo de PCR en tiempo real (P < 0,0001). En este estudio, solo 2 de las 28 muestras de individuos asintomáticos fueron positivas por frotis de gota gruesa; sin embargo, las 28 muestras fueron positivas por alt-S&T. Conclusiones. Los ensayos alt-Gen y alt-S&T son adecuados para detectar infecciones submicroscópicas con distintos fines epidemiológicos, como su uso en investigaciones y laboratorios de referencia y el cribado en bancos de sangre, lo que contribuirá a los esfuerzos mundiales para eliminar la malaria.

RESUMO Objectivo. Avaliar ferramentas moleculares para detectar parasitemia de baixo nível e as cinco espécies de Plasmodium que infectam humanos, para utilização em programas de controlo e eliminação e em laboratórios de referência. Métodos. Avaliámos 145 amostras de sangue de doentes que testaram positivo por reacção em cadeia da polimerase aninhada (nPCR), de indivíduos assintomáticos, e do Programa Global de Paludismo da Organização Mundial de Saúde/Serviço Nacional de Avaliação da Qualidade Externa do Reino Unido. As amostras foram ensaiadas utilizando o RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) e o RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). Os resultados dos testes moleculares foram comparados com os resultados da PCR quantitativa (qPCR), nPCR e exame da gota espessa. Resultados. Os níveis de parasitemia variaram de 1 a 518 000 parasitas/µL, dependendo da espécie. Em comparação com a nPCR, alt-S&T tinha uma sensibilidade de 100%, excepto na identificação de P. falciparum, para a qual a sensibilidade era de 93,94%. Todas as amostras positivas por alt-Gen foram também positivas por nPCR. Ao comparar alt-Gen com qPCR, a sensibilidade foi de 100% para P. vivax, P. malariae e P. falciparum. Para todas as espécies Plasmodium, a correlação entre os valores limiares de ciclo de alt-S&T e alt-Gen comparados com qPCR foi significativa (P < 0,0001, teste de Spearman), com r = 0,8621 para alt-S&T e r = 0,9371 para alt-Gen. Quando todas as espécies de Plasmodium foram consideradas, houve uma correlação negativa entre o nível de parasitemia e os valores limiares do ciclo de PCR em tempo real (P < 0,0001). Neste estudo, apenas 2 de 28 amostras de indivíduos assintomáticos foram positivas por exame da gota espessa; no entanto, todas estas 28 amostras foram positivas por alt-S&T. Conclusões. Os ensaios alt-Gen e alt-S&T são adequados para a detecção de infecções submicroscópicas para fins epidemiológicos distintos, tais como para utilização em inquéritos e laboratórios de referência e o rastreio em bancos de sangue, o que contribuirá para os esforços globais de eliminação da malária.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination

In vitro evaluation of erythromycin in chloroquine resistant brazilian P. falciparum freshly isolates: modulating effect and antimalarial activity evidence

Erythromycin, a reversal agent in multidrug-resistant cancer, was assayed in chloroquine resistance modulation. The in vitro microtechnique for drug susceptibility was employed using two freshly isolates of Plasmodium falciparum from North of Brazil. The antimalarial effect of the drug was confirmed, with an IC50 estimates near the usual antimicrobial therapy concentration, and a significant statistical modulating action was observed for one isolate