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A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.

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BACKGROUND: Pregnancy poses specific challenges for the diagnosis of Plasmodium falciparum infection due to parasite sequestration in the placenta, which translates in low circulation levels in peripheral blood. The aim of this study is to assess the performance of a new highly sensitive rapid diagnostic test (HS-RDT) for the detection of malaria in peripheral and placental blood samples from pregnant women in Colombia. METHODS: This is a retrospective study using 737 peripheral and placental specimens collected from pregnant women in Colombian malaria-endemic regions. Light microscopy (LM), conventional rapid diagnostic tests (Pf/Pv RDT and Pf RDT), and HS-RDT testing were performed. Diagnostic accuracy endpoints of LM, HS-RDT and RDTs were compared with nested polymerase chain reaction (nPCR) as the reference test. RESULTS: In comparison with nPCR, the sensitivity of HS-RDT, Pf RDT, Pf/Pv RDT and LM to detect infection in peripheral samples was 85.7% (95% CI = 70.6-93.7), 82.8% (95% CI = 67.3-91.9), 77.1% (95% CI = 61.0-87.9) and 77.1% (95% CI = 61.0-87.9) respectively. The sensitivity to detect malaria in asymptomatic women, was higher with HS-RDT, where LM and Pf/Pv RDT missed half of infections detected by nPCR, but differences were not significant. Overall, specificity was similar for all tests (>99.0%). In placental blood, the prevalence of infection by P. falciparum by nPCR was 2.8% (8/286), by HS-RDT was 1% and by conventional RDTs (Pf RDT and Pf/Pv RDT) and LM was 0.7%. The HS-RDT detected placental infections in peripheral blood that were negative by LM and Pf/Pv RDT, however the number of positive placentas was low. CONCLUSIONS: The sensitivity of HS-RDT to detect P. falciparum infections in peripheral and placental samples from pregnant women was slightly better compared to routinely used tests during ANC visits and at delivery. Although further studies are needed to guide recommendations on the use of the HS-RDT for malaria case management in pregnancy, this study shows the potential value of this test to diagnose malaria in pregnancy in low-transmission settings.

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BACKGROUND: Pregnant women frequently show low-density Plasmodium infections that require more sensitive methods for accurate diagnosis and early treatment of malaria. This is particularly relevant in low-malaria transmission areas, where intermittent preventive treatment is not recommended. Molecular methods, such as polymerase chain reaction (PCR) are highly sensitive, but require sophisticated equipment and advanced training. Instead, loop mediated isothermal amplification (LAMP) provides an opportunity for molecular detection of malaria infections in remote endemic areas, outside a reference laboratory. The aim of the study is to evaluate the performance of LAMP for the screening of malaria in pregnant women in Colombia. METHODS: This is a nested prospective study that uses data and samples from a larger cross-sectional project conducted from May 2016 to January 2017 in three Colombian endemic areas (El Bagre, Quibdó, and Tumaco). A total of 531 peripheral and placental samples from pregnant women self-presenting at local hospitals for antenatal care visits, at delivery or seeking medical care for suspected malaria were collected. Samples were analysed for Plasmodium parasites by light microscopy (LM), rapid diagnostic test (RDT) and LAMP. Diagnostic accuracy endpoints (sensitivity, specificity, predictive values, and kappa scores) of LM, RDT and LAMP were compared with nested PCR (nPCR) as the reference standard. RESULTS: In peripheral samples, LAMP showed an improved sensitivity (100.0%) when compared with LM 79.5% and RDT 76.9% (p < 0.01), particularly in afebrile women, for which LAMP sensitivity was two-times higher than LM and RDT. Overall agreement among LAMP and nPCR was high (kappa value = 1.0). Specificity was similar in all tests (100%). In placental blood, LAMP evidenced a four-fold improvement in sensitivity (88.9%) when compared with LM and RDT (22.2%), being the only method, together with nPCR, able to detect placental infections in peripheral blood. CONCLUSIONS: LAMP is a simple, rapid and accurate molecular tool for detecting gestational and placental malaria, being able to overcome the limited sensitivity of LM and RDT. These findings could guide maternal health programs in low-transmission settings to integrate LAMP in their surveillance systems for the active detection of low-density infections and asymptomatic malaria cases.

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BACKGROUND: Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. METHODS: Overall, we recruited 1167 individuals from terrestrial ('road') and hydric ('riverine') communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. RESULTS: LAMP had a sensitivity of 91.8% (87.7-94.9) and specificity of 91.9% (87.8-95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12-24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. CONCLUSIONS: LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.

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Malaria remains endemic in 21 countries of the American continent with an estimated 427,000 cases per year. Approximately 10% of these occur in the Mesoamerican and Caribbean regions. During the last decade, malaria transmission in Mesoamerica showed a decrease of ~85%; whereas, in the Caribbean region, Hispaniola (comprising the Dominican Republic [DR] and Haiti) presented an overall rise in malaria transmission, primarily due to a steady increase in Haiti, while DR experienced a significant transmission decrease in this period. The significant malaria reduction observed recently in the region prompted the launch of an initiative for Malaria Elimination in Mesoamerica and Hispaniola (EMMIE) with the active involvement of the National Malaria Control Programs (NMCPs) of nine countries, the Regional Coordination Mechanism (RCM) for Mesoamerica, and the Council of Health Ministries of Central America and Dominican Republic (COMISCA). The EMMIE initiative is supported by the Global Fund for Aids, Tuberculosis and Malaria (GFATM) with active participation of multiple partners including Ministries of Health, bilateral and multilateral agencies, as well as research centers. EMMIE's main goal is to achieve elimination of malaria transmission in the region by 2020. Here we discuss the prospects, challenges, and research needs associated with this initiative that, if successful, could represent a paradigm for other malaria-affected regions.

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BACKGROUND: Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance. METHODOLOGY/PRINCIPAL FINDINGS: First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms. CONCLUSIONS/SIGNIFICANCE: mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.

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Las metacaspasas son proteasas de cisteína similares estructuralmente a las caspasas y cuyos genes están presentes en organismos carentes de caspasas, tales como plantas, levaduras y parásitos protozoarios. El bien conocido papel de varias caspasas en la apoptosis de células de mamíferos, ha motivado el estudio del posible rol de las metacaspasas en la muerte celular programada de los organismos en los cuales se expresan. Así, por ejemplo, las metacaspasas de los parásitos protozoarios humanos de los géneros Trypanosoma y Leishmania han sido implicadas en muerte celular programada. Sin embargo, algunos estudios en T. brucei señalan que estas proteínas podrían estar involucradas en otras funciones biológicas tales como procesos de reciclaje. Los parásitos del género Plasmodium también expresan metacaspasas, pero aún se sabe poco sobre su función. La presencia de más de una metacaspasa en algunos de estos microorganismos, ha llevado a especular que estas proteínas podrían tener funciones fisiológicamente redundantes o diferentes de acuerdo con su localización e interacción con otras moléculas. Los estudios recientes han demostrado que la especificidad catalítica de las metacaspasas difiere de aquélla de las caspasas. Esta característica, sumada a la ausencia de sus genes codificadores en el genoma de mamíferos, hace de las metacaspasas y de sus mecanismos de activación un área interesante de investigación en la identificación de nuevos blancos terapéuticos en los parásitos causantes de tripanosomiasis, leishmaniasis y malaria. La presente revisión resume la información disponible sobre las metacaspasas y busca motivar la generación de masa crítica en este tema en los países hispanohablantes.

Metacaspases are caspase-related cysteine-proteases that are present in organisms devoid of caspases such as plants, yeast, and protozoan parasites. Since caspases are important effector molecules in mammalian apoptosis, the possible role of metacaspases in programmed cell death has been evaluated in the organisms where they are expressed. In some species of the human protozoan parasites Trypanosoma spp. and Leishmania spp., metacaspases have been involved in programmed cell death, although a role of metacaspases in recycling processes in T. brucei has also been suggested. Metacaspases are also expressed in Plasmodium spp., however their role in these organisms is still unknown. More than one metacaspase gene is present in some of these parasites, which suggests that these proteins are physiologically redundant or have different functions depending on their localization and protein interactions. The catalytic activity of metacaspases is different from that of caspases—again noting that metacaspase genes are absent in mammals. These characteristics make metacaspases and their activating mechanisms interesting subjects of research in the development of new drug targets for the treatment of trypanosomiasis, leishmaniasis, and malaria. A summary of the literature on metacaspases is provided, and Latin American researchers are encouraged to more actively explore the metacaspase potential.

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Metacaspases are caspase-related cysteine-proteases that are present in organisms devoid of caspases such as plants, yeast, and protozoan parasites. Since caspases are important effector molecules in mammalian apoptosis, the possible role of metacaspases in programmed cell death has been evaluated in the organisms where they are expressed. In some species of the human protozoan parasites Trypanosoma spp. and Leishmania spp., metacaspases have been involved in programmed cell death, although a role of metacaspases in recycling processes in T. brucei has also been suggested. Metacaspases are also expressed in Plasmodium spp., however their role in these organisms is still unknown. More than one metacaspase gene is present in some of these parasites, which suggests that these proteins are physiologically redundant or have different functions depending on their localization and protein interactions. The catalytic activity of metacaspases is different from that of caspases-again noting that metacaspase genes are absent in mammals. These characteristics make metacaspases and their activating mechanisms interesting subjects of research in the development of new drug targets for the treatment of trypanosomiasis, leishmaniasis, and malaria. A summary of the literature on metacaspases is provided, and Latin American researchers are encouraged to more actively explore the metacaspase potential.

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INTRODUCTION: The current antimalarial drug policy in Colombia has been based on studies conducted in Antioquia and the Pacific Coast. However, the efficacy of antimalarial drugs in other endemic regions is unknown. OBJECTIVE: The therapeutic efficacy of three monotherapies was assessed: amodiaquine and sulfadoxine/pyrimethamine for uncomplicated Plasmodium falciparum malaria, and chloroquine for Plasmodium vivax malaria in the municipality of Tarapacá, located in the Colombian province of Amazonas. MATERIALS AND METHODS: Treatment was supervised and clinical/parasitological follow-up was undertaken through a 28-day period following to World Health Organization standard protocols for subjects with a single P. falciparum or P. vivax infection. RESULTS: Due to a decrease in malaria transmission at the time of the study, the sample size was very small. The treatment failed for two subjects who received amodiaquine, and treatment with sulfadoxine/pyrimethamine was discontinued due to a high frequency of therapeutic failures (7/8). Most subjects (18/20) with P. vivax infections showed an adequate therapeutic response. CONCLUSIONS: The use of sulfadoxine/pyrimethamine in Tarapacá, and possibly in the Amazon region of Colombia, needs to be reviewed. Therapeutic efficacy studies in other endemic areas in the Amazon and Orinoco regions in Colombia are desirable but not feasible. Alternative methods such as in vitro assays or detection of molecular markers for resistance in the parasite can provide a basis for decisions concerning antimalarial drug policy for the Amazon and Orinoco regions in Colombia.

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Introducción. La selección de los esquemas de tratamiento para malaria en Colombia se ha basado principalmente en estudios realizados en Antioquia y en la costa Pacífica, pero se desconoce la eficacia de los antimaláricos en la Orinoquia y Amazonia colombianas. Objetivo. Evaluar la eficacia terapéutica de tres monoterapias: amodiaquina y sulfadoxina/pirimetamina para el tratamiento de malaria no complicada por Plasmodium falciparum y de cloroquina para el tratamiento de malaria por Plasmodium vivax en Tarapacá, departamento del Amazonas. Materiales y métodos. Suministro de tratamiento supervisado y seguimiento clínico y parasitológico durante 28 días según protocolos estandarizados de sujetos con infección única por P. falciparum o P. vivax. Resultados. No fue posible completar el tamaño de muestra por disminución marcada en la transmisión. En el grupo de P. falciparum, el tratamiento con amodiaquina fracasó en dos sujetos; se suspendió el grupo de sulfadoxina/pirimetamina por alto número de fracasos terapéuticos (7/8). En el grupo de P. vivax, la mayoría (18/20) presentó respuesta adecuada al tratamiento. Conclusiones. Es necesario reconsiderar el uso de sulfadoxina/pirimetamina en Tarapacá y posiblemente en el Amazonas colombiano. Se requieren estudios de eficacia terapéutica en otras áreas endémicas, o la utilización de métodos in vitro o moleculares, para definir el esquema de tratamiento para malaria por P. falciparum en la Orinoquia y Amazonia colombianas.

Introduction. The current antimalarial drug policy in Colombia has been based on studies conducted in Antioquia and the Pacific Coast. However, the efficacy of antimalarial drugs in other endemic regions is unknown. Objective. The therapeutic efficacy of three monotherapies was assessed: amodiaquine and sulfadoxine/pyrimethamine for uncomplicated Plasmodium falciparum malaria, and chloroquine for Plasmodium vivax malaria in the municipality of Tarapacá, located in the Colombian province of Amazonas. Materials and methods. Treatment was supervised and clinical/parasitological follow-up was undertaken through a 28-day period following to World Health Organization standard protocols for subjects with a single P. falciparum or P. vivax infection. Results. Due to a decrease in malaria transmission at the time of the study, the sample size was very small. The treatment failed for two subjects who received amodiaquine, and treatment with sulfadoxine/pyrimethamine was discontinued due to a high frequency of therapeutic failures (7/8). Most subjects (18/20) with P. vivax infections showed an adequate therapeutic response. Conclusions. The use of sulfadoxine/pyrimethamine in Tarapacá, and possibly in the Amazon region of Colombia, needs to be reviewed. Therapeutic efficacy studies in other endemic areas in the Amazon and Orinoco regions in Colombia are desirable but not feasible. Alternative methods such as in vitro assays or detection of molecular markers for resistance in the parasite can provide a basis for decisions concerning antimalarial drug policy for the Amazon and Orinoco regions in Colombia.

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The resurgence and spread of antimalarial drug resistance is one of the causes of the worldwide increase of malaria. In Colombia, uncomplicated Plasmodium falciparum malaria has been treated with a combination of amodiaquine (AQ) and sulfadoxine/pyrimethamine (SP) since 2000. The efficacy of these two antimalarials was evaluated after the implementation of the new malaria treatment scheme. In the municipalities of El Charco and Tumaco (Nariño) on the Pacific Coast region, the standard PAHO protocol was used to evaluate antimalarial efficacy in areas of low to moderate malaria transmission. Patients were randomly allocated to treatment regime in two cities of Nariño, El Charco (n = 48) and Tumaco (n = 50). After 14 days none of El Charco patients presented therapeutic failure to either antimalarial. However, in Tumaco after 28 days, 12 of 24 (95% CI: 30.6-69.4) patients presented AQ treatment failure while 4 of 26 (95% CI: 5.1-33.1) patients had SP treatment failure. The high level of AQ treatment failure in Tumaco was unexpected because it had been introduced only recently as an antimalarial treatment in Colombia. The results suggest that the use of the current dose of AQ in combination with SP will be therapeutically useful for less time than expected. Use of combined therapies is a key strategy to delay antimalarial resistance. Unfortunately, its success depends on the efficacy of antimalarial drugs individually.

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La resistencia a los antimaláricos es una de las causas del aumento de casos de malaria en el mundo. Desde el año 2000, el tratamiento de malaria no complicada por Plasmodium falciparum en Colombia ha sido la combinación de amodiaquina (AQ) y sulfadoxina/pirimetamina (SP). La eficacia de estos dos medicamentos se evaluó después de la implementación del nuevo esquema. El estudio se realizó en los municipios de El Charco y Tumaco (Nariño) en la Costa Pacífica. Se utilizó el protocolo estándar de la OPS para la evaluación de la eficacia de antimaláricos en reas de baja a moderada transmisión. Los pacientes incluidos fueron asignados al azar a los dos medicamentos de estudio y seguidos hasta el día 14 en El Charco y hasta el día 28 en Tumaco. Ninguno de los 48 pacientes en El Charco presentó falla terapéutica a los medicamentos en estudio. En Tumaco, por el contrario, 12 de 24 pacientes (IC95 por ciento: 30,6 a 69,4) presentaron falla a la AQ y 4 de 26 (IC95 por ciento:5,1-33,1) presentaron falla a SP. Los altos niveles de falla a AQ en Tumaco fueron inesperados por su reciente introducción oficial al tratamiento de malaria, mientras que los niveles de falla a SP aumentaron respecto a lo encontrado en estudios anteriores. Estos hallazgos sugieren que el uso de AQ a las dosis actuales en combinación con SP tendrá un tiempo de vida útil más corto que el esperado. El uso combinado de antimaláricos como estrategia para retardar la aparición de resistencia ser efectivo en la medida en que las monoterapias sean eficaces.

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Polymorphisms in Plasmodium falciparum cg2 and pfcrt genes and their association with chloroquine resistance in vitro in Colombian parasites were evaluated in this study. Association of chloroquine resistance with resistance to other antimalarial drugs in vitro was also examined. Polymerase chain reactions (PCR) for kappa and omega cg2 regions and nested PCR and digestion with ApoI enzyme for K-76T pfcrt point mutation defined corresponding polymorphisms in 83 samples collected between 1995 and 1999. The isotopic microtest was used to evaluate sensitivity in vitro in a subgroup of 18 isolates. The predominant cg2 pattern observed was 13K/14omega repeats (46/83 [55.4%]) and all samples presented the K-76T mutant allele. Seventy-eight percent of samples were resistant to chloroquine in vitro, 35.3% to amodiaquine, 16.7% to mefloquine, and 5.6% to quinine. Significant correlations (P < 0.05) were observed between the IC50s of chloroquine and arteether, and among IC50s of arteether, mefloquine, and quinine. These results suggest the development of multiple and cross-resistance of Colombian P. falciparum isolates to second- and third-line antimalarials and new alternative drugs.

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Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL(R), is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL(R) in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL(R) for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL(R) in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.

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Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL®, is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL® in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL® for both sets of patients was 96.9 percent. Efficiency was higher for P. vivax (98.1 percent) than for P. falciparum (94.9 percent). These results corroborate the diagnostic utility of OptiMAL® in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility

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