Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis.

The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring less genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to changes in drug regime. Using newly sequenced whole genomes, we measure relatedness between 166 parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore past selection events in the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in small populations, as will occur in any region nearing malaria elimination.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept.

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.

Detection of Leishmania RNA Virus in Clinical Samples from Cutaneous Leishmaniasis Patients Varies according to the Type of Sample.

Leishmania RNA virus (LRV) is a double-stranded RNA virus belonging to the Totiviridae family detected as cytoplasmic inclusions in some strains of the human parasite Leishmania spp. Experimental evidence supports the hypothesis that human coinfection with Leishmania spp.-LRV triggers an exacerbated immune response in the host that can be responsible for the observed complicated outcomes in cutaneous leishmaniasis (CL), such as mucosal leishmaniasis (ML) and treatment failure of CL. However, the reported frequencies of LRV associated with complicated outcomes in patient's series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. To assess whether or not the inconsistent information about the frequency of LRV associated with CL complicated outcomes could be related to the virus detection approach, the present study evaluated the LRV presence in clinical samples using a diagnostic algorithm according to the type of the sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and six samples with non-Leishmania spp. infection, the LRV presence was assessed by RT-PCR, RT-qPCR, and nested RT-PCR. Viral load was estimated in parasite clinical isolates. By combining the methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 37.5% (6/16) of the incisional biopsies. Remarkably, in some cases (4/8), LRV1 was undetectable in the isolates but present in their respective biopsies, and less frequently, the opposite was observed (1/8), suggesting the possibility of loss of parasites harboring LRV1 during the in vitro growth.

Spatio-temporal dynamics of Plasmodium falciparum transmission within a spatial unit on the Colombian Pacific Coast.

As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests.

In vitro susceptibility of P. falciparum populations from Colombia and Tanzania to a new synthetic peroxide (OZ277).

Sensitivity of Plasmodium falciparum populations from Colombia (N = 38) and Tanzania (N = 45) to the newly developed, fully synthetic peroxide OZ277 was investigated using a standard isotopic microtest. OZ277 showed excellent activity against chloroquine-resistant isolates in Colombia with median IC(50) [range] values of 2.5 ng/mL [0.34-8] (4.4 nM [0.6-14]) and Tanzania with 1.5 ng/mL [0.22-10] (2.65 nM [0.4-17.7]). The potency of OZ277 was similar to artesunate, showing median IC(50) values of 1.5 ng/mL [0.42-8.6] (3.8 nM [1.1-22.3]) and 1.8 ng/mL [0.2-10] (4.7 nM [0.5-26.04]) in Colombia and Tanzania, respectively. These results support the development of this new antimalarial compound.

Baseline in vivo, ex vivo and molecular responses of Plasmodium falciparum to artemether and lumefantrine in three endemic zones for malaria in Colombia.

Background: Colombia began using artemisinin-based combination therapies for the treatment of uncomplicated Plasmodium falciparum malaria in 2006. It is necessary to implement resistance surveillance to antimalarial drugs in order to promptly detect changes in parasite susceptibility. The aim of this study was to establish a susceptibility baseline of P. falciparum to artemether-lumefantrine using three monitoring tools. Methods: Patients with uncomplicated malaria treated with artemether-lumefantrine underwent clinical and parasitological follow-up over 28 days. Ex vivo test was performed using the microtest technique for chloroquine, arthemeter, dihydroartemisinin and lumefantrine. Pfmdr1 copy number and polymorphisms in Pfk13, Pfatp6, Pfcrt and Pfmdr1 genes were analyzed. Results: From a total of 150 screened patients, 49 completed follow-up for 28 days. All treated patients had adequate clinical and parasitological responses. Parasitic clearance showed a drastic reduction of parasite biomass at 24 hours and complete elimination at 48 hours. One hundred eleven isolates were processed, all exhibited high susceptibility to artemisinins and a slight decrease in susceptibility to lumefantrine. No genetic polymorphisms associated with resistance to artemisinin were found. Conclusion: This study generated a susceptibility baseline in response to therapy with Coartem (artemether-lumefantrine) with numerical reference values, which will allow data comparison with future studies to systematically monitor changes in the parasite and to provide an early alert to the health authorities.

Differentiation of an adult neuron cell line increases susceptibility to rabies infection.

A wide variety of in vitro models have been used for studying rabies infection, however, currently, no central nervous system (CNS) adult neuron cultures are available. The current study determined the susceptibility to rabies infection in an adult CNS neuron cell line (CAD-R1). Cultures of CAD-R1 cells were held for 5 days in medium containing serum (undifferentiated CAD-R1 cells) or in serum-free medium (differentiated CAD-R1 cells). They were then infected with highly neurotropic rabies virus (RV) strain (CVS), obtained from fibroblastic cells (CVS-BHK) or from adult mouse brain (CVS-MB). Undifferentiated and differentiated cells were infected with the two RV strains, but the percentage of infected cells in differentiated cultures was significantly greater (83% and 79%, respectively) than in undifferentiated cells (51% and 60%) (Student's t test<0.05). Susceptibility to infection apparently depended on cellular differentiation state, possibly due to acquisition of additional morphological and biochemical characteristics during the differentiation process that made them more susceptible to RV infection. Therefore, CAD R1 cells may represent a good model for RV infection, making them a useful tool for studying RV neurotropism, infection pathogeny, isolation of street virus or producing safer and most potent vaccines.

[Field evaluation for diagnostic accuracy of the rapid test SD Bioline Malaria Antigen Pf/Pv® in Colombia]. / Evaluación de campo de la precisión de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia.

INTRODUCTION: Rapid diagnostic tests (RDT) have been postulated as a way to ensure access to malaria diagnosis in remote areas. Despite its widespread use, there are no field studies to evaluate the accuracy of the SD Bioline Malaria Antigen Pf/Pv in Colombia RDT. OBJECTIVE: To evaluate the diagnostic accuracy of the SD Bioline Malaria Antigen Pf/Pv® RDT in two departments endemic for malaria, comparing diagnosis with thick film corrected with PCR. MATERIALS AND METHODS: A retrospective study was carried out to evaluate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), concordance and sensitivity limits according to parasitemia ranges for the SD Bioline Malaria Antigen Pf/Pv ® test in Cordoba and Choco. The results were compared with microscopy corrected by PCR. RESULTS: A total of 383 samples processed, 121 were positive (75 for P. vivax , 42 for P. falciparum and 4 for mixed infection) and 262 negative samples. P. vivax: sensitivity 92.0% (95% CI: 83.6-96.3), specificity 98.7% ( 95% CI: 96.7-99.5), PPV 94.5% (95% CI: 86.7-97.9), NPV 98.1% (95% CI: 95.8-99.1), Cohen's kappa coefficient was 0.90 (0.80-1.00). P. falciparum: sensitivity 88.1% (95% CI: 75.0-94.8), specificity 97.9% (95% CI: 95.8-99.0), PPV 84.1% (95% CI: 70.6-92.1), NPV 98.5% (95% IC: 96.6-99.4), Cohen's kappa coefficient 0.80 (95% CI: 0.70-0.90). CONCLUSIONS: The test performed well, being better for P. vivax as compared to P. falciparum. There are still difficulties of RDT to detect low parasitemias. The non amplification of Pfhrp2 and Pfhrp3 genes in two samples diagnosed as mixed infection, suggest a possible deletion of these two genes together.

Evaluación de campo de la precisión de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia / Field evaluation for diagnostic accuracy of the rapid test SD Bioline Malaria Antigen Pf/Pv® in Colombia

Introducción. Las pruebas de diagnóstico rápido han sido postuladas como una forma de garantizar el diagnóstico de malaria, o paludismo, en zonas de difícil acceso. A pesar de su uso difundido, no hay estudios de campo que evalúen la precisión de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia. Objetivo. Evaluar la precisión diagnóstica de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv ®, en dos departamentos endémicos para malaria, comparando el diagnóstico con la gota gruesa corregida por reacción en cadena de la polimerasa (PCR). Materiales y métodos. Se trata de un estudio retrospectivo para evaluar sensibilidad, especificidad, valor diagnóstico positivo (VPP) y negativo (VPN), concordancia y límites de sensibilidad por rangos de parasitemia, de la prueba SD Bioline Malaria Antigen ® Pf/Pv, en Córdoba y Chocó. Los resultados fueron comparados con la gota gruesa corregida por PCR. Resultados. De 383 muestras procesadas, 121 fueron positivas (75 para Plasmodium vivax, 42 para P. falciparum y 4 para infección mixta) y 262 muestras negativas; los resultados obtenidos fueron los siguientes: P. vivax: sensibilidad, 92,0 % (IC 95% 83,6-96,3); especificidad, 98,7 % (IC 95% 96,7-99,5); VPP, 94,5 % (IC 95% 86,7-97,9); VPN, 98,1 % (IC 95% 95,8-99,1); IK, 0,90 (0,80-1,00). P. falciparum: sensibilidad, 88,1 % (IC 95% 75,0-94,8); especificidad, 97,9 % (IC 95% 95,8-99,0); VPP, 84,1% % (IC 95% 70,6-92,1); VPN, 98,5 % (IC 95% 96,6-99,4); IK, 0,80 (0,70-0,90). Conclusiones. La prueba tuvo un buen desempeño, siendo mejor para P. vivax en comparación con que para P. falciparum. Persisten dificultades en la detección de bajas parasitemias. La falta de amplificación de los genes Pfhrp2 y Pfhrp3 en dos muestras con diagnóstico de como infección mixta, sugiere una posible deleción conjunta de estos genes.

Introduction: Rapid diagnostic tests (RDT) have been postulated as a way to ensure access to malaria diagnosis in remote areas. Despite its widespread use, there are no field studies to evaluate the accuracy of the SD Bioline Malaria Antigen Pf/Pv in Colombia RDT. Objective: To evaluate the diagnostic accuracy of the SD Bioline Malaria Antigen Pf/Pv® RDT in two departments endemic for malaria, comparing diagnosis with thick film corrected with PCR. Materials and methods: A retrospective study was carried out to evaluate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), concordance and sensitivity limits according to parasitemia ranges for the SD Bioline Malaria Antigen Pf/Pv ® test in Cordoba and Choco. The results were compared with microscopy corrected by PCR. Results: A total of 383 samples processed, 121 were positive (75 for P. vivax , 42 for P. falciparum and 4 for mixed infection) and 262 negative samples. P. vivax: sensitivity 92.0% (95% CI: 83.6-96.3), specificity 98.7% ( 95% CI: 96.7-99.5), PPV 94.5% (95% CI: 86.7-97.9), NPV 98.1% (95% CI: 95.8-99.1), Cohen´s kappa coefficient was 0.90 (0.80-1.00). P. falciparum: sensitivity 88.1% (95% CI: 75.0-94.8), specificity 97.9% (95% CI: 95.8-99.0), PPV 84.1% (95% CI: 70.6-92.1), NPV 98.5% (95% IC: 96.6-99.4), Cohen´s kappa coefficient 0.80 (95% CI: 0.70-0.90). Conclusions: The test performed well, being better for P. vivax as compared to P. falciparum. There are still difficulties of RDT to detect low parasitemias. The non amplification of Pfhrp2 and Pfhrp3 genes in two samples diagnosed as mixed infection, suggest a possible deletion of these two genes together.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.