Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-3 Alpha and Beta from Diverse Geographic Areas of Thailand.

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.

BackgroundMalaria is a significant public health concern in Haiti where approximately 30,000 cases are reported annually with CDC estimates as high as 200,000. Malaria infections in Haiti are caused almost exclusively by Plasmodium falciparum, while a small number of Plasmodium malariae and an even smaller number of putative Plasmodium vivax infections have been reported. The lack of confirmed P. vivax infections in Haiti could be due to the genetic background of native Haitians. Having descended from West African populations, many Haitians could be Duffy negative due to a single nucleotide polymorphism from thymine to cytosine in the GATA box of the promoter region of the Duffy antigen receptor for chemokines (DARC) gene. This mutation, encoded by the FYES allele, eliminates the expression of the Duffy antigen on erythrocytes, which reduces invasion by P. vivax. This study investigated the frequency of the FYES allele and P. vivax infections in malaria patients with the goal of uncovering factors for the lack of P. vivax infections reported in Haiti.MethodsDNA was extracted from dried blood spots collected from malaria patients at four clinic locations in Haiti. The samples were analysed by polymerase chain reaction (PCR) for the presence of the P. vivax small subunit ribosomal RNA gene. PCR, sequencing, and restriction enzyme digestion were used to detect the presence of the FYES allele. Matched samples were examined for both presence of P. vivax and the FYES allele.ResultsNo cases of P. vivax were detected in any of the samples (0/136). Of all samples tested for the FYES allele, 99.4% had the FYES allele (163/164). Of the matched samples, 99% had the FYES allele (98/99).ConclusionsIn this preliminary study, no cases of P. vivax were confirmed by PCR and 99% of the malaria patients tested carried the FYES allele. The high frequency of the FYES allele that silences erythroid expression of the Duffy antigen offers a biologically plausible explanation for the lack of P. vivax infections observed. These results provide insights on the host susceptibility for P. vivax infections that has never before been investigated in Haiti.

Genetic diversity of Merozoite surface protein 1-42 (MSP1-42) fragment of Plasmodium vivax from Indonesian isolates: Rationale implementation of candidate MSP1 vaccine.

Genetic diversity and molecular evolution of Plasmodium vivax Duffy Binding Protein and Merozoite Surface Protein-1 in northwestern Thailand.

Cerebral malaria (CM) is a diffuse encephalopathy associated with coma and seizures commonly caused by Plasmodium falciparum (P. falciparum) in children with severe malaria. We present a case of a 19-year-old man with CM due to Plasmodium vivax (P. vivax) infection. Cerebrospinal fluid (CSF) was negative for Japanese B encephalitis, enterovirus, herpes simplex 1 and 2, varicella and mumps viruses as determined by real-time polymerase chain reaction (PCR). P. falciparum and P. vivax species were analysed by microscopy, immunochromatography and PCR assays and confirmed mono-infection of P. vivax in the patient's blood, and P. falciparum infection was established to be negative. The patient was discharged after intensive supportive care and antimalarial treatment (intravenous artesunate and oral doxycycline). We conclude that P. vivax infection is associated with CM, a life-threatening complication rarely seen in coastal districts of Karnataka. In endemic areas, the possibility of CM should be considered even with P. vivax infection.

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996-2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.

BackgroundPlasmodium falciparum and Plasmodium vivax infections compromise dendritic cell (DC) function and expand regulatory T (Treg) cells in both clinical disease (malaria) and experimental human sub-microscopic infection. Conversely, in asymptomatic microscopy-positive (patent) P. falciparum or P. vivax infection in endemic areas, blood DC increase or retain HLA-DR expression and Treg cells exhibit reduced activation, suggesting that DC and Treg cells contribute to the control of patent asymptomatic infection. The effect of sub-microscopic (sub-patent) asymptomatic Plasmodium infection on DC and Treg cells in malaria-endemic area residents remains unclear.MethodsIn a cross-sectional household survey conducted in Papua, Indonesia, 162 asymptomatic adults were prospectively evaluated for DC and Treg cells using field-based flow cytometry. Of these, 161 individuals (99 %) were assessed retrospectively by polymerase chain reaction (PCR), 19 of whom had sub-microscopic infection with P. falciparum and 15 with sub-microscopic P. vivax infection. Flow cytometric data were re-analysed after re-grouping asymptomatic individuals according to PCR results into negative controls, sub-microscopic and microscopic parasitaemia to examine DC and Treg cell phenotype in sub-microscopic infection.ResultsAsymptomatic adults with sub-microscopic P. falciparum or P. vivax infection had DC HLA-DR expression and Treg cell activation comparable to PCR-negative controls. Sub-microscopic P. falciparum infection was associated with lower peripheral CD4+ T cells and lymphocytes, however sub-microscopic Plasmodium infection had no apparent effect on DC sub-set number or Treg cell frequency.ConclusionsIn contrast to the impairment of DC maturation/function and the activation of Treg cells seen with sub-microscopic parasitaemia in primary experimental human Plasmodium infection, no phenotypic evidence of dysregulation of DC and Treg cells was observed in asymptomatic sub-microscopic Plasmodium infection in Indonesian adults. This is consistent with DC and Treg cells retaining their functional capacity in sub-microscopic asymptomatic infection with P. falciparum or P. vivax in malaria-endemic areas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1382-7) contains supplementary material, which is available to authorized users.

Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country. Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR). samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively. Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.

Short tandem repeats (STR) typing is an essential analysis method for human identification in forensic field. When DNAs obtained from the field as evidences are severely degraded or in too small amounts, STR analysis often shows allele drop-out. To improve STR analysis for degraded DNA or trace DNA, reduced-size STR (rSTR) polymerase chain reaction (PCR) system was devised by selecting relatively large-size STR loci. The rSTR PCR system consisted of 8 loci (amelogenin, SE33, CSF1PO, D7S820, D13S317, D2S1338, TPOX, and FGA). The size of PCR product was reduced by designing new primers in the flanking region. The efficiency of this system was verified against existing kits through concordance study, sensitivity study, efficiency study, and casework sample study. The size of PCR product in the rSTR PCR system was reduced to be less than 322bp. The amplicon of each locus was reduced by about 100bp on average. Results of this rSTR PCR system were confirmed using 146 Korean samples and other commercial kits. The rSTR PCR system was capable of analyzing DNA samples with a minimum amount of DNA of 16pg and a degradation index of 4.215. The rSTR PCR system was more effective than other PCR kits for obtaining genetic profiles from a small amount of DNA or degraded DNA. The combination of this new system and other commercial kits is more effective than existing systems. This combination is expected to be helpful for the identification of unidentified bodies and skeletal samples.

BackgroundMalaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such as microscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and Plasmodium falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia.MethodsThree hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr–Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodium species and parasitaemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analysed using Fisher’s exact test and kappa statistics.ResultsThe Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated with Plasmodium infection (P < 0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections. Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitaemia.ConclusionsThe present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitaemia. In this study, an improved molecular diagnostic tool was used to detect and characterize Plasmodium infections, with the goal of quantifying P. vivax infection in Duffy-negative individuals. Advanced molecular diagnostic techniques, such as multiplex real-time PCR, loop-mediated isothermal amplification (LAMP), and CRISPR-based diagnostic methods. These techniques offer increased sensitivity, specificity, and the ability to detect low-parasite-density infections compared to the employed methodologies.

The Philippines is targeting malaria elimination by 2020. To reach this goal, it is important to locate all residual foci of malaria and where possible, aggressively diagnose and treat every malaria infection. In low endemic provinces malaria transmission becomes focal in hard-to-reach areas where asymptomatic people do not actively seek treatment and thus, continue to perpetuate transmission. This study aimed (a) to estimate prevalence of Plasmodium species in three malaria-endemic provinces in Mindanao, (b) to measure malaria transmission intensity in these provinces using antibody markers of exposure to P. falciparum and P. vivax AMA-1 and MSP-119 antigens, (c) to determine polymorphisms in pfcrt, pfmdr1 and pvmdr1 genes, and (d) to discuss implications of these findings to malaria elimination in Mindanao. Cross-sectional surveys were conducted to a total of 2,628 consenting participants across all ages in the provinces of Sarangani, South Cotabato and Tawi-Tawi from 2010 to 2013. The RDT FalciVax™ was used for field diagnosis of malaria in Sarangani Province and South Cotabato Province for P. falciparum and P. vivax infection while microscopy was used in Tawi-Tawi Province for field diagnosis of malaria. Finger-prick blood spots on filter paper were collected from participants for PCR diagnosis, genotyping of pfcrt, pfmdr1 and pvmdr1 genes, and screening antibodies to P. falciparum and P. vivax AMA-1 and MSP-119 antigens using indirect ELISA. Blood spots were also collected from patients presenting with malaria symptoms from selected municipalities of Sarangani Province and South Cotabato Province as a pilot survey. Overall malaria prevalence by PCR was 3.7% in Sarangani Province, 10% in South Cotabato Province and 4.2% in Tawi-Tawi Province. P. falciparum prevalence by PCR was higher than P. vivax prevalence in Sarangani Province and Tawi-Tawi Province but the opposite was found in South Cotabato Province. There was one imported case of P. malariae in South Cotabato and there were no P. knowlesi and P. ovale infections found in the three provinces surveyed. There were disagreements in diagnosing P. falciparum and P. vivax using antigen detection, microscopy and PCR and these were attributed to sampling low parasite-density infections from small volume of peripheral blood spotted on filter paper. The pfcrt codons 72-76 haplotypes CVMNK (27.4%), CVIET (59.7%) and SVMNT (9.7%) were described in 62 P. falciparum isolates from Mindanao. The pfcrt mutant A144T and L160Y alleles were not found among P. falciparum isolates with pfcrt K76T mutant allele but lacked the pfcrt A220S mutation. The pfmdr1 86N- 184F-1034S-1042N-1246D haplotype, which was repeatedly associated with higher parasite survival following artemether-lumefantrine treatment, was found in seven P. falciparum isolates from Mindanao. Genotyped P. vivax isolates from Mindanao have the wild type pvmdr1 91N allele, which corresponded to pfmdr1 codon 86. The pvmdr1 Y976F mutant allele, which has been reported in chloroquine-resistant P. vivax in other countries, was found in 55.6% (5/9) P. vivax isolates successfully genotyped in this codon while the pvmdr1 1076L wild-type allele was found in three P. vivax isolates successfully genotyped in this codon. Combined seroprevalence to P. falciparum and P. vivax AMA-1 and MSP-119 antigens suggested that exposure to P. falciparum was higher than exposure to P. vivax in Sarangani Province and Tawi-Tawi Province. Overall seroprevalence to P. falciparum and P. vivax was 18.9% and 14.6% in Sarangani Province respectively. In Tawi-Tawi Province the overall seroprevalence to P. falciparum and P. vivax was 18.2% and 12.9% respectively. The opposite was observed in South Cotabato Province where overall seroprevalence to P. falciparum (3.4%) was lower than the overall seroprevalence to P. vivax. The seroconversion rates (λ) for P. falciparum and P. vivax malaria were estimated using simple reversible catalytic models. In Sarangani Province the SCR for P. falciparum (0.014, 95%CI 0.010-0.020) was lower than SCR for P. vivax (0.019, 95% CI 0.010-0.036). A model allowing two forces of infection was used to estimate SCR for P. falciparum in Tawi-Tawi. Results suggested that there was a change in P. falciparum transmission in Tawi-Tawi Province approximately 25 years before the survey was conducted. The estimated SCR for P. falciparum was 0.041 (95% CI 0.017-0.098) in Tawi-Tawi Province before 1987. The model suggested that SCR was reduced to 0.007 (95% CI 0.005-0.009) after 1987 to the time of survey. In South Cotabato the SCR for P. falciparum was very low (0.004, 95% CI 0.001-0.016). There was no SCR estimated for P. vivax in South Cotabato because seropositivity was equally distributed across age groups. Findings in this study were held back by sample size and low-density parasite infections in small number of infected humans. Nevertheless, this provided important baseline data for malaria epidemiology in Mindanao.

Genetic diversity and distribution patterns of PfEMP1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

BackgroundIn malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation.ResultsFor the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses.ConclusionThe statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

Although Plasmodium vivax has been assumed to be absent from sub-Saharan Africa because of the protective mutation conferring the Duffy-negative phenotype, recent evidence has suggested that P. vivax cases are prevalent in these regions. We selected 292 dried blood spots from children who participated in the 2013-2014 Demographic and Health Survey of the Democratic Republic of the Congo (DRC), to assess for P. vivax infection. Four P. vivax infections were identified by polymerase chain reaction, each in a geographically different survey cluster. Using these as index cases, we tested the remaining 73 samples from the four clusters. With this approach, 10 confirmed cases, three probable cases, and one possible case of P. vivax were identified. Among the 14 P. vivax cases, nine were coinfected with Plasmodium falciparum. All 14 individuals were confirmed to be Duffy-negative by sequencing for the single point mutation in the GATA motif that represses the expression of the Duffy antigen. This finding is consistent with a growing body of literature that suggests that P. vivax can infect Duffy-negative individuals in Africa. Future molecular and sequencing work is needed to understand the relationship of these isolates with other P. vivax samples from Asia and South America and discover variants linked to P. vivax virulence and erythrocyte invasion.

Frequent T and B Cell Oligoclones in Histologically and Immunophenotypically Characterized Angioimmunoblastic Lymphadenopathy