Vivax Parasites Research Articles

An Uncommon Case of Plasmodium vivax Malaria with Disseminated Intravascular Coagulation: Rare Case Report with Review of Literature.

Malaria is a severe health issue in India despite government efforts. Malaria has many complications, some of which can be life-threatening. Disseminated intravascular coagulation (DIC) is a prominent malaria complication, besides renal failure, cerebral malaria, pulmonary edema, and anemia. We report a young man with Plasmodium vivax malaria who developed septic shock due to DIC. A 25-year-old male had a fever, abdominal pain, vomiting, and nausea for 5 days. On clinical examination, the patient was in distress and had a fever of 102.6°F with a blood pressure of 86/46 mmHg. A complete blood count revealed anemia, leukocytopenia, and severe thrombocytopenia. Peripheral blood smear findings showed a P. vivax parasite in red blood cells with the presence of a few schistocytes. Further coagulation tests showed raised prothrombin, partial thromboplastin time, and d-dimer. DIC and septic shock required critical care unit admission. Conservative treatment with artesunate and lumefantrine completely cured him. This unusual case shows that P. vivax causes severe malaria and its morbid consequences. It stresses early diagnosis and treatment to prevent mortality and morbidity.

First Report of Trypanosoma vivax (Duttonella), Babesia bovis and Babesia bigemina DNA in Cattle from the Galapagos Islands, Ecuador, and Its Relationship with Anaplasma marginale.

Bovine trypanosomoses, caused by Trypanosoma vivax, is a disease present in African and South American countries. This haemoflagellate protozoan parasite, as well as Anaplasma marginale and Babesia spp., are microorganisms that have a blood tropism, mainly causing fever and anaemia, which reduces the productive capacity of dairy or meat farms. This study aimed to detect T. vivax and other blood parasites in bovine herds in the Galapagos Islands. A total of 170 blood samples from bovines in 19 farms on Santa Cruz Island (the most populated) were collected and analyzed using different PCR techniques: Da-PCR and CatL-PCR to detect Trypanosoma vivax, CatL-PCR to detect Trypanosoma theileri, ESAG-PCR to detect Trypanosoma evansi, 18S rRNA-PCR to detect Babesia spp., rap-1-PCR to detect Babesia bovis, hyp-PCR to detect Babesia bigemina, and msp5-PCR to detect A. marginale. The prevalence of T. vivax, B. bovis, B. bigemina, and A. marginale was estimated as 14.7%, 11.2%, 14.7%, and 67.1%, respectively. In this study, the presence of four haemotropic agents was evidenced in 26.3% (5/19) of the farms. Coinfected cattle (A. marginale, B. bovis and B. bigemina) had significantly higher body temperatures compared to others (two-sample Wilcoxon rank-sum test; p-value = 0.047). The molecular techniques used in this study demonstrated the presence of T. vivax and B. bovis in cattle from Santa Cruz Island in the Galapagos for the first time. The study also investigates the relationship between T. vivax, A. marginale and Babesia spp., making a significant contribution to the field of veterinary medicine.

Exploring Quinoline Derivatives: Their Antimalarial Efficacy and Structural Features

Objectives: Malaria continues to be the primary cause of mortality worldwide, and timely recognition and prompt intervention are crucial in mitigating adverse consequences. This review article aims to examine the effectiveness and structural characteristics of quinoline-based compounds as antimalarial agents. It specifically focuses on their therapeutic effects as well as potential prospects for exploring structure-activity relationship (SAR). In addition, this study aims to identify lead compounds that can efficiently battle multidrug-resistant forms of Plasmodium falciparum and Plasmodium vivax Methods: A comprehensive review was conducted to evaluate the effectiveness of quinoline-based antimalarial medications in eradicating P. falciparum and P. vivax. The mechanism of action and SAR of these compounds were analyzed Results: Quinoline-based antimalarials demonstrated significant effectiveness in eliminating P. falciparum parasites, particularly in regions severely impacted by malaria, including Africa and Asia. These compounds were found to exhibit tolerance and immune-modulating properties, indicating their potential for more widespread utilization. The investigation identified various new quinoline compounds with improved antimalarial activity, including metal-chloroquine complexes, diaminealkyne chloroquines, and cinnamoylated chloroquine hybrids. This study explored different mechanisms by which these compounds interact with parasites, including their ability to accumulate in the parasite’s acidic food vacuoles and disrupt heme detoxification. The derivatives demonstrated strong efficacy against chloroquine-resistant strains and yielded positive results. Conclusion: Quinoline-based compounds represent a promising avenue for combating malaria due to their demonstrated efficacy against P. falciparum and P. vivax parasites. Further research on their mechanisms of action and SAR could lead to the development of more effective antimalarial medications.

Cell Death of P. vivax Blood Stages Occurs in Absence of Classical Apoptotic Events and Induces Eryptosis of Parasitized Host Cells.

Elucidation of pathways regulating parasite cell death is believed to contribute to identification of novel therapeutic targets for protozoan diseases, and in this context, apoptosis-like cell death has been reported in different groups of protozoa, in which metacaspases seem to play a role. In the genus Plasmodium, apoptotic markers have been detected in P. falciparum and P. berghei, and no study focusing on P. vivax cell death has been reported so far. In the present study, we investigated the susceptibility of P. vivax to undergo apoptotic cell death after incubating mature trophozoites with the classical apoptosis inducer staurosporine. As assessed by flow cytometry assays, staurosporine inhibited parasite intraerythrocytic development, which was accompanied by a decrease in cell viability, evidenced by reduced plasmodial mitochondrial activity. However, typical signs of apoptosis, such as DNA fragmentation, chromatin condensation, and nuclear segregation, were not detected in the parasites induced to cell death, and no significant alteration in metacaspase gene expression (PvMCA1) was observed under cell death stimulus. Interestingly, dying parasites positively modulated cell death (eryptosis) of host erythrocytes, which was marked by externalization of phosphatidylserine and cell shrinkage. Our study shows for the time that P. vivax blood stages may not be susceptible to apoptosis-like processes, while they could trigger eryptosis of parasitized cells by undergoing cell death. Further studies are required to elucidate the cellular machinery involved in cell death of P. vivax parasites as well as in the modulation of host cell death.

Plasmodium vivax populations in the western Greater Mekong Subregion evaluated using a genetic barcode.

An improved understanding of the Plasmodium vivax populations in the Great Mekong Subregion (GMS) is needed to monitor the progress of malaria elimination. This study aimed to use a P. vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics and explore the gene flow among P. vivax parasite populations in the western GMS (China, Myanmar and Thailand). A total of 315 P. vivax patient samples collected in 2011 and 2018 from four regions of the western GMS were genotyped for 42 SNPs using the high-throughput MassARRAY SNP genotyping technology. Population genetic analysis was conducted to estimate the genetic diversity, effective population size, and population structure among the P. vivax populations. Overall, 291 samples were successfully genotyped at 39 SNPs. A significant difference was observed in the proportion of polyclonal infections among the five P. vivax populations (P = 0.0012, Pearson Chi-square test, χ2 = 18.1), with western Myanmar having the highest proportion (96.2%, 50/52) in 2018. Likewise, the average complexity of infection was also highest in western Myanmar (1.31) and lowest in northeast Myanmar (1.01) in 2018. The older samples from western China in 2011 had the highest pairwise nucleotide diversity (π, 0.388 ± 0.046), expected heterozygosity (He, 0.363 ± 0.02), and the largest effective population size. In comparison, in the neighboring northeast Myanmar, the more recent samples in 2018 showed the lowest values (π, 0.224 ± 0.036; He, 0.220 ± 0.026). Furthermore, the 2018 northeast Myanmar parasites showed high and moderate genetic differentiation from other populations with FST values of 0.162-0.252, whereas genetic differentiation among other populations was relatively low (FST ≤ 0.059). Principal component analysis, phylogeny, and STRUCTURE analysis showed that the P. vivax population in northeast Myanmar in 2018 substantially diverged from other populations. Although the 42 SNP barcode is a valuable tool for tracking parasite origins of worldwide parasite populations, a more extended barcode with additional SNPs is needed to distinguish the more related parasite populations in the western GMS.

Genetic diversity of the PvMSP-3α gene in Plasmodium vivax isolates circulating in the National Capital Region (NCR) of India

Malaria is still a public health problem in tropical countries like India; major malaria parasite species are Plasmodium falciparum and P. vivax. Of which, P. vivax is responsible for ∼40% of the malaria burden at least in the Indian scenario. Unfortunately, there is limited data on the population structure and genetic diversity of P. vivax parasites in India. In this study, we investigated the genetic diversity of P. vivax strains in the South-west district, Delhi and, Nuh district, Haryana [National Capital Region (NCR)], using a polymorphic marker- P. vivax merozoite surface protein-3α (PvMSP-3α) gene. Dried blood spots from microscopically confirmed P. vivax patients were used for investigation of the PvMSP-3α gene. PCR-RFLP was performed on the PvMSP-3α gene to investigate the genotypes and allelic variability with HhaI and AluI restriction enzymes. In total, 40 successfully PCR amplified PvMSP-3α gene segments were subjected to RFLP analysis. Amplified products showed three different base pair size variations viz. genotype A in 31(77.5%), genotype B in 4(10%) and genotype C in 5(12.5%) P. vivax specimens. RFLP with HhaI and AluI revealed 17 (H1-H17) and 25 (A1-A25) allelic variants, respectively. Interestingly, two similar sub-allelic variants, ie. H8 (with HhaI), and A4 (with AluI) clustered within the rural area of Nuh district, Haryana in two samples. With this study, we propose to commission such type of genetic diversity analysis of P. vivax to investigate the circulating genotypes of the parasites from distinct geographical locations across India, that can have significant implications in understanding the population structures of P. vivax.

A SYSTEMATIC REVIEW ON MALARIA DISEASE AND ITS TREATMENTS FOCUS ON ARTEMETHER DRUG

Malaria is one of the infectious illnesses of the highest scientific significance and importance to international health organizations. Plasmodium falciparum, the parasite responsible for a highly severe form of the disease in Africa, has traditionally received the most attention. However, in the last two decades, the Plasmodium vivax parasite, which is linked to a large number of cases in Latin America, the Middle East, South and Southeast Asia, the Horn of Africa, and Oceania, has sparked enormous interest, owing to published evidence that it can cause severe malaria, among other things. Malaria is a worldwide public health problem, with a saw 247 million cases recorded in 2021. African countries accounted for approximately 94% of all reported cases. So far, over 200 distinct varieties of protozoa have been discovered and identified, with at least 13 of them being pathogenic to humans. The malaria parasite's life cycle is a complex process involving a mosquito with the species Anopheles and a vertebrate host.
 Artemether is a lipid-soluble artemisinin derivative. It is available in both oral and intramuscular forms. It is also available as a fixed-dose formulation with lumefantrine. Artemether-lumefantrine is one of the ACTs authorized by the WHO and is being used by the majority of countries transitioning from less effective medications to ACTs. However, the emergence of antimalarial medication resistance poses a significant challenge to malaria control. It works by killing the pathogenic organisms responsible for malaria. It is known as an antimalarial medication, and it is more effective than quinine therapies.

Dynamics of asymptomatic Plasmodium falciparum and Plasmodium vivax infections and their infectiousness to mosquitoes in a low transmission setting of Ethiopia: a longitudinal observational study

ObjectiveA 15-month longitudinal study was conducted to determine the duration and infectivity of asymptomatic qPCR-detected Plasmodium falciparum and Plasmodium vivax infections in Ethiopia. MethodTotal parasite and gametocyte kinetics were determined by molecular methods; infectivity to Anopheles arabiensis mosquitoes by repeated membrane feeding assays. Infectivity results were contrasted with passively recruited symptomatic malaria cases. ResultsFor P. falciparum and P. vivax infections detected at enrolment, median durations of infection were 37 days (95% confidence interval [CI], 15-93) and 60 days (95% CI, 18-213), respectively. P. falciparum and P. vivax parasite densities declined over the course of infections. From 47 feeding assays on 22 asymptomatic P. falciparum infections, 6.4% (3/47) were infectious and these infected 1.8% (29/1579) of mosquitoes. No transmission was observed in feeding assays on asymptomatic P. vivax mono-infections (0/56); one mixed-species infection was highly infectious. Among the symptomatic cases, 4.3% (2/47) of P. falciparum and 73.3% (53/86) of P. vivax patients were infectious to mosquitoes. ConclusionThe majority of asymptomatic infections were of short duration and low parasite density. Only a minority of asymptomatic individuals were infectious to mosquitoes. This contrasts with earlier findings and is plausibly due to the low parasite densities in this population.

A Survey on Trypanosoma evansi (Kinetoplastida, Trypanosomatidae) Infection in Domestic Animals in a Surra Endemic Area of Southern Algeria.

Background: Trypanosoma (T.) evansi infection is endemic in dromedary camels (Camelus dromedaries) of southern Algeria. Materials and Methods: In order to assess the presence of T. evansi in other domestic animals living together with dromedary camels, a study was conducted in the wilayate of Béchar, El Bayadh, Ouargla and Tamanrasset, between 2015 and 2017. Authorisation to conduct the survey was obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development and Fisheries). A total of 190 animals were sampled, including 42 cattle (Bos taurus), 11 dogs (Canis familiaris), 44 horses (Equus caballus), 3 donkeys (Equus asinus) and 1 mule, 49 goats (Capra hircus) and 40 sheep (Ovis aries). These animals were examined by parasitological (Giemsa stained thin smear, GST), serological (card agglutination test for trypanosomosis (CATT/T. evansi), enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2 [ELISA/VSG RoTat 1.2], immune trypanolysis [TL]) and molecular tests (T. evansi type A specific RoTat 1.2 PCR). Results and Conclusions: The CATT/T. evansi was positive in 10/42 cattle, 0/11 dogs, 2/48 equids, 27/49 goats and 15/40 sheep. On the other hand, 20/38 cattle, 1/9 dogs, 21/42 equids, 17/44 goats and 31/39 sheep were positive in ELISA/VSG RoTat 1.2. However, no single animal was positive in TL. In addition, the T. evansi parasite could not be demonstrated by either GST or RoTat 1.2 PCR in any of the examined animals. This may suggest cross-reactions of CATT/T. evansi and ELISA/VSG RoTat 1.2 with other pathogenic or commensal trypanosome species such as T. vivax or other parasites. Based on these data, in particular taking into account the high specificity of the TL for T. evansi type A, this study does not support the hypothesis that T. evansi circulates in the studied domestic animal species and that they would act as reservoirs for the parasite that causes trypanosomosis in dromedary camels.

Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45.

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

Species composition, infection rate and feeding preference of Anopheles mosquito species (Diptera: Culicidae) in the west Amhara Region, northwest Ethiopia.

Reports showed that Amhara Region is accounting for 31% of Ethiopia`s malaria burden. Reports also depicted that despite the existing malaria vector control tools implemented by the regional government, currently there is an increment in malaria prevalence in the region. This might be due to lack of entomological monitoring and comprehensive information on the prevailing species composition, infection rate and feeding presences of An. mosquito species in the study areas. Therefore, this study aimed to address this information. A community-based cross-sectional study was conducted from 18 February to 30 March 2023 at three districts of the west Amhara Region. For this, six CDC light traps (three indoor and three outdoor) were used to collect adult female An. mosquito species. In this study, 261 An. mosquito species comprising seven species were collected. Their morphological identification and abdominal status determination were carried out using standard morphological identification keys under a dissection microscope. While their infection rates and blood meal sources were determined, using circumsporozoite protein-ELISA and blood meal-ELISA based on the standard protocol. Data were entered to SPSS versions 20 for analysis and p- value < 0.05 was considered as statistically significant. In our study, total of 261 An. mosquito species were identified. An. demeilloni was the most abundant species accounted for 112(42.9%) of all captures. It was also the most predominant species at Ayehu-Guagusa followed by Jabitehnan districts. An. demeilloni and An. cinereus altogether accounting for infection rate of 3(1.1%) for P. vivax parasite. Furthermore, our study showed that more than 50% of An. mosquito species collected fed on cattle blood showing shifting of feeding behaviors. Our study depicted that An. demeilloni and An. cinereus were the most abundant species and infected with P. vivax parasite. Therefore, further comprehensive study should be done in the future.

Review on Malaria Disease

Malaria continues to be the main community health problem in numerous nations. Six species of Plasmodium are documented as the cause of human malaria infection. Among others, Plasmodium falciparum and Plasmodium vivax parasites produce an immense challenge in the public health. Anopheles funestus and Anopheles gambiae are the major transimmiter of the disease (malaria) from one person to another. The disease parasite has a complicated cycle of life that occurs in human and mosquitoes. In general, malaria diagnosis is divided into parasitological and clinical diagnosis. Internationally, the death rate of malaria becomes reduced although few records from Ethiopia describe the presence of raised prevalence of malaria in certain areas. Apart from reduction in incidence and prevalence, transmission of malaria is continued throughout the globe. Hence, its control needs a combined approach comprising treatment with effective antimalarial agents. A lot of novel compounds are under pre-clinical and clinical studies that are triggered by the occurrence of resistance among commonly used antimalarial drugs. In addition to the already known new compounds and targets for drug discovery, scientists from all corner of the world are in search of novel targets and chemical entities.

The Darc Side of Vivax Malaria in Africa: Unveiling Invasion Pathways into Duffy-Negative Erythroblasts

Background &amp; Objectives: Plasmodium vivax malaria was long thought to be absent from sub-Saharan Africa owing to the high prevalence of people lacking the Duffy antigen receptor for chemokines (DARC) on their erythrocytes. The interaction between P. vivax Duffy binding protein (PvDBP) and DARC is assumed to be the main mechanism used by P. vivax merozoites to invade human erythrocytes. However, the increasing numbers of vivax malaria cases in Duffy-negative African individuals has raised questions about alternative P. vivax invasion pathway(s) other than PvDBP-DARC interaction. Since P. vivax has a tropism for CD71+ immature reticulocytes and the hematopoietic niches of the bone marrow may be a hidden reservoir of P. vivax parasites, we hypothesized that P. vivax merozoites may be able to invade erythroblasts derived from Duffy-positive (DP) and Duffy-negative (DN) individuals. Therefore, our objectives were to (i) investigate the expression of DARC during DP and DN erythropoiesis and (ii) examine the infection of DP and DN erythroblasts with P. vivax merozoites in vitro to study and identify host receptors used by P. vivax to invade erythroblasts. Methods: We performed in vitro erythropoiesis assays (two-phase culture) of genotypically DN and DP erythroid progenitors. Erythroid differentiation, CD71 and DARC expression profiles were monitored using flow cytometry, microscopy, and western blot over time during phase 2. In vitro invasion assays were conducted using DP and DN erythroblasts to investigate whether genotypically DN erythroblasts could be invaded by P. vivax. At D7 of phase 2, we thawed and cultured parasite isolates obtained from two Malagasy and seven Ethiopian patients infected with P. vivax. After approximately 24-30 hours of in vitro maturation (at D8 of phase 2), each P. vivax parasite culture was placed in co-culture with either DP or DN erythroblasts for 24-48 hours. We examined the infection of DP and DN erythroblasts by P. vivax parasites by light and confocal microscopy. Results: Using in vitro erythropoiesis, we show that both genotypically DP and DN erythroblasts have similar kinetics of terminal erythroid differentiation. Moreover, we found similar CD71 expression profiles in both DP and DN erythroblasts. Interestingly, we observed that a subset of DN erythroblasts transiently expressed DARC during erythropoiesis. This was confirmed using both flow cytometry and western blot analysis. In vitro invasion assays conducted on both DP and DN erythroblasts using parasite isolates, showed that P. vivax merozoites were able to invade both DP and DN erythroblasts (Figure 1). We found that all the DP and DN erythroblasts that were infected with P. vivax were CD71+ and DARC+. These results confirm that P. vivax can invade DP and DN erythroblasts expressing both DARC and CD71, irrespective of the origin of the isolates. Conclusion: This study provides the first biological evidence for the ability of P. vivax to invade genotypically DN erythroblasts and highlights that African populations may represent a silent reservoir of P. vivax infections, with major implications for the control and elimination of vivax malaria in sub-Saharan Africa.

Determination of Plasmodium vivax and Plasmodium falciparum Malaria Exposure in Two Ethiopian Communities and Its Relationship to Duffy Expression.

Despite historical dogma that Duffy blood group negativity of human erythrocytes confers resistance to Plasmodium vivax blood stage infection, cases of P. vivax malaria and asymptomatic blood stage infection (subclinical malaria) have recently been well documented in Duffy-negative individuals throughout Africa. However, the impact of Duffy negativity on the development of naturally acquired immunity to P. vivax remains poorly understood. We examined antibody reactivity to P. vivax and P. falciparum antigens at two field sites in Ethiopia and assessed Duffy gene expression by polymerase chain reaction amplification and sequencing of the GATA-1 transcription factor-binding site of the Duffy antigen receptor for chemokines (DARC) gene promotor region that is associated with silencing of erythroid cell transcription and absent protein expression. Antibodies to three of the four P. vivax blood stage antigens examined, RBP2b, EBP2, and DBPIISal-1, were significantly lower (P < 0.001) in Duffy-negative individuals relative to Duffy-positive individuals. In stark contrast, no clear pattern was found across Duffy-negative and Duffy-positive genotypes for P. falciparum antibodies. We conclude that lack of erythroid Duffy expression is associated with reduced serologic responses, indicative of less naturally acquired immunity and less cumulative exposure to blood stage P. vivax parasites relative to Duffy positive individuals living in the same communities.

Investigation of Trypanosoma-induced vascular damage sheds insights into Trypanosoma vivax sequestration

Multiple blood-borne pathogens infecting mammals establish close interactions with the host vascular endothelium as part of their life cycles. In this work, we investigate differences in the interactions of three Trypanosoma species: T. brucei, T. congolense and T. vivax with the blood vasculature. Infection with these species results in vastly different pathologies, including different effects on vascular homeostasis, such as changes in vascular permeability and microhemorrhages. While all three species are extracellular parasites, T. congolense is strictly intravascular, while T. brucei is capable of surviving both extra- and intravascularly. Our knowledge regarding T. vivax tropism and its capacity of migration across the vascular endothelium is unknown. In this work, we show for the first time that T. vivax parasites sequester to the vascular endothelium of most organs, and that, like T. congolense, T. vivax Y486 is largely incapable of extravasation. Infection with this parasite species results in a unique effect on vascular endothelium receptors including general downregulation of ICAM1 and ESAM, and upregulation of VCAM1, CD36 and E-selectin. Our findings on the differences between the two sequestering species (T. congolense and T. vivax) and the non-sequestering, but extravasating, T. brucei raise important questions on the relevance of sequestration to the parasite’s survival in the mammalian host, and the evolutionary relevance of both sequestration and extravasation.

Surveillance of drug resistance molecular markers in Plasmodium vivax before and after introduction of dihydroartemisinin and piperaquine in Thailand: 2009-2019.

Resistance to antimalarial drugs is a serious issue around the world. Widespread Plasmodium vivax and P. falciparum coinfections are commonly found in Thailand. Dihydroartemisinin and piperaquine (DHA-PPQ) have been used as first-line treatments for P. falciparum since 2015, and chloroquine (CQ) and primaquine (PQ) have remained first-line drugs for P. vivax for more than 60 years. Coinfections may lead parasites to evolve with regard to genetics under selective drug pressure. This study is aimed at investigating genes linked to antimalarial resistance in P. vivax before and after introduction of DHA-PPQ as a new drug regimen in Thailand. A total of 400 P. vivax isolates were collected from samples along the Thai-Myanmar and Thai-Malaysian borders before (2009-2015) and after (2016-2019) introduction of DHA-PPQ. Genomic DNA of P. vivax was obtained and subjected to analysis of five drug resistance-associated genes (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o, and PvK12) by nested polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and nucleotide sequencing. A high prevalence of Pvdhfr was found in both endemic areas over the period. The quadruple (57I/58R/61M/117T) Pvdhfr haplotype was predominant in both periods in both endemic areas. Although the wild-type haplotype of Pvdhps was predominant in Thai-Malaysian isolates in both periods, a single mutant haplotype (383G) was dominant in Thai-Myanmar isolates during both periods. A low prevalence of the Pvmdr1 976F mutation was found in both periods among Thai-Myanmar isolates. A significant decrease in Pvmdr1 976F was identified in Thai-Malaysian isolates from the second period (p < 0.01). Only one nonsynonymous mutation of Pvcrt-o (193E) and one synonymous mutation of PvK12 (R584) were detected in four isolates (4.7%) and one isolate (0.5%) in the first period among Thai-Myanmar isolates, respectively. Thus, with limited clinical efficacy data, the low prevalence of drug-resistance markers may suggest that there is a low prevalence of P. vivax-resistant strains and that the current drug regimen for P. vivax is still effective for treating this P. vivax parasite population. Continued surveillance of antimalarial drug resistance markers and monitoring of clinical drug efficacy should be conducted for epidemiological and policy implications.

Nationwide spatiotemporal drug resistance genetic profiling from over three decades in Indian Plasmodium falciparum and Plasmodium vivax isolates

BackgroundDrug resistance is a serious impediment to efficient control and elimination of malaria in endemic areas.MethodsThis study aimed at analysing the genetic profile of molecular drug resistance in Plasmodium falciparum and Plasmodium vivax parasites from India over a ~ 30-year period (1993–2019). Blood samples of P. falciparum and/or P. vivax-infected patients were collected from 14 regions across India. Plasmodial genome was extracted and used for PCR amplification and sequencing of drug resistance genes in P. falciparum (crt, dhps, dhfr, mdr1, k13) and P. vivax (crt-o, dhps, dhfr, mdr1, k12) field isolates.ResultsThe double mutant pfcrtSVMNT was highly predominant across the country over three decades, with restricted presence of triple mutant CVIET from Maharashtra in 2012. High rates of pfdhfr-pfdhps quadruple mutants were observed with marginal presence of “fully resistant” quintuple mutant ACIRNI-ISGEAA. Also, resistant pfdhfr and pfdhps haplotype has significantly increased in Delhi between 1994 and 2010. For pfmdr1, only 86Y and 184F mutations were present while no pfk13 mutations associated with artemisinin resistance were observed. Regarding P. vivax isolates, the pvcrt-o K10 “AAG” insertion was absent in all samples collected from Delhi in 2017. Pvdhps double mutant SGNAV was found only in Goa samples of year 2008 for the first time. The pvmdr1 908L, 958M and 1076L mutations were highly prevalent in Delhi and Haryana between 2015 and 2019 at complete fixation. One nonsynonymous novel pvk12 polymorphism was identified (K264R) in Goa.ConclusionsThese findings support continuous surveillance and characterization of P. falciparum and P. vivax populations as proxy for effectiveness of anti-malarial drugs in India, especially for independent emergence of artemisinin drug resistance as recently seen in Africa.

Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in ethiopia

BackgroundMalaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of an invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum parasites was first reported in Ethiopia in 2016. The ecology of An. stephensi is different from that of Anopheles arabiensis, the primary Ethiopian malaria vector, and this suggests that alternative control strategies may be necessary. Larviciding may be an effective alternative strategy, but there is limited information on the susceptibility of Ethiopian An. stephensi to common larvicides. This study aimed to evaluate the efficacy of temephos and Bacillus thuringiensis var. israelensis (Bti) larvicides against larvae of invasive An. stephensi.MethodsThe diagnostic doses of two larvicides, temephos (0.25 ml/l) and Bti (0.05 mg/l) were tested in the laboratory against the immature stages (late third to early fourth stages larvae) of An. stephensi collected from the field and reared in a bio-secure insectary. Larvae were collected from two sites (Haro Adi and Awash Subuh Kilo). For each site, three hundred larvae were tested against each insecticide (as well as an untreated control), in batches of 25. The data from all replicates were pooled and descriptive statistics prepared.ResultsThe mortality of larvae exposed to temephos was 100% for both sites. Mortality to Bti was 99.7% at Awash and 100% at Haro Adi site.ConclusionsLarvae of An. stephensi are susceptible to temephos and Bti larvicides suggesting that larviciding with these insecticides through vector control programmes may be effective against An. stephensi in these localities.

Naturally acquired antibodies to gametocyte antigens are associated with reduced transmission of Plasmodium vivax gametocytes to Anopheles arabiensis mosquitoes.

Naturally acquired antibodies may reduce the transmission of Plasmodium gametocytes to mosquitoes. Here, we investigated associations between antibody prevalence and P. vivax infectivity to mosquitoes. A total of 368 microscopy confirmed P. vivax symptomatic patients were passively recruited from health centers in Ethiopia and supplemented with 56 observations from asymptomatic P. vivax parasite carriers. Direct membrane feeding assays (DMFA) were performed to assess mosquito infectivity; for selected feeds these experiments were also performed after replacing autologous plasma with malaria naïve control serum (n=61). The prevalence of antibodies against 6 sexual stage antigens (Pvs47, Pvs48/45, Pvs230, PvsHAP2, Pvs25 and PvCelTOS) and an array of asexual antigens was determined by ELISA and multiplexed bead-based assays. Gametocyte (ρ< 0.42; p = 0.0001) and parasite (ρ = 0.21; p = 0.0001) densities were positively associated with mosquito infection rates. Antibodies against Pvs47, Pvs230 and Pvs25 were associated with 23 and 34% reductions in mosquito infection rates (p<0.0001), respectively. Individuals who showed evidence of transmission blockade in serum-replacement DMFAs (n=8) were significantly more likely to have PvsHAP2 or Pvs47 antibodies. Further studies may demonstrate causality for the observed associations, improve our understanding of the natural transmission of P. vivax and support vaccine development.

Genetic diversity of Plasmodium vivax populations from the China–Myanmar border identified by genotyping merozoite surface protein markers

BackgroundParasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China–Myanmar border.MethodsWe collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3β, by PCR-restriction fragment length polymorphism (PCR–RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations.ResultsPCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3β among the 370 and 378 samples, respectively. PCR–RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3β with Pst I. PCR–RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3β. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion.ConclusionsMigrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR–RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.