Time to stimulate Plasmodium vivax research in India: A way forwards

India bears the largest Plasmodium (P.) vivax (Pv) malaria burden and contributes 48%of Pv cases globally[1]. The efforts of government and private bodies to control malaria have successfully reduced the number of Plasmodium falciparum (Pf) malaria cases in several countries, including India.

Conserved regions of Plasmodium vivax potential vaccine candidate antigens in Sri Lanka:Conscious in silico analysis of prospective conformational epitope regions

Objective:To do mapping and modeling of conformational B cell epitope regions of highly conserved and protective regions of three merozoitecandidate vaccine proteins of Plasmodium vivax(P.vivax),ie.merozoite purface protein-1(PvMSP-1),apical membrane antigen-1 domainⅡ(PvAMA1-DⅡ)and regionⅡof the Duffy binding protein(PvDBPⅡ).and to analyze the immunogenie properties of these predicted epitopes.Methods:3-D structures of amino acid haplotypes from Sri Lanka(available in GeneBank)of PvMSP-1_(19)(n=27),PvAMA1-DⅡ(n=21)and PvDBPⅡ(n=33)were modeled.SEPPA,selected as the best online server was used for conformational epitope predictions,while prediction and moodeling of protein structuve and properties related to immunogenicity was carried out with Geno3D server.SCRATCH Protein Server,NetSurfP Server and standaloneroftware,Genious 5.4.4.Results:SEPPA revealed that regions of predicted conformational epitopes formed 4 clusters in PvMSP-I_(19),and 3 clusters each in PvAMA1-DⅡand PvDBPⅡ,all of which displayed a high degree of hydrophilicity,contained solveut exposed residues,displayed high probability of antigenicity and showed positive antigenic propensity values,that indicated high degree of immunogenicity.Conclusions:Findings of this study revealed and confirmed that different parts of the sequences of each of the conserved regions of the three selected potential vaccine candidate antigens of P.vivax are important with regard to conformational epitope prediction that warrants further laboratory experimental invertigations in in vivo animal models.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.Methods:The study was conducted at Mae Tao Clinic for migrant workers,Tak Province during April 2009 to July 2010.A total of 64 blood samples(1 mL blood collected into sodium heparinized plastic tube) were collected from patients with monoinfection with P.vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P.vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-Ⅰ assays.Results:A total of 30 out of 64 blood samples collected from patients with P.vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays.The failure rates of the schizont maturation inhibition assay(50%) and the SYBR Green-I assay(54%) were similar(P=0.51).The median IC_(10)s,IC_(50)s and IC_(90)s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P.vivax tested.Based on the cut-off of 100 nM,the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates,respectively.The strength of agreement between the two methods was very poor for both chloroquine and WR992I0.Conclusions:On the basis of this condition and its superior sensitivity,the microscopic method appears better than the SYUK Green-I Green assay for assessing in vitro sensitivity of fresh P.vivax isolates to antimalarial drugs.

Plasmodium vivax cerebral malaria complicated with venous sinus thrombosis in Colombia

Complicated malaria is usually due to Plasmodium falciparum.Nevertheless,Plasmodium vivax is infrequently related with life-threatening complications.Few cases have been reported of severe Plasmodium vivax infection,and most of them from Southeast Asia and India.We report the first case of cerebral malaria due to Plasmodium vivax in Latin America,complicated with sagittal sinus thrombosis and confirmed by a molecular method.

Spectrum of complications associated with Plasmodium vivax infection in a tertiary hospital in South-Western India

Objective:To determine the range and incidence of complications associated with Plasmodium vivax[P.vivax) malaria.Methods:A retrospective analysis was performed of all patients of P.vivax malaria admitted in kasturba Medical College.Manipal between January and December,2010.Patients with mixed malarial infection were excluded by appropriate tests. Clinical presentation and laboratory parameters were studied.Results:Medical records of 213 individuals who satisfied the inclusion criteria were reviewed.Anaemia was seen in 65 (30.5%),leucopenia in 38(17.8%]and thrombocytopenia in 184(86.4%l patients.Aspartate and alanine aminotransferases were elevated in 86(40.4%).and 89(41.9%) patients respectively. Hypoalbuminemia was observed in 157(73.6%) cases.Elevated serum creatinine was noted in in 59(27.5%) patients.Creatine kinase was elevated in 30 nut of 59 patients(50.8%).Overall.107 (50.2%) patients fulfilled WHO criteria for sever=e malaria.None of the patients succumbed to the disease.Conclusion:P.vivax malaria is a potentially severe disease,and the term ’ benign" tertian malaria is a misnomer.Despite significant morbidity,with timely and appropriate treatment P.rirax malaria has an excellent outcome.

The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand

Objective:To investigate the genetic polymorphism of Plasmodium vivax(P.vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions(North,East,West and South) of Thailand.Methods:The 152 P.vivax injected cases from dried blood spots were DMA extracted and confirmed by species-specific primer sets using multiplex PCR method.PvMSPI fragments F2 and F3:PvCSP were gcnotvped using RFLP-PCR method.Resuits:Totally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50%and 8.55%.respectively while PvCSP was 3.95%.The overall frequency of multiple genotypes was 25%.There were 12 allele tvpes of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in P\MSP1 F3.The isolates from western region was highly genetic diverse when compare among all isolates.The predominant variant type of PvCSP gene was \ K2I0 type.Conclusions:The niulliple genotypes are common found in Thailand and it might hide the real genotype.PvCSP does not have extensive genetic diversity in this study.However.PvMSPI marker due to multiple genotypes is difficult In be analyzed.The multiple genotypes findings might stem from population migration and vector species findings.

A study on the clinical profile of complicated Plasmodium vivax mono-infections

Objective:To identify cases of severe Plasmodium vivax(P.vivax) mono-infections among adults.Methods:In this retrospective study,30 adult patients admitted to medical wards of a tertiary hospital in a malaria endemic urban area from March 2010 to April 2010 were included. The diagnosis of P.vivax malaria was established by peripheral blood film(PBF) examination, and severe malaria was categorized as per World Health Organization guidelines.Results: Complications observed were thrombocytopenia in 28(93.3%),hepatic dysfunction and jaundice in 13(433%),renal dysfunction in 8(26.7%),severe anaemia in 3(10.0%),cerebral malaria in 2 (6.7%),and acute respiratory distress syndrome(ARDS) in 1(3.3%) of 30 patients.Conclusions:P. vivax malaria with severe complications is common in the investigated area,and an intensive and large-scale study of the disease is necessary.

Duffy blood group genotypes among malaria Plasmodium vivax patients of Baoulch population in southeastern Iran

Objective:To determine the distribution of Duffy blood group genotypes in Balouch population as a major ethnic group that living in a sub-tropical area in south East of Iran.Methods:In this study,the Duffy blood group FY phenolypes were determined using indireet anti-globulin technique and also genotype by PCR-RFLP in 160 vivax malaria patients and 160 control individuals.Results:The results showed that the most common Duffy geitolype was FYA/FYB(46.6%)followed by FYA/FYA(15.3%),FYA/FYO(14.4%),FYB/FYO(11.9%,),FYB/FYB(10%)and FYO/FYO(1.9%).In case individuals,frequency of FY A.FYB and FYO alleles were 0.471,0.431and 0.097,respectively compaired to 0.444,0.353 and 0.203,respectively in control(non-infected)group.Conclusions:This data provide evidence that individuals with the FYA/FYB genotype have higher susceptibility to malaria and there are significant associations between Duffy blood group variants and susceptibility or resistance to vivax malaria.

Multiple splenic infarcts in acute Plasmodium vivax malaria:A rare case report

In tropical countries like India,malaria has been one of the most common parasitic illnesses leading to frequent hospitalization and causing major economic burden among the masses. Although Plasmodium vivax infection is considered to be benign,in contrast to Plasmodium, falciparum infection which is notorious for its severe splenic complications can occur frequently. Splenomegaly tends not to receive special attention,as it is not usually accompanied by any symptoms and can be gradually resolved via standard antimalarial therapy.Splenic infarction, although rarely attributable to malaria in an endemic region with high parasitemia,can be a rare presentation of this disease entity.

Sequential check and computer analysis of a cloned DNA fragment specific for Plasmodium vivax

A cloned DNA fragment specific for Plasmodium vivax was cleaved from theplasmid pVA1 and ligated into phage M13mp18.The nucleotides of the insert were se-quenced by the dideoxy-chain termination procedure.The fragment was composed of 261base pairs and 3 Hinf Ⅰ sites but no tandem repeat sequences.Computer retrieval andanalysis show that the nucleotide sequence of the cloned DNA fragment is unique,forthere has not been any significant homologue with that of other Plasmodium genes pub-lished as yet.

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes

Objective To detect malaria DNA in mosquitoes.Methods A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used.Results In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121?bp band was seen with DNA templates from other malaria parasites or negative mosquitoes.Conclusion Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

Epidemiological characterization of imported recurrent Plasmodium vivax and Plasmodium ovale in China,2013–2020

Background China has reached important milestones in the elimination of malaria.However,the numbers of imported recurrent cases of Plasmodium vivax and P.ovale are gradually increasing,which increases the risk of malaria re-establishment in locations where Anopheles mosquitoes exist.The aim of this study is to characterize the epidemiological profiles of imported recurrent P.vivax and P.ovale cases,quantifying the recurrence burden and guiding the development of appropriate public health intervention strategies.Methods Individual-level data of imported recurrent P.vivax and P.ovale cases were collected from 2013 to 2020 in China via the Parasitic Diseases Information Reporting Management System.Demographic characteristics,temporal and spatial distributions,and the interval from previous infection to recurrence were analyzed by SAS,ArcGIS and GraphPad Prism software,respectively,to explore the epidemiological profiles of imported recurrent cases.Results A total of 307 imported recurrent cases,including 179 P.vivax and 128 P.ovale cases,were recorded.The majority of cases occurred in males(P.vivax 91.1%,P.ovale 93.8%)and migrant workers(P.vivax 43.2%,P.ovale 44.7%).Individuals aged 30–39 years had the highest P.vivax and P.ovale recurrent infection rates,respectively.The number of imported recurrent cases of infection by these two malaria species increased from 2013 to 2018,and P.vivax infection showed well-defined seasonality,with two peaks in February and June,respectively.More than 90%of patients with recurrent cases did not receive radical treatment for previous infection.Most imported recurrent P.vivax cases were reported in Yunnan Province and were imported from Myanmar,Ethiopia,and Pakistan,while most recurrent P.ovale cases were reported in southern China and primarily imported from Cameroon,Ghana,and Nigeria.The intervals from previous malaria infection to recurrence among different continents were significantly different(P=0.0016)for P.vivax malaria but not for P.ovale malaria(P=0.2373).Conclusions The large number of imported recurrent cases has been a major challenge in the prevention of malaria re-establishment in China.This study provides evidence to guide the development of appropriate public health intervention strategies for imported recurrent P.vivax and P.ovale cases.

Polymorphisms of potential drug resistant molecular markers in Plasmodium vivax from China-Myanmar border during 2008-2017

Background:Plasmodium vivax remains the predominant species at the China–Myanmar border,imposing a major challenge to the recent gains in regional malaria elimination.To closely supervise the emerging of drug resistance in this area,we surveyed the variations in genes potentially correlated with drug resistance in P.vivax parasite and the possible drug selection with time.Methods:A total of 235 P.vivax samples were collected from patients sufering uncomplicated malaria at Yingjiang,Tengchong,and Longling counties,and Nabang port in China,Yunnan province,and Laiza sub-township in Myanmar,from 2008 to 2017.Five potential drug resistance genes were amplifed utilizing nested-PCR and analyzed,including pvdhfr,pvdhps,pvmdr1,pvcrt-o,and pvk12.The Pearson’s Chi-squared test or Fisher’s exact test were applied to determine the statistical frequency diferences of mutations between categorical data.Results:The pvdhfr F57I/L,S58R,T61M and S117T/N presented in 40.6%,56.7%,40.1%,and 56.0% of the sequenced P.vivax isolates,and these mutations signifcantly decreased with years.The haplotype formed by these quadruple mutations predominated in Yingjiang,Tengchong,Longling and Nabang.While a mutation H99S/R(56.6%)dominated in Laiza and increased with time.In pvdhps,the A383G prevailed in 69.2% of the samples,which remained the most prevalent haplotype.However,a signifcant decrease of its occurrence was also noticed over the time.The S382A/C and A553G existed in 8.4% and 30.8% of the isolates,respectively.In pvmdr1,the mutation Y976F occurred at a low frequency in 5/232(2.2%),while T958M was fxed and F1076L was approaching fxed(72.4%).The K10 insertion was detected at an occurrence of 33.2% in pvcrt-o,whereas there was no signifcant diference among the sites or over the time.No mutation was identifed in pvk12.Conclusions:Mutations related with resistance to antifolate drugs are prevalent in this area,while their frequencies decrease signifcantly with time,suggestive of increased susceptibility of P.vivax parasite to antifolate drugs.Resistance to chloroquine(CQ)is possibly emerging.However,since the molecular mechanisms underneath CQ resistance is yet to be better understood,close supervision of clinical drug efciency and continuous function investigation is urgently needed to alarm drug resistance.

Prevalence of antifolate drug resistance markers in Plasmodium vivax in China

The dihydrofolate reductase(dhfr)and dihydropteroate synthetase(dhps)genes of Plasmodium vivax,as antifolate resistance-associated genes were used for drug resistance surveillance.A total of 375 P.vivax isolates collected from different geographical locations in China in 2009–2019 were used to sequence Pvdhfr and Pvdhps.The majority of the isolates harbored a mutant type allele for Pvdhfr(94.5%)and Pvdhps(68.2%).The most predominant point mutations were S117T/N(77.7%)in Pvdhfr and A383G(66.8%)in Pvdhps.Amino acid changes were identified at nine residues in Pvdhfr.A quadruple-mutant haplotype at 57,58,61,and 117 was the most frequent(57.4%)among 16 distinct Pvdhfr haplotypes.Mutations in Pvdhps were detected at six codons,and the double-mutant A383G/A553G was the most prevalent(39.3%).Pvdhfr exhibited a higher mutation prevalence and greater diversity than Pvdhps in China.Most isolates from Yunnan carried multiple mutant haplotypes,while the majority of samples from temperate regions and Hainan Island harbored the wild type or single mutant type.This study indicated that the antifolate resistance levels of P.vivax parasites were different across China and molecular markers could be used to rapidly monitor drug resistance.Results provided evidence for updating national drug policy and treatment guidelines.

Genetic diversity of Plasmodium vivax revealed by the merozoite surface protein-1 icb5-6 fragment

Background:Plasmodium vivax remains a potential cause of morbidity and mortality for people living in its endemic areas.Understanding the genetic diversity of P.vivax from different regions is valuable for studying population dynamics and tracing the origins of parasites.The PvMSP-1 gene is highly polymorphic and has been used as a marker in many P.vivax population studies.The aim of this study was to investigate the genetic diversity of the PvMSP-1 gene icb5-6 fragment and to provide more genetic polymorphism data for further studies on P.vivax population structure and tracking of the origin of clinical cases.Methods:Nested PCR and sequencing of the PvMSP-1 icb5-6 marker were performed to obtain the nucleotide sequences of 95 P.vivax isolates collected from Zhejiang province,China.To investigate the genetic diversity of PvMSP-1,the 95 nucleotide sequences of the PvMSP-1 icb5-6 fragment were genotyped and analyzed using DnaSP v5,MEGA software.Results:The 95 P.vivax isolates collected from Zhejiang province were either indigenous cases or imported cases from different regions around the world.A total of 95 sequences ranging from 390 to 460 bp were obtained.The 95 sequences were genotyped into four allele-types(Sal I,Belem,R-III and R-IV)and 17 unique haplotypes.R-III and Sal I were the predominant allele-types.The haplotype diversity(Hd)and nucleotide diversity(Pi)were estimated to be 0.729 and 0.062,indicating that the PvMSP-1 icb5-6 fragment had the highest level of polymorphism due to frequent recombination processes and single nucleotide polymorphism.The values of dN/dS and Tajima’s D both suggested neutral selection for the PvMSP-1icb5-6 fragment.In addition,a rare recombinant style of R-IV type was identified.Conclusions:This study presented high genetic diversity in the PvMSP-1 marker among P.vivax strains from around the world.The genetic data is valuable for expanding the polymorphism information on P.vivax,which could be helpful for further study on population dynamics and tracking the origin of P.vivax.

Plasmodium vivax in the Elimination Phase—China,2013–2020

Summary What is already known about this topic?Plasmodium vivax(P.vivax)was the most widely distributed and major human malaria parasite in China,considered the last parasite to be eliminated.What is added by this report?The last domestic P.vivax case was reported in 2016,while hundreds of imported cases were reported annually from 2013–2020,predominantly from Southeast Asia.What are the implications for public health practice?In the post-elimination phase,adaptive and practical strategies focusing on imported P.vivax cases should be updated and adopted to prevent malaria resurgence.

Effects of mosquito hemocytes on normal and degenerated oocysts of Plasmodium yoelii and their role in oocyst melanization

Anopheles stephenst, infected with Plasinodium yoelii, was fed with 10% sucrose solution contatning 1% difluoromethylornithine (DFMO) to induce oocyst degeneration of the malaria parasites. On the 11th day after infection, the effects of mosquito hemocytes On the normal and degenerated oocysts were observed under transmission electron microscope. In the control group, no hemocyte could be found around the normally-developed oocysts. In the DFMO-treated group, all the oocysts underwent degeneration in various degrees and some of them were melanized. All the oocysts were attached by one or more hemocytes of the kind of granulocytes.There were many granules with microtubular structure in the cytoplasm of the hemocytes and in the space between the hemocyte and the oocyst. The findings suggest that the degeneration of oocysts can attract the hemocytes to attach around them and the latter can release granules and possibly other substances to cause encapsulation and melanization of the oocvsts.

A case of hemophagocytic syndrome as a complication of <i>Plasmodium</i>vivax malaria

Hemophagocytic lymphohistiocytosis syndrome (HPS) is a potentially fatal hyperinflammatory response characterized by a generalized histiocytic proliferation with marked hemophagocytosis in bone marrow [1]. Hemophagocytic syndrome has been associated with genetic mutations, autoimmune diseases, hematological malignancies or infections [2,3]. According to the data from Centre for Disease Control and prevention (CDC) Plasmodium falciparum has been associated with HPS but not the Plasmodium vivax [4-7]. We report a case of hemophagocytic syndrome as a complication of Plasmodium vivax malaria which is a rare presentation according to the data. This patient presented with high grade fever with chills (P. vivax positive), fever however did not respond to anti-malarials. The patient continued to have high grade fever with altered sensorium and deranged liver function with pancytopenia. Since she fulfilled the criteria of (HPS), patient was put on injectable steroids and responded dramatically. Hemophagocytic syndrome is a potentially fatal syndrome and therefore high index suspicion and early treatment is the key to reduce the mortatlity.

A Protocol for a Highly Consistent, High Level Production <i>in Vivo</i>of <i>Plasmodium falciparum</i>Oocysts and Sporozoites

Investigation of the intimate relationship between the human malaria parasite Plasmodium falciparum and its Anopheles vector requires the reliable production and isolation of successive sexual stages of the parasite from infected mosquitoes. Such an advance in propagation would benefit a range of molecular, cellular, immunochemical and transmission-blocking research studies. Parasite cultivation, mosquito rearing, infection and subsequent dissection of mosquitoes are all highly technical procedures that require both skill and experience to perform with competence. Furthermore, to produce mosquitoes of an appropriate age to infect during the short period in which parasites are viable for infection demands precise planning in order to coordinate the interacting life cycles of the parasite and vector. Here, a protocol is described for the complete development of P. falciparum within Anopheles stephensi. A very consistent, high level production in vivo of P. falciparum oocysts and sporozoites is demonstrable by dissection of the mosquito midgut and salivary glands, respectively.

Association of ABO blood group and Plasmodium falciparum malaria in Dore Bafeno Area,Southern Ethiopia

Objective:To assess the distribution of ABO blood group and their relationship with Plasmodium falciparum(P.falciparum) malaria among febrile outpatients who sought medical attention at Dore Bafeno Health Center,Southern Ethiopia.Methods:A total of 269 febrile outpatients who visited Dore Bafeno Health Center,Southern Ethiopia,were examined for malaria and also tested for ABO blood groups in January 2010.The blood specimens were collected by finger pricking,stained with Geimsa,and examined microscopically.Positive cases of the parasitemia were counted.CareStart^(TM) Malaria PflPv Combo was also used to test the blood specimens for malaria.ABO blood groups were determined by agglutination test using ERYCLONE antisera.Data on socio-demographic characteristics and treatment status of the participants were also collected.Chi-square and ANOVA tests were used to assess the difference between frequencies and means,respectively.Results:Out of a total of 269 participants,178(66.2%) febrile patients were found to be infected with Plasmodium parasites,among which 146(54.3%),28(10.4%),and 4(1.5%) belonged to P.falciparum,P.vivax,and mixed infections,respectively.All febrile patients were also tested for ABO blood groups and 51.3%,23.5%,21.9%and 3.3%were found to be blood types of 0,A,B and AB,respectively.Both total malaria infection and P.falciparum infection showed significant association with blood types(P<0.05).The proportion of A or B but not 0 phenotypes was higher(P<0.05) in individuals with P.falciparum as compared with non-infected individuals.The chance of having P.falciparum infection in patients with blood groups A,B and AB was 2.5,2.5 and 3.3times more than individuals showing blood 0 phenotypes,respectively.The mean P.falciparum malaria parasitemia for blood groups A,B,AB,and 0 were 3 744/μ L,1 805/ μ L,5 331/μ L,and1 515/μ L,respectively(P<0.01).Conclusions:The present findings indicate that individuals of blood groups A,B and AB are more susceptible to P.falciparum infection as compared with individuals of blood group O.Nevertheless,further in depth studies are required to clearly establish the role that ABO blood group plays in P.falciparum malaria.