Genetic diversity and natural selection analysis of VAR2CSA and vir genes: implication for vaccine development.

Variable surface antigens (VSAs) encoded by var and vir genes in Plasmodium falciparum and Plasmodium vivax, respectively, are known to be involved in malaria pathogenesis and host immune escape through antigenic variations. Knowledge of the genetic diversity of these antigens is essential for malaria control and effective vaccine development. In this study, we analysed the genetic diversity and evolutionary patterns of two fragments (DBL2X and DBL3X) of VAR2CSA gene and four vir genes (vir 4, vir 12, vir 21 and vir 27) from different endemic regions, including Southeast Asia and sub-Saharan Africa. High levels of segregating sites (S) and haplotype diversity (Hd) were observed in both var and vir genes. Among vir genes, vir 12 (S = 131, Hd = 0.996) and vir 21 (S = 171, Hd = 892) were found to be more diverse as compared to vir 4 (S = 11, Hd = 0.748) and vir 27 (S = 23, Hd = 0.814). DBL2X (S = 99, Hd = 0.996) and DBL3X (S = 307, Hd = 0.999) fragments showed higher genetic diversity. Our analysis indicates that var and vir genes are highly diverse and follow the similar evolutionary pattern globally. Some codons showed signatures of positive or negative selection pressure, but vir and var genes are likely to be under balancing selection. This study highlights the high variability of var and vir genes and underlines the need of functional experimental studies to determine the most relevant allelic forms for effective progress towards vaccine formulation and testing.

Genotyping var Gene DBL1α Domain of Severe and Non-severe Plasmodium falciparum Patients.

This study analysed the genetic diversity of DBL1α domain of Plasmodium falciparum var gene in severe and non-severe malaria patients from Delhi and Mewat in Northern India. After confirming P. falciparum infection, samples were cloned and the var gene DBL1α domain was sequenced. Out of 377 cloned DBL sequences, 194 were from severe samples and 183 from non-severe samples. Proportion of DBL1α sequences belonging to groups 1, 4 and 5 were significantly higher in severe isolates as compared to non-severe isolates-group 1 (4.1% vs 1.09%, P = 0.0333), group 4 (69.58% vs 74.31%, P < 0.0001), and group 5 (19.58% vs 10.38%, P < 0.0001). Conversely, higher proportion of group 2 was observed in non-severe isolates (0% vs 3.82%, P = 0.0350). Highest diversity was seen in PoLV4 motif of severe and non-severe isolates and like other DBL1α sequences reported from several geographical areas (Africa, Americas, Asia, and Oceania). A total of 247 DBL1α domain haplotypes were found in this study where 139 (56.27%) haplotypes are novel and not reported from India till date. These findings could aid in developing effective malaria interventions, including vaccine and drug targets, by understanding the existing antigenic diversity and vulnerabilities in the parasite's genetic makeup. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01200-1.

Comparison of SYBR green I and lactate dehydrogenase antimalarial in vitro assay in Plasmodium falciparum field isolates.

Several assay methods are in use for monitoring the drug sensitivity of malaria parasites and screening new antimalarial drugs. Plasmodium lactate dehydrogenase (pLDH) and SYBR Green I in vitro assays were used to evaluate the drug efficacy of Chloroquine, Artemisinin and Azadirachta indica silver nano particles against Plasmodium falciparum 3D7 strain. The half-maximal inhibitory concentration (IC50) of each compound was estimated with non-linear regression model - dose-response analysis. The consistency between two methods was analysed with Cohen's kappa coefficient, interclass correlation and Bland-Altman plots. No statistical difference was found between IC50 values determined by both assays (p = 0.714). The proportion of resistant isolates to chloroquine according to SYBR green I (43.48%) and pLDH (34.78%) assays were similar (z = 0.302; p = 0.762) with significant concordant between methods (k = 0.819, p < 0.001). The results of pLDH Qualisa assay was comparable with classic SYBR green I assay and can be potentially useful in antimalarial drug efficacy surveillance.

Trends in clinical features and severity of Plasmodium vivax malaria among children at tertiary care center in North India.

BACKGROUND: Malaria is a significant cause of morbidity and mortality in adults and children. Plasmodium falciparum is the primary cause of severe malaria, but recently Plasmodium vivax is also recognized to cause severe malaria-associated morbidity and mortality. The study focuses on determining the mortality related to severity parameters in individuals under 12 years and their critical presentation in P.vivax malaria-infected children. METHODS: A prospective cross-sectional hospital-based study was conducted at Safdarjung Hospital, New Delhi, and ICMR-NIMR, New Delhi. All clinically suspected cases were admitted for screening. Exclusion criteria (rapid malaria antigen test, microscopy and medication history) were applied to all the admitted patients (n = 221) to obtain P.vivax patients only. Patients aged ≤ 12 years were included in the study. DNA was extracted from dried blood spots and amplified by nested PCR, followed by visualization on gel electrophoresis. RESULT: A total of 221 clinically suspected cases of malaria were screened for P.vivax. After implementing various exclusion criteria, 45/221 cases were enrolled for the study, among which 44.4% (20/45) of children had the symptoms of severe malaria in terms of cerebral malaria, thrombocytopenia, anemia, pancytopenia, acute respiratory distress syndrome and hemophagocytic lymphohistiocytosis. CONCLUSION: Plasmodium vivax mono-infection can cause severe manifestation and must be treated as P.falciparum without any delay because it may lead to increased morbidity and mortality. A changing trend in clinical symptoms has shown in P.vivax which was an earlier phenomenon of P.falciparum.

Intermittent preventive treatment with Sulfadoxine pyrimethamine for malaria: a global overview and challenges affecting optimal drug uptake in pregnant women.

Malaria in Pregnancy (MiP) leading to morbidity and mortality is a major public health problem that poses significant risk to pregnant women and their fetus. To cope with this alarming situation, administration of Sulfadoxine-pyrimethamine (SP) drugs to pregnant women as an intermittent preventive treatment (IPT) from 16 weeks of gestation is recommended by the World Health Organization (WHO) guidelines. We conducted a comprehensive search of published articles related to MiP in last 10 years with predefined keywords or their synonyms. The mapping of malaria in pregnant women showed a prevalence rate up to 35% in many countries. Although IPTp-SP has been implemented in endemic regions since several years but the IPTp-SP coverage percentage vary from country to country and continue to remain below the target of 80%. Major reasons for low IPTp-SP involve gestational age at first prenatal visit, level of education, place of residence, knowledge of IPTp-SP benefits, and use of antenatal services. Several challenges including the emergence of septuple and octuple SP-resistant parasites is reported from many countries which make the prophylactic use of IPTp-SP currently debatable. This narrative review addresses the barriers for optimal use of IPTp-SP and discusses alternative approaches to increase the use and effectiveness of SP intervention for preventing MiP. The COVID pandemic has drastically affected the public health disrupting the management of diseases worldwide. In view of this, a brief summary of COVID impact on MiP situation is also included.

Molecular characterization of the severe falciparum malaria with typhoid co-infection: A case report.

Malaria and typhoid co-infections can be a serious public health issue in tropical countries leading to incorrect diagnosis due to overlapping clinical presentations of malaria and typhoid and hence, causing a delay in implementing the appropriate treatment regimen for these concurrent infections. This study reports a case of six-year-old female child co-infected with severe malaria (Plasmodium falciparum) and typhoid (Salmonella typhi) diagnosed by rapid malaria antigen test (RMAT) and blood culture respectively. Further, analysis of the chloroquine resistance gene Pfcrt for the falciparum demonstrated the presence of K76T mutant allele in pfcrt gene with high IC50 (150nM) for chloroquine (CQ) drug. The present case highlights the significance of timely identification and treatment of co-infections and also provides information about the circulating P. falciparum clinical strains.

Studying the disease severity in clinical isolates of Plasmodiumvivax.

Among the human malaria Plasmodium species, Plasmodium vivax is the most widespread species globally. In recent times, this historically benign species is now being recognized as also responsible for severe malaria infections in humans. Hence, a deeper insight of P.vivax immunopathogenesis in clinical patients is essential for malaria control and elimination strategies. Certain genes like vir genes, merozoite surface protein 3α genes (msp3α) and biomarkers like super oxide dismutase (SOD-1), tumor necrosis factor (TNF- α), interleukin (IL-10) are speculated to have some role in disease severity and thus can be useful as diagnostic markers. In the reported study, the clinical samples of P.vivax were genotyped for msp3α gene and cytokine analysis, expression profiling of vir genes were also carried out in these samples. A total of 84 P.vivax samples were collected (39 severe and 45 non-severe samples) and no correlation of parasitemia with severity of disease was seen in these samples (p-value = 0.38). On analysis four genotypes of msp3α were found, with type B (1.5 kb) as the predominant genotype. Cytokine analysis revealed SOD-1 and TNF-α levels to be significantly more in the severe group than in non-severe group, whereas for IL-10 no significant difference was observed between two clinical groups. The vir gene profiling revealed increased level of expression for vir-12, vir-14 related, and vir-17 like in severe group and vir-10 related gene expression was more in non-severe samples. There are multiple factors that bring phenotypic and genotypic changes in P.vivax malaria and thus, it is important to assess the potential diagnostic markers for detection of disease severity. In future, studies with more number of clinical samples should be undertaken for better insight of P.vivax disease severity.

A systematic review on genetic diversity of var gene DBL1α domain from different geographical regions in Plasmodium falciparum isolates.

Background The major variant surface antigen (VSA) in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by var gene family has an important role in cytoadhesion/sequestration and rosetting by adhesion of uninfected erythrocytes to infected erythrocytes leading to disease severity. DBL1α domain in the PfEMP-1, protein is crucial in the cytoadhesion phenomena in P. falciparum infections and this review aims to analyse the genetic diversity of DBL1α domain sequences in PfEMP-1 from different geographical regions globally. Methods All available DBL1α sequence data was reviewed by using the electronic database PubMed, ResearchGate, Google, Google scholar, MEDLINE with the following Keywords-Plasmodium falciparum", "var gene", "DBL1α", "field isolate", "diversity", "polymorphism", "Africa", "America", "Asia" and "Caribbean" from different geographical regions across the world. Results A total of 240 studies were identified initially but only 20 studies qualified for this systematic review. The overall ratio of distinct sequences DBL1α domain was 24.62/1167 the highest in African region (33.59/766 isolates) and lowest in South America (5.6/215 isolates). In the 18 included studies, the presence of distinct DBL1α sequences was the highest in Oceania 55.32% (1186/2144) followed by Africa (38.43%), Asia (22.45%) and South America (16.48%), though the sample size in Oceania was comparatively smaller to that of Africa and South America. Conclusion This review highlights the ratio and percentage of distinct sequences of DBL1α domain of var gene in different geographical regions giving an idea of the existing diversity prevalent in this potential vaccine target gene which may contribute to designing the preventive measures towards disease severity.

Pancytopenia with hemophagocytic lymphohistiocytosis in Plasmodium falciparum: A unusual presentation.

Hematological manifestations such as anemia and thrombocytopenia are known complications in malaria. Here, we report two cases presented as pancytopenia with hepatosplenomegaly and initial diagnosis kept as hematological malignancy like leukemia but later on its diagnosed as malaria-associated hemophagocytic lymphohistiocytosis which is a rare entity. The aim of this report is to draw the attention of physicians, especially in tropical countries such as India and Sub-Saharan nations to keep in mind this uncommon presentation of malaria, though the exact pathophysiological mechanism still remains obscure.

Artemisinin-based combination therapy (ACT) and drug resistance molecular markers: A systematic review of clinical studies from two malaria endemic regions - India and sub-Saharan Africa.

Artemisinin-based combination therapies (ACT) are currently used as a first-line malaria therapy in endemic countries worldwide. This systematic review aims at presenting the current scenario of drug resistance molecular markers, either selected or involved in treatment failures (TF) during in vivo ACT efficacy studies from sub-Saharan Africa (sSA) and India. Eight electronic databases were comprehensively used to search relevant articles and finally a total of 28 studies were included in the review, 21 from sSA and seven from India. On analysis, Artemether + lumefantrine (AL) and artesunate + sulfadoxine-pyrimethamine (AS + SP) are the main ACT in African and Indian regions with a 28-day efficacy range of 54.3-100% for AL and 63-100% for AS + SP respectively. It was observed that mutations in the Pfcrt (76T), Pfdhfr (51I, 59R, 108N), Pfdhps (437G) and Pfmdr1 (86Y, 184F, 1246Y) genes were involved in TF, which varied with respect to ACTs. Based on studies that have genotyped the Pfk13 gene, the reported TF cases, were mainly linked with mutations in genes associated with resistance to ACT partner drugs; indicating that the protection of the partner drug efficacy is crucial for maintaining the efficacy of ACT. This review reveals that ACT are largely efficacious in India and sSA despite the fact that some clinical efficacy and epidemiological studies have reported some validated mutations (i.e., 476I, 539T and 561H) in circulation in these two regions. Also, the role of PfATPase6 in ART resistance is controversial still, while P. falciparum plasmepsin 2 (Pfpm2) in piperaquine (PPQ) resistance and dihydroartemisinin (DHA) + PPQ failures is well documented in Southeast Asian countries but studied less in sSA. Hence, there is a need for continuous molecular surveillance of Pfk13 mutations for emergence of artemisinin (ART) resistance in these countries.