Sri Krishna

Prevalence of pfhrp2 and/or pfhrp3 Gene Deletion in Plasmodium falciparum Population in Eight Highly Endemic States in India

Background Plasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions. Methods This is the first prospective field study carried out at 16 sites located in eight endemic states of India to assess the performance of PfHRP2 based RDT kits used in the national malaria control programme. In this study, microscopically confirmed P. falciparum but RDT negative samples were assessed for presence of pfhrp2, pfhrp3, and their flanking genes using PCR. Results Among 1521 microscopically positive P. falciparum samples screened, 50 were negative by HRP2 based RDT test. Molecular testing was carried out using these 50 RDT negative samples by assuming that 1471 RDT positive samples carried pfhrp2 gene. It was found that 2.4% (36/1521) and 1.8% (27/1521) of samples were negative for pfhrp2 and pfhrp3 genes, respectively. However, the frequency of pfhrp2 deletions varied between the sites ranging from 0–25% (2.4, 95% CI; 1.6–3.3). The frequency of both pfhrp2 and pfhrp3 gene deletion varied from 0–8% (1.6, 95% CI; 1.0–2.4). Conclusion This study provides evidence for low level presence of pfhrp2 and pfhrp3 deleted P. falciparum parasites in different endemic regions of India, and periodic surveillance is warranted for reliable use of PfHRP2 based RDTs.

Detection of Mixed Infections with Plasmodium spp. by PCR, India, 2014.

In 8 malaria-endemic states in India, mixed Plasmodium spp. infections were detected by PCR in 17.4% (265/1,521) of blood samples that microscopy had shown to contain only P. falciparum. The quality of microscopy must be improved because use of PCR for detection of malaria parasites is limited in rural areas.

Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation.

Therapeutic efficacy of artemether–lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria from three highly malarious states in India

BackgroundAnti-malarial drug resistance continues to be a leading threat to malaria control efforts and calls for continued monitoring of waning efficacy of artemisinin-based combination therapy (ACT). Artesunatexa0+xa0sulfadoxine/pyrimethamine (ASxa0+xa0SP) is used for the treatment of uncomplicated Plasmodium falciparum malaria in India. However, resistance against ASxa0+xa0SP is emerged in northeastern states. Therefore, artemether–lumefantrine (AL) is the recommended first line treatment for falciparum malaria in north eastern states. This study investigates the therapeutic efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in three malaria-endemic states in India. The data generated through this study will benefit the immediate implementation of second-line ACT as and when required.MethodsThis was a one-arm prospective evaluation of clinical and parasitological responses for uncomplicated falciparum malaria using WHO protocol. Patients diagnosed with uncomplicated mono P. falciparum infection were administered six-dose regimen of AL over 3xa0days and subsequent follow-up was carried out up to 28xa0days. Molecular markers msp-1 and msp-2 were used to differentiate recrudescence and re-infection and K13 propeller gene was amplified and sequenced covering the codon 450–680.ResultsA total of 402 eligible patients were enrolled in the study from all four sites. Overall, adequate clinical and parasitological response (ACPR) was 98xa0% without PCR correction and 99xa0% with PCR correction. At three study sites, ACPR rates were 100xa0%, while at Bastar, cure rate was 92.5xa0% on day 28. No early treatment failure was found. The PCR-corrected endpoint finding confirmed that one late clinical failure (LCF) and two late parasitological failures (LPF) were recrudescences. The PCR corrected cure rate was 96.5xa0%. The mean fever clearance time was 27.2xa0hxa0±xa08.2 (24–48xa0h) and the mean parasite clearance time was 30.1xa0hxa0±xa011.0 (24–72xa0h). Additionally, no adverse event was recorded. Analysis of total 186 samples revealed a mutation in the k13 gene along with non-synonymous mutation at codon M579T in three (1.6xa0%) samples.ConclusionAL is an efficacious drug for the treatment of uncomplicated falciparum malaria. However, regular monitoring of AL is required in view of malaria elimination initiatives, which will be largely dependent on therapeutic interventions, regular surveillance and targeted vector control.

Sequence variation in Plasmodium falciparum Histidine Rich Proteins 2 and 3 in Indian isolates: Implications for Malaria Rapid Diagnostic Test Performance

Commercial malaria rapid diagnostic tests (RDTs) detect P. falciparum histidine rich protein 2 (PfHRP2) and cross react with PfHRP3, a structural homologue. Here, we analysed natural variations in PfHRP2 and PfHRP3 sequences from Indian isolates and correlated these variations with RDT reactivity. A total 1392u2009P. falciparum positive samples collected from eight endemic states were PCR amplified for Pfhrp2 and Pfhrp3 genes and were sequenced. The deduced protein sequences were analysed for repeat variations and correlated with RDT reactivity. Out of 1392 PCR amplified samples, a single sample was Pfhrp2 negative and two samples were Pfhrp3 negative. Complete Pfhrp2 and Pfhrp3 sequences were obtained for 769 samples and 750 samples, respectively. A total of 16 distinct repeat motifs were observed for Pfhrp2 and 11 for Pfhrp3, including some new repeat types. No correlation was found between variations in the size of Pfhrp2 repeat types 2 and 7, nor between any combinations of repeat motifs, and performance of a commercial RDT at low parasite densities. The findings suggest that sequence diversity in Pfhrp2 and Pfhrp3 genes in Indian isolates is not likely to negatively influence performance of currently used PfHRP2 RDTs.

A rare case of quadruple malaria infection from the highly malaria-endemic area of Bastar, Chhattisgarh, India

The past decade has seen tremendous progress in malaria control worldwide, as 57 out of 106 countries have shown a sharp reduction of about 75% in malaria incidence [1]. Despite this progress, many febrile patients are still treated with antimalarial drugs without a confirmed malaria diagnosis, because malaria diagnosis is mainly based on microscopic examination of blood smears [2, 3]. Although rapid diagnostic tests (RDTs) are easy and reliable tools for diagnosis of Plasmodium falciparum and P. vivax, RDTs are unable to differentiate mixed infection with uncommon parasite species, i.e., P. ovale and P. malariae [4, 5]. In this report, we are presenting a rare case having all 4 species of Plasmodium in peripheral blood of a young boy from a remote community health centre (CHC), Darbha, in Bastar district of Chhattisgarh state, India (Fig 1). Malaria is a major health problem in Bastar district [6]. This area is also having serious problems of insurgency, which are affecting the health services of the area adversely [7, 8]. This is the first report from India of 4 Plasmodium species in 1 case. Such rare cases of malaria are a diagnostic and clinical challenge.

Prevalence of malaria in two highly endemic Community Health Centers in the Bastar district, Chhattisgarh showing mixed infections with Plasmodium species

Malaria is a major public health problem in India and in the Chhattisgarh state. The diagnosis of malaria presents a major challenge in remote areas The prevalence of malaria in Darbha and Kilepal Community Health Centers (CHCs) of the Jagdalpur district, Chhattisgarh affected by conflict was determined using microscopy and polymerase chain reaction (PCR). In the year 2015, 29.4% and 21.5% cases were found to be positive for malaria at the Darbha and Kilepal CHCs, respectively, by microscopy, and 7.4% and 1.6% of cases had mixed infections, respectively. Among the suspected cases of mixed infections and doubtful diagnoses, 21% had mixed infections with two or more species at the Darbha CHC, and 17% from the Kilepal CHC, as determined by PCR. Both the P. vivax subspecies Pv210 (56%) and Pv247 (44%) and the P. ovale curtisi subspecies were found in this area. The high proportion of mixed malaria parasitic infections detected in this study indicate the need to adequately train health staff involved in diagnosing malaria. This study showed that there is a need for site-specific data to understand the epidemiological picture and to develop appropriate intervention strategies and management guidelines for controlling and eliminating malaria in India.

Prevalence of afebrile parasitaemia due to Plasmodium falciparum & P. vivax in district Balaghat (Madhya Pradesh): Implication for malaria control

Background & objectives: Balaghat district in Central India is a highly malarious district where both Plasmodium falciparum and P. vivax are prevalent. In this district, the persistence of malaria was on an increase and not responsive to intervention measures even though there was no drug resistance. This study was undertaken by conducting mass screening to determine the prevalence of malaria among particularly vulnerable tribe of Balaghat, for developing evidence-based intervention measures for malaria control in hard to reach areas. Methods: This prospective study was carried out during 2013-2014 by conducting mass survey of the population in 10 villages of Birsa community health centre (CHC) and 12 villages of Baihar CHC. Finger-pricked blood smears were collected from all consenting individuals with or without fever for microscopic examination. Results: In the febrile group, the slide positivity rate (SPR) and slide falciparum rate (SFR) were 32.4 and 28.9 per cent, respectively, with 89.4 per cent P. falciparum, while in the afebrile individuals also, the SPR and SFR were high (29 and 26%, respectively), but these were significantly lower than that of febrile group. The gametocyte carriers were significantly higher (odds ratio 1.67, 95% confidence interval 1.25-2.25, P =0.0004) in afebrile patients when compared with febrile group. Vector incrimination showed the presence of four sporozoite-positive Anopheles culicifacies out of 1953 assayed. Interpretation & conclusions: Plasmodium falciparum malaria was high in young children (up to 8 years) as compared to the adult in both afebrile and febrile group in Balaghat district. High prevalence of gametocyte was observed in all age groups among the afebrile cases. The identification of afebrile malaria parasitaemia is an important challenge for the malaria elimination initiatives. A strong malaria surveillance system is fundamental to both programme design and implementation.