Epidemiology of malaria and anemia in high and low malaria-endemic North-Eastern districts of India.

Anemia and malaria are the two major public health problems that lead to substantial morbidity and mortality. Malaria infection destroys erythrocytes, resulting in low hemoglobin (Hb) levels known as anemia. Here we report the determinants of anemia in high and low malaria-endemic areas that would help understand which parasite densities, age, and gender-associated low Hb levels. Therefore, a cross-sectional mass survey (n = 8,233) was conducted to screen anemia and malaria in high and low malaria-endemic districts (HMED and LMED) of North-East India. Axillary body temperature was measured using a digital thermometer. The prevalence of anemia was found to be 55.3% (4,547/8,233), of which 45.1% had mild (2,049/4,547), 52.1% moderate (2,367/4,547) and 2.9% had severe anemia (131/4,547). Among anemic, 70.8% (3,219/4,547) resided in LMED and the rest in HMED. The median age of the anemic population was 12 years (IQR: 7-30). Overall, malaria positivity was 8.9% (734/8,233), of which HMED shared 79.6% (584/734) and LMED 20.4% (150/734) malaria burden. The village-wise malaria frequency was concordant to asymptomatic malaria (10-20%), which showed that apparently all of the malaria cases were asymptomatic in HMED. LMED population had significantly lower Hb than HMED [standardized beta (ß) = -0.067, p < 0.0001] and low-density Plasmodium infections had higher Hb levels than high-density infections (ß = 0.113; p = 0.031). Women of reproductive age had higher odds for malaria (OR: 1.42; 95% CI: 1.00-2.05; p = 0.04). Females (ß = -0.193; p < 0.0001) and febrile individuals (ß = -0.029; p = 0.008) have shown lower Hb levels, but malaria positivity did not show any effect on Hb. Young children and women of reproductive age are prone to anemia and malaria. Although there was no relation between malaria with the occurrence of anemia, we found low-density Plasmodium infections, female gender, and LMED were potential determinants of Hb.

Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

Mutations in Pfcrt and Pfmdr1 genes of Plasmodium falciparum isolates from two sites in Northcentral and Southwest Nigeria.

The ability of malaria parasites to develop resistance to antimalarial drugs has made it necessary to continuously survey malaria parasite populations for resistance markers. Mutations in specific malaria parasite genes confer resistance to antimalarial drugs. The study compared mutations in Pfcrt and Pfmdr1 genes of P. falciparum from two ecologically different areas of Nigeria. Plasmodium falciparum dried blood spots collected from New Bussa (Northcentral Nigeria) and Ijede (Southwest Nigeria) were analysed by PCR-RFLP for Pfcrt, K76 T, Pfmdr1, N86Y and Y184F mutations. Pfmdr1 copy number was determined by quantitative-PCR. A total of 145 blood spots [Ijede = 55; New Bussa = 90 blood spots] were analysed, but Pfcrt gene was successfully amplified in 144 samples while Pfmdr1 was amplified in 132 samples. Overall, prevalence of mutant forms of Pfcrt 76 T,Pfmdr1 86Y and 184F were 74.3% (95% CI: 66.4-81.2%), 18.2% (95% CI: 12.0-25.8%) and 35.6% (95% CI: 27.5-44.4%). The frequency of Pfcrt 76 T was similar in both study sites [Ijede: 81.8% (95%CI: 69.1-90.9%); New Bussa: 69.7% (95%CI: 59.0-79.0), p = 0.105]. However, the frequencies of Pfmdr1 86Y and 184F were significantly higher in Ijede (28.3% and 62.3%) than in New Bussa (11.4% and 17.7%), respectively (P < 0.05). Eight parasite genotypes based on three codons of the two genes were identified. The most frequent genotype was TNY 53(40.5%) while the least was KYF 1 (0.8%). The most frequent genotype in Ijede and New Bussa were TNF 18(34.0%) and TNY 40 (51.3%) respectively. The frequency of wild strain KNF in Ijede and New Bussa were 3 (5.7%) and 18 (23.1%), respectively. The distribution of the genotypes differed significantly by location. The genotypes with more than two or more mutations were more in Ijede 32 (60.4%) than in New Bussa 16 (20.5%) (p < 0.001). Amplification of Pfmdr1 copy number was not observed in the two study sites. The prevalence of Pfcrt 76 T was similar in both locations while Pfmdr1 86Y and 184F differed in both locations. Single nucleotide polymorphisms in the three codons assessed were more in Ijede than in New Bussa.

Diagnostic performance of rapid diagnostic test, light microscopy and polymerase chain reaction during mass survey conducted in low and high malaria-endemic areas from two North-Eastern states of India.

An early and accurate diagnosis followed by prompt treatment is pre-requisite for the management of any disease. Malaria diagnosis is routinely performed by microscopy and rapid diagnostic tests (RDTs) in the field settings; however, their performance may vary across regions, age and asymptomatic status. Owing to this, we assessed the diagnostic performance of conventional and advanced molecular tools for malaria detection in low and high malaria-endemic settings. We performed mass blood surveys in low and high endemic regions of two North-Eastern districts from the states of Assam and Meghalaya. A total of 3322 individuals were screened for malaria using RDT, microscopy and PCR and measures of diagnostic accuracy were estimated. Out of 3322 individuals, 649 (19.5%) were detected with malaria parasite. Asymptomatic were 86.4% (2872/3322), of which 19.4% (557/2872) had Plasmodium infection. The sensitivity and specificity of microscopy were 42.7% and 99.3%, and RDT showed 49.9% and 90.4%, respectively, considering PCR as standard. RDT (AUC: 0.65 vs 0.74; p = 0.001) and microscopy (AUC: 0.64 vs 0.76; p < 0.0001) performances were significantly lower in low compared to high endemic areas. True positive rate was lower in asymptomatics but true negative rate was found similar to symptomatic individuals. The conventional diagnostic tools (RDT and microscopy) had detected malaria in children with nearly twofold greater sensitivity than in the adults (p < 0.05). To conclude, asymptomatics, adults and low malaria-endemic regions require major attention due to mediocre performance of conventional diagnostic tools in malaria detection.

Asymptomatic low-density Plasmodium infection during non-transmission season: a community-based cross-sectional study in two districts of North Eastern Region, India.

BACKGROUND: Malaria elimination requires targeting asymptomatic and low-density Plasmodium infections that largely remain undetected. Therefore we conducted a cross-sectional study to estimate the burden of asymptomatic and low-density Plasmodium infection using conventional and molecular diagnostics. METHODS: A total of 9118 participants, irrespective of age and sex, were screened for malaria using rapid diagnostic tests (RDTs), microscopy and polymerase chain reaction. RESULTS: Among the participants, 707 presented with symptoms and 8411 without symptoms, of which Plasmodium was present in 15.6% (110/707) and 8.1% (681/8411), respectively. Low-density infection was found in 5.1% (145/2818) of participants and 8327 of 9118 were Plasmodium negative. Endemicity was propotional to asymptomatic infections (high endemicity 11.1% [404/3633] vs low endemicity 5.8% [277/4778]; odds ratio [OR] 2.0 [95% confidence interval {CI} 1.7 to 2.4]) but inversely related to low-density infection (high endemicity 3.7% [57/1545] vs low endemicity 6.9% [88/1273]; OR 1.9 [95% CI 1.4 to 2.7]). The spleen rate in children 2-9 y of age was 17.9% (602/3368) and the enlarged spleen index was 1.6. Children between 8 and 14 y showed higher odds for asymptomatic (adjusted OR [aOR] 1.75 [95% CI 1.4 to 2.2]) and low-density infections (aOR 0.63 [95% CI 0.4 to 1.0)] than adults. CONCLUSIONS: The prevalence of asymptomatic and low-density Plasmodium infection undermines the usefulness of standard diagnostic tools used by health agencies. This necessitates deploying molecular tools in areas where malaria microscopy/RDTs indicate a dearth of infection.

Redox interactome in malaria parasite Plasmodium falciparum.

The malaria-causing parasite Plasmodium falciparum is a severe threat to human health across the globe. This parasite alone causes the highest morbidity and mortality than any other species of Plasmodium. The parasites dynamically multiply in the erythrocytes of the vertebrate hosts, a large number of reactive oxygen species that damage biological macromolecules are produced in the cell during parasite growth. To relieve this intense oxidative stress, the parasite employs an NADPH-dependent thioredoxin and glutathione system that acts as an antioxidant and maintains redox status in the parasite. The mutual interaction of both redox proteins is involved in various biological functions and the survival of the erythrocytic stage of the parasite. Since the Plasmodium species is deficient in catalase and classical glutathione peroxidase, so their redox balance relies on a complex set of five peroxiredoxins, differentially positioned in the cytosol, mitochondria, apicoplast, and nucleus with partly overlapping substrate preferences. Moreover, Plasmodium falciparum possesses a set of members belonging to the thioredoxin superfamily, such as three thioredoxins, two thioredoxin-like proteins, one dithiol, three monocysteine glutaredoxins, and one redox-active plasmoredoxin with largely redundant functions. This review paper aims to discuss and encapsulate the biological function and current knowledge of the functional redox network of Plasmodium falciparum.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

Prevalence and spectrum of mutations causing G6PD deficiency in Indian populations.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human erythroenzymopathy affecting around 10% of the world population. India is endemic for malaria and antimalarial drugs are known to induce haemolysis in G6PD deficient individuals. Here we report the prevalence as well as the molecular diversity of G6PD deficiency in geographical regions of India. METHODS AND RESULTS: A total of 20,896 individuals (11,838 males and 9058 females) were screened by DPIP dye decolorisation method followed by quantitation of G6PD enzyme activity on the suspected samples. Molecular analysis was undertaken in a total of 350 G6PD deficient individuals by PCR-RFLP and DNA sequencing. A structural characteristic of the novel variant was deduced by using DynaMut web-server. The prevalence rate of G6PD deficiency varied between 0.8 and 6.3% with an overall prevalence of 1.9%. A total of twelve mutations were identified. Of the total deleterious alleles detected G6PD Orissa (56.5%) was found to be the most predominant variant followed by G6PD Mediterranean (23.6%). G6PD Mediterranean, G6PD Kaiping and G6PD Mahidol were found to be severely deficient variant and 14.1% of them showed undetectable activity. A novel mutation c.544C➔G (R182G) in exon 6 was identified in one tribal male where substitution of arginine by glycine, likely causes the alteration in the alpha helix leading to disruption of secondary structure of the protein. CONCLUSION: There are large differences in the distribution of G6PD causal variants between Indian states, and this may have implications for the treatment in the malaria endemic areas.

Evaluation of Implementation of Intermittent Screening and Treatment for Control of Malaria in Pregnancy in Jharkhand, India.

This study evaluated intermittent screening and treatment during pregnancy (ISTp) for malaria using rapid diagnostic tests (RDTs) at antenatal care (ANC) compared with passive case detection within the routine health system. The mixed-method evaluation included two cross-sectional household surveys (pre- and post-implementation of ISTp), in-depth interviews with health workers, and focus group discussions (FGDs) with pregnant women. Differences in proportions between surveys for a number of outcomes were tested; 553 and 534 current and recently pregnant women were surveyed (pre- and post-implementation, respectively). In-depth interviews were conducted with 29 health providers, and 13 FGDs were held with pregnant women. The proportion of pregnant women who received an RDT for malaria at ANC at least once during their pregnancy increased from pre- to post-implementation (19.2%; 95% CI: 14.9, 24.3 versus 42.5%; 95% CI: 36.6, 48.7; P < 0.0001), and the proportion of women who had more than one RDT also increased (16.5%; 95% CI: 13.1, 20.5 versus 27.7%; 95% CI: 23.0, 33.0; P = 0.0008). Post-implementation, however, only 8% of women who had completed their pregnancy received an RDT on three visits to ANC. Health workers were positive about ISTp mainly because of their perception that many pregnant women with malaria were asymptomatic. Health workers perceived pregnant women to have reservations about ISTp because of their dislike of frequent blood withdrawal, but pregnant women themselves were more positive. Intermittent screening and treatment during pregnancy was not sufficiently adopted by health workers to ensure the increased detection of malaria infections achievable with this strategy in this setting.

Geographic Plasmodium falciparum sarcoplasmic reticulum Ca2+ ATPase (PfSERCA) genotype diversity in India.

Plasmodium falciparum sarcoplasmic reticulum Ca2+ ATPase (PfSERCA) is sarcoplasmic reticulum membrane bound transporter to regulate cytosol Ca2+ ions. Ca2+ act as secondary messenger and play important role in differentiation of parasite during its life cycle. Present study is epidemiological surveillance of PfSERCA (Pf3D7_0106300) gene fragment harboring 263, 402, 431 codon to look for its single nucleotide polymorphism which is well documented to be associated with Artemisinin tolerance. Filter paper with finger pricked blood samples for Plasmodium falciparum infected uncomplicated malaria patients were obtained for region as diverse as down the longitude from east to west of India i.e. Mizoram, Tripura, Meghalaya, Jharkhand, Odhisa. There observed no mutation for codon 263 at all study sites. Mizoram showed highest PfSERCA diversity with well known SNPs of L402 V, E431 K, A438 V and novel mutations as well i.e. A338 V, S357Y, S379Y. Tripura reported highest proportion of Plasmodium isolates (18.5%) with E431 K single nucleotide polymorphism. Moving towards the west i.e. Meghalaya, Jharkhand, Odhisa showed no occurrence of most prevalent PfSERCA 431, 402 polymorphism worldwide but some novel mutations and its haplotypes. In present study, significantly increased proportion of novel PfSERCA polymorphism among children suggests the susceptibility of these Plasmodium falciparum strains to acquired immunity. Mizoram, sharing open international border with south east asia, demonstrated highest PfSERCA diversity. Spatial PfSERCA diversity from far north east India to moving towards west implies its association with antimalarial susceptibility.

First report of glucose-6-phosphate dehydrogenase (G6PD) variants (Mahidol and Acores) from malaria-endemic regions of northeast India and their functional evaluations in-silico.

G6PD deficiency results from numerous mutations in the G6PD gene and can cause alterations in enzyme function up to varying degrees. P. vivax malaria infections require G6PD deficiency screening because of the potential risk of haemolysis by the gametocytocidal drug (primaquine) during the radical treatment. The present study investigated the incidence of G6PD deficiency from northeast India and further, molecular characterization was performed. During 2014-16, a total of 1,015 patients from four north-eastern states of India (Tripura, Mizoram, Meghalaya & Arunachal Pradesh), were screened for G6PD deficiency, using Beutler's fluorescence spot test (FST) and confirmed with SPAN G6PD kit. The deficient individuals (55/1015, 5.4%) were further characterized by PCR-RFLP and DNA sequencing except one case of lost to follow up. As observed by FST, the frequency of G6PD deficient males (42/538, 7.8%) were found to be higher than females (13/477, 2.73%), (p < 0.0001). Two non-synonymous mutations; G6PD-D (Mahidol)487A (48/54, 88.9%; 36 hemizygous males, 8 homozygous and 4 heterozygous females) and G6PD-D (Acores)595T (2/54, 3.7%) were identified. Remaining (4/54, 7.4%) individuals could not be characterized. Molecular modeling and dynamic simulations were performed for the G6PD wild-type (G6PD-WT) enzyme and its variants. The in-silico results demonstrated alterations in the secondary structures & crucial loss of ligand-protein interactions, which might result in reduced enzyme function, leading to enzyme deficiency. To the best of our knowledge, this is the first report to document G6PD-Mahidol and G6PD-Acores variants from malaria-endemic regions of northeast India, and provided molecular insights on the varied genetic makeup of the studied population.

Novel molecular diagnostic technique for detecting the different species of Plasmodium.

BACKGROUND: Sensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes. METHOD: Plasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes. RESULT: A total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method. CONCLUSION: This method identifies and distinguishes four species of human malaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques.

Effectiveness of intermittent screening and treatment for the control of malaria in pregnancy: a cluster randomised trial in India.

BACKGROUND: The control of malaria in pregnancy (MiP) in India relies on testing women who present with symptoms or signs suggestive of malaria. We hypothesised that intermittent screening and treatment for malaria at each antenatal care visit (ISTp) would improve on this approach and reduce the adverse effects of MiP. METHODS: A cluster randomised controlled trial comparing ISTp versus passive case detection (PCD) was conducted in Jharkhand state. Pregnant women of all parities with a gestational age of 18-28 weeks were enrolled. Women in the ISTp group were screened with a rapid diagnostic test (RDT) for malaria at each antenatal clinic visit and those in the PCD group were screened only if they had symptoms or signs suggestive of malaria. All RDT positive women were treated with artesunate/sulfadoxine-pyrimethamine. The primary endpoint was placental malaria, determined by placental histology, and the key secondary endpoints were birth weight, gestational age, vital status of the newborn baby and maternal anaemia. RESULTS: Between April 2012 and September 2015, 6868 women were enrolled; 3300 in 46 ISTp clusters and 3568 in 41 PCD clusters. In the ISTp arm, 4.9% of women were tested malaria positive and 0.6% in the PCD arm. There was no difference in the prevalence of placental malaria in the ISTp (87/1454, 6.0%) and PCD (65/1560, 4.2%) groups (6.0% vs 4.2%; OR 1.34, 95% CI 0.78 to 2.29, p=0.29) or in any of the secondary endpoints. CONCLUSION: ISTp detected more infections than PCD, but monthly ISTp with the current generation of RDT is unlikely to reduce placental malaria or impact on pregnancy outcomes. ISTp trials with more sensitive point-of-care diagnostic tests are needed.

A Simple and Cost-Effective Freeze-Thaw Based Method for Plasmodium DNA Extraction from Dried Blood Spot.

BACKGROUND: Available DNA isolation methods for Plasmodium involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of Plasmodium DNA from dried filter paper spot (DBS), appropriate for resource-limited setups. METHODS: The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed in terms of DNA quantity, PCR detection sensitivity, time requirement, cost effectiveness, labor intensiveness and degree of shearing. The reliability of this method was confirmed by comparing it with other in use methods for Plasmodium DNA isolation. RESULTS: Pure DNA was obtained with this method, as exemplified by the absorbance ratio (260nm /280nm) of 1.2. The protocol produced digestible, PCR-grade genomic DNA, also found to be suitable for sequencing. DNA isolated remained stable and retained its integrity after storage for one month at 4 °C. CONCLUSION: Our process substantiated as efficient, reproducible, simple, fast, and inexpensive. Development of this optimized freeze-thaw based DNA extraction method for malaria parasite may provide a valuable tool for molecular analysis in resource-limited setups. This is the first report of DNA extraction from DBS of Plasmodium utilizing freeze-thaw.

Efficacy of two artemisinin-based combinations for the treatment of malaria in pregnancy in India: a randomized controlled trial.

BACKGROUND: In India, the recommended first-line treatment for malaria in the second and third trimester of pregnancy is artesunate + sulfadoxine-pyrimethamine (AS+SP). However, data on safety and efficacy of artemisinin-based combination therapy (ACT) in pregnancy is limited. This study assessed the safety and efficacy of AS+SP and artesunate + mefloquine (AS+MQ) for treatment of Plasmodium falciparum in pregnancy in India. METHODS: This open-label, randomized clinical trial was conducted from October 2010 to December 2013 at three sites in India (Ranchi and Jamshedpur in Jharkhand state, and Rourkela in Odisha state). Pregnant women in the second or third trimester who had P. falciparum mono-infection of any parasite density with or without fever were randomized to receive AS+SP or AS+MQ. Blood slides and filter paper samples for Polymerase Chain Reaction (PCR) were collected on days 0, 1, 2, 3, 14, 21, 28, 42 and 63 post treatment. Women were followed up at delivery and at day 42 postpartum. FINDINGS: Two hundred and forty-eight women of 7064 pregnant women (3.5%) who were screened at monthly antenatal clinics had a P. falciparum mono-infection and were randomized to receive AS+SP (125) or AS+MQ (123) and all of these women were included in the intention to treat (ITT) analysis. The primary endpoint of an adequate clinical and parasite response (ACPR) on day 63 was not available for 9 women who were counted as treatment failure in the ITT analysis. In the ITT population, the ACPR was 121/125 (96.8%; 95% Confidence interval (CI) 92.0-99.1%) in the AS+SP group and 117/123 (95.1%; 95% CI 89.7-98.2) in the AS+MQ group. Among the 239 women (121 from the AS+SP arm and 118 from the AS+MQ arm) who completed the day 63 follow up (per protocol analysis) the ACPR was 100% in the AS+SP group and 99.2% (117/118) in the AS+MQ group. There were five serious adverse events (SAE) among pregnant women (4 in the AS+SP group and 1 in the AS+MQ group) and 13 fetal/neonatal SAEs (7 in the AS+SP group and 6 in the AS+MQ) but none of them were related to the study drugs. A higher proportion of women in the AS+MQ arm reported vomiting within 7 days post-treatment than did women in the AS+SP arm (6.9 vs. 1.6%; p = 0.001). CONCLUSION: Both AS+SP and AS+MQ are safe and effective for treatment of uncomplicated falciparum malaria in pregnancy in India. Trial registration CTRI This study is registered with Clinical Trial Registry India (CTRI), number CTRI/2009/091/001055. Date of Registration 11 January 2010, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=1185&EncHid=&userName=anvikar.

Spatio-temporal distribution of PfMDR1 polymorphism among uncomplicated Plasmodium falciparum malaria cases along international border of north east India.

PfMDR1 single nucleotide polymorphisms (SNP) are good correlate markers for antimalarial drug resistance worldwide. Present study is a comprehensive view of screening of PfMDR1 polymorphism to antimalarials practiced with geography and time. Study sites Mizoram, Tripura, Meghalaya chosen are at multivariate drug pressure due to cross border migration and transmission. Mizoram is gateway to south east Asia through Myanmar whereas Tripura, Meghalaya share porous border with Bangladesh. Baseline finger pricked blood stained filter paper for confirmed uncomplicated Plasmodium falciparum infected patients (year 2015) were obtained from National Institute of Malaria Research, New Delhi, India. PfMDR1 polymorphism for codon N86Y, Y184F, D1246Y was determined by PCR-RFLP, further confirmed by sequencing. There observed marked predominance of Plasmodium isolates with PfMDR1 wild type alleles for all codons under study i.e. 86, 184, 1246. Spatially, Plasmodium isolates from Mizoram were most diverse with co-existence of PfMDR1 genotype with NYD, YYD, NFD haplotypes, followed by Tripura. Isolates from Meghalaya were of all NYD haplotype. Reports, referring to screening of PfMDR1 SNPs to CQ/SP/AS-SP across India, were archived. Temporal study show distinct rise in proportion of PfMDR1 wild type N86 allele since introduction of Artemether-Lumefantrine as first line antimalarial. Hence spatio-temporal screening of Plasmodium population with PfMDR1 single nucleotide polymorphism accounts for its association with antimalarial susceptibility and validate PfMDR1 SNPs as antimalarial drug resistant marker.

Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum.

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/µl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Correlation of in vitro sensitivity of chloroquine and other antimalarials with the partner drug resistance to Plasmodium falciparum malaria in selected sites of India.

BACKGROUND: Antimalarial drug resistance is a potential threat for control and elimination of malaria. To ascertain the status of antimalarial drug resistance at the study sites, correlation between in vitro drug sensitivity pattern and drug resistance molecular markers in Plasmodium falciparum malaria was undertaken. MATERIALS AND METHODS: Polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt) K76T and pfmdr1 N86Y were studied in relation to the in vitro susceptibility of P. falciparum in culture (n = 10) and field isolates (n = 40) to chloroquine (CQ), amodiaquine (AQ), quinine (QN), mefloquine (MQ) and artemisinin (ART). The prevalence of drug resistance molecular markers, pfdhfr (codon S108N, C59R, N51I, I164 L and A16V), pfdhps (codon S436F and A437G), pfATPase6 (codon D639G and E431K) and mutation in the propeller domain of pfK13 gene were also analysed. Chi-square test and parametric Pearson correlation test were performed using SPSS version 17. RESULTS: In vitro assay showed 18% resistance to CQ, 8% to AQ and 4% to QN. However, no resistance was observed towards MQ and ART. The mutations in pfcrt and pfmdr1 were statistically not significantly associated with susceptibility responses for antimalarials; however, increased IC50values of drugs were reflected as mutant and/or mixed isolates for both gene polymorphisms. CQ was found as independent predictor for other antimalarials, i.e., AQ, QN and ART, with r2 score 0.241, 0.241 and 0.091, respectively. Mutation in the pfATPase6 gene at codon E431K was observed in only one sample from Tripura which also had increased IC50value of 6.28 nM. However, moderate numbers of mutations at codon S108N, C59R and I164 L for pfdhfr gene and S436F and A437G for pfdhps gene were also observed. None of the samples showed mutation in propeller domain of pfK13 gene. CONCLUSION: The correlation between IC50and molecular markers for antimalarial drug resistance is reported for the first time through this study. A positive correlation between in vitro drug resistance with molecular markers for antimalarial drug resistance could make in vitro assay a reliable tool to predict drug efficacy which is needed for detection of emerging resistance in the country.

Comparison of WHO Mark III and HRP II ELISA for in vitro sensitivity of Plasmodium falciparum.

BACKGROUND & OBJECTIVES: Antimalarial drug resistance is a serious challenge to malaria control worldwide. In vitro sensitivity assays provide an early indication of emerging drug resistance. In vitro susceptibility of field and culture adapted Plasmodium falciparum isolates to different antimalarials was compared using two Methods: World Health Organization (WHO) micro-test (MARK III) and histidine rich protein II (HRP II) based enzyme- linked immunosorbent assay (ELISA). METHODS: In total, 50 P. falciparum isolates were collected from five states, viz. Chhattisgarh, Meghalaya, Mizoram, Tripura and Odisha of India during December 2011-September 2014. The isolates were revived and evaluated for their susceptibility to chloroquine (CQ), monodesethylamodiaquine (AQ), mefloquine (MQ), quinine (QN) and artemisinin (ART) using the WHO micro-test (Mark III) and HRP II ELISA. The data were analyzed using non- linear regression analysis. RESULTS: The geometric mean (GM) IC50 values of different antimalarials for WHO Mark III assay were comparatively lower than HRP II ELISA assay. The GM IC50 value for CQ was 59.5 nM (95% confidence interval [CI]: 49.35-71.73 nM) and 78.34 nM (95% CI: 64.57-95.03 nM) for Mark III and HRP II ELISA, respectively. Similarly, the values of GM IC50 for AQ, MQ, QN and ART by Mark III and HRP II ELISA were 13.31, 7.07, 146.4, 0.43 nM and 22.02, 11.46, 258.7, 1.00 nM, respectively. On analyzing statistically, the results of both assays were comparable (R2 = 0.96, p < 0.001; mean log difference at IC50= 0.037). INTERPRETATION & CONCLUSION: The HRP II ELISA assay showed a reliable sensitivity in comparison to WHO Mark III micro-test complemented with distinguishing features such as high specificity, ease of performance, and notable consistency.