Search

In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.

Plasmodium falciparum sarcoplasmic reticulum Ca 2+ ATPase (PfSERCA) is sarcoplasmic reticulum membrane bound transporter to regulate cytosol Ca 2+ ions. Ca 2+ 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sulphadoxine and pyrimethamine (SP) have been used as long-acting partner antimalarial drugs in artemisinin combination therapy (ACT) for falciparum malaria. The emergence and increasing spread of SP resistance in malaria-endemic areas have become a challenge for the control of malaria. Therefore, regular monitoring of the mutation status of partner drugs is important for the better management of drug policy. There are limitations with traditional molecular methods and there is an urgent need for an easy method for diagnosis of drug resistance. In this study we have introduced and developed a novel single nucleotide polymorphism loop-mediated isothermal amplification (SNP-LAMP) approach based on a hydroxynaphthol blue (HNB) indicator for the easier and quicker detection of pyrimethamine resistance in Plasmodium falciparum malaria. To implement this novel approach, many sets of LAMP primers were designed and tested. Finally, one set of forward inner primer M1 (FIPM1) of LAMP primer was selected that specifically distinguishes pyrimethamine-resistant P. falciparum malaria. The LAMP reactions were optimized at 60-66 °C for 45 min. High sensitivity (7 parasites/µL) was observed with 10 -4 fold dilutions (<2 ng DNA) of genomic DNA. Moreover, this approach has the potential to be applied even in laboratories unfamiliar with PCR or other molecular methods, and in future, this can be helpful for the better management of anti-malarial policy.

Novel 4-amino-7-chloroquinoline-based 1,2,3-triazole hybrids were synthesised in good yields by Cu I -catalysed Huisgen 1,3-dipolar cycloaddition reactions of 2-azido-N-(7-chloroquinolin-4-ylaminoalkyl)acetamides with various terminal alkynes. These new hybrids were screened in vitro against asexual blood stages of the chloroquine-sensitive 3D7 strain of P. falciparum. The most active compounds were further screened against asexual and sexual stages (gametocytes) of the chloroquine-resistant RKL-9 strain of P. falciparum. Although all compounds were less potent than chloroquine against the 3D7 strain, the three best compounds were appreciably more active than chloroquine against the RKL-9 strain, displaying IC 50 values of <100 nm, with one of them having an IC 50 of 2.94 nm. Further, the lead compounds were gametocytocidal with IC 50 values in the micromolar range, and were observed to induce morphological deformations in mature gametocytes. Most compounds demonstrated little or no cytotoxicity and exhibited good selectivity indices. The most active compounds represent promising candidates for further evaluation of their schizonticidal and gametocytocidal potential., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient best method for the rapid, sensitive, cost-effective, and fairly user friendly tool for detection of K76T mutants of P. falciparum and therefore presents an alternative to PCR-based assays. Based on our comparative analysis, better field based LAMP visualization method can be chosen easily for the monitoring of other important drug targets (Kelch13 propeller region)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening., Methods: Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination., Results: Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC 50 (early stages) as 424.1 nM and mean IC 50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations., Conclusions: Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field isolates and is proposed to be used as a gametocytocidal adjunct with artemisinin-based combination therapy. Further exploration of methylene blue in clinical studies amongst Indian population, including G6PD deficient patients, is recommended.

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.

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., Competing Interests: The authors declare no competing financial interests.

Background & Objectives: Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid based diag- nostic approach that is easily adaptable to the field settings with limited technical resources. This study was aimed to evaluate the LAMP assay for the detection and identification of Plasmodium falciparum and P. vivax infection in malaria suspected cases using genus and species-specific assay., Methods: The 18S rRNA-based LAMP assay was evaluated for diagnosis of genus Plasmodium, and species- specific diagnosis of P. falciparum and P. vivax, infection employing 317 malaria suspected cases, and the results were compared with those obtained by 18S nested PCR (n-PCR). All the samples were confirmed by microscopy for the presence of Plasmodium parasite., Results: The n-PCR was positive in all Plasmodium-infected cases (n=257; P. falciparum=133; P. vivax=124) and negative in microscopy negative cases (n=58) except for two cases which were positive for P. vivax, giving a sen- sitivity of 100% (95% CI: 97.04-100%) and a specificity of 100% (95% CI: 88.45-99.5%). Genus-specific LAMP assay missed 11 (3.2%) microscopy and n-PCR confirmed vivax malaria cases. Considering PCR results as a refer- ence, LAMP was 100% sensitive and specific for P. falciparum, whereas it exhibited 95.16% sensitivity and 96.7% specificity for P. vivax. The n-PCR assay detected 10 mixed infection cases while species-specific LAMP detected five mixed infection cases of P. vivax and P. falciparum, which were not detected by microscopy., Interpretation & Conclusion: Genus-specific LAMP assay displayed low sensitivity. Falciparum specific LAMP assay displayed high sensitivity whereas vivax specific LAMP assay displayed low sensitivity. Failed detection of vivax cases otherwise confirmed by the n-PCR assay indicates exploitation of new targets and improved detection methods to attain 100% results for P. vivax detection.

Background: Recent reports of emergence and spread of artemisinin resistance in the Southeast Asia region, including Myanmar, pose a greater threat to malaria control and elimination in India. Whole genome sequencing studies have associated mutations in the K13 propeller gene (k13), PF3D7&#95;1343700 with artemisinin resistance both in vitro and in vivo. The aim of the present study was to find the k13 gene polymorphisms in Plasmodium falciparum parasites from the three sites in the Northeast region of India, bordering Bangladesh and Myanmar., Methods: A total of 254 samples collected during 2014-2015 from Tripura, Mizoram and Arunachal Pradesh states in the Northeast region of India were used to obtain the full-length k13 gene sequences., Results: Three non-synonymous (NS) mutations: two in the propeller region, namely at codon 446 and 578, were observed besides one at codon 189 in the non-propeller region. The treatment outcome was not affected by these mutations at any of the sites. In addition, microsatellite variation in the N-terminus of the k13 protein was observed at all the study sites., Conclusion: This is the first study to document the presence of F446I NS mutation in the k13 propeller region from Changlang district, Arunachal Pradesh, a site adjoining the Indo-Myanmar border region, where this mutation is highly prevalent. In addition, NS mutation A578S has been observed only at Lunglei district, Mizoram, a site bordering Bangladesh and K189T mutation with relatively higher frequency in Mizoram and Tripura states. The presence of F446I mutation in a region close to the Myanmar border is notable. Considering the spread of anti-malarial drug resistance from Southeast Asia to the Northeast region of India in the past, there is an urgent need to undertake systematic mapping studies to ascertain the role and extent of this mutation in artemisinin resistance in this region of country.

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.

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.

Background & Objectives: Malaria has become endemic and subject of concern in most part of the India especially Northeastern states of India. Surveys before 2000 revealed that Anopheles minimus was major vector responsible for transmission of malaria in this region followed by An. dirus and An. fluviatilis. However, recent studies indicate replacement of An. minimus vector by An. culicifacies due to different ecological changes and change in landuse pattern etc. The objective of present study was to explore the vectorial role of An. culicifacies in transmission of malaria in four malaria endemic states, viz. Assam, Meghalaya, Manipur and Sikkim of India., Methods: The seven surveys were conducted in 176 selected villages belonging to eight districts of the four states in both pre-monsoon (March-April) and post-monsoon (September-October) seasons from 2010 to 2013. However, in 2011 surveys could not be carried out due to public inconvenience in pre-monsoon season. For vectorial role of all vector species collected, ELISA and PCR were assayed., Results: A total of 19,173 specimens belonging to 30 anopheline species were collected, out of which 4315 belonged to four established vector species. In total, 4183 specimens were processed through ELISA, out of which 236 specimens were found positive for circumsporozoite (CS) protein. Further, infectivity was confirmed by PCR in 35 samples, of which 12 samples were found positive for Plasmodium falciparum and three for P. vivax. Out of 12 Plasmodium falciparum positive samples, nine samples were positive for An. culicifacies, two for An. fluviatilis and one for An. minimus. While out of three Plasmodium vivax positive samples, two samples were positive for An. dirus and one sample was positive for An. culicifacies., Interpretation & Conclusion: Anopheles culicifacies replaced the An. minimus, the vector of malaria in Northeastern states of India, as it was found to be highly infected with malaria parasite as compared to An. minimus by ELISA and PCR analysis, and thus playing a major role in malaria transmission in this region. The ecological changes like deforestation, development of irrigation channels and change in landuse pattern, have helped in evolution of An. culicifacies in the study area. Therefore, modified vector control strategies are required on urgent basis.

Background & Objectives: To combat the problem of antimalarial drug resistance, monitoring the changes in drug efficacy over time through periodic surveillance is essential. Since 2009, systematic and continuous monitoring is being done through nationwide sentinel site system. Potential early warning signs like partner drug resistance markers were also monitored in the clinical samples from the study areas., Methods: A total of 1864 patients with acute uncomplicated malaria were enrolled in therapeutic efficacy studies of artesunate plus sulphadoxine-pyrimethamine (AS+SP) for Plasmodium falciparum; those infected with P. vivax were given chloroquine (CQ). Polymerase chain reaction (PCR) was used to distinguish post-treatment reinfection from treatment failures. Isolates of P. falciparum were also analysed for dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) gene mutations., Results: Overall, 1687 (91.7%) patients completed the follow-up. In most of the falciparum patients the parasitaemia was cleared within 24 h of treatment, except 12 patients who remained parasite positive after 72 h. Presence of dhfr and dhps quintuple mutation was observed predominantly in treatment failure samples. A daily dose of artesunate of < 3 mg/kg of body weight, age of <5 yr, and fever at enrolment were associated with an increased risk of treatment failure. The AS+SP in P. falciparum was effective in > 95% cases in all the sentinel sites except in Northeastern region (NE). Chloroquine remained 100% efficacious in case of P. vivax infections., Interpretation & Conclusion: Till 2012, India's national antimalarial drug resistance monitoring system proved highly efficacious and safe towards first-line antimalarials used in the country, except in Northeastern region where a decline in efficacy of AS+SP has been observed. This led to change in first-line treatment for P. falciparum to artemether-lumefantrine in Northeastern region.

Background: Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought., Methods: Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media., Results: At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients., Conclusions: Culture adaptation of P. falciparum in a field setting is formally shown to be robust. Parasites were found to have intrinsic variations in adaptability to culture conditions, with some lines requiring longer attempt periods for successful adaptation. Quantitative approaches described here can help describe phenotypic diversity of field parasite collections with precision. This is expected to improve population-based extrapolations of findings from field-derived fresh culture-adapted parasites to broader questions of public health importance.

Genotyping the sulfadoxine-pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Sulfadoxine-pyrimethamine (SP) combination drug is currently being used in India for the treatment of Plasmodium falciparum as partner drug in artemisinin-based combination therapy (ACT). Resistance to sulfadoxine and pyrimethamine in P. falciparum is linked with mutations in dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes respectively. This study was undertaken to estimate the prevalence of such mutations in pfdhfr and pfdhps genes in four states of India., Methods: Plasmodium falciparum isolates were collected from two states of India with high malaria incidence i.e., Jharkhand and Odisha and two states with low malaria incidence i.e., Andhra Pradesh and Uttar Pradesh between years 2006 to 2012. Part of sulfadoxine-pyrimethamine (SP) drug resistance genes, pfdhfr and pfdhps were PCR-amplified, sequenced and analyzed., Results: A total of 217 confirmed P. falciparum isolates were sequenced for both Pfdhfr and pfdhps gene. Two pfdhfr mutations 59R and 108N were most common mutations prevalent in all localities in 77 % of isolates. Additionally, I164L was found in Odisha and Jharkhand only (4/70 and 8/84, respectively). Another mutation 51I was found in Odisha only (3/70). The pfdhps mutations 436A, 437G, 540E and 581G were found in Jharkhand and Odisha only in 13, 26, 14 and 13 % isolates respectively, and was absent in Uttar Pradesh and Andhra Pradesh. Combined together for pfdhps and pfdhfr locus, triple, quadruple, quintuple and sextuple mutations were present in Jharkhand and Odisha while absent in Uttar Pradesh and Andhra Pradesh., Conclusion: While only double mutants of pfdhfr was present in low transmission area (Uttar Pradesh and Andhra Pradesh) with total absence of pfdhps mutants, up to sextuple mutations were present in high transmission areas (Odisha and Jharkhand) for both the genes combined. Presence of multiple mutations in pfdhfr and pfdhps genes linked to SP resistance in high transmission area may lead to fixation of multiple mutations in presence of high drug pressure and high recombination rate.

Antimalarial drug resistance including artemisinin resistance in Plasmodium falciparum malaria is a major concern in combating malaria throughout the world. Delayed parasite clearance time (PCT) is indicative of emergence of artemisinin resistance. Herein, PCT has been monitored with the help of gold standard technique microscopy accompanied by a more sensitive real-time assay for academic purpose. After the administration of artemisinin based combination therapy, artesunate + sulfadoxine pyrimethamine (AS + SP), all the subjects were followed up to day 42 for monitoring the therapeutic efficacy of AS + SP in Bisra Community Health Centre (CHC), Sundergarh district in the state of Odisha in India. Further, representative samples were analyzed for L263E, E431K, A623E and S769N SNPs in Pfatpase6 gene and copy number polymorphisms in Pfmdr1 gene. Though all the samples were found parasite negative according to microscopy by the end of day 3 and attained adequate clinical and parasitological response (ACPR) at the end of day 42, real-time PCR showed day 3 positivity in 12 of the total analyzed samples (n = 43). This was further validated by end-point diagnostic PCR and correlated with high initial parasite load. E431K mutation was observed in 2 of the 12 samples (16.7 %) while the controls (n = 18) were all wild. L263E, A623E and S769N were wild in all the analyzed samples (n = 30). Pfmdr1 copy number analysis showed no change in the said trait. Conclusively, real-time PCR could support microscopy for better monitoring of PCT and may provide as an additional but useful research tool for artemisinin resistance studies.

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies., Methods: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined., Results: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days., Conclusions: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

Background: Anti-malarial drug resistance in Plasmodium falciparum in India has historically travelled from northeast India along the Myanmar border. The treatment policy for P. falciparum in the region was, therefore, changed from chloroquine to artesunate (AS) plus sulphadoxine-pyrimethamine (SP) in selected areas in 2005 and in 2008 it became the first-line treatment. Recognizing that resistance to the partner drug can limit the useful life of this combination therapy, routine in vivo and molecular monitoring of anti-malarial drug efficacy through sentinel sites was initiated in 2009., Methods: Between May and October 2012, 190 subjects with acute uncomplicated falciparum malaria were enrolled in therapeutic efficacy studies in the states of Arunachal Pradesh, Tripura, and Mizoram. Clinical and parasitological assessments were conducted over 42 days of follow-up. Multivariate analysis was used to determine risk factors associated with treatment failure. Genotyping was done to distinguish re-infection from recrudescence as well as to determine the prevalence of molecular markers of antifolate resistance among isolates., Results: A total of 169 patients completed 42 days of follow-up at three sites. The crude and PCR-corrected Kaplan-Meier survival estimates of AS + SP were 60.8% (95% CI: 48.0-71.4) and 76.6% (95% CI: 64.1-85.2) in Gomati, Tripura; 74.6% (95% CI: 62.0-83.6) and 81.7% (95% CI: 69.4-89.5) in Lunglei, Mizoram; and, 59.5% (95% CI: 42.0-73.2) and 82.3% (95% CI: 64.6-91.6) in Changlang, Arunachal Pradesh. Most patients with P. falciparum cleared parasitaemia within 24 hours of treatment, but eight, including three patients who failed treatment, remained parasitaemic on day 3. Risk factors associated with treatment failure included age < five years, fever at the time of enrolment and AS under dosing. No adverse events were reported. Presence of dhfr plus dhps quintuple mutation was observed predominantly in treatment failure samples., Conclusion: AS + SP treatment failure was widespread in northeast India and exceeded the threshold for changing drug policy. Based on these results, in January 2013 the expert committee of the National Vector Borne Disease Control Programme formulated the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast. Continued monitoring of anti-malarial drug efficacy is essential for effective malaria control.

Background: The resistance of human malaria parasites to anti-malarial compounds has become considerable concern, particularly in view of the shortage of novel classes of anti-malarial drugs. One way to prevent resistance is by using new compounds that are not based on existing synthetic antimicrobial agents., Results: Sensitivity of 100 Plasmodium falciparum isolates to chloroquine, quinine, amodiaquine, mefloquine, sulphadoxine/pyrimethamine, artemisinin, Momordica charantia ('Ejirin') Diospyros monbuttensis ('Egun eja') and Morinda lucida ('Oruwo') was determined using the in vitro microtest (Mark III) technique to determine the IC50 of the drugs. All the isolates tested were sensitive to quinine, mefloquine and artesunate. Fifty-one percent of the isolates were resistant to chloroquine, 13% to amodiaquine and 5% to sulphadoxine/pyrimethamine. Highest resistance to chloroquine (68.9%) was recorded among isolates from Yewa zone while highest resistance to amodiaquine (30%) was observed in Ijebu zone. Highest resistance to sulphadoxine/pyrimethamine was recorded in Yewa and Egba zones, respectively. A positive correlation was observed between the responses to artemisinin and mefloquine (P<0.05), artemisinin and quinine (P<0.05) and quinine and mefloquine (P<0.05). A negative correlation was observed between the responses to chloroquine and mefloquine (P>0.05). Highest anti-plasmodial activity was obtained with the ethanolic extract of D. monbuttensis (IC50 = 3.2 nM) while the lowest was obtained from M. lucida (IC50 = 25 nM)., Conclusions: Natural products isolated from plants used in traditional medicine, which have potent anti-plasmodial action in vitro, represent potential sources of new anti-malarial drugs.

The use of antimalarial drugs in India has evolved since the introduction of quinine in the 17 th century. Since the formal establishment of a malaria control programme in 1953, shortly after independence, treatments provided by the public sector ranged from chloroquine, the mainstay drug for many decades, to the newer, recently introduced artemisinin based combination therapy. The complexity of considerations in antimalarial treatment led to the formulation of a National Antimalarial Drug Policy to guide procurement as well as communicate best practices to both public and private healthcare providers. Challenges addressed in the policy include the use of presumptive treatment, the introduction of alternate treatments for drug-resistant malaria, the duration of primaquine therapy to prevent relapses of vivax malaria, the treatment of malaria in pregnancy, and the choice of drugs for chemoprophylaxis. While data on antimalarial drug resistance and both public and private sector treatment practices have been recently reviewed, the policy process of setting national standards has not. In this perspective on antimalarial drug policy, this review highlights its relevant history, analyzes the current policy, and examines future directions.

The positive selection of a nucleotide substitution in exon 2 of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene (mutation responsible for chloroquine resistance) causes a reduction in variation of neutral loci close to the gene. This reduction in allelic diversity around flanking regions of pfcrt gene was reported in worldwide chloroquine resistant isolates and referred as selective sweep. In Plasmodium falciparum isolates of India, the selective sweep in flanking loci of pfcrt gene is well established, however, high allelic diversity observed in intragenic microsatellites of pfcrt gene implied an ongoing genetic recombination. To understand, if molecular evolution of chloroquine-resistant P. falciparum isolates in India follow a selective sweep model, we analyzed genetic diversity at both seven intragenic and seven flanking microsatellites of pfcrt (-24 to +106kb) gene in chloroquine sensitive and resistant parasites originating from high and low transmission areas. We observed low expected heterozygosity at all loci of resistant pfcrt-haplotypes (He=0-0.77) compared to the wild-type (He=0.38-0.96). Resistant SVMNT from high transmission areas showed significantly higher mean He (P=0.03, t-test) at both intragenic and pfcrt-flanking loci (-24 to +22 kb) in comparison to low transmission areas. Our observation of reduction in variation at both intragenic and flanking loci of mutant pfcrt gene confirmed the selective sweep model of natural selection in chloroquine resistant P. falciparum isolates in India., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background & Objectives: India has switched over to artemisinin-based combination therapy (ACT) for the treatment of acute uncomplicated Plasmodium falciparum malaria and the ACT used in the national programme is artesunate + sulphadoxine-pyrimethamine. Since the efficacy of ACT is dependent also on the partner drug, there is a need to evaluate and deploy multiple ACTs., Methods: This multicentre, single-arm, open-label clinical trial was carried out to assess the efficacy, safety and population pharmacokinetics of a fixed dose combination (FDC) artesunate mefloquine (ASMQ) in P. falciparum infected, Indian adults at Panjim, Goa, and Mangalore, Karnataka between December 2007 and November 2008., Results: A total of 77 patients (males 74) were screened and enrolled: 42 at Goa and 35 at Mangalore with a median age of 25 yr (range 18-55 yr). One patient failed in treatment on D53, a PCR proven new infection, seven developed recurrent vivax parasitaemia and 11 did not have a parasitological endpoint. By per protocol analysis, the D63 cure rate was 58/59 (98.3; 95% C.I. 90.9-99.9%), and 58/58, with PCR correction. ASMQ was well-tolerated and no serious adverse events were reported., Interpretation & Conclusion: The study showed that the ASMQ FDC was efficacious and well-tolerated for the treatment of acute, uncomplicated P. falciparum malaria in highly endemic, chloroquine resistant areas of Goa and Mangalore. It is a viable option for India.

Background: Artesunate + sulphadoxine-pyrimethamine (AS + SP) is recommended throughout India as the first-line treatment for uncomplicated falciparum malaria. Due to the presence of several eco-epidemiological zones of malaria and variable drug pressure, it is necessary to evaluate the efficacy of this combination in different regions of India. The objective of this study was to use clinical and molecular methods to monitor the efficacy of AS + SP in three diverse sites., Methods: The study was undertaken in three high endemic sites of central and eastern India. Patients with uncomplicated falciparum malaria were enrolled and followed for 28 days. Molecular genotyping was conducted for merozoite surface protein (msp1 and msp2) to differentiate between re-infection and recrudescence and for the dhfr and dhps genes to monitor antifolate drug resistance., Results: In all, 149 patients were enrolled at the three sites. The crude cure rates were 95.9%, 100%, and 100% in Ranchi, Keonjhar, and West Garo Hills respectively. PCR-corrected cure rates were 100% at all sites. In dhfr, 27% of isolates had triple mutations, while 46% isolates were double-mutants. The most prevalent mutation was S108N followed by C59R. 164 L mutation was observed in 43/126 (34%) isolates. In dhps, most (76%) of the isolates were wild-type. Only 2.5% (2/80) isolates showed double mutation. dhfr-dhps two locus mutation were observed in 16% (13/80) isolates. Parasite clearance time was not related with antifolate mutations., Conclusions: AS + SP combination therapy remained effective against falciparum malaria despite common mutations promoting resistance to antifolate drugs. Although the prevalence of double and triple mutations in dhfr was high, the prevalence of dhfr-dhps two locus mutations were low. Even isolates with dhfr triple and dhfr-dhps two locus mutations achieved adequate clinical and parasitological response.

Objective: To characterise the epidemiology of Plasmodium falciparum gametocytemia and determine the prevalence, age structure and the viability of a predictive model for detection., Methods: We collected data from 21 therapeutic efficacy trials conducted in India during 2009-2010 and estimated the contribution of each age group to the reservoir of transmission. We built a predictive model for gametocytemia and calculated the diagnostic utility of different score cut-offs from our risk score., Results: Gametocytemia was present in 18% (248/1 335) of patients and decreased with age. Adults constituted 43%, school-age children 45% and under fives 12% of the reservoir for potential transmission. Our model retained age, sex, region and previous antimalarial drug intake as predictors of gametocytemia. The area under the receiver operator characteristic curve was 0.76 (95%CI:0.73,0.78), and a cut-off of 14 or more on a risk score ranging from 0 to 46 provided 91% (95%CI:88,95) sensitivity and 33% (95%CI:31,36) specificity for detecting gametocytemia., Conclusions: Gametocytemia was common in India and varied by region. Notably, adults contributed substantially to the reservoir for potential transmission. Predictive modelling to generate a clinical algorithm for detecting gametocytemia did not provide sufficient discrimination for targeting interventions., (© 2013 Blackwell Publishing Ltd.)

Artemisinin combination therapies eliminate immature Plasmodium falciparum gametocytes but not mature gametocytes, which may persist for up to 1 month posttreatment. A single dose of primaquine, which is inexpensive and effective against mature gametocytes, could be added to further reduce the potential for posttreatment parasite transmission. Currently, we have few data regarding the effectiveness or safety of doing so. We collected data from 21 therapeutic efficacy trials of the National Antimalarial Drug Resistance Monitoring System of India conducted during 2009 to 2010, wherein 9 sites used single-dose primaquine (0.75 mg/kg of body weight) administered on day 2 along with artesunate plus sulfadoxine-pyrimethamine (AS+SP) while 12 did not. We estimated the effect of primaquine on posttreatment gametocyte clearance and the total number of gametocyte-weeks as determined by microscopy. We compared the median area under the curve for gametocyte density and reported adverse events. One thousand three hundred thirty-five patients completed the antimalarial drug treatment. Adjusting for region, primaquine increased the rate of gametocyte clearance (hazard ratio, 1.9; 95% confidence interval [CI], 1.1 to 3.3), prevented 45% (95% CI, 19 to 62) of posttreatment gametocyte-weeks, and decreased the area under the gametocyte density curve over the 28-day follow-up compared to AS+SP alone (P value = 0.01). The results were robust to other adjustment sets, and the estimated effect of primaquine increased during sensitivity analysis on the measurement of exposure time. No serious adverse events were detected. In conclusion, the addition of primaquine to AS+SP was effective in reducing the posttreatment presence of P. falciparum gametocytes. Primaquine was well tolerated and could be administered along with an artemisinin combination therapy as the first-line therapy.

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Plasmodium falciparum the main causative agent of malaria is an important public health vector. With the use of PCR, its genetic diversity has been extensively studied with dearth information from Nigeria., Methods: In this study, 100 P. falciparum strains merozoite surface protein 1(msp-1), merozoite surface protein 2 (msp-2) and Glutamate rich protein (Glurp) from Ogun State General Hospitals were characterized. The genetic diversity of P. falciparum isolates was analyzed by restriction fragment length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) of their respective allelic families KI, MAD 20, RO33 (MSP-1);FC27, 3D7 (MSP-2) and Glutamate rich protein respectively., Results: Majority of the patients showed monoclonal infections while multiplicity of the infection for msp-1 and msp-2 were 1.1 and 1.2 respectively. The estimated number of genotypes was 8 msp-1 (4 KI; 3 MAD; 1 RO33) and 6 msp-2 (3 FC27; 3 3D7). 80% of the isolates coded for Glurp with allelic size ranged between 700 and 900 bp., Conclusion: The allelic distributions however were similar to those previously reported in other endemic malaria countries. Future studies will be designed to include other malaria endemic regions of Nigeria such as the oil exploration regions.

Background: Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates., Methods: One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined., Results: Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs., Conclusion: The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.

Background & Objectives: In vitro assays are an important tool to assess baseline sensitivity and monitor the drug response of Plasmodium falciparum over time and place and, therefore, can provide background information for the development and evaluation of drug policies. This study was aimed at determining the in vitro sensitivity of P. falciparum isolates to antimalarials., Methods: The in vitro activity of 108 P. falciparum isolates obtained from five States of India was evaluated using WHO microtest (Mark III) to chloroquine, monodesethylamodiaquine, dihydroartesunate and mefloquine. Samples were collected from the States of Orissa, Jharkhand, Karnataka, Goa and Chhattisgarh from September 2007 to August 2009. In addition, representative samples from different States of India cryopreserved and culture adapted in the Malaria Parasite Bank of National Institute of Malaria Research, New Delhi, were also evaluated., Results: The proportion of isolates resistant to chloroquine and monodesethylamodiaquine was 44.4 and 25 per cent, respectively. Of the 27 isolates resistant to monodesethylamodiaquine, 16 (59.3%) were cross-resistant to chloroquine. No isolate showed resistance to dihydroartesunate and mefloquine. Isolates from Orissa showed the highest degree of resistance to chloroquine and amodiaquine followed by Jharkhand. Forty two isolates were genotyped for pfcrt T76K chloroquine resistant mutation; mutations were seen in 38 (90.47%) isolates., Interpretation & Conclusions: The Indian P. falciparum isolates showed a high degree of resistance to chloroquine followed by monodesethylamodiaquine. No resistance was recorded to mefloquine and dihydroartesunate.

After the launch of the National Malaria Control Programme in 1953, the number of malaria cases reported in India fell to an all-time low of 0·1 million in 1965. However, the initial success could not be maintained and a resurgence of malaria began in the late 1960s. Resistance of Plasmodium falciparum to chloroquine was first reported in 1973 and increases in antimalarial resistance, along with rapid urbanisation and labour migration, complicated the challenge that India's large geographical area and population size already pose for malaria control. Although several institutions have done drug-resistance monitoring in India, a complete analysis of countrywide data across institutions does not exist. We did a systematic review of P falciparum malaria drug-efficacy studies in India to summarise drug-resistance data and describe changes over the past 30 years to inform future policy. Continued use of chloroquine for treatment of P falciparum malaria in India will likely be ineffective. Resistance to sulfa-pyrimethamine should be closely monitored to protect the effectiveness of treatment with artesunate plus sulfadoxine-pyrimethamine, which is the new first-line treatment for P falciparum malaria. Strategies to reduce the emergence and spread of future drug resistance need to be proactive and supported by intensive monitoring., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Drug-resistant Plasmodium falciparum malaria necessitates development of novel drugs for treatment.The present study assessed the efficacy and safety of 3 dose levels of arterolane (RBx 11160), a synthetic trioxolane, for treatment of acute uncomplicated falciparum malaria., Methods: In this randomized, double-blind, multicenter, parallel-group, dose-finding, phase II trial, 230 patients from 4 centers in Thailand, India, and Tanzania (mainland and Zanzibar) received either 50 mg (n=78), 100mg (n=76), or 200 mg (n=76) of arterolane once daily for 7 days. Patients (aged 13-65 years) with asexual parasite density of 1000-100,000 parasites/microL were included and were followed up for 28 days. The median time to 90% parasite clearance (PC90) was evaluated., Results: The median PC90 was longer in the group receiving the 50-mg dose (19.4 h), compared with the groups receiving the 100-mg dose (12.8 h) and 200-mg dose (12.6 h) (P < .01). The polymerase chain reaction-corrected adequate clinical and parasitological responses on day 28 were 63%, 71%, and 72% for the groups receiving the 50-mg, 100-mg, and 200-mg doses, respectively, by intention-to-treat analysis (odds ratio, 1.55; 95%confidence interval, 0.78-3.06, for comparison of the 200-mg and 50-mg dose groups). Treatment was generally well tolerated. No patient died or experienced any serious adverse event. Mild complaints were reported in <10%of the patients and were similar in the 3 groups. Biochemistry and hematological analyses did not show any signof drug toxicity in any patient., Conclusion: Arterolane at daily doses of 100 and 200 mg is a rapidly acting, effective, and safe synthetic antimalarial drug, which may potentially represent an alternative to artemisinin derivatives in antimalarial combination therapy., Trial Registration: ClinicalTrials.gov identifier NCT00362050.

Drug resistance in Plasmodium falciparum poses a major threat to malaria control globally; including India. Chloroquine is still the most widely used drug in the country because of its safety and cost effectiveness. Although chloroquine resistance was first reported in 1973 in North Eastern India, the extent of the problem was realized only after the more intensive 28-day drug efficacy studies were used to monitor drug resistance. In the present study, efficacy of chloroquine in treatment of uncomplicated falciparum malaria was investigated using standard World Health Organization (WHO) procedures in three distinct epidemiological settings. The prevalence of molecular markers of drug resistance, Pfcrt K76T, Pfmdr1 N86Y, was also studied. A total of 374 children and adults with uncomplicated P. falciparum malaria were enrolled at six sites in four states, treated with chloroquine and follow-up was done for 28 days. The cumulative incidence of success of chloroquine at Day 28 by the Kaplan Meier analysis in the state of Orissa (District Sundargarh, CHC Bisra and Kuarmunda) was 57 (95% CI 43-68) and 54 (95% CI 40-66); in the state of Jharkhand (District Ranchi, PHC Angara and District Simdega, PHC Jaldega) it was 72 (95% CI 59-81) and 65 (95% CI 50-76); in the state of Goa (District North-Goa, Panaji Town), it was 20 (95% CI 10-2) and in the state of Rajasthan (District Udaipur, PHC Rishabdev), it was 96 (95% CI 85-99). Treatment failure was related to Pfcrt mutations but not Pfmdr mutations. Early treatment failure was observed only in 15.8% out of total failures, probably due to the semi-immune nature of the population. This type of response may give false perception about efficacy of the failing drug to patients, clinicians and National Authorities. In a large country like India it is not feasible to conduct in vivo studies in all districts and lack of direct correlation between molecular markers, in vitro studies and treatment outcome makes it difficult to predict the areas requiring change of policy. In this scenario, it is a challenge for National Programmes to make evidence-based revisions in the drug policy. However, considering the global, especially Southeast Asian, scenario and interpretation of available in vivo data, trends of mutations, availability of effective drugs and support of international donors, India should consider changing the first line treatment, at least for all diagnosed P. falciparum cases.

Background: Artemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 x 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required., Methods: One hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers msp-1 and msp-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in pfmdr1 was also carried out in the samples obtained from patients before and after the treatment., Results: The PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of pfmdr1 gene., Conclusion: AL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in pfmdr1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.

Background: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India., Methods: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa., Results: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains., Conclusion: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Four xanthones were isolated from the roots of Andrographis paniculata using a combination of column and thin-layer chromatographic methods. They were characterized as (i) 1,8-di-hydroxy-3,7-dimethoxy-xanthone, (ii) 4,8-dihydroxy-2,7-dimethoxy-xanthone, (iii) 1,2-dihydroxy-6,8-dimethoxy-xanthone and (iv) 3,7,8-trimethoxy-1-hydroxy xanthone by IR, MS and NMR spectroscopic methods. In vitro study revealed that compound 1,2-dihydroxy-6,8-dimethoxy-xanthone possessed substantial anti-plasmodial activity against Plasmodium falciparum with its IC(50) value of 4 microg ml(-1). Xanthones bearing hydroxyl group at 2 position demonstrated most potent activity while xanthones with hydroxyl group at 1,4 or 8 position possessed very low activity. In vivo anti-malarial sensitivity test of this compound on Swiss Albino mice with Plasmodium berghei infection using Peters' 4-day test gave substantial reduction (62%) in parasitaemia after treating the mice with 30 mg kg(-1) dose. In vitro cytotoxicity against mammalian cells revealed that 1,2-dihydroxy-6,8-dimethoxy-xanthone is non-cytotoxic with its IC(50) > 32 microg ml(-1).

A standardised protocol has been developed by World Health Organization (CDS/RBM/2002) to assess the efficacy of common antimalarials in the treatment of clinically manifested infection with uncomplicated P. falciparum malaria for areas with low to moderate transmission. The therapeutic efficacy protocol is based on clinical and parasitological responses of the patients and it has the purpose of determining the practical efficacy of the drug regimen in study areas with the ultimate objective of ascertaining its continued usefulness or the necessity for replacing it in the routine treatment. Present study has been conducted at seven sites--Kathiatali and Simonabasti of District Nowgaon, Assam; Sonapur and Boko of District Kamrup, Assam; Keonjhar Town, Padampur and Basudebpur of District Keonjhar, Orissa. In order to reduce the patient recruitment time, health centre close to well-defined community was identified to conduct the activities at peak malaria season by selecting local pockets and organising mobile clinics. Microscopically confirmed cases of P. falciparum were enrolled according to the criteria for inclusion and exclusion. Treatment with recommended drug was given under supervision and a follow-up schedule at various intervals for 28 days was maintained. In chloroquine (CQ) study areas, wherever patients showed treatment failure, they were treated with second line drug--sulphadoxine-pyrimethamine (SP) combination and then followed-up as per study protocol. It was observed that 30% cases showed treatment failure to CQ in District Nowgaon, where revised drug policy has already been introduced. In Kamrup district, treatment failure with CQ was found to be less than 25%, which denotes the said regimen is still effective. Almost all the patients from Padampur and Basudebpur of District Keonjhar responded to CQ, treatment failure was noticed only in two patients (3%). The antifolate combination found to be fully effective as second line and also as first line wherever revised drug policy has been introduced.

A rapid new immunochromatographic test (ICT malaria P.f/P.v) for diagnosis of Plasmodium falciparum and P.vivax was evaluated against thick blood smears in forest villages of Chhindwara, Madhya Pradesh, where both Plasmodium falciparum and P.vivax are prevalent. 344 symptomatic patients (Gond ethnic tribe) in five villages were screened by field staff of the Malaria Research Centre in October 1999. For P.falciparum, the ICT was 97.5% sensitive and 88% specific, with a positive predictive value (PPV) of 87.6% and a negative predictive value (NPV) of 97.6%. For P.vivax the sensitivity was only 72%, the specificity 99%, with a PPV of 92% and an NPV of 96%. Although a negative test result was inadequate to exclude parasitaemia < or = 300/microl for P.falciparum and < or = 1500/microl for P.vivax, the test is potentially useful in remote areas.

Several Baylis-Hillman adducts and their derivatives were synthesized and evaluated as targeted potential anti-malarials. The compounds 4, 7 and 9 were found to have highest potency against P. falciparum in vitro. The in vivo test result of compound 4 and 9 against P. berghei demonstrated activity at 80 mg/Kg dose level.

A non-radioactive, thymidine analogue-bromodeoxyuridine (Brdu), derivative of uridine has been used for incorporation in DNA in culture of P. falciparum at various dosages and at different time period. Parasite growth rate and effect of chloroquine in culture were monitored by microscopic observation of stained smears and incorporation of Brdu molecules were visualized by immunofluorescence and measured by enzyme immuno assay using anti-Brdu. Uptaking of Brdu in parasite is slower unlike tumour cells. A positive correlation between parasite growth and Brdu uptake measurement by ELISA has been observed.

Synthetic analogues of a monoterpenic fragment of aplasmomycin were tested for their antimalarial activity in Plasmodium falciparum culture in vitro. The antimalarial activities of these agents were evaluated in chloroquine sensitive strains. Parasite growth was inhibited in a dose dependent manner in the presence of the synthetic compounds (3-9).

The in vitro activity of three fluoroquinolones--ciprofloxacin, norfloxacin and ofloxacin--was studied on four laboratory-adapted strains (one chloroquine-resistant) and one fresh isolate of P. falciparum from Delhi by the schizont maturation inhibition microtest. The IC50 concentrations (mean +/- SD) were found to be as: ciprofloxacin 6.38 +/- 1.34 micrograms/ml, norfloxacin 11.24 +/- 1.27 micrograms/ml, and ofloxacin 22.3 +/- 3.11 micrograms/ml, while the MIC values were 32 micrograms/ml, 64 micrograms/ml and 128 micrograms/ml for the three drugs in the same order. The IC50 and MIC values for chloroquine-resistant strain were not significantly different from those for the chloroquine-sensitive strains. We conclude that there is little interstrain variability in the in vitro susceptibility of P. falciparum to fluoroquinolones, and that there is no cross resistance between them and chloroquine. The reported variability in clinical response of falciparum malaria to fluoroquinolones is not likely to be due to variation in parasite sensitivity.

Abstract Background Resistance in Plasmodium falciparum to commonly used anti-malarial drugs, especially chloroquine, is being increasingly documented in India. By 2007, the first-line treatment for uncomplicated malaria has been revised to recommend artemisinin-based combination therapy (ACT) for all confirmed P. falciparum cases. Objective The objective of this study was to compare the efficacy, safety and tolerability between dihydroartemisinin-piperaquine (DP) and artesunate plus mefloquine (A + M) drug combinations in the treatment of uncomplicated P. falciparum malaria in India. Methods Between 2006 and 2007, 150 patients with acute uncomplicated P. falciparum malaria were enrolled, randomized to DP (101) or A + M (49) and followed up for 63 days as part of an open-label, non-inferiority, randomized, phase III multicenter trial in Asia. Results The heterogeneity analysis showed no statistically significant difference between India and the other countries involved in the phase III study, for both the PCR-corrected and uncorrected cure rates. As shown at the whole study level, both forms of ACT were highly efficacious in India. In fact, in the per protocol population, the 63-day cure rates were 100% for A + M and 98.8% for DP. The DP combination exerted a significant post-treatment prophylactic effect, and compared with A + M a significant reduction in the incidence of new infections for DP was observed (respectively 17.1% versus 7.5% of patients experienced new infection within follow up). Parasite and fever clearance was rapid in both treatment arms (median time to parasite clearance of one day for both groups). Both DP and A + M were well tolerated, with the majority of adverse events of mild or moderate severity. The frequencies of individual adverse events were generally similar between treatments, although the incidence of post treatment adverse events was slightly higher in patients who received A + M with respect to those treated with DP. Conclusion DP is a new ACT displaying high efficacy and safety in the treatment of uncomplicated P. falciparum malaria and could potentially be considered for the first-line treatment of uncomplicated falciparum malaria in India. Trial registration Current Controlled Trials ISRCTN 81306618

In 2003–2005, following an increase in the local incidence of human malaria, the therapeutic efficacy of chloroquine (CQ) in the treatment of Plasmodium vivax and P. falciparum malaria was evaluated in the Anand district of Gujarat state, in western India. After oral administration of CQ, clinical and parasitological responses were measured over a follow-up period of 28 days, following the standard protocol of the World Health Organization. Most of the recurrent infections were checked, by genotyping, to see whether they were the result of treatment failure or re-infection during the follow-up. At the primary health centre (PHC) in Deva, all 57 P. vivax cases included in the study responded to CQ within 3 days. At the Pansora PHC, however, only 59 [90.8%, with a 95% confidence interval (CI) of 83.7%–97.8%] of the 65 P. vivax cases appeared to respond completely, recurrent infections being observed in the other six cases (9.2%; CI=2.2%–16.3%). Of the four recurrent infections checked by genotyping, however, only two appeared to be the result of true treatment failure. Twenty-seven (81.8%; CI=67.2%–94.4%) of the 33 P. falciparum cases who were enrolled in the study, all from Pansora PHC also showed apparent treatment failure, with one early failure, 17 late clinical failures and nine late parasitological failures. All 23 P. falciparum cases that showed apparent treatment failure and were investigated by genotyping appeared to be true cases of failure, none showing any evidence of re-infection during follow-up. The mean parasite-clearance times for those infected with P. falciparum, both those considered CQ-sensitive and the treatment failures, exceeded 2 days. These results indicate the presence of CQ-resistant P. vivax and P. falciparum in Anand district. The high frequency of CQ failure against P. falciparum observed in this study led to a change in the drug policy at the Pansora PHC, with artemisinin-based combination therapy now being used for the first-line treatment of P. falciparum malaria. Chloroquine remains the recommended first-line treatment for P. vivax infections in the area but the treatment failure seen in at least two P. vivax cases indicates a need for further monitoring of the therapeutic efficacy of CQ against such infections, in central Gujarat and elsewhere. [ABSTRACT FROM AUTHOR]