Defeating malaria in the North-East region: the forerunner for malaria elimination in India.

India is a malaria endemic country which is targeting malaria elimination by 2027. Transmission intensities are low-to-moderate depending on the region supported by multiple disease vectors. Among these, comparatively North-East India contributes to high proportions of malaria cases annually, the majority of which are due to Plasmodium falciparum (90%). Anopheles minimus and An. baimaii (sibling species in the An. dirus complex) are widely prevalent and incriminated as vectors of malaria. Number of intervention tools were field-evaluated beginning 1988 to date against disease vectors and causative parasites to contain the spread of malaria. These included (i) insecticide-treated netting materials (ITNs) for vector control, (ii) rapid diagnostic tests (RDTs) for in situ diagnosis, and (iii) therapeutic efficacy of artemisinin-based combination therapies (ACTs) for improved drug-policy; all of which were incorporated in healthcare services resulting in substantial disease transmission reduction. Populations of both An. minimus and An. baimaii were observed depleting, instead An. culicifacies s.l. recorded to be fast invading degraded forests and assessed to be resistant to multiple insecticides. Of the two prevalent Plasmodium species, while P. vivax continued to be susceptible to chloroquine therapy, P. falciparum had emerged resistant to most available antimalarial drugs except ACTs over space and time and spreading to peninsular India threatening elimination efforts. Disease transmission trends were observed to be declining for which the state of Assam has made huge strides reporting steady fall in cases each passing year vis-à-vis Meghalaya, Mizoram and Tripura (all sharing international border with Bangladesh), in which malaria transmission remained uninterrupted. Consequently, control of malaria in the North-East region of India is of immediate importance and needs prioritization for intensified disease surveillance and control interventions coupled with improved access to healthcare services mitigating risk of disease outbreaks and spread of drug-resistant malaria helping realize the goal of malaria elimination in the country.

Blue fluorescence as a frequency offset reference in the rubidium 5S-5P-5D transition.

In this work, we address the issue of the suitability of blue fluorescent light as a frequency offset reference during the ladder-level excitation 5S1/2→5P3/2→5D3/2 of Rb85 atoms. A simple pump-probe spectroscopy experiment is performed to generate non-degenerate blue light emission produced by the spontaneous decay route 5D3/2→6P3/2→5S1/2. By varying the relative intensity of the pump-probe combination, we can hover between regimes dominated by mechanisms such as double resonance optical pumping (DROP) and Autler-Townes splitting. The efficacy of the blue fluorescence (420 nm) as a frequency offset reference is pronounced under the DROP regime due to its narrow (∼natural) linewidth. To provide further insight into the stability performance, the pump laser connecting the 5P3/2→5D3/2 transition is stabilized to the peak of the blue light with the help of a probe stabilized to the saturation absorption signal of the 5S1/2→5P3/2 transition. For this purpose, we used the coherence-induced modulation transfer technique. The error frequency noise power spectra and the subsequent Allan variance calculation clearly show that probe fluctuations are transferred to the pump laser because of an existing two-photon coherent coupling.

Biology, distribution and control of Anopheles (Cellia) minimus in the context of malaria transmission in northeastern India.

Among six dominant mosquito vector species involved in malaria transmission in India, Anopheles minimus is a major species in northeast India and held responsible for focal disease outbreaks characterized by high-rise of Plasmodium falciparum infections and attributable death cases. It has been now genetically characterized that among the three-member species of the Minimus Complex spread in Asia, An. minimus (former species A) is prevalent in India including northeastern states and east-central state of Odisha. It is recorded in all seasons and accounts for perennial transmission evidenced by records of sporozoite infections. This species is highly anthropophilic, and largely endophilic and endophagic, recorded breeding throughout the year in slow flowing seepage water streams. The populations of An. minimus in India are reported to be highly diverse indicating population expansion with obvious implications for judicious application of vector control interventions. Given the rapid ecological changes due to deforestation, population migration and expansion and developmental activities, there is scope for further research on the existence of potential additional sibling species within the An. minimus complex and bionomics studies on a large geographical scale for species sanitation. For control of vector populations, DDT continues to be applied on account of retaining susceptibility status even after decades of residual spraying. Anopheles minimus is a highly adaptive species and requires continuous and sustained efforts for its effective control to check transmission and spread of drug-resistant malaria. Anopheles minimus populations are reportedly diminishing in northeastern India whereas it has staged comeback in east-central State of Odisha after decades of disappearance with its eco-biological characteristics intact. It is the high time to siege the opportunity for strengthening interventions against this species for its population diminution to sub-optimal levels for reducing transmission in achieving malaria elimination by target date of 2030.

Malaria elimination in India and regional implications.

The malaria situation in India is complex as a result of diverse socio-environmental conditions. India contributes a substantial burden of malaria outside sub-Saharan Africa, with the third highest Plasmodium vivax prevalence in the world. Successful malaria control in India is likely to enhance malaria elimination efforts in the region. Despite modest gains, there are many challenges for malaria elimination in India, including: varied patterns of malaria transmission in different parts of the country demanding area-specific control measures; intense malaria transmission fuelled by favourable climatic and environment factors; varying degrees of insecticide resistance of vectors; antimalarial drug resistance; a weak surveillance system; and poor national coordination of state programmes. Prevention and protection against malaria are low as a result of a weak health-care system, as well as financial and socioeconomic constraints. Additionally, the open borders of India provide a potential route of entry for artesunate-resistant parasites from southeast Asia. This situation calls for urgent dialogue around tackling malaria across borders-between India's states and neighbouring countries-through sharing of information and coordinated control and preventive measures, if we are to achieve the aim of malaria elimination in the region.

Monitoring the efficacy of antimalarial medicines in India via sentinel sites: Outcomes and risk factors for treatment failure.

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.

A cross-sectional study assessing the residual bio-efficacy and durability of field-distributed long-lasting insecticidal nets in malaria endemic ethnic communities of Assam, Northeast India.

Long-lasting insecticidal nets (LLINs) are being promoted for malaria vector control in the northeastern Indian state of Assam. A cross-sectional study was conducted to assess the current residual bio-efficacy and durability of both the Olyset(®) and PermaNet(®)2.0 LLINs that were distributed earlier in 2009, 2011 and 2013 to help formulate informed policy regarding net procurement, supplies and replacement. The study was undertaken in three different malaria endemic blocks of Assam during the period of June to October of 2014. The residual bio-efficacies were ascertained using the WHO cone-bioassay method for mosquito mortality post-exposure and corroborated with the ring-net assay for the median knockdown times of both types of LLINs in use by these communities. Cross-sectional community surveys were distributed to assess net ownership, utilization, community practices and the physical conditions of the nets in terms of being torn and the numbers of holes per position. Both the Olyset(®) and PermaNet(®)2.0 LLINs that were distributed in 2009 (i.e., nearly after five years of community usage) were completely torn, worn out and obsolete. However, the LLINs distributed in 2011 (i.e., three years of community usage) retained their residual bio-efficacies in susceptibility ranges that varied from 57% to 79%. However, for the LLINs that were distributed in 2013, the observed residual efficacy was adequate and resulted in a mosquito mortality rate >80 percent. Of the two types of LLINs inspected, the Olyset(®)nets were more durable and robust in terms of being torn less frequently (37.1%, 39/105) compared with the PermaNet(®)2.0 nets (51.8%, 204/394). Regarding the LLINs that were distributed in 2013, all were physically intact and in good condition. The majority of the distributed LLINs (99.2%, 639/644) were still in the possession of the householders of the surveyed populations. This study revealed that the serviceable life of the nets was slightly less than three years in terms of waning residual bio-efficacy and durability that warranted replacement. The communities were aware of the benefits of the use of mosquito net for personal protection and regularly used the nets; thus, LLIN-based interventions for sustained vector control should be scaled up.

First evidence of dengue virus infection in wild caught mosquitoes during an outbreak in Assam, Northeast India.

BACKGROUND & OBJECTIVES: Dengue is one of the major public health problems worldwide, transmitted mainly by Aedes aegypti and Ae. albopictus mosquitoes. Rapid urbanisation and industrialisation have led to an increase in vector population in Northeastern states of India. In 2013, Guwahati, the capital city of Assam, India experienced an outbreak of dengue. This study was undertaken with an objective to determine infection rates of dengue viruses (DENV) in both the established vectors present in this region. METHODS: During the outbreak (2013), adults and larvae of both the vector species were collected from different container habitats found in case reporting areas and container index was also recorded. The mosquitoes were first pooled, homogenised and processed for NS1-ELISA. This was followed by RT-PCR of the mosquito pools. RESULTS: Both Ae. aegypti and Ae. albopictus were found breeding in containers with container index in the range of 29.41 to 80%. Six pools of Ae. aegypti were found to be positive for NS1 antigen. RT-PCR assay revealed positivity in only the NS1-ELISA positive pools, exhibiting circulation of serotype DENV-2. Minimum infection rate of female and male Ae. aegypti was recorded as 10.87 and 11.03 respectively. INTERPRETATION & CONCLUSION: This is the maiden report of detection of DENV in wild caught Ae. aegypti mosquitoes from Northeastern Region of India. The study also demonstrates the presence of transovarial transmission of dengue virus in this part of country. This information is useful in respect of both entomological as well as epidemiological point of view for taking appropriate vector control measures.

Neglected Plasmodium vivax malaria in northeastern States of India.

BACKGROUND & OBJECTIVES: The northeastern States of India are co-endemic for Plasmodium falciparum and P. vivax malaria. The transmission intensity is low-to-moderate resulting in intermediate to stable malaria. Malaria control prioritized P. falciparum being the predominant and life threatening infection (>70%). P. vivax malaria remained somewhat neglected. The present study provides a status report of P. vivax malaria in the northeastern States of India. METHODS: Data on spatial distribution of P. vivax from seven northeastern States (Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura) were analysed retrospectively from 2008-2013. In addition, cross-sectional malarial surveys were conducted during 1991-2012 in malaria endemic pockets across the States of Assam, Meghalaya, Mizoram and Tripura to ascertain the prevalence of P. vivax in different age groups. RESULTS: Vivax malaria was encountered in all northeastern States but there existed a clear division of two malaria ecotypes supporting ≤30 and >30 per cent of total malaria cases. High proportions of P. vivax cases (60-80%) were seen in Arunachal Pradesh and Nagaland in the north with alpine environment, 42-67 per cent in Manipur, whereas in Assam it varied from 23-31 per cent with subtropical and tropical climate. Meghalaya, Tripura and Mizoram had the lowest proportion of P. vivax cases. Malaria cases were recorded in all age groups but a higher proportion of P. vivax consistently occurred among <5 yr age group compared to P. falciparum (P<0.05). P. vivax cases were recorded throughout the year with peak coinciding with rainy season although transmission intensity and duration varied. INTERPRETATION & CONCLUSIONS: In northeast India, P. vivax is a neglected infection. Estimating the relapsing pattern and transmission dynamics of P. vivax in various ecological settings is an important pre-requisite for planning malaria elimination in the northeastern States.

Surveillance of artemisinin resistance in Plasmodium falciparum in India using the kelch13 molecular marker.

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.

Malaria transmission in Tripura: Disease distribution & determinants.

BACKGROUND & OBJECTIVES: Malaria is a major public health problem in Tripura and focal disease outbreaks are of frequent occurrence. The state is co-endemic for both Plasmodium falciparum and P. vivax and transmission is perennial and persistent. The present study was aimed to review data on disease distribution to prioritize high-risk districts, and to study seasonal prevalence of disease vectors and their bionomical characteristics to help formulate vector species-specific interventions for malaria control. METHODS: Data on malaria morbidity in the State were reviewed retrospectively (2008-2012) for understanding disease distribution and transmission dynamics. Cross-sectional mass blood surveys were conducted in malaria endemic villages of South Tripura district to ascertain the prevalence of malaria and proportions of parasite species. Mosquito collections were made in human dwellings of malaria endemic villages aiming at vector incrimination and to study relative abundance, resting and feeding preferences, and their present susceptibility status to DDT. RESULTS: The study showed that malaria was widely prevalent and P. falciparum was the predominant infection (>90%), the remaining were P. vivax cases. The disease distribution, however, was uneven with large concentration of cases in districts of South Tripura and Dhalai coinciding with vast forest cover and tribal populations. Both Anopheles minimus s.s. and An. baimaii were recorded to be prevalent and observed to be highly anthropophagic and susceptible to DDT. Of these, An. minimus was incriminated (sporozoite infection rate 4.92%), and its bionomical characteristics revealed this species to be largely indoor resting and endophagic. INTERPRETATION & CONCLUSIONS: For effective control of malaria in the state, it is recommended that diseases surveillance should be robust, and vector control interventions including DDT spray coverage, mass distribution of insecticide-treated nets/ long-lasting insecticidal nets should be intensified prioritizing population groups most at risk to avert impending disease outbreaks and spread of drug-resistant malaria.

Impact of deforestation on known malaria vectors in Sonitpur district of Assam, India.

BACKGROUND & OBJECTIVES: An alarming rate of deforestation has been reported from Sonitpur district of Assam, India therefore, a study was initiated during 2009 using remote sensing (RS) to assess deforested areas in the district and to study the impact on malaria vectors in order to formulate appropriate control strategy. METHODS: RS imageries of 2000 and 2009 were used to assess deforested areas in the selected district. Entomological data were collected in four surveys during 2009-2011. The data were analyzed statistically using test of single proportions (χ 2 ) and pair-wise comparison. Vector incrimination was done using enzyme-linked immunosorbent assay (ELISA) and entomological inoculation rate (EIR) was calculated to estimate transmission intensity. RESULTS: The deforested areas were identified in north-western parts of Sonitpur district falling in Dhekiajuli Primary Health Centre (PHC). The forest cover of the PHC decreased >50% during 2000-2009. Five species of anopheline vectors were collected. Anopheles minimus sensu lato (s.l.) was collected least abundantly while An. culicifacies s.l. prevailed most abundantly and significant difference was observed between proportions of the collected vector species. Pair-wise comparison between An. culicifacies s.l. and An. minimus s.l. was also found statistically significant indicating that An. culicifacies s.l. is establishing its population in deforested areas. An. culicifacies s.l. was found ELISA positive and EIR was measured as 4.8 during transmission season. CONCLUSION: An. culicifacies s.l. replaced An. minimus s.l., the vector of malaria in northeast India and was found ELISA positive, therefore could have possible role in malaria transmission in the deforested areas of the district.

Declining efficacy of artesunate plus sulphadoxine-pyrimethamine in northeastern India.

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.

Therapeutic efficacy and safety of dihydroartemisinin-piperaquine versus artesunate-mefloquine in uncomplicated Plasmodium falciparum malaria in India.

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.

Malaria control in Bhutan: case study of a country embarking on elimination.

BACKGROUND: Bhutan has achieved a major reduction in malaria incidence amid multiple challenges. This case study seeks to characterize the Bhutan malaria control programme over the last 10 years. METHODS: A review of the malaria epidemiology, control strategies, and elimination strategies employed in Bhutan was carried out through a literature review of peer-reviewed and grey national and international literature with the addition of reviewing the surveillance and vector control records of the Bhutan Vector-Borne Disease Control Programme (VDCP). Data triangulation was used to identify trends in epidemiology and key strategies and interventions through analysis of the VDCP surveillance and programme records and the literature review. Enabling and challenging factors were identified through analysis of socio-economic and health indicators, corroborated through a review of national and international reports and peer-review articles. FINDINGS: Confirmed malaria cases in Bhutan declined by 98.7% from 1994 to 2010. The majority of indigenous cases were due to Plasmodium vivax (59.9%) and adult males are most at-risk of malaria. Imported cases, or those in foreign nationals, varied over the years, reaching 21.8% of all confirmed cases in 2006. Strategies implemented by the VDCP are likely to be related to the decline in cases over the last 10 years. Access to malaria diagnosis in treatment was expanded throughout the country and evidence-based case management, including the introduction of artemisinin-based combination therapy (ACT) for P. falciparum, increasing coverage of high risk areas with Indoor Residual Spraying, insecticide-treated bed nets, and long-lasting insecticidal nets are likely to have contributed to the decline alongside enabling factors such as economic development and increasing access to health services. CONCLUSION: Bhutan has made significant strides towards elimination and has adopted a goal of national elimination. A major challenge in the future will be prevention and management of imported malaria infections from neighbouring Indian states. Bhutan plans to implement screening at border points to prevent importation of malaria and to targeted prevention and surveillance efforts towards at-risk Bhutanese and migrant workers in construction sites.

Differential genetic hitchhiking around mutant pfcrt alleles in the Indian Plasmodium falciparum population.

OBJECTIVES: To study the origin and spread of the chloroquine-resistant Plasmodium falciparum population in the Indian subcontinent. METHODS: Fourteen microsatellites spanning a ∼120 kb region, flanking the P. falciparum chloroquine resistance transporter (pfcrt) gene, were analysed in 185 parasite isolates. RESULTS: The Indian P. falciparum population exhibited a selective valley of reduced genetic variation in the flanking microsatellites of the mutant pfcrt alleles (up to ±29 kb) as compared with the wild-type allele. This valley is much narrower than the ±200 kb valley reported from African and South-East Asian countries. The majority of the isolates showed asymmetry in the selective valley, where upstream microsatellites showed less genetic variation than the downstream microsatellites. Regional variation in the width and symmetry of the selective valley was noticed, which seems to be related to the number of pfcrt alleles present in the parasite population of a region. Forty-six different microsatellite haplotypes were observed among the P. falciparum isolates containing mutant pfcrt alleles. Parasite populations from different regions of mainland India shared microsatellite haplotypes between them, but they shared none with the isolates from the Andaman and Nicobar Islands, and vice versa. Indian isolates shared microsatellite haplotypes with the isolates from Papua New Guinea and Thailand. CONCLUSIONS: With regard to chloroquine there is regional variation in the selection pressure on the P. falciparum population in India. These findings will help the regional implementation of drug policy in India's malaria control programme.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship.

BACKGROUND: Multi-drug resistance and severe/complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. METHODS: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. RESULTS: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 ± 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 ± 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. CONCLUSIONS: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Molecular epidemiology of Plasmodium vivax anti-folate resistance in India.

BACKGROUND: Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent. METHODS: Microscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes. RESULTS: Sequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p<0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent. CONCLUSION: Study suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.

Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India.

With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine- and pyrimethamine-resistant parasites in the past.

Persistent transmission of malaria in Garo hills of Meghalaya bordering Bangladesh, north-east India.

BACKGROUND: Malaria is endemic in Garo hills of Meghalaya, and death cases are reported annually. Plasmodium falciparum is the major parasite, and is solely responsible for each malaria-attributable death case. Garo hills are categorized high-risk for drug-resistant malaria; however, there exists no data on malaria transmitting mosquitoes prevalent in the region. Included in this report are entomological observations with particular reference to vector biology characteristics for devising situation specific intervention strategies for disease transmission reduction. METHODS: The epidemiological data of the West Garo hills have been reviewed retrospectively for 2001-2009 to ascertain the disease transmission profile given the existing interventions. Point prevalence study was conducted in Dalu Community Health Centre that lies in close proximity to international border with Bangladesh to ascertain the true prevalence of malaria, and parasite species. Mosquito collections were made in human dwellings of malaria endemic villages aiming at vector incrimination, and to study relative abundance, resting and feeding preferences, and their present susceptibility status to DDT. RESULTS: Investigations revealed that the West Garo hill district is co-endemic for Plasmodium falciparum and Plasmodium vivax, but P. falciparum was the predominant infection (> 82%). Malaria transmission was perennial and persistent with seasonal peak during May-July corresponding to months of high rainfall. Entomological collections revealed that Anopheles minimus was the predominant species that was incriminated by detection of sporozoites in salivary glands (infection rate 2.27%), and was ascertained to be fully susceptible to DDT. CONCLUSION: For the control of malaria, improved diagnosis and sustained supply of drugs for artemisinin-based combination therapy are strongly advocated, which should be enforced for treatment of every single case of P. falciparum. Greater political commitment is called for organized vector control operations along border/high-risk areas to contain the spread of drug-resistant malaria, and averting impending disease outbreaks.

An open-label, randomised study of dihydroartemisinin-piperaquine versus artesunate-mefloquine for falciparum malaria in Asia.

BACKGROUND: The artemisinin-based combination treatment (ACT) of dihydroartemisinin (DHA) and piperaquine (PQP) is a promising novel anti-malarial drug effective against multi-drug resistant falciparum malaria. The aim of this study was to show non-inferiority of DHA/PQP vs. artesunate-mefloquine (AS+MQ) in Asia. METHODS AND FINDINGS: This was an open-label, randomised, non-inferiority, 63-day follow-up study conducted in Thailand, Laos and India. Patients aged 3 months to 65 years with Plasmodium falciparum mono-infection or mixed infection were randomised with an allocation ratio of 2:1 to a fixed-dose DHA/PQP combination tablet (adults: 40 mg/320 mg; children: 20 mg/160 [DOSAGE ERROR CORRECTED] mg; n = 769) or loose combination of AS+MQ (AS: 50 mg, MQ: 250 mg; n = 381). The cumulative doses of study treatment over the 3 days were of about 6.75 mg/kg of DHA and 54 mg/kg of PQP and about 12 mg/kg of AS and 25 mg/kg of MQ. Doses were rounded up to the nearest half tablet. The primary endpoint was day-63 polymerase chain reaction (PCR) genotype-corrected cure rate. Results were 87.9% for DHA/PQP and 86.6% for AS+MQ in the intention-to-treat (ITT; 97.5% one-sided confidence interval, CI: >-2.87%), and 98.7% and 97.0%, respectively, in the per protocol population (97.5% CI: >-0.39%). No country effect was observed. Kaplan-Meier estimates of proportions of patients with new infections on day 63 (secondary endpoint) were significantly lower for DHA/PQP than AS+MQ: 22.7% versus 30.3% (p = 0.0042; ITT). Overall gametocyte prevalence (days 7 to 63; secondary endpoint), measured as person-gametocyte-weeks, was significantly higher for DHA/PQP than AS+MQ (10.15% versus 4.88%; p = 0.003; ITT). Fifteen serious adverse events were reported, 12 (1.6%) in DHA/PQP and three (0.8%) in AS+MQ, among which six (0.8%) were considered related to DHA/PQP and three (0.8%) to AS+MQ. CONCLUSIONS: DHA/PQP was a highly efficacious drug for P. falciparum malaria in areas where multidrug parasites are prevalent. The DHA/PQP combination can play an important role in the first-line treatment of uncomplicated falciparum malaria. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN81306618.