Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India.

BACKGROUND: Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. METHODS: This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450-680 was also carried out to identify the polymorphism. RESULTS: A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. CONCLUSIONS: The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Clinical and molecular monitoring of Plasmodium falciparum resistance to antimalarial drug (artesunate+sulphadoxine-pyrimethamine) in two highly malarious district of Madhya Pradesh, Central India from 2012-2014.

The spread of P. falciparum resistant strain has led to a significant resurgence of malaria morbidity and mortality. The current cornerstone in malaria treatment in India is Artemisinin based Combination (Artesunate + Sulphadoxine-Pyrimethamine) Therapy (ACT) for treatment of uncomplicated P. falciparum malaria since 2010. In the present study we assessed the therapeutic efficacy of ACT and molecular monitoring of antimalarial resistance. Therapeutic efficacy was determined by in vivo method using 28 days follow-up. Molecular genotyping of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps) and kelch13 genes were analyzed. msp-1 and msp-2 genotyping were used to differentiate recrudescence. Therapeutic efficacy of ACT was determined in 237 patients over the three year period. Most of the patients showed adequate clinical and parasitological response (99.6%). Molecular study revealed that 72% parasites were of mutant genotype (27.2% single mutants, 43.5% double mutants and 1.3% triple mutants) for pfdhfr while pfdhps showed 78.2% wild type alleles and 21.8% mutants (18.1% single mutants and 3.7% double mutants). Analysis of total 135 samples revealed mutation in k13 gene along with non-synonymous single mutation at codon M579T (1.5%) and double mutations at codon M579T & N657H in 37%. ACT remains effective for the treatment of uncomplicated P. falciparum malaria in Madhya Pradesh, Central India. However, increasing mutation in pfdhfr (particularly triple mutations) and pfdhps may reduce susceptibility to partner drug SP and mutation in k13 propeller gene, highlighting the need for continuous monitoring of the efficacy of ACT.

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

BACKGROUND: Anti-malarial drug resistance continues to be a leading threat to malaria control efforts and calls for continued monitoring of waning efficacy of artemisinin-based combination therapy (ACT). Artesunate + sulfadoxine/pyrimethamine (AS + SP) is used for the treatment of uncomplicated Plasmodium falciparum malaria in India. However, resistance against AS + SP is emerged in northeastern states. Therefore, artemether-lumefantrine (AL) is the recommended first line treatment for falciparum malaria in north eastern states. This study investigates the therapeutic efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in three malaria-endemic states in India. The data generated through this study will benefit the immediate implementation of second-line ACT as and when required. METHODS: This was a one-arm prospective evaluation of clinical and parasitological responses for uncomplicated falciparum malaria using WHO protocol. Patients diagnosed with uncomplicated mono P. falciparum infection were administered six-dose regimen of AL over 3 days and subsequent follow-up was carried out up to 28 days. Molecular markers msp-1 and msp-2 were used to differentiate recrudescence and re-infection and K13 propeller gene was amplified and sequenced covering the codon 450-680. RESULTS: A total of 402 eligible patients were enrolled in the study from all four sites. Overall, adequate clinical and parasitological response (ACPR) was 98 % without PCR correction and 99 % with PCR correction. At three study sites, ACPR rates were 100 %, while at Bastar, cure rate was 92.5 % on day 28. No early treatment failure was found. The PCR-corrected endpoint finding confirmed that one late clinical failure (LCF) and two late parasitological failures (LPF) were recrudescences. The PCR corrected cure rate was 96.5 %. The mean fever clearance time was 27.2 h ± 8.2 (24-48 h) and the mean parasite clearance time was 30.1 h ± 11.0 (24-72 h). Additionally, no adverse event was recorded. Analysis of total 186 samples revealed a mutation in the k13 gene along with non-synonymous mutation at codon M579T in three (1.6 %) samples. CONCLUSION: AL is an efficacious drug for the treatment of uncomplicated falciparum malaria. However, regular monitoring of AL is required in view of malaria elimination initiatives, which will be largely dependent on therapeutic interventions, regular surveillance and targeted vector control.

Field evaluation of a real-time fluorescence loop-mediated isothermal amplification assay, RealAmp, for the diagnosis of malaria in Thailand and India.

BACKGROUND: To eliminate malaria, surveillance for submicroscopic infections is needed. Molecular methods can detect submicroscopic infections but have not hitherto been amenable to implementation in surveillance programs. A portable loop-mediated isothermal amplification assay called RealAmp was assessed in 2 areas of low malaria transmission. METHODS: RealAmp was evaluated in 141 patients from health clinics in India (passive surveillance) and in 127 asymptomatic persons in Thailand (active surveillance). The diagnostic validity, precision, and predictive value of RealAmp were determined using polymerase chain reaction (PCR) as the reference method. A pilot study of RealAmp was also performed on samples from patients presenting at a Thai health center. RESULTS: A total of 96 and 7 positive cases were detected in India and Thailand, respectively, via PCR. In comparison with nested PCR, the sensitivity and specificity of RealAmp in India were 94.8% (95% confidence interval [CI], 88.3%-98.3%) and 100% (95% CI, 92.1%-100%), respectively, with correct identification of all 5 Plasmodium vivax cases. In Thailand, compared with pooled real-time PCR, RealAmp demonstrated 100% sensitivity (95% CI, 59.0%-100%) and 96.7% specificity (95% CI, 91.7%-99.1%). Testing at the health center demonstrated RealAmp's potential to serve as a point-of-care test with results available in 30-75 minutes. CONCLUSION: RealAmp was comparable to PCR in detecting malaria parasites and shows promise as a tool to detect submicroscopic infections in malaria control and elimination programs worldwide.

Dynamics of forest malaria transmission in Balaghat district, Madhya Pradesh, India.

BACKGROUND: An epidemiological and entomological study was carried out in Balaghat district, Madhya Pradesh, India to understand the dynamics of forest malaria transmission in a difficult and hard to reach area where indoor residual spray and insecticide treated nets were used for vector control. METHODS: This community based cross-sectional study was undertaken from January 2010 to December 2012 in Baihar and Birsa Community Health Centres of district Balaghat for screening malaria cases. Entomological surveillance included indoor resting collections, pyrethrum spray catches and light trap catches. Anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite protein. FINDINGS: Plasmodium falciparum infection accounted for >80% of all infections. P. vivax 16.5%, P. malariae 0.75% and remaining were mixed infections of P. falciparum, P. vivax and P. malariae. More than, 30% infections were found in infants under 6 months of age. Overall, an increasing trend in malaria positivity was observed from 2010 to 2012 (chi-square for trend  =  663.55; P<0.0001). Twenty five Anopheles culicifacies (sibling species C, D and E) were positive for circumsporozoite protein of P. falciparum (44%) and P. vivax (56%). Additionally, 2 An. fluviatilis, were found positive for P. falciparum and 1 for P. vivax (sibling species S and T). An. fluviatilis sibling species T was found as vector in forest villages for the first time in India. CONCLUSION: These results showed that the study villages are experiencing almost perennial malaria transmission inspite of indoor residual spray and insecticide treated nets. Therefore, there is a need for new indoor residual insecticides which has longer residual life or complete coverage of population with long lasting insecticide treated nets or both indoor residual spray and long lasting bed nets for effective vector control. There is a need to undertake a well designed case control study to evaluate the efficacy of these interventions.

Comparative evaluation of bivalent malaria rapid diagnostic tests versus traditional methods in field with special reference to heat stability testing in Central India.

BACKGROUND: Malaria presents a diagnostic challenge in areas where both Plasmodium falciparum and P.vivax are co-endemic. Bivalent Rapid Diagnostic tests (RDTs) showed promise as diagnostic tools for P.falciparum and P.vivax. To assist national malaria control programme in the selection of RDTs, commercially available seven malaria RDTs were evaluated in terms of their performance with special reference to heat stability. METHODOLOGY/PRINCIPAL FINDINGS: This study was undertaken in four forested districts of central India (July, 2011- March, 2012). All RDTs were tested simultaneously in field along with microscopy as gold standard. These RDTs were stored in their original packing at 25°C before transport to the field or they were stored at 35°C and 45°C upto 100 days for testing the performance of RDTs at high temperature. In all 2841 patients with fever were screened for malaria of which 26% were positive for P.falciparum, and 17% for P.vivax. The highest sensitivity of any RDT for P.falciparum was 98% (95% CI; 95.9-98.8) and lowest sensitivity was 76% (95% CI; 71.7-79.6). For P.vivax highest and lowest sensitivity for any RDT was 80% (95% CI; 94.9 - 83.9) and 20% (95% CI; 15.6-24.5) respectively. Heat stability experiments showed that most RDTs for P.falciparum showed high sensitivity at 45°C upto 90 days. While for P.vivax only two RDTs maintained good sensitivity upto day 90 when compared with RDTs kept at room temperature. Agreement between observers was excellent for positive and negative readings for both P.falciparum and P.vivax (Kappa >0.6-0.9). CONCLUSION: This is first field evaluation of RDTs regarding their temperature stability. Although RDTs are useful as diagnostic tool for P.falciparum and P.vivax even at high temperature, the quality of RDTs should be regulated and monitored more closely.

Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat, Central India: implications for the diagnosis of malaria and its control.

OBJECTIVE: During an epidemiological study (January-July 2012) on malaria in forest villages of Central India, Plasmodium malariae-like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species-specific nested polymerase chain reaction (PCR) and DNA sequencing. METHODS: All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae. RESULTS: Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3-day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum. DISCUSSIONS: Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re-affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.

Genetic diversity in the block 2 region of the merozoite surface protein-1 of Plasmodium falciparum in central India.

BACKGROUND: Malaria continues to be a significant health problem in India. Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic. The genetic diversity of P. falciparum merozoite surface protein-1 (MSP-1) has been extensively studied from various parts of the world. However, limited data are available from India. The aim of the present study was a molecular characterization of block 2 region of MSP-1 gene from the tribal-dominated, forested region of Madhya Pradesh. METHODS: DNA sequencing analysis was carried out in 71 field isolates collected between July 2005 to November 2005 and in 98 field isolates collected from July 2009 to December 2009. Alleles identified by DNA sequencing were aligned with the strain 3D7 and polymorphism analysis was done by using Edit Sequence tool (DNASTAR). RESULTS: The malaria positivity was 26% in 2005, which rose to 29% in 2009 and P. falciparum prevalence was also increased from 72% in 2005 to 81% in 2009. The overall allelic prevalence was higher in K1 (51%) followed by MAD20 (28%) and RO33 (21%) in 2005 while in 2009, RO33 was highest (40%) followed by K1 (36%) and MAD20 (24%). CONCLUSIONS: The present study reports extensive genetic variations and dynamic evolution of block 2 region of MSP-1 in central India. Characterization of antigenic diversity in vaccine candidate antigens are valuable for future vaccine trials as well as understanding the population dynamics of P. falciparum parasites in this area.

Epidemic of Plasmodium falciparum malaria in Central India, an area where chloroquine has been replaced by artemisinin-based combination therapy.

India contributes greatly to the global incidence of malaria. The factors influencing malaria in India are highly diverse and vary greatly from the epidemiological setting of any other country. Central India is the most vulnerable area to malaria in India. This study was carried out in three community health centres in Dindori District, Madhya Pradesh (Central India). Dindori District is mesoendemic for malaria, with both Plasmodium falciparum and P. vivax being present in all age groups. Anopheles culicifacies and A. fluviatilis are highly efficient vectors of malaria. In this study, an epidemic of malaria among indigenous ethnic group Baigas was investigated to determine the causes of the epidemic and the population involved in order to aid in disease containment. The existence of sporozoite-positive A. culicifacies and A. fluviatilis indicates either that spraying had not been done properly or the presence of insecticide resistance. A combination of factors propagated the epidemic. Evidence suggests that the non-availability of artemisinin-based combination therapy and rapid diagnostic tests along with an immunogenically vulnerable population each played an important role. As the global prevalence of malaria decreases owing to initiatives to control or eliminate the disease, more areas will become mesoendemic or hypoendemic for malaria. Detection and control of epidemics requires greater attention, and mechanisms to ensure the quality of interventions are essential.

Field and laboratory comparative evaluation of rapid malaria diagnostic tests versus traditional and molecular techniques in India.

BACKGROUND: Malaria presents a diagnostic challenge in most tropical countries. Microscopy remains the gold standard for diagnosing malaria infections in clinical practice and research. However, microscopy is labour intensive, requires significant skills and time, which causes therapeutic delays. The objective of obtaining result quickly from the examination of blood samples from patients with suspected malaria is now made possible with the introduction of rapid malaria diagnostic tests (RDTs). Several RDTs are available, which are fast, reliable and simple to use and can detect Plasmodium falciparum and non-falciparum infections or both. A study was conducted in tribal areas of central India to measure the overall performance of several RDTs for diagnosis of P. falciparum and non-falciparum infections in comparison with traditional and molecular techniques. Such data will be used to guide procurement decisions of policy makers and programme managers. METHODS: Five commercially available RDTs were tested simultaneously in field in parallel with peripheral blood smears in outbreak-affected areas. The evaluation is designed to provide comparative data on the performance of each RDT. In addition, molecular method i.e. polymerase chain reaction (PCR) was also carried out to compare all three methods. RESULTS: A total of 372 patients with a clinical suspicion of malaria from Bajag Primary Health Centre (PHC) of district Dindori and Satanwada PHC of district Shivpuri attending the field clinics of Regional Medical Research Centre were included in the study. The analysis revealed that the First Response Malaria Antigen pLDH/HRP2 combo test was 94.7% sensitive (95% CI 89.5-97.7) and 69.9% specific (95% CI 63.6-75.6) for P. falciparum. However, for non-falciparum infections (Plasmodium vivax) the test was 84.2% sensitive (95% CI 72.1-92.5) and 96.5% specific (95% CI 93.8-98.2). The Parascreen represented a good alternative. All other RDTs were relatively less sensitive for both P. falciparum and non-falciparum infections. CONCLUSIONS: The results in this study show comparative performance between microscopy, various RDTs and PCR. Despite some inherent limitation in the five RDTs tested, First Response clearly has an advantage over other RDTs. The results suggest that RDTs could play and will play an important role in malaria diagnosis.