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Background: Malaria elimination requires targeting asymptomatic and low-density Plasmodium infections that largely remain undetected. Therefore we conducted a cross-sectional study to estimate the burden of asymptomatic and low-density Plasmodium infection using conventional and molecular diagnostics., Methods: A total of 9118 participants, irrespective of age and sex, were screened for malaria using rapid diagnostic tests (RDTs), microscopy and polymerase chain reaction., Results: Among the participants, 707 presented with symptoms and 8411 without symptoms, of which Plasmodium was present in 15.6% (110/707) and 8.1% (681/8411), respectively. Low-density infection was found in 5.1% (145/2818) of participants and 8327 of 9118 were Plasmodium negative. Endemicity was propotional to asymptomatic infections (high endemicity 11.1% [404/3633] vs low endemicity 5.8% [277/4778]; odds ratio [OR] 2.0 [95% confidence interval {CI} 1.7 to 2.4]) but inversely related to low-density infection (high endemicity 3.7% [57/1545] vs low endemicity 6.9% [88/1273]; OR 1.9 [95% CI 1.4 to 2.7]). The spleen rate in children 2-9 y of age was 17.9% (602/3368) and the enlarged spleen index was 1.6. Children between 8 and 14 y showed higher odds for asymptomatic (adjusted OR [aOR] 1.75 [95% CI 1.4 to 2.2]) and low-density infections (aOR 0.63 [95% CI 0.4 to 1.0)] than adults., Conclusions: The prevalence of asymptomatic and low-density Plasmodium infection undermines the usefulness of standard diagnostic tools used by health agencies. This necessitates deploying molecular tools in areas where malaria microscopy/RDTs indicate a dearth of infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Sensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes., Method: Plasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes., Result: A total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method., Conclusion: This method identifies and distinguishes four species of human malaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest. This work has been published as patent WO/2016/120886 with international application number PCT/IN2016/000023., (Copyright © 2019 Elsevier B.V. All rights reserved.)

G6PD deficiency results from numerous mutations in the G6PD gene and can cause alterations in enzyme function up to varying degrees. P. vivax malaria infections require G6PD deficiency screening because of the potential risk of haemolysis by the gametocytocidal drug (primaquine) during the radical treatment. The present study investigated the incidence of G6PD deficiency from northeast India and further, molecular characterization was performed. During 2014-16, a total of 1,015 patients from four north-eastern states of India (Tripura, Mizoram, Meghalaya & Arunachal Pradesh), were screened for G6PD deficiency, using Beutler's fluorescence spot test (FST) and confirmed with SPAN G6PD kit. The deficient individuals (55/1015, 5.4%) were further characterized by PCR-RFLP and DNA sequencing except one case of lost to follow up. As observed by FST, the frequency of G6PD deficient males (42/538, 7.8%) were found to be higher than females (13/477, 2.73%), (p < 0.0001). Two non-synonymous mutations; G6PD-D (Mahidol) 487A (48/54, 88.9%; 36 hemizygous males, 8 homozygous and 4 heterozygous females) and G6PD-D (Acores) 595T (2/54, 3.7%) were identified. Remaining (4/54, 7.4%) individuals could not be characterized. Molecular modeling and dynamic simulations were performed for the G6PD wild-type (G6PD-WT) enzyme and its variants. The in-silico results demonstrated alterations in the secondary structures & crucial loss of ligand-protein interactions, which might result in reduced enzyme function, leading to enzyme deficiency. To the best of our knowledge, this is the first report to document G6PD-Mahidol and G6PD-Acores variants from malaria-endemic regions of northeast India, and provided molecular insights on the varied genetic makeup of the studied population., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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: 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.

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: 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.

The study was conducted to assess the magnitude of oxidative stress and lung function abnormalities in 34 male pesticide sprayers on exposure to pesticides in mango plantations. Biochemical studies on blood antioxidant enzymes revealed an unchanged glutathione level and increased level of malondialdehyde (P < 0.001), which indicates that pesticide sprayers may have suffered from oxidative stress. Decreased acetyl-cholinesterase levels (P < 0.001) in sprayers compared to the controls suggest inhibition of cholinesterase activity. The present study shows that pesticide toxicity might lead to oxidative stress and airway narrowing resulting in decreased peak expiratory flow rate.

59 workers exposed to different chemicals during the manufacture of quinalphos, an organophosphate pesticide (OP) and 17 control subjects were studied. Despite similar blood acetylcholinestarase (AChE) levels in both the exposed and control subjects, a significant number of exposed subjects had altered plantar and ankle reflexes. Higher nervous functions such as memory, learning and vigilance were also found to be affected in these subjects. These findings were attributed to chronic low dose combined exposure to different chemicals used/formed in the manufacture of quinalphos. The study raises the doubt that monitoring of AChE alone among subjects engaged in the manufacture of OP pesticides may not be an adequate safeguard as regards to their health.

A cross-sectional survey of 489 male subjects in the age group 15 to 65 years engaged directly or indirectly in mango cultivation along with 208 control subjects was carried out to find their socio-economic, environmental and health conditions. The conditions like high illiteracy rate (49.5%), poverty (PCI less than Rs 100 per month, 52.2%), poor housing (mud houses, 66.7%) unsafe water supply (78.6%) were prevailing in the surveyed population. The high respiratory morbidity may be attributed to high prevalence of smoking and prolonged inhalation of organic dusts during farming operation associated with illiteracy and poor socio-economic status. Gastrointestinal disorders were related to poor hygienic conditions, smoking and consumption of contaminated water. The symptoms pertaining to CNS, skin and eyes were found to be associated with exposure to pesticides.

Twenty-six farm workers handling about 4 kg of commercial grade hexachlorocyclohexane (HCH)/y for 2-5 y and 21 control subjects were studied for chronic effects in clinical, hematological and cardiac variables. No clinically apparent morbidity was detected, but changes in hemoglobin and electrocardiograms were early effects of HCH exposure.

We examined the serum levels of thyroxine and thyroid stimulating hormone in 103 rural subjects with respect to blood levels of organochlorine pesticide and occupation. We found that 24.3% of study subjects had depleted thyroxine levels in association with significantly lower organochlorine pesticide residues in blood. Sex, nutritional status, thyromegaly, or handling of pesticides in the course of work were not found to be factors contributing to depleted thyroxine levels.

To study their socioeconomic and health status, 1,770 workers in the organized electronics industry in India were surveyed. Mean age of the workers employed in this industry was 32.5 +/- 6.01 years. The average per capita income/month was Rupees 333, indicating a higher socioeconomic status, compared with the status of the national population, as well as compared with their counterparts in the unorganized sectors. Respiratory symptoms and impairment rates were significantly higher in workers exposed to soldering fumes. The high prevalence of congested or inflamed throat was also related to chemical exposure specially among solderers and workers exposed to metal oxides. The ocular symptoms and signs were also related to chemical exposure. Musculoskeletal disorders were related to erratic ergonomic postures.

Thirty subjects engaged in maintenance and repair of moulds used for producing watermarks on paper were studied, along with 27 control subjects from the same paper mill. The workers were simultaneously exposed to copper, chromium, zinc, xylol, and fumes of sulphuric acid in the course of their work. The study subjects were investigated for clinical, occupational, radiological haematological and psychological details. The aforementioned combined exposure was found to be responsible for a high prevalence of symptoms pertaining to the respiratory system and higher nervous functions. Breathlessness (26.7%), expectoration (10.0%) and emphysema (10.0%) were significantly higher among the exposed subjects. The exposed subjects also showed lowered visuomotor coordination and delayed reaction to light and sound stimuli.

Clinical, haematological and biochemical studies of 34 subjects, occupationally exposed to different types of pesticides, were conducted. The findings have been compared with those observed in 14 control subjects. Inhibition of cholinesterase activity was observed in the exposed group. Serum alkaline phosphatase was also found to be raised. Radiological examination revealed pneumonitic patches in the chest skiagrams of three exposed subjects. Paraesthesia with hyporeflexia was also found in 8.8% of exposed subjects. The findings suggest that exposure to multiple pesticides over many years affects the normal functioning of different organ systems and may produce characteristic clinical effects.

Ninety-four male pump workers, employed in 41 petrol filling stations in Kanpur (formerly Cawnpore) and Lucknow--both in the Ganges plain of north India--were clinically examined. Headache, redness in eyes, lacrimation and signs like coated and/or furred tongue, throat and/or conjunctival congestion and carious teeth were significantly higher (P less than 0.05) in workers exposed to petrol fumes than in controls, as also was the level of phenol in urine (P less than 0.01). Environmental measurements revealed higher concentrations of benzene, sulphur dioxide and photoionizable dust in the air at petrol stations, and most of the symptoms and signs could be attributed to petrol fumes and other environmental pollution.