Prevalence and Pattern of Morbidity, Febrile Illness, and Treatment-Seeking Behavior in a Tribal-Dominated District in Odisha, India: An Observational Study.

Background Tribal populations constitute a major portion of India's total population, especially in the eastern and northeastern states. We lack comprehensive information on the community burden of general morbidity and febrile illness in tribal population-dominated areas, which is quite essential for the microplanning of healthcare expenditure and implementation. This study aimed to provide evidence on the prevalence and pattern of general morbidity and febrile illness at the community level as well as the treatment-seeking behaviour in a tribal-dominated area. Methods The study was undertaken as an observational study in the community setting; looking into seasonal cross-sectional evidence on period prevalence (two weeks) of morbidity and qualitative/semiquantitative information on treatment-seeking behaviour of the selected community during 2012 and 2013. Result This study involved 5541, 5482, and 5638 individuals during the rainy season 2012, winter 2012-13, and rainy season 2013 seasons, respectively, from 25 tribal villages of Odisha, India. A period prevalence (two weeks) of overall morbidities was shown to be 27.28% and 28.9% during the rainy seasons of 2012 and 2013, respectively, of which 13% and 11.5%, respectively, were febrile, with low prevalence (6.44% overall morbidity and 1.81% febrile illness) in the winter of 2012-13. It indicated inadequacy in skills of the village-level health staff, monitoring of supplies/logistics, and population awareness for early reporting of fever to healthcare providers at the community level. Conclusion The evidence provided by the study would be helpful in making public health plans in tribal settings and also highlighted the opportunity to improve tribal health status through community awareness, especially in areas and populations with limited health access.

High Prevalence of Hepatitis C Virus Infection in Primitive Tribes of Eastern India and Associated Sociobehavioral Risks for Transmission: A Retrospective Analysis.

Purpose: The primitive tribal groups (PTGs) need special attention because of their low population growth: declining population size with high mortality rates. Scanty reports are available on the prevalence of hepatitis C virus (HCV) infection in primitive tribes of the country emphasizing their cultural and social practices associated with transmission of the disease. Methods: The study was conducted on 1765 tribal individuals covering 5 PTGs, namely Lodha, Saora, Khadia, Juanga, and Mankidia, from 6 districts of Odisha. Serum samples were tested for the anti-HCV antibody using commercially available enzyme immunoassays. HCV RNA was detected based on the 5' NCR region and genotyping was done by direct sequencing of the core region. Potential risk factors for HCV transmission were collected using a questionnaire and subjected to regression analysis through SPSS, version 17.0. Results: Antibody to HCV was detected in 0%, 3.3%, 5.7%, 8.5%, and 13.4% in Saora, Lodha, Khadia, Juanga, and Mankidia tribes, respectively. HCV RNA was detected in 8.6% (11/127) of the samples tested. Genotyping of HCV isolates in all HCV RNA-positive samples revealed genotype 1b. Sharing of razors and shaving by the village barber were found to be significantly (p<0.05) associated with HCV transmission in males, whereas tattooing and multiple injections were found to be significant risk factors for females. Conclusion: This study indicated a high prevalence of HCV infection in Mankidia and Juanga tribes in comparison with the national scenario, which needs public health attention.

An outbreak of hepatitis E virus infection caused by genotype 1 in an urban setting in eastern India: a probe into risk factors for transmission.

The study was undertaken to find out the cause and etiology of an outbreak presumed to be due to water contamination that caused high morbidity in the western part of the state of Odisha during May, 2014. In this investigation 56 blood samples were collected and tested for HEV IgM through ELISA. Blood sample of 22 patients collected within 1 weeks post onset of symptoms and were subjected to RT-PCR and sequencing followed by phylogenetic analysis. Water samples were also analyzed for viral and bacterial contamination. A total of 290 individuals were examined for suspected jaundice. Out of 56 blood samples in 41 (73.2%) IgM for HEV was found. 12 samples out of 22 early phase samples were positive for RT-PCR and through phylogenetic analysis all were found to be of Genotype 1 and subtype A. This large outbreak was confirmed due to Hepatitis E virus and transmission was fast due to contamination of drinking water sources and lack of hygienic practices. The outcome of this investigation has created alertness among state health and municipal authorities to be prepared for the similar kind of situation for other part of the state.

Molecular dynamics insights into the structure, function, and substrate binding mechanism of mucin desulfating sulfatase of gut microbe Bacteroides fragilis.

The complex and dynamic consortia of microbiota that harbors the human gastrointestinal tract contributes ominously to the maintenance of health, the onset and progression of diverse spectrum of disorders. The capability of these enteric microbes to bloom within the gut mucosal milieu is often associated to the glycan metabolism of mucin-degrading bacteria. Accruing evidences suggests that the desulfation of mucin is a rate-limiting step in mucin degradation mechanism by colonic bacterial mucin-desulfating sulfatase enzymes (MDS) enzymes. Till date no experimental evidence is available on how conformational flexibility influences structure and substrate specificity by MDS of gut microbe Bacteroides fragilis. Henceforth, to gain deep insights into the missing but very imperative mechanism, we performed a comprehensive molecular dynamics study, principal component analysis and MM/PBSA binding free energies to gain insights into (i) the domain architecture and mode of substrate binding (ii) conformational dynamics and flexibility that influence the orientation of substrate, (iii) energetic contribution that plays very decisive role to the overall negative binding free energy and stabilities of the complexes (iv) critical residues of active site which influence binding and aid in substrate recognition. This is the first ever report, depicting the molecular basis of recognition of substrates and provides insights into the mode of catalysis by mucin desulfating sulfatase enzymes in gut microbiota. Overall, our study shed new insights into the unmapped molecular mechanisms underlying the recognition of various substrates by mucin desulfating sulfatase, which could be of great relevance in therapeutic implications in human gut microbiota associated disorders.

HPV genotypes co-infections associated with cervical carcinoma: Special focus on phylogenetically related and non-vaccine targeted genotypes.

HPV is the major causative agent for cervical cancer. Study on the risk of cervical cancer associated with different hr-HPV genotypes would be useful for disease management and new vaccine strategy. With limited reports available, the present study aimed to investigate the pattern of HPV genotypes coinfections and risk of cervical carcinoma associated with them in Indian population. 15 HPV genotypes were detected by E6/E7 multiplex nested type-specific PCR in the HPV-positive cervical samples of 172 cervical cancer cases and 174 subjects with normal cytology. Association between the genotypes and cervical cancer was estimated by calculating the Odds ratio and 95% confidence interval. Risk of cervical carcinoma was associated with multiple genotypes excluding HPV16 (OR:5.87; 95% CI-1.28-26-29; p = .02), multiple genotypes excluding HPV18 (OR = 2.5; 95% CI = 1.09-6.05; p = .03), multiple genotypes of α9 species(OR = 5.3 95% CI = 1.14-24.03; p = .007), and multiple genotypes of α7 species (OR = 2.5; 95% CI = .49-13.45; p = .2). Genotypes not targeted by quadrivalent vaccine types (OR = 2.94 95% CI = 1.48-5.80; p = .001) conferred 2.94 fold higher risk of cervical carcinoma. Cases those coinfected with phylogenetically related genotypes (OR = 2.29; 95% CI(.69-7.59) p = .17) were at 2.9 fold higher risk of invasive cervical carcinoma than those infected with other genotypes although it is not statistically significant. Whereas phylogenetically unrelated genotypes coinfection is negatively associated with cervical carcinoma (OR = .44 95% CI (.244-.8) p = .007) and it is statistically significant.Genotypes not targeted by 9-valent vaccines (OR = .40; 95% CI = .19-.85; p = .017) associated with lesser risk of cervical carcinoma as compared to other genotypes. Subjects infected with any HPV genotype/genotypes excluding HPV16 in association with HPV 18 (OR = 4.1; 95% CI = 1.81-9.25 P = < .001) were at 4.1 fold higher risk of developing invasive cervical carcinoma.In conclusion, the risk of development of cervical cancer is genotype specific and might be associated with type-specific interactions between the genotypes in multiple infections.

Lacto-Vegetarian Diet and Correlation of Fasting Blood Sugar with Lipids in Population Practicing Sedentary Lifestyle.

Rising burden of diabetes in India requires quick intervention that integrates policies and programs for effective prevention and control of disease. This retrospective cross-sectional study was conducted to observe effect of diet in two Indian communities practicing sedentary lifestyle. Fasting blood samples were analyzed for blood sugar, glycated-hemoglobin (HbA1C), and lipid profile. Body mass index (BMI) and waist circumference (WC) measurements were recorded. Diabetes incidence was lower in lacto-vegetarian (1.7%) than in non-vegetarian group (5.3%) despite similar lipid profiles and BMI/WC between the groups. Fasting blood sugar (FBS) was positively correlated with LDL and VLDL levels and negatively correlated with HDL, only in lacto-vegetarian group. Study suggests: (1) Indian lacto-vegetarian diet has beneficial effects on diabetes incidence irrespective of high body weight and sedentary lifestyle; (2) intervention to reduce body lipids, such as lipid-lowering drugs and exercise, may have greater effect in reducing FBS levels in this lacto-vegetarian group.

HPV Genotypes distribution in Indian women with and without cervical carcinoma: Implication for HPV vaccination program in Odisha, Eastern India.

BACKGROUND: Considering the limited cross protection offered by the current HPV vaccines, understanding the HPV genotype distribution among the different population is essential in predicting the efficacy of current vaccine and devising new vaccine strategy. The present work aimed at investigating the HPV genotypes distribution among women with and without cervical carcinoma in Odisha, Eastern India. METHODS: A total of 607 participants have been enrolled between January 2014 and June 2016. L1-PCR, sequencing, and E6/E7 nested multiplex type- specific PCR were performed for HPV detection and genotyping. Cytological distribution of 440 cases includes invasive cervical carcinoma or ICC (n = 210), inflammatory smear (n = 162), normal cytology (n = 68). Statistical analyses were performed by using SPSS version 20.0 software and MediCal version 14.10.2(7). A p-value of ≤ 0.05 was considered statistically significant. RESULTS: The overall prevalence of HPV infection was (359/595) 60.33%. Prevalence of HPV infection was 93.80% (197/210) in invasive cervical cancer (ICC) cases, 54.32% (88/162) in inflammatory smear and 19.11% (13/68) in normal cervical cytology. The most prevalent genotype was HPV16 (87.28%) followed by HPV18 (24.56%) and HPV 51(3.46%). The overall prevalence of single type was 76.58% and highest (78.9%) among ICC cases. The most frequent genotype combination after HPV16 + 18(9.4%) was HPV16 + 66 + 68(2.7%) which was frequently observed in inflammatory cytology. Age > 45years, parity ≥3, low socio-economic condition, rural residential area and post menopause state were significantly associated with HPV infection. Multiple infections did not have a significant association with any of the clinicopathological variables (stage, LN metastasis, cell type) except tumor size ≥ 2cm in ICC cases. The impact of 2v, 4v, and 9v vaccines in preventing cervical cancer in Odisha were 89.99, 91.65, and 92.16% respectively. CONCLUSION: This data would help planning an appropriate strategy for disease monitoring and provides baseline data for post-vaccination surveillance in the region. The nonavalent vaccine would be significant in preventing cervical carcinoma in Odisha. Hence an effective vaccination program based on regional HPV epidemiological profile along with the cervical cancer screening is necessary to reduce the cervical cancer burden in India.

Identification of the hot-spot areas for sickle cell disease using cord blood screening at a district hospital: an Indian perspective.

Sickle cell disease (SCD), a genetic disorder often reported late, can be identified early in life, and hot-spot areas may be identified to conduct genetic epidemiology studies. This study was undertaken to estimate prevalence and to identify hot spot area for SCD in Kalahandi district, by screening cord blood of neonates delivered at the district hospital as first-hand information. Kalahandi District Hospital selected for the study is predominated by tribal population with higher prevalence of SCD as compared to other parts of Odisha. Cord blood screening of SCD was carried out on 761 newborn samples of which 13 were screened to be homozygous for SCD. Information on area of parent's residence was also collected. Madanpur Rampur area was found to be with the highest prevalence of SCD (10.52 %) and the gene distribution did not follow Hardy-Weinberg Equation indicating un-natural selection. The approach of conducting neonatal screening in a district hospital for identification of SCD is feasible and appropriate for prioritizing area for the implementation of large-scale screening and planning control measures thereof.

A randomized controlled trial of increased dose and frequency of albendazole with standard dose DEC for treatment of Wuchereria bancrofti microfilaremics in Odisha, India.

Although current programmes to eliminate lymphatic filariasis have made significant progress it may be necessary to use different approaches to achieve the global goal, especially where compliance has been poor and 'hot spots' of continued infection exist. In the absence of alternative drugs, the use of higher or more frequent dosing with the existing drugs needs to be explored. We examined the effect of higher and/or more frequent dosing with albendazole with a fixed 300 mg dose of diethylcarbamazine in a Wuchereria bancrofti endemic area in Odisha, India. Following screening, 104 consenting adults were randomly assigned to treatment with the standard regimen annually for 24 months (S1), or annually with increased dose (800 mg albendazole)(H1) or with increased frequency (6 monthly) with either standard (S2) or increased (H2) dose. Pre-treatment microfilaria counts (GM) ranged from 348 to 459 mf/ml. Subjects were followed using microfilaria counts, OG4C3 antigen levels and ultrasound scanning for adult worm nests. Microfilarial counts tended to decrease more rapidly with higher or more frequent dosing at all time points. At 12 months, Mf clearance was marginally greater with the high dose regimens, while by 24 months, there was a trend to higher Mf clearance in the arm with increased frequency and 800 mg of albendazole (76.9%) compared to other arms, (S1:64%, S2:69.2% & H1:73.1%). Although higher and/or more frequent dosing showed a trend towards a greater decline in antigenemia and clearance of "nests", all regimens demonstrated the potential macrofilaricidal effect of the combination. The higher doses of albendazole did not result in a greater number or more severe side effects. The alternative regimens could be useful in the later stages of existing elimination programmes or achieving elimination more rapidly in areas where programmes have yet to start.

An outbreak of Japanese encephalitis after two decades in Odisha, India.

Sudden deaths in children due to acute encephalitis syndrome (AES) from a tribal dominated district of Malkangiri in Odisha, India, was reported during September-November, 2012. The investigation was carried out to search for the possible viral aetiology that caused this outbreak. Clinico-epidemiological survey and seromolecular investigation were carried out to confirm the viral aetiology. Two hundred seventy two suspected cases with 24 deaths were observed. The patients presented with low to moderate grade fever (87%), headache (43%), vomiting (27%), cold (18%), cough (17%), body ache (15%), joint pain (15%), rash (15%), abdomen pain (9%), lethargy (5%), altered sensorium (8%), convulsion (2%), diarrhoea (3%), and haematemesis (3%). Laboratory investigation showed Japanese encephalitis virus (JEV) IgM in 13.8 per cent (13/94) in blood samples and JEV RNA in one of two cerebrospinal fluid (CSF) samples. Paddy fields close to the houses, high pig to cattle ratio, high density (33 per man hour density) of Culex vishnui mosquitoes, low socio-economic status and low health awareness in the tribal population were observed. This report confirmed the outbreak of JEV infection in Odisha after two decades.

Occult HBV infection in multi transfused thalassemia patients.

OBJECTIVE: To determine the prevalence of Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infection in multitransfused thalassemic patients, with an aim to further highlight the need for donor screening strategy with supplementary molecular diagnostic tools for high risk population. METHODS: The study was conducted in 174 thalassemic subjects from Thalassemia unit of Central Red Cross Blood Bank, Cuttack, Odisha, India. Sero molecular diagnosis was followed to detect antigen, antibody and DNA in the study subjects. RESULTS: Prevalence of antibody to Hepatitis C, HBsAg, Anti HBs and Anti HBc were found to be 3.4 %, 0.5 %, 30.4 % and 21.8 % respectively. HBV seropositivity increased with increase in number of transfusions. Anti HBc was 12 %, 26.8 % and 71.4 % in subjects who received <40, 40-80 and >80 units of transfusions respectively. HBV DNA was detected in 50 % (3/6) of subjects having anti HBc as the only marker (Occult HBV infection). More so, it was detected in 16.12 % (5/31) of cases who were sero positive for both Anti HBs and Anti HBc. CONCLUSIONS: These results indicate that thalassemic subjects need detailed screening of transfusion products. Fifty percent of occult HBV infection is a major concern suggesting inclusion of viral DNA amplification test along with antigen/antibody detection.

Transportation of sputum samples in cetylpyridinium chloride for drug resistance studies from remote areas of Odisha, India.

INTRODUCTION: Antimicrobial susceptibility testing of Mycobacterium tuberculosis is required for successful treatment of patients, mainly in retreatment cases which necessitate isolation of mycobacteria from sputum samples within 24-48 hours. In situations where transportation of sputum is required, the use of cetylpyridinium chloride (CPC) effectively sustains the viability of mycobacteria up to two weeks. METHODOLOGY: Sputum samples were collected from pulmonary TB patients attending designated microscopy centres (DMC), stored in CPC solution and transported to a culture drug susceptibility testing laboratory using overnight bus transport facilities. For culture, the sputum specimens were processed and inoculated in Lowenstein- Jensen (LJ) medium. Growth on LJ was identified by colony morphology, growth rate and biochemical tests, and transit time was calculated as the time taken from the date of sample collection to the inoculation date. RESULTS: Out of the 816 sputum samples collected in CPC, 691 (84.7%) yielded M. tuberculosis, 97 (11.9%) yielded no growth, 21(2.6%) grew contaminants and 7 (0.8%) were nontuberculous mycobacteria. CPC containing sputum samples processed within two weeks showed 88.6% culture positivity, while positivity was significantly affected beyond two weeks. CONCLUSIONS: CPC is cheap, easy to use, inhibits the growth of other organisms and can effectively be used to transport sputum specimens within two weeks from hard to reach areas to central locations without compromising culture positivity. Bus transport services can also help in reducing delay and the cost of transportation from remote areas.

Fat-soluble antioxidant vitamins, iron overload and chronic malnutrition in ß-thalassemia major.

OBJECTIVE: To assess the antioxidant vitamins A (retinol) and E (α-tocopherol) levels, iron status and growth retardation in children with ß-thalassemia major in Odisha, an eastern state of India. METHODS: Forty three children aged 1-15 y diagnosed with ß-thalassemia major (28 males and 15 females) and 42 age-matched healthy controls (22 males and 20 females) were studied. ß-thalassemia was detected by using Bio-rad variant assay. Measurement of blood hemoglobin (Hb), hematocrit, serum vitamins (A and E) and ferritin was carried out by standard methods. RESULTS: Mean hemoglobin (6.60 ± 1.84 vs. 11.8 ± 2.29 g/dL, p < 0.01), serum retinol (28.0 ± 17.67 vs. 54.4 ± 36.56 µg/dL, p < 0.001) and α-tocopherol (0.2 ± 0.34 vs. 1.1 ± 0.82 mg/dL, p < 0.001) were significantly lower in children with thalassemia compared with control group, however, serum ferritin (storage iron) was elevated in thalassemia patients (553.7 ± 176.80 vs. 57.3 ± 40.73 ng/mL, p < 0.001). Vitamin E had significantly correlated with hemoglobin and hematocrit values in the patients. Growth retardation in terms of stunting (79 % vs. 24 %, p < 0.0001) and thinness (32.6 % vs. 9.5 %, p < 0.05) was significantly higher in thalassemic children compared with normal children. CONCLUSIONS: This study shows that children with ß-thalas-semia major are in a state of oxidative stress of hyperfer-ritinemia with deprived antioxidant vitamins (A and E) and poor growth status suggesting a possible need for reduction in iron overload and additional antioxidant supplementation.

Molecular monitoring of antimalarial drug resistance among Plasmodium falciparum field isolates from Odisha, India.

In the absence of definite marker for artemisinin (ART) resistance, molecular monitoring of its partner drug sulfadoxine pyrimethamine (SP) in artemisinin based combination therapy (ACTs) together with chloroquine (CQ) for which ART is negatively correlated, may predict the effectiveness of ACT. We analyzed 201 Plasmodium falciparum field isolates for drug resistance markers for CQ (pfcrt and pfmdr1), pyrimethamine (pfdhfr) and sulfadoxine (pfdhps). Our study reveals high prevalence and non-random association of resistant mutants (K76T and N86Y) of CQ markers (pfcrt and pfmdr1). The predominance of highly resistant pfdhfr genotypes for SP with intragenic and intergenic pair-wise linkage disequilibrium between single nucleotide polymorphisms of resistant mutants of pfdhfr (C59R and S108N) and pfdhps (S436A, A437G, K540E) warn on further inclusion of SP in ACT. These findings suggest the replacement of SP in ACT with alternative partner drug for better efficacy.

An outbreak of hand, foot and mouth disease in Bhubaneswar, Odisha.

OBJECTIVE: To describe the epidemiology and clinical features of cases in an outbreak of Hand, Foot and Mouth Disease (HFMD). DESIGN: Descriptive epidemiological study. SETTING: Hospitals and community in urban areas of Bhubaneswar city, Odisha. METHODS: Upon clinical suspicion of the first case as HFMD, local pediatricians and dermatologists were sensitized for case referral to Dermatology department of Institute of Medical Science and SUM hospital (IMS and SH) for evaluation and follow up. Community survey was undertaken by household visit by the team from Regional Medical Research Centre, Bhubaneswar in an outbreak area through hospital case tracing. Blood samples were tested for hematological counts and RT PCR assay done in a subset of samples for confirmation. RESULTS: Seventy eight cases of HFMD were detected between September 7 and November 6, 2009. Mean age (SD) was 5.13 (4.94) years (range 4 mo-31 yrs) and both sexes were equally affected. Fever and rash were the most common presenting symptoms with the rash distributed mostly over buttocks (83.3%), knees (77.5%), both surfaces of hands and oral mucosa (78.2%). Lesions healed in Mean (SD) 8.6 (1.5) days (range 7-15 d). Recovery was complete with minimal supportive treatment but, nail shedding was noted in three children within 4-5 weeks. CA16 was confirmed as the viral agent. CONCLUSION: Children (5-14 yrs) were majorly affected and complete recovery without neurological complications were noted. The characteristic clinical features described will be useful for early clinical diagnosis where laboratory confirmation is not feasible.

Vitamin A status and hematological values in sickle cell disorder cases.

BACKGROUND: Sickle cell anemia (SCA), which is an inherited blood disorder characterized primarily by chronic anemia and oxidative stress plays a major role in pathophysiology. OBJECTIVE: This study aims to evaluate vitamin A (serum retinol) status and hematological parameters in children with homozygous and heterozygous sickle cell disorders and compared with age- and sex-matched healthy controls. MATERIALS AND METHODS: A sample of 80 referred cases (37 sickle cell disorders and 43 normal cases) aged 2-40 years were included in the study. Hematological parameters were measured in cell counter and serum retinol by high-performance liquid chromatography. RESULTS: The mean hemoglobin (Hb) and serum retinol were significantly lower among cases with sickle cell disease than in sickle cell trait and normal. Vitamin A deficiency (retinol < 20 µg/dl) reported to be higher in homozygous cases (46.2%) as compared to either heterozygous (29.2%) or control (23.2%) groups. Serum retinol was correlated directly with Hb, RBC count, and hematocrit levels, and inversely with percentage of sickling among sickle cell disorder cases. CONCLUSION: The results indicate that deprived vitamin A status with inductive oxidative stress is mainly due to sickling and hemolysis in SCA cases.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India.

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.