Molecular detection of drug-resistant Plasmodium falciparum mutants in Assam.

Background & objectives: The spread of drug-resistant Plasmodium falciparum (Pf) poses a serious threat to the control and elimination of malaria. The objective of this study was to detect the molecular biomarkers of antimalarial drug resistance in Pf in patients visiting a tertiary care hospital in Assam. Methods: Malaria was first detected in fever cases using microscopy and a rapid diagnostic test (RDT), and then confirmed using PCR. Pf chloroquine resistance transporter (Pfcrt), Pf multidrug resistance-1 (Pfmdr-1), and single-nucleotide polymorphisms linked to delayed parasite clearance after treatment with artemisinin MAL 10-688956 and MAL 13-1718319 and Kelch-13 propeller (PfK-13) genes were evaluated by PCR-restriction fragment length polymorphism (RFLP). Results: Sixty nine cases of malaria were found among 300 cases of fever. Of these, 54 were positive for Pf, 47 of which were confirmed by PCR. Pfcrt-K76T mutation was seen in 96.6 per cent and Pfmdr1-N86Y mutation in 84.2 per cent of cases. Mutation was not detected in MAL10 and MAL13 genes. Sequence analysis of Kelch-13 gene showed the presence of a novel mutation at amino acid position 675. Statistically, no significant association was found between the molecular biomarkers and demographic profile, clinical presentation and outcome of the cases. Interpretation & conclusions: Molecular surveillance is essential to assess the therapeutic efficacy of the drugs against circulating Pf isolates in Assam which are found to be highly resistant to CQ. The role of the new mutation found in the Kelch-13 gene in the development of artemisinin resistance in Assam needs to be thoroughly monitored in future research.

Subgroups of patients with young-onset type 2 diabetes in India reveal insulin deficiency as a major driver.

AIM/HYPOTHESIS: Five subgroups were described in European diabetes patients using a data driven machine learning approach on commonly measured variables. We aimed to test the applicability of this phenotyping in Indian individuals with young-onset type 2 diabetes. METHODS: We applied the European-derived centroids to Indian individuals with type 2 diabetes diagnosed before 45 years of age from the WellGen cohort (n = 1612). We also applied de novo k-means clustering to the WellGen cohort to validate the subgroups. We then compared clinical and metabolic-endocrine characteristics and the complication rates between the subgroups. We also compared characteristics of the WellGen subgroups with those of two young European cohorts, ANDIS (n = 962) and DIREVA (n = 420). Subgroups were also assessed in two other Indian cohorts, Ahmedabad (n = 187) and PHENOEINDY-2 (n = 205). RESULTS: Both Indian and European young-onset type 2 diabetes patients were predominantly classified into severe insulin-deficient (SIDD) and mild obesity-related (MOD) subgroups, while the severe insulin-resistant (SIRD) and mild age-related (MARD) subgroups were rare. In WellGen, SIDD (53%) was more common than MOD (38%), contrary to findings in Europeans (Swedish 26% vs 68%, Finnish 24% vs 71%, respectively). A higher proportion of SIDD compared with MOD was also seen in Ahmedabad (57% vs 33%) and in PHENOEINDY-2 (67% vs 23%). Both in Indians and Europeans, the SIDD subgroup was characterised by insulin deficiency and hyperglycaemia, MOD by obesity, SIRD by severe insulin resistance and MARD by mild metabolic-endocrine disturbances. In WellGen, nephropathy and retinopathy were more prevalent in SIDD compared with MOD while the latter had higher prevalence of neuropathy. CONCLUSIONS /INTERPRETATION: Our data identified insulin deficiency as the major driver of type 2 diabetes in young Indians, unlike in young European individuals in whom obesity and insulin resistance predominate. Our results provide useful clues to pathophysiological mechanisms and susceptibility to complications in type 2 diabetes in the young Indian population and suggest a need to review management strategies.

Effectiveness of a single dose of Japanese encephalitis vaccine among adults, Assam, India, 2012-2018.

BACKGROUND: Japanese encephalitis virus (JEV) remains the major etiology of encephalitis throughout Asia. In India, the state of Assam alone contributes more than one-third of the national burden of JE. Between 2011 and 2014, a single dose of JE vaccine SA 14-14-2 (LAJEV) was administered among adults aged 15-65 years residing in Sivasagar and Dibrugarh districts of Assam, India. We monitored the trend of JE incidence between 2009 and 2018 using JE surveillance data, estimated the long-term effectiveness of the single dose of LAJEV and estimated the coverage of JE vaccine in two districts. METHODS: We compared the JE vaccination status of laboratory-confirmed hospitalized JE patients (case) and age, sex and locality matched healthy individuals (controls) to estimate the effectiveness of single dose of JE vaccine. We used surveillance data for 2009-2018 to calculate the incidence of JE among adults. We conducted a community-based survey to estimate the coverage of JE vaccine in the two districts. RESULTS: A total of 452 laboratory-confirmed JE case-patients and 904 matched healthy controls were enrolled in the study between 2012 and 2018. The effectiveness of a single dose of JE vaccine over the 7-year period was 77.0 (95% CI: 67.0-83.0). Vaccine effectiveness decreased from 91% (95% CI: 73.0-97.0) in first year of vaccination to 71% (95% CI: 21.0-90.0) at six years post-vaccination. The incidence of adults JE cases declined from 10.5 per 100,000 in the pre-vaccination period to 5.7 per 100,000 in the years following vaccination. The coverage of vaccine among adults in two districts was 40.1% (36.8-43.5). CONCLUSIONS: A single dose of JE vaccine offered adequate protection for at least six years. Conducting mass vaccination campaigns periodically would further reduce the incidence of JE in endemic districts in Assam.

Demographic and clinical profile of youth onset diabetes patients in India-Results from the baseline data of a clinic based registry of people with diabetes in India with young age at onset-[YDR-02].

BACKGROUND: We here report the demographic and clinical profile of the patients enrolled in the Indian Council of Medical Research funded Registry of people with diabetes in India with young age at onset (YDR) from 1 January 2000 to 31 July 2011. METHODS: The YDR registry recruits all diabetes cases (newly diagnosed or treated) reporting on or after 1 January 2000 with age of diagnosis ≤25 years, and residing within the assigned geographical area of the reporting centres. A baseline proforma was used to obtain information on demographic and clinical details at registration. RESULTS: The registry has enrolled 5546 patients (49.5% male; 50.5% female) with youth onset diabetes from 205 reporting centres linked to 8 regional collaborating centres (RCC) across India. T1DM (63.9%; n = 3545) and T2DM (25.3%; n = 1401) were the commonest variants of youth onset diabetes, though their relative proportion varied across RCCs. The mean (SD) age at diagnosis for T1DM was 12.9 (6.5) years, while that for T2DM was 21.7 (3.7) years. Nearly half the T1DM patients were registered within 6 months of the onset of disease. Most cases of T2DM (47.3%) were registered after 3 years from their date of diagnosis. 56.1% of patients had at least one episode of hospitalization at registration. CONCLUSION: The observations from YDR registry indicate the need to establish a surveillance system in India to monitor diabetes in youth, not only to understand its complex etiology and natural history but also due to its detrimental socio economic impact.

Vitamin D status and its relationship with systemic lupus erythematosus as a determinant and outcome of disease activity.

Background The importance of vitamin D (VD) in systemic lupus erythematosus (SLE) is being increasingly appreciated, with studies suggesting a relationship between VD deficiency and SLE onset/disease activity. We investigated VD status in SLE patients and its associations with disease activity in a geographical region of India receiving low solar ultraviolet-B (UV-B) index. Materials and methods We enrolled 109 SLE patients along with 109 healthy controls belonging to same ethnicity and localities. Demographic and clinico-laboratory information were recorded. VD status was assessed by estimating serum 25-hydroxyvitamin D (25-OH-D) concentrations (deficient: <20 ng/mL, insufficient: 21-29 ng/mL, and sufficient/normal: ≥30 ng/mL) using an enzyme-linked fluorescent assay (ELFA). The SLE Disease Activity Index (SLEDAI) scoring system was used to evaluate disease activity. The association between VD status and disease activity was assessed by univariate and multivariate approaches. Results Hypovitaminosis D was prevalent in 90.83% SLE patients [vs. 77.98% healthy controls; chi-squared (χ2) = 10.125, df = 2, p < 0.01]. SLEDAI scores and 25-OH-D values were inversely associated, which extended in a two-way manner as revealed by multiple logistic regression models. SLE patients with VD deficiency were more likely to have high/very high disease activity [adjusted odds ratio (OR) = 3.5, 95% confidence intervals (CI): 1.4-8.9]. Conversely, patients with high SLEDAI scores (>10) also had greater risks of being VD deficient (adjusted OR = 3.9, 95% CI: 1.5-10.8). Conclusion VD deficiency is widespread in SLE. The relationship appears to be bidirectional, with VD status associated both as determinant and outcome of disease activity in SLE.

Incidence of Japanese Encephalitis amongst acute encephalitis syndrome cases in upper Assam districts from 2012 to 2014: A report from a tertiary care hospital.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a major public health problem in India because of high mortality rate and residual neuropsychiatric damage in the survivors. The present study was undertaken to investigate JE positivity amongst patients admitted with acute encephalitis syndrome (AES) in upper Assam districts and different parameters with their changing trends related to it. METHODS: It was a hospital-based prospective cross-sectional study conducted from January 2012 to December 2014. A total of 1707 consecutive non-repetitive hospitalized patients, satisfying the clinical case definition of AES as per the WHO guidelines, were included in the study. Cerebrospinal fluid (CSF) and serum samples were tested for JEV-specific IgM antibodies. RESULTS: Of the 1707 patients admitted, 696 (40.77 %) were diagnosed as JE with male-to-female ratio 1.7:1 and adult to paediatric ratio 2.2:1. Fever (100%), change in mental status (100%), headache (80.02%), neck rigidity (52.01%), unconsciousness (48.99%), seizure (37.64%) and paralysis (11.06%) were the major clinical findings. The majority of cases (94%) were from rural areas. There was a significant association of JE cases with rainy season of the year i.e., June to August (P<0.001). Overall, 14.94 per cent deaths were reported in JE positive cases. INTERPRETATION & CONCLUSIONS: A higher occurrence of JE was observed in above 15 yr age group. Cases were mainly from rural areas, and there was clustering of cases in rainy season.

Neurological Manifestations in Systemic Lupus Erythematosus: A Single Centre Study from North East India.

INTRODUCTION: Neurological manifestations although common in Systemic Lupus Erythematosus (SLE), are often not recognized due to their diversed and varied presentation. Therefore, the study was planned to highlight the pattern of neurological involvement in SLE to help in early recognition. AIM: To study the pattern of neurological involvement in SLE and its correlation with disease activity and different investigation. MATERIALS AND METHODS: This hospital based prospective observational study was carried out from August 2009 to July 2010. Diagnosed cases of SLE [based upon American Rheumatism Association (ARA) criteria] who presented with neurological manifestations at the time of diagnosis or develop during the course of the disease were included in the study. They were assessed clinically and investigated with neuroimaging and neurophysiological tests as applicable. RESULTS: In total, 52 consecutive patients with SLE were evaluated, 92% were female. The most common age group was 21 to 25 years. Nervous system involvement was found in 19 (36.54%) patients. Cognitive impairment was the most frequent manifestation, present in 11 (57.89%) patients followed by seizure disorder in eight patients (42.1%). Peripheral neuropathy was diagnosed in eight (42.1%), acute confusional state in six (31.57%) and headache and depression was diagnosed in five (26.31%) patients each. Less common manifestations were psychosis, movement disorder and aseptic meningitis. Percentage of neurological manifestations directly correlated with disease activity. A significant difference was found in Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score between the patients with Neuro Psychiatric Systemic Lupus Erythematosus (NPSLE) and those without NPSLE (32.42±16.34 Vs 17.3±10.6). CONCLUSION: Neurological involvement in SLE is seen relatively early in the course of the disease with cognitive impairment being the most common manifestation and correlate with disease activity.

Registry of Youth Onset Diabetes in India (YDR): Rationale, Recruitment, and Current Status.

BACKGROUND: With the aim of addressing the relative scarcity of information on youth-onset diabetes in India, the Indian Council of Medical Research (ICMR) decided to establish the Registry of People with Diabetes with Young Age at Onset (YDR) in 2006. The major objectives of YDR are to generate information on disease pattern or types of youth-onset diabetes including their geographical variations within India and to estimate the burden of diabetes complications. METHODS: YDR is an observational multicenter clinic based registry enlisting physician diagnosed diabetes in individuals below 25 years of age. Diabetes was classified using symptom based clinical criteria. YDR data collection is coordinated through regional collaborating centers and their interacting reporting centers across India. A baseline and an annual follow-up proformas are used to obtain information on sociodemographic details, clinical profile, and anthropometric and laboratory measurements of the patients. RESULTS: In phase 1, the registry has enrolled 5546 patients, in which type 1 diabetes mellitus (T1DM) was the most prevalent (63.9%), followed by youth-onset type 2 diabetes mellitus (T2DM) (25.3%). CONCLUSION: This registry provides a unique opportunity to study the natural history of youth-onset diabetes in India.

Immunogenicity & safety of a single dose of live-attenuated Japanese encephalitis vaccine SA 14-14-2 in adults.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) caused by mosquito-borne Flavivirus is one of the leading causes of viral encephalitis in Asia. Control strategies include vector control and human vaccination. Due to lack of immunization programmes in endemic regions, there are still high mortality and morbidity. A live-attenuated SA 14-14-2 JE vaccine (LAJEV) has been licensed and used in Asian countries, including India. We report the assessment of immunogenicity and safety of the vaccine in adults during the first mass adult vaccination campaign carried out in Assam, India. METHODS: One thousand and seventy five adults (aged ≥15 yr) who received LAJEV were monitored for adverse events following immunization for one year. The safety assessment of vaccinated population was evaluated till 28 days and at 6 and 12 months. Blood samples collected from the enrolled participants were tested by plaque reduction neutralization test (PRNT 50 ) to assess the neutralizing antibody titres (NATs) before vaccination and 28 days, six and 12 months post-vaccination (PV). RESULTS: Among the 1075 vaccinated individuals, four reported minor adverse effects from 30 min to 28 days PV. Based on the pre-vaccination NAT, the study participants were categorized as seronegative, moderately seropositive and strongly seropositive. Nearly 85.5 per cent of JE seronegative participants seroconverted by 28 days PV. The geometric mean titre (GMT) in all the three groups increased by 28 days and decreased by six and 12 months PV. Nearly 60 per cent of the moderately positive individuals exhibited four-fold rise in GMT, 28 days PV. Almost 95.5 per cent of the participants in the study population remained seroprotected at the end of 12 months PV. INTERPRETATION & CONCLUSIONS: This study on immunogenicity and safety of LAJEV in adults showed that a single dose of the live-attenuated vaccine was safe and induced protective immunity to both JE seronegative and naturally seropositive adults. Further study is required to find out long term protective efficacy of this vaccine.

Heightened inflammation in severe malaria is associated with decreased IL-10 expression levels and neutrophils.

Dysregulation of the cytokine network in severe malaria owing to variations in factors like parasite load, strains and host factors is well documented but the key cytokines that are dysregulated remain poorly elucidated. Longitudinal changes in cytokine levels in an individual with parasitemia and disease resolution is likely to identify the key cytokines. We have analyzed the mRNA expression of cytokines over a 7-d period in severe (SM) and uncomplicated (UM) Plasmodium falciparum malaria. We found up-regulated expression of TNF-α, IL-1ß, IFN-γ and TGF-ß in SM, with decreased expression of IL-10 on d 0. Further, we observed a negative correlation of IL-10 expression with parasitemia and pro-inflammatory cytokines, suggesting IL-10 to be the key cytokine in tilting the balance to an inflammatory response. Longitudinal analysis revealed that the key cytokines associated with disease were TNF-α, IL-1ß, IFN-γ, IL-12α, RANTES and TGF-ß, while TNF-α, IL-10 and TGF-ß discriminated between SM and UM. A higher neutrophil count in SM and its positive association with parasite density and IL-1ß and IL-8 provides support for neutrophils in inflammation in malaria. Our findings suggest subversion of anti-inflammatory response in SM by parasite factors towards an exaggerated pro-inflammatory response with involvement of neutrophils, the classical inflammatory cells.

The association of IL-8-251T/A polymorphism with complicated malaria in Karbi Anglong district of Assam.

Amongst host genetic factors, cytokine gene polymorphism can be anticipated to be an important factor as qualitative, quantitative and time of secretion play an important role in disease outcome. We have investigated association of cytokine promoter SNPs with risk of Plasmodium falciparum malaria and disease severity in a case control study in malaria endemic Karbi Anglong district of Assam, India. Frequency of IL-8-251T/A (p=0.03 and p=0.01) and TGF-ß1-509C/T (p=0.02 and p=0.03) was higher in malaria in comparison to control participants and non-malarial fever controls. Interestingly, a higher frequency of mutant allele of IL-10-819T/C was observed in non-malarial fever controls compared to malaria thus suggesting its role as a distinguishing marker of the two disease groups. Higher IL-8 expression and increased frequency of IL-8-251T/A in complicated malaria (p=0.002) was reported indicating its role in susceptibility to complicated malaria. In conclusion, our study suggests the role of mutant genotype of IL-8-251T/A as a marker of complicated malaria in our population. Surprisingly, decreased expression of TGF-ß1 in uncomplicated malaria even in presence of high expressing mutant genotype was observed and needs to be investigated in context of the pool of activated cells producing the cytokine.

Malaria in South Asia: prevalence and control.

The "Malaria Evolution in South Asia" (MESA) program project is an International Center of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. This US-India collaborative program will study the origin of genetic diversity of malaria parasites and their selection on the Indian subcontinent. This knowledge should contribute to a better understanding of unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. In this first of two reviews, we highlight malaria prevalence in India. In particular, we draw attention to variations in distribution of different human-parasites and different vectors, variation in drug resistance traits, and multiple forms of clinical presentations. Uneven malaria severity in India is often attributed to large discrepancies in health care accessibility as well as human migrations within the country and across neighboring borders. Poor access to health care goes hand in hand with poor reporting from some of the same areas, combining to possibly distort disease prevalence and death from malaria in some parts of India. Corrections are underway in the form of increased resources for disease control, greater engagement of village-level health workers for early diagnosis and treatment, and possibly new public-private partnerships activities accompanying traditional national malaria control programs in the most severely affected areas. A second accompanying review raises the possibility that, beyond uneven health care, evolutionary pressures may alter malaria parasites in ways that contribute to severe disease in India, particularly in the NE corridor of India bordering Myanmar Narayanasamy et al., 2012.