Seroprevalence of Dengue Infection Using IgG Capture ELISA in India, 2017-2018.

We conducted a nationally representative population-based survey in 60 districts from 15 Indian states covering all five geographic regions during 2017-2018 to estimate the age specific seroprevalence of dengue. Of the 12,300 sera collected, 4,955 were positive for IgG antibodies against dengue virus using IgG Indirect ELISA indicating past dengue infection. We tested 4,948 sera (seven had inadequate volume) positive for IgG antibodies on indirect ELISA using anti-dengue IgG capture ELISA to estimate the proportion of dengue infections with high antibody titers, suggestive of acute or recent secondary infection. Of the 4,948 sera tested, 529 (10.7%; 95% CI: 9.4-12.1) were seropositive on IgG capture ELISA. The proportions of dengue infections with high titers were 1.1% in the northeastern, 1.5% in the eastern, 6.2% in the western, 12.2% in the southern, and 16.7% in the northern region. The distribution of dengue infections varied across geographic regions, with a higher proportion of infections with high antibody titer in the northern and southern regions of India. The study findings could be useful for planning facilities for clinical management of dengue infections.

Improved kala-azar case management through implementation of health facility-based sentinel sites surveillance system in Bihar, India.

BACKGROUND: Visceral leishmaniasis (VL), also known as kala-azar (KA), is a neglected vector-borne disease, targeted for elimination, but several affected blocks of Bihar are posing challenges with the high incidence of cases, and moreover, the disease is spreading in newer areas. High-quality kala-azar surveillance in India, always pose great concern. The complete and accurate patient level data is critical for the current kala-azar management information system (KMIS). On the other side, no accurate data on the burden of post kala-azar dermal leishmaniasis (PKDL) and co-infections are available under the current surveillance system, which might emerge as a serious concern. Additionally, in low case scenario, sentinel surveillance may be useful in addressing post-elimination activities and sustaining kala-azar (KA) elimination. Health facility-based sentinel site surveillance system has been proposed, first time to do a proper accounting of KA, PKDL and co-infection morbidity, mortality, diagnosis, case management, hotspot identification and monitoring the impact of elimination interventions. METHODOLOGY/PRINCIPAL FINDINGS: Kala-azar sentinel site surveillance was established and activated in thirteen health facilities of Bihar, India, using stratified sampling technique during 2011 to 2014. Data were collected through specially designed performa from all patients attending the outpatient departments of sentinel sites. Among 20968 symptomatic cases attended sentinel sites, 2996 cases of KA and 53 cases of PKDL were registered from 889 endemic villages. Symptomatic cases meant a person with fever of more than 15 days, weight loss, fatigue, anemia, and substantial swelling of the liver and spleen (enlargement of spleen and liver).The proportion of new and old cases was 86.1% and 13.9% respectively. A statistically significant difference was observed for reduction in KA incidence from 4.13/10000 in 2011 to 1.75/10000 in 2014 (p<0.001). There were significant increase (0.08, 0.10 per 10 000 population) in the incidences of PKDL and co-infection respectively in the year 2014 as compared to that of 2011 (0.03, 0.06 per 10 000 population). The proportion of HIV-VL co-infection was significantly higher (1.6%; p<0.05) as compared to other co-infections. Proportions of male in all age groups were higher and found statistically significant (Chi-square test = 7.6; P = 0.026). Utilization of laboratory services was greatly improved. Friedman test showed statistically significant difference between response of different anti kala-azar drugs (F = 25.0, P = 0.004).The initial and final cure rate of AmBisome was found excellent (100%). The results of the signed rank sum test showed significant symmetry of unresponsiveness rate (P = 0.03). Similarly, relapse rate of sodium antimony gluconate (SAG) was also found significantly higher as compared to other drugs (95%CI 0.2165 to 19.7035; P = 0.03). A statistically significant difference was found (p<0.001) between villages having 1-2 cases (74%) and villages with 3-5 cases (15%). Significantly higher proportion (95%) of cases were captured by existing Govt. surveillance system (KMIS) (p<0.001), as compared to private providers (5%). CONCLUSIONS/SIGNIFICANCE: Establishment of a sentinel site based kala-azar surveillance system in Bihar, India effectively detected the rising trend of PKDL and co-infections and captured complete and accurate patient level data. Further, this system may provide a model for improving laboratory services, KA, PKDL and co-infection case management in other health facilities of Bihar without further referral. Program managers may use these results for evaluating program's effectiveness. It may provide an example for changing the practices of health care workers in Bihar and set a benchmark of high quality surveillance data in a resource limited setting. However, the generalizability of this sentinel surveillance finding to other context remains a major limitation of this study. The justifications for this; the sentinel sites were made in the traditionally high endemic PHC's. The other conditions were Program commitment for diagnostic (rk-39) and the first line anti kala-azar drug i.e. miltefosine throughout the study period in the sentinel sites. In addition, there were clause of fulfillment of readiness criteria at each sentinel site (already described in the line no 171 to 180 at page no-8, 181-189 at page no-9 and 192-212 at page no-10). Rigorous efforts were taken to improve all the sentinel sites to meet the readiness criteria and research activities started only after meeting readiness criteria at the site. Therefore sentinel site surveillance described under the present study cannot be integrated into other set up (medium and low endemic areas). However, it can be integrated into highly endemic areas with program commitment and fulfillment of readiness criteria.

A randomized, open-label study to evaluate the efficacy and safety of liposomal amphotericin B (AmBisome) versus miltefosine in patients with post-kala-azar dermal leishmaniasis.

BACKGROUND: Treatment of post-kala-azar dermal leishmaniasis cases is of paramount importance for kala-azar elimination; however, limited treatment regimens are available as of now. AIM: To compare the effectiveness of liposomal amphotericin B vs miltefosine in post-kala-azar dermal leishmaniasis patients. METHODOLOGY: This was a randomized, open-label, parallel-group study. A total of 100 patients of post kala azar dermal leishmaniasis, aged between 5 and 65 years were recruited, 50 patients in each group A (liposomal amphotericin B) and B (miltefosine). Patients were randomized to receive either liposomal amphotericin B (30 mg/kg), six doses each 5 mg/kg, biweekly for 3 weeks or miltefosine 2.5 mg/kg or 100 mg/day for 12 weeks. All the patients were followed at 3rd, 6th and 12th months after the end of the treatment. RESULTS: In the liposomal amphotericin B group, two patients were lost to follow-up, whereas four patients were lost to follow-up in the miltefosine group. The initial cure rate by "intention to treat analysis" was 98% and 100% in liposomal amphotericin B and miltefosine group, respectively. The final cure rate by "per protocol analysis" was 74.5% and 86.9% in liposomal amphotericin B and miltefosine, respectively. Twelve patients (25.5%) in the liposomal amphotericin B group and six patients (13%) in the miltefosine group relapsed. None of the patients in either group developed any serious adverse events. LIMITATIONS: Quantitative polymerase chain reaction was not performed at all the follow-up visits and sample sizes. CONCLUSION: Efficacy of miltefosine was found to be better than liposomal amphotericin B, hence, the use of miltefosine as first-line therapy for post-kala-azar dermal leishmaniasis needs to be continued. However, liposomal amphotericin B could be considered as one of the treatment options for the elimination of kala-azar from the Indian subcontinent.

Immunity against diphtheria among children aged 5-17 years in India, 2017-18: a cross-sectional, population-based serosurvey.

BACKGROUND: Diphtheria is re-emerging as a public health problem in several Indian states. Most diphtheria cases are among children older than 5 years. In this study, we aimed to estimate age-specific immunity against diphtheria in children aged 5-17 years in India. METHODS: We used residual serum samples from a cross-sectional, population-based serosurvey for dengue infection done between June 19, 2017, and April 12, 2018, to estimate the age-group-specific seroprevalence of antibodies to diphtheria in children aged 5-17 years in India. 8309 serum samples collected from 240 clusters (122 urban and 118 rural) in 60 selected districts of 15 Indian states spread across all five geographical regions (north, northeast, east, west, and south) of India were tested for the presence of IgG antibodies against diphtheria toxoid using an ELISA. We considered children with antibody concentrations of 0·1 IU/mL or greater as immune, those with levels less than 0·01 IU/mL as non-immune (and hence susceptible to diphtheria), and those with levels in the range of 0·01 to less than 0·1 IU/mL as partially immune. We calculated the weighted proportion of children who were immune, partially immune, and non-immune, with 95% CIs, for each geographical region by age group, sex, and area of residence (urban vs rural). FINDINGS: 29·7% (95% CI 26·3-33·4) of 8309 children aged 5-17 years were immune to diphtheria, 10·5% (8·6-12·8) were non-immune, and 59·8% (56·3-63·1) were partially immune. The proportion of children aged 5-17 years who were non-immune to diphtheria ranged from 6·0% (4·2-8·3) in the south to 16·8% (11·2-24·4) in the northeast. Overall, 9·9% (7·7-12·5) of children residing in rural areas and 13·1% (10·2-16·6) residing in urban areas were non-immune to diphtheria. A higher proportion of girls than boys were non-immune to diphtheria in the northern (17·7% [12·6-24·2] vs 7·1% [4·1-11·9]; p=0·0007) and northeastern regions (20·0% [12·9-29·8] vs 12·9% [8·6-19·0]; p=0·0035). INTERPRETATION: The findings of our serosurvey indicate that a substantial proportion of children aged 5-17 years were non-immune or partially immune to diphtheria. Transmission of diphtheria is likely to continue in India until the immunity gap is bridged through adequate coverage of primary and booster doses of diphtheria vaccine. FUNDING: Indian Council of Medical Research.

Seroprevalence of chikungunya virus infection in India, 2017: a cross-sectional population-based serosurvey.

BACKGROUND: Since its re-emergence in 2005, chikungunya virus (CHIKV) transmission has been documented in most Indian states. Information is scarce regarding the seroprevalence of CHIKV in India. We aimed to estimate the age-specific seroprevalence, force of infection (FOI), and proportion of the population susceptible to CHIKV infection. METHODS: We did a nationally representative, cross-sectional serosurvey, in which we randomly selected individuals in three age groups (5-8, 9-17, and 18-45 years), covering 240 clusters from 60 selected districts of 15 Indian states spread across all five geographical regions of India (north, northeast, east, south, and west). Age was the only inclusion criterion. We tested serum samples for IgG antibodies against CHIKV. We estimated the weighted age-group-specific seroprevalence of CHIKV infection for each region using the design weight (ie, the inverse of the overall probability of selection of state, district, village or ward, census enumeration block, and individual), adjusting for non-response. We constructed catalytic models to estimate the FOI and the proportion of the population susceptible to CHIKV in each region. FINDINGS: From June 19, 2017, to April 12, 2018, we enumerated 117 675 individuals, of whom 77 640 were in the age group of 5-45 years. Of 17 930 randomly selected individuals, 12 300 individuals participated and their samples were used for estimation of CHIKV seroprevalence. The overall prevalence of IgG antibodies against CHIKV in the study population was 18·1% (95% CI 14·2-22·6). The overall seroprevalence was 9·2% (5·4-15·1) among individuals aged 5-8 years, 14·0% (8·8-21·4) among individuals aged 9-17 years, and 21·6% (15·9-28·5) among individuals aged 18-45 years. The seroprevalence was lowest in the northeast region (0·3% [95% CI 0·1-0·8]) and highest in the southern region (43·1% [34·3-52·3]). There was a significant difference in seroprevalence between rural (11·5% [8·8-15·0]) and urban (40·2% [31·7-49·3]) areas (p<0·0001). The seroprevalence did not differ by sex (male 18·8% [95% CI 15·2-23·0] vs female 17·6% [13·2-23·1]; p=0·50). Heterogeneous FOI models suggested that the FOI was higher during 2003-07 in the southern and western region and 2013-17 in the northern region. FOI was lowest in the eastern and northeastern regions. The estimated proportion of the population susceptible to CHIKV in 2017 was lowest in the southern region (56·3%) and highest in the northeastern region (98·0%). INTERPRETATION: CHIKV transmission was higher in the southern, western, and northern regions of India than in the eastern and northeastern regions. However, a higher proportion of the population susceptible to CHIKV in the eastern and northeastern regions suggests a susceptibility of these regions to outbreaks in the future. Our survey findings will be useful in identifying appropriate target age groups and sites for setting up surveillance and for future CHIKV vaccine trials. FUNDING: Indian Council of Medical Research.

Acute uveitis: A rare adverse effect of miltefosine in the treatment of post-kala-azar dermal leishmaniasis

Abstract Post-kala-azar dermal leishmaniasis is a skin disorder occurring in 5-10% of visceral leishmaniasis patients after treatment with miltefosine,the first-line drug for this skin disorder. We reported a case of acute anterior uveitis,a rare adverse effect, experienced by a patient treated with miltefosine for post-kala-azar dermal leishmaniasis. This adverse effect developed after 15 days of miltefosine consumption, and the patient himself discontinued the treatment. The ophthalmic complication was completely resolved with antibiotics and steroid eye drops. After recovery from the ophthalmic complication, the patient was successfully treated with liposomal amphotericin B for the skin lesions.

Acute uveitis: A rare adverse effect of miltefosine in the treatment of post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis is a skin disorder occurring in 5-10% of visceral leishmaniasis patients after treatment with miltefosine,the first-line drug for this skin disorder. We reported a case of acute anterior uveitis,a rare adverse effect, experienced by a patient treated with miltefosine for post-kala-azar dermal leishmaniasis. This adverse effect developed after 15 days of miltefosine consumption, and the patient himself discontinued the treatment. The ophthalmic complication was completely resolved with antibiotics and steroid eye drops. After recovery from the ophthalmic complication, the patient was successfully treated with liposomal amphotericin B for the skin lesions.

False Positivity of rK39 Test in Five Chronic Myeloid Leukemia Cases from Bihar, India: A Possible Challenge to Leishmaniasis Diagnosis.

A rapid and noninvasive rK39 rapid diagnostic test (RDT) is the best and most reliable tool for visceral leishmaniasis (VL) screening in the field. However, splenic and bone marrow aspiration remain two gold standard methods for microscopic identification of Leishmania donovani (LD) bodies and confirmatory diagnosis of VL. Five patients with signs and symptoms of fever, loss of appetite, loss of weight, hepatomegaly, and massive splenomegaly were found to be false positive with the rK39 RDT. These patients were suspected to have chronic myeloid leukemia (CML) because their blood pictures showed a total white blood cell count of > 100,000/mm3 and abnormal cells such as stab, segmented promyelocytes, myelocytes, metamyelocytes, and blast cells. Splenic aspirate and bone marrow were negative for Leishmania donovani bodies. The bone marrow showed myeloid series of cells, that is, myelocytes, metamyelocytes, stab and segmented cells, blast cells, and markedly increased myeloid:erythroid ratio. Later, the CML diagnosis was confirmed in all cases by breakpoint cluster region-tyrosine protein kinase (BCR-ABL) gene positive test results. In this study, the rK39 RDT's false positivity was observed in CML cases. It could have important implications for the differential diagnosis of VL with CML. The rK39 positive test result in CML cases was a serendipitous occurrence; this should be validated further to determine the utility of the rK39 test in the differential diagnosis of VL with CML.

Hepatitis-B virus infection in India: Findings from a nationally representative serosurvey, 2017-18.

INTRODUCTION: India introduced a hepatitis-B (HB) vaccine in the Universal Immunization Program in 2002-2003 on a pilot basis, expanded to ten states in 2007-2008 (phase-1), and the entire country in 2011-2012 (phase-2). We tested sera from a nationally representative serosurvey conducted duing 2017, to estimate the seroprevalence of different markers of HB infection among children aged 5-17 years in India and to assess the impact of vaccination. METHODS: We tested sera from 8273 children for different markers of HB infection and estimated weighted age-group specific seroprevalence of children who were chronically infected (HBsAg and anti-HBc positive), and immune due to past infection (anti-HBc positive and HBsAg negative), and having serological evidence of HB vaccination (only anti-HBs positive). We compared the prevalence of serological markers among children born before (aged 11-17 years) and after (aged 5-10 years) introduction of HB-vaccine from phase-1 states. RESULTS: Among children aged 5-8 years, 1.1% were chronic carriers, 5.3% immune due to past infection, and 23.2% vaccinated. The corresponding proportions among children aged 9-17 years were 1.1%, 8.0%, and 12.0%, respectively. In phase-1 states, children aged 5-10 years had a significantly lower prevalence of anti-HBc (4.9% vs. 7.6%, p<0.001) and higher prevalence of anti-HBs (37.7% vs. 14.7%, p<0.001) compared to children aged 11-17 years. HBsAg positivity, however, was not different in the two age groups. CONCLUSIONS: Children born after the introduction of HB vaccination had a lower prevalence of past HBV infection and a higher prevalence of anti-HBs. The findings of our study could be considered as an interim assessment of the impact of the hepatitis B vaccine introduction in India.

Conversion of asymptomatic infection to symptomatic visceral leishmaniasis: A study of possible immunological markers.

INTRODUCTION: Presence of asymptomatic individuals in endemic areas is common. The possible biomarkers in asymptomatic individuals once they get exposed to infection as well as following conversion to symptomatic disease are yet to be identified.We identified asymptomatic Visceral leishmaniasis (VL) infection amongst rK39+sorted direct agglutination test positive (DAT+) endemic healthy population and confirmed it by quantitative PCR(qPCR).The immunological determinants such as Adenosine deaminase (ADA), Interferon gamma (IFN-γ), Tumour Necrosis Factor alpha (TNF-α) and Interleukin 10 (IL-10)were examined to predict probable biomarkers for conversion to symptomatic VL. METHODS: Sample size was 5794 healthy individuals from VL endemic region. Antibody tests(DAT &rK39) were performed and later a qPCR assay was employed using kDNA specific primers and probes. Immunological biomarkers examined were ADA level by ADA-MTP kit and quantitative cytokines(IFN-γ, IL-10 and TNF-α) by ELISA. RESULTS: 120 asymptomatic individuals of 308 rK39 sero-positives were DAT positive comprising of 56 with previous history and 64 with no history of VL. RT-PCR confirmed asymptomatic VL in 42 sero-positives. These were followed up through repeated qPCR and evaluation of immunological determinants. We observed10 symptomatic cases converted from a total of 42 asymptomatic individuals identified at base-line. The level of ADA, IL-10 and IFN-γ remained consistently high in asymptomatic cases and amongst these, ADA and IL-10 but not IFN-γ remained higher at the development of clinical symptoms into active VL. On the contrary, there was no significant change in the mean concentration of TNF-α at both stages of the disease. DISCUSSION: We surmise from our data that considerable proportion of asymptomatic cases can be a reservoir and may play a crucial role in transmission of visceral leishmaniasis in endemic areas. The data also suggests that ADA and IL-10 can serve as a potential biomarker during the conversion of asymptomatic into symptomatic VL.

Kala-azar elimination in a highly-endemic district of Bihar, India: A success story.

BACKGROUND: Visceral leishmaniasis (VL) or Kala-azar has been a major public health problem in Bihar, India, for several decades. A few VL infected districts including Vaishali have reported >600 cases annually. Hence, in 2015, the Government of India entrusted ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, to implement an integrated control strategy for achieving the VL elimination target (<1 case per 10,000 people at the block level) in the Vaishali District of Bihar. METHODOLOGY: This study was conducted between January 2015 and December 2016. An integrated control strategy including the spatio-temporal mapping of VL-case distribution, active case detection, chemical-based vector control using indoor residual spraying (IRS), community awareness campaigns, the training of IRS members, the training of medical doctors for effective treatment, daily monitoring and the supervision of IRS activities, logistic management, post-IRS quality assurance, epidemiological surveillance, and entomological monitoring was performed. An insecticide quantification test was performed for evaluating the IRS quality on sprayed walls. A modern compression pump was used to maintain spray quality on different wall surfaces. The impact of IRS was assessed through sand fly collection in human dwellings and cattle sheds in pre- and post-IRS. The insecticide susceptibility of local P. argentipes was performed before each IRS round (in February and June) during 2015-2016. Statistical analysis such as the mean, percentage, and 95% CI were used to summarize the results. FINDINGS: All 16 blocks of the Vaishali District achieved the VL elimination target in 2016. The integrated VL control strategy helped reduce the number of VL cases from 664 in 2014 to 163 in 2016 and the number of endemic villages from 282 in 2014 to 142 in 2016. The case reduction rate was increased from 22.6% in 2014 to 58.8% in 2016. On average, 74 VL infected villages became Kala-azar free each year from 2015 to 2016. CONCLUSIONS: The results of this study suggest that the elimination of VL is possible from all endemic blocks of Bihar if the integrated Vaishali VL control strategy is applied under strong monitoring and supervision.

Advanced case of PKDL due to delayed treatment: A rare case report.

Post-kala-azar dermal leishmaniasis (PKDL) is clinical outcome of visceral leishmaniasis (VL) and is thought to be the potential reservoir of parasite. Miltefosine (MF) is the only oral drug existing for treatment of post-kala-azar dermal leishmaniasis (PKDL). Increased miltefosine tolerance in clinical isolates of Leishmania donovani has been reported and is one of the major concerns in the treatment of PKDL. Here, we report a highly ulcerated PKDL case that was successfully cured after miltefosine treatment.

Evaluating the immunomodulatory responses of LdODC-derived MHC Class-II restricted peptides against VL.

In a bid to develop a novel immunoprophylactic measure against visceral leishmaniasis (VL), MHC class-II-restricted epitopes of LdODC were identified by reverse vaccinology approach. Five consensus HLA-DRB1*0101-restricted epitopes were screened. The analysis revealed that the set of epitopes was presented by at least 54 diverse MHC class-II alleles. Based on in silico screening, followed by molecular dynamics simulation, population coverage analysis, and HLA cross-presentation ability, five best epitopes were evaluated. PBMCs isolated from treated VL subjects, when stimulated with synthetic peptide alone or as a cocktail of peptides, triggered a secretory IFN-γ, but not the IL-10 level. Support in this notion came from intracellular cytokine level with a considerable up-regulated IFN-γ produced by CD4+ T cells. Also, the enhanced IFN-γ seemed to be augmented with the activation of macrophages with prominent IL-12 production. Therefore, it can be concluded that the screened MHC class-II-restricted epitope hotspots derived from Leishmania ODC can trigger CD4+ T cells, which can skew macrophage functions towards protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.

Molecular docking studies of chloroquine and its derivatives against P23pro-zbd domain of chikungunya virus: Implication in designing of novel therapeutic strategies.

The arthropod-transmitted chikungunya virus has emerged as an epidemic menace that causes debilitating polyarthritis. With this life-threatening impact on humans, the possible treatment requires to cure the viral infectivity. But, devoid of any vaccine against the chikungunya virus (CHIKV), there is a need to develop a novel chemotherapeutic strategy to treat this noxious infection. CHIKV carries highly compact P23pro-zbd structure that possesses potential RNA-binding surface domains which extremely influences the use of RNA template during genome replication at the time of infection and pathogenesis. Therefore, computational approaches were used to explore the novel small molecule inhibitors targeting P23pro-zbd domain. The tertiary structure was modeled and optimized using in silico approaches. The results obtained from PROCHECK (93.1% residues in favored regions), ERRAT (87.480 overall model quality) and ProSA (Z-score: -11.72) revealed the reliability of the proposed model. Interestingly, a previously reported inhibitor, chloroquine possesses good binding affinities with the target domain. In-depth analysis revealed that chloroquine derivatives such as didesethyl chloroquine hydroxyacetamide, cletoquine, hydroxychloroquine exhibited a better binding affinity. Notably, MD simulation analysis exhibited that Thr1312, Ala1355, Ala1356, Asn1357, Asp1364, Val1366, Cys1367, Ala1401, Gly1403, Ser1443, Tyr1444, Gly1445, Asn1459, and Thr1463 residues are the key amino acid responsible for stable ligand-protein interaction. The results obtained from this study provide new insights and advances the understanding to develop a new approach to consider effective and novel drug against chikungunya. However, a detailed in vivo study is required to explore its drug likeliness against this life-threatening disease.

Burden of dengue infection in India, 2017: a cross-sectional population based serosurvey.

BACKGROUND: The burden of dengue virus (DENV) infection across geographical regions of India is poorly quantified. We estimated the age-specific seroprevalence, force of infection, and number of infections in India. METHODS: We did a community-based survey in 240 clusters (118 rural, 122 urban), selected from 60 districts of 15 Indian states from five geographical regions. We enumerated each cluster, randomly selected (with an Andriod application developed specifically for the survey) 25 individuals from age groups of 5-8 years, 9-17 years, and 18-45 years, and sampled a minimum of 11 individuals from each age group (all the 25 randomly selected individuals in each age group were visited in their houses and individuals who consented for the survey were included in the study). Age was the only inclusion criterion; for the purpose of enumeration, individuals residing in the household for more than 6 months were included. Sera were tested centrally by a laboratory team of scientific and technical staff for IgG antibodies against the DENV with the use of indirect ELISA. We calculated age group specific seroprevalence and constructed catalytic models to estimate force of infection. FINDINGS: From June 19, 2017, to April 12, 2018, we randomly selected 17 930 individuals from three age groups. Of these, blood samples were collected and tested for 12 300 individuals (5-8 years, n=4059; 9-17 years, n=4265; 18-45 years, n=3976). The overall seroprevalence of DENV infection in India was 48·7% (95% CI 43·5-54·0), increasing from 28·3% (21·5-36·2) among children aged 5-8 years to 41·0% (32·4-50·1) among children aged 9-17 years and 56·2% (49·0-63·1) among individuals aged between 18-45 years. The seroprevalence was high in the southern (76·9% [69·1-83·2]), western (62·3% [55·3-68·8]), and northern (60·3% [49·3-70·5]) regions. The estimated number of primary DENV infections with the constant force of infection model was 12 991 357 (12 825 128-13 130 258) and for the age-dependent force of infection model was 8 655 425 (7 243 630-9 545 052) among individuals aged 5-45 years from 30 Indian states in 2017. INTERPRETATION: The burden of dengue infection in India was heterogeneous, with evidence of high transmission in northern, western, and southern regions. The survey findings will be useful in making informed decisions about introduction of upcoming dengue vaccines in India. FUNDING: Indian Council of Medical Research.

Field safety and effectiveness of new visceral leishmaniasis treatment regimens within public health facilities in Bihar, India.

BACKGROUND: In 2010, WHO recommended the use of new short-course treatment regimens in kala-azar elimination efforts for the Indian subcontinent. Although phase 3 studies have shown excellent results, there remains a lack of evidence on a wider treatment population and the safety and effectiveness of these regimens under field conditions. METHODS: This was an open label, prospective, non-randomized, non-comparative, multi-centric trial conducted within public health facilities in two highly endemic districts and a specialist referral centre in Bihar, India. Three treatment regimens were tested: single dose AmBisome (SDA), concomitant miltefosine and paromomycin (Milt+PM), and concomitant AmBisome and miltefosine (AmB+Milt). Patients with complicated disease or significant co-morbidities were treated in the SDA arm. Sample sizes were set at a minimum of 300 per arm, taking into account inter-site variation and an estimated failure risk of 5% with 5% precision. Outcomes of drug effectiveness and safety were measured at 6 months. The trial was prospectively registered with the Clinical Trials Registry India: CTRI/2012/08/002891. RESULTS: Out of 1,761 patients recruited, 50.6% (n = 891) received SDA, 20.3% (n = 358) AmB+Milt and 29.1% (n = 512) Milt+PM. In the ITT analysis, the final cure rates were SDA 91.4% (95% CI 89.3-93.1), AmB+Milt 88.8% (95% CI 85.1-91.9) and Milt+PM 96.9% (95% CI 95.0-98.2). In the complete case analysis, cure rates were SDA 95.5% (95% CI 93.9-96.8), AmB+Milt 95.5% (95% CI 92.7-97.5) and Milt+PM 99.6% (95% CI 98.6-99.9). All three regimens were safe, with 5 severe adverse events in the SDA arm, two of which were considered to be drug related. CONCLUSION: All regimens showed acceptable outcomes and safety profiles in a range of patients under field conditions. Phase IV field-based studies, although extremely rare for neglected tropical diseases, are good practice and an important step in validating the results of more restrictive hospital-based studies before widespread implementation, and in this case contributed to national level policy change in India. TRIAL REGISTRATION: Clinical trial is registered at Clinical trial registry of India (CTRI/2012/08/002891, Registered on 16/08/2012, Trial Registered Prospectively).

The potential HLA Class I-restricted epitopes derived from LeIF and TSA of Leishmania donovani evoke anti-leishmania CD8+ T lymphocyte response.

To explore new protective measure against visceral leishmaniasis, reverse vaccinology approach was employed to identify key immunogenic regions which can mediate long-term immunity. In-depth computational analysis revealed nine promiscuous epitopes which can possibly be presented by 46 human leukocyte antigen, thereby broadening the worldwide population up to 94.16%. This is of reasonable significance that most of the epitopes shared 100% sequence homology with other Leishmania species and could evoke a common pattern of protective immune response. Transporter associated with antigen processing binding affinity, molecular docking approach followed by dynamics simulation and human leukocyte antigen stabilization assay suggested that the best five optimal set of epitopes bind in between α1 and α2 binding groove with sufficient affinity and stability which allows the translocation of intact epitope to the cell surface. Fascinatingly, the human leukocyte antigen stabilization assay exhibited a modest correlation with the positive immunogenicity score predicted by class I pMHC immunogenicity predictor. A support for this notion came from ELISA and FACS analysis where the epitopes as a cocktail induced CD8+ IFN-γ and Granzyme B levels significantly in treated visceral leishmaniasis subject which suggests the immunogenic ability of the selected epitopes.

Noninvasive Sweat-Based Diagnosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis.

The diagnosis of visceral leishmaniasis (VL) is one of the foremost barriers in the control of this disease, as demonstration of the parasite by splenic/bone marrow aspiration is relatively difficult and requires expertise and laboratory support. The aim of the present study was to find a noninvasive diagnostic approach using the existing recombinant kinesine-39 (rK-39) immunochromatographic nitrocellulose strips test (ICT) with a human sweat specimen for the diagnosis of VL. The investigation was carried out on specimens (blood, sweat, and urine) collected from 58 confirmed VL, 50 confirmed post kala-azar dermal leishmaniasis (PKDL), 36 healthy control, and 35 patients from other diseases. The data obtained from this study reveal that 96.6% clinically confirmed active VL participants were found to be positive when tested against a sweat specimen. Interestingly, the scenario was similar when tested against a blood specimen (96.6% positive by rK-39). Moreover, a test of both sweats and blood specimens from 50 PKDL participants resulted in 100% positivity, whereas no healthy control participants were found to be rK-39 positive. The sensitivity of the rK-39 ICT in sweat specimen was 94.7%, whereas the specificity was 100% in healthy controls from endemic, nonendemic, and other infectious diseases, respectively. No difference was observed in sweat specimen of VL and PKDL cases which signifies its reliability. However, further evaluation of this method on a larger scale could enhance the reliability of the proposed model so that it could be used efficiently in VL management and eradication.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host's cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.