Development of real time PCR for detection and quantitation of Dengue Viruses

<p>Abstract</p> <p>Background</p> <p>Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study.</p> <p>Results</p> <p>The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, <it>Plasmodium vivax</it>, <it>Plasmodium falciparum </it>and Rickettsia positive clinical samples.</p> <p>Conclusion</p> <p>We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.</p

Strain improvement for the production of antioxidant activity from Bacillus sp. by induced stress

In the present study, water samples were collected from different beaches in Chennai such as Marina beach, Elliott beach, VGP Golden beach and Kovalam beach to evaluate the best source for antioxidants. The bacteria were isolated on Starch Casein Agar media and screened for the antimicrobial activity. Among 11 isolates, 5 isolates showed antimicrobial potential which were further evaluated for the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity. Among 5 isolates, one sample showed significant DPPH scavenging activity with half minimal inhibitory concentration of (IC50)344.754µg/mL. The maximum antioxidant production was observed at pH 7 and at temperature of 37ºC with an IC50 of 188.66µg/mL and 293.76µg/mL respectively. The potent antioxidant producing strain was subjected for mutagenesis. In physical mutagenesis, the organism exposed for UV light for 25 minutes showed maximum antioxidant production with an IC50 of 133.55µg/mL. This mutant strain was then subjected for chemical mutagenesis with the addition of different concentrations of Ethidium bromide such as 10µL, 20µL, 30µL, 40µL and 50µL. The mutant strain obtained with the addition of 20µL Ethidium bromide (EtBr) showed significant antioxidant activity with an IC50 of 325.4µg/mL. The sample was purified by solvent extraction method and was evaluated for antioxidant production. The analyte was subjected to HPLC (High Performance Liquid Chromatography) analysis to avince the presence of antioxidants. The ethyl acetate extract showed the total phenolic content of 0.892 mg GAE/g of dry extract. It also showed the total flavonoid content of 0.522mg RE/g of dry extract

AYURVEDA SUCCESSFUL MANAGEMENT OF HEPATITIS B – A CASE STUDY

A successful case study of Ayurveda management of Hepatitis B was reported. A 19 year old patient with classical symptoms of hepatitis B assessed subjectively and objectively and meticulously recorded at all stages. Yellow discoloration of eye, urine, nails and tongue, loss of appetite, generalized itching and fatigue was noted. After enhancing digestive capacity, medicated ghee –‘Vyoshadi grta,’ was administered as per Shaman principles in a dose of 10gm twice daily. Mrudu virechana (mild purgation) induced by Trivrth lehya 20gm which resulted in 5 to 6 Vegas (bouts of loose stools). Agni (digestive capacity) has slowly but gradually increased. Diet quantity and quality was rectified as required. Rasayan prescribed in one single dose daily was Draskhavlehya, after patient showed clinically significant clearance of all symptoms. Rasayana and Ahara vihara management was continued until blood serum hepatitis antigen B reported negative. Liver Function Test, HB percent in laboratory registered normalization and clinical confirmation showed significant results. Photographs of urine and eye documented periodically. Blood reports showed radical improvements. Physically, mentally and spiritually patient has reported wellbeing. Patient moved towards Swasthya (complete wellbeing). This case study guaranteed Ayurveda management as quick, safe and effective. Large scale study was required to reconfirm reports and can be initiated on similar principles and practices

Expression of NLRC4 Inflammasome and its Correlation with Treponema Denticola in Stage III/IV Periodontitis with Type II Diabetes mellitus

To evaluate the expression of NLRC4 inflammasome and correlate with Treponema denticola (T. denticola) levels, so as to comprehend their role in mediating chronic inflammation in individuals with periodontitis (PD) and Type 2 Diabetes Mellitus (T2DM). Materials and Methods: A total of fifty-one subjects were recruited and grouped as those systemically and periodontally healthy (PH, n=17), those systemically healthy with PD (PD, n=17), and those with T2DM with PD (PD+T2DM, n=17). Site specific probing pocket depth, clinical attachment level, plaque index, and gingival index were recorded. Thereafter, samples of subgingival plaque and gingival tissue taken with biopsy using an internal bevel incision were procured at sites evidencing the disease state. T. denticola was quantified using qPCR, and NLRC4 expression was evaluated with immunohistochemistry. Results: Compared to the PH controls, significantly higher expression and intensity of NLRC4 inflammasome was observed in the PD and PD with T2DM groups, with a significantly greater expression in the PD+T2DM group (p<0.05). In all three groups, NLRC4 expression (mean percentage, intensity) and T. denticola levels showed a significant positive correlation (p<0.05). Conclusion: The subgingival plaque, T. denticola levels in PD were significantly related to NLRC4 expression, both in the presence and absence of T2DM. NLRC4 activation possibly plays a role in establishing a hyperinflammatory state in diabetes mellitus modified PD

Impact of COVID-19 pandemic on postpartum contraception services in women delivering at a tertiary care centre in South India

Background: Contraception and sexual health are a fundamental human right and an integral part of women’s health services. Postpartum period is the unique opportunity for counselling and availing contraception. COVID-19 pandemic hindered routine and special services like postpartum clinics has negative impact on family planning services. Objectives were to assess the effect of the COVID-19 pandemic on postpartum contraceptive care services received and to describe the challenges faced in availing these services among pregnant women delivering at a tertiary care centre in South India.Methods: A descriptive study carried out in 422 women who had delivered at our hospital from July 2020 to October 2020. Data was collected in Epicollect version 5 and analysed using Stata version 14.2.Results: A total of 422 women were interviewed. Only one-third of the participants received contraceptive counselling in the antenatal period compared to 90% postpartum. 39% (primiparous-34%/multiparous-5%) had adopted barrier methods followed by post-partum intrauterine uterine contraceptive devices in 33% (primiparous-19.6%/multiparous-13.4%) and 5% had sterilisation concurrent with caesarean section. Around 30-40% of women faced challenges in accessing the family planning methods due to closure of elective services like postpartum clinics, operation theatres, nationwide lockdown, and non-availability of field health workers.Conclusions: Contraceptive choices for postpartum women appear to be largely restricted to temporary methods with additional challenges of availing these services during the pandemic. With the ongoing COVID-19 crisis and continuous need for contraception, there is a need to refocus and motivate eligible couples for long-acting reversible contraceptive methods (LARC) with significantly lower failure rates

Clinical Findings and Pro-Inflammatory Cytokines in Dengue Patients in Western India: A Facility-Based Study

BACKGROUND: Descriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: In 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-gamma, TNF-alpha, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version. Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-gamma were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-gamma and IL-8 were higher in early samples collected 2-5 days after onset than late samples collected 6-15 days after onset. IFN-gamma showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-gamma and IL-8 were positively associated with alanine transaminase levels. CONCLUSIONS/SIGNIFICANCE: Rash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-gamma and IL-8 levels had greater significance than absolute values in DHF pathogenesis

Awareness regarding eye donation among stakeholders in Srikakulam district in South India

Background There is a huge need for the availability of transplantable donor corneas worldwide to reduce the burden of corneal blindness due to corneal opacity. Voluntary eye donation depends on the awareness levels of various stakeholders in the community. This study aimed to assess the awareness level regarding eye donation among various stakeholders in Srikakulam district in the state of Andhra Pradesh, India. Methods 355 subjects were selected from the district using multi stage random sampling. A pre tested semi structured questionnaire was used to collect information regarding each individual’s awareness, knowledge, and perception regarding eye donation. Each response was scored individually and a total score was calculated. Univariate and multivariate regression analysis was used to determine the factors associated with willingness towards eye donation and increased awareness levels. Results Of the 355 subjects interviewed, 192 (54%) were male and 163 (46%) were female. The mean age of the stakeholders was 35.9 years (SD ±16.1) and all the study subjects were literate. Ninety-three percent of subjects were aware of the concept of eye donation. Knowledge levels were similar among the teaching community and persons engaged in social service, but lower among students (p < 0.05). Among the stakeholders, there was considerable ambiguity regarding whether persons currently wearing spectacles or suffering from a chronic illnesses could donate their eyes. Older age group (p < 0.001), female gender (p < 0.001) and education (p < 0.001) were associated with increased knowledge levels. 82% of the subjects were willing to donate their eyes and this was unaffected by gender or geographical location (rural vs urban). Conclusions Awareness levels and willingness to donate eyes are high among the stakeholders in Srikakulam district in India. The services of stakeholders could be utilized, in conjunction with other community based eye donation counselors, to promote awareness regarding eye donation among the general population

Variability in Tuberculosis Granuloma T Cell Responses Exists, but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas