Integrated immune monitoring of HCMV infection in pregnant women with complications and its association with adverse pregnancy outcomes.

Human Cytomegalovirus (HCMV) infection is associated with bad obstetric history (BOH) and adverse pregnancy outcomes (APO). Here, we characterized antiviral humoral profiles, systemic and virus specific cellular immune responses concurrently in pregnant women (n = 67) with complications including BOH and associated these signatures with pregnancy outcomes. Infection status was determined using nested blood PCR, seropositivity and IgG avidity by ELISA. Systemic and HCMV specific (pp65) cellular immune responses were evaluated by flow cytometry. Seropositivity was determined for other TORCH pathogens (n = 33) on samples with recorded pregnancy outcomes. This approach was more sensitive in detecting HCMV infection. Blood PCR positive participants, irrespective of their IgG avidity status, had higher cytotoxic potential in circulating CD8+ T cells (p < 0.05) suggesting that infection associated cellular dysfunction was uncoupled with avidity maturation of antiviral humoral responses. Also, impaired anamnestic degranulation of HCMV-pp65-specific T cells compared to HCMV blood PCR negative participants (p < 0.05) was observed. APO correlated with HCMV blood PCR positivity but not serostatus (p = 0.0039). Most HCMV IgM positive participants (5/6) were HCMV blood PCR positive with APO. None were found to be IgM positive for other TORCH pathogens. Multiple TORCH seropositivity however was significantly enriched in the APO group (p = 0.024). Generation of HCMV specific high avidity IgG antibodies had no bearing on APO (p = 0.9999). Our study highlights the utility of an integrated screening approach for antenatal HCMV infection in the context of BOH, where infection is associated with systemic and virus specific cellular immune dysfunction as well as APO.

The dynamics of SARS-CoV-2 infection in unvaccinated and vaccinated populations in Mumbai, India, between 28 December 2020 and 30 August 2021.

The emergence and evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants that could compromise vaccine efficacy (VE) with re-infections in immunized individuals have necessitated continuous surveillance of VE. Here, the occurrence and dynamics of SARS-CoV-2 infections in the context of vaccination during the second wave of infection in Mumbai were evaluated. RT-PCR cycle threshold (Ct) values of the open reading frame (ORF)/envelope (E)/nucleocapsid (N) genes obtained from a total of 42415 samples, comprising unvaccinated (96.88%) and vaccinated cases (3.12%) were analyzed between December 28, 2020, and August 30, 2021. A lower incidence of SARS-CoV-2 infection in fully vaccinated cases (5.07%) compared to partially vaccinated cases (6.5%) and unvaccinated cases (13.453%) was recorded. VE was significant after the first dose of vaccination (ORF gene p-value = 0.003429, and E/N gene p-value = 0.000866). Furthermore, VE was observed to be significant when the post-immunization (first dose) period was stratified to within 30 days (ORF gene p-value = 0.0094 and E/N gene p-value = 0.0023) and to 60 days following the second dose of vaccination (ORF gene p-value = 0.0238). Also, significantly higher efficacy was observed within individuals receiving two doses compared to a single dose (ORF gene p-value = 0.0132 and E/N gene p-value = 0.0387). The emergence of breakthrough infections was also evident (odds ratio= 0.34; 95% confidence interval= 0.27-0.43). Interestingly, viral loads trended towards being higher in some groups of partially vaccinated individuals compared to completely vaccinated and unvaccinated populations. Finally, our results delineated a significantly higher incidence of SARS-CoV-2 acquisition in males, asymptomatic individuals, individuals with comorbidities, and those who were unvaccinated.

Synthesis and biological evaluation of benzamide-chalcone hybrids as potential c-Met kinase and COX-2 inhibitors.

c-Met kinase and cyclooxygenase 2 (COX-2) enzymes are two significant targets in tumor progression. Chalcone and benzamide moieties were combined using molecular hybridization to assess their potential as c-Met kinase and COX-2 inhibitors. 4-Methylbenzamide and 4-chlorobenzamide chalcone analogs were synthesized, characterized, and evaluated for antiproliferative activity on Michigan Cancer Foundation-7 (MCF-7), HT-29, MDA-MB-231, COLO-205, and A549 cell lines by sulforhodamine-B stain (SRB) assay. Following the SRB assay, compounds were evaluated for their c-Met kinase and COX-2 inhibitory potential. All compounds inhibited COX-2 with half-maximal inhibitory concentration (IC50 ) <10 µM. Compounds 7h, 7i, 7j, 8f, and 8j inhibited c-Met with IC50 <10 µM. Compound 7h was evaluated for its long-term antiproliferative and anti-migratory effects by colony formation and wound healing assay. It exerted these effects in a concentration-dependent manner. Compounds 7j and 8j were further evaluated for in vitro antiangiogenic effects. Compound 7j exhibited moderate antiangiogenic effect while compound 8j exhibited strong effect. Compounds 7h, 7i, 7j, 8f, and 8j were evaluated for the serum protein binding, using the in vitro bovine serum albumin binding assay. The results indicated that the tested compounds bind to bovine serum albumin (BSA) and can be further explored by other studies.

Membrane vesicles of Lactobacillus gasseri ATCC 19992 disrupt biofilms of vaginal pathogens.

Membrane vesicles (MVs) are bioactive, nano-sized entities produced by all organisms. MVs of L. gasseri ATCC 19992 were isolated and their effect on the biofilms of vaginal pathogens, G. vaginalis and S. aureus was studied. The L. gasseri MVs resulted in significant disruption of biofilms of the vaginal pathogens.

A literature review on correlation between HPV coinfection with C. trachomatis and cervical neoplasia - coinfection mediated cellular transformation.

Cervical cancer is the fourth most common cause of mortality worldwide. Persistent infection with high-risk human papillomaviruses (hrHPV) is a known significant risk factor in cervical neoplasia development (CN). Though HPV contributes to carcinogenesis, other factors provide an ideal niche for persistence of HPV, especially, coinfection with Chlamydia trachomatis (CT) which has been linked to CN development. CT infection is associated with inflammation, cell proliferation, EMT transition and anti-apoptotic processes. To better understand the correlation between HPV-CT coinfection and CN development, a literature review was conducted on the prevalence of HPV-CT coinfection focusing on the role of infection-induced inflammation as HPV-CT coinfection creates an environment for cellular transformation, activates an innate immune response and triggers EMT transition. Moreover, inflammation plays a crucial role in developing neoplasia as there is a decrease in effector cells and a change in the levels of players like ROS and miRs. CT infection induces chronic inflammation followed by cervical epithelial cell damage and increases susceptibility to HPV infection which may lead to cellular transformation. The literature search was performed based on a comprehensive investigation of publications in the PubMed journal database and Scopus, on the development of CN. We have reviewed the prevalence of HPV-CT infection and the factors increasing the risk of developing CN.

Evaluation of cytokine profile in cervicovaginal lavage specimens of women having asymptomatic reproductive tract infections.

Reproductive tract infections (RTIs) such as vaginal candidiasis and bacterial vaginosis (BV) are common among sexually active women and can be both symptomatic or asymptomatic. The microbiota of the reproductive tract triggers immune response at the cervicovaginal interface resulting in secretion of cytokines during the course of these RTIs. The objective of this study was to evaluate the cytokine profile in cervicovaginal lavage of women having asymptomatic vaginal infections. Measurement of vaginal cytokines was done for various interleukins including IL-1ß, IL-6, IL-8, IL-10, IL-12/IL23p40, IL-17A, tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) by ProcartaPlex™ Multiplex Immunoassay. Women having vaginal Candida infection had increased concentration of IL-1ß (p=.01), IL-6 (p=.007), IL-8 (p=.327), IL-12/IL23p40 (p=.049) and IFN-γ (p=.125). The results of our study suggest that evaluation of these cytokines could be explored as an additional measure to determine host inflammatory response in women having asymptomatic vaginal candidiasis.Impact StatementWhat is already known on this subject? Studies assessing the vaginal cytokine profile to assess the vaginal milieu in various cohorts such as post-menopausal women, pregnant women, women with history of preterm birth, CIN and scheduled IVF cycle are being undertaken. Variable cytokine response has been reported in literature in women with symptomatic bacterial vaginosis and Candida infection. However, much less is known about vaginal cytokine profile in asymptomatic infection.What do the results of this study add? The results of the study show increased concentration of the pro-inflammatory cytokines IL-1ß, IL-6 IL-8, IL-12/IL23p40 and interferon gamma (IFN-γ) in women having asymptomatic Candida, vaginal leucocytosis and raised vaginal pH.What are the implications of these findings for clinical practice and/or further research? Evaluation of vaginal cytokine profile (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be explored as an additional measure to determine inflammation in asymptomatic women. Vaginal cytokines (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be used further for development of a point of care test.

Cycle threshold values in RT-PCR to determine dynamics of SARS-CoV-2 viral load: An approach to reduce the isolation period for COVID-19 patients.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.

Persistence of SARS-CoV-2 in the first trimester placenta leading to transplacental transmission and fetal demise from an asymptomatic mother.

Coronavirus disease 2019 (COVID-19) is caused by infection of the respiratory tract by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which survives in the tissues during the clinical course of infection but there is limited evidence on placental infection and vertical transmission of SARS-CoV-2. The impact of COVID-19 in first trimester pregnancy remains poorly understood. Moreover, how long SARS-CoV-2 can survive in placenta is unknown. Herein, we report a case of a pregnant woman in the first trimester who tested positive for SARS-CoV-2 at 8 weeks of gestation, although her clinical course was asymptomatic. At 13 weeks of gestation, her throat swab tested negative for SARS-CoV-2 but viral RNA was detected in the placenta, and the Spike (S) proteins (S1 and S2) were immunolocalized in cytotrophoblast and syncytiotrophoblast cells of the placental villi. Histologically, the villi were generally avascular with peri-villus fibrin deposition and in some areas the syncytiotrophoblast layer appeared lysed. The decidua also had fibrin deposition with extensive leukocyte infiltration suggestive of inflammation. The SARS-CoV-2 crossed the placental barrier, as the viral RNA was detected in the amniotic fluid and the S proteins were detected in the fetal membrane. Ultrasonography revealed extensively subcutaneous edema with pleural effusion suggestive of hydrops fetalis and the absence of cardiac activity indicated fetal demise. This is the first study to provide concrete evidence of persistent placental infection of SARS-CoV-2 and its congenital transmission is associated with hydrops fetalis and intrauterine fetal demise in early pregnancy.

Association of cervicovaginal dysbiosis mediated HPV infection with cervical intraepithelial neoplasia.

Cellular transformation to malignancy is a multifactorial process strongly linked with microbiome dysbiosis. The female reproductive tract (FRT) is inhabited by specific Lactobacillus spp which play a significant role in maintaining a homeostatic balance and providing resistance to perturbation. Any imbalance in the resident microbiota of the FRT results in cervicovaginal dysbiosis and increased predisposition to viral and bacterial infections. In the present review, we discuss the critical role played by the cervicovaginal microbiome in maintaining cervicovaginal homeostasis. Loss of the mutualistic relationship between cervicovaginal microbiota and the host leads to increased susceptibility to Human papilloma virus (HPV) infection. HPV in coinfection with Chlamydia trachomatis has been linked with increased risk for cellular transformation. The progression to cervical neoplasia is a multistep process regulated by cellular and epigenetic changes mediated by oncogenes and miRNA. Exosomes derived from the infected cells play an important role in the pathological development and progression to cervical neoplasia as they harbor the regulatory molecules like miRNA, proteins and prooncogenic factors which may facilitate cellular transformation.

Discordance between TST and QFT-TBGold Plus for Latent Tuberculosis Screening among Under-Five Children: An Interim Analysis.

AIM: To analyze the agreement between tuberculin skin test (TST) and fourth-generation QuantiFERON (QFT)-TB Gold Plus [interferon gamma (INF-γ) release assays (IGRA)] for latent tuberculosis infection (LTBI) diagnosis among under-five children who are undernourished and/or who have history of contact with active tuberculosis (TB) patients. METHODS: Children from the age group of 6 months to 5 years (undernourished or tuberculosis household contacts) were screened through anganwadis (government playschools) and TB Health posts from Mumbai, India during September 2019 to January 2021. Both TST and QFT-TB Gold Plus test were carried out to diagnose LTBI. RESULTS: Out of the total 299, 35 (11.7%) (95% CI 8.1-15.3%) children tested positive by IGRA (QFT-TB Gold Plus) and 68 (22.7%) (95% CI 18.0-27.4%) by TST, suggestive of moderate concordance (κ = 0.483) between both tests. IGRA and TST showed moderate concordance in children <24 months (κ = 0.478). Moreover, 26 (21.1%) children with TB contact had both TST and IGRA positive with moderate concordance (κ = 0.550). A fair concordance (κ = 0.396) was observed between IGRA and TST in undernourished children. Also, 45 (15.0%) children showed discordance of which 39 (13.0%) had positive TST but negative IGRA and 6 (2.0%) had negative TST but positive IGRA. CONCLUSIONS: The study strongly recommends both TST and QFT-TB Gold Plus test for the diagnosis of LTBI in under-five children. A moderate concordance in children <24 months endorses the reliability of QFT-TB Gold Plus in diagnosing LTBI in this age group. This study highlights the need for screening undernourished children for LTBI to consider repeating IGRA testing for TST positives as per the window period and risk of ongoing exposure.

The current study focuses on discordance and concordance between tuberculin skin test (TST) and fourth-generation QuantiFERON (QFT)-TB Gold Plus [interferon gamma (INF-γ) release assays (IGRA)] for latent tuberculosis infection (LTBI) diagnosis among at-risk under-five children who are underweight and/or who have history of contact with active tuberculosis patients. The IGRA prevalence came out to be 11.7% (95% CI 8.1­15.3%) whereas the TST prevalence turned out to be 22.7% (95% CI 18.0­27.4%). A stronger concordance was observed between IGRA and TST among the age group of 2 to 5 years, and a relatively fair one for children below the age of 1 year. The present study strongly recommends to include both TST and IGRA test for the diagnosis of LTBI with respect to Indian pediatric population. This study also suggests the importance of repetition of IGRA for TST positive patients. Another vital opinion that is showcased in the present study is the inclusion of undernourished pediatric population residing in at-risk areas like urban slums for routine LTBI screening programs.

Characterization of Gardnerella vaginalis membrane vesicles reveals a role in inducing cytotoxicity in vaginal epithelial cells.

Bacterial vaginosis (BV) is a common polymicrobial infection affecting women in the reproductive age and is associated with adverse obstetric and gynaecological outcomes. Gardnerella vaginalis is the most virulent anaerobic bacterial species predominantly associated with BV. However, a clear understanding of the mechanisms by which it contributes to the pathogenesis and persistence of BV is lacking. In this report, we demonstrate for the first time, the isolation of membrane vesicles (MVs) from G. vaginalis ATCC 14019. These MVs are approximately 120-260 nm in diameter. Proteomic characterization of the MVs by LC-MS/MS led to the identification of 417 proteins, including proteins involved in cellular metabolism as well as molecular chaperones and certain virulence factors. Immunoblot analysis of the MVs confirmed the presence of vaginolysin, the most well-characterized virulence factor of G. vaginalis. The exposure of the vaginal epithelial cells, VK2/E6E7 to the G. vaginalis MVs resulted in the internalization of the MVs. The MVs induced cytotoxicity and an increase in the levels of the pro-inflammatory cytokine, IL-8 in VK2 cells as well lysis of erythrocytes. The results of the study indicate that G. vaginalis MVs may be involved in the delivery of cytotoxic proteins and other virulence factors to the host cells and could thereby contribute towards enhancing the cellular damage associated with pathogenesis of BV.

HLA-G*01:01:01:34 allele discovered in two individuals from a tribal community of Western Maharashtra, India.

HLA-G*01:01:01:34 differs from HLA-G*01:01:01:01 by one nucleotide in intron 3 at position 1432 (G to A).

Mapping the vaginal microbiota variations in women from a community clinic in Mumbai, India.

PURPOSE: The vaginal microbiome contributes significantly to women's reproductive health and fluctuates due to various physiological and pathological factors. The study's objective is to map the vaginal microbiome of non-pregnant women and evaluate variations based on various potential factors influencing vaginal milieu. METHODS: Fifty-two sexually active, non-pregnant women between 18 and 45 years were recruited from a community clinic and clinical history was recorded. Vaginal swabs were collected to assess the vaginal microbiome by sequencing the V3-V4 region of the 16S rRNA using the Illumina HiSeq platform, followed by data analysis with QIIME 2. Vaginal milieu was assessed by Nugent score and profiling cytokines in the cervico-vaginal lavage. RESULTS: Lactobacillus iners (34.3%) were the most abundant species in all women. Significant changes in abundance of genera (Lactobacillus, Prevotella and Anaerococcus), expression of pro-inflammatory cytokine IFN-γ and changes in alpha and beta diversity was observed in women having asymptomatic bacterial vaginosis (BV). Differences in beta diversity were seen between healthy women and women exhibiting presence of Candida spp. Variations in the abundance of genera (Lactobacillus, Bifidobacterium, Porphyromonas) were observed in women who had delivery less than twelve months back, probably as more of these women (50%, 53.7%) had higher abnormal Nugent score. CONCLUSION: Lactobacillus iners was the most prevalent vaginal species in women from a Mumbai community clinic. Maximum variations in the vaginal microbiome characterized by a perturbation of the Lactobacillus predominant vaginal microbiota are seen in those women who have asymptomatic BV and childbirth within last twelve months.

A novel gut microbiome-immune axis influencing pathology in HCMV infected infants with neonatal cholestasis.

The interplay of active HCMV infection with gut dysbiosis in the immunopathology of cholestasis in neonates and infants remains unexplored. In this study, we evaluated gut microbiome profiles and immune dysfunction in a cohort of HCMV infected cholestatic infants (IgM positive, N = 21; IgM negative, N = 25) compared to healthy infants, N = 10. HCMV infected IgM positive individuals exhibited increased clinical severity in terms of liver dysfunction, altered CD4+: CD8+ ratio, and elevated Granzyme B levels in cellular immune subsets. Gut microbiome analysis revealed distinct and differential diversity and composition within infected groups aligned with clinical severity reflected through the increased abundance of Gammaproteobacteria, reduced Bifidobacteria, and a unique signature mapping to the HCMV infected IgM negative group. Correlation analyses revealed associations between Bifidobacterium breve, Gammaproteobacteria, Firmicutes, Clostridia, Finegoldia magna, Veillonella dispar, and Granzyme B expressing immune cell subsets. Our study describes a novel gut microbiome-immune axis that may influence disease severity in cholestatic infants with active HCMV infection.

Cytomegalovirus reactivation in a SARS-CoV-2 infected woman experiencing fetal demise in the first trimester with fetal trisomy 21: A case report.

Cytomegalovirus (CMV) is the most common cause of congenital viral infections. Women seropositive for CMV prior to pregnancy can develop a non-primary CMV infection. Here, we present a case of first trimester pregnancy loss during active SARS-CoV-2 infection. There was no evidence of SARS-CoV-2 RNA in placenta and fetal tissue, but there was presence of congenital cytomegalovirus infection by nested PCR. To the best of our knowledge, this is the first report demonstrating association of early congenital CMV infection due to reactivation and fetal demise in a SARS-CoV-2 positive woman with fetal trisomy 21.

Opposing roles for sMAdCAM and IL-15 in COVID-19 associated cellular immune pathology.

Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.

pH Stress Mediated Alteration in Protein Composition and Reduction in Cytotoxic Potential of Gardnerella vaginalis Membrane Vesicles.

The vagina of healthy women is predominantly colonized by lactobacilli but it also harbors a limited proportion of certain anaerobes such as Gardnerella vaginalis. An increase in G. vaginalis along with other anaerobes on account of perturbation in the vaginal microbiota is associated with bacterial vaginosis (BV). Although strategies adopted by G. vaginalis for survival and pathogenesis in a conducive environment (i.e., high vaginal pH, characteristic of BV) have been previously studied, the approaches potentially employed for adaptation to the low pH of the healthy vagina are unknown. In the present study, we investigated the effect of acidic stress on the modulation of the production and function of membrane vesicles (MVs) of G. vaginalis. pH stress led to a distortion of the bacterial cell morphology as well as an altered biogenesis of MVs, as revealed by transmission electron microscopy (TEM). Both qualitative and quantitative differences in protein content of MVs produced in response to pH stress were observed by flow cytometry. A significant change in the protein composition characterized by presence of chaperones despite a reduction in number of proteins was also noted in the stress induced MVs. Further, these changes were also reflected in the reduced cytotoxic potential toward vaginal epithelial cells. Although, these findings need to be validated in the in vivo settings, the modulation of G. vaginalis MV biogenesis, composition and function appears to reflect the exposure to acidic conditions prevailing in the host vaginal mileu in the absence of vaginal infection.

Detection of SARS-CoV-2 infection by RT-PCR test: factors influencing interpretation of results.

In this current pandemic of coronavirus disease 2019 (COVID-19), prompt interventions in terms of early detection and clinical management along with isolation of positive cases is of utmost importance. This helps to limit not only the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections but also the morbidity and mortality associated with it. Different strategies for screening of COVID-19 in containment zones and non-containment areas include testing of symptomatic patients and their contacts in fever clinics, hospital-based testing, testing on demand and population-based screening. The choice of tests like reverse-transcription polymerase chain reaction (RT-PCR), rapid antigen testing (RAT) or antibody test depends upon these strategies and also the turnaround time. Currently, RT-PCR is considered the gold standard for COVID-19 detection. This commentary provides the insights and experiences on COVID-19 diagnosis by RT-PCR. The utility of this test is limited by several false positive, false negative and inconclusive results at early stages of infection, scarcity of reagents and lack of well-equipped labs including trained staff. Moreover, appropriate sample collection and transport, standard laboratory protocols, stringent quality control norms, good quality RNA extraction kits, PCR kits with suitable primers can help in improving accuracy of the test results. A careful assessment of clinical, radiological and molecular findings is required for identifying potential cases of COVID-19.