Th2-predominant immune response underlies the pathogenesis of Dengue.

BACKGROUND: Dengue is a rapidly emerging pandemic-prone disease, whose manifestations range from asymptomatic infection to life-threatening complications like Dengue Hemorrhagic Fever and Dengue Shock Syndrome. This study investigates and compares the immune response in clinically defined cohorts of Dengue with and without warning signs, with the aim of identifying immunological correlates of clinical disease and potential markers of disease severity. METHODS: Blood samples, collected from study participants fulfilling the WHO definition of Dengue with and without warning signs and healthy volunteers, were analyzed using flow cell-based fluorometric methods for cytokines and chemokines. Gene expression analysis, using RT-PCR, was conducted on T helper cell subset-specific transcription factors and cytokines. Demographic details, virological markers, serotype distribution, and hematological parameters were also investigated in all the subjects. RESULTS: The 35 participants recruited in the study, included 11 healthy volunteers and 12 patients each fulfilling the WHO criteria of Dengue with and without warning signs. While the demographic characteristics and serotype distribution was similar in Dengue with and without warning signs cohorts of the disease, platelet counts and Aspartate Aminotransferase (AST) levels changed significantly between Dengue with and without warning signs patients. Plasma cytokine analysis showed up-regulation of IL-4, IL-10, IP-10, and MCP-1 in Dengue patients compared to healthy volunteers. Disease severity was associated with elevated levels of IL-10, IP-10, IL-4, MCP-1, and MIP-1α. IL-8 and MIP-1α were significantly up-regulated in Dengue with warning sign compared to Dengue without warning signs cases. Transcription factor analysis indicated increased expression of RORα, FoxP3, and GATA3 in Dengue patients. mRNA expression of TGFß and IL-4 was also elevated in Dengue patients. A positive correlation between mRNA expression of IL-4 and plasma IL-4 was observed. CONCLUSION: The study reveals a Th2-predominant immune response in all Dengue patients, regardless of disease severity, with overexpression of IL-8 and MIP-1α being observed in patients with warning signs.

Role of real-time polymerase chain reaction in diagnosing Hepatitis E, the commonest cause of acute hepatitis in adult patients seeking institutional care.

Background: This cross-sectional study was performed with the aim of determining the prevalence of hepatitis E virus (HEV) infection among acute hepatitis patients attending a tertiary care teaching hospital in a developing country and to determine the relative performance of prevalent diagnostic assays in establishing its diagnosis. Materials and Methods: A total of 46 adult patients were included in this study, all of whom presented with jaundice of <4 weeks' duration and elevation of AST and ALT above 500 U/L. The prevalence of HEV among patients with acute hepatitis was calculated on the basis of the proportion of recruited patients reacting positively in serum anti-HEV immunoglobulin M (IgM) and real-time polymerase chain reaction (RT-PCR) assays. Results: Among the recruited patients, 11 (23.91%) and 15 (32.6%) patients were positive for anti-HEV IgM and RT-PCR, respectively. The two tests demonstrated poor inter-test agreement, thereby implying the necessity of performing both tests for reliable diagnosis of acute HEV virus infection. We also observed a significant difference in the duration of illness between RT-PCR positive and negative patients (P = 0.008). The mean (±SD) duration of illness in the two groups was 8.6 (±3.50) and 11.66 (± 5.15) days, respectively. Combining the results of IgM ELISA and RT-PCR, we observed that 23 out of 46 patients (50%) had evidence of acute HEV virus infection among our patients. Conclusion: Our study suggests that HEV is the commonest cause of acute hepatitis in adult patients attending a tertiary care teaching hospital and that the diagnostic algorithm for its confirmation should include both IgM ELISA and RT-PCR assays.

Unilateral complicated pleural empyema in a patient with bronchial asthma due to clindamycin-resistant Prevotella buccae.

Prevotella buccae (P. buccae) is a gram-negative obligate anaerobe mainly associated with infections of odontogenic origin. Non-oral monomicrobial infection by these obligate anaerobic bacteria is rare. Only a few cases of monomicrobial non-oral infections by P. buccae have been reported in the literature. We are reporting a case of unilateral complicated pleural empyema in a patient with bronchial asthma infected by P. buccae. Pleural fluid aerobic culture and blood culture reports were sterile. No acid-fast bacilli were detected by Acid Fast Bacilli (AFB) staining, and cartridge-based nucleic acid assay test (CBNAAT) reports were negative for Mycobacterium tuberculosis. The isolate, P. buccae was found susceptible to Metronidazole (MIC = 3 µg/ml) and resistant to Clindamycin (MIC = 256 µg/ml). In view of rising trends of antimicrobial resistance among anaerobes, it is recommended to perform anaerobic culture and sensitivity testing in clinically suspected cases of pleuropulmonary infection for appropriate diagnosis and optimal patient management. Clindamycin should be used with caution for empiric treatment.

Antibiotic associated diarrhea due to Clostridioides difficile in a tertiary care teaching hospital, central India.

Background and Objectives: The misuse of antibiotics in recent years has led to an increase in antibiotic associated diarrheas (AAD). Out of several implicated pathogens, Clostridioides difficile is responsible for causing 15-25% of all cases of AAD. However, it has remained under diagnosed for a long time. The current study is planned to explore prevalence of C. difficile amongst AAD patients and to study clinical presentation and associated risk factors. Materials and Methods: Hospital based cross sectional study conducted in patients above 2 years of age. Diagnosis of C. difficile was done by two modalities i.e. glutamate dehydrogenase test followed by toxin detection using enzyme immunoassay and stool culture followed by toxin gene detection. Results: Twelve of 65 patients (18.4%) were positive for C. difficile. Maximum cases were found in younger age group. Abdominal pain and fever were most common complaints. 12 (18.4%) out of 65 study subjects were found to be positive by ELISA. 2/65 (3%) patients were positive for culture with presence of only tcdB gene. Ceftriaxone was the most commonly used antibiotic (25%). Conclusion: C. difficile is significant pathogen implicated in AAD with a prevalence rate of 18.4%. GDH antigen detection followed by Toxin A/B ELISA for C. difficile yielded better detection rate as compared to stool culture.

Genome-wide mutational analysis of Chikungunya strains from 2016 to 2017 outbreak of central India: An attempt to elucidate the immunological basis for outbreak.

Chikungunya re-emerged in India in 2016-2017, as the first major outbreak since 2006. In our previous study, we undertook partial E1 gene sequencing and phylogenetic/mutational analysis of strains from the 2016-2017 outbreak of Chikungunya in central India and reported important mutations associated with the outbreak. This study was performed to validate the previous findings and to identify key mutations that had emerged throughout the entire genome of Chikungunya virus that could be driving the enormity of this outbreak. The phylogenetic analysis revealed the closeness of our isolates with ECSA genotype, specifically with the Singapore 2015 strain. We found 2 mutations in C and E2 genes, which were present in our isolates but were non-existent during the period of 2010-2016. Furthermore, re-emergence of Arg amino acid in place of stop codon in nsP3 gene and Thr at E2:312 positions was observed after 2011. We also used computational tools to assess the effect of the identified mutations on the T cell and B cell epitopes that could influence the protective immune response against this infection.

Presence of entry receptors and viral markers suggest a low level of placental replication of hepatitis B virus in a proportion of pregnant women infected with chronic hepatitis B.

The transplacental route of vertical transmission of Hepatitis B Virus (HBV) has been known for over a decade. Here we present evidence which suggest HBV can replicate in placenta. Forty-one HBsAg positive and 10 control pregnant women were enrolled in the study after obtaining informed consent. HBV positives were further divided in the High Viral Load (HVL) Group and Low Viral Load (LVL) Group according to INASL guidelines 2018. The Presence of the HBV DNA and expression of NTCP in the placenta was analyzed by qPCR/RT-qPCR and/or immunohistochemistry (IHC). The presence of cccDNA was assessed using Digital Droplet PCR while the presence of pre-genomic (pg) RNA was assessed through qRT-PCR and sequencing. The presence of HBeAg and HBcAg in the placenta was assessed by IHC. Immunostaining of NTCP, HBeAg and HBcAg on trophoblasts along with the presence of total HBV DNA, cccDNA and pgRNA indicated, that these cells are not only susceptible to HBV infection but may also support viral replication. This is further supported by the finding that trophoblasts of the several HBeAg seronegative samples harbored the HBeAg. Although, we did not find any correlation in NTCP expression and viral markers with viral load indicates placental replication may not aping hepatocytes. The presence of the HBV receptor, NTCP along with the presence of cccDNA, pgRNA, and HBeAg in placenta of HBV infected females without circulating HBeAg suggest that placenta act as a replication host.

Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India.

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the "bridging variant" between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

Testing of four-sample pools offers resource optimization without compromising diagnostic performance of real time reverse transcriptase-PCR assay for COVID-19.

Quick identification and isolation of SARS-CoV-2 infected individuals is central to managing the COVID-19 pandemic. Real time reverse transcriptase PCR (rRT-PCR) is the gold standard for COVID-19 diagnosis. However, this resource-intensive and relatively lengthy technique is not ideally suited for mass testing. While pooled testing offers substantial savings in cost and time, the size of the optimum pool that offers complete concordance with results of individualized testing remains elusive. To determine the optimum pool size, we first evaluated the utility of pool testing using simulated 5-sample pools with varying proportions of positive and negative samples. We observed that 5-sample pool testing resulted in false negativity rate of 5% when the pools contained one positive sample. We then examined the diagnostic performance of 4-sample pools in the operational setting of a diagnostic laboratory using 500 consecutive samples in 125 pools. With background prevalence of 2.4%, this 4-sample pool testing showed 100% concordance with individualized testing and resulted in 66% and 59% reduction in resource and turnaround time, respectively. Since the negative predictive value of a diagnostic test varies inversely with prevalence, we re-tested the 4-sample pooling strategy using a fresh batch of 500 samples in 125 pools when the prevalence rose to 12.7% and recorded 100% concordance and reduction in cost and turnaround time by 36% and 30%, respectively. These observations led us to conclude that 4-sample pool testing offers the optimal blend of resource optimization and diagnostic performance across difference disease prevalence settings.

Laboratory diagnosis of COVID-19: current status and challenges.

The magnitude and pace of global affliction caused by Coronavirus Disease-19 (COVID-19) is unprecedented in the recent past. From starting in a busy seafood market in the Chinese city of Wuhan, the virus has spread across the globe in less than a year, infecting over 76 million people and causing death of close to 1.7 million individuals worldwide. As no specific antiviral treatment is currently available, the major strategy in containing the pandemic is focused on early diagnosis and prompt isolation of the infected individuals. Several diagnostic modalities have emerged within a relatively short period, which can be broadly classified into molecular and immunological assays. While the former category is centered around real-time PCR, which is currently considered the gold standard of diagnosis, the latter aims to detect viral antigens or antibodies specific to the viral antigens and is yet to be recommended as a stand-alone diagnostic tool. This review aims to provide an update on the different diagnostic modalities that are currently being used in diagnostic laboratories across the world as well as the upcoming methods and challenges associated with each of them. In a rapidly evolving diagnostic landscape with several testing platforms going through various phases of development and/or regulatory clearance, it is prudent that the clinical community familiarizes itself with the nuances of different testing modalities currently being employed for this condition.

Evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of COVID-19: A pilot study.

Timely diagnosis of COVID-19 infected individuals and their prompt isolation are essential for controlling the transmission of SARS-CoV-2. Though quantitative reverse transcriptase PCR (qRT-PCR) is the method of choice for COVID-19 diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. In this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in 3 central Indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. From 545 nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of 109 pools were created with 5 consecutive samples in each pool. The diagnostic performance of qRT-PCR on pooled sample was compared with that of individual samples in a blinded manner. While pooling reduced the cost of diagnosis by 68% and the laboratory processing time by 66%, 5 of the 109 pools showed discordant results when compared with induvial samples. Four pools which tested negative contained 1 positive sample and 1 pool which was positive did not show any positive sample on deconvolution. Presence of a single infected sample with Ct value of 34 or higher, in a pool of 5, was likely to be missed in pooled sample analysis. At the reported point prevalence of 4.8% in this study, the negative predictive value of qRT-PCR on pooled samples was around 96% suggesting that the adoption of this strategy as an effective screening tool for COVID-19 needs to be carefully evaluated.

Pyonephrosis by Prevotella disiens and Escherichia coli coinfection and secondary peritonitis in an obstructive uropathy patient: A case report and review of the literature.

Pyonephrosis is an uncommon condition that is associated with suppurative destruction of the renal parenchyma. Upper urinary tract obstruction by renal stones plays an important role in its aetiology. The majority of pyonephrosis is reported to be caused by aerobic bacteria but the role of anaerobes, especially black-pigmented gram-negative anaerobes, namely, Prevotella and Porphyromonas in renal infections, remain poorly defined. In view of the rarity of the event, a case of pyonephrosis by Prevotella disiens and Escherichia coli coinfection complicated by secondary peritonitis in an obstructive uropathy patient is hereby presented. An attempt is being made to review the literature on the infective aetiologies of renal abscess with special reference to anaerobes.

Association of Antimicrobial Susceptibility and Treatment Outcome in Acne Vulgaris Patients: A Pilot Study.

Purpose Cutibacterium acnes ( C. acnes ) is an emerging pathogen that is highly resistant to antibiotics and is capable of causing persistent infections that are difficult to treat. Methods & Materials Acne vulgaris patients visiting dermatology OPD of our tertiary care hospital during the study period of 2 months were recruited. Skin swabs were collected, and the sample was processed on 5% sheep-blood agar for anaerobic culture by the GasPak method. Isolates were identified by the standard biochemical test. Antimicrobial susceptibility testing was performed for clinically relevant antibiotics by the E-strip method. The clinical response was evaluated after 1-month follow-up to the prescribed antibiotics. Results Minocycline, doxycycline, ceftriaxone, and tetracycline were the most effective antibiotics. Nonsusceptibility to clindamycin and erythromycin were observed in 11.9% and 31% isolates, respectively, with 9.5% isolates being nonsusceptible to both. For none of the antibiotics we found significant difference in the proportion of susceptible and nonsusceptible isolates between mild, moderate, and severe grades of acne vulgaris. For none of the antibiotic regimens, significant difference was observed between nonresponders and responders. Twenty-seven patients received clindamycin and among them 16 of 19 responders and 6 of 8 nonresponders yielded growth of clindamycin-susceptible isolates ( p = 0.57). Conclusion We observed significant prevalence of resistant strains of C. acnes among patients with acne vulgaris. No association was observed between in vitro susceptibility results and treatment outcome.