Role of the Laboratory in the Diagnosis of Poxvirus Infections.

Although WHO-led global efforts led to eradication of smallpox over four decades ago, other poxviruses, especially monkeypox, have re-emerged to occupy the ecological niche vacated by smallpox. Many of these viruses produce similar lesions thus mandating a prompt laboratory confirmation. There has been considerable evolution in the techniques available to diagnose these infections and differentiate between them. With the 2022 multi-country outbreak of monkeypox, significant efforts were made to apprise the laboratory diagnosis of the virus and numerous real-time-PCR-based assays were made commercially available. This chapter discusses the sample collection and biosafety aspects along with the repertoire of diagnostic modalities, both traditional and emerging, for poxviruses which a special focus on monkeypox. The advantages and disadvantages of each technique have been illustrated. We have also reflected upon the newer advances and the existing lacunae.

Rise in the Pathogenic Status of Coagulase-Negative Staphylococci Causing Bloodstream Infection.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are one of the frequently isolated bacteria from blood cultures. Since they are part of the normal skin flora, they were previously considered contaminants. But now, they can be considered as established pathogens causing bloodstream infection (BSI). This study aims to estimate the prevalence of CoNS in BSI cases. METHODS: This study was conducted at the Microbiology Department, All India Institute of Medical Sciences (AIIMS), Raipur, India, for eight months (January 2022 to August 2022). Data were collected retrospectively from medical and laboratory records. Paired blood cultures from 5085 clinically suspected sepsis cases were subjected to aerobic culture for five days in the BacT ALERT 3D system. Pathogenicity was established after recovery of CoNS from paired blood cultures of symptomatic patients. RESULTS: CoNS were isolated from 2.35% of patients, the most common species being Staphylococcus haemolyticus (51.67%). About 90% of isolates were methicillin-resistant. All the isolates were susceptible to linezolid, teicoplanin, and vancomycin, except one isolate of S. haemolyticus which was intermediate to vancomycin. Minimum inhibitory concentration (MIC) 50 and MIC 90 for vancomycin were 1 ug/ml and 2 ug/ml, respectively.  Conclusion: Paired blood cultures are necessary to determine the pathogenicity of CoNS in BSI cases. A high prevalence of methicillin resistance, accompanied by high resistance rates to other non-beta lactam antibiotics, warrants the strict implementation of antimicrobial stewardship practices.

The lurking threat of monkeypox in current times.

BACKGROUND: Since the eradication of smallpox in 1980, monkeypox has been the most prevalent zoonosis caused by orthopoxviruses. The virus has been largely endemic to the rainforests of Central and West Africa with occasional exportation to other countries. The disease typically runs a self-limited course with case fatality rates ranging from 4 to 11%. Currently, the world is faced with a multi-country outbreak of monkeypox, whose extent and impact remains to be seen. OBJECTIVE: The objective of this article is to discuss the changing epidemiology of the monkeypox virus, with special reference to the current outbreak. CONTENT: Since the beginning of this outbreak which started on May 7th, till 14th of June 2022, a total of 1879 cases of monkeypox have been reported worldwide, spanning across 35 non-endemic, and commonly involving men who have sex with men. The magnitude of this unprecedented outbreak has highlighted the lacunae in our understanding of the viral epidemiology and ecology. What was earlier a rare sporadic zoonotic disease is now an emergent pathogen with documented human-to-human transmission potential, both in household as well as nosocomial settings. Waning immunity due to cessation of smallpox vaccination, wide host range of the virus, undetected circulation in wildlife in pan-geographic areas, emergence of better adapted strains of the virus due to unchecked replication in the HIV positive immunocompromised population along with deforestation and human encroachment of reservoir areas are some of the plausible reasons for an increased incidence of the disease. Unflinching government commitment, healthcare worker training, education of the masses, stockpiling of the available vaccine and drug, intersectoral co-ordination in lines of the One Health approach are simultaneously needed to avert the current spread of monkeypox. There is also a compelling need to strengthen surveillance systems to curb future outbreaks.

Epidemiological Characteristics of the COVID-19 Pandemic During the First and Second Waves in Chhattisgarh, Central India: A Comparative Analysis.

Background There is a scarcity of reports of comparative analysis between the first and second waves of the pandemic from any part of India. This article aims to comprehensively investigate the epidemiology of coronavirus disease 2019 (COVID-19) during the course of the pandemic in the state of Chhattisgarh, central India. Methodology A comparative epidemiological analysis of the first and second waves of COVID-19 across Chhattisgarh was conducted on the vital parameters of total tests performed, cases diagnosed, age and gender distribution, case fatality ratio (CFR), and mitigation strategy reported by the state and central government health agencies using the data from Indian Council of Medical Research and National Informatics Centre portals. Results The second wave was shorter than the first wave but the absolute number of cases increased by 2.4 times and deaths by 2.7 times. There was a significant increase in cases per million, deaths per million, and test positivity rate. The hospitalization rate and test per case ratio dropped in the second wave from 33 to 20 and from 12.6 to 7.2, respectively. Both infection and deaths were higher among males in both the waves (p < 0.001). CFR increased from 1.2% in the first wave to 1.4% in the second wave (p < 0.001; odds ratio = 1.14 (1.1-1.19)). Increased mortality was seen in all ages except the young (≤20 years) and the old (>60 years). Conclusions The significantly high number of cases and deaths during the second wave provides evidence to undertake preparedness measures for mitigating any future waves. Regular surveillance, monitoring, and analysis of epidemiological data are pertinent for continued situational awareness.

Genetic analysis of human papilloma virus 16 E6/E7 variants obtained from cervical cancer cases in Chhattisgarh, a central state of India.

Human papilloma virus genotype 16 (HPV-16), a predominant etiological cause of cervical cancer (CC) vary in inflicting oncogenicity according to their geographical distribution and mutational changes. With no published data from central India, the present study aimed to genetically analyze HPV-16 E6/E7 variant obtained from CC women of Chhattisgarh. In twenty one CC patients, PCR amplified E6/E7 genes were decoded by DNA sequencing to study phylogenetic relatedness, mutational changes and their in-silico effect on protein structure. E6 analysis revealed nineteen sequences exhibited intratypic variation. L83V mutation was observed in 76.2% sequences followed by S71C seen in 28.6% sequences. Mutations of E41G, A46G, F47V, R77S, L99V and Q107K were observed in three sequences each. C140 Stop codon mutation has caused early truncation of E6 in three sequences to produce the conformational structural change. In contrast, E7 was relatively more conserved showing D4E (4.7%), G88R (23.8%), I93T (9.5%) and C94S (9.5%) mutations. Other than L83V and S71C, E6 and E7 mutations were reported for the first time from India. E6/E7 nonsynonmous mutations have a spectrum of biological effect in progression of CC. Phylogenetic analysis revealed ten sequence belonged to Asian while eleven to European sublineage to show CC cases in Chhattisgarh are a mix of Asian and European lineage. Asian sequences showing higher frequency of L83V mutations and exclusive presence of S71C and C140 Stop codon mutations may be linked with higher oncogenicity. Various E6/E7 mutational data may prove useful for development of better diagnostic and vaccine for the region of Chhattisgarh.

Correction: Comparative analysis of various clinical specimens in detection of SARS-CoV-2 using rRT-PCR in new and follow up cases of COVID-19 infection: Quest for the best choice.

[This corrects the article DOI: 10.1371/journal.pone.0249408.].

The dilemma of differentiating between acute hepatitis B and chronic hepatitis B with acute exacerbation: Is quantitative serology the answer?

BACKGROUND/AIMS: Acute exacerbations of chronic hepatitis B (CHB-AEs) are common in endemic areas and are often presumed to be acute hepatitis B (AHB) due to their similarities in clinical and serological pictures, presenting a major diagnostic dilemma. This study aimed to identify laboratory markers for differentiating between the two groups, and to establish the cut-off value for significant markers.
. METHODS: A retrospective analysis of records was conducted for patients who presented with clinical features of acute hepatitis along with hepatitis B surface antigen (HBsAg) and IgM antibody to hepatitis B core antigen (IgM anti-HBc) positivity from May 2015 to May 2017. A total of 172 patients were enrolled and grouped as AHB (n=89) and CHB-AE (n=83) based on their history of hepatitis B virus infection and duration of HBsAg persistence. Virological and biochemical parameters were analyzed and compared. Cut-off values, sensitivity, and specificity of the variables were calculated.
. RESULTS: The median value of signal by cut-off (S/Co) ratio for IgM anti-HBc was significantly higher in AHB group (30.44) compared to CHB-AE group (8.63) with a sensitivity and specificity of 97% and 84%, respectively, at a cut-off of 20.5 (P<0.01). The mean international normalized ratio (INR) was significantly greater in CHB-AE (1.88±1.24) group compared to AHB group (1.62±0.17) with a sensitivity and specificity of 57.9% and 45.1%, respectively, at a cut-off value of 1.27.
. CONCLUSION: A value of 20.5 S/Co of IgM anti-HBc and 1.27 INR could be helpful in differentiating between AHB and CHB-AE. (Clin Mol Hepatol 2020;26:187-195).

Scrub typhus and leptospirosis in rural and urban settings of central India: a preliminary evaluation.

Scrub typhus and leptospirosis are bacterial zoonotic diseases reported from different parts of India, whose prevalence in Chhattisgarh is unknown. Our study was carried out to delineate the prevalence of these illnesses there and to assess the clinical profiles of rural and urban patients. A total of 169 patients with acute febrile illnesses (AFI) was enrolled in our study from May to December 2018, of whom 35 (20.7%) tested positive for scrub typhus and only one tested positive for leptospirosis by respective IgM ELISA. Scrub typhus seropositivity was higher in rural patients (25.0%) than in urban (18.1%). Patients in the age group 16-30 years were the most commonly affected. The commonest presenting symptoms were fever with headache (68.57%), extreme weakness (57.14%), myalgia/arthralgia (54.29%) and abdominal pain (51.43%). The preliminary evidence for the presence of scrub typhus in Chhattisgarh necessitates its inclusion in the panel of tests for AFI.

Hepatitis E virus infection: An old virus with a new story!

Hepatitis E virus (HEV) infection is an important public health problem. HEV infection has been identified as a major cause of enterically transmitted acute sporadic hepatitis in India especially in adult age group. India is hyperendemic for HEV, with the disease presenting both as outbreaks and as cases of acute sporadic viral hepatitis. Most of these outbreaks can be traced to contamination of drinking water supplies with human fecal matter. The last decade has witnessed tremendous change in our understanding of the virus in its epidemiology, clinical features, diagnostic approaches, treatment options and the need for vaccination. With the identification of culture systems for HEV and development of animal models for its replication, knowledge regarding its replication and pathogenesis has evolved. This review attempts to discuss the nuances in our understanding of this virus, its pathogenesis and diagnosis, especially with reference to the Indian scenario.

Absence of chronic hepatitis E virus infection in liver transplant recipients: Report from a hyperendemic region.

Most cases of chronic hepatitis E virus (HEV) infection in solid organ transplant recipients are attributable to genotype 3. Although India is hyperendemic for HEV genotype 1, chronic infection in transplant patients has not been reported. In this study, 30 liver transplant recipients were followed up by systematic testing for various markers of HEV (IgM, IgG, HEV-Ag, and RNA) on blood and stool samples obtained pre-transplant, and then at 3 and 6 months post-transplant to look for HEV exposure and persistence. Evidence of HEV infection was found in 6 (20%) cases post-transplant but none of the recipients demonstrated active viremia or antigenemia. This suggests that the circulating genotype of HEV in our population might have limited potential to cause chronic infections.

Genetic signatures coupled with lineage shift characterise endemic evolution of Dengue virus serotype 2 during 2015 outbreak in Delhi, India.

OBJECTIVE: In 2015, New Delhi witnessed a massive outbreak of Dengue virus (DENV) resulting in high morbidity and mortality. We report the molecular characterisation of the dominant circulating DENV strain to understand its evolution and dispersal. MATERIALS AND METHODS: DENV infections were diagnosed by detection of IgM/NS1 antigen, and serotyping was performed by C-PrM PCR. Envelope gene was amplified, and variation(s) in envelope gene were analysed. Phylogenetic tree construction, time-based phylogeny and origin of DENV were analysed. Site-specific selection pressure of envelope gene variants was analysed. RESULTS: Confirmed DENV infection was observed in 11.34% (32 of 282) cases, while PCR positivity for C-PrM region was observed in 54.16% (13 of 24) of NS1 antigen-positive cases. All samples belonged to serotype 2 and cosmopolitan genotype. Phylogenetic analysis using envelope gene revealed segregation of cosmopolitan genotype strains into specific lineages. The Indian strains clustered separately forming a distinct monophyletic lineage (lineage III) with a signature amino acid substitution viz., I162V and R288K. Selection pressure analysis revealed that 215D, 288R and 304K were positively selected sites. The rate of nucleotide substitution was 6.93 × 10-4 substitutions site-1 year-1 with time to most common ancestor was around 10 years with JX475906 (Hyderabad strain) and JN030345 (Singapore strain) as its most probable ancestor. CONCLUSION: We observed evolution of a distinct lineage of DENV-2 strains on the Indian subcontinent with possible changes in endemic circulating dengue strains that might give rise to more pathogenic strains.

Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection.

BACKGROUND: Molecular testing at the point-of-care may turn out to be game changer for HCV diagnosis and treatment monitoring, through increased sensitivity, reduced turnaround time, and ease of performance. One such assay GeneXpert® has recently been released. OBJECTIVES: Comparative analysis between performances of GeneXpert® and Abbott HCV-RNA was done. STUDY DESIGN: 174 HCV infected patients were recruited and, one time plasma samples from 154 patients and repeated samples from 20 patients, obtained at specific treatment time-points (0, 4, 12 and 24) weeks were serially re-tested on Xpert®. RESULTS: Genotype 3 was the commonest, seen in 80 (66%) of the cases, genotype 1 in 34 (28.3%), genotype 4 in 4 (3.3%) and genotypes 2 and 5 in 1 (0.8%) each. Median HCV RNA load was 4.69 log10 (range: 0-6.98log10) IU/ml. Overall a very good correlation was seen between the two assays (R2=0.985), concordance of the results between the assays was seen in 138 samples (89.6%). High and low positive standards were tested ten times on Xpert® to evaluate the precision and the coefficient of variation was 0.01 for HPC and 0.07 for the LPC. Monitoring of patients on two different regimes of treatment, pegylated interferon plus ribavirin and sofosbuvir plus ribavirin was done by both the systems at baseline, 4, 12 and 24 weeks. Perfect correlation between the assays in the course of therapy at different treatment time- point in genotypes 3 and 1 was seen. CONCLUSION: The study demonstrates excellent performance of the Xpert® HCV assay in viral load assessment and in treatment course monitoring consistency.