Peripheral lymphocyte subset alteration in patients with COVID-19 having differential clinical manifestations

Background & objectives: The COVID-19 disease profile in Indian patients has been found to be different from the Western world. Changes in lymphocyte compartment have been correlated with disease course, illness severity and clinical outcome. This study was aimed to assess the peripheral lymphocyte phenotype and subset distribution in patients with COVID-19 disease from India with differential clinical manifestations. Methods: Percentages of peripheral lymphocyte subsets were measured by flow cytometry in hospitalized asymptomatic (n=53), mild symptomatic (n=36), moderate and severe (n=30) patients with SARS-CoV-2 infection, recovered individuals (n=40) and uninfected controls (n=56) from Pune, Maharashtra, India. Results: Percentages of CD4+Th cells were significantly high in asymptomatic, mild symptomatic, moderate and severe patients and recovered individuals compared to controls. Percentages of Th memory (CD3+CD4+CD45RO+), Tc memory (CD3+CD8+CD45RO+) and B memory (CD19+CD27+) cells were significantly higher in the recovered group compared to both asymptomatic, mild symptomatic patient and uninfected control groups. NK cell (CD56+CD3-) percentages were comparable among moderate +severe patient and uninfected control groups. Interpretation & conclusions: The observed lower CD4+Th cells in moderate+severe group requiring oxygen support compared to asymptomatic+mild symptomatic group not requiring oxygen support could be indicative of poor prognosis. Higher Th memory, Tc memory and B memory cells in the recovered group compared to mild symptomatic patient groups might be markers of recovery from mild infection; however, it remains to be established if the persistence of any of these cells could be considered as a correlate of protection.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety

Background & objectives: Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS- CoV-2 used in these programmes. Methods: Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin–Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription–polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control. Results: SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per ?l. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively. Interpretation & conclusions: Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories

Central Venous Catheter-related Blood Stream Infections: Incidence, Risk Factors and Associated Pathogens in a University Hospital ICU

Central Venous Catheters (CVCs) are indispensable in current intensive care treatment; also pose a greater riskof device related infections in comparison to any other type of medical device and are major cause of morbidity,mortality and increased expense. A cross sectional prospective study of one year duration was conducted inthe tertiary care University Hospital ICU located in the rural region of Haryana, India, to determine the incidenceof the central venous catheter related bloodstream infection (CRBSI), rate of catheter colonization and toidentify the associated risk factors and the microbial spectrum of CRBSI along with the antimicrobial sensitivitypattern of microbial isolates. Sixty patients with central venous catheter inserted and admitted under ICUhaving signs and symptoms of septicaemia post 48 hours of central venous catheter insertion were included.The rate of CRBSI was assessed by paired quantitative blood culture method in the CVC and peripheral vein.The CRBSI incidence was 16.67% and catheter colonization was found to be 53.3%. Methicillin-resistantstaphylococcus aureus and Acinetobacter baumanni were the predominant isolates. A statistically significantassociation of duration of catheterization with CRBSI was found. It is concluded that CRBSI incidence ishigh, with significant association of prolonged duration of catheterization with CRBSI. By knowing the changingtrends of microbial flora, empirical therapy can be formulated for early and effective management of CRBSI.

​Assessment For Performance Of Middlebrook 7h9 With Oleic-Albumin-Dextrose-Catalase Culture Media For Isolation Of Mycobacterium Tuberculosis In Ziehl–Neelsen Smear-Positive Sputum Samples In Clinically Suspected Cases Of Tuberculosis

Introduction: Pulmonary tuberculosis (TB) is a second foremost cause of death from a communicable disease, after the HIV.Being communicable should be diagnosed at the earliest. Smear examination is preliminary step for the confirm diagnosis, butculture is still a gold standard method.Materials and Methods: The present study was carried out in the Department of Microbiology on a total of 600 smear sputumsamples from clinically suspected cases of pulmonary TB attending the outpatient and inpatient departments of MMIMSR,Mullana, Ambala, from December 2016 to June 2018. Specimens were subjected to ZN and LED staining before and afterdecontamination. After microscopy, specimens were subjected to culture on LJ and Middlebrook 7H9.Results: Mycobacterium tuberculosis was isolated in 23.33% of samples. 110 (78.57%) were detected by microscopy (ZN andLED), respectively. ZN smear positivity before and after decontamination was maximum in mucopurulent 78% and 76.63% andLED 73.63% and 72.03%. Culture positivity on Middlebrook 7H9 was 100% while 87.85% on LJ media. The rate of contaminationwas 5% and 7% on Middlebrook 7H9 and LJ media, respectively.Conclusions: Middlebrook media was superior to the conventional LJ medium in being rapid, easy to use and interpret, andsignificantly low time-to-growth detection and had lesser contamination rate because the liquid media contains growth supplementoleic-albumin–dextrose-catalase, provides additional nutrition.

Bacterial Isolates and their Antibiotic Sensitivity Pattern in Clinically Suspected Cases of Fever of Unknown Origin.

The present study was conducted on 100 suspected cases of fever of unknown origin to identify the prevalence of predominant bacterial microorganisms and their drug sensitivity pattern. The blood samples were subjected to conventional blood culture and BACTEC 9050 culture system. Out of 100 suspected cases, culture positivity was seen in 46% cases with 80.43% pathogenic bacterial isolates comprising of 54.05% gram positive and 45.94% gram negative isolates. Predominant gram positive isolates were coagulase negative Staphylococcus 35% followed by 30% Staphylococcus aureus with sensitivity to vancomycin (100%) and resistance to ampicillin, cloxacillin & cefalexin. Gram negative isolates were Salmonella typhi (29.41%) followed by E coli (17.64%) showing sensitivity to piperacillin/tazobactum and cefoperazone/sulbactum (90%) each and resistance to amoxicillin. BACTEC 9050 was observed to be sensitive(100%) as compared to conventional blood culture(67.56%) for cultural isolation of pathogenic organisms in clinical specimens.

Extended-spectrum β-lactamase and AmpC β-lactamase Production among Gram-negative Bacilli Isolates Obtained from Urinary Tract Infections and Wound Infections.

Extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases continue to be a major problem in healthcare settings. Due to the scarcity of information regarding the antibiotic susceptibility patterns particularly from urinary tract infections (UTIs) and wound infections, the current study was carried out to assist the clinicians to prescribe appropriate antibiotics against gram-negative clinical isolates. In the current study, urine (n = 620) and pus (n = 228) samples were collected from different sites (at various clinical departments) and subjected to direct microscopic examination, culture and antibiotic susceptibility testing (AST). In the AST testings, the isolates that exhibited reduced zone of inhibition to one or more of the antibiotics such as cefotaxime (≤27 mm), ceftriaxone (≤25 mm), ceftazidime (≤22 mm), cefpodoxime (≤17 mm) and aztreonam (≤27 mm) were considered as potential ESBL producers and the ESBL production was confirmed using phenotypic screening test (doubledisk synergy test) and phenotypic confirmatory test (combined-disk test). However, isolates showing resistance or decreased sensitivity to cefoxitin, cefotaxime, ceftriaxone, ceftazidime, cefpodoxime or aztreonam and sensitive to cefepime were considered as a screen positive AmpC producer and subjected to AmpC disk tests. The current study concluded that 72.41% and 21.76% of ESBL and AmpC producers were detected, respectively in our hospital. It was also observed that the double-disk synergy and combined-disk tests were equally effective for ESBL detection. Further, AmpC disk test is simple, easy to perform and interpret, requiring less expertise for the rapid detection of AmpC isolates.

Oseltamivir-resistant influenza A(H1N1) pdm09 virus: first reported case from India.

Background: Recent studies on antiviral susceptibiliy from South-East Asia, Europe and the United States have shown sporadic neuraminidase inhibitor (NAI) resistance in A(H1N1)pdm09 viruses. We undertook a study to evaluate NAI resistance in these viruses isolated in India. Methods: Pandemic influenza viruses, isolated from 2009 to 2013, along with clincal samples were genetically analysed for known resistance markers in the neuraminidase (NA) gene. Clinical samples (n=1524) were tested for H275Y (N1 numbering; H274Y in N2 numbering) mutation by real time reverse transcriptase PCR (rRT-PCR). One hundred and ten randomly selected resistant and sensitive viruses were analysed by phenotypic assay. Results: All but one of the 2013 A(H1N1)pdm09 isolates were sensitive to oseltamivir. Genetic analysis of this isolate as well as the original clinical material showed that the presence of H275Y mutation was responsible for reduced susceptibility to oseltamivir in the patient. This was confirmed by phenotypic assay. Conclusion: The emergence of a pandemic influenza strain resistant to oseltamivir emphasizes the need for monitoring antiviral resistance as part of the National Influenza Programme in India.

Extrapulmonary Tuberculosis In M.M.I.M.S.R, Muallana Ambala: A Microbiological & Histopathological Study.

Clinical microbiological and histopathological confirmation plays a key role in the diagnosis of tuberculosis. The present study correlates Zeihl Nelson staining , Lowenstein Jensen culture media, Montoux test and histopathology in the diagnostic yield of extrapulmonary tuberculosis.Result of current study shows out of total 255 samples, 24 (9.4 %.) showed the presence of mycobacteria by either of the following methods: LJ culture media, ZN staining, histopathology and montoux test.

Pandemic influenza A(H1N1) 2009 outbreak in a residential school at Panchgani, Maharashtra, India.

Background & objectives: An outbreak of influenza was investigated between June 24 and July 30, 2009 in a residential school at Panchgani, Maharashtra, India. The objectives were to determine the aetiology, study the clinical features in the affected individuals and, important epidemiological and environmental factors. The nature of public health response and effectiveness of the control measures were also evaluated. Methods: Real time reverse transcriptase polymerase chain reaction was performed on throat swabs collected from 82 suspected cases to determine the influenza types (A or B) and sub-types [pandemic (H1N1) 2009, as well as seasonal influenza H1N1, H3N2]. Haemagglutination inhibition assay was performed on serum samples collected from entire school population (N = 415) to detect antibodies for pandemic (H1N1) 2009, seasonal H1N1, H3N2 and influenza B/Yamagata and B/Victoria lineages. Antibody titres ≥ 10 for pandemic (H1N1) 2009 and ≥ 20 for seasonal influenza A and B were considered as positive for these viruses. Results: Clinical attack rate for influenza-like illness was 71.1 per cent (295/415). The attack rate for pandemic (H1N1) 2009 cases was 42.4 per cent (176/415). Throat swabs were collected from 82 cases, of which pandemic (H1N1) 2009 virus was detected in 15 (18.3%), influenza type A in (6) 7.4 per cent and influenza type B only in one case. A serosurvey carried out showed haemagglutination inhibition antibodies to pandemic (H1N1) 2009 in 52 per cent (216) subjects in the school and 9 per cent (22) in the community. Interpretation & conclusion: Our findings confirmed an outbreak of pandemic (H1N1) 2009 due to local transmission among students in a residential school at Panchgani, Maharashtra, India.