Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity.

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. Identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

Antibiotic resistance of uropathogens among the community-dwelling pregnant and nonpregnant female: a step towards antibiotic stewardship.

BACKGROUND: Indiscriminate and widespread use of antibiotics has resulted in emergence of many antibiotic-resistant organisms. Antibiotic administration during pregnancy is mostly avoided, unless there is compelling medical condition. We hypothesized that the uropathogens isolated from pregnant women would be more susceptible to antibiotics compared to those isolated from nonpregnant women, thus will be helpful in formulating separate empiric guideline for pregnant women based on the resistance pattern. METHODS: This was a prospective cross-sectional study conducted over a period of 2 years in which females with the clinical diagnosis of either cystitis or asymptomatic bacteriuria during pregnancy were included from the community settings. Uropathogen species and their antimicrobial resistance pattern were compared between the pregnant and nonpregnant groups. After accounting for centre-to-centre variation and adjusting for age and socio-economic status, the adjusted odds ratio for antibiotic resistance was calculated and compared between pregnant and nonpregnant women using logistic regression analysis. RESULTS: A total of 1758 women (pregnant: 43.3%; nonpregnant: 56.6%) were screened in the study over a period of 2 years, out of which 9.3% (163/1758) were having significant bacteriuria. Escherichia coli and Klebsiella pneumoniae were the two commonest uropathogen in both the groups; their prevalence being 83.6% in pregnant women and 85.2% in nonpregnant women, respectively. Resistance against ampicillin, cefixime, cefoxitin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid were found significantly lower in the pregnant women compared to nonpregnant. After adjusting the age and socio-economic status accounting for centre-to-centre variation, the odds of resistance for cefixime, amoxicillin-clavulanic acid and co-trimoxazole were found lower and statistically significant among the pregnant women group. CONCLUSIONS: The antimicrobial resistance was significantly higher among the community-dwelling nonpregnant women compared to pregnant women in case of few antibiotics. The study highlighted the need of building local antibiogram that could help to initiate the empirical treatment and thus prevent emergence of antimicrobial resistance.

Simple and low-cost nucleic acid extraction methods for detection of SARS-CoV2 in self-collected saliva and dry oral swabs.

Background: Ongoing need of alternative strategies for SARS-CoV-2 detection is undeniable. Self-collected samples without viral transport media (VTM), coupled with simple nucleic acid extraction methods for SARS-CoV-2 PCR are beneficial. Objectives: To evaluate results of SARS-CoV-2 PCR using simple nucleic acid extraction methods from self -collected saliva and oral swabs without VTM. Methods: A cross-sectional single-centre study was conducted on 125 participants (101 SARS-CoV-2 positive cases and 24 controls). PCR was performed following five simple nucleic acid extraction methods on self -collect saliva and oral swabs without VTM and results were compared with gold standard PCR. For saliva, kit-based extraction (SKE), Proteinase K and Heat extraction (SPHE), only Heat extraction (SHE) methods and for dry oral swabs, Proteinase K and Heat extraction (DPHE) and only Heat extraction (DHE) was performed. Results: SARS-CoV-2 was detected in self-collected saliva and oral swabs. 93.07% were correctly classified as positive by SKE, 69.31% by SHE, 67.33% by SPHE, 67.33% by DPHE and 55.45% by DHE. Discriminant power of SKE was significantly higher than other methods (p-value < 0.001) with good- fair agreement of alternate extraction methods against gold standard. Conclusion: Combination of self-collected saliva/ oral-swab without VTM and alternative RNA extraction methods offer a simplified, economical substitute strategy for SARS-CoV-2 detection.

A 9-gene biomarker panel identifies bacterial coinfections in culture-negative COVID-19 cases.

Confirmatory diagnosis of bacterial coinfections with COVID-19 is challenging due to the limited specificity of the widely used gold-standard culture sensitivity test despite clinical presentations. A misdiagnosis can either lead to increased health complications or overuse of antibiotics in COVID-19 patients. With a multi-step systems biology pipeline, we have identified a 9-gene biomarker panel from host blood that can identify bacterial coinfection in COVID-19 patients, even in culture-negative cases. We have also formulated a qPCR-based score that diagnoses bacterial coinfection with COVID-19 with the accuracy, specificity, and sensitivity of 0.93, 0.96, and 0.89, respectively. This gene signature and score can assist in the clinical decision-making process of necessary and timely prescription of antibiotics in suspected bacterial coinfection cases with COVID-19 and thereby help to reduce the associated morbidity and mortality.

Evaluation of hospital environment for presence of Mucorales during COVID-19-associated mucormycosis outbreak in India - a multi-centre study.

BACKGROUND: An unprecedented rise in the number of COVID-19-associated mucormycosis (CAM) cases has been reported in India. Myriad hypotheses are proposed for the outbreak. We recently reported uncontrolled diabetes and inappropriate steroid therapy as significant risk factors for the outbreak. However, Mucorales contamination of hospital environment was not studied. AIM: To perform a multi-centre study across India to determine possible Mucorales contamination of hospital environment during the outbreak. METHODS: Eleven hospitals from four zones of India representing high to low incidence for mucormycosis cases were included in the study. Samples from a variety of equipment used by the patients and ambient air were collected during May 19th, 2021 through August 25th, 2021. FINDINGS: None of the hospital equipment sampled was contaminated with Mucorales. However, Mucorales were isolated from 11.1% air-conditioning vents and 1.7% of patients' used masks. Other fungi were isolated from 18% of hospital equipment and surfaces, and 8.1% of used masks. Mucorales grew from 21.7% indoor and 53.8% outdoor air samples. Spore counts of Mucorales in air were significantly higher in the hospitals of North and South zones compared to West and East zones (P < 0.0001). Among Mucorales isolated from the environment, Rhizopus spp. were the most frequent genus. CONCLUSION: Contamination of air-conditioning vents and hospital air by Mucorales was found. Presence of Mucorales in these areas demands regular surveillance and improvement of hospital environment, as contamination may contribute to healthcare-associated mucormycosis outbreaks, especially among immunocompromised patients.

Predicted CTL responses from pressured epitopes in SARS-CoV-2 correlate with COVID-19 severity

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Protective cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we focus on the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients and dictating mechanisms of immune evasion adopted by SARS-CoV-2 due to the imposed immune pressure at global and cohort levels. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that asymptomatic individuals recognize a larger number of pressured epitopes which could facilitate emergence of mutations at these epitopic regions to overcome the protectivity they offer to the host. Based on the severity of COVID-19, we also identify HLA-alleles and epitopes that offer higher protectivity against severe disease in infected individuals. Finally, we shortlist a set of pressured and protective epitopes that represent regions in the viral proteome that are under higher immune pressure across SARS-CoV-2 variants due to the protectivity they offer. Identification of such epitopes could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens, defined by the distribution of HLA-genotypes among members of a population.

Laboratory preparedness for SARS-CoV-2 testing in India: Harnessing a network of Virus Research & Diagnostic Laboratories.

Background & objectives: An outbreak of respiratory illness of unknown aetiology was reported from Hubei province of Wuhan, People's Republic of China, in December 2019. The outbreak was attributed to a novel coronavirus (CoV), named as severe acute respiratory syndrome (SARS)-CoV-2 and the disease as COVID-19. Within one month, cases were reported from 25 countries. In view of the novel viral strain with reported high morbidity, establishing early countrywide diagnosis to detect imported cases became critical. Here we describe the role of a countrywide network of VRDLs in early diagnosis of COVID-19. Methods: The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune, established screening as well as confirmatory assays for SARS-CoV-2. A total of 13 VRDLs were provided with the E gene screening real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay. VRDLs were selected on the basis of their presence near an international airport/seaport and their past performance. The case definition for testing included all individuals with travel history to Wuhan and symptomatic individuals with travel history to other parts of China. This was later expanded to include symptomatic individuals returning from Singapore, Japan, Hong Kong, Thailand and South Korea. Results: Within a week of standardization of the test at NIV, all VRDLs could initiate testing for SARS-CoV-2. Till February 29, 2020, a total of 2,913 samples were tested. This included both 654 individuals quarantined in the two camps and others fitting within the case definition. The quarantined individuals were tested twice - at days 0 and 14. All tested negative on both occasions. Only three individuals belonging to different districts in Kerala were found to be positive. Interpretation & conclusions: Sudden emergence of SARS-CoV-2 and its potential to cause a pandemic posed an unsurmountable challenge to the public health system of India. However, concerted efforts of various arms of the Government of India resulted in a well-coordinated action at each level. India has successfully demonstrated its ability to establish quick diagnosis of SARS-CoV-2 at NIV, Pune, and the testing VRDLs.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India. Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases. Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups. Interpretation & conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India.

Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.

Comparison of microbiome isolated from the conjunctiva, contact lens and lens storage case of symptomatic and asymptomatic contact lens users.

BACKGROUND AND OBJECTIVES: Contact lenses (CLs) are increasingly being used for cosmetic or therapeutic purposes. Lack of compliance and poor hygiene towards lens care is strongly associated with microbial contamination and has been proved to result in eye infections. The present study was done to compare the microbial flora between symptomatic and asymptomatic contact lens users. The study also attempts to analyze the contact lens hygiene practices of CL users. MATERIALS AND METHODS: Six samples each were collected from both the eyes, CLs and lens cases of 40 CL users (n=240) divided into two groups based on symptoms present asasymptomatic CL users and symptomatic CL users. Organisms were identified using standard microbiological techniques. RESULTS: The proportion GNB obtained in symptomatic CL users was significantly higher when compared to asymptomatic CL users (p-value= <0.003). In 56.2% eyes, the microbial flora of conjunctiva was similar to either the contact lens isolate/storage case. Enterococcal microbial keratitis was seen in one case. CONCLUSION: There was significant microbial contamination present in CL users despite compliance to contact lens hygiene practices. There were a significant number of bacteria (p-value <0.001) present which were resistant to ampicillin, amoxicillin-clavulanate, and cefotaxime in both the groups.

Outbreak of Pichia kudriavzevii fungaemia in a neonatal intensive care unit.

PURPOSE: Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, Pichia kudriavzevii (a teleomorph of Candida krusei). To the best of our knowledge, this is the first report of neonatal sepsis due to P. kudriavzevii. METHODOLOGY: Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed. RESULTS: Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as P. kudriavzevii and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices. CONCLUSION: This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.