Structural Analysis and Epitope Prediction of S2 Domain of SARS-CoV-2, Conservation Analysis Among Major Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. There are four structural proteins of the virus: spike, envelope, membrane, and nucleocapsid proteins. Various vaccines were designed and are effectively being used against the spike protein of the virus. However, several vaccine-related complications have been reported worldwide. Assuming that the structural integrity of the whole protein might be contributing to these complications, this study was performed to design epitopes using the S2 domain of the spike protein, which could trigger a strong immune response. We have also predicted antigenic and allergenic properties of the selected epitopes. A total of 49 B cell epitopes passing antigenicity and other assessment filters were found using three methods. Among them, RDLICAQ had the highest antigenicity score (1.1443). However, only one cytotoxic T lymphocyte epitope, RSFIEDLLF, passed the essential filters with an antigenicity score of 0.5782 to show an appropriate immune response for T cells, while among 21 helper T cell lymphocyte epitopes that were filtered, FAMQMAYRFNGIGVT showed the highest (1.3688) antigenicity score. Conservation analysis revealed that the S2 domain is significantly conserved, thus making it an ideal candidate for vaccine development. We have also designed a vaccine construct based on the best suiting components found during the whole study. This construct and S2 domain solely can be future subjects of interest or might be included in a subunit cocktail formulation for attaining unabridged immunogenicity.

blaOXA, blaSHV-, and blaTEM- extended-spectrum ß-lactamases in Gram-negative strains from burn patients in Lahore, Pakistan.

INTRODUCTION: Gram-negative bacterial pathogens are associated with complications of post-burn infections that showed significant association with different genotypic variants of extended-spectrum ß-lactamases. In this study, we aimed to determine the distribution of ESBL gene variants among bacterial pathogens from post-burn infections. METHODOLOGY: Cephalosporin-resistant isolates were collected from Jinnah Burn and Reconstructive Surgery Centre, Lahore. Phenotypic testing: double-disk synergy test, combination disk test, multiplex PCR for blaOXA, blaTEM, and blaSHV genes were performed. RESULTS: Of the 53.5% cephalosporin-resistant isolates, 50.7% were community-acquired and 49.3% were nosocomial pathogens. Seventy-two percent of post-burn infections were found in males (p-value = 0.92, OR = 1.04). The age of burn victims ranged from 4 to 85 years (mean = 28.9, SD = 15.6). Pseudomonas aeruginosa was most predominant at a rate of 49%. There were 83.3% multidrug-resistant isolates, which showed susceptibility to meropenem, imipenem, and amikacin in 28.7%, 25.3%, and 26% respectively. In phenotypic tests 16% ESBLs detected by the DDST and 14% confirmed by the CDT. Molecular detection proved effective for the detection of 79.7% blaTEM, 37.7% blaOXA, and 18.8% blaSHV isolates. blaTEM genes were confirmed in 18.1% CDT-positive isolates, with 62.6% diagnostic accuracy (95% CI = 54.7-70) and 88% specificity (95% CI = 80.4-93.4). CONCLUSIONS: The antimicrobial resistance associated with the ESBL-producing Pseudomonas spp. and Enterobacteriaceae is becoming a challenge for the treatment and survival of burn patients. The high rate blaTEM, blaOXA, and blaSHV genes confirm the need to improve the management of burn patients in order to prevent post-burn infections.

Systematic surveillance and meta-analysis on the prevalence of metallo-ß-lactamase producers among carbapenem resistant clinical isolates in Pakistan.

BACKGROUND: Emergence of carbapenem resistance (CR) is a health concern of pertinent importance. Epidemiological surveillance of CR at global and indigenous level (Pakistan) can help to improve infection control and establish pharmacovigilance programs. This study evaluates the prevalence of clinically significant CR isolates, and its genetic variant distribution among geographical regions of Pakistan. METHODS: A meta-analysis was conducted to present the current rate of CR infections and prevalence of Metallo-ß-lactamases (MBLs). The proposed subject was researched using electronic databases to identify the available literature. Thereafter, relevant data was extracted and statistical analysis was performed using STATA version 14. RESULTS: A total of 110 relevant studies were identified with 19 meeting the inclusion criteria for the meta-analysis of CR, while 22 for MBLs. Pooled rate for carbapenem resistance was determined to be 0.28 (95% CI: 0.26-0.31) with overall significant heterogeneity (I2=99.61%, P<0.001) and significant estimated score ES=0 (Z=22.65, P<0.001). In Pakistan, the pooled proportion of MBL producers was 0.34 (95% CI: 0.29-0.39) with overall heterogeneity significance (I2=99.62%, P<0.001) and significant ES=0 (Z=13.17, P<0.001). CONCLUSION: Conclusively, diverse variants of carbapenemases (VIM, IMP, NDM, KPC, GIM) along with other ß-lactamase variants (OXA, TEM, SHV, CTX-M) have been reported across the country. However, New Delhi Metallo-ß-lactamase (NDM)-variants were reported in predominant literature. The prevalence of CR isolates in Pakistan is alarming, associated with MBL production primarily evident from the studies. The study emphasizes the need for regular surveillance, pharmacovigilance and antibiotic stewardship programs to ensure the availability of data to the authorities for preemptive measures of infection control.

Distribution of blaCTX - M , blaTEM , blaSHV and blaOXA genes in Extended-spectrum-ß-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan.

Background: Frequency of extended-spectrum-ß-lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-ß-lactamase-producing Clinical isolates in Pakistan. Methods: A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results: A total of 497/995 (50%) isolates including Escherichia coli 65% (n = 321), Klebsiella spp. 25% (n = 124) and Pseudomonas. 5% (n = 24), Enterobacter spp. 4% (n = 20) and Acinetobacter spp. 2% (n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% (n = 278) followed by pus 20% (n = 99) and wound swab 6% (n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% (n = 392). PCR revealed blaCTX - M (76%) gene followed by blaOXA (52%), blaTEM (28%) and blaSHV (21%) were most prevalent among ESBL-producers detected by CDST. blaCTX - M - 1(65%), blaOXA (78%) and blaTEM (57%) genes were carried on plasmids. Amplicon sequencing revealed blaCTX - M - 15 (75%), blaOXA - 1 (49%) and blaTEM - 1B (34%) and 21 (n = 28) isolates carried three genes in them. Conclusion: Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding blaCTXM - 15 gene and narrow spectrum blaOXA - 1 and blaTEM - 1B were also prevalent.

High frequency and molecular epidemiology of metallo-ß-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan.

Background: Metallo-ß-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the ß-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem). Methods: A total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of ß-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection. Results: The rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of bla TEM, bla SHV, bla OXA, bla IMP-1, and bla VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla MBL (bla IMP and bla VIM) and bla ESBL (bla TEM, bla SHV, bla OXA) was also detected through multiplex and singleplex PCR. bla OXA, bla TEM and bla SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion: To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla OXA, bla TEM and bla SHV with bla IMP-1 and bla VIM in MBL-producing gram-negative bacilli.

Prevalence of extended-spectrum-ß-lactamase-producing Enterobacteriaceae: first systematic meta-analysis report from Pakistan.

Background: South-Asia is known as a hub for multidrug-resistant (MDR) bacteria. Unfortunately, proper surveillance and documentation of MDR pathogens is lacking in Pakistan. The alarming increase in the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a serious problem. From this perspective, we analysed published data regarding ESBL-producing Enterobacteriaceae in different regions of Pakistan. Methods: A meta-analysis was performed to determine the prevalence of ESBL-producing Enterobacteriaceae in Pakistan. A Web-based search was conducted in electronic databases, including PubMed, Scopus and PakMedi Net (for non-indexed Pakistani journals). Articles published (in either indexed or non-indexed journals) between January 2002 and July 2016 were included in the study. Relevant data were extracted, and statistical analysis was performed using the Metaprop command of STATA version 14.1. Results: A total of 68 studies were identified from the electronic data base search, and 55 of these studies met our inclusion criteria. Pakistan's overall pooled proportion of ESBL-producers was 0.40 (95% CI: 0.34-0.47). The overall heterogeneity was significant (I2 = 99.75%, p < 0.001), and significant ES = 0 (Z = 18.41, p < 0.001) was found. OXA, SHV, TEM and CTX-M were the most commonly found gene variants for ESBLs in these studies. Conclusion: The prevalence of ESBL-producing Enterobacteriaceae is high in Pakistan. Little is known about the annual frequency of ESBLs and their prevalence in different provinces of Pakistan. No data are available regarding ESBL frequency in Baluchistan. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance problem. Surveillance to better understand the annual ESBL burden is crucial to improve national and regional guidelines.

Distribution of CTX-M group I and group III ß-lactamases produced by Escherichia coli and klebsiella pneumoniae in Lahore, Pakistan.

Extended-spectrum-lactamases (ESBLs) of the CTX-M type is worrisome issue in many countries of the world from past decade. But little is known about CTX-M beta-lactamase producing bacteria in Pakistan. Therefore, this study was carried out to investigate the distribution of CTX-M beta-lactamase producing E. coli and Klebsiella pneumoniae using phenotypic and molecular techniques. A total of 638 E. coli and 338 Klebsiella pneumoniae were isolated from patients attending two hospitals and one diagnostic Centre in Pakistan during 2013-2015. ESBL production was screened by double disc synergism, combination disc (cefotaxime and ceftazidime with clavulanic acid) and E-test. These strains were further characterized by PCR (CTX-M I, CTX-M III) and sequencing. After ribotyping of strains accession numbers were obtained. These isolates were highly resistant to cephalosporins, ceftazidime, cefotaxime, aztreonam, and cefuroxime but susceptible to carbapenems, sulfzone, amikacin and tazocin. Multiple antibiotic resistances index (MAR) revealed that 51% of E. coli strains fell in the range of 0.61-0.7 and 39% of Klebsiella pneumoniae strains fell in the range of 0.71-0.8. 64% Double disc synergism (DDS), 76.4% combination disc (CD), 74% E-test showed ESBL positivity in strains. In E. coli ESBL genes blaCTX-M-I and blaCTX-M-III were detected in 212 (72.1%) and 25 (8.5%) respectively. In Klebsiella pneumoniae ESBL genes blaCTX-M-I and blaCTX-M-III were detected in 89 (82.4%) and 10 (9.2%). Combination of both genes blaCTX-M-I and blaCTX-M-III were found in 16 (5.4%) of E. coli strains and 5 (4.6%) of Klebsiella pneumoniae strains. Sequencing revealed that CTXM-15 was predominately present in the CTX-M-I group. The prevalence of ESBL producing E. coli and Klebsiella pneumoniae isolates was high and the majority of them positive for blaCTX-M-I as compared to blaCTX-M-III. These findings highlight the need to further investigate the epidemiology of other CTX-M beta-lactamases in Pakistan.