Diagnostic performance of an in-house multiplex PCR assay and the retrospective surveillance of bacterial respiratory pathogens at a teaching hospital, Kelantan, Malaysia.

Respiratory tract infections (RTIs), including pneumonia and pulmonary tuberculosis, are among the leading causes of death worldwide. The use of accurate diagnostic tests is crucial to initiate proper treatment and therapy to reduce the mortality rates for RTIs. A PCR assay for simultaneous detection of six respiratory bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, was developed in our lab. The current study aimed to evaluate the performance of this assay along with the retrospective surveillance of respiratory pathogens at a teaching hospital in Kelantan, Malaysia. Leftover sputa (n = 200) from clinical laboratories were collected and undergone DNA template preparation for PCR analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR assay were determined in comparison with the gold standard sputum culture. Overall, the accuracy performance of this assay was 94.67% (95% CI: 90.87% to 97.21%) with sensitivity, specificity, PPV and NPV of 100%, 91.67%, 87.1% and 100%, respectively. Based on the organisms detected from sputa, K. pneumoniae ranked as the top isolate (n = 48), followed by P. aeruginosa (n = 13) and H. influenzae (n = 10). Surveillance among the patients showed that the associations of bacterial positive with gender and means of acquisition were found significant (p values = 0.049 and 0.001, respectively). Besides the promising performance of this ready-to-use molecular-based assay for the rapid detection of selected bacteria pathogens, this study also highlighted significant spread of K. pneumoniae RTIs in the community.

Prevalence of Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Producing Shigella Species in Asia: A Systematic Review and Meta-Analysis.

Shigellosis remains one of the leading causes of morbidity and mortality worldwide and is the second leading cause of diarrheal mortality among all age groups. However, the global emergence of antimicrobial-resistant Shigella strains, limiting the choice of effective drugs for shigellosis, has become the major challenge in the treatment of Shigella infections. The aim of this systematic review and meta-analysis was to provide an updated picture of the prevalence of antimicrobial-resistant Shigella species in Asia. A comprehensive and systematic search was performed on three electronic databases (PubMed, ScienceDirect and Scopus), in which 63 eligible studies published between 2010 and 2022 were identified. From our meta-analysis of proportions using a random-effects model, the overall prevalence of Shigella spp. in Asian patients was estimated to be 8.0% (95% CI: 5.5-10.5). The pooled prevalence rates of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Shigella strains were 68.7% (95% CI: 59.9-77.5) and 23.9% (95% CI: 12.9-34.8), respectively. Concerning recommended antimicrobial drugs for Shigella, the prevalence of resistance was highest for ciprofloxacin (29.8%) and azithromycin (29.2%), followed by ceftriaxone (23.8%), in spite of their importance as first- and second-line treatments for shigellosis. In contrast, resistance to carbapenems, such as ertapenem (0.0%), imipenem (0.1%) and meropenem (0.0%), was almost non-existent among the 49 tested antibiotics. The significantly high prevalence estimation suggests that the multidrug-resistant Shigella is a pressing threat to public health worthy of careful and justified interventions. Effective antibiotic treatment strategies, which may lead to better outcomes for the control and treatment of shigellosis in Asia, are essential.

Prevalence of Multidrug-Resistant Diarrheagenic Escherichia coli in Asia: A Systematic Review and Meta-Analysis.

Diarrhea is one of the leading causes of morbidity and mortality in developing countries. Diarrheagenic Escherichia coli (DEC) is an important bacterial agent for diarrhea in infants, children, and international travelers, and accounts for more than 30% of diarrheal cases in children less than 5 years old. However, the choices of antimicrobial agents are now being limited by the ineffectiveness of many first-line drugs, in relation to the emergence of antimicrobial-resistant E. coli strains. The aim of this systematic review and meta-analysis was to provide an updated prevalence of antimicrobial-resistant DEC in Asia. A comprehensive systematic search was conducted on three electronic databases (PubMed, ScienceDirect, and Scopus), where 40 eligible studies published between 2010 and 2022 were identified. Using meta-analysis of proportions and a random-effects model, the pooled prevalence of DEC in Asian diarrheal patients was 22.8% (95% CI: 16.5-29.2). The overall prevalence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing DEC strains was estimated to be 66.3% (95% CI: 58.9-73.7) and 48.6% (95% CI: 35.1-62.1), respectively. Considering antimicrobial drugs for DEC, the resistance prevalence was highest for the penicillin class of antibiotics, where 80.9% of the DEC isolates were resistant to amoxicillin and 73.5% were resistant to ampicillin. In contrast, resistance to carbapenems such as imipenem (0.1%), ertapenem (2.6%), and meropenem (7.9%) was the lowest. The relatively high prevalence estimation signifies that the multidrug-resistant DEC is a public health threat. Effective antibiotic treatment strategies, which may lead to better outcomes for the control of E. coli infections in Asia, are necessary.

Low Seroprevalence of SARS-CoV-2 among Healthcare Workers in Malaysia during the Third COVID-19 Wave: Prospective Study with Literature Survey on Infection Prevention and Control Measures.

Healthcare workers (HCWs) are at greater risk for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This serology surveillance study aimed to investigate the prevalence of SARS-CoV-2 antibodies among the HCWs who were asymptomatic during the third wave of COVID-19 in Malaysia. HCWs from the Universiti Sains Malaysia (USM) Health Campus were prospectively recruited between August 2020 and March 2021 on a voluntary basis. Data on socio-demographics, possible risk factors and travel history were recorded. Serological diagnoses from serum samples were examined for total antibodies against SARS-CoV-2 using an immunoassay kit. A literature survey was performed on the compliance with infection and prevention control (IPC) practices for COVID-19 among HCWs. The majority of the total 617 HCWs participating in this study were nurses (64.3%, n = 397), followed by health attendants (20.9%, n = 129), medical doctors (9.6%, n = 59) and others (6.3%, n = 39). Of those, 28.2% (n = 174) claimed to have exposure to COVID-19 cases, including history of close contact and casual contact with infected patients. Most importantly, all serum samples were found to be non-reactive to SARS-CoV-2, although nearly half (40.0%, n = 246) of the HCWs had been involved directly in the management of acute respiratory illness cases. A proportion of 12.7% (n = 78) of the HCWs reported having underlying health problems, such as diabetes mellitus, hypertension and hyperlipidemia. Despite the presence of medical and sociological risks associated with SARS-CoV-2 infections, the current study found zero prevalence of antibodies against SARS-CoV-2 among the HCWs of USM. Based on the literature survey, the vast majority of Malaysian HCWs demonstrated good IPC practices during the pandemic (average percentage ranged between 92.2% and 99.8%). High compliance with IPC measures may have led to the low seroprevalence of SARS-CoV-2 among the HCWs.

Development of Multiplex PCR Assay for Screening of T6SS-5 Gene Cluster: The Burkholderia pseudomallei Virulence Factor.

Despite the advanced understanding of the disease, melioidosis, an infection caused by Burkholderia pseudomallei, continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for B. pseudomallei that is associated with internalization and intracellular survival of the pathogen. To detect the virulence gene cluster, this study has successfully developed a novel seven-gene (tssC-5, tagD-5, tssA-5, hcp-5, tssB-5, tssF-5, and vgrG-5) multiplex PCR assay. The optimum annealing temperature for this assay ranged between 59 and 62 °C. The limit of detection for this assay was 103 CFU/mL for all genes, excluding tssF-5, which was found at 105 CFU/mL of the bacterial concentration. In sensitivity and specificity tests, this multiplex assay was able to amplify all of the seven target genes from 93.8% (n = 33/35) clinical and 100% (n = 2/2) environmental isolates of B. pseudomallei. Whereas only four genes (tssC-5, tagD-5, tssF-5, and vgrG-5) were amplified from Bukholderia thailandesis, two genes (tagD-5 and tssB-5) were amplified from Bukholderia stagnalis, and zero target genes were amplified from Bukholderia ubonensis. No amplification of any genes was obtained when tested against isolated DNA from non-Bukholderia species (n = 20), which include Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of B. pseudomallei.

Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens.

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

Molecular detection of selected zoonotic respiratory pathogens and the presence of virulence and antibiotic resistance genes via PCR among Kelantan Hajj pilgrims

Aims@#Respiratory tract infections (RTIs) among Malaysian pilgrims are caused by exposure to zoonotic-potential respiratory pathogens, symptomatically and asymptomatically affected by rigorous pilgrimage rituals, overcrowding and other stressors. This study aimed to determine the prevalence, virulence and antibiotic resistance genes of selected zoonotic respiratory pathogens using polymerase chain reaction (PCR) assays among Hajj pilgrims from Kelantan state, Malaysia.@*Methodology and results@#Throat swab specimens were obtained from 189 Kelantan Hajj pilgrims in 2016 and examined by PCR for the identification of respiratory pathogens. Thirteen samples (6.88%) were positive for Streptococcus pneumoniae and four (2.11%) were positive for Klebsiella pneumoniae. All the samples were negative for Influenza A virus, MERS-CoV and Mycobacterium bovis. One sample was positive for S. pneumoniae virulence lytA gene. One sample was positive for K. pneumoniae virulence magA and K2A genes respectively, and three samples were positive for K. pneumoniae rmpA genes. Ten and seven samples were positive for S. pneumoniae mefA and pbpA antibiotic resistance genes respectively. Two samples were positive for K. pneumoniae blaKPC and blaOXA-48 antibiotic resistance genes. @*Conclusion, significance and impact of study@#This work provided insight into the existence of zoonotic respiratory pathogens inducing Hajj RTIs in Kelantan pilgrims. It showed promising findings for zoonotic studies in Hajj settings. The findings could be relevant in potential control measures for the management of zoonotic infections among Hajj pilgrims.