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

Nik Zuraina NMN, Goni MD, Amalina KN, Hasan H, Mohamad S, Suraiya S

Diagnostics (Basel), 2021 Apr 22;11(5).
PMID: 33922299 DOI: 10.3390/diagnostics11050753

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.
Fulltext High frequency of Haemophilus influenzae associated with respiratory tract infections among Malaysian Hajj pilgrims

Nik Zuraina NMN, Sarimah A, Suharni M, Hasan H, Suraiya S

J Infect Public Health, 2018 08 07;11(6):878-883.
PMID: 30097415 DOI: 10.1016/j.jiph.2018.07.010

BACKGROUND: Overcrowding during the annual Hajj pilgrimage has been known to increase the risk of infectious diseases transmission. Despite the high prevalence of respiratory illness among Malaysian Hajj pilgrims, knowledge about the etiologic pathogens is yet very limited. Thus, this study aimed to determine the spectrum of bacterial respiratory pathogens among the Hajj pilgrims returning to Malaysia in year 2016.
METHODS: Expectorated sputum specimens were collected from the Hajj pilgrims with symptomatic respiratory tract infections (RTIs). Subsequently, the bacterial pathogens were identified using the standard bacteriological culture method and Vitek II system.
RESULTS: This study indicated that 255 (87.33%) out of 292 cultured sputa were positive with at least one potential pathogenic bacteria. Out of 345 total bacterial isolates, 60% (n=207) were Haemophilus influenzae, which was associated with both single bacterium infection (132/173, 76.3%) and multiple bacterial infections (75/82, 91.5%). The other bacterial isolates included; Klebsiella pneumoniae (n=37, 10.7%), Moraxella catarrhalis (n=27, 7.8%), Haemophilus parainfluenzae (n=25, 7.2%), Streptococcus group G (n=18, 5.2%), Klebsiella spesies (n=16, 4.6%), Streptococcus pneumoniae (n=11, 3.2%) and few other organisms.
CONCLUSION: High frequency of H. influenzae was isolated from Malaysian Hajj pilgrims, especially those with respiratory symptoms. Further study should evaluate the actual pathogenicity of the organism and the interactions between the respiratory microbiota towards developing effective prevention strategies of RTIs among the local pilgrims.
Fulltext Diagnostic performance of an in-house multiplex PCR assay and the retrospective surveillance of bacterial respiratory pathogens at a teaching hospital, Kelantan, Malaysia

Nik Zuraina NMN, Mohamad S, Hasan H, Goni MD, Suraiya S

Pathog Glob Health, 2023 Feb;117(1):63-75.
PMID: 35331083 DOI: 10.1080/20477724.2022.2028378

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

Salleh MZ, Nik Zuraina NMN, Hajissa K, Ilias MI, Banga Singh KK, Deris ZZ

Antibiotics (Basel), 2022 Nov 18;11(11).
PMID: 36421297 DOI: 10.3390/antibiotics11111653

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.