RESUMO
Bacterial respiratory tract infections (RTIs) are prone to be associated with serious health problems during the annual Hajj pilgrimage and are a public health concern due to the potential of pathogens transmission across continents. This study aimed to perform a diagnostic screening of intended bacteria associated with RTIs among Malaysian Hajj pilgrims by using a newly developed PCR assay. Expectorated sputum specimens (n = 202) and sociodemographic characteristics of the returning Hajj pilgrims were collected upon arrival in Kelantan, Malaysia. Diagnostic screening of bacterial respiratory pathogens was performed using a thermostabilized multiplex PCR assay in parallel with the sputum culture. Of the six intended bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, the sputum specimens were found positive for H. influenzae (n = 139), K. pneumoniae (n = 20), and S. pneumoniae (n = 19) by the multiplex PCR assay. The sensitivity, specificity, positive- and negative predictive values (PPV and NPV) of this assay were 100% (95% confidence interval (CI): 97.85% to 100.00%), 92.23% (95% CI: 85.27% to 96.59%), 95.51% (95% CI: 91.61% to 97.64%) and 100.00%, respectively. The accuracy of this assay was 97.07% (95% CI: 94.31% to 98.73%). Overall, H. influenzae was found to be the predominant organism in the pilgrims' sputa by both molecular and microbial culture methods. The multiplex PCR assay would enable a simple, faster and reliable means for the massive screening of intended bacteria compared to the sputum culture, especially during the Hajj pilgrimage.
RESUMO
Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.
RESUMO
Cholera, a severe form of gastroenteritis, is one of the most widespread diseases in developing countries. The mechanism of intestinal infection caused by V. cholerae O139 remains unclear. In order to explore some morphological aspects of its infection in the intestine including Peyer's patches, we investigated the V. cholerae O139 infection at intestinal site of the rabbit gut-loop model. The electron microscopic analysis revealed denuded mucosal surface with loss of microvilli and integrity of the surface epithelium. Infection of the intestine with V. cholerae O139 induces destruction of villi, microvilli and lining epithelium with exposure of crypts of Lieberkuhn.
