METHODOLOGY: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay.

RESULTS: A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics.

METHODS: Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells.

RESULTS: Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques.

STUDY DESIGN: Eighty-six postnasal biopsy samples and 71 fine-needle aspirate samples of neck masses were obtained from patients who were clinically suspect for NPC. Genomic DNA was extracted from the samples, and EBNA1, EBNA2, and LMP genes of EBV were detected by PCR. PCR results were compared with NPC histopathology findings.

RESULTS: The sensitivity of PCR to detect EBNA1 (97.14%), EBNA2 (88.57%), and LMP (91.43%) genes of EBV in nasopharyngeal biopsy samples were higher than those in fine-needle aspirate samples.

CONCLUSION: Detection of EBV by PCR in tissue obtained from nasopharyngeal biopsy and fine-needle aspirate samples of neck masses is a relatively inexpensive, reliable, and accurate method of diagnosing NPC. Detection of EBV genes is on par with histopathological examination (HPE) and superior to fine-needle aspirate cytology.