MATERIALS AND METHODS: We studied the samples of the pharyngeal mucosa smears taken from children aged 1-15 years with X-ray confirmed pneumonia. The selection of DNA probes for specific detection of community-acquired pneumonia pathogens (S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumonia, and L. pneumophila) and development of the microarray design were carried out using the disprose program. The nucleotide sequences of pathogens were obtained from NCBI Nucleotide database. In the research we used CustomArray microarrays (USA). For a pooled sample containing S. pneumoniae and H. influenzae DNA, we performed a sequential selection of the best combinations of hybridization parameters: DNA fragment size, DNA amount, hybridization temperature. The selection criteria were: the percentage of effective probes with a standardized hybridization signal (SHS) ≥3 Z, and the excess of SHS levels of effective specific probes compared to SHS of effective nonspecific probes. We selected the probes to detect of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under optimal conditions. The developed microarray was tested under the selected conditions on clinical samples containing S. pneumoniae or H. influenzae DNA. Using ROC analysis there were established threshold values for the signals of specific probes at optimal sensitivity points and the test specificity, the excess of which was interpreted as the evidence of pathogen presence in a sample.

RESULTS: A microarray design included 142 DNA probes to detect S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, and L. pneumophila, the probes being synthesized onto slides. Using the example of clinical samples containing S. pneumoniae and/or H. influenza DNA, we selected optimal parameters for DNA hybridization on microarrays, which enabled to identify bacterial pathogens of community-acquired pneumonia with sufficient efficiency, specificity and reproducibility: the amount of hybridized DNA was 2 μg, the DNA fragment size: 300 nt, hybridization temperature: 47°C. There was selected a list of probes for specific detection of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under the identified conditions. We determined the threshold values of standardized probe signals for specific detection of S. pneumoniae (4.5 Z) and H. influenzae (4.9 Z) in clinical samples.

METHODOLOGY: A retrospective study of all positive Streptococcus pneumoniae isolates consistent with invasive disease from children below 14 years of age hospitalised in two tertiary hospitals; between year 2012 and 2016 was conducted. IPD cases were defined as isolates of S. pneumoniae from a normally sterile body fluid site.

RESULTS: Fifty-four patients were identified in both centres, 35 (65%) from HRPB as compared to 19 (35%) from HS. Majority of cases (14/35, 40 %) in HRPB were of Orang Asli in comparison to Malay children (16/19, 84%) in HS. Septicaemia, pneumonia and meningitis were the most common clinical presentation of IPD in both centres. There was a noticeably higher percentage of isolates found to be non-susceptible (NS) in HS (62.5%) as compared to HRPB (37.5%) although of no statistical significance. Mortality rate was higher in HRPB (26%) in comparison to 11% in HS.