Faranak REZAEI, Mehri HAEILI, Abbasali Imani FOOLADI and Mohammad Mehdi FEIZABADI

ABSTRACT

Objectives: Development of molecular techniques for rapid detection of drug resistant tuberculosis allows for the prompt initiation of appropriate anti-TB treatment. We aimed to assess high-resolution melting (HRM) analysis for the detection of rpsL, rrs and embB mutations to identify streptomycin and ethambutol resistance in Mycobacterium tuberculosis.
Materials and Methods: A total of 76 clinical isolates of M. tuberculosis including 25 SM-R, 21 EB-R and 30 drug susceptible – determined by the proportion method of drug susceptibility testing (DST) – were analyzed by HRM analysis, and the results were confirmed using DNA sequencing.
Results: The sensitivity and specificity of the HRMA compared to phenotypic DST were 88% and 100.0%, respectively for the detection of streptomycin resistance (SM-R), and 90.4% and 96.6%, respectively for ethambutol resistance (EB-R). Three SM-R and two EB-R isolates had no mutations in the studied regions of rpsL, rrs and embB genes determined by DNA sequencing and therefore were not identified as resistant by HRM assay. Interestingly, one phenotypic EM-S isolate was found by sequencing to have a mutation at codon 423 (Met → Ilu) of embB gene and was clustered as resistant by HRM as well.
Conclusions: The sensitivity and specificity of HRM curve assay was consistent with DNA sequencing, which is the gold standard method for genotypic DST. This assay can be utilized as a screening method for detection of drug-resistant tuberculosis, offering the advantages of a high throughput, single step, cost effectiveness, and rapid work flow method.
Keywords: HRM analysis; Mycobacterium tuberculosis; rpsL; rrs; embB

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Zahra GHARAILOO, Seyed Fazlollah MOUSAVI and Mohammad Mehdi FEIZABADI

ABSTRACT

Background: Streptococcus pneumoniae (pneumococcus) is an important pathogen of respiratory tract in developing countries. It also causes meningitis, otitis media, community-acquired pneumoniae, and bacteremia. Nasopharyngeal carriage is the paramount importance in the transmission of these bacteria. The pathogenesis of disease begins with colonization of the nasopharynx. Due to lack of pneumococcal disease surveillance and epidemiological data on the carriage in Sistan-Baluchestan, this study was done to determine the frequency of common pneumococcal capsular types among non-vaccinated children under 6 years old by Multiplex PCR.
Materials and method: In this study 260 nasopharyngeal swabs were taken from non-vaccinated healthy children between 6 months to 6 years old at medical centers in Sistan-Baluchestan during August 2013 to January 2014. These samples were cultured on blood agar. Primary identification of bacterial isolated was determined by biochemical analysis and molecular tests. Capsular typing was performed by Multiplex PCR using primers targeting cps locus that is highly conserved among different capsular types. The master mixes for PCR were grouped them into six multiplex reactions.
Results: Out of 260 nasopharyngeal swabs, 42 isolates of Streptococcus pneumoniae were detected and identified. The overall pneumococcal carriage rate was 16.1%. The most frequently isolated capsular types were: 6A/B, 19A, 19F and 23F. These capsular types accounted for 49.9% of all strains detected.
Conclusion: We found that the prevalence of pneumococcal carriage among non-vaccinated children under six years old is about 16%. Our study provides much data about carriage rate and pneumococcal capsular types in preschool children, which is necessary for predicting the different valent pneumococcal conjugated vaccines in Iran.
Keywords: Streptococcus pneumoniae, Nasopharynx, Multiplex PCR, Capsular typing.

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