Pneumocystis jiroveci, Mycoplasma pneumoniae and Mycobacterium tuberculosis are respiratory pathogens associated with pneumonia, with increasing prevalence of Pneumocystis pneumonia (PcP) and tuberculosis (TB) in AIDS patients. Increased resistance of M. tuberculosis has emphasized the need for rapid susceptibility testing, such as flow cytometry. Sputum specimens (102) were assessed by PCR employing primers directed at the following genes: P. jiroveci: mitochondrial large subunit ribosomal RNA (mtLSUrRNA), dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), and for M. pneumoniae: 16S rRNA and P1 adhesin. Positive P. jiroveci samples were genotyped by PCR-SSCP (single-strand conformation polymorphism) targeting the: internal transcribed spacer region (ITS), intron of the nuclear 26S rRNA gene (26S), variable region of the mitochondrial 26S rRNA gene (mt26S) and β-tubulin gene (β-tub). Multi-drug resistant (MDR-TB) cultures grown in the presence and absence of four antibiotics (rifampicin, isoniazid, ethambutol and ofloxacin) were heat killed, stained with SYTO16 and Propidium Iodide and analysed using flow cytometry. Rifampicin resistance gene mutations were screened by PCR and DNA sequencing. Details of patient’s gender, age, HIV and M. tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 55.1 percent (54/102 patients). P. jiroveci was mainly associated with HIV (25/102 P. jiroveci positive patients for which clinical data was available) and co-colonisation with M. tuberculosis was observed in 11 cases. Sequence analysis of DHPS and DHFR products found no resistance associated mutations. M. pneumoniae was detected in one patient. Four simple SSCP patterns were identified and there were no co-infections with other P. jiroveci strains. Nine M. tuberculosis samples [8 MDR-TB isolates (NHLS) and M. tuberculosis ATCC® 27294TM] were tested. There was a 53 percent (19 out of 36 tests) agreement of flow cytometry with the BACTEC MGIT 960. Mutations (at two specific codons, namely 516 and 531) in the rifampicin resistance-determining region (RRDR) of the rpoB gene were observed in eight M. tuberculosis isolates. Evaluation of methods for genotyping and drug susceptibility testing of PcP and TB are imperative for epidemiology and drug resistance studies, and impact on treatment protocols.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10328 |
| Date | January 2008 |
| Creators | Du Plessis, Sarah Jane |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | x, 95 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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