Direct Manual Antibiotic Susceptibility Tests (DMAST) compared with Minimal Inhibitory Concentration (MIC) System: Reliable results in 8 to 18 hours

Abstract

Background: Pneumocystis pneumonia (PCP), caused by the fungus Pneumocystis jirovecii, is a major cause of hospitalization and mortality in HIV-infected patients presenting to public-sector hospitals in South Africa. Microbiological diagnosis currently relies on immunofluorescent (IF) staining of lower respiratory tract (LRT) samples, which may be difficult to obtain. Diagnosis on upper respiratory tract (URT) specimens using PCR technology has been reported useful but cost is a common limitation to implementing many of these PCR methods in resource-limited settings. The main objectives of the study was (1) to optimise and validate an inexpensive, SYBR green-mediated (probe-free), real-time PCR assay, targeting themajor surface glycoprotein (MSG) gene, for the detection of P. jirovecii in a variety of respiratory samples compared to IF, and (2) to sequence the dihydropteroate synthase (DHPS) locus of PCR positive samples and characterize the genotypes of the circulating strains. Methods & Materials: Upper and lower respiratory samples referred to the laboratory at Tygerberg Hospital between August and October 2013, from adult and paediatric patients with suspected PCP, were submitted for real-time PCR and IF. Specimens from patients with confirmed viral lower respiratory tract infections were used as a control group. The fas gene, encoding the enzyme DHPS, was amplified and sequenced from the PCR positive samples to determine wild-type strain versus with those non-synonymous point mutations associated with sulphonamideresistance. Results: 70 respiratory samples were collected for the validation. The overall detection rate by PCR (43%) was higher than by IF (22%). The assay had a sensitivity of 100% (95% CI: 100 100%) and a specificity of 74.1% (95% CI: 60.5 87.7%). The control group had a 16.7% positivity rate with PCR. Sequencing performed on 30 PCR positive samples revealed that 73.3% were wild-type strains. Conclusion: SYBR green-mediated, real-time PCR is a reliable and rapid PCR method that may be used to replace IF for the routine diagnosis of PCP. A more detailed molecular epidemiological study, to includea larger sample size, informationon sulphonamide exposure and clinical outcome, is warranted in our setting.