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Author(s): Derrick Banda
Pneumocystis jirovecii (formerly Pneumocystis carinii) is a common, atypical opportunistic fungal pathogen, causing a severe, life-threatening disease called Pneumocystis pneumonia (PCP) in patient’s immunosuppressed by HIV infection, malignancy, transplantation, or therapeutic immunosuppression (Saric et al., 1994; Aderaye et al., 2003). PCP is associated with substantial morbidity, and mortality rates range from 10% to 40%. The diagnosis of PCP relies on the microscopic detection of P. jirovecii in stained clinical samples. Polymerase chain reaction (PCR) may provide better sensitivity than microscopy; therefore, evaluation and implementation of PCR assays are required for the detection of Pneumocystis infection.
P. jirovecii is not cultivatable, therefore molecular tools are used for characterizing P. jirovecii genotypes; common targets are the dihydropteroate synthase (DHPS) and mitochondrial large subunit rRNA (mtLSU rRNA) genes. DHPS is a therapeutic target; mutations may be associated with co-trimoxazole prophylaxis and treatment failure. Polymorphisms in mtLSUrRNA have been used for phylogenetic studies.
Aims: to evaluate a real time PCR (rtPCR) assay for diagnosis of PCP by comparing the performance to immunofluorescence (IF) and 2) to describe the molecular epidemiology of P. jirovecii isolates from Tygerberg Hospital by analyzing DHPS and mtLSU rRNA genes.
Objectives: To compare 2 diagnostic real-time PCR assays (one using a hydrolysis probe, one using SYBR-green intercalating dye) for the detection of PCP.
To describe the molecular epidemiology of P. jirovecii in patients in Cape Town, South Africa by:
Characterizing polymorphisms at DHPS and mtLSU-rRNA regions of the P. jirovecii genome b) Analyzing the different combinations of the 2 alleles (DHPS and mtLSU-rRNA) and evaluating the genetic diversity of P. jirovecii in study isolates
Materials and Methods:
Three hundred and five residual respiratory samples from adult and pediatric patients with clinical suspicion of PCP were included in the study. These samples had been sent to the NHLS Immunology laboratory at Tygerberg Hospital for routine diagnostic PCP investigations during the time period of March 2014 to January 2015. The Tygerberg hospital serves a drainage area of approximately half of Cape Town. The hospital acts as a referral centre for 4 regional hospitals (Karl Bremer, Paarl, Worcester and Helderberg Hospitals), 17 district hospitals and over 120 primary health care clinics. The population served is approximately 2.6 million, representing just under half the population of the Western Cape.
DNA was extracted using the NucliSens easyMAG platform (Biomérieux). The rtPCR assay targeting the major surface glycoprotein (MSG) gene was evaluated to detect P. jirovecii DNA. The DHPS and mtLSU rRNA genes were amplified by nested PCR and analyzed by DNA sequencing.
Results:
The SYBR Green rtPCR detected P.jirovecii in 57% of samples (175/305) compared to the 7% (21/305) detected by IF. Our rtPCR had a sensitivity
of 100% and specificity of 46%, although this increased if the detection threshold increased. Of the 50 negative control samples used in this study, none tested positive
for P.jirovecii. There were 237 lower respiratory tract (LRT) and 58 upper respiratory tract (URT) samples. The yield of PCR in LRT samples was 55.3% (131/237) compared to 70.6% (41/58) in URT samples (p=0.03). In contrast, none of the URT samples were positive using IF, and 8.9% (21/237) of LRT samples were positive on IF. Stellenbosch University https://scholar. sun.ac.za iii DHPS was successfully amplified in 123 (70.3%) samples; and mtLSU in 126 (72%) samples. Genotype 1 (wild type) was the predominant DHPS genotype, and a mutation rate of 42.3% was recorded for this gene. The mtLSU genotype 3 was present in 50.8% of samples, genotype 1 (42%) was the next most common genotype. Mixed genotypes were detected in 2.4% of the samples analyzed for each gene. There was no clear association between DHPS polymorphisms and mtLSU genotype.
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