The molecular epidemiology of pneumocystis jirovecii in Cape Town, South Africa

Banda, Derrick (2016-12)

Thesis (M Med)--Stellenbosch University, 2016

Thesis

ENGLISH ABSTRACT : Pneumocystis jirovecii is an opportunistic fungal pathogen that causes Pneumocystis pneumonia (PCP) in immunocompromized hosts. 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: 1) 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. Methods: Clinical samples from 305 children and adult patients at Tygerberg Hospital were collected, after testing using IF. 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. 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. Conclusions: The SYBR Green rtPCR was more sensitive than IF for detection of P. jirovecii; especially in URT samples, which is comparable to previous studies. The DHPS mutation rate increased to 42% from 27% recorded in 2013 from our division. The increase in DHPS mutation rate may be a result of on-going co-trimoxazole use, for prophylaxis or treatment of PCP or other infections. Our findings need to be linked to clinical data to better understand transmission dynamics and potential impact of strain variation on clinical outcome, and further studies are required to better describe the local strain diversity.

AFRIKAANSE OPSOMMING : Pneumocystis jirovecii is 'n opportunistiese swampatogeen wat Pneumosistis longontsteking (PCP) veroorsaak in immuunonderdrukte pasiënte. PCP gaan gepaard met hoë morbiditeit; en sterftesyfers wissel van 10% tot 40%. PCP word tans gediagnoseer deur middle van mikroskopiese ontleding van kliniese monsters. Die polimerasekettingreaksie (PCR) bied egter hoër sensitiwiteit vir die ontleding opsporing van Pneumosistis as mikroskopie. Dit is dus nodig om PCR toetse verder te evalueer voor suksessvolle implementasie daarvan in routine kliniese laboratoriums te bevorder. P. jirovecii kan nie gekweek word nie, dus word daar ook van molekulêre metodes gebruik gemaak vir die genotipering van P. jirovecii. Die twee mees algemene teikengene is die dihidropteroaatsintase (DHPS) en mitochondriale groot subeenheid rRNA (mtLSU rRNA) gene in. DHPS is 'n terapeutiese teiken; mutasies in die geen gaan gepaard met kotrimoksasool profilakse en mislukte behandeling. Polimorfismes in mtLSU rRNA word gebruik vir filogenetiese tipering studies. Doelwitte: 1) om 'n “real-time” PCR (rtPCR) toets vir die diagnose van PCP te evalueer deur dit met immunofluoressensie (IF) te vergelyk; en 2) om die molekulêre epidemiologie van P. jirovecii afkomstig van Tygerberg-hospitaal te beskryf, deur die DHPS en mtLSU rRNA gene te ontleed. Metodes: Kliniese monsters (met IF resultate) van 305 kinders en volwasse pasiënte by Tygerberg-hospitaal is ingesamel. DNS is deur middel van die NucliSens easyMAG platform (BIOMERIEUX) geïsoleer. Die SYBR Green rtPCR toets wat die “major surface” glikoproteïen (MSG) geen amplifiseer, is evalueer. Laastens is die DHPS en mtLSU rRNA gene amplifiseer vir DNA volgorde bepaling. Resultate: 57% van die monsters (175/305) het P. jirovecii DNS bevat, in vergelyking met die 7% (21/305) waargeneem deur IF. Die rtPCR het 'n sensitiwiteit van 100% en spesifisiteit van 46% gehad, waarvan die laasgenoemde verhoog kom word deur die deteksiedrumpel te verhoog. Nie een van die 50 negatiewe kontrole monsters het positief getoets vir P. jirovecii nie Van die 237 laer lugweg (LRT) monsters, het 55.3% daarvan positief getoets vir P. jirovecii in vergelyking met die 70.6% van boonste lugweg (URT) monsters (n=58). In teenstelling hiermee het geen van die URT monsters positief getoets met die IF toets nie, waar 8.9% van die LRT wel positief getoets het. DHPS is suksesvol amplifiseer in 123 (70.3%) monsters; en mtLSU in 126 (72%) monsters. Genotipe 1 (wildetipe) was die oorheersende DHPS genotipe, en 'n mutasie tempo van 42.3% is vir hierdie geen aangeteken. Die mtLSU genotipe 3 was teenwoordig in 50.8% van die monsters, en genotipe 1 in 42%. Gemengde genotipes is in 2.4% van die monsters gevind vir elk van die twee gene. Geen verband is tussen DHPS polimorfismes en mtLSU genotipes gevind nie. Gevolgtrekkings: Die SYBR Green rtPCR is meer sensitief as IF vir die opsporing van P. jirovecii; veral in URT monsters, wat vergelykbaar is met vorige studies. Die DHPS mutasietempo het toegeneem vanaf 27% tot 42%in 2013. Die toename in DHPS mutasie frekwensie kan moontlik toegeskryf word aan voortgesette kotrimoksasool gebruik, vir profilakse of behandeling van PCP of ander infeksies. Ons bevindinge moet gekoppel word aan kliniese data om die oordragsdinamika en effek van stamvariasie op kliniese uitkoms te beter verstaan. Verdere studies word benodig om 'n beter beskrywing van die plaaslike stam diversiteit te beskryf.

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