Article

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.

Unidade de Parasitologia Médica, Grupo de Protozoários Oportunistas/VIH e Outras Protozooses--CMDT, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
Clinical Microbiology and Infection (Impact Factor: 4.58). 03/2012; 18(6):E177-84. DOI: 10.1111/j.1469-0691.2012.03828.x
Source: PubMed

ABSTRACT Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia.

0 Bookmarks
 · 
77 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A pneumonia por Pneumocystis jirovecii (pneumocistose ou PPc) é um importante factor de morbilidade e mortalidade nos doentes com a síndroma de imunodeficiência adquirida (sida). A pneumocistose é, também, um problema emergente nos imunocomprometidos, seronegativos para vírus da imunodeficiência humana (VIH), devido ao número crescente de doentes imunocomprometidos, incluindo aqueles sob terapêutica imunossupressora. Na década de 80 do século passado, com a epidemia da sida, a pneumocistose era responsável por cerca de dois terços das infecções definidoras de sida, sendo que 80% dos infectados por VIH desenvolvia, pelo menos, um episódio de pneumocistose que, em 20 a 25% dos casos, era mortal. O uso de quimioprofilaxia anti-P. jirovecii, estabelecida no início dos anos 90, do século passado, e a introdução da terapêutica antirretrovírica (TARV), em meados da mesma década, permitiram um melhor controlo da doença na América do Norte e na Europa Ocidental. No entanto, parte dos casos de pneumocistose ilustram desigualdades no acesso aos cuidados de saúde naquelas regiões do Mundo ou em alguns grupos sociais, mas também, dificuldades na implementação de métodos de diagnóstico adequados, devido à falta recorrente de recursos. Assim, o diagnóstico definitivo da pneumocistose baseia-se em métodos citoquímicos de coloração, imunofluorescência indirecta com anticorpos monoclonais (IFI/AcM) e técnicas moleculares, aplicados a produtos biológicos de obtenção mais ou menos invasiva, processo que exige recursos e pessoal especializado. Recentemente, diversos marcadores serológicos de infecção por P. jirovecii têm sido estudados, com o objectivo de desenvolver um método de diagnóstico da pneumocistose, com recurso a amostras biológicas obtidas de forma minimamente invasiva, como é o caso do sangue.
    Revista Portuguesa de Doenças Infecciosas. 01/2014; 10:16-22.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.
    European Journal of Clinical Microbiology 02/2014; · 3.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pneumocystis jirovecii is a symbiotic respiratory fungus that causes pneumonia (PcP) in immunosuppressed patients. Because P. jirovecii cannot be reliably cultured in vitro, it has proven difficult to study and gaps in our understanding of the organism persist. The release of a draft genome for the organism opens the door for the development of new genotyping approaches for studying its molecular epidemiology and global population structure. We identified and validated 8 putatively neutral microsatellite markers and 1 microsatellite marker linked to the dihydropteroate synthase gene (dhps), the enzymatic target of sulfa drugs used for PcP prevention and treatment. Using these tools, we analyzed P. jirovecii isolates from HIV-infected patients from three geographically distant populations: Uganda, the United States, and Spain. Among the 8 neutral markers, we observed high levels of allelic heterozygosity (average He = 0.586 - 0.842). Consistent with past reports, we observed limited global population structuring, with only Ugandan isolates showing minor differentiation from the other two populations. In Ugandan isolates that harbored mutations in dhps, the microsatellite locus linked to dhps demonstrated a depressed He, consistent with positive directional selection for sulfa-resistance mutations. Using a subset of these microsatellites, analyses of individual and paired samples from infections in San Francisco showed reliable typeability within a single infection and high discriminatory power between infections. These features suggest this novel microsatellite typing approach will be an effective tool for molecular-epidemiological investigations into P. jirovecii population structure, transmission, and drug resistance.
    Journal of clinical microbiology 02/2014; · 4.16 Impact Factor

Full-text (2 Sources)

Download
6 Downloads
Available from
Sep 17, 2014