Detection of Pneumocystis carinii with DNA amplification

The opportunistic fungal pathogen Pneumocystis carinii sp. f. hominis is a frequent cause of pneumonia in the immunocompromised host. Analysis of genetic variation among isolates of P. carinii sp. f. hominis from 12 human immunodeficiency virus (HIV)-infected persons with single and multiple episodes of P. carinii pneumonia was undertaken at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon. In samples from 24 episodes of pneumonia, 10 different types of P. carinii sp. f. hominis were identified. More than 1 sequence type was observed in 8 samples, indicating that mixed infection with different types of P. carinii sp. f. hominis is not uncommon. In 4 of 7 patients with recurrent episodes of pneumonia, the sequence types observed at the second episode were different from those of the first, suggesting the occurrence of both reactivation of a previously acquired infection and reinfection from an exogenous source.

Genetic Diversity at the Internal Transcribed Spacer Regions of the rRNA Operon among Isolates of Pneumocystis carinii from AIDS Patients with Recurrent Pneumonia

The possible transmission of Pneumocystis carinii f. sp. hominis from patients with P. carinii pneumonia to asymptomatic health care workers (HCW), with or without occupational exposure to human immunodeficiency virus (HIV)-infected patients with P. carinii pneumonia, was examined. HCW in a specialist inpatient HIV-AIDS facility and a control group in the general medical-respiratory service in the same hospital provided induced sputum and/or nasal rinse samples, which were analyzed for the presence of P. carinii f. sp. hominis DNA by using DNA amplification (at the gene encoding the mitochondrial large subunit rRNA [mt LSU rRNA]). P. carinii f. sp. hominis DNA was detected in some HCW samples; those with the closest occupational contact were more likely to have detectable P. carinii DNA. P. carinii DNA was detected in one HCW who carried out bronchoscopy over a 2-year period. P. carinii-positive samples were genotyped by using DNA sequence variations at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, along with bronchoalveolar lavage samples from patients with P. carinii pneumonia hospitalized at the same time. Genotyping identified 31 different P. carinii f. sp. hominis ITS genotypes, 26 of which were found in the patient samples. Five of the eight ITS genotypes detected in HCW samples were not observed in the patient samples. The results suggested that HCW in close occupational contact with patients who had P. carinii pneumonia may have become colonized with P. carinii. Carriage was asymptomatic and did not result in the development of clinical disease.

Pneumocystis carinii f. sp. hominis DNA in Immunocompetent Health Care Workers in Contact with Patients with P. carinii Pneumonia

Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about development of resistant organisms. The inability to culture human Pneumocystis, Pneumocystis jirovecii, in a standardized culture system prevents routine susceptibility testing and detection of drug resistance. In other microorganisms, sulfa drug resistance has resulted from specific point mutations in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone.

/pdf/dihydropteroate-synthase-gene-mutations-in-pneumocystis-and-1swxxbw68y.pdf

Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

Background: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. Objectives and methods: To use an extensively optimized real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P. jirovecii heat-shock protein 70 (HSP70) gene to quantify P. jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP and to compare this assay with conventional PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA).
 Results: Sixty-one patients had 62 episodes of PCP (defined by detection of P. jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62; range ~13-18608 [median ~332] copies/reaction and detectable, below the limit of quantification (~5 copies/reaction, 10 copies/reaction) showed clinical sensitivity =98% (95% Confidence Interval (CI) =91-100%) and specificity =96% (95%CI =87-99%), for diagnosis of PCP. By contrast, clinical sensitivity and specificity of mt LSU rRNA PCR was 97% (95%CI =89-99%) and 68% (95 CI =56-78%), respectively.
 Interpretation: The HSP70 real-time PCR assay detects P. jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P. jirovecii DNA by real-time PCR may also discriminate between colonization with P. jirovecii and infection.

https://thorax.bmj.com/content/thoraxjnl/63/2/154.full.pdf

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients

Sputum induction for the diagnosis of pulmonary disease in hiv positive patients

Isolates of Pneumocystis jiroveci from sulfa-exposed and nonexposed patients from London, United Kingdom, and Harare, Zimbabwe, were genotyped. At the dihydropteroate synthase (DHPS) locus, there was evidence of selection pressure from sulfa drug exposure, and reversal of DHPS genotype ratios occurred when selection pressure was absent or was removed.

Genotypes of Pneumocystis jiroveci Isolates Obtained in Harare, Zimbabwe, and London, United Kingdom

Pneumocystis jirovecii is the cause of Pneumocystis pneumonia (PCP) in humans. Isolates of P. jirovecii obtained from patients in Harare, Zimbabwe were genotyped at the superoxide dismutase locus. High genotypic similarity to isolates of P. jirovecii obtained from patients in London, UK was observed. These data provide additional support for the hypothesis that P. jirovecii is genetically indistinguishable in isolates from geographically diverse locations.

Adam S. Malin

Papers

Sputum induction for the diagnosis of pulmonary disease in hiv positive patients

Genotypes of Pneumocystis jiroveci Isolates Obtained in Harare, Zimbabwe, and London, United Kingdom

Isolates of Pneumocystis jirovecii from Harare show high genotypic similarity to isolates from London at the superoxide dismutase locus.