Effect of Oral Washes on the Diagnosis of Pneumocystis carinii Pneumonia with a Low Parasite Burden and on Detection of Organisms in Subclinical Infections

Matos, Olga; Costa, Marina C.; Lundgren, Bettina; Caldeira, L.; Aguiar, Pedro; Antúnes, Francisco

doi:10.1007/s100960100563

Cited by 44 publications

(38 citation statements)

References 16 publications

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“…Other loci such as the cdc2 gene are also under investigation in an attempt to maximize the sensitivity and specificity of these molecular assays (88,319). PCR techniques have also been applied to analyses of oral washes (gargles) in an attempt to establish a noninvasive test for PCP (9,83,198). Quantitative touchdown PCR of the Msg locus in oral washes from HIV-infected patients has a sensitivity of 88% and a specificity of 85% (169).…”

Section: New Diagnostic Methodsmentioning

confidence: 99%

Colonization by Pneumocystis jirovecii and Its Role in Disease

2012

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SUMMARY Although the incidence of Pneumocystis pneumonia (PCP) has decreased since the introduction of combination antiretroviral therapy, it remains an important cause of disease in both HIV-infected and non-HIV-infected immunosuppressed populations. The epidemiology of PCP has shifted over the course of the HIV epidemic both from changes in HIV and PCP treatment and prevention and from changes in critical care medicine. Although less common in non-HIV-infected immunosuppressed patients, PCP is now more frequently seen due to the increasing numbers of organ transplants and development of novel immunotherapies. New diagnostic and treatment modalities are under investigation. The immune response is critical in preventing this disease but also results in lung damage, and future work may offer potential areas for vaccine development or immunomodulatory therapy. Colonization with Pneumocystis is an area of increasing clinical and research interest and may be important in development of lung diseases such as chronic obstructive pulmonary disease. In this review, we discuss current clinical and research topics in the study of Pneumocystis and highlight areas for future research.

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Section: New Diagnostic Methodsmentioning

confidence: 99%

Colonization by Pneumocystis jirovecii and Its Role in Disease

2012

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“…The detection of P. jirovecii organisms was performed by the amplification of the mtLSU rRNA gene by nested PCR (4,23). Parasite burden was quantified by scoring the number of cysts observed by applying indirect immunofluorescence with monoclonal antibodies (MonoFluo kit P. jirovecii; Bio-Rad, France) and designated low (no cysts identified but positive by PCR), moderate (1 to 3 cysts in 30 fields at ϫ1,000), and high (4 to 30 or more cysts in 1 field at ϫ1,000) (7).…”

Section: Methodsmentioning

confidence: 99%

Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

et al. 2011

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Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

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“…The result suggests that patients with higher Pneumocystis load in NPA specimens have a higher likelihood of true PCP rather than mere colonization. The use of upper respiratory tract specimens in the diagnosis of Pneumocystis infection has been evaluated in previous studies (18)(19)(20)(21)(22)(23)(24). Our study differs from these studies in several aspects.…”

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confidence: 95%

Use of Nasopharyngeal Aspirate for Diagnosis of Pneumocystis Pneumonia

Wong

et al. 2013

J Clin Microbiol

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Quantitative PCR on nasopharyngeal aspirate (NPA) can achieve high sensitivity and specificity in diagnosing Pneumocystis pneumonia (PCP) compared to microscopic examination of bronchoscopic specimens in a population with low HIV prevalence. Since NPA is a minimally invasive procedure, it is ideal as a screening test for PCP. C urrently, microbiological confirmation of Pneumocystis pneumonia (PCP) requires the identification of Pneumocystis cysts or trophozoites using microscopy. Bronchoscopic specimens are preferred because Pneumocystis resides mainly in the alveolar space (1). However, both transbronchoscopic bronchoalveolar lavage and sputum induction pose significant risk to the patient (2-4). Upper respiratory tract specimens are not recommended because of low sensitivity when traditional microscopic examination is used (5). PCR has higher sensitivity than microscopic examination in the detection of Pneumocystis (6) and may overcome the problem of low sensitivity associated with microscopic examination of upper respiratory tract specimens. In this study, we sought to determine whether nasopharyngeal aspirate (NPA) specimens could be used for the diagnosis of PCP.Patients of the Hong Kong West Cluster Hospitals with bronchoscopic specimens (bronchoalveolar lavage fluid [BALF] or bronchial aspirate) submitted to our laboratory for Pneumocystis examination from 1 January 2010 to 31 March 2012 were identified using the laboratory information system. In our laboratory, Pneumocystis cysts were visualized with methenamine silver stain by using standard procedures (7). With this method, Pneumocystis trophozoites or sporozoites could not be detected. Archived NPA specimens from these patients collected during the same hospitalization were retrieved. Only the earliest NPA specimen was tested if multiple specimens were available from the same patient. NPA specimens were collected as described previously (8). Clinical and laboratory data were obtained using the clinical management system. Patients without archived NPA specimens were excluded. A patient was considered to have definite PCP if Pneumocystis was identified in the bronchoscopic specimen by microscopic examination (9). This study has been approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.DNA was extracted from 200 l of NPA specimens and from other fungi using the QIAamp DNA kit (Qiagen, Hilden, Germany) and DNeasy plant minikit (Qiagen, Hilden, Germany), respectively, according to the manufacturer's instructions (10). Real-time quantitative PCR (qPCR) targeting a 124-bp fragment of the mitochondrial large subunit rRNA (mt LSU rRNA) gene of Pneumocystis jirovecii was performed as described previously (11). Plasmid suspensions were used as standards for quantification and positive controls (Fig. 1).We verified the positive results from the mitochondrial (mt) large-subunit (LSU) rRNA qPCR (LSU-qPCR) by a conventional PCR targeting the mitochondrial small-subunit rRNA (mt SSU rRNA) gene of P. jiro...

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Effect of Oral Washes on the Diagnosis of Pneumocystis carinii Pneumonia with a Low Parasite Burden and on Detection of Organisms in Subclinical Infections

Cited by 44 publications

References 16 publications

Colonization by Pneumocystis jirovecii and Its Role in Disease

Colonization by Pneumocystis jirovecii and Its Role in Disease

Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

Use of Nasopharyngeal Aspirate for Diagnosis of Pneumocystis Pneumonia

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