PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients

Pinlaor, Somchai; Mootsikapun, Piroon; Pinlaor, Porntip; Phunmanee, Anakapong; Pipitgool, Vichit; Sithithaworn, Paiboon; Chumpia, Worawan; Sithithaworn, Jiraporn

doi:10.1007/s00436-004-1200-y

Cited by 53 publications

(45 citation statements)

References 26 publications

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“…Of the positive samples, 13 were microscopy-positive and PCR-negative, whereas 24 samples were microscopy-negative and PCRpositive. Pinlaor et al (2004) compared Wright-Giemsa and Gomori's methenamine silver staining methods with PCR targeting 5S rRNA in 160 respiratory specimens. The incidence of P. jirovecii was 10 % by both microscopy methods and 15.6 % by PCR.…”

Section: Discussionmentioning

confidence: 99%

“…To diagnose PcP, various P. jirovecii genes were targeted by PCR, such as major surface glycoprotein (MSG) (Huang et al, 1999;Fischer et al, 2001;Helweg-Larsen et al, 2002;Larsen et al, 2002bLarsen et al, , 2004Flori et al, 2004;Linssen et al, 2006;Fillaux et al, 2008), dihydropteroate synthase (Demanche et al, 2001;AlvarezMartínez et al, 2006;Linssen et al, 2006;Jiancheng et al, 2009), dihydrofolate reductase (Schluger et al, 1991;Lu et al, 1995;Larsen et al, 2002a;Bandt & Monecke, 2007), internal transcribed spacer regions of the rRNA (Lu et al, 1995;Torres et al, 2000), mitochondrial large subunit rRNA (mtLSU rRNA) (Wakefield et al, 1990;Leibovitz et al, 1995;Lu et al, 1995;Wakefield, 1996;Huang et al, 1999;Kaiser et al, 2001;Olsson et al, 2001;Helweg-Larsen et al, 2002;Flori et al, 2004;Gupta et al, 2009;Jiancheng et al, 2009;Choukri et al, 2010), mitochondrial smallsubunit rRNA (Hunter & Wakefield, 1996;Helweg-Larsen et al, 2002), 5S rRNA (Kitada et al, 1991;Wakefield et al, 1991;Lu et al, 1995;Ribes et al, 1997;Sandhu et al, 1999;Pinlaor et al, 2004), 18S rRNA …”

Section: Introductionmentioning

confidence: 99%

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Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Döşkaya

Caner

Değirmenci

et al. 2011

Journal of Medical Microbiology

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Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Döşkaya

Caner

Değirmenci

et al. 2011

Journal of Medical Microbiology

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“…However, lack of standardization and clinical validation of the laboratory-developed assays is the primary reason why the EORTC/MSG group decided to omit all fungal PCR from the definitions of invasive fungal diseases (7). In addition, several papers attest to the fact that PCR diagnosis of PCP is more sensitive than microscopy (4,10,11,13,22,24). The generally higher sensitivity of PCR assays than of microscopy may be disproportionately important for non-AIDS patients and may result in fewer missed clinical diagnoses.…”

mentioning

confidence: 99%

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay

et al. 2011

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Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations.Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, <3.5 copies/l of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

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“…TBO: azul de toluidina O; PM: metenamina de plata; IFD: inmunofluorescencia directa Las razones mencionadas no parecen afectar la inmunofluorescencia directa para detectar P. jirovecii. Además, esta técnica permite identificar casos adicionales porque los anticuerpos monoclonales dirigidos contra los antígenos de superficie de Pneumocystis, detectan tanto formas quísticas como formas tróficas (éstas constituyen las formas más abundantes al inicio de la neumonía), mientras que el azul de toluidina O identifica sólo las formas quísticas (29,30,(41)(42)(43). Asimismo, debido a que la especificidad de la reacción inmunitaria resalta las estructuras del hongo en la placa, su lectura es más sencilla que la del azul de toluidina O (44).…”

Section: Inmunofluorescencia Directa Enunclassified

Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumonía

Rodiño

Rincón²,

Aguilar

et al. 2011

biomedica

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Introducción. El diagnóstico de neumonía por Pneumocystis jirovecii se fundamenta en la visualización microscópica del hongo en secreciones respiratorias. Técnicas moleculares recientes también lo han detectado en muestras de orofaringe, pero su utilidad diagnóstica es discutible. En Colombia, hay poca información al respecto. Objetivo. Comparar el rendimiento de dos coloraciones, azul de toluidina O e inmunofluorescencia directa, en muestras de lavado broncoalveolar y lavado orofaríngeo en pacientes inmunocomprometidos con neumonía. Materiales y métodos. Se llevó a cabo un estudio transversal de evaluación de pruebas diagnósticas en 166 pacientes inmunocomprometidos con sospecha de neumonía por P. jirovecii. Por protocolo, las muestras de lavado broncoalveolar y orofaríngeo se citocentrifugaron y se colorearon con azul de toluidina e inmunofluorescencia. Se determinó la proporción de resultados positivos con ambas tinciones en cada una de las muestras, y la concordancia entre ellas. Resultados. Se detectaron 24 casos de neumonía por P. jirovecii en las muestras de lavado broncoalveolar (14,5 %), 21 de los cuales fueron positivos por ambas pruebas, mientras que tres casos se detectaron sólo por inmunofluorescencia. Ninguna de las 166 muestras de lavado orofaríngeo fue positiva por cualquiera de estas técnicas. Al comparar las proporciones de resultados positivos, no se encontraron desacuerdos significativos (p=0,63). La concordancia (coeficiente kappa) entre ellas fue de 0,92 (IC 95% : 0,84-1). Conclusiones. Ambas coloraciones son útiles para diagnosticar neumonía por P. jirovecii en muestras de lavado broncoalveolar. Sin embargo, el azul de toluidina no detecta, aproximadamente, el 12 % de los casos positivos por inmunofluorescencia. Las muestras de lavado orofaríngeo no son apropiadas para detectar microscópicamente P. jirovecii.Palabras clave: neumonía por Pneumocystis/diagnóstico, técnica del anticuerpo fluorescente directa, cloruro de tolonio, orofaringe, lavado broncoalveolar, huésped inmunocomprometido. Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumoniaIntroduction. The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests. Objective. The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia. Materials and methods. Cross-sectional evaluation diagnostic tests were used in 166 immunossupressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were st...

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PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients

Cited by 53 publications

References 26 publications

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay

Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumonía

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