Oropharyngeal samples for detection of Pneumocystis carinii by DNA amplification
Pneumocystis carinii pneumonia is a major complication of T-lymphocyte immune deficiency. Restriction of the disease to the alveolar spaces and failure to culture R. carinii has hindered simple diagnostic methods. We have developed a specific DNA amplification method for P. carinii and shown diagnostic sensitivity and specificity exceeding 95% for pneumocystis pneumonia when applied to bronchoscopic lavage and hypertonic saline induced sputum. We here report application of DNA amplification to simple oropharyngeal samples in 31 HIV-positive patients with respiratory illness. P. carinii-specific DNA was detected in 10 of 18 (56%) patients with pneumocystis pneumonia by ethidium bromide stained gels and 14 of 18 (78%) patients by the more sensitive technique of oligoblotting. P. carinii DNA was not detected in samples from 13 patients with other respiratory diagnoses. An oropharyngeal sample offers a simple specimen for detecting P. carinii by DNA amplification; refinements of technique and calibration may allow its development for accurate diagnostic and epidemiological work.
Available from: Brenda Morrow
- "The development of polymerase chain reaction (PCR) techniques has provided a more reliable diagnostic method. In adult studies, PCR is as specific as and more sensitive than microscopy for diagnosis when performed on respiratory specimens, including oral washes [7,11-21]. In a study of oropharyngeal washes from HIV-infected adult patients, P. jirovecii DNA-amplification had a sensitivity of 44% using a nested PCR protocol compared to trans-bronchial biopsy,  increasing to 90% when touch-down real-time PCR was utilised [7,11]. "
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ABSTRACT: Pneumocystis pneumonia (PCP) is a major cause of hospitalization and mortality in HIV-infected African children. Microbiologic diagnosis relies predominantly on silver or immunofluorescent staining of a lower respiratory tract (LRT) specimens which are difficult to obtain in children. Diagnosis on upper respiratory tract (URT) specimens using PCR has been reported useful in adults, but data in children are limited. The main objectives of the study was (1) to compare the diagnostic yield of PCR with immunofluorescence (IF) and (2) to investigate the usefulness of upper compared to lower respiratory tract samples for diagnosing PCP in children.
Children hospitalised at an academic hospital with suspected PCP were prospectively enrolled. An upper respiratory sample (nasopharyngeal aspirate, NPA) and a lower respiratory sample (induced sputum, IS or bronchoalveolar lavage, BAL) were submitted for real-time PCR and direct IF for the detection of Pneumocystis jirovecii. A control group of children with viral lower respiratory tract infections were investigated with PCR for PCP.
202 children (median age 3.3 [inter-quartile range, IQR 2.2 - 4.6] months) were enrolled. The overall detection rate by PCR was higher than by IF [180/349 (52%) vs. 26/349 (7%) respectively; p < 0.0001]. PCR detected more infections compared to IF in lower respiratory tract samples [93/166 (56%) vs. 22/166 (13%); p < 0.0001] and in NPAs [87/183 (48%) vs. 4/183 (2%); p < 0.0001]. Detection rates by PCR on upper (87/183; 48%) compared with lower respiratory tract samples (93/166; 56%) were similar (OR, 0.71; 95% CI, 0.46 - 1.11). Only 2/30 (6.6%) controls were PCR positive.
Real-time PCR is more sensitive than IF for the detection of P. jirovecii in children with PCP. NPA samples may be used for diagnostic purposes when PCR is utilised. Wider implementation of PCR on NPA samples is warranted for diagnosing PCP in children.
BMC Infectious Diseases 11/2011; 11(1):329. DOI:10.1186/1471-2334-11-329 · 2.61 Impact Factor
Available from: Valeria Micheli
- "Oropharyngeal samples. The application of PCR techniques on oropharyngeal samples was originally proposed with mt-rRNA primers by Wake¢eld et al. . The application of ITS nested PCR on garglings showed a moderate/high sensitivity (74.7%) and high speci¢city. "
FEMS Immunology & Medical Microbiology 10/1998; 22(1-2):93-5. DOI:10.1111/j.1574-695X.1998.tb01192.x · 3.08 Impact Factor
Available from: bmj.com
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ABSTRACT: Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.
Journal of Clinical Pathology 08/1994; 47(7):664-6. DOI:10.1136/jcp.47.7.664 · 2.92 Impact Factor
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