Detection of Pneumocystis carinii DNA in blood by PCR is not of value for diagnosis of P. carinii pneumonia.

A nested PCR which amplified a portion of the mitochondrial large-subunit rRNA gene of Pneumocystis carinii was used to detect P. carinii DNA in blood from patients with P. carinii pneumonia. P. carinii DNA was not detected in serum and was detected at low levels of blood cells.     

Monoclonal immunofluorescence compared with silver stain for investigating Pneumocystis carinii pneumonia.

Two hundred and eighty two specimens from 220 patients positive for HIV with respiratory tract symptoms, or febrile illness, or both, were examined for the presence of Pneumocystis carinii. Specimens were either induced sputum samples or bronchoalveolar lavage fluids. To establish the optimal method for laboratory diagnosis a comparison was made of detection of the organism by use of monoclonal antibody and immunofluorescence with conventional silver staining methods. Three commercially available reagents for immunofluorescence were also compared. Immunofluorescence was significantly more sensitive than the silver stain and the best results for immunofluorescence were obtained using. Northumbria Biologicals Ltd reagents.     

Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups.

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.     

A search for Pneumocystis carinii in post-mortem lungs by DNA amplification.

DNA amplification of specific sequences and subsequent oligonucleotide hybridization were used to search for Pneumocystis carinii in post-mortem lung samplings from non-immunosuppressed individuals ranging from 15 to 70 years of age. No P. carinii-specific DNA was detected in 45 DNA amplification reactions from 15 lungs.     

Granulomatous Pneumocystis carinii pneumonia: DNA amplification studies on bronchoscopic alveolar lavage samples.

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.     

Usefulness of PCR for detection of Pneumocystis carinii DNA.

Diagnosis of Pneumocystis carinii pneumonia is based on the identification of the various stages of the parasite in lung samples by standard staining techniques. We therefore assessed the value of the PCR for detection of P. carinii in bronchoalveolar lavage, induced sputum, and blood samples relative to that of standard staining techniques.     

环介导等温扩增技术检测卡氏肺孢子虫的研究

目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论 检测Pc的LAMP方法敏感、特异及简便.     

Pneumocystis carinii pneumonia in thoroughbred foals: identification of a genetically distinct organism by DNA amplification.

Genetically distinct forms of Pneumocystis carinii infect several mammalian hosts. We report the amplification of P. carinii DNA from samples of two infected thoroughbred foal lungs by using primers designed from the sequence of a P. carinii mitochondrial rRNA gene; these primers also prime the amplification of P. carinii DNA from other hosts. The nucleotide sequence of part of the mitochondrial rRNA gene amplified from P. carinii infecting one of the foals was determined and found to be distinct from that of published rat-, rabbit-, ferret-, and human-derived P. carinii sequences.     

Pneumocystis carinii pneumonia. An approach to rapid laboratory diagnosis.

Laboratory procedures used to establish the diagnosis of Pneumocystis carinii pneumonia were evaluated using an experimental murine model. Touch preparations and suspension smears were prepared from lung tissue know to contain Pneumocystis cysts. These preparations were stained by a variety of methods known to demonstrate either cyst forms or sporozoites and trophozoites. Suspension smears proved to be superior to touch preparations in terms of cyst content and homogeneity of staining. Also, methods that stain cyst forms were superior to those that stain sporozoites and trophozoites for location and identification of organisms. The authors believe that suspension smears prepared from lung tissue and stained with toluidine blue O should be examined initially as a rapid screening method for Pneumocystis cysts. When the results of this initial screen are negative or inconclusive, additional suspension smears stainded by the modified Gomori methenamine silver nitrate method should be examined, pending availability of histologic sections.     

Acridine orange staining of Pneumocystis carinii.

Acridine orange was used to stain smears of mouse lung which contained cyst and trophozoite forms of Pneumocystis carinii. Trophozoite forms stained yellow to orange; however, cyst forms did not stain. Acridine orange is a rapid and sensitive method for demonstrating trophozoites of P. carinii in mouse lung tissue.     

Laboratory diagnosis of Pneumocystis carinii: comparison of cytological, immunofluorescent and immunocytochemical staining]

Authors review several methods for diagnosis of Pneumocystis carinii on BAL fluid in AIDS patients aiming to identify ideal routine technic. Giemsa, Gomori and Toluidine Blue staining. Direct and Indirect Immunofluorescence. Immunocytochemical methods were tested and advantages/disadvantages compared on BAL fluid from AIDS patients. Pneumocystis carinii was detected in 7 out 21 cases (33%). Gomori staining and Toluidine Blue staining were chosen for routine Pneumocystis carinii detection while IF technics are deserved as very useful in fields showing high fungal ++ contamination (inducted sputum).     

Giemsa staining for cysts and trophozoites of Pneumocystis carinii.

Although Giemsa staining has been routinely used for the detection of trophozoites and intracystic bodies in smears of bronchoalveolar lavage fluid (BAL) from patients with Pneumocystis carinii pneumonia, it does not normally stain the cyst wall. For detection of the cysts other stains such as toluidine Blue 'O' and methenamine silver must be used as well. Sulphation of smears before staining with Giemsa allows cysts to be visualised, thus enabling a single stain to be used to show all the stages of BAL or sputum, which is particularly useful, considering the increase in the prevalence of P carinii pneumonia in conjunction with the spread of AIDS.     

Rapid diagnosis of Pneumocystis carinii infection in AIDS by cytocentrifugation and rapid hematoxylin-eosin staining.

Pneumocystis carinii pneumonia (PCP) is the major pulmonary complication in patients with the acquired immunodeficiency syndrome. While fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsy (TBBX) is regarded as the procedure of choice to identify PCP, these techniques, particularly TBBX, pose potential risks to the already compromised patient. To reduce the duration of bronchoscopy and, hence, lessen the chance for complications, we describe a rapid technique to identify PCP in BAL fluid by cytocentrifugation and hematoxylin-eosin staining. By this method, PCP can be easily diagnosed within 10 min. The sensitivity of this rapid diagnostic procedure is 95.7%, and the specificity is 100%.     

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia

Objective   To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii .
Methods   The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR.
Results   Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample.
Conclusions   IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii .     

Pathological characteristics for the diagnosis of Pneumocystis carinii pneumonia. A retrospective autopsy study

The clinical records of 63 patients who during the period 1980-1983 showed pneumocysts in lung specimen imprints obtained at autopsy have been reviewed in order to establish possible characteristics for the diagnosis of Pc pneumonia. Autopsies were performed at 2 Copenhagen hospitals, Rigshospitalet and the Finsen Institute. The 63 Pc-positive cases included 9 with blood diseases, 27 with solid tumors, 5 with immunological disorders and 22 with other diseases. The characteristic extensive changes in the lungs in Pc pneumonia included increased firmness, dark red/brown or black/grey colour of sectioned areas, stasis and edema sometimes excessive, and foci of white/grey or red/brown areas giving the lung a marbled or speckled appearance. The content of air was always decreased. The microscopic changes reported included intra-alveolar eosinophilic material, an intra-alveolar transudate containing macrophages and very few neutrophilic granulocytes, dilated capillary tubes, stasis and edema, thickened alveolar septae and peri-alveolar or diffuse fibrosis. The chest X-rays showed no specific features, and the infection had been subclinical in all cases; the only evidence of Pc infection was the demonstration of pneumocysts in imprints of lung specimens stained with toluidine blue in combination with observations during pathology. The diagnosis depends to a great extent upon a keen alertness in addition to the information given in the clinical record.     

Improved rat model for studying Pneumocystis carinii pneumonia.

Sprague-Dawley rats treated for 8 weeks with cortisone acetate (25 mg per rat twice weekly) were immunosuppressed to variable degrees. A total of 55% lost over 12% of their initial body weight, had cortisol concentrations in serum more than five times greater than those of the controls, and had markedly depressed ratios of helper to non-helper T cells, in both the spleen and peripheral blood. Animals that gained weight during immunosuppression had cortisol concentrations in serum only three times higher than those of the controls, had normal ratios of helper to non-helper T cells in the spleen, and had only modestly reduced T-cell ratios in peripheral blood. The degree of Pneumocystis pneumonia was evaluated in impression smears and sections of lungs taken from immunosuppressed rats. Pneumocystis infections were more severe in the rats that showed the greatest weight loss. Weight change during immunosuppression may therefore be used as a reliable means for predicting the degree of Pneumocystis infection in living rats. This protocol allows the selection of uniformly infected rats for studies assessing drug therapy of Pneumocystis pneumonia.     

Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers

The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.