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.     

Comparison of PCR and standard cytological staining for detection of Pneumocystis carinii from respiratory specimens from patients with or at high risk for infection by human immunodeficiency virus.

The detection of Pneumocystis carinii DNA by PCR was compared with routine cytologic staining techniques (CYT). A total of 284 clinical respiratory specimens, including 137 bronchoalveolar lavage (BAL), 63 bronchoalveolar washing, 63 sputum, and 21 induced sputum samples, obtained from patients with or at high risk for human immunodeficiency virus infection were evaluated. Eighty specimens were positive by PCR, and 69 were positive by CYT. PCR was able to detect P. carinii in more bronchoalveolar washing specimens (15 versus 11) and in comparable BAL specimens (53 versus 54) compared with CYT. PCR was particularly more sensitive than CYT in detecting P. carinii in expectorated sputum (12 versus 4 samples). Of the 19 patients whose respiratory specimens were positive for P. carinii by PCR but negative by CYT, 5 had P. carinii pneumonia (PCP) confirmed by subsequent BAL and transbronchial or mediastinal lymph node biopsy and 9 had a clinical course highly suggestive of acute PCP. Eleven (58%) of the 19 patients with discordant PCR and CYT results had received prior anti-PCP prophylaxis. In this clinical setting in particular and in the evaluation of sputum specimens, the ability of PCR to detect a low parasitic load suggests that this technique may become an important additional tool, along with current cytological methods, for the detection of P. carinii.     

The use of mucolysed induced sputum for the identification of pulmonary pathogens associated with human immunodeficiency virus infection

We describe a system for diagnosis of pulmonary disease in the human immunodeficiency virus-infected patient using induced sputum and other diagnostic procedures. This system has been successfully used at San Francisco (Calif) General Hospital for more than 2 years. It utilizes outpatient facilities and reduces the need for bronchoscopy. Sputum induced by inhalation of 3% saline mist, mucolysed, concentrated by centrifugation, and stained by a rapid modified Giemsa stain was the first diagnostic specimen examined in 404 episodes of suspected human immunodeficiency virus-associated pulmonary disease in 358 patients. Pneumocystis carinii was found in 222 (55%) sputum specimens. In 118 episodes in which the sputum did not contain P carinii, bronchoscopy with transbronchial biopsy and/or bronchoalveolar lavage was performed and P carinii was found in 50 (42%). These 118 bronchoscopy results, as well as evaluation of the subsequent clinical course of those patients who accounted for 64 episodes of lung disease and who did not have bronchoscopy following examination of nondiagnostic induced sputum, indicated a range of sensitivity for detection of P carinii in induced sputum of 74% to 77% and a negative predictive value of 58% to 64%. Mycobacteria were recovered from 11 (6%) of the induced sputum and 6 (12%) of the bronchoscopic specimens containing P carinii. However, only oral or environmental fungi were recovered from P carinii-containing induced sputum or bronchoscopic specimens. For those patients in whom P carinii was not detected, only the bronchoscopic specimens were cultured for Mycobacteria and fungi. Potentially pathogenic Mycobacteria and fungi were recovered from 16 (23.5%) and 34 (50%), respectively, of these P carinii-negative specimens. Analysis of these results, obtained under routine practice conditions, indicates that bronchoscopy should be reserved for those patients whose induced sputum examinations do not show P carinii and that mycobacterial and fungal cultures be performed only on bronchoscopic specimens in which P carinii is not detected.     

Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens.

Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.     

PCR detection of Pneumocystis carinii in bronchoalveolar lavage specimens: analysis of sensitivity and specificity.

Although PCR detection of Pneumocystis carinii DNA has been described, little is known about the sensitivity or specificity of the assay in routine laboratory practice. We had the unique opportunity to use a bronchoalveolar lavage (BAL) specimen bank with samples for which the direct examination results for P. carinii were known. DNA purified from 129 selected specimens was amplified by using the primers described previously (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moton, and J. M. Hopkin, Mol. Biochem. Parasitol. 43:69-76, 1990). Of the 129 specimens, 37 were positive for P. carinii by direct examination. All 37 specimens were positive for P. carinii by PCR, yielding a 100% sensitivity and 100% negative predictive value for the assay. An additional 23 specimens were repeatedly positive for P. carinii by PCR but were not positive by direct examination. Review of the patient charts for these specimens with discordant results demonstrated that five of the patients were actually positive for P. carinii, as determined by either biopsy or examination of repeat or prior BAL specimens. A response to empiric therapy for P. carinii pneumonia was seen in an additional two patients. Of the remaining specimens, 8 produced no significant isolates other than P. carinii, while 12 contained culture-confirmed significant respiratory pathogens in addition to P. carinii (two fungal, nine bacterial, and one viral pathogen). Cytomegalovirus, which was of unknown significance, was isolated from 16 additional specimens. Overall, the specificity of the PCR assay was 79.3% compared to the results of direct examination. We hypothesized that the apparently poor specificity of the PCR assay was due to the increased sensitivity of the assay compared to that of direct examination. The sensitivity of the PCR assay was therefore assessed with BAL specimens containing P. carinii cysts. Serial dilutions of this preparation were evaluated by direct examination and PCR. PCR was found to be 100-fold more sensitive than direct examination, which detected one to two cysts per amplification. No false-positive results were detected in controls containing no DNA or by using target DNA from various fungal, viral, or bacterial respiratory pathogens. We conclude that PCR detection of P. carinii in BAL specimens is very sensitive and should be considered for patients whose specimens do not yield a diagnosis. The increased sensitivity of the PCR assay may help to identify those patients with low-titer infections who might benefit from directed antibiotic therapy for P. carinii and would otherwise be missed by direct examination alone.     

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.     

Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences.

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.     

Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system.

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.     

Development and evaluation of a rapid and simple procedure for detection of Pneumocystis carinii by PCR.

We report the development of a simplified PCR-based assay for the detection of Pneumocystis carinii DNA in clinical specimens. The adoption of a rapid DNA extraction procedure and the introduction of a type of enzyme-linked immunosorbent assay for PCR product detection enabled this procedure to be carried out in a single working day in a clinical microbiology laboratory. The PCR assay was prospectively compared with an immunofluorescent-antibody (FA) staining method for the detection of P. carinii in induced sputum and bronchoalveolar lavage (BAL) specimens. The results of the study showed that, for induced sputum specimens, FA staining had a sensitivity of 78% (32 of 41 specimens) and a specificity of 100% (166 of 166 specimens); PCR was 100% (41 of 41 specimens) sensitive and 98% (162 of 166 specimens) specific. For BAL specimens, FA staining was 100% sensitive (21 of 21 specimens) and 100% specific (133 of 133 specimens), and PCR had a sensitivity of 100% (21 of 21 specimens) and a specificity of 99% (132 of 133 specimens). These results strongly suggest that use of our PCR-based assay could effect clinically useful improvements in the sensitivity of induced sputum specimens for the detection of P. carinii.     

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

Detection of Pneumocystis carinii sequences by polymerase chain reaction: animal models and clinical application to noninvasive specimens.

Pneumocystis carinii is a eukaryotic microbe which causes fatal pneumonia in patients with AIDS. Oligonucleotide primers were used to amplify the 5S rDNA sequence of P. carinii by the polymerase chain reaction (PCR) in various clinical and animal samples. Of 35 independent lung specimens tested, PCR detected the P. carinii sequence in all 23 cases which were known to be P. carinii infected, i.e., 15 from mice, 1 from rat, 3 from human autopsy, and 4 from biopsy of AIDS patients by needle aspiration. The results were consistent with clinical and microscopic diagnosis. The detection was highly sensitive and specific. Direct sequencing of these amplified DNAs revealed homogeneity of 5S rDNA sequences of independent isolates from mice, rats, and humans. Preliminary trials manifested efficacy of the PCR method to detect P. carinii sequences in induced sputum or blood from AIDS patients, the latter case suggesting that P. carinii might enter peripheral blood via phagocytosis or direct intrusion. Development of less-invasive or noninvasive PCR diagnostic techniques to detect P. carinii infection would greatly facilitate therapeutic and prophylactic management of P. carinii pneumonia.     

Detection of Pneumocystis carinii in respiratory specimens by PCR-solution hybridization enzyme-linked immunoassay.

By using a recently developed PCR-solution hybridization enzyme-linked assay (PCR-SHELA), we investigated Pneumocystis carinii in bronchoalveolar lavage fluid samples and induced sputa of patients with pneumocystosis. In detecting P. carinii, PCR-SHELA proved more sensitive than immunofluorescence staining or a single PCR and significantly more diagnostically specific than a nested PCR. Our data suggest that PCR-SHELA could be used to detect P. carinii organisms in respiratory samples, particularly in patients with uncertain diagnoses.     

Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification

A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.     

mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。     

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 .     

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.     

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.     

Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens.

Three monoclonal antibody staining kits, from Genetic Systems, Disease Detection International, and Meridian Diagnostics, were compared with calcofluor white for the direct detection of Pneumocystis carinii in respiratory specimens. Of the 150 specimens tested, 23 were found positive for P. carinii by any of the four stains; 13 were bronchoalveolar lavage, 7 were induced sputum and 3 were expectorated sputum specimens. All stains detected the positive bronchoalveolar lavage specimens, the Genetic Systems stain detected six induced sputum specimens, and the other stains each detected four induced sputum specimens. The monoclonal antibody stains detected all three expectorated specimens, while the calcofluor stain detected only one. Overall, the sensitivities of the stains were 96% for Genetic Systems, 87% for both Disease Detection International and Meridian Diagnostics, and 78% for calcofluor.     

Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients with Pneumocystis carinii pneumonia.

A nested PCR, amplifying a portion of the gene encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) of Pneumocystis carinii sp. f. hominis was applied to oropharyngeal samples obtained on repeated occasions from 12 HIV-infected patients with P. carinii pneumonia (PCP) to monitor response to anti-P. carinii treatment. Genotyping of P. carinii sp. f. hominis was also performed on paired samples of oropharyngeal and broncho-alveolar lavage samples before the start of treatment, and on oropharyngeal samples during the course of treatment, by analysis of sequence variation at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon. When a simple dilutional method was used, a reduction in the amount of amplification product was observed in samples from all patients during the course of treatment. In eight of the 12 patients, a single ITS sequence type was found in the oropharyngeal samples and also in the paired broncho-alveolar lavage sample. A mixed infection was identified in the samples from three patients. In eight patients, the ITS sequence types identified in the oropharyngeal sample were the same as in the broncho-alveolar lavage sample. Nested PCR amplifying the mt LSU rRNA on oropharyngeal samples provides a non-invasive method of monitoring response to treatment of PCP. ITS sequence typing of P. carinii sp. f. hominis from oropharyngeal samples appears to be a reliable alternative to broncho-alveolar lavage samples and provides a non-invasive tool for further epidemiological studies.     

Identification of Pneumocystis carinii f. sp. hominis Gene Sequences in Filtered Air in Hospital Environments

To evaluate the risk of a nosocomial spread of Pneumocystis carinii f. sp. hominis (P. carinii hominis), air filter samples from rooms of P. carinii pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of P. carinii hominis. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the P. carinii hominis genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used. P. carinii hominis DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The P. carinii hominis genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of P. carinii hominis from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of P. carinii hominis from the environment.