Pneumocystis carinii diagnosis: an update.

From 1994 to date we have been using the internal transcribed spacers (ITSs) nested polymerase chain reaction (PCR) to investigate the possibility of diagnosing Pneumocystis carinii pneumonia on non-invasive samples collected from HIV-positive patients with pulmonary involvement. The objectives were: (1) to test the sensitivity, specificity and prognostic value of PCR in diagnosis and follow up of PCP; (2) to investigate the eventual occurrence and role of asymptomatic carriers of P. carinii; (3) to evaluate the prognostic significance of blood PCR positivity versus respiratory samples; (4) to verify the occurrence of exogenous infections or endogenous reactivations in cases of recurrent P. carinii pneumonia; and (5) to study the possible correlation between P. carinii genotype identified and capability of blood dissemination, prior prophylactic treatments, clinical parameters and outcome of the patients.

In vitro activity of human immunodeficiency virus protease inhibitors against Pneumocystis carinii.

Since 1996, the introduction of protease inhibitors (PIs) has led to a dramatic decrease of human immunodeficiency virus-related Pneumocystis carinii pneumonia. This effect is clearly due, in large part, to the induction of immune reconstitution by highly active antiretroviral therapy (HAART). However, it is conceivable that PIs had other beneficial effects, including direct activity against Pneumocystis. In this study, the occurrence of specific aspartyl proteases in Pneumocystis is described. These protease targets seemed to be affected in vitro by antiretroviral PIs. These data suggest intriguing implications for the possible antipneumocystis benefit of receiving indinavir, ritonavir, nelfinavir, or saquinavir during HAART.

Detection of rat Pneumocystis carinii proteinases and elastase and antipneumocystis activity of proteinase inhibitors in vitro.

Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.

In vitro drug assays and statistical analysis.

The development of in vitro drug tests to assess the efficacy of drugs against Pneumocystis carinii has been hindered by the lack of efficient methods for continuous cultivation of the microorganism. However, different short-term culture systems have been proposed by many teams. In the present contribution an in vitro microplate drug assay and two statistical programs allowing the analysis of results are presented.

Combined use of blood and oropharyngeal samples for noninvasive diagnosis of Pneumocystis carinii pneumonia using the polymerase chain reaction.

To evaluate the clinical use of a polymerase chain reaction (PCR) assay for diagnosis of Pneumocystis carinii pneumonia (PCP) using samples collected noninvasively, the Internal Transcribed Spacers (ITSs) nested PCR was performed on 148 samples from 40 subjects. Bronchoalveolar lavage (BAL) fluid sera, gargled oropharyngeal washes, and peripheral blood mononuclear cells (PBMC) from 14 AIDS patients (mean age, 35.6 years; mean CD4+ cell count, 49.2 cells/mm3) with proven PCP and from 13 HIV-seropositive controls (mean age, 34.6 years; mean CD4+ cell count, 107.3 cells/mm3) with other AIDS-related opportunistic infections were evaluated. Sera and oropharyngeal samples were also collected from 13 HIV-seronegative health care personnel working in an infectious disease ward for use as negative controls. The ITSs nested PCR confirmed the morphological diagnosis of PCP in all patients when BAL fluid was tested (100% sensitivity). This technique also detected Pneumocystis carinii DNA in oropharyngeal samples from 78.6% of patients, in sera from 71.4% of patients, in PBMC from 35.7% of patients. When all results obtained after ITSs nested PCR were considered together for the same patient, the sensitivity for PCP diagnosis was 100% for blood and oropharyngeal samples (gargled saline), as confirmed by subsequent BAL. All samples collected noninvasively from 26 of 26 controls were negative using ITSs nested PCR (100% specificity).

Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome.

OBJECTIVES: To verify the clinical value of two different polymerase chain reactions (PCRs) for noninvasive diagnosis and follow-up during Pneumocystis carinii f. sp. hominis pneumonia (PCP) and to analyze the P. carinii f. sp. hominis genotypes involved. METHODS: Internal transcribed spacers (ITSs) nested PCR was applied to 630 samples (bronchoalveolar lavage, sera, peripheral blood mononuclear cells, and oropharyngeal samples) from 122 patients with acquired immunodeficiency syndrome and pneumonia and 40 control samples from 20 subjects seronegative for human immunodeficiency virus. One hundred and eighty samples also were examined by mt-rRNA PCR. Bronchoalveolar lavage samples and 33 sera were analyzed by type-specific oligonucleotide hybridization. RESULTS: On bronchoalveolar lavage samples, the two PCRs consistently confirmed the morphologic diagnosis of PCP. The sensitivity of ITSs nested PCR versus mt-rRNA PCR was 57.3% versus 14.3% on sera, 32.3% versus 22. 8% on peripheral blood mononuclear cells, and 69.1% versus 48.6% on oropharyngeal samples (garglings). Both PCRs had 100% specificity. Type-specific oligonucleotide hybridization revealed in 72.2% of bronchoalveolar lavage samples a single P. carinii f. sp. hominis genotype, whereas in 27.8% co-infection with more than one strain was detected. CONCLUSION: On noninvasive samples, ITSs nested PCR was more sensitive than mt-rRNA PCR, and it confirmed the diagnosis in all patients with PCP. For each patient with PCP at least one noninvasive sample was positive for P. carinii f. sp. hominis DNA.

ITSs genotypes and blood dissemination during acute PCP.

The presence of P. carinii DNA in serum and in Peripheral Blood Mononuclear Cells (PBMC) during acute phase of PCP in AIDS patients was previously demonstrated by several authors using different specific primers. Amplification by ITSs nested PCR followed by TSO hybridization of P. carinii isolates derived from BAL and blood samples allows to compare genotypes involved in the disease and genotype-related dynamics of Pc-DNA clearance from blood during therapy. Different virulence characteristics among P. carinii genotypes could explain the various spectrum of clinical presentation (pulmonary and extrapulmonary) and susceptibility to classic antipneumocystic drugs during PCP.

Algorithmic analysis of in vitro chemotherapy against Pneumocystis carinii.

The development of in vitro experimental chemotherapy against P.carinii has been hindered by the lack of efficient methods for continuous cultivation of the microorganism. Various short-term systems, allowing the production of infectious forms of Pneumocystis, can be employed for in vitro experimental chemotherapy. The purpose of the present study is to describe a statistically relevant algorithmic model for the evaluation of P.carinii in short-term in vitro culture and drug screening.

Diagnosis of PCP by ITSs nested PCR on notinvasive oropharingeal samples.

Pneumocystis carinii pneumonia is an opportunistic disease usually affecting immunocompromised hosts. Diagnosis is based on the microscopical detection of microrganism on BAL, an invasive sample which requires pt compliance for collection and skilled health care personnel for examination. In order to verify the possible diagnostic utility of noninvasive specimens collected in the upper respiratory tract we examined by the Internal Transcribed Spacers (ITSs) nested PCR, 39 oropharingeal samples (garglings) collected from 20 HIVAb positive pts and from 15 healthy controls.

P.carinii DNA detected by ITSs nested PCR in serum and PBMC of AIDS patients with PCP.

Pneumocystis carinii pneumonia (PCP) is usually diagnosed by examination of BAL, a sample often unpleasant to be collected from immunocompromised host affected by acute respiratory disease. We studied by the Internal Transcribed Spacers (ITSs) nested PCR the presence of P.carinii DNA in serum and Peripheral Blood Mononuclear Cells (PBMC) during acute episodes of PCP to test blood as a possible noninvasive diagnostic tool.

ITSs typing of P.carinii samples from Italy, The Netherlands and Tanzania.

P.carinii molecular epidemiology appears a new interesting investigational field to understand distribution and incidence of isolates from different geographical locations. Recently a typing system, the Type Specific Oligoblotting (TSO) based on 6 different sequences of the Internal Transcribed Spacers (ITSs) of P.carinii rRNA has been developed [1]. By using P.carinii ITSs nested PCR followed by TSO hybridization we have typed 55 lung derived specimens collected in Italy, The Netherlands and sub-Saharian Africa from pts with microscopically detected P.carinii pneumonia.

Hemorrhagic infarction: risk factors, clinical and tomographic features, and outcome. A case-control study.

A radiological diagnosis of hemorrhagic infarction (HI) was made in 41 of 2726 cases with cerebrovascular lesions (1.9%). The clinical records of the cases and those of 82 age- and gender-matched subjects with ischemic infarction were examined, and notes of the principal risk factors of cerebrovascular disorders, the clinico-radiologic features and the outcome of the disease were taken for comparison. Cardiac sources of emboli (atrial fibrillation, native or prosthetic valve disorders, recent myocardial infarction) were present in 44% of cases and in 24% of controls. Diabetes mellitus was recorded in 31% and 18% respectively. Thirteen percent of cases and 35% of controls gave a history of transient ischemic attacks. Stupor or coma during the acute phase and a more severe course were more common among cases. In general, HIs were significantly larger than ischemic infarcts, with mass-effect, although the size of the lesion did not seem to be related to the presence of cardiogenic embolism.

Cranial computed tomography findings in children with acute lymphoblastic leukemia at diagnosis.

Forty children newly diagnosed with acute lymphoblastic leukemia (ALL) were examined by computed tomography (CT) of the central nervous system (CNS) on hospital admission before any medication was started. The results of the CT scans were defined as normal, borderline (slight or moderate dilatation of the ventricular system and/or basal cisterns and/or convolutional sulci), or pathologic (severe cerebral atrophy). The mean age of the patients was 5.8 years (range 1.7-15 years). Sixteen of the 40 patients (40%) had CT scan abnormalities with 14 patients having borderline scans and two patients pathologic scans. No child presented with neurologic symptoms or CNS leukemia. These data suggest that CT abnormalities of the brain are common in children with ALL at diagnosis and may represent clinically unsuspected lesions secondary to leukemia.