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A New PCR-Based Assay for Testing Bronchoalveolar Lavage Fluid Samples from Patients with Suspected Pneumocystis jirovecii Pneumonia.

ABSTRACT: To support the clinical laboratory diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial PJ-specific real-time quantitative PCR (qPCR) assays are todays' reliable options. The performance of these assays depends on the type of PJ gene (multi-copy mitochondrial versus single-copy nuclear) targeted by the assay. We described the development of a PJ-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the PJ-PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by PJ-PCR. Of 18 PJ-PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ-PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (n = 18) and not having (n = 182) proven (PJ-PCR+/IFA+) or probable (PJ-PCR+/IFA-) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our PJ-PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.

SUBMITTER: Liotti FM

PROVIDER: S-EPMC8466016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evaluation of PCR in bronchoalveolar lavage fluid for diagnosis of Pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review.

Project description:BackgroundAs a promising tool, PCR in bronchoalveolar lavage fluid (BALF) has not been accepted as a diagnostic criterion for PJP.ObjectiveWe undertook a systematic review of published studies to evaluate the diagnostic accuracy of PCR assays in BALF for PJP.MethodsEligible studies from PubMed, Embase and Web of Science reporting PCR assays in BALF for diagnosing PJP were identified. A bivariate meta-analysis of the method's sensitivity, specificity, and positive and negative likelihood ratios with a 95% confidence interval (CI) were analyzed. The post-test probability was performed to evaluate clinical usefulness. A summary receiver operating characteristics (SROC) curve was used to evaluate overall performance. Subgroup analyses were carried out to analysis the potential heterogeneity.ResultsSixteen studies published between 1994 and 2012 were included. The summary sensitivity and specificity values (95% CI) of PCR in BALF for diagnosis of PJP were 98.3% (91.3%-99.7%) and 91.0% (82.7%-95.5%), respectively. The positive and negative likelihood ratios were 10.894 (5.569-21.309) and 0.018 (0.003-0.099), respectively. In a setting of 20% prevalence of PJP, the probability of PJP would be over 3-fold if the BALF-PCR test was positive, and the probability of PJP would be less than 0.5% if it was negative. The area under the SROC curve was 0.98 (0.97-0.99).ConclusionsThe method of PCR in BALF shows high sensitivity and good specificity for the diagnosis of PJP. However, clinical practice for the diagnosis of PJP should consider the consistent respiratory symptoms, radiographic changes and laboratory findings of the suspected patients.

| S-EPMC3762835 | biostudies-literature

Pneumocystis jirovecii Colonization in Non-HIV-Infected Patients Based on Nested-PCR Detection in Bronchoalveolar Lavage Samples.

Project description:BackgroundPneumocystis jirovecii causes Pneumocystis pneumonia (PCP) in immunocompromised patients with a high rate of morbidity and mortality. Colonization with this fungus may stimulate pulmonary inflammation or lead to PCP in susceptible patients. The epidemiology of this infection and routs of its transmission has poorly studied in Iran. We examined Pneumosystis colonization in patients with various lung underlying diseases.MethodsBronchoalveolar lavage (BAL) fluids of 458 patients with different underlying diseases or pulmonary signs were collected between August 2010 and January 2012. Patients were divided into four groups: transplant recipients, malignant patients, immunosuppressive drug recipients and patients with other different lung diseases. A sensitive nested-PCR method targeted 18S ribosomal RNA gene was used for investigating P. jirovecii in the specimens.ResultsP. jirovecii DNA was detected in 57 out of 458 (12.5%) BAL samples by nested-PCR. Colonization rate in malignant patients, transplant recipients, immunosuppressive therapy recipients and patients with other various lung diseases was 21.7%, 20.3%, 12.7% and 7.3%, respectively. The enzyme BanI cuts all PCR products producing fragments with the size of 228 and 104 base pair. This finding as well as sequencing of four random positive samples validated and reconfirmed the PCR results. P. jirovecii cysts were found in 5 out of 57 PCR positive samples.ConclusionA significant number of patients with pulmonary diseases were colonized by P. jirovecii that can develop to PCP in these patients or they may transmit the fungus to other susceptible patients.

| S-EPMC3633800 | biostudies-literature

Prognostic implication of bronchoalveolar lavage fluid analysis in patients with Pneumocystis jirovecii pneumonia without human immunodeficiency virus infection.

Project description:BackgroundThe prognostic value of bronchoalveolar lavage (BAL) fluid analysis in non-human immunodeficiency virus (HIV)-infected patients with Pneumocystis jirovecii pneumonia (PJP) has not been well elucidated. We aimed to investigate the prognostic implication of BAL fluid analysis in non-HIV patients with PJP.MethodsThe data of 178 non-HIV patients diagnosed with PJP based on the results of the polymerase chain reaction assay of BAL fluid specimens between April 2018 and December 2020 were retrospectively reviewed. The clinical characteristics, laboratory findings, and BAL fluid analysis results of patients who died within 90 days after hospital admission were compared.ResultsTwenty patients (11.2%) died within 90 days from admission. The neutrophil count in BAL fluid was significantly higher (median 22.0%, interquartile range [IQR] 2.0-46.0% vs. median 6.0%, IQR 2.0-18.0%, P = 0.044), while the lymphocyte count was significantly lower (median 24.0%, IQR 7.0-37.0% vs. median 41.0%, IQR 22.5-60.5%, P = 0.001) in the non-survivor group compared with that in the survivor group. In the multivariate analysis, the C-reactive protein level (odds ratio [OR] 1.093, 95% confidence interval [CI] 1.020-1.170, P = 0.011) and a BAL fluid lymphocyte count of ≤ 30% (OR 3.353, 95% CI 1.101-10.216, P = 0.033) were independently associated with mortality after adjusting for albumin and lactate dehydrogenase levels.ConclusionA low lymphocyte count in BAL fluid may be a predictor of mortality in non-HIV patients with PJP.

| S-EPMC9233854 | biostudies-literature

Cellular profiles of bronchoalveolar lavage fluid and their prognostic significance for non-HIV-infected patients with Pneumocystis jirovecii pneumonia.

Project description:The usefulness of bronchoalveolar lavage (BAL) fluid cellular analysis in non-human immunodeficiency virus (HIV)-infected patients with Pneumocystis jirovecii pneumonia (PCP) has not been adequately evaluated. The objective of this study was to analyze the cellular profiles of BAL fluid and to evaluate their prognostic significance in non-HIV-infected patients with PCP. A 7-year retrospective cohort study of 166 non-HIV-infected adult patients with PCP who underwent BAL was performed in a tertiary care hospital. The median total BAL fluid white blood cell count was 180/μl (interquartile range, 80 to 330) and was unaffected by the severity of PCP. The median percentages of BAL fluid neutrophils, lymphocytes, and alveolar macrophages were 13.1%, 31.7%, and 30.2%, respectively. The median percentage of BAL fluid neutrophils was significantly higher in severe than in mild-to-moderate PCP (20.4% versus 6.0%, P<0.001), as was the absolute neutrophil count (24/μl versus 13/μl, P=0.001). The percentage of BAL fluid neutrophils was an independent predictor of 30-day (adjusted odds ratio [aOR], 1.02; 95% confidence interval [CI], 1.01 to 1.03) and 60-day (aOR, 1.02; 95% CI, 1.01 to 1.04) mortalities. The 30-day and 60-day mortalities increased at rates of 15% (P=0.006) and 21% (P<0.001) per 10% increment of BAL fluid neutrophil levels, respectively. The degree of BAL fluid pleocytosis was relatively low without regard to the severity of PCP. The percentage of BAL fluid neutrophils can be used as a prognostic marker in non-HIV-infected patients with PCP.

| S-EPMC4365245 | biostudies-literature

Clinical significance of quantifying Pneumocystis jirovecii DNA by using real-time PCR in bronchoalveolar lavage fluid from immunocompromised patients.

Project description:Quantitative PCR (qPCR) is more sensitive than microscopy for detecting Pneumocystis jirovecii in bronchoalveolar lavage (BAL) fluid. We therefore developed a qPCR assay and compared the results with those of a routine immunofluorescence assay (IFA) and clinical data. The assay included automated DNA extraction, amplification of the mitochondrial large-subunit rRNA gene and an internal control, and quantification of copy numbers with the help of a plasmid clone. We studied 353 consecutive BAL fluids obtained for investigation of unexplained fever and/or pneumonia in 287 immunocompromised patients. No qPCR inhibition was observed. Seventeen (5%) samples were both IFA and qPCR positive, 63 (18%) were IFA negative and qPCR positive, and 273 (77%) were both IFA and qPCR negative. The copy number was significantly higher for IFA-positive/qPCR-positive samples than for IFA-negative/qPCR-positive samples (4.2 ± 1.2 versus 1.1 ± 1.1 log(10) copies/?l; P < 10(-4)). With IFA as the standard, the qPCR assay sensitivity was 100% for ?2.6 log(10) copies/?l and the specificity was 100% for ?4 log(10) copies/?l. Since qPCR results were not available at the time of decision-making, these findings did not trigger cotrimoxazole therapy. Patients with systemic inflammatory diseases and IFA-negative/qPCR-positive BAL fluid had a worse 1-year survival rate than those with IFA-negative/qPCR-negative results (P < 10(-3)), in contrast with solid-organ transplant recipients (P = 0.88) and patients with hematological malignancy (P = 0.26). Quantifying P. jirovecii DNA in BAL fluids independently of IFA positivity should be incorporated into the investigation of pneumonia in immunocompromised patients. The relevant threshold remains to be determined and may vary according to the underlying disease.

| S-EPMC3264196 | biostudies-literature

De novo assembly of the Pneumocystis jirovecii genome from a single bronchoalveolar lavage fluid specimen from a patient.

Project description:UnlabelledPneumocystis jirovecii is a fungus that causes severe pneumonia in immunocompromised patients. However, its study is hindered by the lack of an in vitro culture method. We report here the genome of P. jirovecii that was obtained from a single bronchoalveolar lavage fluid specimen from a patient. The major challenge was the in silico sorting of the reads from a mixture representing the different organisms of the lung microbiome. This genome lacks virulence factors and most amino acid biosynthesis enzymes and presents reduced GC content and size. Together with epidemiological observations, these features suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, which causes disease only in immune-deficient individuals. This genome sequence will boost research on this deadly pathogen.ImportancePneumocystis pneumonia is a major cause of mortality in patients with impaired immune systems. The availability of the P. jirovecii genome sequence allows new analyses to be performed which open avenues to solve critical issues for this deadly human disease. The most important ones are (i) identification of nutritional supplements for development of culture in vitro, which is still lacking 100 years after discovery of the pathogen; (ii) identification of new targets for development of new drugs, given the paucity of present treatments and emerging resistance; and (iii) identification of targets for development of vaccines.

| S-EPMC3531804 | biostudies-literature

Pneumocystis jirovecii with high probability detected in bronchoalveolar lavage fluid of chemotherapy-related interstitial pneumonia in patients with lymphoma using metagenomic next-generation sequencing technology.

Project description:BackgroundPrevious studies achieved low microbial detection rates in lymphoma patients with interstitial pneumonia (IP) after chemotherapy. However, the metagenomic next-generation sequencing (mNGS) is a comprehensive approach that is expected to improve the pathogen identification rate. Thus far, reports on the use of mNGS in lymphoma patients with chemotherapy-related IP remain scarce. In this study, we summarized the microbial detection outcomes of lymphoma patients with chemotherapy-related IP through mNGS testing of bronchoalveolar lavage fluid (BALF).MethodsFifteen lymphoma patients with chemotherapy-related IP were tested for traditional laboratory microbiology, along with the mNGS of BALF. Then, the results of mNGS and traditional laboratory microbiology were compared.ResultsOf the 15 enrolled patients, 11 received rituximab and 8 were administered doxorubicin hydrochloride liposome. The overall microbial yield was 93.3% (14/15) for mNGS versus 13.3% (2/15) for traditional culture methods (P ≤ 0.05). The most frequently detected pathogens were Pneumocystis jirovecii (12/15, 80%), Cytomegalovirus (4/15, 26.7%), and Epstein-Barr virus (3/15, 20%). Mixed infections were detected in 10 cases. Five patients recovered after the treatment with antibiotics alone without glucocorticoids.ConclusionOur findings obtained through mNGS testing of BALF suggested a high microbial detection rate in lymphoma patients with IP after chemotherapy. Notably, there was an especially high detection rate of Pneumocystis jirovecii. The application of mNGS in patients with chemotherapy-related IP was more sensitive.

| S-EPMC10698987 | biostudies-literature

Molecular diagnosis and genotyping of <i>Pneumocystis jirovecii</i> in bronchoalveolar lavage samples obtained from patients with pulmonary disorder.

Project description:Background and purposePneumocystis pneumonia (PCP) is one of the most common and life-threatening fungal diseases in patients with human immunodeficiency, treated with immunosuppressive medications. Immunocompetent people can also be a spreading agent for PCP. Regarding this, the aim of the present study was to diagnose and identify Pneumocystis jirovecii in bronchoalveolar lavage (BAL) samples obtained from patients with pulmonary disorder using a molecular method.Materials and methodsFor the purpose of the study, BAL samples (n=138) were collected from patients, undergoing bronchoscopy at the different departments of university hospitals affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, during a period of one year (i.e., April 2014 until May 2015). Giemsa staining and molecular identification were carried out for each sample. The samples were also subjected to nested polymerase chain reaction (PCR), sequencing, and genotyping based on mitochondrial ribosomal large subunit (mtLSU rRNA) of P. jirovecii. The phylogenic tree was constructed by MEGA6 software.ResultsThe results of direct microscopic examination revealed the presence of P. jirovecii in 3 (2.2%) out of 138 samples; in addition, nested PCR and sequencing led to the detection of species in 17 (12.3%) samples. Out of patients with positive results, 10 (25%) and 7 (7.1%) cases were immunosuppressed and immunocompetent, respectively. The most common clinical symptoms among patients with pneumocystis were fever, dyspnea, and dry cough. In addition, genotypes III and II were the dominant genotypes in our dataset.ConclusionNested PCR and sequencing methods showed higher sensitivity and specificity as compared with a direct staining technique. Genotype III was identified as the most dominant type in patients with pulmonary disorder in Mashhad.

| S-EPMC6910711 | biostudies-literature

Pathogenic Analysis of the Bronchoalveolar Lavage Fluid Samples With Pediatric Refractory <i>Mycoplasma pneumoniae</i> Pneumonia.

Project description:Background: We conducted a pathogenic analysis in the bronchoalveolar lavage fluid (BALF) samples from refractory Mycoplasma pneumoniae pneumonia (RMPP) children. Methods: A total of 150 BALF samples from 60 RMPP patients were analyzed to investigate pathogenic changes. The characteristics of M. pneumoniae were analyzed through culture, real-time PCR, genotyping, antimicrobial susceptibility testing and proteomics. The other pathogens were determined using culture, sequencing and nucleic acid detection. Results: In 60 RMPP cases, the bacterial co-infection rate was 5%, while that of virus was 33.3%. The poor prognosis rate was 61.7%. The DNA positive rate among the 150 samples was 98.7%, while the culture positive rate was 56.7% for M. pneumoniae. Significant differences were noticed in the positivity of M. pneumoniae culture obtained from samples with a disease course of at least 3 weeks compared with those within 3 weeks. The genotype 1 M. pneumoniae strains showed a macrolide resistant (MLr) rate of 100%, and that for genotype 2 was 90.1%. Proteomics showed that there were 57 proteins up-regulated in the MLs M. pneumoniae, half of which were membrane-associated protein with adhesion or toxicity. Conclusions: Pediatric RMPP usually presented with viral co-infection, but it caused limited effects on the progression and prognosis of RMPP. Persistent presence of viable M. pneumoniae is not necessary in the later stage of RMPP. The expression of virulence factor in the MLr M. pneumoniae was higher than that of the MLs M. pneumoniae, which was more common in the RMPP children.

| S-EPMC7655529 | biostudies-literature

Detection of Streptococcus pneumoniae antigen in bronchoalveolar lavage fluid samples by a rapid immunochromatographic membrane assay.

Project description:We conducted a retrospective study to evaluate an immunochromatographic membrane test (ICT), applied to bronchoalveolar lavage (BAL) fluid samples obtained in patients with suspected pneumonia, for the detection of Streptococcus pneumoniae antigen. The NOW Streptococcus pneumoniae test was assessed on 96 BAL fluid samples. Sensitivity was tested in 20 samples obtained from patients diagnosed as having pneumococcal pneumonia (growth of S. pneumoniae in blood cultures and/or in BAL fluid samples of > or =10(4) CFU/ml). Specificity was tested in BAL fluid samples of nonpneumococcal etiology (n = 41) and in samples with no respiratory pathogen and a total bacterial count of <10(4) CFU/ml (n = 35). Using the ICT, pneumococcal antigen was detected in 29 (30.2%) BAL fluid samples, with a sensitivity of 95.0% (95% confidence interval [CI], 90.6% to 99.4%) and a specificity of 86.8% (95% CI, 80.1% to 93.8%). The ICT was easy to perform and revealed unequivocal and reproducible results. No interference was observed with high cell counts, red blood cells, or elevated protein levels. Four out of 10 false-positive readings occurred in samples with S. pneumoniae counts below the 10(4) CFU/ml threshold limit of pneumonia. In BAL fluid samples obtained after pneumococcal bacteremia, positive test results were found for up to 35 days after bacteremia. The ICT test applied to BAL fluid specimens is reproducible and accurate in the diagnosis of pneumococcal antigen. Further studies are required to establish the impact of the ICT on patient care.

| S-EPMC1233991 | biostudies-literature

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