Pneumocystis Colonization Research Articles

Rapid Diagnosis of Pneumocystis jirovecii Pneumonia and Respiratory Tract Colonization by Next-Generation Sequencing

ObjectivesTo describe the epidemiology of Pneumocystis jirovecii pneumonia and colonization diagnosed by next-generation sequencing (NGS) and explore the usefulness of the number of P. jirovecii sequence reads for the diagnosis of P. jirovecii pneumonia.MethodsWe examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics.ResultsAmong 285 respiratory samples collected over a 12-month period (January to December 2022), P. jirovecii sequences were detected in 56 samples from 53 patients. Fifty (94.3%) of the 53 patients were HIV-negative. Following our case definitions, 37 (69.8%) and 16 (30.2%) of the 53 patients had P. jirovecii infection and colonization respectively. P. jirovecii infection was associated with presence of underlying disease with immunosuppression (94.6% vs 18.8%, P < 0.05), positive serum 1,3-β-D-glucan (41.2% vs 0%, P < 0.01) and higher number of P. jirovecii sequence reads (P < 0.005). In contrast, P. jirovecii colonization was associated with the male sex (93.8% vs 54.1%, P < 0.01), another definitive infectious disease diagnosis of the respiratory tract (43.8% vs 2.7%, P < 0.001) and higher survival (100% vs 67.6%, P < 0.01). Although P. jirovecii pneumonia was associated with higher number of P. jirovecii reads in respiratory samples, only a sensitivity of 82.14% and a specificity of 68.75% could be achieved.ConclusionDetection of P. jirovecii sequences in respiratory samples has to be interpreted discreetly. A combination of clinical, radiological and laboratory findings is still the most crucial in determining whether a particular case is genuine P. jirovecii pneumonia.

Clinical Performance of BALF Droplet Digital PCR for Differential Diagnosis of Pneumocystis jirovecii Pneumonia and Pneumocystis jirovecii Colonization

BackgroundAccurate differentiation between Pneumocystis jirovecii (Pj) infection and colonization is crucial for effective treatment. MethodsFrom September 2016 to June 2022, 89 immunocompromised patients with unexplained lung infiltrates and clinical suspicion of Pj pneumonia were enrolled at Peking University People's Hospital. Bronchoalveolar lavage fluid (BALF) of these patients were detected by quantitative PCR (qPCR) and droplet digital PCR (ddPCR). ResultsThe performance of ddPCR was superior to qPCR in detecting Pj infection. Area under the curve was 0.97 (95%CI: 0.94–1) for ddPCR of the BALF in all patients. The optimal threshold value for discriminating Pj infection from colonization was 13.98 copies/test, with a sensitivity of 97.96%, specificity of 85.71%. No obvious correlation between ddPCR copy number and disease severity was observed. ConclusionBALF ddPCR exhibits robust potential in detecting Pj and effectively discriminating colonization and infection.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n=62), probable PCP (n=87), Pneumocystis colonization (n=37), and no PCP (n=53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

Usefulness of ß-d-Glucan Assay for the First-Line Diagnosis of Pneumocystis Pneumonia and for Discriminating between Pneumocystis Colonization and Pneumocystis Pneumonia.

According to the immunodepression status, the diagnosis of Pneumocystis jirovecii pneumonia (PjP) may be difficult. Molecular methods appear very sensitive, but they lack specificity because Pj DNA can be detected in Pneumocystis-colonized patients. The aim of this study was to evaluate the value of a serum ß-d-Glucan (BDG) assay for the diagnosis of PjP in a large cohort of HIV-negative and HIV-positive patients, either as a first-line diagnostic test for PjP or as a tool to distinguish between colonization and PjP in cases of low fungal load. Data of Pj qPCR performed on bronchopulmonary specimens over a 3-year period were retrieved retrospectively. For each result, we searched for a BDG serum assay performed within ±5 days. Among the 69 episodes that occurred in HIV-positive patients and the 609 episodes that occurred in immunocompromised HIV-negative patients, we find an equivalent sensitivity of BDG assays compared with molecular methods to diagnose probable/proven PjP, in a first-line strategy. Furthermore, BDG assay can be used confidently to distinguish between infected and colonized patients using a 80 pg/mL cut-off. Finally, it is necessary to search for causes of false positivity to increase BDG assay performance. BDG assay represents a valuable adjunctive tool to distinguish between colonization and infection.

Pneumocystis Colonization in Dogs Is as in Humans.

Pneumocystis is an atypical fungus that resides in the pulmonary parenchyma of many mammals, including humans and dogs. Immunocompetent human hosts are usually asymptomatically colonised or show subtle clinical signs, but some immunocompromised people can develop florid life-threatening Pneumocystis pneumonia (PCP). Since much less is known concerning Pneumocystis in dogs, we posit the question: can Pneumocystis colonization be present in dogs with inflammatory airway or lung disease caused by other pathogens or disease processes? In this study, Pneumocystis DNA was detected in bronchoalveolar lavage fluid (BALF) of 22/255 dogs (9%) with respiratory distress and/or chronic cough. Although young dogs (<1 year-of-age) and pedigree breeds were more often Pneumocystis-qPCR positive than older dogs and crossbreds, adult dogs with other infectious conditions and/or a history of therapy-resistant pulmonary disease could also be qPCR-positive, including two patients with suppression of the immune system. Absence of pathognomonic clinical or radiographic signs render it impossible to convincingly discriminate between overt PCP versus other lung/airway disease processes colonised by P. canis. It is possible that colonisation with P. canis might play a certain role as a co-pathogen in some canine patients with lower respiratory disease.

Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Factors associated with Pneumocystis colonization and circulating genotypes in chronic obstructive pulmonary disease patients with acute exacerbation or at stable state and their homes.

Chronic obstructive pulmonary disease patients tend to be more frequently colonized by Pneumocystis during exacerbation (19.0%) than at stable state (10.7%). Factors associated with colonization include lower BMI, higher IL-17, and CD62P. Pneumocystis detection in patients' dwellings suggests potential persistence in indoor dust.

Quantitative PCR to Discriminate Between Pneumocystis Pneumonia and Colonization in HIV and Non-HIV Immunocompromised Patients.

Pneumocystis pneumonia (PCP) is an opportunistic infection that commonly occurs in immunocompromised individuals. A definite diagnosis of PCP can be made only when the organism is identified in a respiratory specimen. It remains unclear whether qPCR can differentiate patients with PCP from those with Pneumocystis jirovecii colonization. In this study, we retrospectively collected data from HIV and non-HIV patients during 2013–2019. A diagnosis of definite, probable PCP, or PCP excluded was made based on clinical criteria, radiological reports, and three standard laboratory staining methods with blinding to qPCR data. Data from qPCR that was performed to determine the fungal burden (DNA copies/μl) in the BAL specimens of 69 HIV and 286 non-HIV patients were then obtained and reviewed. Receiver Operating Characteristic (ROC) curve analysis was performed to determine the upper and lower cut-off values for PCP diagnosis in HIV and non-HIV groups. In the non-HIV group, the lower cut-off value of 1,480 DNA copies/μl yielded a sensitivity of 100% (95% confidence interval [CI], 91.0–100), specificity of 72.9% (95% CI, 64.0–80.7), a positive predictive value (PPV) of 54.9% (95% CI, 47.6–62.1), and a negative predictive value (NPV) of 100% with Youden index of 0.73 for PCP diagnosis. In this group, the upper cut-off value of 9,655 DNA copies/μl showed the sensitivity of 100% (95% CI, 91.0–100) and specificity of 95.8% (95% CI, 90.4–98.6) with PPV of 88.6% (95% CI, 76.8–94.8) and a NPV of 100% with Youden index of 0.96 for PCP diagnosis. Regarding the HIV group, the lower cut-off value of 1,480 DNA copies/μl showed the sensitivity of 100% (95% CI, 92.5–100%) and specificity of 91.7% (95% CI, 61.5–99.8) with PPV of 97.9% (95% CI, 87.8–99.7) and a NPV of 100% with Youden index of 0.92 for PCP diagnosis. The sensitivity and specificity of the upper cut-off value of 12,718 DNA copies/μl in this group were 97.9% (95%CI, 88.7–100) and 100% (95%CI, 73.5–100), respectively. The values above the upper cut-off point had a PPV of 100% (95% CI, N/A) and a NPV of 92.3% (95% CI, 63.3–98.8) with Youden index of 0.98 for PCP diagnosis in the HIV group.

The prevalence of laboratory-confirmed Pneumocystis jirovecii in HIV-infected adults in Africa: A systematic review and meta-analysis.

The epidemiology of Pneumocystis jirovecii, known to colonize the respiratory tract and cause a life-threatening HIV-associated pneumonia (PCP), is poorly described in Africa. We conducted a systematic review to evaluate P. jirovecii prevalence in African HIV-positive adults with or without respiratory symptoms. We searched Medline, Embase, Cochrane library, Africa-Wide, and Web of Science for studies employing PCR and/or microscopy for P. jirovecii detection in respiratory samples from HIV-positive adults in Africa between 1995 and 2020. Prevalence with respiratory symptoms was pooled using random-effect meta-analysis, and stratified by laboratory method, sample tested, study setting, CD4 count, and trimethoprim/sulfamethoxazole prophylaxis. Colonization prevalence in asymptomatic adults and in adults with non-PCP respiratory disease was described, and quantitative PCR (qPCR) thresholds to distinguish colonization from microscopy-confirmed PCP reviewed. Thirty-two studies were included, with 27 studies (87%) at high risk of selection bias. P. jirovecii was detected in 19% [95% confidence interval (CI): 12-27%] of 3583 symptomatic and in 9% [95% CI: 0-45%] of 140 asymptomatic adults. Among symptomatic adults, prevalence was 22% [95% CI: 12-35%] by PCR and 15% [95% CI: 9-23%] by microscopy. Seven percent of 435 symptomatic adults had PCR-detected Pneumocystis colonization without evidence of PCP [95% CI: 5-10%, four studies]. One study established a qPCR cutoff of 78 copies/5μl of DNA in 305 induced sputum samples to distinguish Pneumocystis colonization from microscopy-confirmed PCP. Despite widened access to HIV services, P. jirovecii remains common in Africa. Prevalence estimates and qPCR-based definitions of colonization are limited, and overall quality of studies is low.

Investigation of Pneumocystis jirovecii Infection and Colonization in Immunocompromised Patients with Pneumonia

Pneumocystis jirovecii is an atypical fungus that causes P.jirovecii pneumonia (PCP) in immunocompromised patients. Currently, while the incidence of AIDS-related PCP is decreasing, PCP has become more common in HIV-negative immunosuppressive patients as a result of increased diseases requiring immunosuppressive therapy. In this study, it was aimed to investigate PCP and colonizations by microscopy, polymerase chain reaction (PCR) and Krebs von den Lungen-6 (KL-6) tests in symptomatic immunosuppressive inpatients with the sign of radiologically atypical pneumonia in Mersin University Hospital. A total of 96 patients, between August 2016 and February 2018 were included in the study. Seventy two (75%) of the 96 patients were under immunosuppressive therapy. P.jirovecii was investigated in the respiratory tract samples [sputum (n= 88), tracheal aspirate (n= 6) and bronchoalveolar lavage (n= 2)] by mtLSUrRNA nested PCR and microscopic staining methods [immunofluorescence assay (IFA), Toluidine Blue O (TBO)], and KL-6 levels were tested in serum samples. P.jirovecii was detected in 16 (16.7%) samples by PCR, in five (5.2%) samples by IFA, in three (3.1%) samples by TBO stain method. When IFA was taken as a reference test, sensitivity and specificity of TBO and PCR were calculated as 60% and 100%; 100% and 87.9%, respectively. In P.jirovecii PCR positive patients, the distribution of underlying diseases; cancer (n= 6), hematological malignancy (n= 3), HIV/AIDS (n= 3), COPD (n= 2), and interstitial lung disease (n= 2) were found as 11 (68.75%) of the 16 positive patients, received immunosuppressive therapy (HIV positive non-Hodgkin lymphoma); of the 3 (18.75%) patients of were immunocompetent, and only 2 (12.5%) were HIV/AIDS. Five of the 16 PCR positive the patients that have positive microscopic examination were definited PCP [HIV/AIDS (n= 3), lung cancer (n= 1), interstitial lung disease (n= 1)]; three patients were PCR positive and microscopy negative probable PCP [multiple myeloma (n= 1), interstitial lung disease (n= 1), cholangiocellular carcinoma (n= 1)] and eight other patients were identified as colonized. In the study, when the frequency of the detection of P.jirovecii was evaluated according to the underlying diseases, it was found statistically significantly higher only in HIV/AIDS patients (p= 0.012). When KL-6 was evaluated among the patients defined as PCP/possible PCP and colonization, sensitivity and specificity were determined as 62.5% and 75%, respectively. As a result, nested PCR method was found as sensitive and successful for the detection of P.jirovecii from sputum samples. KL-6 test was not found sufficient for the differentiation of colonization and the infection in PCR positive patients. The results obtained in the study showed that PCP should be on the differential diagnosis list according to the immune status and the clinical features of the inpatients. More researchs are required with more patients to achieve for detailed reliable results in these groups. In addition, molecular epidemiological studies related to genotyping and resistance against anti-PCP drugs are needed to understand P.jirovecii infections in our region and country.

CO-INFECTION WITH HISTOPLASMA CAPSULATUM AND PNEUMOCYSTIS JIROVECII IN A PATIENT WITH LIVER CIRRHOSIS: A DIAGNOSTIC DILEMMA

SESSION TITLE: Medical Student/Resident Chest Infections Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Histoplasma capsulatum and Pneumocystis jirovecii pulmonary coinfection are unusual in HIV negative populations. We present the case of a man in respiratory failure with H. capsulatum and P. jirovecii detected in bronchial washings. CASE PRESENTATION: A 64-year-old man with T2DM, moderate COPD, and NASH cirrhosis with pancytopenia presented with 2-month history of progressive hypoxic respiratory failure. CT chest revealed multifocal consolidations with mediastinal lymphadenopathy which progressed despite antimicrobial therapy (Fig. 1). Bronchoalveolar lavage (BAL) was remarkable for 2800 copies/mL of P. jirovecii PCR with beta-D-glucan of 286. BAL specimen also grew H. capsulatum on culture and the FNAC specimen taken from lymph node 7 during the transbronchial biopsy was indicative of fungal elements similar to Histoplasma (Fig. 2A-2C). The patient was started on itraconazole for Histoplasmosis and trimethoprim-sulfamethoxazole with steroids for P. jirovecii pneumonia (PJP). He was tested during the hospitalization and found to be negative for HIV but had CD4 and CD8 counts at 86 and 70 x10E9/L respectively. Due to progression of his disease the patient was transitioned to comfort measures and passed on hospital day 36. DISCUSSION: Histoplasmosis is an endemic mycosis in the Ohio and Mississippi river valleys often resulting in asymptomatic infection [1]. PJP has also been reported as an asymptomatic infection or colonization in immunocompetent hosts [2]. Co-infection with both H. capsulatum and PJP ranges from 0.5-13% in studies on HIV positive patient groups [3,4]. In the sample population studied by Carreto-Binaghi et al, a coinfection rate of 1.5% was noted in the HIV negative group [5]. Irrespective of host immune status, patients with coinfection have a higher mortality rate than with a single infection alone [5], thus warranting treatment for both pathogens. Our patient had a history of chronic lymphopenia which had been attributed to his cirrhosis, with tests showing extremely low CD4 counts. Patients with end stage liver disease are recognized as being relatively immunocompromised when compared to healthy individuals due to coexisting leucopenia, lymphopenia and CD4/CD8 ratio abnormalities [6]. This case raises the question if patients with cirrhosis and lymphopenia should be treated with prophylactic antimicrobial agents based on their CD4 counts. CONCLUSIONS: This case highlights the importance of recognizing and treating co-infection of PJP and Histoplasmosis in non-HIV infected individuals. Additionally, it reminds us that patients with liver disease have important deficiencies in their cellular and humoral immunity which make them more susceptible to unusual opportunistic infections. Reference #1: [1] Goodwin RA, Loyd JE, Des Prez RM. Histoplasmosis in Normal Hosts. Medicine. 1981; 60(4): 231-266. Reference #2: [2] Ponce CA, Gallo M, Bustamante R, et al. Pneumocystis colonization is highly prevalent in the autopsied lungs of the general population. Clin Infect Dis. 2010; 50(3):347-353. [3] Baughman RP, Dohn MN, Frame PT. The continuing utility of bronchoalveolar lavage to diagnose opportunistic infection in AIDS patients. Am J Med. 1994; 97(6): 515-522. Reference #3: [4] Wheat JL, Slama TG, Zeckel ML. Histoplasmosis in the acquired immune deficiency syndrome. Am J Med. 1985; 78(2): 203-210. [5] Carreto-Binaghi LE, Morales-Villarreal FR, García-de la Torre G, et al. Histoplasma capsulatum and Pneumocystis jirovecii coinfection in hospitalized HIV and non-HIV patients from a tertiary care hospital in Mexico. Int J of Inf Dis. 2019; 86: 65-72. [6] Tuchendler E, Tuchendler PK, Madej G. Immunodeficiency caused by cirrhosis. Clin Exp Hepatol. 2018;4(3):158-164. DISCLOSURES: No relevant relationships by James Dugan, source=Web Response No relevant relationships by Akisha Glasgow, source=Web Response No relevant relationships by Eduarda Grinsztejn, source=Web Response No relevant relationships by Amrita John, source=Web Response

Recent Advances in the Diagnosis and Management of Pneumocystis Pneumonia.

In human immunodeficiency virus (HIV)-infected patients, Pneumocystis jirovecii pneumonia (PCP) is a wellk-nown opportunistic infection and its management has been established. However, PCP is an emerging threat to immunocompromised patients without HIV infection, such as those receiving novel immunosuppressive therapeutics for malignancy, organ transplantation, or connective tissue diseases. Clinical manifestations of PCP are quite different between patients with and without HIV infections. In patients without HIV infection, PCP rapidly progresses, is difficult to diagnose correctly, and causes severe respiratory failure with a poor prognosis. High-resolution computed tomography findings are different between PCP patients with HIV infection and those without. These differences in clinical and radiological features are due to severe or dysregulated inflammatory responses that are evoked by a relatively small number of Pneumocystis organisms in patients without HIV infection. In recent years, the usefulness of polymerase chain reaction and serum β-D-glucan assay for rapid and non-invasive diagnosis of PCP has been revealed. Although corticosteroid adjunctive to anti-Pneumocystis agents has been shown to be beneficial in some populations, the optimal dose and duration remain to be determined. Recent investigations revealed that Pneumocystis colonization is prevalent and that asymptomatic carriers are at risk for developing PCP and can serve as the reservoir for the spread of Pneumocystis by airborne transmission. These findings suggest the need for chemoprophylaxis in immunocompromised patients as well as infection control measures, although the indications remain controversial. Because a variety of novel immunosuppressive therapeutics have been emerging in medical practice, further innovations in the diagnosis and treatment of PCP are needed.

Colonización por Pneumocystis jirovecii en mujeres gestantes y recién nacidos en Lima, Perú

BackgroundPneumocystisjirovecii primary infection occurs asymptomatically before 6 months of age, suggesting that the infection is acquired very early in life. Furthermore, Pneumocystis pneumonia has been described in newborns, which emphasizes the importance of studying Pneumocystis colonization in mother-infant pairs. AimsTo evaluate the prevalence of Pneumocystis colonization among pregnant women and to determine the potential transplacental transmission. MethodsA cross-sectional study was carried out on HIV-negative women over 18 years-old, and 37 or more weeks of pregnancy attending Hospital Cayetano Heredia Maternity unit during 2016-2017. Clinical and demographical information was collected on them and their newborns. Oropharyngeal washes, nasal swabs, and placenta samples were collected from women, as well as a nasopharyngeal aspirate and nasal swab from newborns. All respiratory samples were analysed by nested-PCR for the detection of Pneumocystis. Placenta samples from women with a positive PCR result in their respiratory samples were also analysed by nested-PCR. ResultsOf the 92 pregnant women included, five of them (5.43%) were colonized by Pneumocystis. Pneumocystis DNA was not found in any of the 87 available newborn samples or in the placentas of the five women who had a positive result by PCR in their upper respiratory samples. ConclusionsIt was found that 5.43% of the pregnant women were colonized by Pneumocystis, there was no evidence of any role of this colonization in the transmission to their newborns, since none of them tested positive for Pneumocystis.

Pneumocystis jirovecii in Spanish Patients With Heart Failure.

Objective: Pneumocystis colonization is frequent in patients with chronic obstructive pulmonary disease (COPD) producing local and systemic inflammation. Heart failure is also a common comorbidity among patients with COPD. Heart failure is a chronic, frequent, and disabling condition with high morbidity and mortality, but with a modifiable course where endothelial dysfunction and pulmonary arterial hypertension have great importance. Animal models have shown that Pneumocystis infection can cause relevant functionally changes in vascular responses in the lung, promoting the development of pulmonary hypertension. Pneumocystis colonization could be a hidden cause of worsening heart failure through it capacity to induce inflammatory response with subsequent endothelial dysfunction and pulmonary hypertension. The aim of the present study was to investigate the prevalence of Pneumocystis jirovecii colonization in heart failure patients and its possible association with reduced or preserved ejection fraction.Methods: A cross-sectional study was carried out including 36 heart failure patients and 36 control cases. Identification of P. jirovecii colonization was performed by means of molecular techniques in oropharyngeal washing.Results: Pneumocystis-DNA was identified in oropharyngeal washing in 1 (2.7%) of 36 heart failure patients and in 3 (8.3%) of 36 controls.Conclusions: Pneumocystis colonization does not seem to have a role in the pathophysiology of heart failure.

A multi-centered study of Pneumocystis jirovecii colonization in patients with respiratory disorders: Is there a colonization trend in the elderly?

Background and Purpose: Pneumocystis jirovecii colonization plays a key role in the progression of pulmonary infection. However, there are limited data regarding the colonization of these fungi in the patients residing in different regions of Iran. Regarding this, the present study was conducted to evaluate the prevalence of P. jirovecii colonization in non-HIV-infected patients with respiratory failure introduced by physicians using nested polymerase chain reaction (PCR).Materials and Methods:This study was conducted on 136 samples obtained from 136 patients with respiratory disorders referring to different hospitals in the capital and north of Iran during 2013-2015. The samples were collected using bronchoalveolar lavage (BAL; n=121) and sputum induction (n=15). Nested PCR method targeting mtLSU rRNA gene was used for the detection of P. jirovecii DNA in the specimens.Results:The nested PCR analysis resulted in the detection of P. jirovecii DNA in 32 (23.5%) patients. The mean age of the participants was 49.04±11.94 years (age range: 14-90 years). The results revealed no correlation between Pneumocystis colonization and gender. The studied patients were divided into two groups of immunocompromised and immunocompetent patients. In the regard, 25.4% of the patients with detectable P. jirovecii DNA were immunocompromised and had cancer, organ transplantation, asthma, sarcoidosis, dermatomyositis, chronic obstructive pulmonary disease, bronchiectasis, and pulmonary vasculitis. On the other hand, Pneumocystis DNA was detected in 21.8% of the immunocompetent patients. Frequencies of P. jirovecii DNA detection in the patients with tuberculosis, hydatid cyst, and unknown underlying diseases were obtained as 20.8%, 25%, and 22%, respectively. The prevalence of Pneumocystis colonization varied based on age. In this regard, P. jirovecii colonization was more prevalent in patients aged above 70 years.Conclusion:As the findings indicated, non-HIV-infected patients, especially the elderly, had a high prevalence of P. jirovecii colonization. Therefore, these patients are probably a potential source of infection for others. Regarding this, it is of paramount importance to adopt monitoring and prophylactic measures to reduce this infection.

Differentiation between pneumocystis jirovecii pneumonia and colonization in immunocompromised patients

BackgroundThe diagnosis of Pneumocystis jirovecii pneumonia (PJP) is mainly based on microscopic detection of P. jirovecii in the respiratory samples. Quantitative PCR (qPCR) can detect low levels of P. jirovecii DNA but cannot differentiate between infection and colonization. Therefore a new and more accurate assay have to be used. 1,3 BD-glucan (BD-glucan) with a threshold value of 100 pg/ml can differentiate P. jirovecii infection from colonization.AimThe aim of this study is to evaluate the diagnostic accuracy of qPCR and BD-glucan assays in differentiating pneumocystis infection from colonization in immunocompromised patients with help of radiological pulmonary infiltrates.Patients and methodsThis study consisted of 75 immunocompromised patients (37 renal transplanted patients and 38 HIV patients) who were admitted for radiological pulmonary infiltrates and who presented a clinical picture suspecting PJP. They were investigated using microscopic staining of their respiratory samples (induced sputum or bronchoalveolar lavage).By applying both qPCR and serum BD-glucan assays we can differentiate between P. jirovecii infection from colonization.ResultsIn this study, the first group of 25 patients were diagnosed as definite PJP, the second group of 20 patients were diagnosed as having pneumonia with P. jirovecii colonization, and a third group of 30 patients were diagnosed as having pneumonia without colonization. The number of copies of fungal DNA detected by qPCR were significantly higher in a definite PJP than in those with pneumonia accompanied with P. jirovecii colonization. Also BD-glucan assays were significantly higher in definite PJP by applying a threshold value of 100 pg/ml. The sensitivity and specificity of qPCR for differentiation of PJP infection from colonization were 100 and 64%, respectively, whereas the sensitivity and specificity of BD-glucan were 100 and 96%, respectively.ConclusionBoth BD-glucan and qPCR assays had high diagnostic values in differentiating definite PJP from P. jirovecii colonization, and by applying qPCR with two cutoff values combined with serum BD-glucan using a threshold value of 100 pg/ml.

2048. Comparison Between Endpoint and Real-Time (RT) Polymerase Chain Reaction (PCR) for the Diagnosis of Pneumocystis Pneumonia (PCP)

BackgroundThe definitive diagnosis of PCP requires direct visualization of the organism by silver or direct fluorescent antibody stain, but in recent years PCR has become a widely used diagnostic tool. Varying results have been noted with different PCR assays; one concern has been that RT-PCR will be more sensitive and not differentiate colonization from infection. For this study, we compared the performance of RT-PCR with that of endpoint PCR for detection of PCP.MethodsAll adult patients who had a bronchoalveolar lavage (BAL) or sputum sample positive for Pneumocystis by PCR at the U. Michigan Hospitals from February 2014–February 2018 were studied. Before February 2017 samples were tested with endpoint PCR followed by agarose gel electrophoresis and after February 2017 with RT-PCR. For each patient, a strict case definition based on host factors, clinical presentation, radiological and pathologic findings, was used to classify PCP as proven, probable, possible, and unlikely. Based on this classification, endpoint PCR and RT-PCR results were designated as true positive or false-positive presumably colonized (FP).ResultsThe number of specimens tested each year was similar, ranging from 751 to 791. One hundred and fifty-three patients tested positive: 77/2318 (3%) by endpoint PCR and 76/783 (10%) by RT-PCR. One hundred and twenty-six patients had risk factors for PCP: hi-dose steroids (39), hematologic malignancy (38), chemotherapy within 3 months (24), HIV (14), solid-organ transplant (12), stem cell transplant (9), and 27 patients had no PCP risk factors. By our definitions, patients were classified as proven (2), probable (70), possible (46) and unlikely (35). RT-PCR gave a higher FP rate (27/76, 35%) than endpoint PCR (8/77, 10%,) P < 0.0001, especially in those with chronic lung disease, P = .001 and those with no known PCP risk factors, P < 0.0001. More patients with no risk factors tested positive with RT-PCR (20) than with endpoint PCR (7), P = .006. FP rates RT-PCR were similar in sputum (34%) and BAL (36%).ConclusionRT-PCR gave significantly more FP results, likely due to increased detection of Pneumocystis colonization. Pretest probability should be considered when ordering a highly sensitive test such as RT-PCR and positive results must be interpreted in the context of the clinical presentation, radiological findings and risk factors.Disclosures All authors: No reported disclosures.

Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients\u2014preliminary report

Pneumocystis jirovecii is an opportunistic fungus occurring in human lungs. The group at highest risk consists of HIV-infected and non-HIV-infected immunosuppressed individuals. In these patients, P. jirovecii infection may lead to Pneumocystis pneumonia; it may, however, persist also in an asymptomatic form. This study aimed to determine the prevalence of P. jirovecii and potential risk factors for infection in a group of renal transplant recipients and to characterize the genetic diversity of this fungus in the studied population. Sputum specimens from 72 patients were tested for presence of P. jirovecii using immunofluorescence microscopy, as well as nested PCR targeting the mtLSU rRNA gene. Genotyping involving analysis of four loci—mtLSU rRNA, CYB, DHPS, and SOD—was used to characterize the diversity of the detected organisms. Pneumocystis DNA was detected in eight (11.11%) patients. It has been shown that low eosinophil count and dual immunosuppressive treatment combining prednisone and calcineurin inhibitors are potential risk factors for colonization. Analysis of genotype distribution showed an association of the wild-type genotype of mtLSU rRNA with lower average age of patients and shorter time after kidney transplantation. Furthermore, CYB 2 genotype was detected only in patients with the ongoing prophylaxis regimen. In conclusion, renal transplant recipients are at risk of Pneumocystis colonization even a long time after transplantation. The present preliminary study identifies specific polymorphisms that appear to be correlated with certain patient characteristics and highlights the need for deeper investigation of these associations in renal transplant recipients.

Epidemiological characteristics of Pneumocystis jirovecii infection and colonization in non-AIDS patients

Objective: To analyze the epidemiological characteristics of Pneumocystis jirovecii infection and colonization in non-AIDS patients. Methods: From January 2010 to December 2017, bronchoalveolar lavage fluid (BALF) was detected by Grocott's methenamine silver (GMS) staining and real-time fluorescence quantitative PCR (qPCR) in non-AIDS patients with bronchoscopic alveolar lavage at Peking University First Hospital. At the same time, Pneumocystis jirovecii was detected in the environment of the hospital. Results: Within 8 years, Pneumocystis jirovecii were detected in BALF of a total of 1 407 non-AIDS patients. GMS staining was performed in all these 1 407 cases, of which 114 (8.10%) cases were with Pneumocystis jirovecii infection and 3 (0.21%) cases with colonization. There were totally 946 non-AIDS immunocompromised patients, of which 113 (11.95%) cases were infected and 2 (0.21%) cases with colonization; there were 461 non-AIDS immunocompetent patients, of which only 1 (0.22%) case was infected and 1 (0.22%) case with colonization. GMS staining and qPCR were both performed in 196 cases, of which 36 (18.37%) cases were infected and 33 (16.84%) cases with colonization. There were totally 175 non-AIDS immunocompromised patients, of which 36 (20.57%) cases were infected and 30 (17.14%) cases with colonization; there were 21 non-AIDS immunocompetent patients, of which no one was infected and 3 (14.29%) cases with colonization. By means of GMS staining and qPCR, no Pneumocystis jiroveci were detected in the environment of Peking University First Hospital. Conclusions:Pneumocystis pneumonia is rare in immunocompetent patients, but not in non-AIDS immunocompromised patients. And colonization is rare in both of them. There may be no Pneumocystis jiroveci in the environment.

Colonization of Pneumocystis jirovecii in Patients who Received and not Received Corticosteroids Admitted to the Intensive Care Unit: Airborne Transmission Approach

Pneumocystis pneumonia (PCP) is responsible for pulmonary infection in immunocompromised patients. The current study aimed at investigating the frequency of Pneumocystis colonization in patients hospitalized in the intensive care unit (ICU) and evaluating the relationship between PCP and Pneumocystis colonization. The current cross sectional study was conducted on bronchoalveolar lavage (BAL) fluids of 100patients collected from surgery and neurosurgery ICUs with different underlying corticosteroid therapy conditions. Patients were divided into 2 groups (patients receiving and not receiving corticosteroids). Direct examination on BAL fluids was performed by the Gomori methenamine silver and Giemsa staining techniques. Additionally, 2 filtered air samples of the 2 above mentioned units were collected. A nested-PCR targeted mtLSUrRNA gene and sequencing were used to identify Pneumocystis spp. In direct microscopy, 31 out of 100 hospitalized patients (31%) showed positive results. Twenty-three (46%) of smear positive patients were from the group of patients receiving corticosteroid, the other 8(16%) were from the group of patients not receiving corticosteroids (P= 0.001). Pneumocystis jirovecii DNA was detected in 77 out of 100 BAL samples by nested-PCR (77%) in which 40 (52%) and 37 (48%) samples were obtained from the patients receiving and not receiving corticosteroids, respectively. Pneumocystis genome was observed in 1 of the 2 filtered air samples. A significant number of patients receiving corticosteroids were also colonized by P. jirovecii that may be predisposed to PCP or be transmitted to susceptible patients. A significant relationship was observed between the mean hospital stay and detection rate.