Prevalence of colonisation and genotypic characterisation of Pneumocystis jirovecii among cystic fibrosis patients in Spain

Pneumocystis jirovecii colonisation may occur among cystic fibrosis (CF) patients because of their underlying pulmonary disease. A wide epidemiological analysis was performed among CF patients from Spain to assess the prevalence of P. jirovecii colonisation and the distribution of different genotypes. P. jirovecii was identified by nested PCR targeting the mitochondrial large-subunit rRNA gene from sputum samples or oropharyngeal washes. The genotype was determined by direct sequencing. The prevalence of P. jirovecii colonisation among 88 consecutive CF patients was 21.5%. The polymorphisms identified were 85C/248C (45.4%), 85T/248C (27.2%) and 85A/248C (18.1%); in one case, a mix of genotypes was found. Colonisation was more frequent in subjects aged < 18 years (25.5% vs. 15.1%). Among the patients studied, 20.8% received treatment with azithromycin; all of these patients were colonised with P. jirovecii, but none developed Pneumocystis pneumonia (PcP) during a 1-year follow-up period. Concordance in the colonisation status of siblings suggested a common source of infection or person-to-person transmission.     

Outbreak of Pneumocystis jirovecii pneumonia in renal transplant recipients on prophylaxis: Our observation and experience

Pneumocystis jirovecii is a life-threatening opportunistic pathogen affecting immunocompromised hosts, especially renal transplant recipients. This study reports an outbreak of seven such cases, both inpatients and outpatients, occurring in our hospital over a period of 4 months (January–April 2013). All patients were male with a median age of 38 years (range, 28–58 years); the median period between transplantation and diagnosis was 39.5 months (range, 11–123 months). One patient succumbed to the infection. Two were breakthrough cases, developing the infection while on prophylaxis, highlighting the need to view prophylaxis in light of the immunosuppression and clinical picture of such patients.     

A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients

Pneumocystis jirovecii pneumonia (PCP) is a leading cause of morbidity and mortality in immunocompromised patients. Despite the sensitivity of the commonly used PCR for diagnosing P. jirovecii with primers pAZ102-H/pAZ102-E and pAZ102-X/pAZ102-Y derived from mtLSU rRNA (conventional PCR), some PCP patients who had demonstrable organisms by staining methods failed to give positive PCR results. Herein, we devised a more sensitive PCR assay derived from the same gene target to circumvent these false-negative tests. Single brochoalveolar lavage (BAL) samples were collected from human immunodeficiency virus (HIV)-infected (n = 66) and non-HIV (n = 36) immunocompromised patients presenting with fever, dyspnoea, cough and pulmonary infiltrates. Pneumocystis jirovecii was diagnosed with Giemsa-stained smear, immunofluorescence assay, conventional single-round and nested PCR, and new single-round and nested PCR in 46 (45.1%), 53 (52.0%), 69 (67.6%), 74 (72.6%), 87 (85.3%) and 91 (89.2%) patients, respectively. The new PCR could detect P. jirovecii DNA in BAL fluids two to three orders of magnitude more dilute than conventional PCR. Sequence analysis revealed one to three nucleotide substitutions within the primers for conventional PCR among clinical isolates. Although both conventional and new PCR assays were highly specific for diagnosing P. jirovecii, the new PCR yielded more positive results than conventional PCR among BAL samples that were negative by both Giemsa stain and immunofluorescence assay. Hence, the new PCR offered a more sensitive detection of P. jirovecii infection and colonization than conventional PCR.     

Pneumocystis jirovecii colonisation in patients with interstitial lung disease

A prospective study was conducted to determine the prevalence of colonisation by Pneumocystis jirovecii in 80 consecutive patients who required bronchoscopy and bronchoalveolar lavage (BAL) following suspicion of interstitial lung disease (ILD). The mtLSU rRNA gene of P. jirovecii was identified by nested PCR in BAL samples. Patients with ILDs were divided into three groups: group A comprised those with idiopathic interstitial pneumonias; group B comprised those with sarcoidosis; and group C comprised those with other ILDs. The overall prevalence of P. jirovecii carriage was 33.8%, with colonisation rates of 37.8%, 18.8% and 37% in groups A, B and C, respectively (p not significant). There were more smokers among the carriers, but there were no other significant differences between carriers and non-carriers. The high prevalence of P. jirovecii carriers found among immunocompetent patients with ILDs in Spain suggests a possible role of P. jirovecii in the natural history of these diseases.     

Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients

Conventional polymerase chain reaction (PCR) in respiratory samples does not differentiate between Pneumocystis pneumonia (PCP) and Pneumocystis jirovecii (Pj) colonization. We used Pj real-time quantitative PCR (qPCR) with the objective to discriminate PCP from Pj colonization in immunocompromised patients. All positive Pj qPCR [targeting the major surface glycoprotein (MSG) gene] obtained in respiratory samples from immunocompromised patients presenting pneumonia at the Grenoble University Hospital, France, were collected between August 2009 and April 2011. Diagnoses were retrospectively determined by a multidisciplinary group of experts blinded to the Pj qPCR results. Thirty-one bronchoalveolar lavages and four broncho aspirations positive for the Pj qPCR were obtained from 35 immunocompromised patients. Diagnoses of definite, probable, and possible PCP, and pneumonia from another etiology were retrospectively made for 7, 4, 5, and 19 patients, respectively. Copy numbers were significantly higher in the “definite group” (median 465,000 copies/ml) than in the “probable group” (median 38,600 copies/ml), the “possible group” (median 1,032 copies/ml), and the “other diagnosis group” (median 390 copies/ml). With the value of 3,160 copies/ml, the sensitivity and specificity of qPCR for the diagnosis of PCP were 100 % and 70 %, respectively. With the value of 31,600 copies/ml, the sensitivity and specificity were 80 % and 100 %, respectively. The positive predictive value was 100 % for results with more than 31,600 copies/ml and the negative predictive value was 100 % for results with fewer than 3,160 copies/ml. qPCR targeting the MSG gene can be helpful to discriminate PCP from Pj colonization in immunocompromised patients, using two cut-off values, with a gray zone between them.     

Dynamic colonisation by different Pneumocystis jirovecii genotypes in cystic fibrosis patients

Although asymptomatic carriers of Pneumocystis jirovecii with cystic fibrosis (CF) have been described previously, the molecular epidemiology of P. jirovecii in CF patients has not yet been clarified. This study identified the distribution and dynamic evolution of P. jirovecii genotypes based on the mitochondrial large-subunit (mt LSU) rRNA gene. The mt LSU rRNA genotypes of P. jirovecii isolates in 33 respiratory samples from CF patients were investigated using nested PCR and direct sequencing. Three different genotypes were detected: 36.3% genotype 1 (85C/248C); 15.1% genotype 2 (85A/248C); 42.4% genotype 3 (85T/248C); and 6% mixed genotypes. Patients studied during a 1-year follow-up period showed a continuous colonisation/clearance cycle involving P. jirovecii and an accumulative tendency to be colonised with genotype 3.     

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.     

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 .     

Effectiveness of a real-time PCR for diagnosis of Pneumocystis pneumonia in immunocompromised patients – Experience from a tertiary care center,India

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening fungal infection in immunocompromised patients. Traditionally, the laboratory diagnosis of PCP relied on the visualization of organisms by microscopy as Pneumocystis cannot be readily cultured in the laboratory. The polymerase chain reaction (PCR) method is preferred over the conventional microscopic methods as PCR is rapid and found to have higher sensitivity. This retrospective study aimed to analyze the diagnostic value of a real-time PCR (qPCR) for routine diagnosis of PCP in immunocompromised patients with various underlying conditions. The qPCR targets a 121 bp fragment of P.jirovecii mitochondrial large subunit rRNA gene. The study was conducted in a 2600-bed tertiary care hospital between January and December 2019. All patients whose respiratory samples were tested for PCP by qPCR were included. The clinical diagnosis was made for each patient and categorized into PCP and non-PCP based on multi-component clinical criteria by a multi-disciplinary team. The performance characteristics of qPCR were analyzed using clinical diagnosis as the reference. A total of 339 respiratory samples from 289 patients were tested for PCP by qPCR during the study period. The overall sensitivity and specificity of qPCR were 84.75% (95% CI, 73.01% to 92.78%) and 96.1% (95% CI, 92.7 to 98.2), respectively. The sensitivity was slightly higher among HIV-infected patients (91%) than the non- HIV group (81%). The PCR exhibited higher sensitivity in bronchoalveolar lavage (BAL) (94%) than in sputum samples (81%). The colonization can be ruled out with the cycle threshold (C_T) value of below 34 with a sensitivity and specificity of 100% and 78%, respectively.The real-time PCR showed good sensitivity and specificity for routine diagnosis of PCP in patients with various underlying conditions. In addition, a cut-off C_T value (≤ 34) was determined to exclude colonization from active pneumonia.     

Community-acquired pneumonia in immunocompromised older patients: incidence,causative organisms and outcome

The number of elderly patients in the community with immunosuppressive conditions has increased progressively over recent decades. We sought to determine the incidence, causative organisms and outcome of community-acquired pneumonia (CAP) occurring in immunocompromised older patients. We prospectively compared cases of CAP in immunocompromised and non-immunocompromised patients admitted to five public hospitals in three Spanish regions. Of 320 cases studied, 115 (36%) occurred in immunocompromised patients, including: solid or hematological malignancy (97), corticosteroids or other immunosuppressive drugs (44), solid organ or stem cell transplant (five), and other conditions (eight). The etiology was established in 44% of immunocompromised patients vs. 32% of non-immunocompromlsed patients (p 0.03). Streptococcus pneumoniae was the most common causative organism in both groups (29% vs. 21%; p 0.08), followed by Legionella pneumophila (3% vs. 6%; p 0.01). Gram-negative bacilli were more frequent among immunocompromised patients (5% vs. 0.5%; p <0.01), particularly Pseudomonas aeruginosa (3% vs. 0%; p 0.04). Nocardiosis was only observed in immunocompromised patients (two cases). Bacteremia occurred similarly in the two groups. No significant differences were found with respect to ICU admission (8%, in both groups) or the length of stay (12.5 vs. 10.4 days). The early (<48 h) (3.5 vs. 0.5%; p 0.04) and overall case-fatality rates (12% vs. 3%; p <0.01) were higher in immunocompromised patients. In conclusion, a substantial number of older patients hospitalized for CAP are immunocompromised. Although relatively uncommon, CAP due to gram-negative bacilli, including P. aeruginosa, is more frequent among these patients. CAP occurring in immunocompromised patients causes significant morbidity and mortality.     

Alveolar macrophages in AIDS patients: increased spontaneous tumour necrosis factor-alpha production in Pneumocystis carinii pneumonia.

In order to assess the role of alveolar macrophages and their products in the control of Pneumocystis carinii pneumonia (PCP) and other infections in AIDS, bronchoalveolar lavage cells and peripheral blood mononuclear cells from HIV-positive AIDS/ARC patients (with and without PCP) and HIV-negative patients were counted and cultured in vitro; spontaneous and LPS-induced tumour necrosis factor-alpha (TNF-alpha) production was measured. Markedly increased spontaneous TNF-alpha production by alveolar macrophages and, to a lesser extent, peripheral blood monocytes was found in HIV-positive patients with active PCP but not in patients without the infection. Higher TNF production was associated with lower counts of Pneumocystis in the bronchoalveolar lavage fluid. These results suggest that TNF-alpha production by macrophages may play an important role in the control of Pn. carinii infection in AIDS.     

Detection of Pneumocystis jirovecii by quantitative real-time PCR in oral rinses from Pneumocystis pneumonia asymptomatic human immunodeficiency virus patients

Pneumocystis pneumonia (PCP) is a potentially life-threatening fungal infection usually seen in immunocompromised patients. Pneumocystis jirovecii can be easily detected from oral rinse samples in HIV patients with suspected PCP. In this study, a quantitative real-time PCR assay was used to establish the frequency of detection of P. jirovecii in oral rinses from HIV patients without respiratory symptoms or suspicion of PCP. Two saline oral rinses were collected from 100 ambulant HIV patients and from 60 COPD patients (comparator group). Four HIV patients were positive for P. jirovecii. In three patients, the first sample was positive and in one the second one was positive. One of these patients was on PCP prophylaxis and had a CD4⁺ count of 76 cells/mm³. The mean CD4⁺ count for all patients was 527 cells/mm³. All qRT-PCR test results for the COPD patients were negative. No patient developed PCP at six months follow-up. The qRT-PCR assay can be used to detect P. jirovecii DNA in oral rinse samples from HIV patients without evident clinical symptoms, however the oral carriage of this fungus was rare in our cohort of patients. In conclusion, although rare, a positive oral rinse P. jirovecii result may reflect colonisation, in particular in patients with HIV. This needs to be kept in mind when using oral rinses and qRT-PCR in the diagnosis of P. jirovecii infection.     

HCMV-DNA is detected more frequently than infectious virus in blood leucocytes of immunocompromised patients: a direct comparison of culture-immunofluorescence and PCR for detection of HCMV in clinical specimens.

In two studies comparing detection of human cytomegalovirus (HCMV) in 118 patients (93 of whom were immunocompromised) by the polymerase chain reaction (PCR) and virus isolation using either early antigen detection by culture-immunofluorescence or conventional cytopathic effect, DNA-PCR was found to be the most sensitive, followed by culture-immunofluorescence, then by cytopathic effect. Urine was inhibitory to the action of Taq polymerase; this was overcome by concentration of HCMV with PEG 6000 prior to gene amplification. Without PEG treatment, HCMV-DNA in 6 of the 11 specimens positive by culture-immunofluorescence was not detectable by PCR. In healthy seropositive individuals, HCMV-DNA was not detected in leucocytes. However, in immunocompromised patients with AIDS or transplants, and therefore at high risk of HCMV infection or reactivation, blood leucocytes were usually positive for HCMV-DNA (19/20), some for as long as 20 weeks after initial detection and persisting for long after culture-immunofluorescence became negative. Neither HCMV-RNA nor infectious HCMV were detected in the follow-up blood leucocyte specimens from immunocompromised patients who had detectable HCMV-DNA in these cells. These data suggest that persistence of HCMV-DNA in blood leucocytes of immunocompromised patients after reactivation or primary infection may be due to persistence of non-viable virus, residual HCMV genomic DNA, or latent HCMV-DNA.     

Usefulness of real-time PCR for lytA,ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.     

Pneumocystis carinii pneumonia in simian immunodeficiency virus infection: immunohistological and scanning and transmission electron microscopical studies

Pneumocystis carinii pneumonia occurred in 6 of 17 rhesus monkeys infected with simian immunodeficiency virus and was studied by immunohistochemistry and by scanning and transmission electron microscopy. A monoclonal antibody/streptavidin-biotin-peroxidase staining method was highly sensitive for detecting the organisms in small, early lesions and was much more sensitive and specific than traditional silver impregnation methods. Reprocessing of paraffin wax-embedded lung tissue for scanning electron microscopy and use of a video printer to produce a photographic montage of light microscopic lesions allowed the same areas of tissue to be examined and compared by both methods. The ultrastructural morphology of P. carinii in the rhesus monkey was identical to that in man, as were the histological and electron microscopic lesions, including pulmonary fibrosis. Trophozoites were seen attached to alveolar type I epithelium mainly by intimate apposition to the plasma membrane, but scanning electron microscopy also showed attachment by elongated filopodia. Few macrophages were present in infected alveoli, and though phagocytosis followed by digestion of P. carinii trophozoites was observed, it appeared to occur at a very low level.     

Leishmania donovani and Pneumocystis jirovecii (carinii) diagnosed on bronchoalveolar lavage cytology in a liver transplant recipient with Cytomegalovirus infection

Liver transplant recipients are prone to several infections, including lung infections, which can lead to substantial morbidity and mortality. Bronchoalveolar lavage (BAL) cytology is a rapid and sensitive diagnostic tool to identify the etiologic agents. We report a rare case of a 24‐year‐old male, post Live donor liver transplantation for autoimmune chronic liver disease, who presented with cough, fever, weight loss, and cavitatory lesion in lung. BAL cytology revealed Leishmania donovani (LD) and Pneumocystis jirovecii/carinii (PCP). Cytomegalovirus deoxyribonucleic acid polymerase chain reaction (CMV DNA PCR) test showed markedly raised levels. Patient was put on treatment for these multiple infections and showed significant improvement. Thus, rapid diagnosis of infections through BAL cytology is crucial in transplant recipients to institute timely therapy and avoid undesirable empirical treatments. Moreover, this case highlights a rare finding of LD bodies along with PCP in BAL cytology.     

实时PCR仪中温度控制的计算与模拟

本研究自行设计了一个PCR反应池的模型，并对该反应池和反应样品的温度进行了理论计算，在此基础上进行了温度场动态分布仿真分析，仿真结果与理论计算相吻合，为自行设计的实时PCR仪的研发提供了理论基础。     

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia.     

Merkel cell polyomavirus DNA in immunocompetent and immunocompromised patients with respiratory disease

Merkel cell polyomavirus (MCPyV) was identified originally in association with a rare but aggressive skin cancer, Merkel cell carcinoma. The virus has since been found in the respiratory tract of some patients with respiratory disease. However, the role of MCPyV in the causation of respiratory disease has not been established. To determine the prevalence of MCPyV in 305 respiratory samples from immunocompetent and immunocompromised patients and evaluate their contribution to respiratory diseases, specimens were screened for MCPyV using single, multiplex, or real-time PCR; co-infection with other viruses was examined. Of the 305 samples tested, 10 (3.27%) were positive for MCPyV. The virus was found in two groups of patients: in 6 (2%) nasopharyngeal aspirate samples from children aged 26 days to 7 months who were immunocompetent; and in 4 (1.3%) of nasopharyngeal aspirate samples taken from patients aged 41 to 69 years who were severely immunosuppressed from leukemia or transplant therapy. Both groups had upper or lower respiratory tract infection. Co-infections with other viruses were found in 30% of the MCPyV positive samples. The data present a pattern of infection similar to that seen with the polyomaviruses JC and BK in which the virus is acquired during childhood, probably by the respiratory route. The viruses then establish latency and become reactivated in the event of immunosuppression.