AIM: In the past, Pneumocystis jirovecii belonged to the Protozoa group, but is currently taxonomically included in the kingdom Fungi. P. jirovecii is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine and evaluate the reliability of methods of laboratory diagnosis of pneumocystosis used in routine laboratories as well as the occurrence of this disease in patients from Slovakia during 19 years. MATERIAL AND METHODS: The diagnosis is based on microscopic examination (Giemsa- and Gram-Weigert-staining) and detection of parasite DNA by classical or real-time PCR in bronchoalveolar lavage and sputum. RESULTS: Pneumocysts were detected in 190 persons (5.7%) from the whole group of patients. Cancer patients represented the riskiest group in terms of pneumocystosis, which was confirmed by the highest percentage (57.9%) of individuals infected with P. jirovecii. Compared with the PCR, 33.7% sensitivity and 100% specificity of microscopy was calculated by using a binary classification test. Molecular methods are more sensitive in the detection of P. jirovecii compared to microscopic evidence and currently represent a reliable detection system in the diagnosis of pneumocystosis. CONCLUSION: In view of the increasing number of immunocompromised persons, diagnostics of P. jirovecii in patients with pulmonary complications is essential. This was also confirmed in our study, where the number of examinations and detection of this opportunistic pathogen increased over the years.
- Klíčová slova
- Pneumocystis jirovecii, microscopic evidence, pneumocystosis, polymerase chain reaction (PCR),
- MeSH
- bronchoalveolární lavážní tekutina mikrobiologie MeSH
- imunokompromitovaný pacient MeSH
- incidence MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- Pneumocystis carinii * genetika MeSH
- pneumocystová pneumonie * diagnóza epidemiologie mikrobiologie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.
- Klíčová slova
- Pneumocystis jirovecii, Cq, threshold, DNA, PCP, diagnosis, efficiency, panel specimens, pneumocystosis, qPCR, quantification, quantification cycles, standardization, whole nucleic acids,
- MeSH
- bronchoalveolární lavážní tekutina mikrobiologie MeSH
- diagnostické techniky molekulární metody normy MeSH
- DNA fungální genetika MeSH
- kvantitativní polymerázová řetězová reakce normy MeSH
- lidé MeSH
- Pneumocystis carinii genetika MeSH
- pneumocystová pneumonie diagnóza mikrobiologie MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- multicentrická studie MeSH
- Názvy látek
- DNA fungální MeSH
The present study aims to evaluate the diagnostic yield of bronchoalveolar lavage (BAL) fluid in patients with hematological malignancies and describe the most common pathogens detected in BAL fluid (BALF.) An analysis of 480 BALF samples was performed in patients with hematological malignancies over a period of 7 years. The results of culture methods, PCR, and immunoenzymatic sandwich microplate assays for Aspergillus galactomannan (GM) in BALF were analyzed. Further, the diagnostic thresholds for Aspergillus GM and Pneumocystis jiroveci were also calculated. Microbiological findings were present in 87% of BALF samples. Possible infectious pathogens were detected in 55% of cases; 32% were classified as colonizing. No significant difference in diagnostic yield or pathogen spectrum was found between non-neutropenic and neutropenic patients. There was one significant difference in BALF findings among intensive care units (ICU) versus non-ICU patients for Aspergillus spp. (22% versus 9%, p = 0.03). The most common pathogens were Aspergillus spp. (n = 86, 33% of BAL with causative pathogens) and Streptococcus pneumoniae (n = 46, 18%); polymicrobial etiology was documented in 20% of cases. A quantitative PCR value of > 1860 cp/mL for Pneumocystis jirovecii was set as a diagnostic threshold for pneumocystis pneumonia. The absorbance index of GM in BALF of 0.5 was set as a diagnostic threshold for aspergillosis. The examination of BAL fluid revealed the presence of pathogen in more than 50% of cases and is, therefore, highly useful in this regard when concerning pulmonary infiltrates.
- MeSH
- Aspergillus genetika izolace a purifikace patogenita MeSH
- bronchoalveolární lavážní tekutina mikrobiologie MeSH
- DNA fungální genetika MeSH
- dospělí MeSH
- galaktosa analogy a deriváty MeSH
- hematologické nádory komplikace mikrobiologie MeSH
- jednotky intenzivní péče MeSH
- lidé středního věku MeSH
- lidé MeSH
- mannany analýza MeSH
- mladiství MeSH
- mladý dospělý MeSH
- neutropenie mikrobiologie MeSH
- Pneumocystis carinii genetika izolace a purifikace patogenita MeSH
- pneumocystová pneumonie diagnóza mikrobiologie MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Streptococcus pneumoniae genetika izolace a purifikace patogenita MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA fungální MeSH
- galactomannan MeSH Prohlížeč
- galaktosa MeSH
- mannany MeSH
Pneumocystis jirovecii is an airborne human-specific ascomycetous fungus responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, affecting >500,000 patients per year (www.gaffi.org). The understanding of its epidemiology is limited by the lack of standardised culture. Recent genotyping data suggests a limited genetic diversity of P. jirovecii. The objective of the study was to assess the diversity of P. jirovecii across European hospitals and analyse P. jirovecii diversity in respect to clinical data obtained from the patients. Genotyping was performed using six already validated short tandem repeat (STR) markers on 249 samples (median: 17 per centre interquartile range [11-20]) from PCP patients of 16 European centres. Mixtures of STR markers (i.e., ≥2 alleles for ≥1 locus) were detected in 67.6% (interquartile range [61.4; 76.5]) of the samples. Mixture was significantly associated with the underlying disease of the patient, with an increased proportion in HIV patients (78.3%) and a decreased proportion in renal transplant recipients (33.3%) (p<0.001). The distribution of the alleles was significantly different (p<0.001) according to the centres in three out of six markers. In analysable samples, 201 combinations were observed corresponding to 137 genotypes: 116 genotypes were country-specific; 12 in two; six in three; and two in four and one in five countries. Nine genotypes were recorded more than once in a given country. Genotype 123 (Gt123) was significantly associated with France (14/15, p<0.001) and Gt16 with Belgium (5/5, p<0.001). More specifically, Gt123 was observed mainly in France (14/15/16 patients) and in renal transplant patient (13/15). Our study showed the wide population diversity across Europe, with evidence of local clusters of patients harbouring a given genotype. These data suggest a specific association between genotype and underlying disease, with evidence of a different natural history of PCP in HIV patients and renal transplant recipients.
- Klíčová slova
- Europe, Genotyping, MLS typing, Microsatellites, Mixed infection, Pneumocystis jirovecii, Transmission,
- MeSH
- DNA fungální genetika MeSH
- dospělí MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- genetická variace MeSH
- genotypizační techniky metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrosatelitní repetice MeSH
- Pneumocystis carinii klasifikace genetika izolace a purifikace MeSH
- pneumocystová pneumonie mikrobiologie MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- pozorovací studie MeSH
- Geografické názvy
- Evropa MeSH
- Názvy látek
- DNA fungální MeSH
For many years Pneumocystis pneumonia was thought to be caused by the reactivation of a latent infection, but several studies have demonstrated that Pneumocystis jirovecii infection can be acquired de novo. On the basis of our results obtained from a patient with recurrent pneumocystosis, we support the hypothesis that recurrent episodes are caused by reinfection.
- MeSH
- geny hub MeSH
- hematologické nádory mikrobiologie MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- mykologické určovací techniky MeSH
- Pneumocystis carinii genetika izolace a purifikace MeSH
- pneumocystová pneumonie komplikace mikrobiologie MeSH
- polymerázová řetězová reakce MeSH
- recidiva MeSH
- RNA mitochondriální MeSH
- RNA ribozomální genetika MeSH
- RNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- RNA mitochondriální MeSH
- RNA ribozomální MeSH
- RNA MeSH
