Reliable differentiation of Pneumocystis pneumonia from Pneumocystis colonisation by quantification of Major Surface Glycoprotein gene using real‐time polymerase chain reaction

Rudramurthy, Shivaprakash M.; Sharma, Megha; Sharma, Manisha; Rawat, Pankaj; Ghosh, Anup; Venkatesan, Lakshmishree; Aggarwal, Ritesh; Singh, Meenu; Chakrabarti, Arunaloke

doi:10.1111/myc.12708

Cited by 9 publications

(14 citation statements)

References 33 publications

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“…The finding that P. jirovecii DNA load was significantly higher in the clinical PCP group than in the other groups shows that real-time PCR can be helpful in discriminating active PCP from colonization. Our data confirm previous findings of a lower fungal burden in colonized patients than in PCP patients [5817181920]. In this context, DNA copy number or Ct values have been suggested as cut-off values of fungal burden for differentiating PCP from colonization [4691721].…”

Section: Discussionsupporting

confidence: 91%

Comparative Evaluation Between the RealStarPneumocystis jiroveciiPCR Kit and the AmpliSensPneumocystis jirovecii(carinii)-FRT PCR Kit for DetectingP. jiroveciiin Non-HIV Immunocompromised Patients

Huh

Lim

et al. 2019

Ann Lab Med

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Background Real-time PCR is more sensitive than microscopic examination for detecting Pneumocystis jirovecii . We compared the performance of two assays for detecting P. jirovecii DNA: the RealStar Pneumocystis jirovecii PCR Kit 1.0 CE (Altona Diagnostics, Hamburg, Germany) and the AmpliSens Pneumocystis jirovecii ( carinii )-FRT PCR kit (InterLabService Ltd., Moscow, Russia). Methods We used 159 samples from the lower respiratory tract (112 bronchoalveolar lavage [BAL] fluid, 37 sputum, and 10 endotracheal aspirate [ETA] samples) of non-HIV immunocompromised patients. Nested PCR and sequencing were used to resolve discordant results. The performance of the two assays was evaluated according to clinical categories (clinical Pneumocystis pneumonia [PCP], possible PCP, or unlikely PCP) based on clinical and radiological observations. Results The positive and negative percent agreement values were 100% (95% confidence interval [CI], 85.4–100%) and 96.6% (95% CI, 90.9–98.9%), respectively, and kappa was 0.92 (95% CI, 0.84–0.99). P. jirovecii DNA load was significantly higher in the clinical PCP group than in the other groups ( P <0.05). When stratified by sample type, the positive rate for BAL fluids from the clinical PCP group was 100% using either assay, whereas the positive rate for sputum/ETA samples was only 20%. Conclusions The two assays showed similar diagnostic performance and detected low P. jirovecii burden in BAL fluids. Both assays may be useful as routine methods for detecting P. jirovecii DNA in a clinical laboratory setting, though their results should be interpreted considering sample type.

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Section: Discussionsupporting

confidence: 91%

Comparative Evaluation Between the RealStarPneumocystis jiroveciiPCR Kit and the AmpliSensPneumocystis jirovecii(carinii)-FRT PCR Kit for DetectingP. jiroveciiin Non-HIV Immunocompromised Patients

Huh

Lim

et al. 2019

Ann Lab Med

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“…To date, we found 31 articles in PubMed that established a cut-off for molecular diagnosis of Pj using qPCR. Among them, 19 were retrospective [4,14,16,19,20,22,23,26,[42][43][44][45][46][47][48][49][50][51], 9 were prospective [13,15,18,21,[23][24][25]52,53], and 3 were mixed [12,54,55]. In 26 of them, authors accepted the diagnosis of PjP when one or more of the following criteria were present: clinical presentation suggestive of PjP, adequate response to PjP treatment, lung images suggestive of pneumonia caused by Pj, and/or a microscopy-positive Pj sample.…”

Section: Discussionmentioning

confidence: 99%

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

Aguilar

Rueda

Maya

et al. 2021

JoF

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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.

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“…Several PCR and real-time PCR assays have been standardized targeting different single-copy genes such as superoxide dismutase ( sod ) ( Morilla et al, 2019 ), dihydrofolate reductase ( dhfr ) ( Alanio et al, 2016 ), cytochrome c oxidase ( cox-1 ), or the kexin-like serine protease ( kex1 ) gene ( Dunaiski et al, 2018 ). Increased detection sensitivity has also been reported using multicopy genes, such as the mitochondrial large subunit ribosomal RNA ( mtLSUrRNA ) ( Morris et al, 2008 ; Alanio et al, 2011 ; Fraczek et al, 2019 ; Chotiprasitsakul et al, 2020 ; Yang et al, 2020 ), the mitochondrial small subunit ribosomal RNA ( mtSSUrRNA ) ( Dellière et al, 2019 ) or the major surface glycoprotein ( Msg ) gene family ( Rudramurthy et al, 2018 ; Bossart et al, 2020 ; Gits-Muselli et al, 2020 ). In addition, multiplex real-time PCR methods have also been developed where more than one gene is detected to characterize P. jirovecii specific single-nucleotide polymorphisms located at different loci ( Esteves et al, 2011 ; Montesinos et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies have attempted to use the burden of P. jirovecii to differentiate between acceptable levels of colonization and those where P. jirovecii pneumonia (PcP) represents a health risk ( Rudramurthy et al, 2018 ). However, even if standardized underlying causes of immunosuppression are met, the Pneumocystis burden in itself is not specific enough to define PcP.…”

Section: Introductionmentioning

confidence: 99%

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A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

et al. 2022

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Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

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Reliable differentiation of Pneumocystis pneumonia from Pneumocystis colonisation by quantification of Major Surface Glycoprotein gene using real‐time polymerase chain reaction

Cited by 9 publications

References 33 publications

Comparative Evaluation Between the RealStarPneumocystis jiroveciiPCR Kit and the AmpliSensPneumocystis jirovecii(carinii)-FRT PCR Kit for DetectingP. jiroveciiin Non-HIV Immunocompromised Patients

Comparative Evaluation Between the RealStarPneumocystis jiroveciiPCR Kit and the AmpliSensPneumocystis jirovecii(carinii)-FRT PCR Kit for DetectingP. jiroveciiin Non-HIV Immunocompromised Patients

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

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

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