Comparison of In-house Real-time Assay and MucorGenius Assay performance for testing of Clinical Samples from Immunocompromised Patients

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F65269705%3A_____%2F19%3A00071805" target="_blank" >RIV/65269705:_____/19:00071805 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.mdpi.com/2309-608X/5/4/95" target="_blank" >https://www.mdpi.com/2309-608X/5/4/95</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3390/jof5040095" target="_blank" >10.3390/jof5040095</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Comparison of In-house Real-time Assay and MucorGenius Assay performance for testing of Clinical Samples from Immunocompromised Patients

Popis výsledku v původním jazyce

Objectives: Diagnostics of infections caused by mucormycetes is still quite complicated because no serological tests (unlike in aspergillosis) are available. Therefore, PCR methods are currently the methods of choice. The aim of this study was to compare MucorGenius assay results (Pathonostics) on samples that previously tested positively using our in-house PCRs. Methods: In our laboratory, we use nested real-time PCR assay followed by high-resolution melting analysis (RT-HRM) for PCR screening of clinical samples from patients at risk of invasive mucormycosis. This assay enables not only detection but also identification of detected species. If the result is positive, we confirm the result and determine the fungal load with species-specific real-time quantitative PCR (RQ-PCR). In this study, we tested 29 selected clinical samples (10 tissues, 15 bronchoalveolar lavage (BAL)samples, 3 cerebrospinal fluid samples and 1 endosecret), 25 that had been tested as positive using our screening assay and 4 mucormycetes negative samples. The results were compared to tests with the MucorGenius assay. Results: Out of 25 RT-HRM positive samples, 21 were positive using species-specific RQ-PCR. A significant fungal load (Ct pounds 35) was detected in 10 samples. Using MucorGenius assay, 17 out of 25 samples (68%) were positive (Ct 18.1 - 37.3). RT-HRM detected Rhizopus sp. (17 samples), Mucor sp. (3 samples), Lichtheimia corymbifera (2 samples), Lichtheimia ramosa (2 samples) and Rhizomucor sp. (1 sample). 12 out of 17 samples with Rhizopus sp. detected revealed low fungal load or were negative. To analyze this discrepancy, sequencing was performed and in 10 samples and R. stolonifer was identified (which is not targeted by in-house RQ-PCR). The MucorGenius assay was positive in 7/10 R. stolonifer positive samples (Ct 31.0-37.3). Conclusion: Using commercial assays for PCR diagnostics is currently preferred because of standardization; therefore we compared our in-house assay results with a commercial kit for mucormycetes detection. In this study, we also analyzed samples with discrepant results and identified R. stolonifer as a frequent finding when testing BAL samples. As this species is rarely described as a cause of infection, its detection most probably represents colonization or contamination. Based on these results, we added R. stolonifer specific RQ-PCR to our diagnostic algorithm (if RT-HRM detects Rhizopus sp. than R. microsporus, R. oryzae and R. stolonifer RQ-PCRs are used to verify the results). Due to our experience, the MucorGenius kit is easy to use, as it is a one tube real-time PCR assay and has very good sensitivity. The main disadvantage is the inability to identify mucormycetes species. Although this information is not critical to initiating treatment, it is essential for epidemiological reasons and might help to distinguish colonization/contamination from a truly ongoing infection.

Název v anglickém jazyce

Comparison of In-house Real-time Assay and MucorGenius Assay performance for testing of Clinical Samples from Immunocompromised Patients

Popis výsledku anglicky

Objectives: Diagnostics of infections caused by mucormycetes is still quite complicated because no serological tests (unlike in aspergillosis) are available. Therefore, PCR methods are currently the methods of choice. The aim of this study was to compare MucorGenius assay results (Pathonostics) on samples that previously tested positively using our in-house PCRs. Methods: In our laboratory, we use nested real-time PCR assay followed by high-resolution melting analysis (RT-HRM) for PCR screening of clinical samples from patients at risk of invasive mucormycosis. This assay enables not only detection but also identification of detected species. If the result is positive, we confirm the result and determine the fungal load with species-specific real-time quantitative PCR (RQ-PCR). In this study, we tested 29 selected clinical samples (10 tissues, 15 bronchoalveolar lavage (BAL)samples, 3 cerebrospinal fluid samples and 1 endosecret), 25 that had been tested as positive using our screening assay and 4 mucormycetes negative samples. The results were compared to tests with the MucorGenius assay. Results: Out of 25 RT-HRM positive samples, 21 were positive using species-specific RQ-PCR. A significant fungal load (Ct pounds 35) was detected in 10 samples. Using MucorGenius assay, 17 out of 25 samples (68%) were positive (Ct 18.1 - 37.3). RT-HRM detected Rhizopus sp. (17 samples), Mucor sp. (3 samples), Lichtheimia corymbifera (2 samples), Lichtheimia ramosa (2 samples) and Rhizomucor sp. (1 sample). 12 out of 17 samples with Rhizopus sp. detected revealed low fungal load or were negative. To analyze this discrepancy, sequencing was performed and in 10 samples and R. stolonifer was identified (which is not targeted by in-house RQ-PCR). The MucorGenius assay was positive in 7/10 R. stolonifer positive samples (Ct 31.0-37.3). Conclusion: Using commercial assays for PCR diagnostics is currently preferred because of standardization; therefore we compared our in-house assay results with a commercial kit for mucormycetes detection. In this study, we also analyzed samples with discrepant results and identified R. stolonifer as a frequent finding when testing BAL samples. As this species is rarely described as a cause of infection, its detection most probably represents colonization or contamination. Based on these results, we added R. stolonifer specific RQ-PCR to our diagnostic algorithm (if RT-HRM detects Rhizopus sp. than R. microsporus, R. oryzae and R. stolonifer RQ-PCRs are used to verify the results). Due to our experience, the MucorGenius kit is easy to use, as it is a one tube real-time PCR assay and has very good sensitivity. The main disadvantage is the inability to identify mucormycetes species. Although this information is not critical to initiating treatment, it is essential for epidemiological reasons and might help to distinguish colonization/contamination from a truly ongoing infection.

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

<a href="/cs/project/TE02000058" target="_blank" >TE02000058: Centrum kompetence pro molekulární diagnostiku a personalizovanou medicínu</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů