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Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
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Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

Published on: November 10, 2023

Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a

F Esteves1, J Gaspar, B De Sousa

  • 1Unidade de Protozoários Oportunistas/VIH e Outras Protozooses-CMDT, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 1349-008 Lisboa, Portugal.

Journal of Clinical Microbiology
|March 11, 2011
PubMed

Insights

Pneumocystis jirovecii pneumonia (PcP) genetic markers, including dihydrofolate reductase (DHFR) and superoxide dismutase (SOD) single-nucleotide polymorphisms (SNPs), correlate with disease severity. A new multiplex PCR/single-base extension method accurately genotypes these SNPs for PcP prognosis.

Area of Science:

  • Medical Mycology
  • Molecular Diagnostics
  • Infectious Diseases

Background:

  • Pneumocystis jirovecii pneumonia (PcP) is a significant respiratory illness in AIDS patients.
  • Genetic variations in P. jirovecii may influence disease presentation and outcome.
  • Accurate genotyping of P. jirovecii is crucial for understanding PcP pathogenesis and prognosis.

Purpose of the Study:

  • To develop and validate a rapid, accurate method for genotyping P. jirovecii single-nucleotide polymorphisms (SNPs).
  • To investigate the correlation between specific P. jirovecii SNPs (DHFR, mtLSU rRNA, SOD) and PcP clinical data, including parasite burden.
  • To assess the potential of these SNPs as predictors for PcP outcomes.

Main Methods:

  • Multiplex-PCR (MPCR) combined with single-base extension (SBE) techniques (acrylamide and capillary electrophoresis) for genotyping P. jirovecii SNPs.
  • Direct sequencing as a standard method for comparison.
  • Statistical and cluster analysis to identify associations among SNPs and correlate them with clinical data.

Main Results:

  • High concordance (96.9%-98.4%) between MPCR/SBE and direct sequencing methods.
  • Identified significant genetic associations between SNPs, such as mt85C-SOD110T and SOD110T-SOD215C.
  • Correlated specific SNP profiles with PcP parasite burden: DHFR312C, mt85C, SOD110T, SOD215C in low/moderate burden; DHFR312T, mt85T, SOD110C, SOD215T in high burden.

Conclusions:

  • The MPCR/SBE protocol is a rapid and accurate method for P. jirovecii SNP genotyping.
  • Studied P. jirovecii polymorphisms are potential genetic predictors of PcP clinical data and outcomes.
  • These findings can aid clinical decision-making and epidemiological studies for PcP management.

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