1887

Journal of Medical Microbiology logo

Volume 73, Issue 9

Development of novel broad-range pan-genus PCR assays for the detection of species No Access

Abstract

is responsible for the classical Whipple’s disease. Recently, a new species was described in a Belgian immunocompromised patient with pleuritis.

There is currently no specific molecular diagnostic test detecting other species than .

To develop and validate two broad-range pan- genus PCRs detecting both and new species.

From shotgun sequencing data of the lung tissue biopsy of the Belgian subject, we designed two PCRs targeting the 23S rRNA and genes. Prospectively, requests for PCR were tested with -specific PCRs and the two broad-range PCRs from January 2019 to November 2022.

In total, 2605 samples were tested using both the pan- 23S rRNA PCR and the -specific PCR. In addition, 833 of the 2605 samples were also tested using the pan- PCRs. Sensitivity was 78.8% and 79.7% for 23S rRNA and PCRs, as compared with the species-specific PCR. Specificity was 99.9% and 99.7% for the 23S rRNA and the PCRs, respectively. We identified a patient whose bronchoalveolar lavage tested positive with the two broad-range PCRs with >10 copies ml. Specific PCRs were negative. Known for panuveitis, this 49-year-old male presented with an eye inflammation recurrence, and a CT scan showed multiple mediastino-hilar necrotic adenopathies. Doxycyclin (1 year), hydroxychloroquin (1 year) and co-trimoxazol (1 month) treatments led to a favourable outcome.

Specific PCR exhibited better sensitivity than the pan- PCRs. However, both broad-range pan- PCRs demonstrated excellent specificity and were pivotal to identifying a new probable case of infection due to another species-level lineage.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): 16S rRNA , colonization , lymphadenopathy , molecular diagnosis , pneumopathy and whole-genome sequencing
Funding
This study was supported by the:
  • Institutional funding (Award N/A)
    • Principle Award Recipient: GilbertGreub
© 2024 The Authors
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001889
2024-09-25
2024-10-13
Loading full text...

Full text loading...

References

  1. Biagi F, Balduzzi D, Delvino P, Schiepatti A, Klersy C et al. Prevalence of Whipple’s disease in north-western Italy. Eur J Clin Microbiol Infect Dis 2015; 34:1347–1348 [View Article]
     [Google Scholar]
  2. Bai JC, Mazure RM, Vazquez H, Niveloni SI, Smecuol E et al. Whipple’s disease. Clin Gastroenterol Hepatol 2004; 2:849–860 [View Article] [PubMed]
     [Google Scholar]
  3. Marth T, Moos V, Müller C, Biagi F, Schneider T. Tropheryma whipplei infection and Whipple’s disease. Lancet Infect Dis 2016; 16:e13–22 [View Article] [PubMed]
     [Google Scholar]
  4. Marth T, Raoult D. Whipple’s disease. Lancet 2003; 361:239–246 [View Article]
     [Google Scholar]
  5. Geissdörfer W, Moos V, Moter A, Loddenkemper C, Jansen A et al. High frequency of Tropheryma whipplei in culture-negative endocarditis. J Clin Microbiol 2012; 50:216–222 [View Article] [PubMed]
     [Google Scholar]
  6. Greub G, Lepidi H, Rovery C, Casalta J-P, Habib G et al. Diagnosis of infectious endocarditis in patients undergoing valve surgery. Am J Med 2005; 118:230–238 [View Article] [PubMed]
     [Google Scholar]
  7. Gruber JR, Sarro R, Delaloye J, Surmely J-F, Siniscalchi G et al. Tropheryma whipplei bivalvular endocarditis and polyarthralgia: a case report. J Med Case Rep 2015; 9:259 [View Article] [PubMed]
     [Google Scholar]
  8. Gabus V, Grenak-Degoumois Z, Jeanneret S, Rakotoarimanana R, Greub G et al. Tropheryma whipplei tricuspid endocarditis: a case report and review of the literature. J Med Case Rep 2010; 4:245 [View Article] [PubMed]
     [Google Scholar]
  9. Greub G. A new piece added to the whipple puzzle: Tropheryma whipplei primary infection with bacteremia and cough. Clin Infect Dis 2010; 51:522–524 [View Article] [PubMed]
     [Google Scholar]
  10. Vankeerberghen A, Jonckheere S, De Raeve H, Caluwe R, De Beenhouwer H. Novel Tropheryma species in a lung biopsy sample from a kidney transplant recipient. Clin Microbiol Infect 2018; 24:548 [View Article]
     [Google Scholar]
  11. Kim M, Oh H-S, Park S-C, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351 [View Article] [PubMed]
     [Google Scholar]
  12. Yarza P, Ludwig W, Euzéby J, Amann R, Schleifer K-H et al. Update of the all-species living tree project based on 16S and 23S rRNA sequence analyses. Syst Appl Microbiol 2010; 33:291–299 [View Article] [PubMed]
     [Google Scholar]
  13. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014; 30:2114–2120 [View Article] [PubMed]
     [Google Scholar]
  14. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article]
     [Google Scholar]
  15. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 2009; 25:1754–1760 [View Article]
     [Google Scholar]
  16. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012; 28:1647–1649 [View Article]
     [Google Scholar]
  17. Schmieder R, Edwards R. Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PLoS One 2011; 6:e17288 [View Article]
     [Google Scholar]
  18. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article] [PubMed]
     [Google Scholar]
  19. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R et al. Circos: an information aesthetic for comparative genomics. Genome Res 2009; 19:1639–1645 [View Article] [PubMed]
     [Google Scholar]
  20. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M et al. Versatile and open software for comparing large genomes. Genome Biol 2004; 5:R12 [View Article] [PubMed]
     [Google Scholar]
  21. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 2013; 29:1072–1075 [View Article]
     [Google Scholar]
  22. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC et al. Primer3--new capabilities and interfaces. Nucleic Acids Res 2012; 40:e115 [View Article] [PubMed]
     [Google Scholar]
  23. Greub G, Sahli R, Brouillet R, Jaton K. Ten years of R&D and full automation in molecular diagnosis. Future Microbiol 2016; 11:403–425 [View Article] [PubMed]
     [Google Scholar]
  24. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. BLAST+: architecture and applications. BMC Bioinform 2009; 10:421 [View Article] [PubMed]
     [Google Scholar]
  25. Abdeldaim G, Svensson E, Blomberg J, Herrmann B. Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene. APMIS Acta Pathol Microbiol Immunol Scand 2016; 124:991–995 [View Article]
     [Google Scholar]
  26. Maeda Y, Goldsmith CE, Coulter WA, Mason C, Dooley JSG et al. Comparasion of five gene loci (rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ) to aid the molecular identification of viridans-group streptococci and pneumococci. Br J Biomed Sci 2011; 68:190–196 [View Article] [PubMed]
     [Google Scholar]
  27. Rubin C-J, Thollesson M, Kirsebom LA, Herrmann B. Phylogenetic relationships and species differentiation of 39 Legionella species by sequence determination of the RNase P RNA gene rnpB. Int J Syst Evol Microbiol 2005; 55:2039–2049 [View Article] [PubMed]
     [Google Scholar]
  28. Tao Y, Zhou Y, Ouyang Y, Lin HC. Association of oral streptococci community dynamics with severe early childhood caries as assessed by PCR-denaturing gradient gel electrophoresis targeting the rnpB gene. J Med Microbiol 2015; 64:936–945 [View Article] [PubMed]
     [Google Scholar]
  29. Täpp J, Thollesson M, Herrmann B. Phylogenetic relationships and genotyping of the genus Streptococcus by sequence determination of the RNase P RNA gene, rnpB. Int J Syst Evol Microbiol 2003; 53:1861–1871 [View Article] [PubMed]
     [Google Scholar]
  30. Moter A, Schmiedel D, Petrich A, Wiessner A, Kikhney J et al. Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years’ experience in a National Reference Laboratory. J Clin Microbiol 2013; 51:3858–3861 [View Article] [PubMed]
     [Google Scholar]
  31. Dreier J, Szabados F, von Herbay A, Kröger T, Kleesiek K. Tropheryma whipplei Infection of an acellular porcine heart valve bioprosthesis in a patient who did not have intestinal Whipple’s disease. J Clin Microbiol 2004; 42:4487–4493 [View Article] [PubMed]
     [Google Scholar]
  32. Fenollar F, Fournier P-E, Robert C, Raoult D. Use of genome selected repeated sequences increases the sensitivity of PCR detection of Tropheryma whipplei. J Clin Microbiol 2004; 42:401–403 [View Article] [PubMed]
     [Google Scholar]
  33. Duss F-R, Jaton K, Vollenweider P, Troillet N, Greub G. Whipple disease: a 15-year retrospective study on 36 patients with positive polymerase chain reaction for Tropheryma whipplei. Clin Microbiol Infect 2021; 27:910 [View Article] [PubMed]
     [Google Scholar]
  34. Salman AR, Salomao DR, Dalvin LA, Olsen TW, Smith WM. Ocular Whipple disease: cases diagnosed over four decades. Ocul Immunol Inflamm 20231–6 [View Article] [PubMed]
     [Google Scholar]
  35. Günther U, Moos V, Offenmüller G, Oelkers G, Heise W et al. Gastrointestinal diagnosis of classical Whipple disease: clinical, endoscopic, and histopathologic features in 191 patients. Medicine 2015; 94:e714 [View Article] [PubMed]
     [Google Scholar]
  36. Grif K, Heller I, Prodinger WM, Lechleitner K, Lass-Flörl C et al. Improvement of detection of bacterial pathogens in normally sterile body sites with a focus on orthopedic samples by use of a commercial 16S rRNA broad-range PCR and sequence analysis. J Clin Microbiol 2012; 50:2250–2254 [View Article] [PubMed]
     [Google Scholar]
  37. Zucol F, Ammann RA, Berger C, Aebi C, Altwegg M et al. Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. J Clin Microbiol 2006; 44:2750–2759 [View Article] [PubMed]
     [Google Scholar]
  38. Rampini SK, Bloemberg GV, Keller PM, Büchler AC, Dollenmaier G et al. Broad-range 16S rRNA gene polymerase chain reaction for diagnosis of culture-negative bacterial infections. Clin Infect Dis 2011; 53:1245–1251 [View Article]
     [Google Scholar]
  39. Rolain J-M, Fenollar F, Raoult D. False positive PCR detection of Tropheryma whipplei in the saliva of healthy people. BMC Microbiol 2007; 7:48 [View Article] [PubMed]
     [Google Scholar]
  40. Fenollar F, Laouira S, Lepidi H, Rolain J-M, Raoult D. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for first-line screening. Clin Infect Dis 2008; 47:659–667 [View Article] [PubMed]
     [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.001889
Loading
Development of novel broad-range pan-genus PCR assays for the detection of Tropheryma species
J. Med. Microbiol. 73, 001889 (2024); https://doi.org/10.1099/jmm.0.001889
/content/journal/jmm/10.1099/jmm.0.001889
/content/journal/jmm/10.1099/jmm.0.001889
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error