1887

Abstract

Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.

Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.

To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.

In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).

Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. spp., s spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.

Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Funding
This study was supported by the:
  • National Institute for Health and Care Research (Award NIHR300448)
    • Principle Award Recipient: KathrynAnn Harris
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001633
2022-12-19
2024-12-14
Loading full text...

Full text loading...

/deliver/fulltext/jmm/71/12/jmm001633.html?itemId=/content/journal/jmm/10.1099/jmm.0.001633&mimeType=html&fmt=ahah

References

  1. Harris KA, Hartley JC. Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol 2003; 52:685–691 [View Article] [PubMed]
    [Google Scholar]
  2. Saglani S, Harris KA, Wallis C, Hartley JC. Empyema: the use of broad range 16S rDNA PCR for pathogen detection. Arch Dis Child 2005; 90:70–73 [View Article] [PubMed]
    [Google Scholar]
  3. Harris KA, Fidler KJ, Hartley JC, Vogt J, Klein NJ et al. Unique case of Helicobacter sp. osteomyelitis in an immunocompetent child diagnosed by broad-range 16S PCR. J Clin Microbiol 2002; 40:3100–3103 [View Article] [PubMed]
    [Google Scholar]
  4. Bharathan B, Backhouse L, Rawat D, Naik S, Millar M. An unusual case of seronegative, 16S PCR positive Brucella infection. JMM Case Rep 2016; 3:e005050 [View Article] [PubMed]
    [Google Scholar]
  5. Paim AC, Baddour LM, Pritt BS, Schuetz AN, Wilson JW. Lyme Endocarditis. Am J Med 2018; 131:1126–1129 [View Article] [PubMed]
    [Google Scholar]
  6. McNicol M, Yew P, Beattie G, Loughlin L. A case of Capnocytophaga canimorsus endocarditis in A non-immunosuppressed host: the value of 16S PCR for diagnosis. Access Microbiol 2021; 3:000235 [View Article] [PubMed]
    [Google Scholar]
  7. Scagnolari C, Turriziani O, Monteleone K, Pierangeli A, Antonelli G. Consolidation of molecular testing in clinical virology. Expert Rev Anti Infect Ther 2017; 15:387–400 [View Article] [PubMed]
    [Google Scholar]
  8. Patel A, Harris KA, Fitzgerald F. What is broad-range 16S rdna PCR?. Arch Dis Child Educ Pract Ed 2017; 102:261–264 [View Article]
    [Google Scholar]
  9. Lampejo T, Ciesielczuk H, Lambourne J. Clinical utility of 16S rRNA PCR in pleural infection. J Med Microbiol 2021; 70: [View Article] [PubMed]
    [Google Scholar]
  10. Harris KA, Turner P, Green EA, Hartley JC. Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility. J Clin Microbiol 2008; 46:2751–2758 [View Article] [PubMed]
    [Google Scholar]
  11. Tann CJ, Nkurunziza P, Nakakeeto M, Oweka J, Kurinczuk JJ et al. Prevalence of bloodstream pathogens is higher in neonatal encephalopathy cases vs. controls using a novel panel of real-time PCR assays. PLoS One 2014; 9:e97259 [View Article] [PubMed]
    [Google Scholar]
  12. Oeser C, Pond M, Butcher P, Bedford Russell A, Henneke P et al. PCR for the detection of pathogens in neonatal early onset sepsis. PLoS One 2020; 15:e0226817 [View Article] [PubMed]
    [Google Scholar]
  13. Jones HE, Harris KA, Azizia M, Bank L, Carpenter B et al. Differing prevalence and diversity of bacterial species in fetal membranes from very preterm and term labor. PLoS One 2009; 4:e8205 [View Article] [PubMed]
    [Google Scholar]
  14. Harris KA, Yam T, Jalili S, Williams OM, Alshafi K et al. Service evaluation to establish the sensitivity, specificity and additional value of broad-range 16S rDNA PCR for the diagnosis of infective endocarditis from resected endocardial material in patients from eight UK and Ireland hospitals. Eur J Clin Microbiol Infect Dis 2014; 33:2061–2066 [View Article] [PubMed]
    [Google Scholar]
  15. Chakravorty S, Helb D, Burday M, Connell N, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J Microbiol Methods 2007; 69:330–339 [View Article] [PubMed]
    [Google Scholar]
  16. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ et al. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 2001; 39:1553–1558 [View Article] [PubMed]
    [Google Scholar]
  17. Chometon S, Benito Y, Chaker M, Boisset S, Ploton C et al. Specific real-time polymerase chain reaction places Kingella kingae as the most common cause of osteoarticular infections in young children. Pediatr Infect Dis J 2007; 26:377–381 [View Article] [PubMed]
    [Google Scholar]
  18. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB et al. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 2000; 38:1747–1752 [View Article] [PubMed]
    [Google Scholar]
  19. Hatrongjit R, Akeda Y, Hamada S, Gottschalk M, Kerdsin A. Multiplex PCR for identification of six clinically relevant streptococci. J Med Microbiol 2017; 66:1590–1595 [View Article] [PubMed]
    [Google Scholar]
  20. Fitzgerald F, Harris K, Henderson R, Edelsten C. Group A streptococcal endophthalmitis complicating A sore throat in A 2-year-old child. BMJ Case Rep 2015; 2015:bcr2014208168 [View Article] [PubMed]
    [Google Scholar]
  21. Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G et al. Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. N Engl J Med 2019; 380:2327–2340 [View Article]
    [Google Scholar]
  22. Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Infect 2018; 76:225–240 [View Article] [PubMed]
    [Google Scholar]
  23. Duan H, Li X, Mei A, Li P, Liu Y et al. The diagnostic value of metagenomic next⁃generation sequencing in infectious diseases. BMC Infect Dis 2021; 21:62 [View Article] [PubMed]
    [Google Scholar]
  24. O’Grady J. A powerful, non-invasive test to rule out infection. Nat Microbiol 2019; 4:554–555 [View Article] [PubMed]
    [Google Scholar]
  25. Greninger AL. The challenge of diagnostic metagenomics. Expert Rev Mol Diagn 2018; 18:605–615 [View Article] [PubMed]
    [Google Scholar]
  26. Deng X, Achari A, Federman S, Yu G, Somasekar S et al. Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance. Nat Microbiol 2020; 5:443–454 [View Article] [PubMed]
    [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.001633
Loading
/content/journal/jmm/10.1099/jmm.0.001633
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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