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Editorial Comment

From 1990 to 2020

The 30-Year History of the Identification of Difficult to Culture Pathogens

Rodriguez-Noriega, Eduardo MD, PhD; Morfin-Otero, Rayo MD, PhD

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Infectious Diseases in Clinical Practice: May 2020 - Volume 28 - Issue 3 - p 121-122
doi: 10.1097/IPC.0000000000000850
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Written as an editorial commentary regarding El-Dalati et al. The Clinical Impact of 16S Ribosomal RNA Polymerase Chain Reaction Bacterial Sequencing in Infectious Endocarditis: A Single Center Experience pages 138–141 of the Journal.

The introduction of molecular techniques, including the use of the 16s-targeted ribosomal ribonucleic acid (rRNA) polymerase chain reactions (16s PCR) have provided the opportunity to more easily identify the causative microorganisms of infectious diseases that were previously unknown, including uncultured pathogens, culture-negative bacterial, and fungal infections. In a landmark study in 1990, Relman and colleagues1 identified Bartonella henselae, the bacterium responsible for bacillary angiomatosis, which occurred most often in immunosuppressed patients, especially those with human immunodeficiency virus. Further, Relman's team identified an uncultured pathogen observed in liver histopathological specimens from patients with peliosis hepatis, where the proliferation of small vessels coexisted with clusters of bacilli.1 These groundbreaking findings opened the doors to a new world of investigation into detection of microorganisms and ultimately, the treatment and prevention of disease, including the microbial etiology of Whipple's disease, Tropheryma whippelii.2

In 2000, 10 years after Relman and colleagues identified the microbes responsible for 3 different diseases, Drancourt et al3 used 16s PCR sequencing to identify 159 (89.9%) of 177 strains of previously unidentifiable microbial isolates with a sequence similarity of more than 97% and with a similarity score of more than 99% in 139 isolates. This new 16s PCR technology also provided the opportunity to better understand clinical problems, including culture-negative bacterial meningitis. For examples, in 227 cerebrospinal fluid samples obtained following antimicrobial therapy for culture-negative meningitis, 16s PCR indicated a sensitivity of 86%, specificity of 97%, positive predictive value of 80%, and negative predictive value of 98% compared with culture techniques.4

In a recent study, metagenomic next-generation sequencing of 204 pediatric and adult cerebrospinal fluid samples accurately identified etiology in 13 (22%) specimens of which previous clinical testing was not capable of identifying.5 In 7 (54%) specimens, results led to the initiation of appropriate and direct treatment.5 In another study of 394 clinical specimens obtained from culture-negative bacterial infections, assessed by 16s PCR, the specimens had over 90% concordance with routine bacterial culture. The results of 231 negative bacterial cultured samples obtained from patients' clinical data suggest 16s PCR had a sensitivity of 42.9%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 80.2%.6

In a study comparing blood culture and universal 16s PCR/pyrosequencing's ability to detect bacteria directly from blood, the sensitivity of 16s PCR was 77.8%, specificity was 99.3%, positive predictive value was 93.3%, and negative predictive value was 97.2%. Further, 16s PCR only required 7.5 hours to complete, whereas 27.9 hours were needed to complete Gram staining, and 81.6 hours were needed for phenotypic identification.7 In a recent study using a universal broad-range (bacterial/mycobacterial 16s rRNA, fungal 26s) PCR amplicon sequencing, in 1062 clinical samples, 107 of the total samples (10.1%) had clinically significant positive results, generating management changes in 44 of the total samples (1062 [4.1%]).8

El-Dalati and colleagues9 performed a 4.5-year retrospective analysis of all microbial 16s PCR results obtained after cardiac valves surgery to determine if treatment plans were altered based on the 16s PCR results. Forty-one (24.8%) of the 165 surgical endocarditis cases were included in the analysis; and of those 41 patients, 18 (43.9%) were positive for endocarditis, 24 were considered negative culture endocarditis, and 5 cases (12.2%) included changes in their management plans based on the 16s PCR findings.9 These results are similar to those described by Marin et al10 in which 16s PCR in heart valve samples with negative blood cultures detected Tropheryma whippelii, Bartonella quintana, and Streptococcus gallolyticus.

In a 2014 report that included 151 patients from 8 hospitals in the United Kingdom and Ireland, 16s PCR detected causative agents in 46 of 69 heart valves of culture-negative infectious endocarditis, with sensitivity, specificity, positive predictive value, and negative predictive value of 67%, 91%, 96%, and of 46%, respectively.11 In 2015, Shrestha and colleagues reported that valve sequencing was more sensitive than valve culturing in identifying the causative pathogen of endocarditis (90% vs 31%, P < 0.001), had fewer false-positive results (3% vs 33%, P < 0.001), and was able to identify 15% of pathogens missed.12 In a 5-year study that included 283 patients with blood culture-negative endocarditis, the addition of real-time PCR increased diagnostic efficacy by 24.3%.13 Further, in 2017, 16s PCR sequencing of excised heart valves from patients with infectious endocarditis indicated that culture sensitivity was 26% compared with 87% sensitivity for blood cultures and PCR.14 In excised cardiac valves, valve sequencing, not culture, should be considered the primary test for bacterial identification, and molecular testing should be a main component of the diagnostic criteria for infectious endocarditis.12,14

Between 2001 and 2007, over 215 novel bacterial species have been discovered using 16s sequencing, opening up the field of metagenomics to new research opportunities.5,15 Although this technology is not yet available for widespread use, its current use in clinical settings provides the opportunity to more accurately identify causative pathogens, leading to better patient diagnostics, treatment management, and follow-up, especially for patients with vital organ infections.


1. Relman DA, Loutit JS, Schmidt TM, et al. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med. 1990;323:1573–1580.
2. Relman DA, Schmidt TM, MacDermott RP, et al. Identification of the uncultured bacillus of Whipple's disease. N Engl J Med. 1992;327:293–301.
3. Drancourt M, Bollet C, Carlioz A, et al. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol. 2000;38:3623–3630.
4. Schuurman T, de Boer RF, Kooistra-Smid AM, et al. Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting. J Clin Microbiol. 2004;42:734–740.
5. Wilson MR, Sample HA, Zorn KC, et al. Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. N Engl J Med. 2019;380:2327–2340.
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7. Moore MS, McCarroll MG, McCann CD, et al. Direct screening of blood by PCR and pyrosequencing for a 16S rRNA gene target from emergency department and intensive care unit patients being evaluated for bloodstream infection. J Clin Microbiol. 2016;54:99–105.
8. Kerkhoff AD, Rutishauser RL, Miller S, et al. Clinical utility of universal broad-range PCR amplicon sequencing for pathogen identification: a retrospective cohort study. Clin Infect Dis. 2020; pii: ciz1245. doi: 10.1093/cid/ciz1245.
9. El-Dalati S, Riddell J, Fagan C, et al. The clinical impact of 16s rRNA PCR bacterial sequencing in infectious endocarditis: a single Center experience. Infect Dis Clin Pract. 2020;28(3):138–141.
10. Marin M, Munoz P, Sanchez M, et al. Molecular diagnosis of infective endocarditis by real-time broad-range polymerase chain reaction (PCR) and sequencing directly from heart valve tissue. Medicine (Baltimore). 2007;86:195–202.
11. Harris KA, Yam T, Jalili S, 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.
12. Shrestha NK, Ledtke CS, Wang H, et al. Heart valve culture and sequencing to identify the infective endocarditis pathogen in surgically treated patients. Ann Thorac Surg. 2015;99:33–37.
13. Fournier PE, Gouriet F, Casalta JP, et al. Blood culture-negative endocarditis: improving the diagnostic yield using new diagnostic tools. Medicine (Baltimore). 2017;96:e8392.
14. Peeters B, Herijgers P, Beuselinck K, et al. Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study. Clin Microbiol Infect. 2017;23:888 e1–888.e5.
15. Woo PC, Lau SK, Teng JL, et al. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect. 2008;14:908–934.
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