CC BY
12MIRJournal
Microbiology Independent Research Journal REVIEW
DOI: 10.18527/2500-2236-2021-8-1-41-49.
A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis
Nikita D. Yolshin
Smorodintsev Research Institute of Influenza, 15/17, Prof. Popov street, Saint Petersburg, 197376, Russia
ABSTRACT
Osteomyelitis is a rare disease that is often caused by an infection. In case of microbiology analyses failure, molecular assay seems appropriate for the identification of the pathogen. Broad-range PCR is a popular tool to amplify the gene of 16S ribosomal RNA - the component of the 30S subunit of the bacterial ribosome present in various species. The subsequent sequencing of the amplified gene enables scientists to determine the bacteria species. In this review, we discuss studies and case reports where the osteomyelitis causative agent was revealed by means of broad-range PCR. The purpose of the analysis is to assess the relevance and significance of this method for the diagnosis of osteomyelitis in patients. Numerous successful applications of wide-range PCR followed by sequencing in order to identify the causative agent of osteomyelitis have proven that this method is a useful tool in cases where the culture analyses showed negative results.
Keywords: Broad-range PCR, 16S ribosomal RNA, 23S ribosomal RNA, osteomyelitis
#For correspondence: Nikita Yolshin, Smorodintsev Research Institute of Influenza, 15/17, Prof. Popov str., Saint Petersburg, 197376, Russia, nikita.yolshin@gmail.com
Citation: Yolshin ND. A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis. MIR J 2021; 8(1), 41-49. doi: 10.18527/2500-2236-2021-8-1-41-49. Received: May 31, 2021 Accepted: November 7, 2021 Published: December 11, 2021
Copyright: © 2021 Yolshin. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License (CC BYNC-SA), which permits the unrestricted use, distribution, and reproduction in any medium, as long as the material is not used for commercial purposes, provided that the original author and source are cited. Acknowledgements: No funding was received for this study. Conflict of interest: The author claims no commercial or financial interests.
INTRODUCTION
Polymerase chain reaction (PCR) is one of the most widely used laboratory methods in biological science because of its simplicity and high sensitivity. The detection method based on the amplification of the gene of 16S ribosomal RNA - the component of the 30S subunit of prokaryotic ribosome - is named 16S or broad-range PCR. This method enables scientists to detect viable bacteria as well as dead or partly ingested by macrophages bacteria that belong to any species of the Bacterium genus. A broad-range PCR technique is used in clinical microbiology to identify bacteria when phenotypic or proteomic identification is problematic in order to facilitate the diagnosis of infectious diseases as well as for phylogenetic analyses [1]. An important application of broad-range PCR is the detection of bacterial DNA from sterile sites such as
blood, bone, cerebrospinal fluid, joint fluid, pleural fluid, peritonea fluid, pericardial fluid as well as from both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. This technique has proven to be useful in the diagnostics of blood culture-negative endocarditis [2], although it is difficult to achieve clear negative control with reagents used for PCR [3]. Broad-range PCR was successfully used for the detection of non-culturable bacteria, such as Mycoplasma spp., Ureaplasma spp., Treponema pallidum (T. pallidum), and others [4]. However, it was proven to be useless for the detection of bacterial DNA in the serum and ascitic samples of patients with cirrhosis [5]. Therefore, broad-range PCR can be often used in the cases where the culture-based identification has failed and can be recommended for the analysis of the samples obtained during antimicrobial treatment [6]. This method alone
should not be used to rule out infection, but provides helpful information if the test for infection is positive and the causative agent needs to be identified [7]. It is also useful in critical life-threatening conditions, e.g. in case of neonatal sepsis, because PCR combined with sequencing could detect and identify the bacteria faster than the culture-based method [8].
Broad-range PCR technique
The amplified DNA fragments of 16S rRNA genes are used for the identification and classification of bacteria, eukaryotes, and fungi [9, 10]. The identification of the investigated microorganisms is accomplished by comparison of the obtained sequences with the data available in sequence databases such as NCBI or SILVA [11] (Fig. 1).
Conserved regions of the 16S rRNA gene (priming sites) are widely used for the design of specific primers for broad-range PCR [12,13]. However, some specific primers that were developed according to the reference sequences from the database versions are now out of date [14]. While sequencing the amplified DNA fragments seems to be appropriate in most cases, the identification of the pathogen becomes more complicated if more than one type of bacteria is present in the studied sample. This situation can be resolved by cloning amplified fragments into a vector before sequencing.
Contamination issues and solution options
The downside of the sensitivity and non-selectivity of broad-range PCR is revealed when analyzing the contaminated samples. Sometimes amplification could occur in the samples that are used as negative controls because of the bacterial DNA presence in PCR reagents: polymerase [15], dNTPs, primers [16], buffer, or water. These sources of contamination can be avoided by using commercial kits for DNA extraction and broad-range PCR [17] as well as highly purified (low DNA) polymerase preparations and forensic grade plastic. Protocols for the reduction of contamination include UV irradiation [18], DNase I treatment [19], ultrafiltration of PCR mix, treatment with ethidium/propidium monoazide (EMA/ PMA) combined with light exposure [20] and the dilution of Taq polymerase [21, 22].
Contamination could be crucial for making the right conclusions of the broad-range PCR analysis. All the analyzed samples should be checked for any possible sources of contamination that could influence the final results of analysis, since the DNA ascribed to the uncultured bacteria may turn out to be intrinsic DNA from the extraction kits [23]. The sequencing of negative control samples could help in this case. Additional confirmation of the
obtained results by a method other than PCR is strongly recommended. The most suitable solution currently is to use the EMA-UV system [20] or primer extension-PCR (PE-PCR) that involves an additional step of annealing probes to a DNA template before PCR and using primers to these probes in subsequent PCR. Automated broad-range PCR can supposedly decrease the contamination risk too [25]. However, at present, there is no single perfect solution for this problem [3].
Bacterial etiology of osteomyelitis
In this review, we only focus on the cases where the causative agent of osteomyelitis has been identified using a broad-range PCR. The literature analysis includes all sources published in the scientific literature available in PubMed and Google Scholar (up to October 2020).
The pathogen that is most often associated with osteomyelitis is Staphylococcus aureus (S. aureus), and it was found in 80% of the culture-positive cases [26]. The bacterial etiology of osteomyelitis varies with age [27]. Infants (<1 year old) with osteomyelitis usually have Group B streptococci, S. aureus, and Escherichia coli (E. coli), while the samples from children (from 1 to 16 years old) are commonly positive for S. aureus, Streptococcus pyogenes (S. pyogenes), and Haemophilus influenzae (H. influenzae). Pathogens isolated from adults with osteomyelitis (>16 years old) mostly include Staphylococcus epidermidis (S. epidermidis), S. aureus, Pseudomonas aeruginosa (P. aeruginosa), Serratia marcescens (S. marcescens), and E. coli.
Less common bacteria associated with a risk of osteomyelitis development include Pasteurella multocida (P. multocida) and Eikenella corrodens (E. corrodens) associated with animal bites, Bartonella henselae (B. henselae) that could be acquired from contact with kittens, Coxiella burnetii (C. burnetii) that could originate from contact with farm animals, and Salmonella spp. associated with sickle cell anemia. Bacterial cultures isolated from immunocom-promised patients could be positive for Aspergillus spp, Mycobacterium avium-intracellulare complex (M. avium and M. intracellulare), or Candida albicans (C. albicans). Osteomyelitis could also be caused by Mycobacterium tuberculosis (M. tuberculosis) as a complication of the primary disease. There are plenty of cases where the unusual bacteria are identified as the causative agents of osteomyelitis. Below we discuss these cases where unusual pathogens have been detected using a broad-range PCR.
PCR amplification for investigating osteomyelitis pathogenesis: experimental data
Broad-range PCR could be used to analyze bone and joint infections, including arthritis, osteomyelitis, and
Fig. 1. Detection and identification of microorganism by broad-range PCR.
infections on orthopedic implants. The most influential and thorough research in this area was conducted by Fenollar et al. who collected samples and made every effort to avoid procedural errors [28]. In the cases where the culture-based identification and 16S rRNA gene PCR showed contradictory results, additional PCR assays were carried out using a different DNA extraction protocol, followed by a second 16S rRNA gene PCR (performed with a different set of primers) and an additional PCR targeting another gene, e.g. the Mycobacterium tuberculosis gene
encoding beta subunit of RNA polymerase (rpoB). If a microorganism was detected only once in the course of 3 assays and appeared to be a potential skin contaminant, it was considered as a contaminant and a false culture positive result. One negative control analysis was included for every 5 analyzed samples. PCR products were cloned into a vector and then ten clones were sequenced. From 525 analyzed samples, 475 showed comparable test results for the culture-based identification and 16S rRNA gene PCR. All the theoretically possible types of results
have been obtained: 9 false-negative PCR results, 5 false-positive PCR results due to contamination, identification results obtained by PCR only (due to the lack of sensitivity of culture-based methods) for 16 cases and contradictory results in 7 cases for culture-based identification and 16S rRNA gene PCR. In the conclusion, Fenollar et al. suggested to use the 16S rRNA gene PCR assay for cases where an infection is suspected, but wherein the culture-based identification gave negative results.
The prospective study of vertebral osteomyelitis [29] showed that broad-range PCR could be used for the etiological diagnosis. S. aureus, S. epidermidis, E. coli, Streptococcus agalactiae (S. agalactiae), M. tuberculosis, Streptococcus dysgalactiae (S. dysgalactiae), Haemophilus parainfluenzae (H. parainfluenzae), Clostridium perfrin-gens (C. perfringens), Staphylococcus capitis (S. capitis), Achromobacter xylosoxidans (A. xylosoxidans), Salmonella enterica (S. enterica), and Klebsiella pneumoniae (K. pneumoniae) bacteria were identified in patients with vertebral osteomyelitis using broad-range PCR. However, in 5 cases where culture-based testing showed positive results, including the cases with the following pathogens: M. tuberculosis (2 cases), S. aureus (1 case), S. epidermidis (1 case), and Enterococcus faecium (E. faecium) (1 case), the results of PCR were negative. The authors claim that, while the sensitivity of broad-range PCR was almost twofold higher than that of the culture-based method, the false positive results, due to the skin contaminants, were more frequent when using PCR in this study.
Lecouvet et al. contributed to the development of new broad-range PCR applications [30] by the identification of causative agents of a disease with different but very close nosology - spondylodiscitis. They conducted a study in 19 patients to compare the broad-range PCR with the conventional Disc Aspiration method that includes the culture analysis of material from a suspected disc. The causative organisms were identified in 14 of 19 patients (74%) by means of microbiological assay, while PCR showed positive results in 19 of 19 patients (100%) and five more pathogens - Staphylococcus simu-lans (S. simulans), Staphylococcus sciuri (S. sciuri), Brucella spp, Actinomyces israelii (A. israelii), and M. tuberculosis complex - were identified with the help of this method. Despite the obtained results, the authors of this study also considered PCR analysis as an additional analysis, but not as an alternative method. They concluded that broad-range PCR could confirm the results of the culture-based method and identify quickly non-growing pathogens when a rare or atypical pathogen is detected, excluding intercurrent sample contamination. A study of Verdier et al. [31] was focused on the application of a broad-range PCR for the diagnosis of osteoarticular
infections caused by Kingella kingae (K. kingae). Among 171 children with different osteoarticular diseases 9 showed positive results for K. kingae according to the culture-based analysis. K. kingae DNA sequences were found in another 15 samples by broad-range PCR making K. kingae the second most common causative agent of infection in this group (30.4%) after Staphylococcus aureus (38%). Interestingly, no other pathogens were found by PCR except K. kingae. When K. kingae was proven to be the causative agent of the disease, cases with positive results by PCR have been added to the positive culture-based cases but did not coincide with them. The authors argued that this fastidious bacterium is difficult to isolate on the solid medium and recommended inoculation of clinical specimen in enriched blood culture systems. The results suggest that the routine use of molecular methods should be considered for the diagnosis of osteo-articular infection caused by K. kingae in children. The presence of oropharyngeal K. kingae is well known to be associated with osteoarticular infection in children [32].
Another study focused on assessing the value of the broad-range PCR method included the testing of a number of different samples and ended up with identification of Propionibacterium acnes (P. acnes) as a causative agent for T10-T11 osteomyelitis [33]. Therefore, the broad-range PCR has been demonstrated to be a clinically useful and important test for the diagnostics of infections in patients with culture-negative results.
Searns [34] resorted to broad-range PCR while searching for bacterial pathogens in children with chronic recurrent multifocal osteomyelitis. After no bacteria were identified in these patients when using PCR, the author confirmed that they do not require antimicrobial therapy. A rabbit experimental model of chronic osteomyelitis was created [35] in order to determine the applicability of molecular diagnostic procedures for the monitoring of chronic osteomyelitis. The molecular diagnostic method was found to be highly sensitive, accurate, and capable of detecting low quantities of the pathogen when it remained undetected by radiographic and microbiological methods.
The second most common causative agent of osteomyelitis is M. tuberculosis. Importantly, the sensitivity of broad-range PCR for mycobacteria has been reported to be lower than that of mycobacterial culture analysis because the mycobacterial species carry low copy numbers of 16S rDNA [36]. To enhance the sensitivity of broad-range PCR, an optimized DNA extraction protocol was suggested in order to improve the lysis of strong and waxy cell walls of mycobacteria [37].
Broad-range PCR followed by sequencing is used also as a tool for pathogen identification and the subsequent selection of appropriate antibiotic treatment [38, 39].
Osteomyelitis pathogens identified by broad-range PCR
In a patient with hypogammaglobulinemia after sple-nectomy, the Mycoplasma pneumoniae (M. peumoniae) was identified as the bacteria causing osteomyelitis using broad-range PCR [40]. The results were confirmed by a PCR assay with the use of M. pneumoniae specific primers.
The use of broad-range 16S rRNA PCR enabled the diagnostics of E. coli as a pathogen of vertebral osteomyelitis [41]. In this case, E. coli was isolated from patient's blood and urine samples, but when osteomyelitis (accompanied with discitis and epidural abscess) was diagnosed, culture analyses of blood, abscess, and soft tissue samples showed negative results. Shibata et al. noted the special effectiveness of broad-range PCR in cases with unavoidable use of antibiotics. In one of these cases, the E. coli-specific DNA from the biopsy specimen was amplified and identified by PCR despite the preceding administration of antibiotics active against this bacterium for more than 50 days.
Bacterial 16S rRNA genes revealing Porphyromon-as gingivalis (P. gingivalis) as the causative agent of osteomyelitis were detected by broad-range PCR in four months after the resection of the end of the molar root of a 41-year-old man who had cortical destruction of the di-aphysis of the elbow joint [42]. The culture analysis gave negative results.
Harris et al. [43] reported the first case of osteomyelitis caused by Helicobacter spp. in a patient diagnosed by broad-range PCR. A good response to an extended course of antimicrobials against Helicobacter spp. confirmed the diagnosis. In other case Helicobacter cinaedi (H. cinaedi) was revealed as the causative agent of osteomyelitis in the Th11-L2 vertebral bodies when the gene amplified from the biopsy specimen showed the highest similarity to that of rRNA of H. cinaedi. The culture-based analysis of discs in this case gave negative result. In the other four described cases of vertebral osteomyelitis, H. cinaedi was detected only by culture method [44]. All of these cases where helicobacter infection was revealed by one method remained unconfirmed.
In a patient with sternal osteomyelitis after open-heart surgery Gordonia bronchialis (G. bronchialis) was identified as a causative agent of an infection by broad-range PCR and that was confirmed by MALDI-TOF mass spectrometry. However, culture analysis in this case did not identify (G. bronchialis) as bacteria that caused the disease [45]. In the case of a pediatric 0 fever osteomyelitis, the testing of the purulent material by broad-range PCR showed the presence of C. burnetti [46], while the culture-based and serological tests gave negative results.
In a boy with osteomyelitis, Fusobacterium nucleatum (F. nucleatum) was detected by broad-range PCR when synovial fluid cultures and Gram stain tests failed to identify the pathogen [47]. After the detection of F. nu-cleatum, antibiotic therapy was switched accordingly with a rapid response.
Gonococcal osteomyelitis was diagnosed by broad-range PCR with sequencing in 2 men with chronic recurrent multifocal osteomyelitis [48]. In both cases, infection emerged by the dissemination of Neisseria gonorrhoeae (N. gonorrhoeae) from joint fluid (first case) or bone (second case). Osteomyelitis was a complication of the diseases that the patients had in the past. The specific PCR was used to diagnose a number of cases of the gonococcal osteomyelitis [49]. The PCR was useful in the identification of the pathogen at early stages of infection in patients with osteomyelitis of traumatized femur when the traditional culture methods were negative at day 10 of the disease [50]. PCR followed by sequencing identified Tissierella carlieri (T. carlieri) as a causative agent. Two weeks later, the Tissierella spp. was revealed in the biopsy samples by means of pure anaerobic growth of bacteria in brain-heart-infusion broth.
DISCUSSION
A bacterial culture test is considered to be the major tool for the diagnosis of osteomyelitis in patients for a long time. However, when the culture-based analysis of biological material isolated from patients with osteomyelitis show negative results, broad-range PCR is a method of choice. While the contamination issue is important for the culture method, it is much more critical for the broad-range PCR although it often remains neglected. Considering the importance of contamination issues for broad-range PCR analysis, it is highly desirable to confirm the presence of the corresponding bacteria by other methods because of the high risk of false-positive results [24]. According to the published data, the pathogens were identified by broad-range PCR in 14-63% cases when other methods failed. Usually, broad-range PCR is the last resort available for the identification of the pathogen (Tables 1, 2).
The culture method, using a different medium, can also be used for confirmation. A second confirmation could be the presence of specific antibodies in serum, but these assays are described only for a few bacterial species. In most cases, bacteria found in normally sterile sites and liquids are the causative agents for the development of disease. However, the identification of
Table 1. Bacteria found only due to broad-range PCR in large studies of osteoarticular infections
Bacteria found by broad-range PCR (number of cases) Nosology Broad-range PCRa References
Staphylococcus spp. (4) Streptococcus spp. (5) Enterobacteriaceae spp. (2) Enterococcus faecalis (3) Prevotella spp. (1) Granulicatella adiacens (1) Bone and joint infection 16/114 (14%) [28]
Staphylococcus spp. (7) Streptococcus spp. (2) E. coli (1) Klebsiella pneumoniae (1) H. Parainfluenzae (1) Clostridium perfringens (1) Vertebral osteomyelitis 13/21 (62%) [29]
Staphylococcus simulans (1) Staphylococcus sciuri (1) Actinomyces israelii (1) Brucella spp. (1) Mycobacterium tuberculosis (1) Spondylodiscitis 5/19 (26%) [30]
Kingella kingae (15) Pediatric osteoarticular infections 15/24 (63%) [31]
a Quantity of pathogens discovered by broad-range PCR only/total number of diagnosed infections. Table 2. Cases of osteomyelitis when the pathogen was detected by broad-range PCR
Bacteria Osteomyelitis localization Case reports References
Propionibacterium acnes Vertebral (T10-T11) 1 [33]
Mycoplasma pneumoniae Femur and sternum 1 [40]
E. coli Vertebral (C5-C6) 1 [41]
Porphyromonas gingivalis Ulna 1 [42]
Helicobacter spp Femur, vertebral (Th11-L2) 2 [43], [44]
Gordonia bronchialis Sternum 1 [45]
Coxiella burnetti Femoral epiphysis 1 [46]
Fusobacterium nucleatum Knee joint 1 [47]
Neisseria gonorrhoeae Metacarpophalangeal joint, knee joint 2 [48]
Tissierella carlieri Femur 1 [50]
bacteria with PCR in these samples can only be considered as indirect evidence of a causative microorganism, since there are no control groups available in these cases.
In conclusion, numerous successful applications of broad-range PCR followed by sequencing were performed for the identification of causative agents of osteomyelitis. This method helps to prescribe the right treatment when a rare or atypical pathogenic agent is found, to
confirm culture-based analyses, to identify bacterial species, and to exclude an intercurrent culture sample contamination. In some cases, broad-range PCR turned out to be the fastest method for the detection of the pathogen. Fastidious bacteria, antimicrobial treatment, and other reasons can lead to a lack of sensitivity of culture-based methods; in these cases, PCR is probably the only method for the identification of the causative agent of the disease.
REFERENCES
1. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 1985; 82, 6955-59. doi: 10.1073/ pnas.82.20.6955.
2. Fournier PE, Thuny F, Richet H, Lepidi H, Casalta JP, Arzouni JP, et al. Comprehensive diagnostic strategy
for blood culture-negative endocarditis: a prospective study of 819 new cases. Clin Infect Dis 2010, 51, 131-40. doi: 10.1086/653675.
3. Philipp S, Huemer HP, Irschick EU, Gassner C. Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase. Transfus Med Hemother 2010; 37(1), 21-8. doi: 10.1159/000265571.
4. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev, 2010, 23, 235-51. doi: 10.1128/CMR.00043-09.
5. Such J, Frances R, Munoz C, Zapater P, Casellas JA, Cifuentes A, et al. Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites. Hepatology 2002, 36, 135-41. doi: 10.1053/jhep.2002.33715.
6. Rantakokko-Jalava K, Nikkari S, Jalava J, Eerola E, Skurnik M, Meurman O, et al. Direct amplification of rRNA genes in diagnosis of bacterial infections. J Clin Microbiol 2000; 38, 32-9. doi: 10.1128/JCM.38.1.32-39.2000.
7. Payne M, Azana R, Hoang LM. Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory. Can J Infect Dis Med Microbiol 2016; 4210129. doi: 10.1155/2016/4210129.
8. Jordan JA, Butchko AR, Durso MB. Use of pyrose-quencing of 16S rRNA fragments to differentiate between bacteria responsible for neonatal sepsis. J Mol Diagn 2005; 7(1), 105-10. doi: 10.1016/S1525-1578(10)60015-3.
9. Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 1988; 71, 491-9. doi: 10.1016/0378-1119(88)90066-2.
10. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, and White TJ (ed.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990, 315-22. doi: 10.1016/B978-0-12-372180-8.50042-1.
11. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glockner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007; 35, 7188-96. doi: 10.1093/nar/gkm864.
12. Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 2003; 55, 541-55. doi: 10.1016/j.mimet.2003.08.009.
13. Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Dymock D, Wade WG. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 1998; 64, 795-9. doi: 10.1128/ AEM.64.2.795-799.1998.
14. Thijs S, Op De Beeck M, Beckers B, et al. Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys. Front Microbiol 2017; 8, 494. doi: 10.3389/fmicb.2017.00494.
15. Schmidt TM, Pace B, Pace NR. Detection of DNA contamination in Taq polymerase. Bio-Techniques 1991; 11, 176-7. PMID: 1931012.
16. Goldenberger D, Altwegg M. Eubacterial PCR: contaminating DNA in primer preparations and its elimination by UV light. J Microbiol Methods 1995; 21, 2732. doi: 10.1016/0167-7012(94)00024-2.
17. Stavnsbjerg C, Frimodt-M0ller N, Moser C, Bjarn-sholt T. Comparison of two commercial broad-range PCR and sequencing assays for identification of bacteria in culture-negative clinical samples. BMC Infect Dis 2017; 17(1), 233. doi: 10.1186/s12879-017-2333-9.
18. Klaschik S, Lehmann LE, Raadts A, Hoeft A, Stuber F. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR. Mol. Biotechnol 2002; 22, 231-42. doi: 10.1385/MB:22:3:231.
19. Heininger A, Binder M, Ellinger A, Botzenhart K, Un-ertl K, Doring G. DNase pretreatment of master mix reagents improves the validity of universal 16S rRNA gene PCR results. J Clin Microbiol 2003; 41, 1763-5. doi: 10.1128/JCM.41.4.1763-1765.2003.
20. Humphrey B, McLeod N, Turner C, Sutton JM, Dark PM, Warhurst G. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR. PLoS One 2015; 10(7), e0132954. doi: 10.1371/journal.pone.0132954.
21. Spangler R, Goddard NL, Thaler DS. Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria. PLoS One 2009; 4(9), e7010. doi: 10.1371/journal.pone.0007010.
22. Silkie SS, Tolcher MP, Nelson KL. Reagent decontamination to eliminate false-positives in Escherichia coli qPCR. J Microbiol Methods 2008; 72, 275-82. doi: 10.1016/j.mimet.2007.12.011.
23. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, et al. Reagent and laboratory contamination can critically impact sequence-based microbi-ome analyses. BMC Biology 2014; 12, 87. doi: 10.1186/ s12915-014-0087-z.
24. Chang SS, Hsu HL, Cheng JC, Tseng CP. An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents. PLoS One 2011; 6(5), e20303. doi: 10.1371/ journal.pone.0020303.
25. Budding AE, Hoogewerf M, Vandenbroucke-Grauls CM, Savelkoul PH. Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples. J Clin Microbiol 2016; 54(4), 934-43. doi: 10.1128/JCM.02886-15.
26. Thakolkaran N, Shetty AK. Acute Hematogenous Osteomyelitis in Children. Ochsner J 2019; 19(2), 11622. doi: 10.31486/toj.18.0138.
27. Carek PJ, Dickerson LM, Sack JL. Diagnosis and management of osteomyelitis. Am Fam Physician 2001; 63, 2413-20. PMID: 11430456.
28. Fenollar F, Roux V, Stein A, Drancourt M, Raoult D. Analysis of 525 Samples To Determine the Usefulness of PCR Amplification and Sequencing of the 16S rRNA Gene for Diagnosis of Bone and Joint Infections. Journal of Clinical Microbiology 2006; 44(3), 1018-28; doi: 10.1128/JCM.44.3.1018-1028.2006.
29. Sang-Ho Choi, Heungsup Sung, Sung-Han Kim, Sang-Oh Lee, Sang Hoon Lee, Yang Soo Kim, et al. Usefulness of a direct 16S rRNA gene PCR assay of percutaneous biopsies or aspirates for etiological diagnosis of vertebral osteomyelitis. Diagn Microbiol Infect Dis 2014; 78(1), 75-8. doi: 10.1016/j.diagmi-crobio.2013.10.007.
30. Lecouvet F, Irenge L, Vandercam B, Nzeusseu A, Hamels S, Gala JL. The etiologic diagnosis of infectious discitis is improved by amplification-based DNA analysis. Arthritis Rheum 2004; 50, 2985-94. doi: 10.1002/art.20462.
31. Verdier I, Gayet-Ageron A, Ploton C, Taylor P, Benito Y, Freydiere AM, et al. Contribution of a broad range polymerase chain reaction to the diagnosis of osteoarticular infections caused by Kingella kingae: description of twenty-four recent pediatric diagnoses. Pediatr Infect Dis J 2005; 24, 692-6. doi: 10.1097/01.inf.0000172153.10569.dc.
32. Gravel J, Ceroni D, Lacroix LE, Renaud C, Grimard G, Samara E, et al. Association between oropharyngeal carriage of Kingella kingae and osteoarticular infection in young children: a case-control study. CMAJ 2017; 189(35), E1107-11. doi: 10.1503/cmaj.170127.
33. Basein T, Gardiner BJ, Andujar Vazquez GM, Joel Chandranesan AS, Rabson AR, Doron S, Snydman DR. Microbial Identification Using DNA Target Amplification and Sequencing: Clinical Utility and Impact on Patient Management. Open Forum Infect Dis 2018; 5(11), ofy257. doi: 10.1093/ofid/ofy257.
34. Searns J. Searching for Bacterial Pathogens in Pedi-atric Patients with Chronic Recurrent Multifocal Osteomyelitis Using 16S rRNA Quantitative Real-Time PCR. Open Forum Infect Dis 2017; 4, Issue suppl_1, S98, doi: 10.1093/ofid/ofx163.077.
35. Mariani D, Martin DS, Chen AF, Yagi H, Lin SS, Tuan RS. Polymerase Chain Reaction molecular diagnostic technology for monitoring chronic osteomyelitis. J Exp Orthop 2014; 1(1), 9. doi: 10.1186/ s40634-014-0009-6.
36. Klappenbach JA, Saxman PR, Cole JR, Schmidt TM. rrndb: the Ribosomal RNA Operon Copy Number Database. Nucleic Acids Res 2001; 29, 181-4. doi: 10.1093/nar/29.1.181.
37. Käser M, Ruf M-T, Hauser J, Marsollier L, Pluschke G. Optimized Method for Preparation of DNA from Pathogenic and Environmental Mycobacteria. Applied and Environmental Microbiology 2009; 75(2), 414-8. doi: 10.1128/AEM.01358-08.
38. Baraboutis IG, Argyropoulou A, Papastamopoulos V, Psaroudaki Z, Paniara O, Skoutelis AT. Primary sternal osteomyelitis caused by Nocardia nova: case report and literature review. Braz J Infect Dis 2008; 12(3), 257-9. doi: 10.1590/S1413-86702008000300018.
39. Miller AO, Buckwalter SP, Henry MW, Fann Wu, Ma-loney KF, Abraham BK, et al. Globicatella sanguinis Osteomyelitis and Bacteremia: Review of an Emerging Human Pathogen with an Expanding Spectrum of Disease. Open Forum Infect Dis 2017; 4(1), ofw277. doi: 10.1093/ofid/ofw277.
40. La Scola B, Michel G, Raoult D. Use of Amplification and Sequencing of the 16S rRNA Gene to Diagnose Mycoplasma pneumoniae Osteomyelitis in a Patient with Hypogammaglobulinemia. Clin Infect Dis 1997; 24(6), 1161-3. doi: 10.1086/513631.
41. Shibata S, Tanizaki R, Watanabe K, Makabe K, Sho-da N, Kutsuna S, et al. Escherichia coli Vertebral Osteomyelitis Diagnosed According to Broad-range 16S rRNA Gene Polymerase Chain Reaction (PCR). Intern Med 2015; 54(24), 3237-40. doi: 10.2169/internal-medicine.54.5066.
42. Welkerling , Geissdörfer W, Aigner T, Forst R. Osteomyelitis of the ulna caused by Porphyromonas gin-givalis. J Clin Microbiol 2006; 44(10), 3835-7. doi: 10.1128/JCM.00793-06.
43. Harris KA, Fidler KJ, Hartley JC, Vogt J, Klein NJ, Monsell F, Novelli VM. Unique case of Helicobacter sp. osteomyelitis in an immunocompetent child diagnosed by broad-range 16S PCR. J Clin Microbiol 2002; 40(8), 3100-3. doi: 10.1128/jcm.40.8.3100-3103.2002.
44. Hase R, Hirooka T, Itabashi T, Endo Y, Otsuka Y. Vertebral Osteomyelitis Caused by Helicobacter cinaedi Identified Using Broad-range Polymerase Chain Reaction with Sequencing of the Biopsied Specimen. Intern Med 2018; 57(10), 1475-7. doi: 10.2169/inter-nalmedicine.0012-17.
45. Chang J-H, Ji M, Hong H-L, Choi S-H, Kim Y-S, Chung CH, et al. Sternal osteomyelitis caused by Gordonia bronchialis after open-heart surgery. Infect Chemother 2014; 46, 110-4. doi: 10.3947/ ic.2014.46.2.110.
46. Khatami A, Sparks RT, Marais BJ. A Case of Pediatric Q Fever Osteomyelitis Managed Without Antibiotics. Pediatrics 2015; 136(6), e1629-31; doi: 10.1542/ peds.2015-0024.
47. Kroon E, Arents NA, Halbertsma FJ. Septic arthritis and osteomyelitis in a 10-year-old boy, caused by Fu-sobacterium nucleatum, diagnosed with PCR/16S ri-bosomal bacterial DNA amplification. BMJ Case Rep 2012; bcr1220115335. doi: 10.1136/bcr.12.2011.5335.
48. Rehnström M, Unemo M, Tunbäck P. Gonococcal Osteomyelitis Resulting in Permanent
Sequelae. Acta Derm Venereol 2016; 96(4), 562-3. doi: 10.2340/00015555-2296.
49. Shibata Y, Kumano T, Shinoda T, Hanada H, Notomi T, Nagayama A, et al. Gonococcal osteomyelitis of the shoulder extended from gonococcal arthritis: diagnosis by a polymerase chain reaction assay. J Shoulder Elbow Surg 2004; 13, 467-71. doi: 10.1016/S1058274604000266.
50. Schweizer M, Bloemberg GV, Graf C, Falkowski AL, Ochsner P, Graber P, et al. Chronic Osteomyelitis Due to Tissierella carlieri: First Case. Open Forum Infect Dis 2016; 3(1), ofw012. doi: 10.1093/ofid/ofw012.
