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A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis

https://doi.org/10.18527/2500-2236-2021-8-1-41-49

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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 analysеs showed negative results.

For citation:


Yolshin N.D. A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis. Microbiology Independent Research Journal (MIR Journal). 2021;8(1):41-49. https://doi.org/10.18527/2500-2236-2021-8-1-41-49

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 broadrange 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).

Fig. 1. Detection and identification of microorganism by broad-range PCR.

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 broadrange 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 immunocompromised 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 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 falsepositive 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 culturebased 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 perfringens (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 simulans (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 culturebased 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 osteoarticular 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 broadrange 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 broadrange 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 splenectomy, 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 Porphyromonas 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 diaphysis 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 openheart surgery Gordonia bronchialis (G. bronchialis) was identified as a causative agent of an infection by broadrange 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 Q 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. nucleatum, antibiotic therapy was switched accordingly with a rapid response.

Gonococcal osteomyelitis was diagnosed by broadrange 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 brainheart-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).

Table 1. Bacteria found only due to broad-range PCR in large studies of osteoarticular infections

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

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 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 culturebased methods; in these cases, PCR is probably the only method for the identification of the causative agent of the disease.

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About the Author

N. D. Yolshin
Smorodintsev Research Institute of Influenza
Russian Federation

Nikita Yolshin 

15/17, Prof. Popov str., Saint Petersburg, 197376



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For citation:


Yolshin N.D. A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis. Microbiology Independent Research Journal (MIR Journal). 2021;8(1):41-49. https://doi.org/10.18527/2500-2236-2021-8-1-41-49

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ISSN 2500-2236 (Online)