Should surgeons doubt the value of cultures in shoulder surgery?

It is well recognized that bacterial infections are important causes of failed shoulder arthroplasy, failed cuff repairs and failed fracture fixation. The organisms most commonly responsible for these infections (Cutibacterium and coagulase negative staphylococcus (CNS)) are also found on and in healthy skin of healthy individuals as well as in the environment.

Of note, these organisms can be isolated from sterile swabs and sponges that are exposed to the air in the operating room, suggesting that specimens can be contaminated by air-borne organisms, specimen handling or microbiology laboratory processing. Knowing the results of control environmental non-clinical cultures for a surgeon's operating room is important to the interpretation of the results of clinical cultures isolated from deep tissue specimens and explants obtained at the time of revision surgery.

Between-hospital variability in the results of sterile cultures is demonstrated by the reported rates of control samples that are positive for Cutibacterium which range from 4% to 9% to 15% to 20%:

In Efficacy of topical benzoyl peroxide on the reduction of Propionibacterium acnes during shoulder surgery  4% (2 of 50) of sterile control specimens were positive for P. acnes (Cutibacterium)

In The Incidence of Propionibacterium acnes in Open Shoulder Surgery: A Controlled Diagnostic Study seven (13.0%) of fifty-four sterile control specimens returned positive for culture growth. Five of these (9%) grew P. acnes (Cutibacterium), while two (4%) grew coagulase-negative S. aureus.

In Cutibacterium acnes is Isolated from Air Swabs: Time to Doubt the Value of Traditional Cultures in Shoulder Surgery? Cutibacterium was identified by culture of sterile control swabs in 6 of 40 cases (15%) swabs and coagulase negative staphylococcus (CNS) was identified in 3 of 40 (7.5%). 

In Preoperative doxycycline does not decolonize Propionibacterium acnes from the skin of the shoulder: a randomized controlled trial 20 sterile control specimens were submitted for culture, four of which (20%) grew P. acnes (Cutibacterium).

This five fold difference among hospitals in the rate of positive cultures for sterile control specimens suggests different risks of specimen contamination in the operating room, during specimen handling and/or in the microbiological laboratory.

The magnitude of contamination from these environmental sources would be expected to be small in comparison to the load of bacteria in an infected shoulder. The authors of Characterizing the Propionibacterium Load in Revision Shoulder Arthroplasty pointed to the value of assessing the amount of bacteria in a sample using a semi-quantitative report rather than simply referring to a culture result as "positive" or "negative". In 137 Cutibacterium culture-positive revision shoulder arthroplasties the culture results were characterized by a specimen Cutibacterium value (SpCV): 0 = no growth, 0.1 = broth only or one colony only, 1 = growth on one plate quadrant, 2 = growth on two plate quadrants, 3 = growth on three plate quadrants and 4 = growth on all 4 of the plate quadrants after standard microbiological plate streaking.

The authors of Preoperative Skin-Surface Cultures Can Help to Predict the Presence of Propionibacterium in Shoulder Arthroplasty Wounds used this semi-quantitative approach to report the results of cultures for sterile control swabs submitted from the operating room in 50 cases. While 2 of the control samples had positive cultures for Cutibacterium (4%), neither (0%) of the positive control samples had a SpCV of 1 or more, indicating a very low level of contamination.

Some authors have proposed that Next Generation Squencing (NGS) may be superior to cultures in detecting bacteria doing harm in shoulders. See for example, Revision shoulder arthroplasty - what is the role of next-generation sequencing?. NGS is very sensitive to the presence of bacterial RNA and DNA. In the referenced study, NGS found evidence of no fewer than 71 different organisms in specimens from cases of revision arthroplasty. including A calcoaceticus, A excentricus, A ferrireducens, A junii, A radioresistens, A rhizogenes, A tetradius, B aggregatus, B casei, B cepacia, B dorei, B fragilis, B fungorum, B mycoides, B nordii, B thermosphacta, B thetaiotaomicron, B virosa, C tuberculostearicum, C circulans, C acidisoli, C aurimucosum, C chromoreductans, C diptheriae, C hominis, C hveragerdense, C kroppenstedtii, C paradoxus, C quinii, C striatum, C testosteroni, C tuberculostearicum, C vibrioides, C xerosis, C. acnes, E coli, E hormaechei, G ruanii, K rosea, K oxytoca, K palustris, K pneumoniae L agilis, L albida L crispatus, L manihotivorans, M catarrhalis, M granosa, M luteus, P aeruginosa P agglomerans, P saccharophilia, R gnavus, R insidiosa, R picettii, S agalactiae, S aureus (MRSA) S aureus, S cohnii S condimenti, S epidermidis S hominis, S maltophilia, S melonis, S mitis, S parasanguinis, S pettenkoferi, S piscifermentans, and S sanguinis. It is not known if these results actually reflect living bacteria in the shoulder and what implications these NGS results have for treatment.

The authors of Cutibacterium acnes is Isolated from Air Swabs: Time to Doubt the Value of Traditional Cultures in Shoulder Surgery? suggest that NGS is capable of identifying pathogens and the relative percent abundance in which they appear within a sample. They state, "due to budgetary limitations and the cost of NGS, our sample size was admittedly small and only included one sample for culture and NGS from each case".

For 40 consecutive cases they opened and exposed sterile swabs to the air in the operating room for 5 seconds.  One swab was sent to their microbiology laboratory for aerobic and anaerobic culture and held for 13 days. The other sample was sent for NGS, where samples were amplified for pyrosequencing using a forward and reverse fusion primer and matched against a DNA library for species identification.

Cutibacterium was identified by culture in 6/40 (15%) swabs and coagulase negative staphylococcus (CNS) was identified in 3/40 (7.5%). When considering the semi-quantitative assessment of bacterial growth, all six samples with isolated Cutibacterium were rated as very light growth, a finding consistent with the very low SpCVs in the two positive control cultures in Preoperative Skin-Surface Cultures Can Help to Predict the Presence of Propionibacterium in Shoulder Arthroplasty Wounds . 

2/40 cases (5%) had positive NGS sequencing with polymicrobial results in the distributions shown below.

Case #6: 

P. aeruginosa (26%), P. melaninogenica (19%),  C. tuscaniense (12%),  S. mitis (9%), B. japonicum (8%), A. wolffii (7%), C. appendicis (4%), B. antiquum (4%), A. schindleri (2%)

Case #20: S. hominis (31%), S. epidermidis (24%), M. subterranean (11%), C. kroppenstedtii (8%), E. biforme (8%), P. submarinus (4%), N. oleivorans (3%), Cutibacterium (3%)

Comment: From the foregoing, it can be concluded that

(1) contamination of sterile swabs can occur between the time of opening of the swab and the final analysis of bacterial cultures by the microbiology lab. The resulting positive cultures are not "false positives" but rather positive cultures resulting from contamination

(2) the rate of contamination from the operating room environment, specimen transport and laboratory procedures varies widely among medical centers

(3) the bacterial load - as reflected by the specimen Cutibacterium value  (SpCV) -  in contaminated sterile swabs is small in comparison to the SpCV typically seen in cultures of deep tissues and explants retrieved from shoulders with periprosthetic infections; thus reporting the culture results using a  semiquantitative system to reflect bacterial load helps identify clinically important positive cultures.

(4) Next Generation Sequencing is an expensive test that reveals RNA and DNA associated with a wide range of bacterial species, the preponderance of which have not been demonstrated to be pathogenetic. Thus the value of NGS in guiding therapy in shoulders with suspected infection remains to be demonstrated.

(5) In the absence of a better alternative, surgeons should continue to use bacterial cultures (assessed for bacterial load) to help guide the management of failed shoulder surgeries potentially caused by infection.

See also: What we think we know today about Cutibacterium 

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Here are some videos that are of shoulder interest
Shoulder arthritis - what you need to know (see this link).
How to x-ray the shoulder (see this link).
The ream and run procedure (see this link).
The total shoulder arthroplasty (see this link).
The cuff tear arthropathy arthroplasty (see this link).
The reverse total shoulder arthroplasty (see this link).
The smooth and move procedure for irreparable rotator cuff tears (see this link).
Shoulder rehabilitation exercises (see this link).

How does surgery for periprosthetic infection fail?

Revision surgery for periprosthetic infection may fail because of

(1) definite septic failure: persistence of infection after revision surgery as documented by positive cultures at the time of re-revision (see Factors associated with failure of surgical revision and IV antibiotics to resolve Cutibacterium periprosthetic infection of the shoulder).

(2) possible septic failure: poor restoration of comfort and function (i.e. pain, stiffness, weakness, instability) after revision in the absence of culture evidence of recurrent infection

Definite septic failure may be overlooked because
(1) a re-revision was not performed so that multiple deep tissue and explant cultures were not obtained
(2) cultures from re-revision surgery were negative even though viable bacteria were present because
    (a) an insufficient number (i.e. fewer than five) deep tissue specimens for culture were obtained at re-revision surgery (see Origin of propionibacterium in surgical wounds and evidence-based approach for culturing propionibacterium from surgical sites) and What do positive and negative Cutibacteriumculture results in periprosthetic shoulder infection mean? A multi-institutional control study
    (b) bacterial growth was suppressed by the preoperative administration of antibiotics
    (c) the concealment of bacteria in a biofilm on the prosthetic implants so that they were not detected by tissue cultures (see Culturing explants for Cutibacterium at revision shoulder arthroplasty: an analysis of explant and tissue samples at corresponding anatomic sites)
    (d) specimens obtained at re-revision were not cultured on aerobic, anaerobic, and broth media (see Origin of propionibacterium in surgical wounds and evidence-based approach for culturing propionibacterium from surgical sites)
    (e) cultures were not observed for 14 days to optimize detection of Cutibacterium (the most common organism causing shoulder periprosthetic infection) (see Prognostic factors for bacterial cultures positive for Propionibacterium acnes and other organisms in a large series of revision shoulder arthroplasties performed for stiffness, pain, or loosening)
    (f) the inability of certain strains of bacteria to form colonies on laboratory media (see Growing Unculturable Bacteria, Viable But Not Culturable (VBNC), Bacterial Viability
    (g) an intracellular location of the organisms so that they do not grow on media (see Cutibacterium acnes is an intracellular and intra-articular commensal of the human shoulder joint)

The authors of An Enhanced Understanding of Shoulder PJI using Next generation Sequencing: Findings at 3-year Clinical Follow-up suggest that a possible cause of septic failure is that cultures at the time of an index revision arthroplasty may not detect the presence of pathogentic bacteria so that the patients do not receive prolonged antibiotics beyond standard perioperative prophylaxis.. They further suggest that next generation sequencing (NGS) of samples obtained at the time of index revision may be able to detect organisms present but not isolated by traditional culture (see Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections).

They identified 6 re-revisions that yielded ≥2 positive cultures with the same bacteria. Four of these six had no growth on cultures at the index revision. NGS detected bacterial DNA at the index revision in all six cases. Patients with positive NGS results had a mean of seven (range: 1-13) unique bacterial species identified per procedure. The table below shows that NGS detected DNA from organisms that are ubiquitous in the environment and not thought to be pathogenetic (such as A. radiorestens). It also confirms that Cutibacterium was the organism commonly isolated in these six confirmed septic revisions (those that had positive cultures at re-revision - see right hand column).


Thus antibiotic and surgical treatment of a failed arthroplasty must be planned in recognition that organisms, especially Cutibacterium, may persist in the shoulder even if cultures at the time of the index surgical revision are negative. While it is suggested that failure of the index procedure to resolve the infection could be attributed to lack of treatment for an organism detected by NGS and not identified by culture at the index revision, this study does not provide evidence of persistent infection after the index revision by organisms other than Cutibacterium and Staph Epidermidis, so that prolonged antibiotic coverage for these organisms should be considered.

It is of importance to note that NGS can fail to detect viable organisms demonstrated by positive cultures (including the two most commonly isolated from shoulder periprosthetic infections: Cutibaterium and S. Epidermidis). In this study, NGS at the index revision did not detect S. Epidermidis in the one case where this organism was cultured at re-revision.


In the table below from Comparative study of cultures and next-generationsequencing in the diagnosis of shoulder prostheticjoint infections, the lack of sensitivity of NGS to Cutibaterium and S. Epidermidis is demonstrated even when these organisms are present in large numbers.



The lack of sensitivity of NGS for Cutibacterium is also pointed out by the authors of Cutibacterium acnes is less commonly identified by next-generation sequencing than culture in primary shoulder surgery, who concluded, "There was limited concordance between culture and next-generation sequencing for C. acnes identification. Further studies are needed to determine the potential for next-generation sequencing as a diagnostic tool."

Our understanding of NGS in the detection of Cutibacterium would be greatly enhanced by submitting samples containing different dilutions of the bacteria as well as control samples with no Cutibacterium or other bacteria for next generation sequencing. This would determine the sensitivity of NGS for Cutibacterium as well as the frequency with which DNAs from other bacteria were detected.

At this point it remains unclear whether next generation sequencing is of value in determining treatment of the failed arthroplasty (see Revision shoulder arthroplasty - what is the role of next-generation sequencing?)

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Here are some videos that are of shoulder interest
Shoulder arthritis - what you need to know (see this link).
How to x-ray the shoulder (see this link).
The ream and run procedure (see this link).
The total shoulder arthroplasty (see this link).
The cuff tear arthropathy arthroplasty (see this link).
The reverse total shoulder arthroplasty (see this link).
The smooth and move procedure for irreparable rotator cuff tears (see this link).
Shoulder rehabilitation exercises (see this link).

Periprosthetic infections with negative cultures - are there bacteria there? What is the value of next generation sequencing?

The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria is shown below.

By this set of criteria, the presence of a periprosthetic hip or knee infection may be inferred without actually demonstrating the presence of live bacteria doing harm. The authors point out that the proposed criteria may be inaccurate in patients with adverse local tissue reaction, crystalline deposition arthroplasty, flare of inflammatory arthropathy, and infection with slow growing organisms such as Cutibaterium and coagulase negative Staphylococcus. 

In a subsequent publication, An Enhanced Understanding of Culture-Negative Periprosthetic Joint Infection with Next-Generation Sequencing: A Multicenter Study the authors identified 301 joints meeting the International Consensus Meeting (ICM) criteria for periprosthetic infections (PJI) shown above. Of these 85 (28.2%) had negative intraoperative cultures. A number of factors could contribute to the reported discordance between the culture results and the ICM criteria for a PJI:

    lack of specificity of the minor ICM criteria, 

    administration of antibiotics prior to sampling, 

    the inability of viable bacteria to form colonies on laboratory media, 

    bacteria concealed in a biofilm on the implants, 

    an intracellular location of the organisms, 

    inadequate tissue sampling, 

    inadequate culture protocols, and 

    a too-short period of culture observation. 

In 56 (65.9%) of the culture-negative patients, bacterial DNA was identified by Next Generation Sequencing (NGS). The DNA of seventeen species was commonly found. NGS revealed polymicrobial DNA in 91.1% of the culture-negative cases that met the ICM criteria, with the commonest species contributing to 82.4% of polymicrobial profiles. Escherichia coli, Cutibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus ranked highest in terms of incidence and study-wide mean relative abundance and were most frequently the dominant organism when occurring in polymicrobial infections.

The authors concluded that in cases meeting the ICM criteria for PJI but without positive tissue cultures, the DNA of multiple organisms was often found in tissue samples.

Comment: In this study, 301 patients met the 2018 International Consensus Meeting (ICM) criteria for PJI. Of these patients, 216 had one or more positive cultures. While not presented in this paper, it would be of great interest to know the NGS data for these patients with obvious PJI to reveal the degree to which NGS findings concurred with the culture findings. This analysis would enable us to assess the effective sensitivity of NGS in detecting PJI. As pointed out in Revision shoulder arthroplasty - what is the role of next-generation sequencing?, NGS may fail to detect the DNA of live bacteria isolated from tissue cultures.

Furthermore, as also discussed in Revision shoulder arthroplasty - what is the role of next-generation sequencing?, NGS often detects DNA of non-pathogenic bacteria and NGS cannot distinguish the DNA from live bacteria from that of dead bacteria.

Our knowledge of the role of NGS in diagnosing PJI would be greatly enhanced by using NGS to seek bacterial DNA from samples from uninfected joints, such as those undergoing primary arthroplasty. This analysis would enable us to assess the effective selectivity of NGS in detecting PJI.

It would also be of interest to know the NGS results of negative control samples, such as a sterile swab or gauze. This is important because some of the DNA recovered by NGS is from common laboratory contaminants.

We need to better understand the implications of NGS for treatment.  What antibiotics would be used to treat a joint with NGS finding of DNA for both E. Coli and S. Aureus?  

Ultimately we need to know if the use of NGS sampling at revision arthroplasty improves patient outcomes to a degree that offsets the cost.

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You can support cutting edge shoulder research that is leading to better care for patients with shoulder problems, click on this link.

Follow on twitter: https://twitter.com/shoulderarth

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Follow on facebook: https://www.facebook.com/frederick.matsen

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Here are some videos that are of shoulder interest

Shoulder arthritis - what you need to know (see this link).

How to x-ray the shoulder (see this link).

The ream and run procedure (see this link).

The total shoulder arthroplasty (see this link).

The cuff tear arthropathy arthroplasty (see this link).

The reverse total shoulder arthroplasty (see this link).

The smooth and move procedure for irreparable rotator cuff tears (see this link).

Shoulder rehabilitation exercises (see this link).

Next generation sequencing: is this innovation of clinical value?

Poor Agreement Between Next-Generation DNA Sequencing and Bacterial Cultures in Orthopaedic Trauma Procedures

Next-generation DNA sequencing (NGS) detects bacteria-specific DNA corresponding to the 16S ribosomal RNA gene and can identify bacterial DNA presence with greater accuracy than traditional culture methods. These authors point out that clinical relevance of these findings is unknown. They sought to compare the results from bacterial culture and NGS in order to characterize the potential use of NGS in orthopaedic trauma patients.

They enrolled three patient groups: (1) patients undergoing surgical treatment of acute closed fractures (presumed to have no bacteria), (2) patients undergoing implant removal at the site of a healed fracture without infection, and (3) patients undergoing a first procedure for the treatment of a fracture nonunion who might or might not have subclinical infection. Surgical site tissue was sent for culture and NGS. The proportions of culture and NGS positivity were compared among the groups.

Bacterial cultures were positive in 9 of 111 surgical sites (110 patients), whereas NGS was positive in 27 of 111 surgical sites (110 patients). Significantly more cases were positive on NGS as compared with culture (24% vs. 8.1%; p = 0.001), primarily in the acute closed fracture group (in which there was no reason to expect infection). No difference was found in terms of the percent positivity of NGS when comparing the acute closed fracture, implant removal, and nonunion groups. With respect to bacterial identification, culture and NGS agreed in 73% of cases indicating only slight agreement compared with expected chance agreement of 50%.

The authors concluded that NGS identified bacterial presence more frequently than culture, but with only slight agreement between culture and NGS. Their data suggest that NGS should not currently substitute for or complement conventional culture in orthopaedic trauma cases with low suspicion of infection.

Here are some other articles on the topic of NGS:

Next-generation sequencing for diagnosis of infection: is more sensitive really better?

These authors  point out that the utility of next-generation sequencing (NGS) in differentiating between active infection and contaminant or baseline flora remains unclear. They conducted a study of primary shoulder arthroplasty patients with no history of infection or antibiotic use within 60 days of surgery was enrolled. All patients received standard perioperative antibiotics. After skin incision, a sample of the medial skin edge was excised. A synovial tissue biopsy was taken from the rotator interval after subscapularis takedown. Each sample set was halved and sent for NGS and standard cultures.

The 3 most common bacteria identified by NGS of the skin samples (all species 10% of bacterial burden) were C acnes (44%, 11 individuals), Staphylococcus epidermidis (24%, 6 individuals), and Escherichia coli (20%, 5 individuals). The following species were each detected at the 10% bacterial load threshold for 1 patient: Enterococcus faecalis, Bacillus senegalensis, Staphylococcus saccharolyticys, Corynebacterium kroppenstedtii, Porphyromonas uenonis, Ureaplasma urealyticum, Arthrobacter sp, and Corynebacterium sp.

The 3 most common bacteria identified by NGS of the deep tissue samples (all species 10% of bacterial burden) were E coli (16%, 4 individuals), C acnes (12%, 3 individuals), and S epidermidis (8%, 2 individuals). The following species were each detected at the 10% bacterial load threshold for 1 patient: Staphylococcus aureus, Neisseria shayeganii, Streptococcus equinus, Catenabacterium mitsuokai, and Megamonas funiformis.

Comment: This study did not actually examine the utility of NGS for the diagnosis of infection; instead it looked at shoulders having primary arthroplasty.

The article did not directly compare the bacterial species identified by (a) culture and (b) NGS for each patient.

Finally, as in the case of the article discussed below, it would have been helpful if the authors had run negative controls for NGS to make sure that the unexpected bacterial DNA found by NGS (e.g. E Coli, Bacilli, Porphyromonas, Ureplasma, Arthrobacter, Neisseria, Megamonas) were not the result of DNA contamination unrelated to the presence of viable organisms.

Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections

In 44 patients undergoing revision shoulder arthroplasty, these authors compared bacterial identification by (1) culturing and (2) next-generation sequencing (NGS). They included patients with and without preoperative clinical signs of infection. Tissue samples were obtained from the anterior capsule, inferior capsule, glenoid, humeral canal, and underneath the prosthetic humeral head was obtained using “fresh” instruments. Culture media included anaerobic sheep blood agar and anaerobically prereduced hemin-thioglycolate broth. Aerobic media were apparently not used.

The total genomic DNA was isolated from tissue samples. These were then amplified for pyrosequencing. Amplification products were visualized, pooled, and subjected to size selection. Size-selected pools were then quantified and hybridized to generate single-stranded DNA. Single-stranded DNA was diluted and analyzed by emulsion-based polymerase chain reaction. The resulting amplification products were subsequently enriched and sequenced. Trimmed sequences were then run through USEARCH to cluster the sequences and mapped using the USEARCH UPARSE operational taxonomic unit (OTU) selection algorithm. Mapped sequences were then grouped by OTU; quality scoring-based sequence correction was then performed. Corrected sequences were then run through the Research and Testing Laboratory Genomics taxonomic analysis pipeline to determine the taxonomic classifications and abundance for each sample. Selected OTUs were then aligned using MUSCLE and a phylogenetic tree generated using FastTree. The selected OTU sequences were then globally aligned using USEARCH against a database of classified 16S sequences. Confidence values were assigned to each OTU classification, and the lowest common ancestor was determined based on these confidence values. The top hit and lowest common ancestor was then reported for each OTU.

Positive cultures were present in more than 50%, and positive NGS results were present in almost 40% of revision arthroplasty cases.

Cutibacterium (formerly Propionibacterium) acnes was the most common bacterial species cultured (8 of 13 [61.5%]) and identified by NGS (12 of 17 [70.1%]) in cases of definite and probable infection. The concordance (κ) between the 2 diagnostic criteria for defining infection that included culture or NGS was 0.333 (fair). Data from the 44 cases is shown below.

Comment: While it has proposed that we can achieve An Enhanced Understanding of Culture-Negative Periprosthetic Joint Infection with Next-Generation Sequencing A Multicenter Study, the studies above indicate that NGS frequently finds bacterial DNA where there is no reason to suspect infection. Thus the observation that "a pathogen could be identified by NGS in 56 (65.9%) of culture-negative patients" does not in and of itself indicate that the  bacterial DNA detected was an indication of infection by the corresponding organism.

You can support cutting edge shoulder research that is leading to better care for patients with shoulder problems, click on this link.

Follow on twitter: https://twitter.com/shoulderarth

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Follow on facebook: https://www.facebook.com/frederick.matsen

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Here are some videos that are of shoulder interest

Shoulder arthritis - what you need to know (see this link).

How to x-ray the shoulder (see this link).

The ream and run procedure (see this link).

The total shoulder arthroplasty (see this link).

The cuff tear arthropathy arthroplasty (see this link).

The reverse total shoulder arthroplasty (see this link).

The smooth and move procedure for irreparable rotator cuff tears (see this link).

Shoulder rehabilitation exercises (see this link).

Next-generation sequencing - what does it detect?

Next-generation sequencing for diagnosis of infection: is more sensitive really better?

These authors  point out that the utility of next-generation sequencing (NGS) in differentiating between active infection and contaminant or baseline flora remains unclear. They conducted a study of primary shoulder arthroplasty patients with no history of infection or antibiotic use within 60 days of surgery was enrolled. All patients received standard perioperative antibiotics. After skin incision, a sample of the medial skin edge was excised. A synovial tissue biopsy was taken from the rotator interval after subscapularis takedown. Each sample set was halved and sent for NGS and standard cultures.

The 3 most common bacteria identified by NGS of the skin samples (all species 10% of bacterial burden) were C acnes (44%, 11 individuals), Staphylococcus epidermidis (24%, 6 individuals), and Escherichia coli (20%, 5 individuals). The following species were each detected at the 10% bacterial load threshold for 1 patient: Enterococcus faecalis, Bacillus senegalensis, Staphylococcus saccharolyticys, Corynebacterium kroppenstedtii, Porphyromonas uenonis, Ureaplasma urealyticum, Arthrobacter sp, and Corynebacterium sp.

The 3 most common bacteria identified by NGS of the deep tissue samples (all species 10% of bacterial burden) were E coli (16%, 4 individuals), C acnes (12%, 3 individuals), and S epidermidis (8%, 2 individuals). The following species were each detected at the 10% bacterial load threshold for 1 patient: Staphylococcus aureus, Neisseria shayeganii, Streptococcus equinus, Catenabacterium mitsuokai, and Megamonas funiformis.

Comment: This study did not actually examine the utility of NGS for the diagnosis of infection; instead it looked at shoulders having primary arthroplasty.

The article did not directly compare the bacterial species identified by (a) culture and (b) NGS for each patient.

Finally, as in the case of the article discussed below, it would have been helpful if the authors had run negative controls for NGS to make sure that the unexpected bacterial DNA found by NGS (e.g. E Coli, Bacilli, Porphyromonas, Ureplasma, Arthrobacter, Neisseria, Megamonas) were not the result of DNA contamination unrelated to the presence of viable organisms.

Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections

In 44 patients undergoing revision shoulder arthroplasty, these authors compared bacterial identification by (1) culturing and (2) next-generation sequencing (NGS). They included patients with and without preoperative clinical signs of infection. Tissue samples were obtained from the anterior capsule, inferior capsule, glenoid, humeral canal, and underneath the prosthetic humeral head was obtained using “fresh” instruments. Culture media included anaerobic sheep blood agar and anaerobically prereduced hemin-thioglycolate broth. Aerobic media were apparently not used.

The total genomic DNA was isolated from tissue samples. These were then amplified for pyrosequencing. Amplification products were visualized, pooled, and subjected to size selection. Size-selected pools were then quantified and hybridized to generate single-stranded DNA. Single-stranded DNA was diluted and analyzed by emulsion-based polymerase chain reaction. The resulting amplification products were subsequently enriched and sequenced. Trimmed sequences were then run through USEARCH to cluster the sequences and mapped using the USEARCH UPARSE operational taxonomic unit (OTU) selection algorithm. Mapped sequences were then grouped by OTU; quality scoring-based sequence correction was then performed. Corrected sequences were then run through the Research and Testing Laboratory Genomics taxonomic analysis pipeline to determine the taxonomic classifications and abundance for each sample. Selected OTUs were then aligned using MUSCLE and a phylogenetic tree generated using FastTree. The selected OTU sequences were then globally aligned using USEARCH against a database of classified 16S sequences. Confidence values were assigned to each OTU classification, and the lowest common ancestor was determined based on these confidence values. The top hit and lowest common ancestor was then reported for each OTU.

Positive cultures were present in more than 50%, and positive NGS results were present in almost 40% of revision arthroplasty cases.

Cutibacterium (formerly Propionibacterium) acnes was the most common bacterial species cultured (8 of 13 [61.5%]) and identified by NGS (12 of 17 [70.1%]) in cases of definite and probable infection. The concordance (κ) between the 2 diagnostic criteria for defining infection that included culture or NGS was 0.333 (fair). Data from the 44 cases is shown below.

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Shoulder infections - is there a problem with PCR?

Polymerase chain reaction (PCR) has been advocated for the identification of organisms associated with periprosthetic shoulder infections. A recent articles is of interest.

Cutibacterium acnes and the shoulder microbiome

These authors collected samples from the skin, subcutaneous fat, anterior supraspinatus tendon, middle glenohumeral ligament, and humeral head cartilage of 23 patients (14 male and 9 female patients) during primary arthroplasty surgery. Total DNA was extracted and microbial 16S ribosomal RNA sequencing was performed using an Illumina MiSeq system.

After stringent removal of contamination, genomic DNA from various Acinetobacter species (Acinetobacter are widely distributed in hospitals, where up to 27% of hospital sink traps and 20% of hospital floor swabs have yielded isolates of Acinetobacter. The Class Acinetobacter includes the Family Propionibacteriaceae which includes the Genus Cutibacterium) and from the Oxalobacteraceae family (Oxalobacteraceae is a family within the order Burkholderiales, not known to be a common cause of shoulder infections) was identified in 74% of rotator cuff tendon tissue samples. C acnes DNA was detected in the skin of only 1 male patient but not in any other shoulder tissues.

In a thoughtful response to this article Letter to the Editor regarding Qui et al: ‘‘Cutibacterium acnes and the shoulder microbiome’’ the authors call attention to the absence of C acnes in the skin and subcutaneous fat samples which is an unexpected finding and contrasts with many prior studies in which C acnes has consistently been cultured from the dermis of the shoulder despite standard skin antisepsis measures.

The authors of the letter suggest that the likely explanation for the discrepancy is that "the polymerase chain reaction (PCR) primers selected for the study are unable to detect most strains of C acnes . As a result, the presence or absence of this organism as an endogenous element of the shoulder microbiome cannot reliably be evaluated with the methods used. PCR with 16S rRNA provides a highly sensitive method for quantifying the relative abundance of numerous bacterial species in a sample, based on the detection of rRNA sequences in 1 or more variable regions of this gene (V1, V2, V3, V4, and so on) that are unique to various bacterial taxonomic groups. This method uses PCR primers that target conserved nucleotides (occurring between variable regions) that are highly similar across most bacterial organisms and contain only a limited number of species-specific polymorphisms. Unfortunately, the primers used in this experiment (F515 and R806, targeting the V4 region) are particularly inefficient in amplifying the common skin organism C acnes because of mismatches between the critical 3’ ends of the primer sequences and the complementary annealing sites in the C acnes genome. Of all 121 fully sequenced C acnes genomes currently in the Reference Sequence (RefSeq) database, fewer than 1% would be expected to amplify robustly with these primers. This limitation as it relates specifically to C acnes has been previously reported but was most comprehensively described in a recent study comparing the use of V1 to V3 vs. V4 variable regions in the analysis of human skin samples (see Skin Microbiome Surveys Are Strongly Influenced by Experimental Design). This work concluded that V4 amplification using standard primers is almost entirely unable to detect C acnes. As emphasized in a corresponding editorial and letter, alternative primers or target regions should be used in studies of the human skin microbiome because of the inability of this commonly used V4 primer set to detect C acnes . For this reason, subsequent studies, including a recent characterization of the microbiome of the human skin follicle, have used primers targeting the V1 to V3 region. The experiments reported by Qui et al include a series of negative controls (open-air collection blanks) but do not include positive controls, except for 1 PCR–restriction fragment length polymorphism assay for the single sample in which C acnes was detected. As such, the limitations of this specific primer pair with respect to C acnes identification may not have been apparent."

Comment: It is evident that PCR needs to be used and interpreted carefully. It cannot distinguish the DNA from live bacteria from that from dead bacteria. The utility of this technique depends on the appropriate choice of primers and on the analysis of both negative controls (submission of a sample exposed to the air of the operating room) and the use of positive controls (submission of a sample known to contain the organisms of interest).

Similar steps would seem appropriate for next-generation sequencing, as reported for example in
Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections. In that study the inclusion of negative controls may have helped assess the possibility of contaminants. For example, Acinetobacter radioresistens (which was found in 6 of 17 [35.3%] of the NGS-positive cases), is commonly found in laboratory reagents.

The chart below from A review of next generation sequencing technologies used in the evaluation of the skin microbiome summarizes the features of different methods for bacterial detection.

In our practice we depend on established culture techniques to identify organisms that we know grow well (Cutibacterium and coagulase-negative Staphylococcus). However, it would be of interest to explore the utility of nucleic acid methods to evaluate so called "culture negative infections".

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We have a new set of shoulder youtubes about the shoulder, check them out at this link.

Be sure to visit "Ream and Run - the state of the art"  regarding this radically conservative approach to shoulder arthritis at this link and this link

Use the "Search" box to the right to find other topics of interest to you.

How you can support progress in shoulder surgery

You may be interested in some of our most visited web pages   arthritis, total shoulder, ream and run, reverse total shoulder, CTA arthroplasty, and rotator cuff surgery as well as the 'ream and run essentials'

Detecting bacteria in revision shoulder arthroplasty: culture and next generation sequencing

Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections

In 44 patients undergoing revision shoulder arthroplasty, these authors compared bacterial identification by (1) culturing and (2) next-generation sequencing (NGS). They included patients with and without preoperative clinical signs of infection. Tissue samples were obtained from the anterior capsule, inferior capsule, glenoid, humeral canal, and underneath the prosthetic humeral head was obtained using “fresh” instruments. Culture media included anaerobic sheep blood agar and anaerobically prereduced hemin-thioglycolate broth. Aerobic media were apparently not used.

The total genomic DNA was isolated from tissue samples. These were then amplified for pyrosequencing. Amplification products were visualized, pooled, and subjected to size selection. Size-selected pools were then quantified and hybridized to generate single-stranded DNA. Single-stranded DNA was diluted and analyzed by emulsion-based polymerase chain reaction. The resulting amplification products were subsequently enriched and sequenced. Trimmed sequences were then run through USEARCH to cluster the sequences and mapped using the USEARCH UPARSE operational taxonomic unit (OTU) selection algorithm. Mapped sequences were then grouped by OTU; quality scoring-based sequence correction was then performed. Corrected sequences were then run through the Research and Testing Laboratory Genomics taxonomic analysis pipeline to determine the taxonomic classifications and abundance for each sample. Selected OTUs were then aligned using MUSCLE and a phylogenetic tree generated using FastTree. The selected OTU sequences were then globally aligned using USEARCH against a database of classified 16S sequences. Confidence values were assigned to each OTU classification, and the lowest common ancestor was determined based on these confidence values. The top hit and lowest common ancestor was then reported for each OTU.

Positive cultures were present in more than 50%, and positive NGS results were present in almost 40% of revision arthroplasty cases.

Cutibacterium (formerly Propionibacterium) acnes was the most common bacterial species cultured (8 of 13 [61.5%]) and identified by NGS (12 of 17 [70.1%]) in cases of definite and probable infection. The concordance (κ) between the 2 diagnostic criteria for defining infection that included culture or NGS was 0.333 (fair). Data from the 44 cases is shown below.

They concluded that culture data from revision shoulder arthroplasty cases commonly yields monomicrobial results; whereas, NGS data suggests that bacterial loads in revision arthroplasty are most commonly polymicrobial.

Comment: These results are interesting for several reasons. 

First, they indicate that, like culturing, NGS cannot provide information of use to the surgeon while the patient is undergoing revision arthroplasty; thus the surgeon must make decisions regarding prosthesis removal and immediate antibiotic therapy without knowledge of the results. 

Second, as can be seen from the chart above, in some cases Cutibacterium and Coagulase Negative Staph we cultured but not detected by NGS. These are the two most commonly recovered organisms in revision of failed shoulder arthroplasty. It is unclear whether NGS was insensitive to these bacteria.

Third, in many of the other cases, NGS commonly suggested the presence of organisms that have not been previously reported in the culture results from cases of revision arthroplasty: A junii, A tetradius, A radioresistens, A calcoaceticus, A rhizogenes, A ferrireducens, B cepacia, B fungorum, B nordic, B doer, B cepacia, B fragilis, C diphtheria, C tuberculostearicum, C hominis,  C chromoreductans, C kroppenstedtii, C quinii, C hveragerdense, C paradoxus, C acidisoli, C vibrioides, C circulars, E hormaechei, G ruanii, K palustris, K rosea, L crispatus, L agilis, L albida, M catarrhalis, M Luteus, R picettii, R insidiosa, P saccharophilia, R gnavus, S maltophilia, S agalactiae, and others. Thus it is uncertain whether live forms of these bacteria are actually present in the wounds of these patients.

NGS is a very, very sensitive test! The question is whether it is TOO sensitive for clinical use, i.e. if these microbes do not grow in culture are they truly pathogenic and of deep shoulder origin? Or could they be present in the skin itself and killed during the prep process but leaving their DNA behind? We suspect the latter, mostly due to the fact that NGS can (in theory) detect a single genetic copy in the specimen.

The paper does not provide information on the cost and time necessary to complete NGS in comparison to standard culturing.

NGS is a powerful tool being put to increasingly broad use. We look forward to further work demonstrating its value in the management of failed arthroplasty
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We have a new set of shoulder youtubes about the shoulder, check them out at this link.

Be sure to visit "Ream and Run - the state of the art" regarding this radically conservative approach to shoulder arthritis at this link and this link

Use the "Search" box to the right to find other topics of interest to you.

How you can support progress in shoulder surgery

You may be interested in some of our most visited web pages  arthritis, total shoulder, ream and run, reverse total shoulder, CTA arthroplasty, and rotator cuff surgery as well as the 'ream and run essentials'

Revision shoulder arthroplasty - what is the role of next-generation sequencing?

Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections

These authors point out that serum and synovial markers used to diagnose lower extremity prosthetic joint infection (PJI) have performed poorly for shoulder PJI. As a result, diagnosis is commonly reliant on the accuracy of positive or negative cultures.

They sought to determine the correlation between next-generation sequencing (NGS) and routine cultures in revision shoulder arthroplasty.

In 44 total revision arthroplasties tissue samples were transferred immediately into sterile specimen containers and transported for culture and NGS.

There were no cases of polymicrobial culture results; cutibacterium (formerly Propionibacterium) acnes was the most common bacterial species cultured (8 of 13 [61.5%]) and identified by NGS (12 of 17 [70.1%]) in cases of definite and probable infection. 

The concordance (κ) between the 2 diagnostic criteria for defining infection that included culture or NGS was 0.333 (fair). 

Culture data from revision shoulder arthroplasty cases commonly reveals the presence of a single organism; whereas, NGS results suggests the presence of multiple organisms (see below)

The authors conclude with the statement, "Finally, and perhaps most importantly, given our limited understanding of the normal shoulder joint microbiota, it is difficult to interpret both the NGS and culture data and to define PJI."

Comment: This paper opens an interesting discussion regarding NGS. For example, does a NGS finding of DNA similar to that from an organism, such as staph aureus, indicate that there were viable staph aureus bacteria in the sample? By contrast the growth of staph aureus in a culture would seem to provide strong evidence for the presence of viable bacteria. Secondly, would an NGS finding of K pneumonia, K oytoca, or K palustris in a sample indicate the need for anti-Klebsiella antibiotics? In other words, is there evidence suggesting that NGS finds might help guide treatment?

A related article is discussed below:
Diagnosis of Periprosthetic Joint Infection: The Potential of Next-Generation Sequencing

These authors sought to evaluate the accuracy of next-generation sequencing in identifying the causative organism(s) in patients with periprosthetic joint infection.

Samples were collected from 65 revision arthroplasties (39 knees and 26 hips) and 17 primary arthroplasties (9 hips and 8 knees). Synovial fluid, deep tissue, and swabs were obtained at the time of the surgical procedure and were submitted for next-generation sequencing. Deep-tissue specimens were also sent to the institutional laboratory for culture.

Sensitivity and specificity were calculated for next-generation sequencing using the Musculoskeletal Infection Society (MSIS) definition of periprosthetic joint infection as the standard.

28 of the 65 revisions met the authors' criteria for infection. 17 of these were cultures positive and 25 were next-generation sequencing positive. There was concordance between next-generation sequencing and culture in 15 cases. Among the 11 cases of culture-negative periprosthetic joint infection, next-generation sequencing was able to identify an organism in 9 cases.

Next-generation sequencing identified microbes in 9 of 36 "aseptic" revisions with negative cultures and in 6 of 17 primary total joint arthroplasties. 

Next-generation sequencing detected several organisms in most positive samples. However, in the majority of patients who were infected, 1 or 2 organisms were dominant. These findings suggest that some cases of monomicrobial periprosthetic joint infection may have DNA resembling that from additional organisms.

Comment: In their introduction the authors state "in up to 50% of periprosthetic joint infection cases, cultures fail to isolate the infecting organism". In the realm of revision shoulder arthroplasty, we and others have pointed out that "culture negativity", especially with regard to Propionibacterium, is often due to inadequate culture techniques: failure to obtain at least 5 deep tissue or explant samples, failure to culture on aerobic and anaerobic media and broth, and failure to observe the cultures for 17 days. Thus, unless the details of the approach to culturing are appropriate for Propionibacterium, the phrase "culture negative" must be used with caution. On the other hand, if specimen harvesting and culturing methods are appropriate and standardized, one can generate a semiquantitative picture of the culture results for the joint in question, estimating the load of bacteria in the joint rather than stating that the culture result as simply "positive" or "negative" - see this link.

Thus, it is of interest that in this study, only 60% of the lower extremity joints meeting the MSIS criteria for infection were "culture positive".

Defining and standardizing the culture practice is essential to the application of the MSIS criteria for infection, which are critically dependent on the number of specimens that are culture positive. The smaller number of specimens submitted, the fewer media used, and the shorter the period of culture observation, the less likely that bacteria will be recovered by culture and the less likely the case is to meet the criteria.

To build on this point, the authors state, "in approximately one-third of supposedly noninfected revision cases in this study, next-generation sequencing had detected bacteria. In many of these cases, Propionibacterium acnes was the predominant organism." Thus it would be important to know whether Propionibacterium-specific culturing techniques were used in the those cases; if not, it is unlikely that standard culturing techniques would have been positive for this organism. Furthermore, the detection of Propionibacterium in failed / revised hip and knee arthroplasty cases raises the concern that cases failing to meet the MSIS criteria for infection (because of the low level of inflammatory response and the difficulty of culturing Propionibacterium) may actually harbor this organism. Of note, none of the hip and knee cases in this series were culture positive for Propionibacterium. This is  in marked contrast to the results of some other studies of revised hip and knee arthroplasty in which Propionibacterium were prominent (see this link).

There is a well know saying, "absence of evidence is not evidence of absence," suggesting we be cautious in using the term 'aseptic'. We could take a number of samples of this lawn and not find any that show a poisonous mushroom, but that doesn't mean Amanita phalloides are not really there.

Finally, these authors found that next-generation sequencing was positive in approximately 35% of primary arthroplasties and 25% of revision of arthroplasties, in which the patients were presumed to be noninfected. In these cases we must wonder if these results were spurious, or (more likely in our view) that these cases actually had bacteria present, but that the presence of the bacteria (often Propionibacterium) had not manifested itself in a manner that was otherwise evident.

These authors are to be congratulated on a robust study that challenges current thinking regarding how to define a "periprosthetic infection". It prompts us to ask what may be the more important question, "what preoperative and intraoperative data do we need to select the appropriate surgical and antibiotic treatment of a patient having a revision of a failed arthroplasty?"

===

We have a new set of shoulder youtubes about the shoulder, check them out at this link.

Be sure to visit "Ream and Run - the state of the art" regarding this radically conservative approach to shoulder arthritis at this link and this link

Use the "Search" box to the right to find other topics of interest to you.

How you can support progress in shoulder surgery

You may be interested in some of our most visited web pages  arthritis, total shoulder, ream and run, reverse total shoulder, CTA arthroplasty, and rotator cuff surgery as well as the 'ream and run essentials'