Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: A pilot study

Early administration of antibiotics is crucial in the management of bacterial meningitis. Rapid pathogen identification helps to make a definite diagnosis of bacterial meningitis and enables tailored antibiotic treatment. We investigated if the 16S amplicon sequencing performed by MinION, a nanopore sequencer, was capable of rapid pathogen identification in bacterial meningitis. Six retrospective cases of confirmed bacterial meningitis and two prospective cases were included. The initial cerebrospinal fluid (CSF) samples of these patients were used for the experiments. DNA was extracted from the CSF, and PCR was performed on the 16S ribosomal DNA (16S rDNA). Sequencing libraries were prepared using the PCR products, and MinION sequencing was performed for up to 3 h. The reads were aligned to the bacterial database, and the results were compared to the conventional culture studies. Pathogenic bacteria were successfully detected from the CSF by 16S sequencing in all retrospective cases. 16S amplicon sequencing was more sensitive than conventional diagnostic tests and worked properly even in antibiotics-treated samples. MinION sequencing significantly reduced the turnaround time, and even 10 min of sequencing was sufficient for pathogen detection in certain cases. Protocol adjustment could further increase the sensitivity and reduce the turnaround time for MinION sequencing. Finally, the prospective application of MinION 16S sequencing was successful. Nanopore 16S amplicon sequencing is capable of rapid bacterial identification from the CSF of the bacterial meningitis patients. It may have many advantages over conventional diagnostic tests and should therefore be applied in a larger number of patients in the future.     

Molecular diagnosis of polymicrobial brain abscesses with 16S-rDNA-based next-generation sequencing

ObjectivesBrain abscesses lead to high mortality despite antibiotic and surgical treatment. Identification of causative bacteria is important to guide antibiotic therapy, but culture-based methods and molecular diagnostics by Sanger sequencing of 16S PCR products are hampered by antibiotic treatment and the often polymicrobial nature of brain abscesses. We have applied 16S-rRNA-based next-generation sequencing (NGS) for metagenomic analysis of intracranial abscess (brain and epidural) and meningitis samples.MethodsSeventy-nine samples from 54 patients with intracranial abscesses or meningitis were included. DNA was subjected to 16S PCR. Amplicons were analysed with the Illumina MiSeq system, sequence reads were blasted versus the NCBI 16S bacterial database and analysed using MEGAN software. Results were compared to those of gram-staining, culture and Sanger sequencing.ResultsThe NGS workflow was successful for 51 intracranial abscesses (46 brain and five epidural) and nine meningitis samples. Inclusion of (mono)bacterial meningitis samples allowed us to establish a cut-off criterion for the exclusion of contaminating sequences. In total 86 bacterial taxa were identified in brain abscesses by NGS, with Streptococcus intermedius and Fusobacterium nucleatum as most prevalent species; Propionibacterium and Staphylococcus spp. were associated with epidural abscesses. NGS identified two or more bacterial taxa in 31/51 intracranial abscesses, revealing the polymicrobial nature of these infections and allowing the discrimination of up to 16 bacterial taxa per sample.ConclusionThese results extend earlier studies showing that NGS methods expand the spectrum of bacteria detected in brain abscesses and demonstrate that the MiSeq platform is suitable for metagenomic diagnostics of this severe infection.     

Microbiology of hip and knee periprosthetic joint infections: a database study

ObjectivesKnowledge of the microbiological aetiology of periprosthetic joint infection (PJI) is essential to its management. Contemporary literature from the United States on this topic is lacking. This study aimed to identify the most common microorganisms associated with types of arthroplasty, the timing of infection, and clues to polymicrobial infection.MethodsWe performed an analytical cross-sectional study of patients 18 years of age or older with hip or knee PJI diagnosed at our institution between 2010 and 2019. PJI was defined using the criteria adapted from those of the Musculoskeletal Infection Society. Cases included PJI associated with primary or revision arthroplasty and arthroplasty performed at our institution or elsewhere.ResultsA total of 2067 episodes of PJI in 1651 patients were included. Monomicrobial infections represented 70% of episodes (n = 1448), with 25% being polymicrobial (n = 508) and the rest (5%, n = 111) culture-negative. The most common group causing PJI was coagulase-negative Staphylococcus species (other than S. ludgunensis) (37%, n = 761). The distribution of most common organisms was similar regardless of arthroplasty type. The S. aureus complex, Gram-negative bacteria, and anaerobic bacteria (other than Cutibacterium species) were more likely to be isolated than other organisms in the first year following index arthroplasty (OR 1.7, 95%CI 1.4–2.2; OR 1.5, 95%CI 1.1–2.0; and OR 1.5, 95%CI 1.0–2.2, respectively). The proportion of culture-negative PJIs was higher in primary than revision arthroplasty (6.5% versus 3%, p 0.0005). The presence of a sinus tract increased the probability of the isolation of more than one microorganism by almost three-fold (OR 2.6, 95%CI 2.0–3.3).ConclusionsJoint age, presence of a sinus tract, and revision arthroplasties influenced PJI microbiology.     

Periprosthetic knee infection reconstruction with a hinged prosthesis: Implant survival and risk factors for treatment failure

BackgroundSevere bone and soft tissue defects are common after failed two-stage exchange arthroplasty for periprosthetic joint infection (PJI). There is a paucity of evidence on the outcomes of using a hinged prosthesis for knee PJI reconstruction during second-stage re-implantation, especially regarding implant survivorship, reinfection risk factors, and functionality after successful reconstruction.MethodsA total of 58 knee PJI patients with Anderson Orthopaedic Research Institute (AORI) type II/III defect and soft tissue insufficiency underwent reconstruction with hinged prosthesis. Enrolled patients adhered to a two-stage exchange arthroplasty protocol and were evaluated for a mean follow up of 65.1 months. Kaplan–Meier analysis was conducted for implant survivorship and infection-free survival. Multivariate analysis was used to determine independent risk factors for recurrent infections. Knee Society Score (KSS) was used to evaluate functional outcomes.ResultsThe survivorship of hinged prosthesis was 86.2% at 2 years and 70.2% at 5 years. Infection-free analysis revealed an estimation of 68.9% at 2 years and 60.6% at 5 years. Of the 58 patients, 13 (22.4%) developed recurrent PJI, three (5.2%) aseptic loosening, and one (1.7%) periprosthetic fracture. Multivariate analysis revealed that obesity (hazard ratio (HR), 3.11), high-virulent pathogen (HR, 3.44), and polymicrobial infection (HR, 3.59) were independent risk factors for reinfection. Patients showed a mean improvement of 32.8 ± 7.7 in Knee Society Clinical Score (KSCS) and 30.8 ± 11.0 in Knee Society Function Score (KSFS) after successful reconstruction (P < 0.001).ConclusionsUsing hinged knee prosthesis for PJI reconstruction provided an overall implant survival of 70.2% and an infection-free survival of 60.6% at mid-term follow up. Obesity, virulent pathogens, and polymicrobial infections were independent risk factors for infection recurrence.     

Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.     

Detection of 16S rRNA gene for rapid identification of bacterial pathogens causing peritonitis in patients on continuous ambulatory peritoneal dialysis

PurposePeritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD.MethodsFifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate.ResultsAmong the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed.ConclusionThe molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.     

Origin and characteristics of haematogenous periprosthetic joint infection

ObjectivesRecognition of infectious origin of haematogenous periprosthetic joint infections (PJI) is crucial. We investigated the primary focus and characteristics of haematogenous PJI.MethodsConsecutive patients who presented with haematogenous PJI between 01/2010 and 01/2018 were retrospectively analysed. Haematogenous PJI was defined by diagnosis of infection ≥1 month after surgery, acute manifestation after a pain-free period and positive blood or prosthetic-site culture and/or evidence of distant infectious focus consistent with the pathogen. Fisher's exact, Student's t and Mann–Whitney U tests were used, as appropriate.ResultsA total of 106 episodes of PJI were included, involving 59 knee, 45 hip, one shoulder and one elbow prostheses. The median time from last surgery until haematogenous PJI was 47 months (range, 1–417 months). The pathogen was identified in 105 episodes (99%), including Staphylococcus aureus (n = 43), streptococci (n = 32), enterococci (n = 13), Gram-negative bacteria (n = 9) and coagulase-negative staphylococci (n = 8). Gram-negative bacteria were significantly more often found in hip joints than in knee joints. Blood cultures grew the pathogen in 43 of 70 episodes (61%). The primary infectious focus was identified in 72 episodes (68%) and included infections of intravascular devices or heart valves (22 episodes), skin and soft tissue (16 episodes), the oral cavity (12 episodes), urogenital (12 episodes) or gastrointestinal tract (seven episodes) and other sites (three episodes).ConclusionsIn acute PJI manifesting after a pain-free period, the haematogenous infection route should be considered and the primary infectious focus should be actively searched for. The cardiovascular system, skin and soft tissue, oral cavity, urogenital and gastrointestinal tracts were common origins of haematogenous PJI.     

PCR and DNA sequencing in establishing the aetiology of bacterial infections in children

The results of partial polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene and subsequent DNA sequencing of clinical samples from children are described. In 13 out of 62 samples, DNA from bacteria likely to be the cause of infection was identified. In the vast majority (11/13) of samples with significant pathogen culture the specimen had been negative. Antibiotics had been given in all cases except for three prior to sampling. PCR and subsequent DNA sequencing is a valuable supplementary tool for establishing the cause of bacterial infections in children when culture is negative.     

The impact of preoperative bacteriuria on the risk of periprosthetic joint infection after primary knee or hip replacement: a retrospective study with a 1-year follow up

ObjectivesPatients who undergo elective joint replacement are traditionally screened and treated for preoperative bacteriuria to prevent periprosthetic joint infection (PJI). More recently, this practice has been questioned. The purpose of this study was to determine whether preoperative bacteriuria is associated with an increased risk of PJI.MethodsPatients who had undergone a primary hip or knee replacement in a tertiary care hospital between September 2002 and December 2013 were identified from the hospital database (23 171 joint replacements, 10 200 hips, and 12 971 knees). The results of urine cultures taken within 90 days before the operation were obtained. Patients with subsequent PJI or superficial wound infection in a 1-year follow-up period were identified based on prospective infection surveillance. The association between bacteriuria and PJI was examined using a multivariable logistic regression model that included information on the operated joint, age, gender and the patients' chronic diseases.ResultsThe incidence of PJI was 0.68% (n = 158). Preoperative bacteriuria was not associated with an increased risk of PJI either in the univariate (0.51% versus 0.71%, OR 0.72, 95% CI 0.34–1.54) or in the multivariable (OR 0.82, 95% CI 0.38–1.77) analysis. There were no cases where PJI was caused by a pathogen identified in the preoperative urine culture. Results were similar for superficial infections.ConclusionsThere was no association between preoperative bacteriuria and postoperative surgical site infection. Based on these results, it seems that the preoperative screening and treatment of asymptomatic bacteriuria is not required.     

The international sinonasal microbiome study: A multicentre,multinational characterization of sinonasal bacterial ecology

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43%; P = .02) was identified. Despite some measured differences in microbiome composition and diversity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short-read amplicon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota.     

Novel Tropheryma species in a lung biopsy sample from a kidney transplant recipient

ObjectivesA kidney transplant recipient with recurrent pleuritis underwent an open lung biopsy, the results of which revealed multiple nodular infiltrates. Grocott and periodic acid–Schiff staining were positive. Fungal and Tropheryma whipplei PCR were, however, negative. Further identification was needed.MethodsFormalin-fixed, paraffin-embedded (FFPE) extraction was performed using an FFPE extraction kit. T. whipplei was searched for using a real-time PCR targeting the noncoding repeat specific for T. whipplei. Identification of the bacteria in the extract was done using 16S rDNA and 23S rDNA sequencing and BLAST analysis. Internal transcribed spacer PCR was used for fungal DNA identification.ResultsThe FFPE extract was negative for fungi and T. whipplei. 16S rDNA sequence analysis of a 1375 bp fragment gave T. whipplei as the best match with 26 mismatches, resulting in only 98% agreement. Sequence analysis of the 23S rDNA gene again gave T. whipplei as the best match, but with only 91% agreement. A pan-Tropheryma 16S rDNA real-time PCR was developed, and both the biopsy sample and a respiratory sample of the patient were strongly positive. The patient received antimicrobial treatment targeting T. whipplei with good clinical outcome.Conclusions16S and 23S rDNA sequencing gave T. whipplei as the best hit, although with limited agreement. These findings suggest that a novel Tropheryma species that lacks the noncoding repeat, most frequently used for molecular detection of Whipple disease, might be the cause of the pulmonary disease. Adaptation of current PCR protocols is warranted in order to detect all Tropheryma species.     

The effect of preoperative oral antibiotic use on the risk of periprosthetic joint infection after primary knee or hip replacement: a retrospective study with a 1-year follow-up

ObjectivesAntibiotics are used for various reasons before elective joint replacement surgery. The aim of this study was to investigate patients' use of oral antibiotics before joint replacement surgery and how this affects the risk for periprosthetic joint infection (PJI).MethodsPatients having a primary hip or knee replacement in a tertiary care hospital between September 2002 and December 2013 were identified (n = 23 171). Information on oral antibiotic courses purchased 90 days preoperatively and patients' chronic diseases was gathered. Patients with a PJI in a 1-year follow-up period were identified. The association between antibiotic use and PJI was examined using a multivariable logistic regression model and propensity score matching.ResultsOne hundred and fifty-eight (0.68%) cases of PJI were identified. In total, 4106 (18%) joint replacement operations were preceded by at least one course of antibiotics. The incidence of PJI for patients with preoperative use of oral antibiotics was 0.29% (12/4106), whereas for patients without antibiotic use it was 0.77% (146/19 065). A preoperative antibiotic course was associated with a reduced risk for subsequent PJI in the multivariable model (OR 0.40, 95% CI 0.22–0.73). Similar results were found in the propensity score matched material (OR 0.34, 95% CI 0.18–0.65).ConclusionsThe use of oral antibiotics before elective joint replacement surgery is common and has a potential effect on the subsequent risk for PJI. Nevertheless, indiscriminate use of antibiotics before elective joint replacement surgery cannot be recommended, even though treatment of active infections remains an important way to prevent surgical site infections.     

Prosthetic joint infection of the knee - arthroscopic biopsy identifies more and different organisms than aspiration alone

BackgroundProsthetic joint infection (PJI) causes significant morbidity and mortality following knee replacement surgery. Identifying causative organisms and antibiotic sensitivities is critical in increasing the chance of infection eradication. This study investigated whether biopsy alone was superior to aspiration alone for serological diagnosis in PJI following knee replacement. Secondly, we investigated whether biopsy identifies the same or new/different microbiological flora as aspiration.MethodsSince December 2014, the Exeter Knee Reconstruction Unit (EKRU) has prospectively collated data regarding all PJIs referred from our local/regional network which have been reviewed via our Multi-Disciplinary Team (MDT). We identified and included consecutive patients from this MDT from Dec.2014-Mar.2020 and analysed their electronic records. Statistical analysis was performed using Stata.Results65/100 patients studied had both pre-operative aspiration and biopsy. 31/65 (48%) had positive aspiration and biopsies. No aspirate samples were positive with corresponding biopsies negative. In 19/65 (29%) of infection positive patients, biopsy identified new (7) or additional (12) organisms not identified by aspiration. Aspiration had a sensitivity of 70%, specificity of 88%, positive predictive value of 90.3% and negative predictive value of 64.7%. Biopsy had a sensitivity of 97.5%, specificity of 88%, positive predictive value of 92.9% and negative predictive value of 95.7%.ConclusionIn 29% of confirmed PJI cases, arthroscopic biopsy identified either additional organisms in a polymicrobial PJI when compared to aspiration, or new positive results when aspiration alone was negative. This study demonstrates the benefits of arthroscopic biopsy for serological diagnosis in cases of knee PJI and aids treatment planning.     

Utilizing nanopore sequencing technology for the rapid and comprehensive characterization of eleven HLA loci; addressing the need for deceased donor expedited HLA typing

The comprehensive characterization of human leukocyte antigen (HLA) genomic sequences remains a challenging problem. Despite the significant advantages of next-generation sequencing (NGS) in the field of Immunogenetics, there has yet to be a single solution for unambiguous, accurate, simple, cost-effective, and timely genotyping necessary for all clinical applications. This report demonstrates the benefits of nanopore sequencing introduced by Oxford Nanopore Technologies (ONT) for HLA genotyping. Samples (n = 120) previously characterized at high-resolution three-field (HR-3F) for 11 loci were assessed using ONT sequencing paired to a single-plex PCR protocol (Holotype) and to two multiplex protocols OmniType (Omixon) and NGSgo®-MX6-1 (GenDx). The results demonstrate the potential of nanopore sequencing for delivering accurate HR-3F typing with a simple, rapid, and cost-effective protocol. The protocol is applicable to time-sensitive applications, such as deceased donor typings, enabling better assessments of compatibility and epitope analysis. The technology also allows significantly shorter turnaround time for multiple samples at a lower cost. Overall, the nanopore technology appears to offer a significant advancement over current next-generation sequencing platforms as a single solution for all HLA genotyping needs.     

Polymerase chain reaction, with sequencing, as a diagnostic tool in culture-negative bacterial meningitis

Objective: To evaluate the feasibility of using 16S rDNA universal primer PCR (followed by sequencing) and 65-kDa heat shock Mycobacterium tuberculosis protein gene PCR as a method to determine a bacterial etiology in culture-negative cerebrospinal fluid (CSF) samples.
Methods: One hundred and forty-nine CSF samples from 128 patients were processed. DNA was extracted from the CSF samples and amplified with the eubacterial 16S rDNA primers P11E and P13B, and with the 65-kDa heat shock protein gene mycobacterial primers. The amplicons were identified by sequencing and specific oligoprobe hybridization.
Results: Overall, a microbiological diagnosis was made in 11 of 125 ultimately culture-negative cases. The use of 65-kDa heat shock protein gene PCR was needed to improve the diagnosis of tuberculous meningitis; in four patients, prospectively studied, the outcome of antituberculous therapy could also be followed.
Conclusions: In culture-negative bacterial meningitis it is possible to improve the microbiological diagnosis by use of 16S rDNA amplification and sequencing, together with amplification of a more specific gene in mycobacteria.     

Bloodstream infections due to Peptoniphilus spp.: report of 15 cases

Peptoniphilus spp. are Gram-positive anaerobic cocci (GPAC) that were formerly classified in the genus Peptostreptococcus. This study describes 15 cases of Peptoniphilus spp. bloodstream infection (BSI) diagnosed from 2007 to 2011 using 16S rDNA sequencing in patients with pneumonia, pre-term delivery, soft tissue infection or colon or bladder disease. Seven out of 15 (47%) of these cases had polymicrobial BSIs. One of the isolates was closely related to P. duerdenii (EU526290), while the other 14 isolates were most closely related to a Peptoniphilus sp. reference strain (ATCC 29743) and P. hareii (Y07839). Peptoniphilus is a rare but important cause of BSI.     

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.     

Pyrosequencing of a short hypervariable 16S rDNA fragment for the identification of nontuberculous mycobacteria--a comparison with conventional 16S rDNA sequencing and phenotyping

Conventional methods for identification of nontuberculous mycobacteria (NTM) are often inexact and time consuming. Sequencing of bacterial 16S rDNA is accurate, rapid and effective. We have retrospectively evaluated the discriminative power of pyrosequencing of a short hypervariable 16S rDNA fragment as a simple and rapid tool for NTM characterization. A series of 312 clinical NTM isolates, excluding the M. avium/intracellulare complex, was investigated. When species could not be resolved by sequencing alone, growth rate and pigment production were also examined. 54% (170/312) of the isolates were unambiguously identified by both methods. An additional 14% (45/312) were directly identified to species by conventional 16S rDNA sequencing but needed complementary phenotypic analysis when examined by pyrosequencing. The remaining 31% (97/312) needed additional phenotypic analysis for both sequencing methods. We consider the pyrosequencing procedure to be a useful alternative for the identification of several NTM species, and a versatile tool for the characterization of clinical NTM isolates. At times it requires additional tests for definite species diagnosis and correct identification.     

Whole-genome sequencing of Staphylococcus epidermidis bloodstream isolates from a prospective clinical trial reveals that complicated bacteraemia is caused by a limited number of closely related sequence types

ObjectivesThe significance of isolating Staphylococus epidermidis from a blood culture is highly heterogeneous, ranging from contamination to an indication of a serious infection. Herein we sought to determine whether there is a relationship between S. epidermidis genotype and clinical severity of bacteraemia.MethodsS. epidermidis bacteraemias from a prospective, multicentre trial at 15 centres in the United States and one in Spain were classified as simple (including possible contamination), uncomplicated, and complicated. Whole-genome sequencing (WGS) was performed on 161 S. epidermidis isolates, and clinical outcomes were correlated with genotypic information.ResultsA total of 49 S. epidermidis sequence types (STs) were identified. Although strains of all 49 STs were isolated from patients with either simple or uncomplicated infection, all strains causing complicated infections were derived from five STs: ST2, ST5, ST7, ST16, and ST32. ST2 and ST5 isolates were significantly more likely to cause uncomplicated and complicated bloodstream infections compared to simple bacteraemia (odds ratio 2.0, 95%CI 1.1–3.9, p 0.04). By multivariate regression analysis, having an ST2 or ST5 S. epidermidis bacteraemia was an independent predictor of complicated bloodstream infection (odds ratio 3.7, 95%CI 1.2–11.0, p 0.02). ST2/ST5 strains carried larger numbers of antimicrobial resistance determinants compared to non-ST2/ST5 isolates (6.34 ± 1.5 versus 4.4 ± 2.5, p < 0.001).ConclusionS. epidermidis bacteraemia was caused by a genetically heterogeneous group of organisms, but only a limited number of STs—particularly multidrug-resistant ST2 and ST5 strains—caused complicated infections.     

Role of universal 16S rRNA gene PCR and sequencing in diagnosis of prosthetic joint infection

The etiological diagnosis of prosthetic joint infection (PJI) requires the isolation of microorganisms from periprosthetic samples. Microbiological cultures often yield false-positive and false-negative results. 16S rRNA gene PCR combined with sequencing (16SPCR) has proven useful for diagnosing various infections. We performed a prospective study to compare the utility of this approach with that of culture to diagnose PJI using intraoperative periprosthetic samples. We analyzed 176 samples from 40 patients with PJI and 321 samples from 82 noninfected patients using conventional culture and 16SPCR. Three statistical studies were undertaken following a previously validated mathematical model: sample-to-sample analysis, calculation of the number of samples to be studied, and calculation of the number of positive samples necessary to diagnose PJI. When only the number of positive samples is taken into consideration, a 16SPCR-positive result in one sample has good specificity and positive predictive value for PJI (specificity, 96.3%; positive predictive value, 91.7%; and likelihood ratio [LR], 22), while 3 positive cultures with the same microorganism are necessary to achieve similar specificity. The best combination of results for 16SPCR was observed when 5 samples were studied and the same microorganism was detected in 2 of them (sensitivity, 94%; specificity, 100%; and LR, 69.62). The results for 5 samples with 2 positive cultures were 96% and 82%, respectively, and the likelihood ratio was 1.06. 16SPCR is more specific and has a better positive predictive value than culture for diagnosis of PJI. A positive 16SPCR result is largely suggestive of PJI, even when few samples are analyzed; however, culture is generally more sensitive.