Broad-range Polymerase Chain Reaction Research Articles

Molecular Diagnostics for Invasive Fungal Diseases: Current and Future Approaches

Invasive fungal diseases (IFDs) comprise a growing healthcare burden, especially given the expanding population of immunocompromised hosts. Early diagnosis of IFDs is required to optimise therapy with antifungals, especially in the setting of rising rates of antifungal resistance. Molecular techniques including nucleic acid amplification tests and whole genome sequencing have potential to offer utility in overcoming limitations with traditional phenotypic testing. However, standardisation of methodology and interpretations of these assays is an ongoing undertaking. The utility of targeted Aspergillus detection has been well-defined, with progress in investigations into the role of targeted assays for Candida, Pneumocystis, Cryptococcus, the Mucorales and endemic mycoses. Likewise, whilst broad-range polymerase chain reaction assays have been in use for some time, pathology stewardship and optimising diagnostic yield is a continuing exercise. As costs decrease, there is also now increased access and experience with whole genome sequencing, including metagenomic sequencing, which offers unparalleled resolution especially in the investigations of potential outbreaks. However, their role in routine diagnostic use remains uncommon and standardisation of techniques and workflow are required for wider implementation.

Coniochaeta hoffmannii and mutabilis Invasive Fungal Keratitis: A Case Series and Literature Review

Purpose: To describe 2 Coniochaeta species (Coniochaeta hoffmannii and Coniochaeta mutabilis) as rare causes of invasive fungal keratitis resulting in enucleations and to provide a review of ocular infections caused by the genus Coniochaeta and underscore changes to its classification over the past 3 decades, which may clarify previous reports with outdated nomenclature. Methods: This is a small case series from a single academic institution (Casey Eye Institute) with a summary of the literature. Results: The first case describes a 53-year-old man who had an indolent but persistent anterior chamber reaction after repair of a penetrating nail injury. Cultures from a second penetrating keratoplasty identified C. hoffmannii, which was determined through phenotypic characterization and DNA sequencing. Despite surgical management, cultures were persistently positive for C. hoffmannii. Two years post-injury, he developed a funnel retinal detachment with proliferative vitreoretinopathy and elected for enucleation. The second case describes a 54-year-old woman with poor contact lens hygiene with a presumed filamentous fungal keratitis that remained refractory to topical natamycin. Broad-range polymerase chain reaction of an anterior chamber aspirate identified C. mutabilis. Therapeutic keratoplasty was performed for worsening infection; however, because of intractable pain, she elected for enucleation. Pathology from the enucleation specimen demonstrated persistent, but sparse, fungal elements. Conclusions: This is the first case series characterizing ocular infections caused by Coniochaeta, a rare cause of invasive fungal keratitis resulting in devastating outcomes for our patients. Identification of Coniochaeta was initially difficult by traditional culturing techniques, and subsequent molecular diagnostic testing proved useful in detection. Our review of ocular Coniochaeta clarifies previous reports with outdated nomenclature.

A decade of clinical microbiology: top 10 advances in 10 years: what every infection preventionist and antimicrobial steward should know.

The past 10 years have brought paradigm-shifting changes to clinical microbiology. This paper explores the top 10 transformative innovations across the diagnostic spectrum, including not only state of the art technologies but also preanalytic and post-analytic advances. Clinical decision support tools have reshaped testing practices, curbing unnecessary tests. Innovations like broad-range polymerase chain reaction and metagenomic sequencing, whole genome sequencing, multiplex molecular panels, rapid phenotypic susceptibility testing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry have all expanded our diagnostic armamentarium. Rapid home-based testing has made diagnostic testing more accessible than ever. Enhancements to clinician-laboratory interfaces allow for automated stewardship interventions and education. Laboratory restructuring and consolidation efforts are reshaping the field of microbiology, presenting both opportunities and challenges for the future of clinical microbiology laboratories. Here, we review key innovations of the last decade.

582. Comparing Broad-range PCR Testing and The Biofire® FilmArray® Pneumonia (PN) Panel in the Diagnosis of Bacterial Pneumonia

Abstract Background Given the low sensitivity of conventional microbial isolation methods for identifying respiratory pathogens in bacterial pneumonia, target-specific syndromic multiplex real-time PCR panels have been used in conjunction with culture methods to improve diagnostic yield. Additionally, broad-range polymerase chain reaction (BR-PCR) targeting bacterial 16s rRNA conserved region has shown higher sensitivity with certain specimen types, so we sought to evaluate the clinical performance of BR-PCR performed on bronchoalveolar lavage (BAL) specimens in comparison to The Biofire® FilmArray® Pneumonia (PN) Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Methods A retrospective chart review was performed on all BAL specimens that had both a PN panel test and BR-PCR performed from January 2020 to May 2022 at all Indiana University affiliated hospitals. The PN panel test was performed in-house as per laboratory protocol, while BR-PCR was performed in a reference laboratory. Outcomes assessed included turn-around times (TAT), sensitivity and specificity of BR-PCR and clinical impact, if any. Results A total of 68 BAL specimens from 53 patients were identified (83% of patients were immunocompromised). Percent positivity for the PN panel was 19% and that of BR-PCR was 18%. With the PN panel used as the gold standard, the sensitivity and specificity of BR-PCR was 85% and 98%, respectively. Only one respiratory organism was detected by BR-PCR but not by the PN panel, and it was not considered pathogenic or to have a significant clinical impact. The median TAT for the PN panel was 2.1 hours (1.8, 3.2) versus 7.8 days (6.9, 10.4) for BR-PCR. Conclusion In our cohort of patients, BR-PCR testing was not superior to the Biofire® FilmArray® Pneumonia (PN) Panel when used to detect certain bacterial etiologies of pneumonia. Additionally, faster TAT for the panel test has the potential to enhance antimicrobial stewardship practices by enabling better antibiotic utilization. Adjunctive BR-PCR testing may be useful for clinical care when conventional testing is negative and patients are at risk for a variety of potential pathogens, including fungi. Disclosures All Authors: No reported disclosures

Bacterial DNA and osteoarthritis in dogs with patellar luxation and cranial cruciate ligament rupture.

The association between bacterial DNA in stifle joints, including those with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL), and osteoarthritis in dogs remains elusive. This study investigated the potential association between the detection of bacterial DNA and osteoarthritis in dogs using a broad-range polymerase chain reaction technique targeting the 16S ribosomal RNA gene. Synovial fluid (35 samples) and knee tissue samples (32 samples) were obtained from 35 dogs diagnosed with CCLR (n = 20; 11 males and nine females) or MPL (n = 15; five males and 10 females) who underwent a surgical operation between October 2014 and April 2015. Dogs with CCLR had a higher average osteoarthritis score than those with MPL (2.0 ± 0.9 vs. 0.5 ± 0.9; p = 0.005). Bacterial DNA was detected in the stifle joints of 60.71% of dogs with MPL. Pelomonas spp. (25.00%), Halomonas spp. (17.86%), and 5 other species (17.86%) were the most frequently identified bacteria. Bacterial DNA was detected in 41.03% of dogs with CCLR. Pelomonas spp. (15.38%), Sphingomonas spp. (10.26%), Halomonas spp. (5.13%), and 4 other species (10.26%) were the most frequently identified bacteria. No significant difference was observed in the prevalence of bacterial DNA obtained from tissue samples (46.88%) or joint fluid samples (51.43%). The presence of bacterial DNA was not associated with the type of knee injury (MPL or CCLR; p = 1.000). There was a higher prevalence of bacterial DNA in samples from dogs with moderate-to-severe osteoarthritis (94.44%) than in those with minimal osteoarthritis (41.18%), and a significant association between the presence of bacterial DNA and moderate-to-severe osteoarthritis was identified (p < 0.01). Dogs with moderate-to-severe osteoarthritis were more likely to have bacterial DNA in their stifle joints than those with no or minimal osteoarthritis. These findings provide valuable insight into the potential role of bacterial DNA in joint tissue or joint fluid and the development of osteoarthritis in dogs.

A Dual-Pathogen Mitral Valve Endocarditis Caused by Coxiella burnetii and Streptococcus gordonii-Which Came First?

Infective endocarditis (IE) is still a life-threatening disease with high morbidity and mortality. While usually caused by a single bacterium, poly-microbial infective endocarditis (IE) is rare. Here, we report a (blood-culture-negative) dual pathogen mitral valve IE caused by Coxiella burnetii and Streptococcus gordonii: A 53-year-old woman was presented to an internal medicine department with abdominal pain for further evaluation. Within the diagnostic work up, transthoracic echocardiography (TTE) revealed an irregularly shaped echogenic mass (5 × 13 mm) adherent to the edge of the posterior mitral valve leaflet and protruding into the left atrium. As infected endocarditis was suspected, blood cultures were initially obtained, but they remained negative. Chronic Q fever infection was diagnosed using serologic testing. After the occurrence of cerebral thromboembolic events, the patient was admitted for mitral valve surgery. Intraoperatively, a massively destructed mitral valve with adhering vegetations was noted. Examination of the mitral valve by broad-range bacterial polymerase chain reaction (PCR) and amplicon sequencing confirmed Coxiella burnetii infection and yielded Streptococcus gordonii as the second pathogen. Based on the detailed diagnosis, appropriate antibiotic therapy of both pathogens was initiated, and the patient could be discharged uneventfully on the 11th postoperative day after a successful minimal-invasive mitral valve replacement.

Development of a scoring system to identify high-yield specimens for bacterial broad-range 16S rRNA gene PCR with sequencing at a tertiary care medical center.

Broad-range bacterial polymerase chain reaction with sequencing (BRBPS) provides valuable diagnostic data, especially in cases of culture-negative infections. However, as BRBPS testing demonstrates generally low positivity, cost per impactful result can be high and commonly involves longer turnaround times compared with other methods, targeting use of this assay to high-yield situations remains a challenging goal. Diagnostic stewardship can help alleviate these challenges and increase clinical utility, yet not all laboratories have a dedicated stewardship team, and little formal guidance exists on identifying high-yield samples outside of specific clinical syndromes. In this study, we performed a retrospective review of 86 BRBPS orders from a tertiary care medical center, with a focus on identifying high-yield cases using an infectious markers scoring system, visualized inflammation or organism (VIO) score, to predict return of actionable diagnostic data. Using chart review, we evaluated how results from high VIO score or low VIO score specimens contributed to clinical management. Testing low VIO score samples identified an organism in only 10% of samples, and of these positive results, 33% were considered to represent contamination. Despite negative routine workup and no identified pathogen via BRBPS, broad antimicrobial treatment was continued in 85% of cases with a low VIO score. In contrast, specimens with high VIO scores were more predictably positive by BRBPS, identified organisms that were universally considered pathogens, and provided opportunities to target or de-escalate antimicrobial therapy. This study describes the VIO scoring system to guide the identification of high-yield samples and steward the appropriate use of BRBPS testing.

Are bacteria, fungi, and archaea present in the midtrimester amniotic fluid?

This study was conducted to determine whether bacteria, fungi, or archaea are detected in the amniotic fluid of patients who underwent midtrimester amniocentesis for clinical indications. Amniotic fluid samples from 692 pregnancies were tested by using a combination of culture and end-point polymerase chain reaction (PCR) techniques. Intra-amniotic inflammation was defined as an interleukin-6 concentration >2,935 pg/mL. Microorganisms were detected in 0.3% (2/692) of cases based on cultivation, 1.73% (12/692) based on broad-range end-point PCR, and 2% (14/692) based on the combination of both methods. However, most (13/14) of these cases did not have evidence of intra-amniotic inflammation and delivered at term. Therefore, a positive culture or end-point PCR in most patients appears to have no apparent clinical significance. Amniotic fluid in the midtrimester of pregnancy generally does not contain bacteria, fungi, or archaea. Interpretation of amniotic fluid culture and molecular microbiologic results is aided by the assessment of the inflammatory state of the amniotic cavity. The presence of microorganisms, as determined by culture or a microbial signal in the absence of intra-amniotic inflammation, appears to be a benign condition.

Prosthetic valve endocarditis secondary to Tropheryma whipplei in a patient with chronic polyarthritis

BackgroundWhipple’s disease is a chronic multisystemic infectious disease that rarely presents as culture-negative endocarditis. Most patients reported with Tropheryma whipplei endocarditis involve a native valve and few describe prosthetic valve disease.Case presentationA patient with chronic polyarthritis and previous mitral valve replacement developed decompensated heart failure without fever. Transesophageal echocardiography revealed a prosthetic mitral valve vegetation and he underwent prosthetic mitral valve replacement. Blood and prosthetic mitral valve cultures were unrevealing. Broad-range polymerase chain reaction (PCR) of the extracted valve and subsequent Periodic-acid-Schiff (PAS) staining established the diagnosis of T. whipplei prosthetic valve endocarditis.ConclusionWhipple’s disease may present as culture-negative infective endocarditis and affect prosthetic valves. Histopathology with PAS staining and broad-range PCR of excised valves are essential for the diagnosis. Greater clinical awareness and implementation of these diagnostic procedures should result in an increased reported incidence of this rare disease.

Polymerase chain reaction test for diagnosis of infectious uveitis

BackgroundTo study the clinical utility of broad-range real-time Polymerase Chain Reaction (PCR) assay in patients suspected for infectious uveitis and to analyze the clinical relevance.MethodsMedical records of patients with uveitis were assessed in whom PCR analysis of intraocular fluids was performed between January 2018 and February 2021. Intraocular samples were investigated for cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), herpes simplex viruses type 1 and 2 (HSV1,2), human T-lymphotropic virus type 1 (HTLV-1), Toxoplasma gondii and also for bacterial 16 S and fungal 18 S/28S ribosomal DNA (rDNA).ResultsAqueous paracentesis and vitreous sampling was done for 151 (81.2%) and 35 (18.8%) patients, respectively. Most of the patients had panuveitis (61.3%). PCR results were positive in 69 out of 186 patients (37%) according to the following order: CMV (18 cases), VZV (18 cases), fungal 18s/28s rDNA (17 cases), HSV (9 cases), bacterial 16s rDNA (3 cases), HTLV-1 (2 cases), and Toxoplasma gondii (2 cases). PCR positivity rate was 5.8% in patients with undifferentiated panuveitis. EBV was not detected at all. Initial treatment was changed in 38 patients (20%) based on PCR results. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PCR test for aqueous samples was 82%, 91%, 96%, and 87%, respectively. No significant adverse effect related to sampling was reported.ConclusionPCR analysis of intraocular fluids in patients with suspected infectious uveitis plays an important role in confirming diagnosis or changing treatment with good predictive value. However, routine PCR test in patients with undifferentiated panuveitis in order to rule out possible underlying infectious etiology had low benefit.

Parapneumonic effusion in children: Rapid pathogen detection in pleural fluid using multiplex bacterial PCR.

Parapneumonic effusion in children: Rapid pathogen detection in pleural fluid using multiplex bacterial PCR.

Next generation sequencing to examine associations between vaginal washing and vaginal microbiota: A cohort study.

The association between vaginal washing and HIV risk may be mediated by vaginal washing-associated changes in vaginal microbiota. Data from a cohort of HIV-negative US and Kenyan women enrolled in the Preventing Vaginal Infections trial were analyzed. Vaginal fluid samples and vaginal washing data were collected every 2months for 12months. Bacterial relative abundances were measured by broad-range 16S rRNA gene polymerase chain reaction with next generation sequencing. Generalized estimating equations were used to evaluate the association between vaginal washing and i) the Shannon Diversity Index (SDI); and ii) mean change in percent bacterial relative abundances, with application of a 10% false discovery rate (FDR). Participants (N = 111) contributed 93/630 (14.8%) vaginal washing visits. Mean SDI was 0.74 points higher (95% CI 0.35, 1.14; p < 0.001) at washing visits among US participants (N = 26). Vaginal washing was not associated with SDI in Kenyan participants (N = 85). There were no associations between vaginal washing and vaginal bacterial relative abundances after applying the FDR. The discordant results in Kenyan versus US women suggests the link between vaginal washing and sub-optimal vaginal microbiota may be context specific. Vaginal microbial shifts may not fully explain the association between vaginal washing and HIV acquisition.

A Meta-Analysis of Polymerase Chain Reaction for Prosthetic Joint Infection Diagnosis.

One of the most dangerous side effects of joint replacement for the hip, knee, shoulder, and elbow is prosthesis joint infection (PJI). Polymerase chain reaction (PCR) has been considered a promising method for PJI diagnosis due to its short diagnostic time and high sensitivity. Although several PCR methods such as multiplex PCR and broad-range PCR are useful diagnostic methods for detecting microorganisms causing PJI, values of different PCR methods for the diagnosis of PJI remain unclear. Thus, the objective of this study was to perform a meta-analysis of different PCR methods in the diagnosis of PJI to determine their diagnostic characteristics including sensitivity and specificity. The following data were extracted: PCR method, number of patients, sample site and type, diagnosis standard, true positive, false positive, false negative, and true negative. Pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were calculated. Meta-regression analysis was conducted to assess heterogeneity. Subgroup analysis was also performed to assess effects of several variables on meta-analysis results. The current study showed that pooled sensitivity and pooled specificity were 0.70 (95% CI: 0.67 - 0.73) and 0.94 (95% CI: 0.92 - 0.95), respectively. Results of subgroup analysis indicated that sequencing method showed the lowest sensitivity (0.63, 95% CI: 0.59 - 0.67). However, after excluding studies using tissue samples directly, sequencing method showed higher sensitivity (0.83, 95% CI: 0.73 - 0.90) than other PCR methods (0.74, 95% CI: 0.69 - 0.78). The main significance of this study was that we attempted to classify accuracies of several PCR methods and found that sequencing with a reliable sampling method could be used as an early screening strategy for PJI. Further comparisons for PCR technologies are needed to evaluate their cost effectiveness and diagnostic procedures, not just diagnostic values, to discover the optimal one for PJI diagnosis.

Bacterial Contamination Is Involved in the Etiology of Soft-Tissue Filler, Late-Onset, Inflammatory Adverse Events.

The treatment algorithm in late-onset inflammatory adverse events with soft-tissue fillers depends primarily on the assumed causative factor: immunologic or bacterial. The authors included 29 patients, 13 of whom experienced late-onset inflammatory adverse events to fillers (inflammatory group) and 16 who did not (reference group). Biopsies were acquired from both groups with an 18-G needle. Before taking the biopsy, the authors acquired skin swabs for 25 of the 29 patients. The IS-pro method-a new and very sensitive method to detect microbiota-was used. This is a novel broad-range polymerase chain reaction technique based on length and sequence variations of the 16S to 23S ribosomal interspacer region. IS-pro can detect bacteria at low abundances and identify them up to species level. To exclude contamination from skin microbiota, the authors compared the microbiota found on skin swabs with that found in the corresponding biopsies. A high level of Gram-positive bacteria was found in biopsies of soft-tissue fillers, predominantly in patients from the inflammation group. This suggests that these bacteria were introduced during the primary filler injection treatment. The composition of the microbiota on the skin differed markedly from that in the filler, indicating that contamination during the sampling process did not influence results. Bacteria adherent to soft-tissue fillers or bacteremia probably play a causative role in adverse events. Contamination of samples in the biopsies with skin microbiota was excluded. Therapeutic, III.

332. Broad range PCR of pleural fluid improves diagnosis of bacterial pneumonia complicated by parapneumonic effusion

Abstract Background Bacterial pneumonia complicated by a para pneumonic effusion is frequently encountered in hospitalized patients. However, identifying the etiology of pneumonia remains elusive. Published data report rates of bacteremia ranging from 13-26% among patients with pneumonia complicated by parapneumonic effusion. Good quality sputum samples are difficult to obtain in children. Culture of pleural fluid is helpful, but most patients are already pretreated before a specimen is obtained limiting utility. Nucleic acid assays of pleural fluid have reported sensitivities from 40-80%. We wanted to review our experience with pleural fluid nucleic acid assay at Akron childrens hospital Methods Using help from our laboratory data support team, we retrospectively reviewed the records of all pleural fluid isolates that were sent out to Washington University Medical Center for 16 S rRNA sequencing (broad range polymerase chain reaction assay). The review covered a 8-year time period ranging from January 2014 to March 2022. Negative blood and pleural fluid cultures were a prerequisite before the send out for nucleic acid assay. Results A total of 29 pleural fluid samples were sent out for 16S rRNA sequencing. 22 of these had a detectable target. This translated into a yield of about 75%. The following table summarizes our findings. Conclusion In our experience, nucleic acid assay of the pleural fluid is valuable in making a bacteriological diagnosis of pneumonia with para pneumonic effusion. Its benefit remains in patients pretreated with antibiotics. For the individual patient it may help narrow antibiotic therapy. At an institutional level it would allow for developing guidelines for empiric antibiotic management. However, in most institutions’ nucleic acid assay of pleural fluid remains a send out test and optimizing workflows to reduce turnaround time remains a challenge. Disclosures All Authors: No reported disclosures.

Management and Outcome of Non-Aneurysmal Primary Aortic Infection.

Aortic infection without prior intervention or aneurysm is exceedingly rare. We report the presentation, diagnosis, management, and outcome of patients with this unusual entity. Retrospective chart and imaging review of patients with primary aortic infection. 5 patients (3 male, mean age 71.2years) presented between 2014 and 2022. All had abdominal, back, or flank pain. Four had constitutional symptoms. All were evaluated with a complete blood count; 3 had leukocytosis. Both serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were elevated in the 4 patients evaluated with these tests. All were studied with peripheral blood culture on the first hospital day prior to any antibiotic administration. Blood culture was positive in only 1 patient. Computed tomography (CT) scan showed periaortic inflammation without aneurysm in all. Fluorodeoxyglucose positron emission tomography (PET) was obtained in 3 and a radiolabeled leukocyte single-photon emission CT (SPECT) scan was performed in 2. All demonstrated periaortic concentration of the radioisotope consistent with inflammation or infection. Intraoperative cultures were positive in 3. One patient who had a negative intraoperative culture was examined with broad range polymerase chain reaction (PCR) and DNA sequencing which identified a causative bacterium. The other patient with a negative intraoperative culture had periaortic abscess but was on antibiotics preoperatively, potentially confounding the culture. All patients underwent in-situ repair with rifampin impregnated polyester (N = 2), cryopreserved aortic allograft (N = 2), or autogenous femoral vein (N = 1). No patient developed recurrent infection or aortic related complications following surgery with an average follow up of 31.8months (range 8-88months). Patients with primary aortic infection present similarly with the triad of abdominal or back pain, laboratory markers of infection, and imaging demonstrating periaortic inflammation. Patients were treated successfully with in-situ repair. Preoperative identification of a causative organism was difficult, and PCR may be useful to help identify an organism.

Characteristics of hepatitis E viral infections in Hungary.

Hepatitis E virus (HEV) is one of the most important causes of hepatitis worldwide. Despite this, limited data published more than ten years ago are only available about HEV epidemiology in Hungary. We aimed to determine the epidemiological features of HEV infections among patients submitted to various departments of our university hospital in Hungary with signs and symptoms referring to acute hepatitis. One thousand four hundred thirty-one sera samples from 1,383 patients were analyzed by enzyme-linked immunosorbent assays (ELISA). In some patients, HEV RNA was detected by broad-range nested polymerase chain reaction (PCR) if acute hepatitis was confirmed. PCR products were sequenced and compared with other available sequences in GenBank. Five hundred eighteen sera from 429 patients proved positive (31.0%) for HEV-specific immunoglobulin G (IgG) with a mean age of 60.0 years. Most sera with anti-HEV IgG antibodies were collected from adults and elderly patients. Anti-HEV IgM positive results were found in the case of 95 sera samples from 70 patients (5.1%). Acute HEV infections were confirmed mostly over 40 (n=67, 95.7%). The number of males (n=47, 67.1%) was higher than females (n=23, 32.9%). We detected HEV-specific PCR products in seven patients (10.9%). Genotyping was successful for 5 out of 7 PCR-positive samples. All sequences belonged to genotype 3 (subgenotypes: e, f). In our survey, we confirmed the constant presence of acute HEV infections in Hungary and an increased seroprevalence of anti-HEV IgG antibodies compared to a previous study.

Nonmarinum, Nontuberculous Mycobacterial Infections of the Upper Extremity: A Multi-Institutional Descriptive Report

We analyzed patient demographic factors involved in the development of nonmarinum, nontuberculous mycobacterial infections (NTMI) involving the upper extremity, and assessed diagnostic and prognostic values of commonly used preoperative laboratory and imaging studies, as well as factors related to recurrence of disease and patient outcomes. Patients from 2 academic, tertiary facilities with culture-proven, nonmarinum NTMI involving the upper extremity were reviewed. Patient-related factors and clinical outcomes were extracted. The analysis was based on pathogen identification (rapid- vs slow-growing subspecies) and immune status. Our 76 patients had a mean age of 59 years, and 65% were male. Forty-eight percent reported an injury, and hands were frequently involved (58%). Forty-one percent were immunosuppressed (19% organ transplant recipients). The mean symptom duration prior to presentation was 203 days. The culture identification took a mean of 33 days, with 25 different species identified (subcategorized as rapid or slow growers). Seventy-seven percent had solitary lesions, with a cutaneous or subcutaneous location most common. Immunosuppressed patients were treated longer with antibiotics (243 vs 155 days in immunocompetent patients) and experienced higher rates of side effects, complications, and recurrence. All patients underwent debridement to control infection, including 4 individuals who required amputations. One-third experienced complications and/or recurrence, regardless of the organism type. Upper-extremity nonmarinum NTMI is often misdiagnosed, causing management delays. Early consideration in differential diagnoses of chronic, painful swelling, nodular or inflammatory lesions, or septic arthritis is crucial. Tissue biopsy with specimens for histopathology and microbiological analysis (mycobacterial smear, cultures, and broad range polymerase chain reaction) and early involvement with an infectious disease specialist are recommended. Empiric antibiotic therapy is not standard. Debridement and prolonged, directed combination antimicrobial therapy is required; however, adverse reactions are commonly encountered. Type of study/level of evidence Prognostic IV.

A case of a renal abscess caused by Salmonella bareilly in a previously healthy boy

BackgroundRenal abscesses are relatively uncommon in children, and usually due to Gram-negative rods or Staphylococcus aureus, whereas abscesses caused by Salmonella are very rare.Case presentationWe present the case of a previously healthy 10-year-old boy who had a renal abscess due to Salmonella bareilly. He responded well to treatment with antibiotics, and computed tomography (CT)-guided drainage of the abscess. His blood, urine and abscess aspirate cultures were sterile, but a broad-range 16S rDNA polymerase chain reaction (PCR) assay of the aspirate followed by analysis of four Salmonella genes (fliC, fliD, sopE2, and spaO) identified S. bareilly as the causative agent.ConclusionTo the best of our knowledge, this is the first report of renal abscess caused by S. bareilly.

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue.

Scedosporium (S.) apiospermum is a typical mold causing cerebral abscesses, often after near-drowning. Infections are associated with high morbidity and mortality due to diagnostic challenges including the need for prolonged incubation of cultures. In addition, histopathological differentiation from other filamentous fungi, including Aspergillus fumigatus, may not be possible, excluding early specific diagnosis and targeted therapy. Polymerase chain reaction (PCR) on tissue samples can rapidly identify fungi, leading to an earlier adequate treatment. Due to an extensive spectrum of causative fungi, broad-range PCRs with amplicon sequencing have been endorsed as the best DNA amplification strategy. We herein describe a case with brain abscesses due to S. apiospermum in a 66-year-old immunocompromised female patient. While broad-range PCR failed to identify a fungal pathogen from a cerebral biopsy demonstrating hyaline mold hyphae, specific quantitative PCR (qPCR) identified Scedosporium and ruled out Aspergillus, the most prevalent agent of central nervous system mold infection. A panel of specific qPCR assays, guided by the morphology of fungal elements in tissue or as a multiplex assay, may be a successful molecular approach to identify fungal agents of brain abscesses. This also applies in the presence of negative broad-range fungal PCR, therefore providing diagnostic and therapeutic potential for early specific management and improvement of patient clinical outcome.