Broad-range PCR Research Articles

Clinical Utility of Broad Range PCR for Infectious Organisms

Abstract Broad range PCR is used to identify infectious organisms when bacterial, fungal, and mycobacterial cultures are negative despite clinical evidence of infection. We performed a 6-month retrospective chart review to assess the actionability of broad range PCR and identify opportunities to improve the utility of this referral test at our institution. One hundred eleven broad range PCR tests were ordered from July to December 2023. Seventy-five (67.6%) results did not affect patient management, 18 (16.2%) results affected patient management, and the treatment impact of 18 (16.2%) results is unknown because they were from outside consult cases. There were no instances in which antibiotic therapy was completely discontinued due to a negative broad range PCR result. Concurrent in-house microbiology testing yielded a positive result in 15 (13.5%) cases, of which 8 (53%) had a negative broad range PCR result and 2 (13.3%) had a conflicting broad range PCR result. Thirty-eight (34.2%) specimens were formalin fixed paraffin embedded tissue, 29 (26.1%) were fresh tissue, 13 (11.7%) were ESWABS, and the remaining 31 specimens were various fluid types (27.9%). While fresh tissue is the preferred specimen, broad range PCR detected an organism in 20 (53%) formalin fixed paraffin embedded tissue specimens, 11 (37.9%) fresh tissue specimens, 3 (23.1%) ESWAB specimens, and 2 (6.5%) fluid specimens (abscess aspirate and cerebrospinal fluid). Improvements in physician communication and laboratory workflow are necessary to improve the specimen quality and thus the utility of this referral test at our institution.

Development of novel broad-range pan-genus PCR assays for the detection of Tropheryma species.

Introduction. Tropheryma whipplei is responsible for the classical Whipple's disease. Recently, a new Tropheryma species was described in a Belgian immunocompromised patient with pleuritis.Gap Statement. There is currently no specific molecular diagnostic test detecting other Tropheryma species than Tropheryma whipplei.Aim. To develop and validate two broad-range pan-Tropheryma genus PCRs detecting both T. whipplei and new Tropheryma species.Methodology. From shotgun sequencing data of the lung tissue biopsy of the Belgian subject, we designed two PCRs targeting the 23S rRNA and rnpB genes. Prospectively, requests for T. whipplei PCR were tested with T. whipplei-specific PCRs and the two Tropheryma broad-range PCRs from January 2019 to November 2022.Results. In total, 2605 samples were tested using both the pan-Tropheryma 23S rRNA PCR and the T. whipplei-specific PCR. In addition, 833 of the 2605 samples were also tested using the pan-Tropheryma rnpB PCRs. Sensitivity was 78.8% and 79.7% for 23S rRNA and rnpB PCRs, as compared with the species-specific T. whipplei PCR. Specificity was 99.9% and 99.7% for the 23S rRNA and the rnpB PCRs, respectively. We identified a patient whose bronchoalveolar lavage tested positive with the two broad-range PCRs with >105 copies ml-1. Specific T. whipplei PCRs were negative. Known for panuveitis, this 49-year-old male presented with an eye inflammation recurrence, and a CT scan showed multiple mediastino-hilar necrotic adenopathies. Doxycyclin (1 year), hydroxychloroquin (1 year) and co-trimoxazol (1 month) treatments led to a favourable outcome.Conclusion. Specific T. whipplei PCR exhibited better sensitivity than the pan-Tropheryma PCRs. However, both broad-range pan-Tropheryma PCRs demonstrated excellent specificity and were pivotal to identifying a new probable case of Tropheryma infection due to another species-level lineage.

An Unusual Case of Blastomycosis Presenting as "Punched Out" Ulcerations

Background: Cutaneous blastomycosis commonly presents as verrucous, hyperkeratotic nodules or plaques. Less commonly, it presents as ulcerations with accompanying scale crusting or verrucous features. Case Report: A healthy 34-year-old incarcerated male with a history of drug abuse presented with several, well-defined (punched out), deep ulcerating cutaneous lesions with undermined borders. There was no crusting, or verrucous features. The clinical differential diagnoses included pyoderma, vasculitic ulcers, and pyoderma gangrenosum. The histopathology from a gluteal ulceration demonstrated pseudoepitheliomatous hyperplasia with acute, chronic, and granulomatous inflammation. PAS stain of the specimen exhibited thick-walled yeast demonstrating broad-based budding typical of blastomycosis and tissue cultures grew Blastomyces dermatitidis after one month. Conclusion: Ulceration without verrucous features can portend cutaneous blastomycosis. Diagnosis using fungal culture of the wound or tissue takes weeks to result. A prompt biopsy and histopathologic examination or broad-range PCR testing of a wound swab, tissue or urine specimen can lead to a prompt diagnosis.

Evaluation of the Allplex GI Parasite and Helminth PCR Assay in a Belgian Travel Clinic.

Recently a number of broad-range stool parasite PCR assays have been developed. However, there is ongoing disagreement regarding their diagnostic performance, as various studies have produced contradictory results. In this study, we compared the diagnostic accuracy of the Seegene Allplex GI-Parasite and Allplex GI-Helminth assays (SA) with the conventional methods used at the travel clinic of the Institute of Tropical Medicine (ITM) including microscopy, antigen testing, and molecular detection in order to provide insights into the strengths and limitations of this diagnostic tool which may be crucial to select the most appropriate diagnostic tools for the suspected pathogen. A total of 97 native stool samples from 95 patients with suspected gastrointestinal illness were analyzed, including 26 from a frozen collection and 71 prospectively collected samples. The diagnostic performance of SA was notably superior to the conventional workflow in detecting Dientamoeba fragilis (sensitivity 100% vs. 47.4%) and Blastocystis hominis (sensitivity 95% vs. 77.5%). SA had a comparable performance with the conventional workflow in detecting pathogenic protozoa (sensitivity 90% vs. 95%). In contrast, SA had a much lower diagnostic performance in detecting helminths (59.1%) compared to the conventional workflow (100%). We conclude that the Seegene Allplex GI-Parasite assay may be useful for protozoa screening in low-endemic industrialized countries. However, the Allplex GI-Helminth assay is not recommended due to its suboptimal performance compared to microscopy.

Herpesviruses in migrating procellariforms, northeastern Brazil

Seabirds are one of the most threatened avian groups. Viruses, including herpesvirus, represent considerable threats to marine avifauna. Herein, our goal was to survey herpesvirus in Procellariiformes that stranded in Brazil between June and July 2021. We analyzed 12 Cory's shearwaters (Calonectris borealis), two Great Shearwaters (Ardenna gravis, syn. Puffinus gravis) and one Yellow-nosed Albatross (Thalassarche chlororynchos) found in an unusual mortality event in Bahía state, northeastern Brazil. After necropsy, selected tissue samples were tested for herpesvirus using a broad-range nested PCR. Overall, 20% (3/15) of the birds were herpesvirus-positive, i.e., two Cory's Shearwaters and one Great Shearwater. One alphaherpesvirus sequence type was identified in each shearwater species, classified into the genus Mardivirus. This study describes two likely novel herpesviruses in shearwaters, contributing to the currently very scarce data regarding infectious agents in Procellariiformes. Further studies are necessary to evaluate the presence and characteristics of herpesvirus in Procellariiformes, and the presence (or not) of related disease in order to understand the epidemiology of this infectious agent and eventually contribute to the conservation of this endangered seabird group.

ROLE OF THE MOLECULAR TESTS FOR PATIENTS WITH INFECTIVE ENDOCARDITIS UNDERGOING CARDIAC SURGERY: AN EXPANDING HORIZON

Abstract Background Molecular tests have been proven to be a highly sensible tool to identify the pathogen of infective endocarditis (IE). However, their usefulness in the management of both blood culture positive and valve culture negative IE remains unclear. Methods We prospectively enrolled 100 consecutive patients with IE who underwent cardiac surgery between April 2020 and June 2023. Molecular testing (broad–range PCR 16S rRNA) was systematically performed and results were collected together with those of preoperative blood culture, valve culture and histopathological analysis of the specimen. Results Blood culture, valve culture and molecular testing were positive in 83%, 47% and 76% of patients, respectively. Both the sensitivity of valve culture and 16S rRNA PCR decreased significantly with increasing duration of preoperative antibiotic therapy. In 7% of cases, molecular test was the only positive test, allowing an etiological diagnosis. In 33% of cases, valve culture was negative but molecular test was positive. In the majority of these patients, histopathological analysis documented acute inflammation. Finally, molecular test clarified discrepancies between the results of blood cultures and those of valve cultures in 12.5% of patients. Conclusions The molecular tests showed high sensitivity. Although it decreased as the duration of preoperative antibiotic therapy increased, it was still higher than that of valve culture 28 days after the start of treatment. In addition to patients without an etiological diagnosis, the molecular test proved useful in other circumstances: 1) when the valve culture was negative because, together to the results of the histopathological analysis, it allowed to identify those patients who could benefit from a prolongation of antibiotic treatment; 2) in cases of discordance between the results of blood cultures and those of valve cultures, because it allowed the type of antibiotic treatment to be directed. Figure 1: Usefulness of molecular tests (PCR) according to the results of blood and valve cultures. Figure 2: Valve cultures are of paramount importance in the management of antibiotic therapy (ABT). However, their sensibility is low and the risk of under–treatment exists. Molecular tests may help targeting the duration of ABT but they could be persistently positive leading to an over–treatment. Figure 3: Flow–chart showing the possible role of molecular test in the management of patients with infective endocarditis.

The microbiological outcomes of culture-negative blood specimens using 16S rRNA broad-range PCR sequencing: a retrospective study in a Canadian province from 2018 to 2022.

The existing literature focuses on its performance compared to blood cultures in patients with sepsis, leaving a gap in the literature regarding other blood specimens in suspected infectious syndrome across the severity spectrum. We aimed to characterize its microbiological outcomes and provide insight into its potential clinical utility.

158. Diagnostic Yield and Clinical Utility of Broad Range PCR Testing at a Tertiary Children’s Hospital

Abstract Background Broad range PCR testing (BR-PCR) targets highly conserved DNA sequences of bacteria, fungi, or mycobacteria to detect a broad range of organisms in various clinical samples. Given its potential impact in providing timely diagnoses that cannot always be made through conventional testing (CT), we evaluated the diagnostic yield and clinical impact of BR-PCR at our institution. Methods We retrospectively evaluated all clinical specimen types obtained for BR-PCR at Riley Hospital for Children from October 2019 to May 2022. Percent positivity (PP) was determined by specific PCR test type (Bacterial/Fungal/NTM/TB), along with median turn-around times (in days) from sample collection. Medical charts were reviewed, and clinical impact of results was determined. Results We identified 956 BR-PCR tests sent from 271 specimens collected from 178 patients. Only 14.5% yielded a positive result with a median days-to-result being the longest for fungal PCR at 8.1 days (7.0, 10.1) and TB PCR being the fastest at 7.8 days (6.8, 9.9). Bacterial BR-PCR yielded an overall PP of 42.6% while Fungal BR-PCR was 10.7%. Positivity rates for NTM and TB were 0% and 0.5%, respectively. Bronchial lavage was the most common specimen type (35.5%) with an overall PP of 19.9%. Of the 271 specimens, 245 returned conclusive results from both BR-PCR and clinical testing (CT) for comparison. A clinically significant organism based on CT was identified in 68 specimens (27.8%), 45 of which were confirmed by BR-PCR. 23 (33.8%) were detected by CT but not BR-PCR. 21 clinically significant organisms not detected by CT were identified by BR-PCR, which led to a change in clinical management in 12 instances: new diagnosis (91.7%); or appropriate initiation (91.7%), escalation (25.0%), or de-escalation (25.0%) of antimicrobial therapy. Conclusion BR-PCR overall had low diagnostic yield at our institution but was influenced by specimen type. The clinical utility was predominantly seen in immunocompromised patients in which conventional testing was negative. Further data is needed to determine which specimen types and diagnoses will increase the yield and clinical value of BR-PCR and thus, aid in enhancing diagnostic stewardship. Disclosures All Authors: No reported disclosures

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

584. Microbiological Outcomes of Culture-Negative Blood Specimens Using 16s rRNA Broad-Range PCR Sequencing: a Retrospective Study in a Canadian Province from 2018 to 2022

Abstract Background Broad-range bacterial PCR sequencing (BRBPS) has emerged as a novel tool to detect fastidious organisms. While its utility has been characterized in different specimen types, its role in culture-negative blood specimens remains poorly understood. Methods We reviewed all clinical specimens sent for culture-negative blood BRBPS (blood, serum and blood culture bottles) to the Laboratoire de santé publique du Québec, the reference laboratory for a large Canadian province, from May 2018 to November 2022. Sanger sequencing of the amplified 16s rRNA gene was performed in all PCR-positive specimens. Data were extracted from the laboratory information system, and the analysis was restricted to the first specimen per patient. Microbiological outcomes were categorized as interpretable sequence, uninterpretable sequence, or negative PCR result. Interpretable sequences were identified and then classified based on the National Healthcare Safety Network database (NHSN; Table 1) and microbiological characteristics.Table 1.Definitions of National Health Safety Network (NHSN) Classification and Examples Results A total of 1199 blood specimens were analyzed using BRBPS from 852 unique patients. Of these, there was no PCR amplification in 152, amplification with uninterpretable sequences in 445 and an interpretable sequence in 255 specimens (Figure 1). Blood specimens received at room temperature, in blood culture bottles, or with positive gram stain were more likely to yield interpretable sequences (Table 2). We identified 174 patients with BRBPS results suggestive of organisms associated with mucosal barrier injury (n=89) or possible pathogens (n=85), summarized in Figure 2. In contrast, 75 patients had results suggestive of contamination from common commensal organisms (n=44) or taxa not in the NSHN database (n=31).Figure 1.Flowchart of the analysis from primary specimens to those specimens with successful amplification of 16s rRNA Classification of interpretable sequences using the National Healthcare Safety Network database classification of microorganisms.Table 2.Characteristics of BRBPS of the 16s rRNA on Culture-Negative Blood SpecimensFigure 2.Tree Map of Mucosal Barrier Injury Organisms and Possible Pathogens by Genera Mucosal barrier injury (A) and possible pathogens (B) are categorized based on the National Healthcare Safety Network database. Microorganisms are colour classified based on microbiological characteristics. Conclusion Our findings demonstrate the potential utility of BRBPS in blood specimens from culture-negative patients, particularly infectious syndromes caused by fastidious gram-negative bacteria associated with animal or arthropod exposures or anaerobic bacteria. However, the frequent recovery of commensal and environmental organisms argues for careful and judicious use. Additional technical optimization is likely required to improve diagnostic yield, particularly with mixed sequences. Disclosures Matthew Cheng, MD, Amplyx Pharmaceuticals: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Honoraria|Cidara Therapeutics: Grant/Research Support|GEn1E lifesciences: Advisor/Consultant|GEn1E lifesciences: Stocks/Bonds|Kanvas Biosciences, Inc.: Board Member|Kanvas Biosciences, Inc.: Pending patents|Kanvas Biosciences, Inc.: Ownership Interest|Merck: Honoraria|nomic bio: Advisor/Consultant|nomic bio: Stocks/Bonds|Pfizer: Honoraria|Scynexis Inc.: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Honoraria

Concordance between culture, Molecular Culture and Illumina 16S rRNA gene amplicon sequencing of bone and ulcer bed biopsies in people with diabetic foot osteomyelitis

BackgroundIn clinical practice the diagnosis of diabetic foot osteomyelitis (DFO) relies on cultures of bone or ulcer bed (UB) biopsies, of which bone biopsy is reference standard. The slow growth or fastidious nature of some bacteria, hamper expeditious detection and identification. Rapid molecular techniques may solve both issues, but their additional value for everyday practice is unknown.We investigated the concordance between conventional culture, the molecular techniques Molecular Culture (MC), and illumina 16S rRNA gene amplicon (16S) sequencing in people with DFO.MethodsIn the BeBoP trial, bone and UB biopsies were obtained from people with DFO who visited Amsterdam UMC. These biopsies were analysed using 1) conventional culture, 2)MC, a rapid broad range PCR analysing the 16S-23S ribosomal-interspace-region, and 3) 16S sequencing, and evaluated concordance among these techniques.ResultsWe analysed 20 samples (11 bone and 9 UB) of 18 people. A total of 84 infectious agents were identified, 45 (54%) by all techniques, an additional 22 (26.5%, overall 80.5%) by both MC and 16S, and the remaining 16 species by culture and MC or 16S, or by a single method only. MC and 16S identified anaerobes not detected by culturing in 5 samples, and the presence of bacteria in 7 of 8 culture-negative (6 bone, 2 UB) samples.ConclusionThe high level of concordance between MC and 16S and the additional ability of molecular techniques to detect various bacteria not detected by culturing opens up prospects for routine use of fast molecular techniques, in clinical settings including DFO.Trial registrationThe BeBoP trial is retrospectively registered on 05–03-2019 in Netherlands Trial Register: NL 7582.

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.

Screening of Ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of San Domenico e dei Serpari (Cocullo, AQ, Italy)

In the Abruzzi village of Cocullo (Italy), each year, on May 1st, local snake hunters (known as Serpari) display colubrids, captured in the wild, to commemorate the ancient ritual of San Domenico. The ascomycete Ophidiomyces ophidiicola (Oo) is the causative agent of ophidiomycosis, an emerging disease with sublethal effects. Skin lesions, such as dysecdysis, edematous, crusty or necrotic scales, swellings, nodules, and ulcers, are the most common clinical manifestation of the disease. The pathogen and its associated disease are well characterized in wild snakes in North America, whereas broad screenings of free ranging wild ophidians in Europe are rare. In 2019, as part of a multi-year snake health monitoring project, all the Cocullo ophidians were carefully examined for integumentary affections and those showing signs consistent with ophidiomycosis were dry swabbed on the skin and on any visible cutaneous lesions with a single applicator. The extracted DNA underwent a broad-range panfungal PCR targeting the D1-D2 region, as well as two conventional PCRs specific to the ITS2 and IGS regions of Oo DNA. Twenty-three out of 129 snakes (13/82 Elaphe quatuorlineata; 7/31 Hierophis viridiflavus; 3/15 Zamenis longissumus; 0/1 Natrix helvetica) resulted clinically affected, but no specific Oo genomic DNA was detected by PCR. The Cocullo ritual celebration provided a unique opportunity for the first systematic testing of a large sample size of a local snake community for the monitoring of this pathogen in Italy.

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

Canine piroplasmids: Molecular detection and laboratory characterization in dogs from Brasilia, Brazil, with the first molecular evidence of dog exposure to a novel opossum-associated Babesia sp.

Canine piroplasmid infections can be caused by Babesia spp., Theileria spp. and Rangelia vitalii. In Brazil, canine babesiosis caused by Babesia vogeli is endemic and reported throughout the country. On the other hand, Rangeliosis caused by R. vitalii has only been described so far in the South and Southeast regions. Despite that, studies analyzing the laboratory and molecular characterization of these hemoprotozoa are still scarce. To investigate the occurrence, the laboratory features, the molecular characterization, and the diversity of piroplasmids from Midwestern Brazil, a survey was performed using blood samples obtained from 276 domestic dogs from Brasília, Federal District, Midwestern Brazil. A broad-range quantitative PCR (qPCR) targeting the mitochondrial large subunit ribosomal DNA (LSU4) was used to detect piroplasmid DNA. The overall molecular occurrence of piroplasmids was 11.2% (31/276), with 9.7% (27/276) of the sequences identified as Babesia vogeli (98–100% identity to B. vogeli isolate from the USA). Based on a partial 18S rRNA sequence pairwise alignment (-250 bp), 1.4% (4/276) of the sequences showed only 76.8% identity with B. vogeli but 100% identity with opossum-associated Babesia sp. (MW290046–53). These findings suggest the exposure of dogs from Brazil to a recently described Babesia sp. isolated from white-eared opossum. None of the analyzed dogs was positive for Theileria spp. or R. vitalii. Subsequently, all positive sequences were submitted to three additional PCR assays based on the 18S rRNA, cox-1, and cytb genes, aiming at performing a haplotype network analysis. Haplotype network using cox-1 sequences showed the presence of six different haplotypes of B. vogeli; one of them was shared with isolates from Brazil, the USA, and India. When including animals co-infected with other vector-borne diseases, piroplasmid-positive dogs had 2.3 times higher chance of having thrombocytopenia than the negative ones. The molecular results demonstrated that the compared Babesia vogeli sequences showed a low variability as well as evidence of exposure to a putative novel opossum-associated Babesia sp. in dogs from Midwestern Brazil.

Optimization of the molecular diagnosis of the acute hepatitis E virus infection.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

Blood Culture Negative Endocarditis: A Review of Laboratory Diagnostic Approaches

Infective endocarditis is a potentially fatal condition, and identifying the pathogen is crucial to optimizing antibiotic treatment. While a blood culture takes time and may yield negative results, it remains the gold standard for diagnosis, blood culture-negative endocarditis, which accounts for up to 20% of infective endocarditis cases, poses a clinical challenge with increasing mortality. To better understand the etiology of blood culture-negative infective endocarditis, we reviewed non-culture-based strategies and compared the results. Serology tests work best in limited pathogens, such as Coxiella burnetii and Bartonella infections. Most of the pathogens identified by broad-range PCR tests are Streptococcus spp, Staphylococcus spp and Propionibacterium spp. adding specific real-time PCR assays to the systematic PCR testing of patients with blood culture-negative endocarditis will increase the efficiency of diagnosis. Recently, metagenomic next-generation sequencing has also shown promising results.

Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Partial genetic characterisation of a novel alloherpesvirus detected by PCR in a farmed wels catfish (Silurus glanis).

By a broad-range PCR, we detected a novel herpesvirus (HV) in the specimen of a wels catfish (Silurus glanis) presenting disseminated, carp pox-like dermal lesions all over its body. The sequence analysis of the 463-bp PCR product from the viral DNA polymerase gene indicated the presence of a hitherto unknown virus, a putative member of the family Alloherpesviridae in the sample. Another PCR, targeting the terminase gene of fish HVs, provided an additional genomic fragment of over 1,000 bp. Surprisingly, the sequence of a co-amplified, off-target PCR product revealed its origin from a putative gene homologous to ORF87 and ORF45 of cyprinid HVs and anguillid herpesvirus 1 (AngHV-1), respectively. With specific primers, designed according to the genomic maps of the cyprinid and anguillid HVs, a genomic fragment of 15 kb was also amplified and sequenced by primer walking. In phylogeny inferences, based on several genes, the putative wels catfish HV clustered closest to various cyprinid HVs or to AngHV-1. The novel virus, named as silurid herpesvirus 2, represents a distinct species in the genus Cyprinivirus. However, its association with the skin disease remains unclear.

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.