Clinical Evaluation of the Alinity m STI Multiplex PCR Assay.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are routinely tested and reported; however, Trichomonas vaginalis (TV) is the most common sexually transmitted infection (STI) in the United States and the prevalence of Mycoplasma genitalium (MG) infections is likely higher than estimated. We examined the clinical performance of the Alinity m STI assay for detection and surveillance of CT/NG/TV/MG in urine specimens from patients at a large academic medical center. METHODS: Urine specimen from 198 patients was tested in this evaluation. Alinity m STI and Aptima Combo 2 CT/NG and TV assay (Panther System) results were compared, with discrepant results run on the cobas 6800 CT/NG, TV/MG assays. Analyzer turnaround times, time from loading the specimen on the analyzer to results reporting, were determined for Alinity m and Panther systems. RESULTS: Overall percent agreements of the Alinity m in comparison with the Aptima and cobas assays for CT, NG, TV, and MG were 99.5% (97.2%, 99.9%), 99.5% (97.2%, 99.9%), 98.4% (95.5%, 99.5%), and 86.4% (66.7%, 95.3), respectively. There were 5 discrepant samples (CT, 1; NG, 1; TV, 3) between the Alinity m and the Aptima assays, and 3 MG discrepant samples between the Alinity m STI and cobas 6800. Two of the 5 Aptima and Alinity m discrepant samples were resolved as they yielded similar results on both Alinity m and cobas 6800. TV and MG infections comprised 54% of the positive samples and were more often asymptomatic than CT and NG infections. Analyzer turnaround time was 3 hours 25 minutes for the Aptima CT/NG, 3 hours 25 minutes for Aptima TV, and 1 hour 55 minutes for Alinity m STI assay. CONCLUSIONS: The Alinity m STI assay allows for fast and simultaneous detection of the 4 major STI pathogens, which can facilitate surveillance and provide accurate results to help clinicians diagnose for initiation of appropriate treatment.

Validation of a new extraction-free real-time PCR test to detect MPOX virus.

The monkeypox (Mpox) virus has raised significant concerns given its recent spread with an increasing number of confirmed cases worldwide. In this study, we evaluated the performance of a laboratory developed test (LDT) using BioGX Xfree hMPXV/OPXV reagents for the qualitative detection of non-variola Orthopoxviruses and Mpox virus DNA, in swabs from human pustular or vesicular rash specimens. Analytical and clinical testing analysis were carried out on two different platforms: the BD MAX™ System (BD Diagnostics) and the new pixl.16 Real-Time PCR Platform (BioGX), using a synthetic Mpox virus DNA (ATCC VR-3270SD) and residual clinical samples previously identified with an EUA approved Mpox real-time PCR assay. In the end, the Xfree hMPXV/OPXV LDT proved to be a sensitive, specific, and reproducible test for the detection of Mpox on both platforms evaluated with the pixl.16 having an advantage of a small footprint and providing faster TAT facilitated by an extraction-free workflow.

Multicenter evaluation of BioCode GPP for syndromic molecular detection of gastrointestinal pathogens from stool specimens.

Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality worldwide across all age groups that disproportionally affects young children in low- and middle-income countries and immunocompromised patients in high-income countries. Regional outbreaks of AGE are typically detected by traditional microbiological detection methods that target limited organisms and are associated with low sensitivity and lengthy time-to-results. Combined, these may result in repeat testing, imprecise or delayed treatment, and delayed recognition of outbreaks. We conducted a multi-site prospective study comparing the BioCode Gastrointestinal Pathogen Panel (BioCode GPP) for the detection of 17 common bacterial, viral, and protozoan causes of gastroenteritis with reference methods, including stool culture, enzyme immunoassays, pathogen-specific PCR assays, and sequencing. One thousand five hundred fifty-eight residual, de-identified stool samples (unpreserved stool and stool in Cary-Blair transport medium) were enrolled and tested for 11 bacterial, 3 viral, and 3 protozoan pathogens. BioCode GPP and reference methods were positive for 392 (25.2%) and 283 (18.2%) samples, respectively (P < 0.0001). In this study, the BioCode GPP and reference methods detected 69 and 65 specimens positive for Clostridioides difficile, 51 and 48 for enteroaggregative Escherichia coli, 33 and 27 for enterotoxigenic E. coli, 50 and 47 for norovirus GI/GII, and 30 and 22 for rotavirus A, respectively. The BioCode GPP showed good positive and negative agreements for each pathogen ranging from 89.5% to 100%, with overall sensitivity and specificity of 96.1% and 99.7%, post adjudication. The BioCode GPP detected >1 pathogens in 49 samples, representing 12.5% of the total 392 positive specimens. IMPORTANCE: This study highlights performance of a novel technology for timely and accurate detection and differentiation of 17 common bacterial, viral, and protozoan causes of gastroenteritis. Utilizing molecular tests such as the BioCode Gastrointestinal Pathogen Panel may improve the detection of gastrointestinal pathogens and provide actionable results, particularly for patient populations at most risk.

Draft genome sequence of a highly proteolytic Staphylococcus aureus USA300 isolate from human urine.

The secreted proteases of Staphylococcus aureus have been shown to be critical during infection. Here, we present the draft genome sequence of S. aureus TGH337, a hyper-proteolytic USA300 strain isolated from human urine.

Performance evaluation of the Aptima CMV quant assay using plasma and non-plasma samples.

BACKGROUND: Cytomegalovirus (CMV) infection has a major negative impact on transplantation and is associated with increased morbidity and mortality in this patient population. Quantitation of CMV infections using a molecular test is the preferred method for monitoring patients post-transplant. For this analysis, we compared the Aptima CMV Quant Assay (Aptima CMV) on the Panther system to the ELITech MGB Alert® CMV 3.0 ASR (MGB CMV) run on the ELITe InGenius®. METHODS: The analytical performance of the assay was assessed using commercially available CMV reference panels that meet the 1st WHO International Standard for Human Cytomegalovirus for nucleic acid amplification techniques. The clinical performance of the assay was determined using 249 plasma and non-plasma samples. RESULTS: The 95% LOD of the Aptima assay was determined to be 50 IU/mL and 200 IU/mL for the MGB CMV assay. A strong linear correlation with the reference panel (R2 = 0.9945), excellent reproducibility, and accuracy (R2 = 0.986) over the detection range of the assay was observed. Of the 249 clinical samples tested, only 17 (6.8%) yielded discordant results which were at or near the lower limit of quantification of the assays. Although the Aptima CMV assay demonstrated excellent concordance of qualitative results to the MGB CMV assay for all samples, the MGB CMV quantified CMV DNA at an average of 0.5 Log IU/mL higher than Aptima CMV. CONCLUSION: The Aptima CMV assay is both sensitive and accurate in quantifying CMV in both plasma and non-plasma specimens on the fully automated Panther system.

The Clinical Performance of the BioCode Respiratory Pathogen Panel for the Detection of Viruses and Bacteria from Nasopharyngeal Swabs.

Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) utilizes reverse transcriptase PCR (RT-PCR) in combination with barcoded magnetic beads to amplify, detect, and identify respiratory pathogens. This panel qualitatively detects and identifies 14 viruses, including influenza virus A with H1 pdm09, H1, and H3 subtyping; influenza B; respiratory syncytial virus (RSV); human metapneumovirus; parainfluenza virus 1; parainfluenza virus 2; parainfluenza virus 3; parainfluenza virus 4; coronavirus (229E, NL63, OC43, and HKU1); adenovirus; and human rhinovirus/enterovirus, and 3 bacteria, including Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Reproducibility, which was assessed with contrived specimens containing 12 targets at 3 clinical sites, with 2 operators at each site for 5 days, was 99.4% for Flu A H3 and Flu B, 98.9% for RSV, and 100% for the remaining 9 targets assayed. A multicenter clinical trial evaluated the performance of the BioCode RPP with 2,647 nasopharyngeal swab specimens from 5 geographically distinct sites and revealed comparable performance between the BioCode RPP and FilmArray Respiratory Panel (FA-RP). Specifically, the positive percent agreements (PPAs) for various pathogens ranged between 80.8% and 100% compared with the FA-RP (1.7 and 2.0). Negative percent agreement ranged from 98.4% to 100% for BioCode RPP. The BioCode RPP also offers scalable automated testing capability of up to 96 specimens in a single run with total sample-to-result time under 5 h. The invalid rate of the BioCode RPP on initial testing was 1.0% (26/2,649). IMPORTANCE Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) is a high-throughput test that utilizes RT-PCR in combination with barcoded magnetic beads to amplify, detect, and identify 17 respiratory pathogens, including 14 viruses and 3 bacteria. This study summarizes data generated from a multicenter clinical trial evaluating the performance of the BioCode RPP on 2,647 nasopharyngeal swab specimens from five geographically distinct sites.

Multicenter Evaluation of the BioFire Respiratory Panel 2.1 (RP2.1) for Detection of SARS-CoV-2 in Nasopharyngeal Swab Samples.

As the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) begins to overlap with the traditional respiratory season in the Northern Hemisphere, simultaneous testing for SARS-CoV-2 and the other common causes of respiratory infections is imperative. This has led to the development of multiplex respiratory assays that include SARS-CoV-2 as a target. One such assay is the BioFire respiratory panel 2.1 (RP2.1), which is an expansion of the original BioFire FilmArray respiratory panel 2 (RP2) to include SARS-CoV-2. In this multicenter evaluation, we assessed the performance characteristics of the BioFire RP2.1 for the detection of SARS-CoV-2. One or more targets on the panel were detected in 19.3% (101/524) of specimens tested, with SARS-CoV-2 detected in 12.6% (66/524) of specimens. Human rhinovirus/enterovirus was also detected in 32.7% (33/101) and adenovirus in 3.0% (3/101) of positive specimens, with one dual positive for both SARS-CoV-2 and adenovirus being detected. A further breakdown of pathogens by age revealed a 4-fold predominance of human rhinovirus/enterovirus in subjects 0 to 18 years of age, whereas in all other age groups, SARS-CoV-2 was clearly the predominant pathogen. Overall, SARS-CoV-2 results obtained from the BioFire RP2.1 were highly concordant with the composite result, exhibiting 98.4% (61/62) positive percent agreement (95% confidence interval [CI], 91.4 to 99.7%) and 98.9% (457/462) negative percent agreement (95% CI, 97.5 to 99.5%) with further analysis of discordant results suggesting that the concentration of SARS-CoV-2 in the specimens was near the limit of detection (LoD) for both the BioFire RP2.1 and the comparator assays. Overall, the BioFire RP2.1 exhibited excellent performance in the detection of SARS-CoV-2.

Validation of a modified CDC assay and performance comparison with the NeuMoDx™ and DiaSorin® automated assays for rapid detection of SARS-CoV-2 in respiratory specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has spread rapidly around the globe since it was first identified in December of 2019 in Wuhan, China. In a race to contain the infection, researchers and healthcare officials have developed several assays to help diagnose individuals with COVID-19. To help laboratories decide what assay to bring into testing lines, factors such as assay availability, cost, throughput, and TAT should be considered. Here we validated a modified version of the CDC assay and used it as a reference to evaluate the performance of the NeuMoDxTM SARS-CoV-2 and DiaSorin SimplexaTM Covid-19 Direct assays. In silico analysis and clinical sample testing showed that the primers/probes designed by the CDC were specific to the SARS-CoV-2 as they accurately detected all reactive samples with an assay LoD of 200 copies/mL. The performance of the three assays were analyzed using 159 nasopharyngeal swabs specimen tested within 1-5 days after routine testing. A 100 % agreement was observed between the commercial assays and the modified CDC SARS-CoV-2 assay. A deeper look at the Ct values showed no significant difference between NeuMoDx and the modified CDC SARS-CoV-2 assay, whereas DiaSorin had lower overall Ct values than the modified CDC SARS-CoV-2 assay. NeuMoDx and DiaSorin workflows were much easier to perform. NeuMoDx has the highest throughput and shortest TAT, whereas although the modified CDC SARS-CoV-2 assay has comparable throughput to DiaSorin, it has the longest hands-on time and highest TAT.

Bacterial Whole Genome Sequencing on the Illumina iSeq 100 for Clinical and Public Health Laboratories.

Bacterial whole-genome sequencing (WGS) provides clinical and public health laboratories an unprecedented level of information on species identification, antimicrobial resistance, and epidemiologic typing. However, multiple barriers to widespread adoption still exist. This research describes bacterial WGS using the Illumina iSeq 100 instrument to overcome some of these barriers. Using an in-house, high-quality single-nucleotide polymorphism analysis pipeline and a commercial whole-genome multilocus sequence typing program, the sequencing of Acinetobacter baumannii, Burkholderia cepacia, Clostridioides difficile, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus isolates was validated. The genome coverage range was 17× to 149×, with a mean of 59×. The limit of detection for single-nucleotide polymorphisms was 30×. Overall platform base calling accuracy was >99.999%. Reproducibility and repeatability of base calling inferred from whole-genome multilocus sequence typing was species dependent and ranged from >97% similarity for P. aeruginosa to >99.9% similarity for S. aureus. Resistance gene and multilocus sequence typing allele identification was 100% concordant with expected results. A simple, modified library preparation reduces the per-sample cost by half to give overall theoretical sample costs ranging from approximately $50 to $100 for library preparation and sequencing. The iSeq 100 provides a cost-effective and easy-to-use platform for clinical and public health laboratories to sequence bacterial isolates for a wide range of potential applications.

Evaluation of Copan FecalSwab™ preserved stool specimens with the BD MAX™ Enteric Bacterial Panel and the BD MAX™ Extended Enteric Bacterial Panel.

The objectives of this study were to assess the ideal volume of Copan FecalSwab™ (FS) preserved stool sample to use with the BD MAX™ Enteric Bacterial Panel and the Extended Enteric Bacterial Panel (BDM GIP) and to compare the performance of FS to the recommended Meridian Para-Pak Cary-Blair medium (PP) for the BDM GIP. Three different input volumes (10, 25, and 50 µL) of stool inoculated with American Type Culture Collection strains representing the targets detected by BDM GIP were tested. Additionally, 144 unpreserved stool samples submitted for gastrointestinal (GI) testing were transferred to PP and FS media and tested by the BDM GIP using 10 µL of PP and 50 µL of FS media. A 100% agreement was observed between PP and FS results. The performance of 50 µL of FS stool preserved sample was equivalent to 10 µL of traditional Cary-Blair PP preserved specimens for GI pathogens detection using the BDM GIP.

A TaqMan Probe-Based Real-Time PCR Assay for the Rapid Identification of the Emerging Multidrug-Resistant Pathogen Candida auris on the BD Max System.

Candida auris is an emerging multidrug-resistant fungal pathogen that has been associated with nosocomial bloodstream and deep wound infections causing a high mortality rate mainly in intensive care unit (ICU) patients. Laboratories currently rely on phenotypic testing using commercial automated systems for identification of yeasts; however, this technique has often led to misidentification of C. auris to other closely related species. We developed and validated a TaqMan-based real-time PCR assay on the BD Max platform targeting ribosomal DNA (rDNA) region nucleotide sequences to quickly and accurately test for C. auris infection from culture and clinical specimens. The assay is highly specific, reproducible, and sensitive, allowing detection of as low as 1 C. auris CFU per reaction within 3 h.

A family of genus-specific RNAs in tandem with DNA-binding proteins control expression of the badA major virulence factor gene in Bartonella henselae.

Bartonella henselae is a gram-negative zoonotic bacterium that causes infections in humans including endocarditis and bacillary angiomatosis. B. henselae has been shown to grow as large aggregates and form biofilms in vitro. The aggregative growth and the angiogenic host response requires the trimeric autotransporter adhesin BadA. We examined the transcriptome of the Houston-1 strain of B. henselae using RNA-seq revealing nine novel, highly-expressed intergenic transcripts (Bartonella regulatory transcript, Brt1-9). The Brt family of RNAs is unique to the genus Bartonella and ranges from 194 to 203 nucleotides with high homology and stable predicted secondary structures. Immediately downstream of each of the nine RNA genes is a helix-turn-helix DNA-binding protein (transcriptional regulatory protein, Trp1-9) that is poorly transcribed under the growth conditions used for RNA-seq. Using knockdown or overexpressing strains, we show a role of both the Brt1 and Trp1 in the regulation of badA and also in biofilm formation. Based on these data, we hypothesize that Brt1 is a trans-acting sRNA that also serves as a cis-acting riboswitch to control the expression of badA. This family of RNAs together with the downstream Trp DNA-binding proteins represents a novel coordinated regulatory circuit controlling expression of virulence-associated genes in the bartonellae.

Characterization of the general stress response in Bartonella henselae.

Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In alpha-proteobacteria, the general stress response uses an alternate sigma factor as the main regulator and incorporates it with a two-component system into a unique regulatory circuit. This system has been described in several alpha-proteobacterial species, including the pathogens Bartonella quintana and Brucella abortus. Most of the studies have focused on characterizing the PhyR anti-anti-sigma factor, the NepR anti-sigma factor, and the alternate sigma factor. However, not enough attention is directed toward studying the role of histidine kinases in the general stress response. Our study identifies the general stress response system in Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate sigma factor have similar sequence and domain structures with other alpha-proteobacteria. Our data showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. Furthermore, we showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the gene encoding the BadA adhesin. Finally, we identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR.

Oroya fever and verruga peruana: bartonelloses unique to South America.

Bartonella bacilliformis is the bacterial agent of Carrión's disease and is presumed to be transmitted between humans by phlebotomine sand flies. Carrión's disease is endemic to high-altitude valleys of the South American Andes, and the first reported outbreak (1871) resulted in over 4,000 casualties. Since then, numerous outbreaks have been documented in endemic regions, and over the last two decades, outbreaks have occurred at atypical elevations, strongly suggesting that the area of endemicity is expanding. Approximately 1.7 million South Americans are estimated to be at risk in an area covering roughly 145,000 km2 of Ecuador, Colombia, and Peru. Although disease manifestations vary, two disparate syndromes can occur independently or sequentially. The first, Oroya fever, occurs approximately 60 days following the bite of an infected sand fly, in which infection of nearly all erythrocytes results in an acute hemolytic anemia with attendant symptoms of fever, jaundice, and myalgia. This phase of Carrión's disease often includes secondary infections and is fatal in up to 88% of patients without antimicrobial intervention. The second syndrome, referred to as verruga peruana, describes the endothelial cell-derived, blood-filled tumors that develop on the surface of the skin. Verrugae are rarely fatal, but can bleed and scar the patient. Moreover, these persistently infected humans provide a reservoir for infecting sand flies and thus maintaining B. bacilliformis in nature. Here, we discuss the current state of knowledge regarding this life-threatening, neglected bacterial pathogen and review its host-cell parasitism, molecular pathogenesis, phylogeny, sand fly vectors, diagnostics, and prospects for control.

Zebrafish embryo model of Bartonella henselae infection.

Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)(y1) zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.