Clinical Performance of Cas13a-based Point-of-Care Lateral Flow Assay for Detecting Neisseria gonorrhoeae.

Background: Diagnosis of Neisseria (N.) gonorrhoeae is dependent on nucleic acid amplification testing (NAAT), which is not available in resource-limited settings where the prevalence of infection is highest. Recent advances in molecular diagnostics leveraging the high specificity of CRISPR enzymes can permit field-deployable, point-of-care lateral flow assays. We previously reported on the development and in vitro performance of a lateral flow assay for detecting N. gonorrhoeae. Here we aimed to pair that assay with point-of-care DNA extraction techniques and assess the performance on clinical urine specimens. Methods: We collected an additional urine specimen among individuals enrolling in an ongoing clinical trial at the Massachusetts General Hospital Sexual Health Clinic who presented with symptoms of urethritis or cervicitis (urethral or vaginal discharge, dysuria, or dyspareunia). We then assessed thermal, detergent, and combination DNA extraction conditions, varying the duration of heat at 95°C and concentration of Triton X. We assessed the efficacy of the various DNA extraction methods by quantitative polymerase chain reaction (qPCR). Once an extraction method was selected, we incubated samples for 90 minutes to permit isothermal recombinase polymerase amplification. We then assessed the performance of lateral flow Cas13a-based detection using our previously designed porA probe and primer system for N. gonorrhoeae detection, comparing lateral flow results with NAAT results from clinical care. Results: We assessed DNA extraction conditions on 3 clinical urine specimens. There was no consistent significant difference in copies per microliter of DNA obtained using more or less heat. On average, we noted that 0.02% triton combined with 5 minutes of heating to 95°C resulted in the highest DNA yield, however, 0.02% triton alone resulted in a quantity of DNA that was above the previously determined analytic sensitivity of the assay. Given that detergent-based extraction is more easily deployable, we selected that as our method for extraction. We treated 23 clinical specimens with 0.02% triton, which we added to the Cas13a detection system. We ran all lateral flow detections in duplicate. The Cas13a-based assay detected 8 of 8 (100%) positive specimens, and 0 of 15 negative specimens. Conclusion: Using point-of-care DNA extraction, isothermal amplification, and Cas13a-based detection, our point-of-care lateral flow N. gonorrhoeae assay correctly identified 23 clinical urine specimens as either positive or negative. Further evaluation of this assay among larger samples and more diverse sample types is warranted.

Borrelia-specific antibody profiles and complement deposition in joint fluid distinguish antibiotic-refractory from -responsive Lyme arthritis.

Lyme arthritis, caused by the spirochete Borrelia burgdorferi, is the most common feature of late disseminated Lyme disease in the United States. While most Lyme arthritis resolves with antibiotics, termed "antibiotic-responsive", some individuals develop progressive synovitis despite antibiotic therapy, called "antibiotic-refractory" Lyme arthritis (LA). The primary drivers behind antibiotic-refractory arthritis remain incompletely understood. We performed a matched, cross-compartmental comparison of antibody profiles from blood and joint fluid of individuals with antibiotic-responsive (n = 11) or antibiotic-refractory LA (n = 31). While serum antibody profiles poorly discriminated responsive from refractory patients, a discrete profile of B.burgdorferi-specific antibodies in joint fluid discriminated antibiotic-responsive from refractory LA. Cross-compartmental comparison of antibody glycosylation, IgA1, and antibody-dependent complement deposition (ADCD) revealed more poorly coordinated humoral responses and increased ADCD in refractory disease. These data reveal B.burgdorferi-specific serological markers that may support early stratification and clinical management, and point to antibody-dependent complement activation as a key mechanism underlying persistent disease.

Cerebrospinal fluid metagenomics has greatest added value as a test for Powassan virus among patients in New England with suspected central nervous system infection.

Cerebrospinal fluid (CSF) metagenomic next generation sequencing (mNGS) can detect diverse pathogens in patients with central nervous system infection. Due to its high cost and unclear clinical utility, it is typically reserved for patients with unrevealing routine workups. A multi-center retrospective analysis of real-world CSF mNGS was performed involving orders between 2017 and 2022 at a large New England healthcare system. CSF mNGS was performed 64 times with 17 positive results (27 %). In 11/17 positive samples (65 %), the infectious agent had not been previously detected using routine methods. Arboviruses (n = 8) were the most frequently detected agents, particularly Powassan virus (n = 6). Results changed therapy in 3/64 cases (5 %). Positive results were associated with immunodeficiency (p = 0.06), especially anti-B-cell therapy (p = 0.02), and earlier sample collection (p = 0.06). The association with compromised humoral immunity was stronger in the arbovirus and Powassan virus subgroups (p = 0.001), whose constituents were older than the overall cohort and had higher mortality rates.

Sensitivity of Two-Tiered Lyme Disease Serology in Children With an Erythema Migrans Lesion.

In our prospective cohort of 192 children with a physician-diagnosed erythema migrans (EM) lesion, two-tier Lyme disease serology had higher sensitivity in children with multiple EM lesions (76.8% multiple lesions vs. 38.1% single EM; difference 38.7%, 95% confidence interval 24.8%-50.4%). The diagnosis of cutaneous Lyme disease should be based on careful physical examination rather than laboratory testing.

Development of Cas13a-based assays for Neisseria gonorrhoeae detection and gyrase A determination.

Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, the diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where the prevalence of infection is highest. Recent advances in molecular diagnostics, such as specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. We designed and optimized RNA guides and primer sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the porA gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A (gyrA) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For porA, we created both a fluorescence-based assay and lateral flow assay using a biotinylated fluorescein reporter. Both methods demonstrated sensitive detection of 14 N. gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyrA, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyrA genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, disproportionately affects resource-limited settings. Such areas, however, lack the technical capabilities for diagnosing the infection. The consequences of poor or absent diagnostics include increased disease morbidity, which, for gonorrhea, includes an increased risk for HIV infection, infertility, and neonatal blindness, as well as an overuse of antibiotics that contributes to the emergence of antibiotic resistance. We used a novel CRISPR-based technology to develop a rapid test that does not require laboratory infrastructure for both diagnosing gonorrhea and predicting whether ciprofloxacin can be used in its treatment, a one-time oral pill. With further development, that diagnostic test may be of use in low-resource settings.

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Evaluation of the rapid Quidel Sofia Lyme fluorescent immunoassay as a first-tier test in a modified 2-tier testing algorithm for Lyme disease: A comparison with the Zeus ELISA Borrelia VlsE1/pepC10 lgG/IgM assay followed by the Zeus monovalent IgM/IgG confirmatory assay.

OBJECTIVES: Recently modified 2-tier testing (MTTT) algorithms using 2 enzyme immunoassays (EIAs) as opposed to an EIA followed by immunoblot have been approved by the US Food and Drug Administration (FDA) for the screening and confirmation of Lyme disease. The Quidel Sofia Lyme fluorescent immunoassay is a rapid lateral-flow method that can be performed in real time, permitting on-demand testing. We evaluated the performance of the Sofia assay as a first-tier test in an MTTT algorithm. METHODS: We compared the Sofia Lyme test with the Zeus ELISA Borrelia VlsE1/pepC10 lgG/IgM test, followed by the Zeus monovalent IgM/IgG EIA as the confirmatory test. RESULTS: When used as a first-tier test compared with a standard Zeus MTTT assay, the positive percentage agreement was 91.4%% (95% CI, 77.6%-97.0%). The negative percentage agreement was 100% (95% CI, 94.0%-100%). The overall agreement was 98.3% (95% CI, 94.2%-99.4%). κ = 0.945, indicating "almost perfect agreement." CONCLUSIONS: The Sofia Lyme test performs well compared with an FDA-approved MTTT.

Biomarkers for Pediatric Bacterial Musculoskeletal Infections in Lyme Disease-Endemic Regions.

OBJECTIVES: Bacterial musculoskeletal infections (MSKIs) are challenging to diagnose because of the clinical overlap with other conditions, including Lyme arthritis. We evaluated the performance of blood biomarkers for the diagnosis of MSKIs in Lyme disease-endemic regions. METHODS: We conducted a secondary analysis of a prospective cohort study of children 1 to 21 years old with monoarthritis presenting to 1 of 8 Pedi Lyme Net emergency departments for evaluation of potential Lyme disease. Our primary outcome was an MSKI, which was defined as septic arthritis, osteomyelitis or pyomyositis. We compared the diagnostic accuracy of routinely available biomarkers (absolute neutrophil count, C-reactive protein, erythrocyte sedimentation rate, and procalcitonin) to white blood cells for the identification of an MSKI using the area under the receiver operating characteristic curve (AUC). RESULTS: We identified 1423 children with monoarthritis, of which 82 (5.8%) had an MSKI, 405 (28.5%) Lyme arthritis, and 936 (65.8%) other inflammatory arthritis. When compared with white blood cell count (AUC, 0.63; 95% confidence interval [CI], 0.55-0.71), C-reactive protein (0.84; 95% CI, 0.80-0.89; P < .05), procalcitonin (0.82; 95% CI, 0.77-0.88; P < .05), and erythrocyte sedimentation rate (0.77; 95% CI, 0.71-0.82; P < .05) had higher AUCs, whereas absolute neutrophil count (0.67; 95% CI, 0.61-0.74; P < .11) had a similar AUC. CONCLUSIONS: Commonly available biomarkers can assist in the initial approach to a potential MSKI in a child. However, no single biomarker has high enough accuracy to be used in isolation, especially in Lyme disease-endemic areas.

Development of Cas13a-based Assays for Neisseria gonorrhoeae Detection and Gyrase A Determination.

Background: Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where prevalence is highest. Recent advances in molecular diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. Methods and Results: We designed and optimized RNA guides and primer-sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the por A gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A ( gyr A) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For por A, we created both a fluorescence-based assay and lateral flow assay using a biotinylated FAM reporter. Both methods demonstrated sensitive detection of 14 N. gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyr A, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyr A genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. Conclusion: We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates.

Multiplex High-Definition Polymerase Chain Reaction Assay for the Diagnosis of Tick-borne Infections in Children.

Background: Ixodes scapularis ticks can carry Borrelia species as well as other pathogens that cause human disease. The frequency of tick-borne infections and coinfections in children with suspected Lyme disease is unknown, creating clinical uncertainty about the optimal approach to diagnosis. Methods: We enrolled children aged 1-21 years presenting to 1 of 8 Pedi Lyme Net emergency departments for evaluation of Lyme disease. We selected cases with serologically or clinically diagnosed Lyme disease (erythema migrans or early neurologic disease) matched by symptoms, age, gender, and center to control subjects without Lyme disease. We tested whole blood samples collected at the time of diagnosis using a multiplex high-definition polymerase chain reaction (HDPCR) panel to identify 9 bacterial or protozoan pathogens associated with human disease. We compared the frequency of tick-borne coinfections in children with Lyme disease to matched controls. Results: Of the 612 selected samples, 594 (97.1%) had an interpretable multiplex HDPCR result. We identified the following non-Borrelia tick-borne infections: Anaplasma phagocytophilum (2), Ehrlichia chaffeensis (1), and Babesia microti (12). Children with Lyme disease were more likely to have another tick-borne pathogen identified than matched controls (15/297 [5.1%] Lyme cases vs 0/297 [0%]; difference, 5.1% [95% confidence interval, 2.7%-8.2%]). Conclusions: Although a substantial minority of children with Lyme disease had another tick-borne pathogen identified, either first-line Lyme disease antibiotics provided adequate treatment or the coinfection was subclinical and did not require specific treatment. Further studies are needed to establish the optimal approach to testing for tick-borne coinfections in children.

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto ( Bbss ) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∻800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Evaluation of the Rapid Quidel Sofia 2 Lyme Immunoassay as a First-Tier Test in a Two-Tier Testing Algorithm for Lyme Disease: Comparison to the Zeus ELISA Borrelia VlsE1/pepC10 IgG/IgM Assay Followed by Immunoblot.

OBJECTIVES: Two-tiered serologic testing for Lyme disease is usually performed using an enzyme-linked immunosorbent assay (ELISA) as the first-tier test. The Quidel Sofia 2 Lyme test is a relatively new lateral flow method to provide more rapid turnaround time. We evaluated its performance in comparison to an established ELISA method. The test can be performed on demand rather than batching assays in a central laboratory. METHODS: We compared the Sofia 2 assay to the Zeus VlsE1/pepC10 IgG/IgM test in a standard two-tiered testing algorithm. RESULTS: Comparison of the Sofia 2 to the Zeus VlsE1/pepC10 IgG/IgM showed an overall agreement of 89.9% (κ statistic of 0.750, indicating "substantial agreement"). When the tests were followed by immunoblot in a two-tier algorithm, the agreement was 98.9% (κ statistic of 0.973, indicating "almost perfect" agreement). CONCLUSIONS: The Sofia 2 Lyme test performs well when compared with the Zeus VlsE1/pepC10 IgG/IgM in a two-tiered testing algorithm.

Babesia microti Variant With Multiple Resistance Mutations Detected in an Immunocompromised Patient Receiving Atovaquone Prophylaxis.

We report Babesia microti genomic sequences with multiple mutations in the atovaquone-target region of cytochrome b, including a newly identified Y272S mutation, plus 1 mutation of undetermined significance in the azithromycin-associated ribosomal protein L4. The parasite was sequenced from an immunocompromised patient on prophylactic atovaquone for Pneumocystis pneumonia before diagnosis of babesiosis.

Fun with fungi: a comprehensive review of common fungal organisms encountered in cytology.

The ability to detect and diagnose infection is essential in the practice of cytopathology. The identification of suppurative or granulomatous inflammation should prompt careful evaluation for infection. Many of the most commonly encountered fungal organisms demonstrate characteristic microscopic appearances that allow accurate identification even with routine cytology stains, particularly when considered in the context of clinical factors such as geographic location, social history, patient immune status, and symptoms. Given the vital role cytopathologists play in the accurate diagnosis or presumptive identification of infections, this review explores the epidemiology, clinical manifestations, and morphologic features of common fungal pathogens in addition to their differential diagnoses and ancillary testing methods.

Using CSF Proteomics to Investigate Herpesvirus Infections of the Central Nervous System.

Herpesviruses have complex mechanisms enabling infection of the human CNS and evasion of the immune system, allowing for indefinite latency in the host. Herpesvirus infections can cause severe complications of the central nervous system (CNS). Here, we provide a novel characterization of cerebrospinal fluid (CSF) proteomes from patients with meningitis or encephalitis caused by human herpes simplex virus 1 (HSV-1), which is the most prevalent human herpesvirus associated with the most severe morbidity. The CSF proteome was compared with those from patients with meningitis or encephalitis due to human herpes simplex virus 2 (HSV-2) or varicella-zoster virus (VZV, also known as human herpesvirus 3) infections. Virus-specific differences in CSF proteomes, most notably elevated 14-3-3 family proteins and calprotectin (i.e., S100-A8 and S100-A9), were observed in HSV-1 compared to HSV-2 and VZV samples, while metabolic pathways related to cellular and small molecule metabolism were downregulated in HSV-1 infection. Our analyses show the feasibility of developing CNS proteomic signatures of the host response in alpha herpes infections, which is paramount for targeted studies investigating the pathophysiology driving virus-associated neurological disorders, developing biomarkers of morbidity, and generating personalized therapeutic strategies.

Lyme neuroborreliosis: known knowns, known unknowns.

Lyme borreliosis affects the nervous system in three principal ways-mononuclear cell meningitis, cranial neuropathies and radiculoneuropathies-the last a broad term encompassing painful radiculopathy, unifocal and multifocal peripheral nerve involvement. Diagnostic tools have been significantly refined-including improved peripheral blood and CSF serodiagnostics-and much has been learned about the interactions between the causative pathogen and the nervous system. Despite these advances in our understanding of this disease, a broad range of other disorders continue to be misattributed to nervous system Lyme borreliosis, supported by, at best, limited evidence. These misattributions often reflect limited understanding not only of Lyme neuroborreliosis but also of what constitutes nervous system disease generally. Fortunately, a large body of evidence now exists to clarify many of these issues, establishing a clear basis for diagnosing nervous system involvement in this infection and, based on well performed studies, clarifying which clinical disorders are associated with Lyme neuroborreliosis, which with non-neurologic Lyme borreliosis, and which with neither.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.