Multicenter Performance Evaluation of the Simplexa Bordetella Direct Kit in Nasopharyngeal Swab Specimens.

Detection of Bordetella pertussis and Bordetella parapertussis using molecular methods is sensitive and specific with a short turnaround time compared to other diagnostic methods. In this multicenter study, we compared the performance of the Simplexa Bordetella Direct kit to those of other molecular assays in detecting and differentiating B. pertussis and B. parapertussis in nasopharyngeal swab specimens. The limits of detection (LODs) were 150 CFU/ml or 3 fg/µl of DNA for B. pertussis and 1,500 CFU/ml or 10 fg/µl of DNA for B. parapertussis A total of 1,103 fresh and residual frozen specimens from eight clinical sites were tested. Combining the data from individual clinical sites using different comparative assays, the overall positive percent agreement (PPA) and negative percent agreement (NPA) for B. pertussis were 98.7% and 97.3%, respectively. The overall PPA and NPA for B. parapertussis were 96.7% and 100%, respectively. For prospective fresh specimens, the overall PPA and NPA for both targets were 97.7% and 99.3%, respectively. For retrospective frozen specimens, the overall PPA and NPA for both targets were 92.6% and 93.2%, respectively. The percentage of invalid results was 1.0%. A cross-reactivity study using 74 non-Bordetella bacterial species and five yeast species revealed that the Simplexa Bordetella Direct kit was 100% specific. The hands-on time and assay run time of the Simplexa Bordetella Direct kit are favorable compared to those of other commercial and laboratory-developed tests. In summary, the Simplexa Bordetella Direct kit has a performance comparable to those of other molecular assays for the detection of B. pertussis and B. parapertussis.

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity- and 4 NAAT-/Clarity+ samples, there were 26 CCNA- and 4 CCNA- samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin- and NAAT-/toxin- patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin- than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

Identification and clinical significance of Helcococcus species, with description of Helcococcus seattlensis sp. nov. from a patient with urosepsis.

Helcococcus spp. are Gram-positive, catalase-negative, facultatively anaerobic cocci that are associated with wound and prosthetic joint infections as well bacteremia and empyema. Five Helcococcus spp. strains were isolated from our patient population, including 2 strains of Helcococcus kunzii from trauma-associated wounds, 2 Helcococcus sueciensis strains from blood and abscess, and a novel Helcococcus spp. strain from blood associated with urosepsis. Based on the phenotypic and phylogenetic evidence, we propose that the unknown bacterium be classified as Helcococcus seattlensis sp. nov. We found that all 5 tested Helcococcus strains grew as satellite colonies around Staphylococcus aureus and, interestingly, both H. kunzii strains were isolated together with S. aureus. In addition to 16S rRNA gene sequencing, conventional methods for leucine aminopeptidase (LAP) and pyrrolidonyl arylamidase (PYR) testing can be cost-effective and efficient for differentiation of Helcococcus spp. from Abiotrophia and Granulicatella species. Using nonstandard methods, we found that all tested Helcococcus spp. had high MICs of >4/76 µg/ml for trimethoprim-sulfamethoxazole, an antibiotic commonly used to treat urinary tract infections. High MICs for erythromycin, azithromycin, and clindamycin, and intermediate to high MICs for moxifloxacin, levofloxacin, and gentamicin were also observed among the Helcococcus strains.

Misidentification and misreporting of antibiotic resistance in Kluyvera bacteremia by blood culture molecular identification panels.

The use of molecular identification panels has advanced the diagnosis for blood stream infections with fast turnaround time and high accuracy. Yet, this technology cannot completely replace conventional blood culture and standardized antibiotic susceptibility testing (AST) given its limitations and occasional false results. Here we present two cases of bacteremia caused by Kluyvera. Its identification and antibiotic resistance were at least partially mispresented by blood culture molecular identification panels on ePlex, Verigene, and Biofire. The detection of CTX-M resistance marker did not align with the susceptibility to the third generation cephalosporins among a wide range of antibiotics for this organism. Conventional extended-spectrum beta-lactamase (ESBL) testing was used to confirm the absence of ESBL. Caution should be taken when managing cases with CTX-M or ESBL detection in blood culture caused by uncommon pathogens. Conventional culture with microbial identification and standardized AST should continue to be the gold standard for routine patient care. IMPORTANCE: This is the first report that highlights the limitations of blood culture molecular identification panels on identifying Kluyvera and its associated antibiotic resistance patterns. Both the false identification and overreporting of antibiotic resistance could mislead the treatment for bacteremia caused by this pathogen. Patient isolation could have been avoided due to the lack of extended-spectrum beta-lactamase (ESBL) activity of the organism. This report emphasizes the importance of confirming rapid identification and antibiotic resistance by molecular technologies with standardized methods. It also provides insight into the development of new diagnostic panels.

Combinations of monoclonal antibodies to anthrax toxin manifest new properties in neutralization assays.

Monoclonal antibodies (MAbs) are potential therapeutic agents against Bacillus anthracis toxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in an in vitro mouse macrophage system but did not provide significant protection in vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection both in vitro and in vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralization in vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection with B. anthracis strain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.

Galactoxylomannans from Cryptococcus neoformans varieties neoformans and grubii are structurally and antigenically variable.

Prior studies have established that the Cryptococcus neoformans capsular polysaccharide component galactoxylomannan (GalXM) manifests serotype-related structural differences that translate into antigenic differences. We analyzed GalXM from acapsular serotype A and D strains by carbohydrate analysis and static and dynamic light scattering to determine mass, effective diameter, polydispersity, and diffusion coefficients. Multiangle laser light scattering showed that GalXM from C. neoformans var. grubii strain cap59 (serotype A) had larger molecular mass (4.21 x 10(6) +/- 0.95 x 10(6) g/mol) and radius of gyration (207 +/- 27 nm) than GalXM from C. neoformans var. neoformans cap67 (serotype D). cap67 GalXM had corresponding values of 0.70 x 10(6) +/- 0.05 x 10(6) g/mol and 120 +/- 22 nm, respectively. The effective diameter for GalXM and polydispersity from the two strains varied depending on temperature and medium growth conditions, indicating that GalXM structure can vary within a strain, depending on its environment. Zeta potential determinations were negative for GalXM from both strains under all conditions, consistent with the recently reported presence of glucuronic acid. These results imply that C. neoformans GalXM, like glucuronoxylomannan, can manifest variety- and growth condition-related variations. Analysis of 16 C. neoformans and 7 Cryptococcus gattii strains with polyclonal antibody to a GalXM strain revealed antigenic similarities among the C. neoformans variety neoformans and grubii strains and no reactivity with C. gattii. As a result of the deleterious effects of GalXM on immune function, structural and antigenic variability between serotypes may translate into differences in immunomodulatory effects.

Prolonged Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA in an Obstetric Patient With Antibody Seroconversion.

BACKGROUND: There is a growing understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) in the general population. The unique immunology of pregnancy may result in variations from the reported course of disease. CASE: A 27-year-old primigravid woman presented with mild COVID-19 symptoms at 28 2/7 weeks of gestation, testing positive for SARS-CoV-2 infection by nasopharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR). Antibody seroconversion was detected at 36 6/7 weeks of gestation. She presented for delivery at 38 1/7 weeks of gestation, and her SARS-CoV-2 RT-PCR test result was positive. Severe acute respiratory syndrome coronavirus 2 RNA remained detectable 34 days postpartum and 104 days from her initial positive test. CONCLUSION: Prolonged viral shedding of SARS-CoV RNA may occur in the pregnant patient. If prevalent, this complicates the interpretation of a positive SARS-CoV-2 RT-PCR test result in the asymptomatic gravid patient.

Fluorine-19 NMR chemical shift probes molecular binding to lipid membranes.

The binding of amphiphilic molecules to lipid bilayers is followed by 19F NMR using chemical shift and line shape differences between the solution and membrane-tethered states of -CF 3 and -CHF 2 groups. A chemical shift separation of 1.6 ppm combined with a high natural abundance and high sensitivity of 19F nuclei offers an advantage of using 19F NMR spectroscopy as an efficient tool for rapid time-resolved screening of pharmaceuticals for membrane binding. We illustrate the approach with molecules containing both fluorinated tails and an acrylate moiety, resolving the signals of molecules in solution from those bound to synthetic dimyristoylphosphatidylcholine bilayers both with and without magic angle sample spinning. The potential in vitro and in vivo biomedical applications are outlined. The presented method is applicable with the conventional NMR equipment, magnetic fields of several Tesla, stationary samples, and natural abundance isotopes.

Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum.

Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection.

Incidental identification of Strongyloides stercoralis infection by broad-range 28S rDNA gene sequencing in a patient with a hematolymphoid malignancy.

Strongyloides stercoralis is an important human parasite, especially in rural areas and developing countries. Infected immunosuppressed patients are at risk for hyperinfection, with severe clinical consequences. Here we describe the incidental detection and diagnosis of an unexpected S. stercoralis infection by methods designed to detect fungal 28S ribosomal DNA.

Necessity of 16S rRNA gene sequencing for identifying Haemophilus parainfluenzae-like strains associated with opportunistic urinary tract infections.

The identification of Haemophilus spp. from urogenital sites can be challenging due to the lack of appropriate media for culturing the organisms and the poor resolution of biochemical methods. By incorporating chocolate agar and 16S rRNA gene sequence analysis in our protocol to identify Haemophilus spp. from urinary specimens, we isolated and characterized 30 genetically homogeneous strains of a cryptic species that is phylogenetically close to, but distinct from, Haemophilus parainfluenzae. Commercial biochemical kits and VITEK 2 could not distinguish between the two species. Over 90 % of the strains were isolated from urine and the urogenital area, made possible with the inclusion of chocolate agar in our urine culture protocol. In contrast, no Haemophilus strains isolated from respiratory specimens were identified as the cryptic genospecies. The cryptic genospecies was associated with urinary tract infections (UTIs) in certain patient populations. Distinct from Haemophilus quentinii that also causes urogenital infection, the cryptic genospecies required V factor (NAD) but not X factor (haemin) to grow. The data indicated that 16S rRNA gene sequencing may be necessary in identifying Haemophilus species and that inaccurate categorization of Haemophilus strains isolated from urogenital specimens based on phenotypic characteristics may prevent accurate diagnosis of UTIs.

Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity.

The effector activity of antibodies is dependent on engagement with Fcγ receptors (FcγRs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with FcγRs is hampered by substantial structural and functional FcγR diversity among species. In this report, we used mice expressing only human FcγRs to evaluate the contribution of FcγR-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon FcγR engagement, with minimal protection against anthrax toxin observed in FcγR-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human FcγRs and assessed their activity in FcγR-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating FcγRs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.