Theel, Elitza S.

Objectives: We evaluated agreement between human papillomavirus (HPV) tests performed on self-collected vaginal and clinician-collected cervical specimens to advance HPV self-collection as a cervical cancer screening option in our health care system. Methods: This diagnostic accuracy study was conducted at an academic medical center. Participants were 25–65 years old with upcoming colposcopy appointments. One cohort (n = 79) self-collected a vaginal specimen in the clinic immediately before a clinician-collected cervical specimen. The second cohort (n = 115) self-collected a vaginal specimen at home, mailed it in, and had a clinician-collected cervical specimen in the clinic. Paired specimens were evaluated for total (TPA), positive (PPA), and negative (NPA) percentage agreement. Extreme temperature stability studies were also conducted. Results: Compared with clinician-collected cervical specimens, in-clinic self-collected vaginal specimens showed a TPA, PPA, and NPA of 98%, 100%, and 97% for HPV16; 99%, 100%, and 99% for HPV18; 86%, 94%, and 79% for high-risk other (HRO) HPV. The TPA, PPA, and NPA for at-home self-collected vaginal specimens compared with clinician-collected cervical specimens were 94%, 79%, and 96% for HPV16; 95%, 43%, and 98% for HPV18; 89%, 80%, and 93% for HRO. Vaginal specimens showed prolonged stability and were unaffected by extreme temperatures. Conclusions: In general, in-clinic and at-home self-collected vaginal HPV specimens for HPV screening demonstrate high agreement with clinician-collected cervical specimens, with the exception of low PPA in the at-home self-collected vaginal HPV18 subgroup. Our findings support the use of HPV self-collection as a practical option to expand access to cervical cancer screening.

Abstract Background Coccidioidomycosis is the most prevalent endemic mycosis in the United States. Diagnosis of coccidioidomycosis can be challenging due to limitations of conventional diagnostics. Detection of fungal plasma cell-free DNA (cfDNA) is a novel noninvasive testing modality for the diagnosis of invasive fungal disease. In this study, we characterized the performance of Coccidioides plasma cfDNA polymerase chain reaction (PCR). Methods A retrospective study was conducted in patients with suspected coccidioidomycosis who underwent plasma cfDNA PCR testing and conventional fungal testing for Coccidioides immitis and Coccidioides posadasii. Patients were categorized per the EORTC/MSGERC case definitions as proven or probable for sensitivity analyses and no coccidioidomycosis for specificity analyses. Results Overall, 362 plasma samples from unique patients were included in this study. The overall sensitivity, specificity, and positive- and negative-predictive values of Coccidioides plasma cfDNA PCR at 0.04% prevalence were 57.5% (27/47; 95% CI, 42.2%–71.7%), 100% (315/315; 95% CI, 98.8%–100%), 100% (95% CI, 87.2%–100%), and 100% (95% CI, 100%–100%), respectively. The sensitivity was 33.3% (2/6; 95% CI, 4.3%–77.7%) in patients with pulmonary focal disease, 66.7% (14/21; 95% CI, 43.0%–85.4%; P≥.05) in patients with pulmonary multifocal disease, and 52.6% (10/19; 95% CI, 39.6%–77.9%; P≥.05) in patients with disseminated coccidioidomycosis. In patients with concurrent antigen results, the sensitivity of cfDNA PCR was 58.3% (14/24; 95% CI, 39.6%–77.9%) compared with 37.5% (9/24; 95% CI, 18.8%–59.4%; P≥.05) for antigen. Conclusions Coccidioides plasma cfDNA PCR is modestly sensitive but highly specific, which can be useful for rapid and noninvasive diagnosis of coccidioidomycosis.

Introduction Asymptomatic individuals with negative Interferon-γ release assay (IGRA) and tuberculin skin test (TST) results are often labeled as tuberculosis-unexposed individuals. We hypothesize that flow cytometric (FC) detection of antigen-stimulated T-cells in peripheral blood can differentiate IGRA(-)/TST(-) individuals with and without prior potential M. tuberculosis (Mtb) exposure. Methods We used FC multiparameter profiling of T-cell activation markers in TST/IGRA-negative individuals at risk for Mtb exposure and in unexposed controls. Results We studied 54 IGRA(-)/TST(-) subjects, including 27 individuals at risk for prior Mtb exposure. While quantitative IGRA results were negative in all patients, FC profiling of CD3/CD4 + IFN-γ + HLA-DR + T-cells after stimulation with a pool of Mtb-specific region of difference-1 (RD1) peptides, mycobacterial (PPD and MTB300), or Candida antigens, statistically differentiated between unexposed and potentially exposed individuals (P<0.05). ROC analysis showed PPD and RD1 peptides responses had AUCs >0.7 (sensitivity of 55.5–74% and specificity of 66.6–85.1%). The presence of a polyfunctional RD1-reactive CD3/CD8 + IFN-γ + TNF-α + T-cell subset also significantly differentiated these groups (AUC 0.66). Other T-cell markers demonstrated no significant differences. Conclusions FC detection of antigen-specific IFN-γ + HLA-DR + CD4+T-cells and IFN-γ + TNF-α + CD8 + T-cells can differentiate IGRA(-)/TST(-) individuals at risk of Mtb exposure from unexposed controls. Further validation is needed to determine whether this immunoprofiling approach can improve detection of Mtb infections and identification of unexposed individuals for future biomarker studies and vaccine trails.

Abstract Background Detecting Histoplasma (HP) and Blastomyces (BM) antigen (Ag) in urine (U) and serum (S) is an essential diagnostic tool for these pathogens. Labs have added bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) as additional HP and BM Ag testing sources. We evaluated the sensitivity of Ag detection in U and S in patients with detected HP or BM Ag in BAL or CSF by enzyme immunoassay (EIA). Methods 14 patients with HP or BM Ag-positive results made up the CSF cohort. The Ag concentrations were below the limit of quantification (< LoQ, N=3), above the limit of quantification ( > LoQ, N=1), and 0.3 to 9.5 ng/mL (x̄ = 2.3 ng/mL, N=10). The BM Ag-positive in BAL group included 24 patients, with concentrations of < LoQ (N=4), >LoQ (N=5), and 0.3 to 11.3 ng/mL (x̄ = 3.5 ng/mL, N=15). HP Ag-positive in BAL comprised 36 patients, with 3 < LoQ, 10 >LoQ, and 23 ranging from 0.8 to 16.9 ng/mL (x̄ = 4.7 ng/mL). 16 patients in the BAL cohorts were Ag-positive for HP and BM, demonstrating the inability to discriminate between these dimorphic fungi through Ag testing. All patients were checked for routine clinical HP and BM EIA Ag test results on U and S. HP BAL patients were also investigated for cross-reacting galactomannan (GM; Aspergillus EIA in S or BAL) and (1→3)-β-d-glucan (BDG; Fungitell EIA in S) antigen test orders. All additional test results over 14 days before or after CSF or BAL Ag testing were excluded. Results 100% (14/14), 96% (22/23), and 83% (30/36) of HP/BM CSF, BM BAL, and HP BAL Ag-positive patients were also positive in U and/or S, when both additional sources were ordered, respectively. Of the 6 HP BAL patients without positive U or S results, 3 had GM and BDG testing performed. All 3 had high signal GM and/or BDG results in S and/or BAL. In conjunction with low HP Ag levels in BAL (1.0 to 1.53), these findings suggest cross-reactivity in the HP BAL Ag test. Classifying these 3 patients as HP BAL false-positive shows a positive agreement of 91% (30/33) between HP BAL and HP U/S. Conclusion HP and BM CSF Ag testing showed no increase in sensitivity when U and S were tested. Minimal increases in sensitivity were observed in HP and BM BAL versus U and S. However, false-positive results in BAL due to cross-reactivity with GM and BDG cannot be ruled out, suggesting limited utility of the invasive CSF and BAL sources for HP and BM EIA Ag testing. Disclosures All Authors: No reported disclosures

Abstract Background Diagnosis of VZV CNS vasculitis (CNS-VZV) is aided by detecting intrathecally (i.t.) synthesized VZV-specific antibodies (Abs) through testing paired serum (S) and CSF specimens and determining an antibody index (AI). Currently, laboratories in the United States do not provide AI testing for VZV. The currently used methods (e.g., EIAs, IFAs) lack clinical utility as they cannot discriminate if VZV Abs in the CSF are i.t. produced due to CNS infection or whether they are present due to passive diffusion across the blood-CSF-barrier. To optimize diagnostic options for CNS-VZV, we evaluated a quantitative VZV IgG ELISA (Euroimmun AG, Lübeck, Germany) to calculate AI values using Reiber’s formula on paired S/CSF specimens to assess the diagnostic accuracy of this method. Methods The clinical sensitivity cohort consisted of four patients diagnosed with CNS-VZV (meningitis or vasculitis) as determined by symptoms, imaging, response to antiviral therapy, and/or supporting test results (e.g., lymphocytic pleocytosis, elevated protein in CSF). The patient group for clinical specificity included 11 patients with suspected CNS-VZV who ultimately had an unclear (n=4) or non-CNS-VZV (n=7) diagnosis. The reference range cohort included paired clinical S/CSF samples from 25 patients without infectious diseases testing. Paired S/CSF samples were processed on the same batch using a fully automated ELISA analyzer capable of onboard, ad hoc runtime dilution preparation (AP22 ELITE, DAS ITALY). The AI values were calculated using the Reiber formula, and sensitivity (Sn), specificity (Sp), and positive (PPV) and negative (NPV) predictive values were calculated. The AI was calculated using the following equations: If Total IgG CSF (mg/dL):Total IgG serum (mg/dL) < Quotient limit Total IgG, then AI = (VZV IgG Ab CSF [units × dilution]:VZV IgG serum [units × dilution])/(Total IgG CSF [mg/dL]:Total IgG serum [mg/dL]). If Total IgG CSF (mg/dL):Total IgG serum (mg/dL) > Quotient limit Total, then AI = (VZV IgG Ab CSF [units × dilution]:VZV IgG serum [units × dilution])/Quotient limit Total IgG. Quotient limit Total IgG = 0.93 × [(Qalbumin2) + (6 × 10-6)]1/2 - 1.7 ×10-3. Qalbumin = Albumin CSF (mg/dL):Albumin serum (mg/dL). Results The VZV IgG ELISA with Reiber AI calculation method showed Sn, Sp, PPV, and NPV values of 100% (4/4), 91% (10/11), 80% (4/5), and ?0 (10/10), respectively. One of the Sp patients had an equivocal AI result and was classified as negative, whereas the Sp patient with a positive AI result had an unclear diagnosis. VZV AI results for 96% (24/25) of the reference range patients were negative, with one equivocal. The mean AI of the 25 patients was 1.05, and the lower and upper 3 standard deviation limits were 0.6 and 1.5, respectively. These values match the AI qualitative interpretive criteria recommended by Reiber (0.6 to <1.3 = negative, 1.3 to 1.5 = equivocal, >1.5 AI = positive). Conclusion We show that the VZV IgG ELISA, alongside the Reiber formula for AI calculation, is effective for identifying patients with i.t. synthesized VZV IgG Abs. This method can assist clinicians in evaluating patients with suspected CNS-VZV infections who are negative by alternative methods.

ABSTRACT Recent decades have seen a rise in the incidence of tick-borne diseases in the US, along with an increased number of pathogens transmitted by ticks, and geographic expansion of tick populations. A variety of laboratory testing methodologies are available for the diagnosis of tick-borne diseases, including serology, microscopy, and molecular-based methods. The preferred approach varies by the specific disease, locally available test options, and the stage of illness at patient presentation. This mini-review focuses on updates in our understanding of the epidemiology of tick-borne diseases in the US and advances in the field of laboratory diagnostics.

SUMMARY Antibody and antigen detection assays continue to play a significant role in the diagnosis of dimorphic fungal pathogens, including Blastomyces dermatitidis complex, Coccidioides immitis/posadasii , Paracoccidioides species, Histoplasma species , Sporothrix species, and Talaromyces marneffei . The performance characteristics of serologic and antigen detection assays for these pathogens are variable, however, influenced by multiple factors, including sample type, disease presentation, patient immunostatus, and timing of specimen collection relative to symptom onset. As a result, there is a need for a centralized document summarizing the accuracy of currently available antibody and antigen detection assays for each of these agents, including discussion of individual assay nuances and caveats that should be considered by clinicians and laboratorians alike. In addition, this review provides expert recommendations for the utilization and interpretation of serologic and antigen detection assays for these dimorphic pathogens.