Grys, Thomas E.

The diagnosis of coccidioidomycosis is often achieved serologically by the detection of antibodies against fungal antigens. While several serologic tests are available for coccidioidomycosis, all of them are performed in a laboratory setting causing delays in diagnosis and therapeutic intervention. Point-of-care testing offers the ability to provide a shorter time to result by avoiding specimen send-out, minimizing processing steps, and employing expeditious immunochemical techniques. A preliminary trial of a rapid anti-coccidioidal antibody lateral flow assay (LFA) using fingerstick blood was performed on 22 patients with coccidioidomycosis at the point of care during outpatient clinic visits. Patients were tested longitudinally over the course of one year. An LFA reader was implemented to provide an objective result by quantifying the intensity of the test line band. There was close qualitative concordance observed between positive LFA results with send-out immunodiffusion (89.5%) and complement fixation (78.4%) standard of care clinical laboratory assays. Additionally, the relationship between LFA test line density values and traditional complement fixation antibody titers was assessed.

Abstract Background While most pulmonary infections with Coccidioides spp. are self-limited, a subset of patients is at higher risk for severe disease and extrapulmonary dissemination, including individuals with certain ethnic backgrounds. Disseminated disease can involve soft tissues, bones, and the central nervous system with high morbidity and mortality in the absence of antifungal treatment. Diagnosis of coccidioidomycosis can be challenged by the availability of specimens for culture or histopathology, confusion with, or lack of availability of various antibody tests and overall lack of consideration of Coccidioides spp. as the etiology of disease, especially in immunocompetent hosts. Methods We set out to characterize cases of disseminated coccidioidomycosis at our institution over a 7-year period, solely in immunocompetent hosts, to highlight diagnostic delays and determine which, if any, primary screening test might be the most useful. Results A total of 40 cases met our inclusion criteria, and 100% of these cases had positive immunoglobulin G antibodies on a US FDA-cleared Coccidioides spp. enzyme immunoassay. Nearly all of the cases (87%) had a delay in diagnosis and associated worsening of disease (71%). Locations of initial presentations that led to delayed recognition included primary care settings (56%), emergency departments (33%), and urgent care centers (11%), all in the region of Coccidioides spp. endemicity. Conclusions These findings highlight the need for interventions to increase awareness of the risk factors for, the symptoms of, and the appropriate testing options to diagnose disseminated coccidioidomycosis.

Abstract Description Coccidiomycosis is an endemic fungal infection caused by Coccidioides spp., and diagnosis is often made with serology. Coccidiomycosis is especially problematic for immunocompromised patients, with greater risk for disseminated infection and mortality. This study evaluated current strategies for detecting antibodies to Coccidioides, including enzyme immunoassay (EIA), immunodiffusion (ID) and complement fixation (CF) and applied a new ID assay for improved sensitivity and specificity. Clinical sensitivity and specificity studies were done using 1544 unique serum specimens for which Coccidioides EIA testing was clinically requested. A full panel of EIA IgG, EIA IgM, ID and CF was performed on each, comparing our in-house immunodiffusion assay using reagents from Cactus Bio, LLC to our current reference laboratory. Full method validation was completed, as prescribed by CLIA. Using existing reference lab testing as the standard, a sensitivity of 96.9%, specificity 85.9%, PPV 54.9%, NPV 99.4% was achieved. Additional chart review demonstrated the in-house ID improved to 98.0% sensitivity, 94.6 specificity, 84.2% PPV, 99.4% NPV. Such high NPV supports using reflex testing, which allowed our lab to eliminate hundreds of unnecessary CF referrals each month, saving diagnostic time, financial waste and lab resources. A reflexive testing algorithm paired with this enhanced ID assay is especially useful for the immunosuppressed, for which Coccidioides infections carry a greater risk. Topic Categories Translational and Interventional Immunology (TI)

Abstract Early diagnosis and treatment are essential for improving outcomes of coccidioidal meningitis. While the detection of IgG by serologic testing of the cerebrospinal fluid (CSF) has been a mainstay of diagnosis for years, the diagnosis of coccidioidal meningitis in clinical practice can be very challenging due to suboptimal sensitivity of laboratory tests. We reviewed the results of the CSF diagnostic test results from the initial lumbar puncture in 110 patients with proven, probable, and likely coccidioidal meningitis from 1989 to 2024. One hundred four patients were diagnosed with coccidioidal meningitis on the initial CSF examination. The positivity rate of the first CSF testing was 89.1% (n = 64) for IgG by enzyme immunoassay, 62.2% (n = 98) for IgG by immunodiffusion, and 70.2% (n = 104) for IgG by complement fixation, 4.4% (n = 90) for fungal culture, 18.8% (n = 69) for polymerase chain reaction, and 33% (n = 21) for Coccidioides antigen. The non-specific fungal marker 1,3-β-d-glucan was positive in the CSF in 70.4% (n = 27) of samples. Contrasted brain magnetic resonance imaging identified leptomeningeal enhancement in 53.1%. Optimal detection of coccidioidal meningitis requires a combination of diagnostic modalities.

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.

Abstract Background Lymphocytic choriomeningitis virus (LCMV) infection in immunocompromised hosts can result in disseminated disease, meningoencephalitis, and death. Published cases in transplant recipients have been traced to transmission from infected donors. We report 2 cases of serious, non–donor-derived LCMV infection in solid organ transplant recipients. Methods Initial identification of LCMV infection was done by using metagenomic next-generation sequencing (mNGS). Subsequent evaluations and confirmatory testing involved molecular diagnostics, serology, and phylogenetic analysis. A detailed epidemiologic investigation was conducted. Results LCMV was detected by mNGS in 2 solid organ transplant recipients from distinct donors. A heart transplant recipient (from donor 1) died of progressive, disseminated LCMV infection, while a kidney transplant recipient (from donor 2) with LCMV meningoencephalitis survived. A multistate laboratory and epidemiologic investigation of both donors and all their organ recipients was initiated. Postmortem samples were obtained from both donors, and pretransplant and/or posttransplant samples were obtained from 5 of the 6 organ recipients. mNGS, serologic, and real-time reverse-transcription polymerase chain reaction testing confirmed LCMV infection in both solid organ transplant recipients. Epidemiologic investigation revealed significant pretransplant rodent exposures for both LCMV-infected recipients. Laboratory studies for the other organ recipients from both donors were negative for LCMV infection. Conclusions Our investigations suggest that LCMV infection in 2 solid organ transplant recipients originated from rodent exposure preceding transplantation and were not donor derived. Although uncommon, healthcare providers should be aware of LCMV-associated serious and life-threatening illness in immunocompromised hosts. Diagnostic modalities are limited to reference laboratories.

Abstract Metagenomic sequencing of plasma has been advertised by Karius, Inc. as a way to diagnose a variety of infectious syndromes. Due to the lack of robust evidence of clinical utility, our laboratory began actively stewarding Karius testing. Microbiology Directors recommended cancelation of Karius orders when certain criteria were identified. We set out to review Karius test requests in a 52-month period of stewardship, during which we recommended cancellation on 21 of 57 orders (37%). Of Karius tests sent on samples with negative conventional testing, only 3 (7%) had positive results for Karius with plausible explanatory etiologies. Of these three cases, two were empirically covered for the positive finding without improvement and one case was never treated. Twelve (29%) had positive results that were noted by infectious diseases (ID) to reflect insignificant detections. Given the 4-fold higher detection of insignificant Karius results, we set out to systematically analyze the literature for the experience of insignificant detections at other centers. When we compared studies that included healthy controls or had clinical adjudication of positive Karius findings by ID physicians, we found a median of 17.5% of individual patients that had positive insignificant detections of potential pathogenic bacteria or fungi. The most frequently detected species were as likely to be clinically adjudicated to be insignificant as they were to be significant within the same studies. Overall, these findings highlight limited utility of Karius testing and a need for careful stewardship, not only to ensure it is sent on patients who may benefit, but also to ensure results of potential pathogens are interpreted cautiously.

Abstract Deep sternal wound infection is a rare complication of cardiac surgery that is typically caused by skin resident flora, such as species of Staphylococcus and Streptococcus. Infections caused by fungi are less common and are generally caused by Candida species. Regardless of etiology, these infections are associated with significant morbidity and mortality. We present a case of postoperative mediastinitis that occurred following a 5-vessel coronary artery bypass graft and was caused by a filamentous fungus of the Rhizopus genus. The patient was treated with serial debridement, liposomal amphotericin B, and isavuconazonium and was discharged from the hospital in stable condition. Fungal mediastinitis is a rare entity, and clinicians must maintain a high level of suspicion to make the diagnosis. A fungal cause of postoperative mediastinitis should be considered in patients with negative bacterial cultures, uncontrolled diabetes, or current immunosuppression or those who present weeks after surgery with a subacute onset of symptoms.

Abstract Background In 2021, the state of Arizona experienced the largest focal outbreak of West Nile virus (WNV) in US history. Timely and accurate diagnostic testing remains a challenge for WNV due to transient viremia and limited immunoassay specificity. Recent studies have identified whole blood (WB) and urine as more sensitive specimen types for the detection of WNV RNA. Methods We evaluated ordering practices, test performance, and patient characteristics of probable and confirmed cases. In total, we identified 190 probable and proven cases, including 127 patients (66.8%) with neuroinvasive disease. Results Among all cases, only 29.5% had WNV polymerase chain reaction (PCR) testing ordered on WB, of which 80.3% resulted as positive, including 7 cases in which WNV serologic testing was negative and 5 cases for which serologic testing was not ordered. In comparison, only 23.7% of cases that had cerebrospinal fluid (CSF) PCR ordered had a positive result, including 3 cases that were negative by PCR on WB. In contrast, WNV PCR on WB detected 12 neuroinvasive cases that were CSF PCR negative. WNV PCR testing in urine was only ordered on 2 patients, both of whom were positive. Crossing cycle threshold (Ct) values were not significantly different between WB and CSF specimen types, nor was there a correlation between Ct value and days from symptom onset at the time of sample collection; all specimen types and time points had Ct values, with 98% above 30. WB was positive by WNV PCR in several patients for >7 days (range, 7–25 days) after symptom onset, as was the CSF PCR. Conclusions Taken together, these findings indicate that WNV PCR testing on WB may be the best initial test for timely diagnosis of WNV infection, irrespective of clinical manifestation; however, if negative in patients with suspected neuroinvasive disease, WNV PCR testing on CSF should be ordered.