A quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis.

Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen. Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

Comparison of three anti-coccidioides antibody enzyme immunoassay kits for the diagnosis of coccidioidomycosis.

Coccidioidomycosis is a common cause of community-acquired pneumonia in endemic areas of the southwestern United States. Clinical presentations range from self-limited disease to severe, disseminated disease. As such, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic testing has variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from patients with coccidioidomycosis and controls were tested for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using the MVista Coccidioides antibody detection EIA and two commonly used commercial enzyme immunoassay (EIA) kits: the IMMY Omega EIA and the Meridian Premier EIA. The sensitivity of the IgG antibody detection was 87.4% using the MVista test compared to 46.6% for IMMY and 70.9% for Meridian. The sensitivity for IgM antibody detection was 61.2% for the MVista test, 22.3% for IMMY and 29.1% for Meridian. For IgG antibody detection, specificity was 90% for the MVista EIA, 94.6% for IMMY, 96.4% for Meridian. For IgM antibody detection, specificity was 95.3% for the MVista test 98.2% for IMMY and 99.1% for Meridian. The MVista Coccidioides antibody EIA offers improved sensitivity, including among high-risk patient populations, for the detection of IgG and IgM antibodies in comparison to other currently available EIAs.

Sensitivity of Serologic Testing for Dogs Diagnosed with Coccidioidomycosis on Histology: 52 Cases (2012-2013).

Coccidioidomycosis, or Valley Fever, is a fungal infection caused by Coccidioides spp., soil-living fungi endemic to the southwest region of the United States. The infection can cause various diseases including respiratory, neurologic, cardiac, dermatologic, and ocular disease as well as osteomyelitis in dogs and many other mammals. Obtaining a definitive diagnosis can be challenging. Serology is commonly used as a screening diagnostic test for disease, but both false-negative and false-positive results have been reported. Fifty-two cases of coccidioidomycosis diagnosed via histopathology were retrospectively evaluated. The sensitivity of serology in the study population was determined to be 87% for immunoglobulin G and 46% for immunoglobulin M. The cases were evaluated for an association between negative serology results and anatomic location of disease, but these variables were found to be independent. This study reports the sensitivity of serology for canine coccidioidomycosis and highlights the importance of using multiple diagnostic tests for definitive diagnosis of infection.

The utility of real-time polymerase chain reaction in detecting Coccidioides immitis among clinical specimens in the Central California San Joaquin Valley.

Coccidioidomycosis, the fungal infection caused by dimorphic Coccidioides species, is typically diagnosed by histopathologic identification of spherules, by culture, or by serology. These tests are reliable but time-intensive, delaying diagnosis and treatment. Rapid real-time polymerase chain reaction (RT-PCR) can be performed and was validated to identify Coccidioides immitis using an in-house developed assay for the Becton Dickinson molecular instrument (BD MAXTM). These studies were performed using patient samples that had been shown to be positive on previously set up fungal cultures. To evaluate this new RT-PCR test in the clinical setting, we conducted a retrospective chart review of patients (N = 1160) who underwent Coccidioides PCR (Cocci PCR) on clinical samples between March 1, 2014, and Dec 31, 2016. We abstracted clinical, microbiologic, serologic, radiographic, treatment, and follow-up data. Specimens of cerebrospinal fluid (CSF), bronchioalveolar lavage fluid (BAL), lung tissue biopsy (LTB), sputum, and pleural fluid were evaluated to determine sensitivity and specificity. Of the 113 specimens that tested positive for Cocci PCR, all had clinical disease defined by traditional clinical criteria, yielding 100% specificity. Overall sensitivity was 74% versus 46% for fungal culture and was available in 4 hours rather than 1-2 weeks. Sensitivities varied by source material and clinical setting. CSF had a sensitivity of 59%, BAL for acute pneumonia 91%, sputum for acute pneumonia 94%, pleural fluid 86%, but LTB for lung nodules only 44%. Overall positive predictive value (PPV) was 100%, while negative predictive value (NPV) was 96%, but again this varied by specimen and clinical setting. Our experience with clinical testing of >1160 specimens over 2-3 years shows we can utilize this technology to improve our ability to diagnose disease but that the sensitivity varies by specimen source and clinical setting.

Selection of Specific Peptides for Coccidioides spp. Obtained from Antigenic Fractions through SDS-PAGE and Western Blot Methods by the Recognition of Sera from Patients with Coccidioidomycosis.

Antigenic fractions of 100, 50, 37, and 28 kDa obtained through the SDS-PAGE method that were more frequently recognized by anti-Coccidioides antibodies in the sera of coccidioidomycosis patients were selected using western blotting. Subsequently, these bands were sequenced, and the obtained proteins were analysed by BLAST to choose peptides specific for Coccidioides spp. from among the shared aligned sequences of related fungi. A peptide specific for C. immitis was selected from the "GPI anchored serine-threonine rich protein OS C. immitis", while from the "uncharacterized protein of C. immitis", we selected a peptide for C. immitis and C. posadasii. These proteins arose from the 100 kDa antigenic fraction. From the protein "fatty acid amide hydrolase 1 of C. posadasii" that was identified from the 50 kDa antigenic fraction, a peptide was selected that recognized C. immitis and C. posadasii. In addition, the analysis of all the peptides (353) of each of the assembled proteins showed that only 35 had 100% identity with proteins of C. immitis and C. posadasii, one had 100% identity with only C. immitis, and one had 100% identity with only C. posadasii. These peptides can be used as diagnostic reagents, vaccines, and antifungals.

Screening Coccidioides serology in kidney transplant recipients: A 10-year cross-sectional analysis.

BACKGROUND: Kidney transplant recipients (KTRs) are at risk for reactivation and complicated infection due to Coccidioides. Pre-transplant serological screening should provide benefit for patients from endemic areas. We evaluated Coccidioides seroprevalence by area of residence in KTRs at a major transplant program in Los Angeles. METHODS: We performed cross-sectional analyses of adult KTRs who underwent transplantation at UCLA between 2007-2016. Patients with Coccidioides serology by enzyme immunoassay (EIA) before or within 14 days from transplantation were included. Patients were classified as living in highly, established, suspected, or not endemic areas by their residential zip code. RESULTS: Overall prevalence of Coccidioides IgG and IgM were 1.4% and 2.8%, respectively. Of patients with positive serology, 31.4% had isolated IgG and 66.3% isolated IgM. Patients from established and highly endemic areas had IgG seropositivity of 3.7% versus 1.3% for patients living in suspected endemic areas(P < .01). Rates of IgM seropositivity were 3.7% compared to 2.8% respectively (P = .28). No patients from non-endemic areas had positive screening serology. CONCLUSIONS: Pre-transplant serological screening for Coccidioides is recommended in kidney transplant candidates from endemic areas. We observed high seroprevalence among patients from highly and established endemic areas, for whom universal prophylaxis is recommended. For residents from less well-established areas of endemicity, serological screening showed benefit in identifying patients at risk. In patients with isolated EIA IgM, performing repeat and confirmatory tests is recommended. Patients from non-endemic areas had low risk of infection, however, a thorough social history is necessary to evaluate risk.

Serum procalcitonin levels in patients with primary pulmonary coccidioidomycosis.

RATIONALE: The serum procalcitonin assay has emerged as a promising biomarker to distinguish between bacterial and viral respiratory tract infections but has not been used to differentiate coccidioidomycosis from bacterial infection. A correlation between procalcitonin serum levels and coccidioidomycosis has never been reported. OBJECTIVE: To determine any association between serum procalcitonin levels and primary pulmonary coccidioidomycosis. METHODS: We identified and enrolled 20 immunocompetent patients with symptomatic primary pulmonary coccidioidomycosis of < 8 weeks' duration and performed a one-time procalcitonin assay, with a cutoff of < 0.25 µg/L indicating a nonbacterial infection. MEASUREMENTS AND MAIN RESULTS: Nineteen of 20 patients (95%) had serum procalcitonin of < 0.25 µg/L. The median procalcitonin level was 0.05 µg/L (range, < 0.05-0.87 µg/L; interquartile range, 0.05-0.05 µg/L). Sixteen of 20 patients (80%) had undetectable procalcitonin of < 0.05 µg/L. The four patients with detectable procalcitonin had a median value of 0.2 µg/L (range, 0.09-0.87 µg/L). CONCLUSIONS: In this pilot study, procalcitonin was not elevated in immunocompetent patients with primary pulmonary coccidioidomycosis at a median of 32 days after symptom onset. Larger prospective studies are needed to confirm this finding.

Update on the diagnosis of pulmonary coccidioidomycosis.

Coccidioidomycosis is a common cause of community-acquired pneumonia in the southwest United States, Mexico, and South America. The disease has seen a marked increase in incidence in the western United States in the last decade and can be acquired by individuals who travel even briefly through an endemic area, presenting a diagnostic dilemma for clinicians who are not familiar with the disease. The clinical and radiographic manifestations of pulmonary coccidioidomycosis often mimic those of other causes of pneumonia. However, because treatment recommendations and the potential for chronic sequelae of acute infection differ substantially from those for bacterial community-acquired pneumonia, accurate, timely diagnosis of coccidioidomycosis is paramount. A number of diagnostic tests are available with varying sensitivity and specificity, making the approach complex. Radiographic features, although nonspecific, sometimes demonstrate patterns more suggestive of coccidioidomycosis than bacterial community-acquired pneumonias. A routine blood count may reveal eosinophilia. Serologic testing is used most widely but may be negative early in the course of disease, potentially leading to misdiagnosis with subsequent inappropriate treatment and follow-up. The sensitivity of serologic testing is lower in immunocompromised patients, a population at the highest risk for developing severe disease. When clinically appropriate, other biologic specimens, such as sputum, bronchoalveolar lavage fluid, or lung biopsies, may allow for rapid, definitive diagnosis. In light of the significantly increased incidence and complexities in diagnosis of coccidioidomycosis, we examine the diagnostic approach and provide examples of classic clinical and radiographic presentations, discuss the utility of serologic testing, and suggest algorithms that may aid in the diagnosis.

Identifying areas of high risk of human exposure to coccidioidomycosis in Texas using serology data from dogs.

Coccidioidomycosis or Valley Fever (VF) is an emerging soil-borne fungal zoonosis affecting humans and animals. Most non-human cases of VF are found in dogs, which we hypothesize may serve as sentinels for estimating the human exposure risk. The objective of this study is to use the spatial and temporal distribution and clusters of dogs seropositive for VF to define the geographic area in Texas where VF is endemic, and thus presents a higher risk of exposure to humans. The included specimens were seropositive dogs tested at a major diagnostic laboratory between 1999 and 2009. Data were aggregated by zip code and smoothed by empirical Bayesian estimation to develop an isopleth map of VF seropositive rates using kriging. Clusters of seropositive dogs were identified using the spatial scan test. Both the isopleth map and the scan test identified an area with a high rate of VF-seropositive dogs in the western and southwestern parts of Texas (relative risk = 31). This location overlapped an area that was previously identified as a potential endemic region based on human surveys. Together, these data suggest that dogs may serve as sentinels for estimating the risk of human exposure to VF.

Physical characterization of the "immunosignaturing effect".

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.

Relationship between radiographic hilar lymphadenopathy and serologic titers for Coccidioides sp. in dogs in an endemic region.

Hilar lymphadenopathy is a common radiographic finding in coccidioides infections. Serologic studies are used most often for the diagnosis of coccidioidomycosis in endemic areas, with IgG titers > 1:8 considered positive for infection and lower IgG titers of < 1:8 considered indicative of exposure and not necessarily related to organism presence. The objective of this study was to determine the relationship of hilar lymphadenopathy to coccidioidomycosis titers for dogs in an endemic area. A positive association between these parameters would allow treatment to be initiated before obtaining titer results. Thoracic radiographs of 131 dogs from an endemic area were reviewed for evidence of hilar lymphadenopathy. These results were compared with serology results. There was a significant association between hilar lymphadenopathy and a positive serology result (P < 0.001). With hilar lymphadenopathy as a predictor of a positive titer result, sensitivity was 28.0%, specificity was 91.5%, the positive predictive value was 43.8%; and the negative predictive value was 84.4%. There was no association between the titer result and gender, age, or weight. The radiographic finding of hilar lymphadenopathy appears to be a useful indicator of coccidioidomycosis infection in an endemic population of dogs supporting the treatment of patients for coccidioidomycosis when hilar lymphadenopathy is present and before obtaining serology results.

Significance of isolated positive IgM serologic results by enzyme immunoassay for coccidioidomycosis.

Serologic testing is important for diagnosis of coccidioidomycosis. Many methods are available for diagnostic testing. Enzyme immunoassay (EIA) can be performed quickly and locally but has the potential for false-positive results in patients manifesting a positive EIA for immunoglobulin M (IgM) antibodies and a negative EIA for immunoglobulin G (IgG). We retrospectively reviewed the charts of 405 patients with coccidioidal serologic testing performed between 1999 and 2003. Of 706 EIAs, 37 (5%) produced test results for 28 patients that showed isolated IgM positivity. Among these 28 patients, 24 (86%) had positive serologic findings by other methods (complement fixation or immunodiffusion or both), and 7 (25%) had positive microbiologic or histopathologic findings. All 4 (14%) patients without other positive serologic results had diagnostic tests with positive microbiologic or histopathologic results. No false-positive IgM assays were observed. We conclude that the false-positive rate of the EIA IgM is low, and that an isolated positive EIA IgM should prompt further follow-up and diagnostic testing.

Coccidioidomycosis in healthy persons evaluated for liver or kidney donation.

Coccidioidomycosis is a fungal infection caused by Coccidioides species endemic to the southwestern United States, where it poses unique challenges for transplant recipients. Donor-derived coccidioidomycosis has been documented, but its risk of transmission is not known. We prospectively screened 568 healthy persons requesting evaluation for possible liver or kidney donation. Twelve (2.1%) of the 568 donor candidates were seropositive (11 initially and 1 with seroconversion and symptomatic illness within 1 week after negative screening). Three of these 12 patients proceeded to kidney donation, and a fourth patient proceeded to liver donation. None of the 4 transplant recipients received special coccidioidal prophylaxis, although all were administered fluconazole according to standard antifungal prophylaxis protocols. At follow-up (7-54 months), no coccidioidomycosis was identified in any recipient. The prevalence of coccidioidal antibodies was low among potential organ donor candidates, but the risk of donor-derived coccidioidomycosis remains unknown and further study is warranted.

Comparison of Coccidioides immitis serological antibody titres between forms of clinical coccidioidomycosis in horses.

A retrospective study was performed to determine if there is an association between serological Coccidioides immitis antibody titres (IgG) and form/severity of coccidioidal disease in horses, and to identify trends in survival and treatment success based on the form of the disease. Data were obtained on horses with positive serological titres tested at the Coccidioidomycosis Serology Laboratory, School of Medicine, University of California, Davis from 1981 to 2004. Thirty-nine cases in which a diagnosis of coccidioidomycosis had been made were selected for inclusion. Six distinct categories were identified including abortion (n=6), miliary/interstitial pneumonia (n=6), pneumonia with thoracic effusion (pleural or pericardial) (n=11), disseminated (n=10), osteomyelitis (n=3) and external abscessation (n=3) both without pulmonary disease. Statistical differences in titre distribution were found between the abortion category and the pulmonary category (P=0.003), the abortion category and pneumonia with thoracic effusion (P=0.001), the abortion category and disseminated disease (P=0.001), and the pulmonary form and pneumonia with effusion (P=0.001). The other categories had overlapping titre results. Higher serological antibody titres seemed to be associated with a poorer prognosis for survival. Categories with the highest titres, disseminated (geometric mean titre=104) and pneumonia with thoracic effusion (geometric mean titre=226), were overwhelmingly fatal (19/21 known deaths) due to severe clinical disease. The categories with lower titres, abortion (geometric mean titre=4), bone involvement only (geometric mean titre=13) and cutaneous (geometric mean titre=5), had a better survival rate (10/12 known survivors) and less severe clinical disease. Measurement of serological titre may be a useful diagnostic aid in establishing form and severity of disease and thus inform prognosis.

Coccidioidomycosis in patients with diabetes mellitus.

PURPOSE: The study reviewed the interrelationships of diabetes mellitus and coccidioidomycosis. SUBJECTS AND METHODS: We conducted a retrospective review of the medical records of immunocompetent patients with coccidioidomycosis who were treated at our academic medical institution between January 1, 1999, and October 31, 2003, to compare those with and without diabetes mellitus and to determine whether glycemia correlates with the course of illness. RESULTS: Of 329 immunocompetent patients with coccidioidomycosis, 44 had diabetes (4 type 1 and 40 type 2) and were divided into 2 groups: those with serum glucose concentrations of less than 12.2 mmol/L (220 mg/dL) and those with glucose concentrations of greater than or equal to 12.2 mmol/L (220 mg/dL). Persons with diabetes in either glucose group were more likely than those without diabetes to have cavitary lung disease (relative risk, 2.94; P<.001) and relapsed infection. However, only the diabetes group with serum glucose concentrations greater than or equal to 12.2 mmol/L (220 mg/dL) were more likely to have disseminated infection (relative risk, 2.8; P=.05) and to require treatment (relative risk, 9.85; P=.005), but their infection was less likely to resolve (relative risk, 0.24; P=.002). CONCLUSION: Because glycemia strongly correlated with clinical characteristics of coccidioidomycosis in this cohort, we recommend routine measurement of serum glucose in persons with coccidioidomycosis to identify patients with an increased risk of complicated infection. Future studies should evaluate the efficacy of tight glycemic control on the outcome of coccidioidal infection.

Disseminated coccidioidomycosis in a liver transplant recipient with negative serology: use of polymerase chain reaction.

Coccidioidomycosis has been previously described in recipients of solid organ transplantation, especially in patients who have lived in or have visited areas endemic for Coccidioides spp. We present a case of coccidioidomycosis in a liver transplant recipient with several unique aspects, including negative serology and positive polymerase chain reaction results.

Preliminary evaluation of whole-blood gamma interferon release for clinical assessment of cellular immunity in patients with active coccidioidomycosis.

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-gamma) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-gamma release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-gamma at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-gamma concentration was <5 IU/ml in all subjects with a clinical severity score of > or =6 (P = 0.02). The release IFN-gamma concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-gamma release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.

Abdominal/pelvic Coccidioidomycosis.

BACKGROUND: Coccidioidomycosis is the second most common endemic fungal infection in the southwestern United States. Rarely, this fungal infection exhibits symptoms suggestive of peritoneal malignancy, such as ascites and abdominal pelvic masses. CASE: We present a case involving a 51-year-old woman who presented with abdominal pain, ascites, and elevated serum CA-125 levels in 1995. She underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Final pathology revealed Coccidioidomycosis. Following prolonged treatment with fluconazole, her fungal symptoms resolved completely. CONCLUSION: Patients with Coccidioidomycosis have a good prognosis if they are optimally diagnosed and treated. Ascites and elevated serum CA-125 levels associated with Coccidioidomycosis are not documented in the literature. Although extremely rare, abdominal Coccidioidomycosis could be considered in the differential diagnosis in patients who present with ascites or elevated serum CA-125 levels.