Comparative Study of Newer and Established Methods of Diagnosing Coccidioidal Meningitis.

Meningitis is the most devastating form of coccidioidomycosis. A convenient, rapid diagnostic method could result in early treatment and avoid many meningitis complications. We studied cerebrospinal fluid (CSF) samples in patients with documented coccidioidal meningitis, and controls, with complement fixation (CF), immunodiffusion (ID) (the "classical" assays), lateral flow assays (LFA; one-strip and two-strip), and two enzyme immunoassays (EIA). The two-strip LFA and EIAs not only enabled separate testing for IgG and IgM antibodies separately, but also could aggregate results for each method. CF with ID or the aggregate use of IgG and IgM tests were considered optimal test uses. LFAs and EIAs were evaluated at 1:21 and 1:441 dilutions of specimens. All assays were compared to true patient status. With 49 patient specimens and 40 controls, this is the largest comparative study of CSF coccidioidal diagnostics. Sensitivity of these tests ranged from 71-95% and specificity 90-100%. IgM assays were less sensitive. Assays at 1:441 were similarly specific but less sensitive, suggesting that serial dilutions of samples could result in assays yielding titers. Agreement of positive results on cases was 87-100%. When kits are available, hospital laboratories in endemic areas can perform testing. LFA assays do not require a laboratory, are simple to use, and give rapid results, potentially even at the bedside.

Coccidioidomycosis Complement Fixation Titer Trends in the Age of Antifungals.

Coccidioidomycosis is associated with a broad spectrum of illness severity, ranging from asymptomatic or self-limited pulmonary infection to life-threatening manifestations of disseminated disease. Serologic studies before the widespread availability of antifungals established current understanding of serologic kinetics and dynamics. Chart histories and complement fixation (CF) titer trends were analyzed for 434 antifungal-treated coccidioidomycosis patients, who were classified by three infectious disease physicians as having either pulmonary uncomplicated coccidioidomycosis (PUC) (n = 248), pulmonary chronic coccidioidomycosis (PCC) (n = 64), disseminated coccidioidomycosis (DC) not including meningitis (n = 86), or coccidioidal meningitis (CM) (n = 36). The median maximal CF titers were 1:4 for PUC patients, 1:24 for PCC patients, 1:128 for DC patients, and 1:32 for CM patients. Approximately 25.4% of PUC patients, 6.2% of PCC patients, 2.3% of DC patients, and 8.3% of CM patients did not develop detectable titers during the study period. Maximal titers developed a mean of 31 days (95% confidence interval [CI], 13 to 50 days) after initial serologic positivity, with no significant differences between groups. Serologic recurrence occurred in 9% of PUC patients, 36% of PCC patients, 50% of DC patients, and 52% of CM patients. Median titer improvement rates were 91 days/dilution for PUC patients, 112 days/dilution for PCC patients, 136 days/dilution for DC patients, and 146 days/dilution for CM patients. Receiver operating characteristic (ROC) analysis revealed that CF testing retains moderate classification value for disseminated infections (area under the curve [AUC], 0.82 [95% CI, 0.78 to 0.87]) and complicated infections (AUC, 0.82 [95% CI, 0.77 to 0.86]). A suitable cutoff value for complicated infections is ≥1:32. Findings update serologic parameters that are relevant for clinical assessment of coccidioidomycosis patients in the triazole era.

Ex Vivo Cytokine Release, Determined by a Multiplex Cytokine Assay, in Response to Coccidioidal Antigen Stimulation of Whole Blood among Subjects with Recently Diagnosed Primary Pulmonary Coccidioidomycosis.

The elements of the cellular immune response in human coccidioidomycosis remain undefined. We examined the ex vivo release of an array of inflammatory proteins in response to incubation with a coccidioidal antigen preparation to ascertain which of these might be associated with diagnosis and outcome. Patients with a recent diagnosis of primary pulmonary coccidioidomycosis and a control group of healthy subjects were studied. Blood samples were incubated for 18 h with T27K, a soluble coccidioidal preparation containing multiple glycosylated antigens, and the supernatant was assayed for inflammatory proteins using the multiplex Luminex system. The presentation and course of illness were compared to the levels of the inflammatory proteins. Among the 31 subjects studied, the median time from diagnosis to assay was 15 days. Of the 30 inflammatory proteins measured, the levels of only 7 proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor alpha (IL-1RA), interleukin-1ß (IL-1ß), interferon gamma (IFN-γ), IL-2, IL-13, and tumor necrosis factor alpha (TNF-α), were more than 10-fold above the levels seen without antigen stimulation. The levels of IFN-γ and IL-2 were significantly elevated in those subjects not receiving triazole antifungal therapy compared to those who were receiving triazole antifungal therapy. While the levels of IL-1RA were nonspecifically elevated, elevated levels of IL-13 were seen only in those with active pulmonary coccidioidomycosis. Only six cytokines were specifically increased in subjects with recently diagnosed primary pulmonary coccidioidomycosis. While IFN-γ, IL-2, and TNF-α have been previously noted, the finding of elevated levels of the innate cytokines GM-CSF and IL-1ß could suggest that these, as well as IL-13, are early and specific markers for pulmonary coccidioidomycosis.IMPORTANCE Coccidioidomycosis, commonly known as Valley fever, is a common pneumonia in the southwestern United States. In this paper, we examined the release of 30 inflammatory proteins in whole-blood samples obtained from persons with coccidioidal pneumonia after the blood samples were incubated with a preparation made from the causative fungus, Coccidioides We found that six of these proteins, all cytokines, were specifically released in high concentrations in these patients. Three of the cytokines were seen very early in disease, and an assay for all six might serve as a marker for the early diagnosis of Valley fever.

Cerebrospinal Fluid (1,3)-Beta-d-Glucan Testing Is Useful in Diagnosis of Coccidioidal Meningitis.

Diagnosing coccidioidal meningitis (CM) can be problematic owing to its infrequency and/or a delay in the positivity of a cerebrospinal fluid (CSF) culture or CSF antibody, particularly if the primary coccidioidal infection is unrecognized. We tested 37 CSF specimens, 26 from patients with confirmed CM and 11 from patients with suspected microbial meningitis without fungal diagnosis, for (1,3)-beta-glucan (BG). BG in CM CSF specimens ranged from 18 to 3,300 pg/ml and in controls ranged from <3.9 to 103 pg/ml. Diagnostic performance was determined using a 31-pg/ml cutoff (the bottom of the serum range according to the directions for the commercial kit, although further serial dilutions of the standard indicated linearity to 3.9). Sensitivity was 96%, specificity was 82%, positive and negative predictive values were 93% and 90%, and the area under the receiver operating characteristic curve was 0.937. Fifteen of 15 samples of >103 pg/ml were CM. The one false-negative specimen was from a patient with a pseudosyrinx, without inflammatory evidence of meningitis activity. Serial samples from some patients were positive at ≤8 years, indicating no loss of positivity with chronicity. Samples stored frozen since 2000 included those with 2 of the 3 highest values, indicating that fresh samples not required. A previous study indicated serum sensitivities of 53% in acute, 50% in resolved, and 83% in disseminated and meningeal coccidioidomycosis. Three studies of other fungal meningitides ranged from 86 to 1,524 pg/ml CSF, with 37 controls of <4 to 115 pg/ml CSF. CSF BG analysis had good diagnostic performance in CM. CSF BG testing can be useful in CM, and a commercial kit is available. It will be of interest to correlate this with course, treatment, outcome, inflammation, and antigen. The only mycoses with common central nervous system (CNS) involvement are cryptococcal and coccidioidal, so CSF BG screening can be useful in meningitis diagnosis.

Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis.

The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.

Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

Coccidioidomycosis and other systemic mycoses of marine mammals stranding along the central California, USA coast: 1998-2012.

A wide range of systemic mycoses have been reported from captive and wild marine mammals from North America. Examples include regionally endemic pathogens such as Coccidioides and Blastomyces spp., and novel pathogens like Cryptococcus gattii, which appear may have been introduced to North America by humans. Stranding and necropsy data were analyzed from three marine mammal stranding and response facilities on the central California coast to assess the prevalence, host demographics, and lesion distribution of systemic mycoses affecting locally endemic marine mammals. Between 1 January 1998 and 30 June 2012, >7,000 stranded marine mammals were necropsied at the three facilities. Necropsy and histopathology records were reviewed to identify cases of locally invasive or systemic mycoses and determine the nature and distribution of fungal lesions. Forty-one animals (0.6%) exhibited cytological, culture- or histologically confirmed locally invasive or systemic mycoses: 36 had coccidioidomycosis, two had zygomycosis, two had cryptococcosis, and one was systemically infected with Scedosporium apiospermum (an Ascomycota). Infected animals included 18 California sea lions (Zalophus californianus), 20 southern sea otters (Enhydra lutris nereis), two Pacific harbor seals (Phoca vitulina richardsi), one Dall's porpoise (Phocoenoides dalli), and one northern elephant seal (Mirounga angustirostris). Coccidioidomycosis was reported from 15 sea lions, 20 sea otters, and one harbor seal, confirming that Coccidioides spp. is the most common pathogen causing systemic mycosis in marine mammals stranding along the central California coast. We also report the first confirmation of C. gattii infection in a wild marine mammal from California and the first report of coccidioidomycosis in a wild harbor seal. Awareness of these pathogenic fungi during clinical care and postmortem examination is an important part of marine mammal population health surveillance and human health protection. Temporal-spatial overlap may be observed for pathogenic mycoses infecting coastal marine mammals and adjacent human populations.

Coccidioides species determination: does sequence analysis agree with restriction fragment length polymorphism?

Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment length polymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

Determination of ribosomal DNA copy number and comparison among strains of Coccidioides.

The use of PCR-based assays to detect fungi and diagnose fungal infections as well as to monitor fungal organ burden with diseases such as coccidioidomycosis is becoming more common. The target of these assays is frequently one or more of the ribosomal DNA (rDNA) gene subunits. The multicopy nature of this gene affords greater sensitivity over single-copy genes. However, there are few studies reporting the precise number of copies of the rDNA gene per genome in pathogenic fungi. Quantitative PCR was used to determine the number of copies of rDNA as well as CTS1, a single-copy gene, in samples of Coccidioides genomic DNA by the absolute quantification method. Variability of rDNA genome copy number was determined using 13 different Coccidioides isolates and was found to vary between 20 and 146 copies per genome. This suggests that detection of rDNA will likely afford an increased sensitivity of at least 20-fold over single-copy genes. However, estimation of the number of organisms present by quantification of the rDNA cannot be made prior to knowledge of each isolate's rDNA copy number because of the strain variation.

Identification, molecular characterization, and expression analysis of a DOMON-like type 9 carbohydrate-binding module domain-containing protein of Coccidioides posadasii.

Previously, we investigated the effect of N-acetylglucosamine (GlcNAc) on Coccidioides posadasii chitinolytic enzymes during in vitro spherule-endospore (S/E) phase culture. During those studies, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of supernatants from S/E phase cultures grown in Converse medium with or without added GlcNAc revealed a ∼ 28-kDa band (CFP28), whose abundance was increased by GlcNAc in parallel with the chitinolytic enzymes. Mass spectrometry (MS) of the CFP28 band revealed peptides that matched an open reading frame found in the tentative consensus sequence, TC20325, retrieved from the Dana Farber Cancer Institute C. posadasii Gene Index Database. The TC20325 cDNA sequence was used to design internal primers based on MS peptides and a full-length cDNA was isolated using a combination of rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. The deduced amino acid sequence of the full-length cDNA consists of 231 amino acid residues with a 19 aa signal peptide. The mature protein has a calculated molecular mass of ∼ 24.5 kDa, a theoretical pI of 6.09, and consists of a single DOMON-like type 9 carbohydrate-binding module (CBM9-like-3) conserved domain. The protein shares the highest sequence similarity (≥57%) to hypothetical proteins from fungi within the Pezizomycotina subphylum of Ascomycota. Antiserum against a recombinant version of CFP28 recognized native CFP28 in S/E phase cells and culture supernatants. CFP28 mRNA and protein expression were detectable in S/E phase in Converse medium, but were increased in the presence of added GlcNAc. Purified native CFP28 reacted with pooled sera from patients with coccidioidomycosis.

Demonstration of Coccidioides immitis and Coccidioides posadasii DNA in soil samples collected from Dinosaur National Monument, Utah.

Soil samples were collected in 2006 from Dinosaur National Monument (DNM), Utah, the site of an outbreak of coccidioidomycosis in 2001. DNA was isolated from two soil samples, and polymerase chain reaction (PCR) amplified Coccidioides DNA present in both samples. Ribosomal RNA genes and internal transcribed spacer (ITS) region PCR products were sequenced. Single-nucleotide polymorphisms indicated that the DNA from sample SS06RH was that of Coccidioides immitis, while the DNA from sample SS06UM was C. posadasii. This is the first report to directly demonstrate Coccidioides in soils from DNM and the first to report the presence of both C. immitis and C. posadasii in the same geographic location.

Detection of coccidioidal antibodies in serum of a small rodent community in Baja California, Mexico.

Coccidioidomycosis (Valley Fever) represents a serious threat to inhabitants of endemic areas of North America. Despite successful clinical isolations of the fungal etiological agent, Coccidioides spp., the screening of environmental samples has had low effectiveness, mainly because of the poor characterization of Coccidioides ecological niche. We explored Valle de las Palmas, Baja California, Mexico, a highly endemic area near the U.S.-Mexico border, where we previously detected Coccidioides via culture-independent molecular methods. By testing the serum from 40-trapped rodents with ELISA, we detected antibodies against Coccidioides in two species: Peromyscus maniculatus and Neotoma lepida. This study comprises the first report of wild rodent serum tested for coccidioidal antibodies, and sets the basis to analyze this pathogen in its natural environment and explore its potential ecological niche.

Immunological characterization of bronchoalveolar lavage fluid in patients with acute pulmonary coccidioidomycosis.

BACKGROUND: The specific cellular immunological characteristics of bronchoalveolar lavage (BAL) fluid in acute pulmonary coccidioidomycosis have not been defined. METHODS: BAL fluid from patients living in a coccidioidomycosis-endemic region of Arizona who were undergoing bronchoscopy because of pulmonary infiltrates was analyzed. Mononuclear cells from BAL fluid and peripheral blood mononuclear cells (PBMCs) were incubated with the coccidioidal antigen T27K in vitro, and cellular immunological assays were performed. RESULTS: Forty-six patients were studied. Twelve received a diagnosis of acute pulmonary coccidioidomycosis, 17 received other diagnoses, and 17 had no diagnosis established. There was an increased proportion of polyfunctional CD8(+) T cells after antigen stimulation from subjects with coccidioidomycosis as compared to those with another diagnosis (P = .025). In cells collected from BAL fluid and in PBMCs, the concentrations of interferon γ, tumor necrosis factor α, and interleukin 17 (IL-17) were all significantly increased in samples from those with acute pulmonary coccidioidomycosis, compared with the other 2 groups (for all, P<.05). CONCLUSIONS: When incubated in vitro with a coccidioidal antigen preparation, cells from both BAL fluid and peripheral blood obtained from patients with pulmonary coccidioidomycosis demonstrated specific cellular immune responses, including expression of IL-17.

Case report: Abortion and disseminated infection by Coccidioides posadasii in an alpaca (Vicugna pacos) fetus in Southern California.

Coccidioidomycosis is a fungal disease affecting humans and other mammals caused by the soil-dwelling fungi Coccidioides immitis and C. posadasii. Abortion due to Coccidioides spp. infection is rare in domestic animals and transplacental transmission is considered uncommon in women. This report describes a case of placental-fetal infection and abortion in an alpaca with disseminated C. posadasii infection. PCR amplification and DNA sequencing were used to confirm the etiology, C. posadasii, in fetal tissues.

Association between serum 25-hydroxyvitamin D level and type of coccidioidal infection.

The clinical manifestations of coccidioidomycosis vary depending upon the extent of exposure and immune status of the host. Recent studies have demonstrated an essential role for vitamin D in both innate and acquired immunity and serum levels strongly correlate with the development of upper respiratory tract infections, including tuberculosis. Despite similar pathophysiologic processes at play in the control of tuberculosis and invasive fungal infections, a possible association of low serum 25(OH) vitamin D levels had not previously been assessed in the latter patient group. Therefore, we performed a case-control study examining serum 25(OH) vitamin D levels in three distinct groups of patients with coccidioidomycosis as compared to healthy uninfected controls. Of the 89 patients included in this study, there were 26 negative controls, 23 who were immune, 22 with primary coccidioidal pneumonia, and 18 who had disseminated/meningeal infection. Serum 25(OH) vitamin D levels varied between groups with lowest levels seen in the group with disseminated/meningeal coccidioidomycosis (P= 0.14). In this evaluation of a diverse group of patients with varying forms of coccidioidomycosis we found no association of vitamin D with the acquisition or resolution of this infection. Vitamin D does not play a significant role in host susceptibility to coccidioidomycosis.

Coccidioidomycosis: adenosine deaminase levels, serologic parameters, culture results, and polymerase chain reaction testing in pleural fluid.

BACKGROUND: In a patient with positive serum serology for coccidioidomycosis, the differential diagnosis of concurrent pleural effusions can be challenging. We, therefore, sought to clarify the performance characteristics of biochemical, serologic, and nucleic-acid-based testing in an attempt to avoid invasive procedures. The utility of adenosine deaminase (ADA), coccidioidal serology, and polymerase chain reaction (PCR) in the evaluation of pleuropulmonary coccidioidomycosis has not been previously reported. METHODS: Forty consecutive patients evaluated for pleuropulmonary coccidioidomycosis were included. Demographic data, pleural fluid values, culture results, and clinical diagnoses were obtained from patient chart review. ADA testing was performed by ARUP Laboratories, coccidioidal serologic testing was performed by the University of California-Davis coccidioidomycosis serology laboratory, and PCR testing was performed by the Translational Genomics Research Institute using a previously published methodology. RESULTS: Fifteen patients were diagnosed with pleuropulmonary coccidioidomycosis by European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria. Pleural fluid ADA concentrations were < 40 IU/L in all patients (range, < 1.0-28.6 IU/L; median, 4.7). The sensitivity and specificity of coccidioidal serologic testing was 100% in this study. The specificity of PCR testing was high (100%), although the overall sensitivity remained low, and was comparable to the experience of others in the clinical use of PCR for coccidioidal diagnostics. CONCLUSION: Contrary to prior speculation, ADA levels in pleuropulmonary coccidioidomycosis were not elevated in this study. The sensitivity and specificity of coccidioidal serologic testing in nonserum samples remained high, but the clinical usefulness of PCR testing in pleural fluid was disappointing and was comparable to pleural fluid culture.

Serum (1->3)-ß-D-glucan measurement in coccidioidomycosis.

The serum (1→3)-ß-d-glucan assay has emerged as an important diagnostic test for invasive fungal disease. The utility of this assay in coccidioidomycosis has not been previously studied. Using a cutoff value of ≥80 pg/ml, we found the sensitivity (43.9%), specificity (91.1%), positive predictive value (81.8%), and negative predictive value (64.1%) to be similar to those of the assay in diagnosing other invasive mycoses.

Fluoride excess in coccidioidomycosis patients receiving long-term antifungal therapy: an assessment of currently available triazoles.

The use of voriconazole, a trifluorinated antifungal, has been associated with the development of fluoride excess and periostitis/exostoses. We evaluated a cohort of patients on long-term triazole therapy and found that other fluorinated triazoles (fluconazole and posaconazole) conferred no risk for the development of hyperfluorosis and its complications in our cohort.

Coccidioidomycosis during pregnancy: a review and recommendations for management.

Pregnancy is an established risk factor for the development of severe and disseminated coccidioidomycosis, particularly when infection is acquired during the later stages of gestation. Although recent studies suggest that the incidence of symptomatic coccidioidomycosis during pregnancy is decreasing and that outcome has improved, management is complicated by the observations that azole antifungal agents can be teratogenic when given to some women, particularly at high doses, early in pregnancy. This article summarizes the data on these issues and offers guidance on the management of coccidioidomycosis during pregnancy.