Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.

Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp.

Valley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii, which grow as mycelia in the environment and as spherules within the lungs of vulnerable hosts. Current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time to diagnosis, and therefore, new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study, we characterized the volatile metabolomes (or volatilomes) of in vitro cultures of Coccidioides Using solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS), we characterized the volatile organic compounds (VOCs) produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least 2-fold more abundant in a Coccidioides culture than in medium controls and found that the volatile metabolome of Coccidioides is more dependent on the growth phase (spherules versus mycelia) than on the species. The volatile profiles of C. immitis and C. posadasii have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species.IMPORTANCE Coccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in highly populated areas of the U.S. desert southwest where the disease is endemic. The infection is difficult to diagnose by standard serological and histopathological methods, which delays appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes (or volatilomes) of six strains each of Coccidioides immitis and C. posadasii, the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth of the fungus-mycelia and spherules-we observed that the life cycle plays a significant role in the volatiles produced by Coccidioides In contrast, we observed no significant differences in the C. immitis versus C. posadasii volatilomes. These data suggest that life cycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test.

Using soil survey data to model potential Coccidioides soil habitat and inform Valley fever epidemiology.

Coccidioidomycosis, also known as Valley fever, is a disease that can result in substantial illness and death. It is most common in the southwestern United States and areas of Latin America with arid climates, though reports increasingly suggest its range is wider than previously recognized. The natural habitat of the causative organisms, Coccidioides spp., have been associated with certain soil properties and climatic conditions. Current understanding of its geographic range is primarily defined by skin test studies and outbreak locations. We developed a fuzzy system model to predict suitable soil habitats for Coccidioides across the western United States based on parameters (electrical conductivity, organic matter content, pH, water holding capacity, temperature, and precipitation) from sites where soil sampling has confirmed the presence of Coccidioides. The model identified high coccidioidomycosis incidence areas as having high suitability and identified pockets of elevated suitability corresponding with outbreak locations outside the traditional range. By providing high-resolution estimates of Coccidioides suitability, including areas without public health surveillance for coccidioidomycosis, this model may be able to aid public health and clinical provider decision making. Awareness of possible Coccidioides soil habitats could help mitigate risk during soil-disturbing activities and help providers improve coccidioidomycosis diagnosis and treatment.

Environmental factors affecting ecological niche of Coccidioides species and spatial dynamics of valley fever in the United States.

Coccidioidomycosis is an understudied infectious disease acquired by inhaling fungal spores of Coccidioides species. While historically connected to the southwestern United States, the endemic region for this disease is not well defined. This study's objective was to estimate the impact of climate, soil, elevation and land cover on the Coccidioides species' ecological niche. This research used maximum entropy ecological niche modeling based on disease case data from 2015 to 2016. Results found mean temperature of the driest quarter, and barren, shrub, and cultivated land covers influential in characterizing the niche. In addition to hotspots in central California and Arizona, the Columbia Plateau ecoregion of Washington and Oregon showed more favorable conditions for fungus presence than surrounding areas. The identification of influential spatial drivers will assist in future modeling efforts, and the potential distribution map generated may aid public health officials in watching for potential hotspots, assessing vulnerability, and refining endemicity.

Population Structure and Genetic Diversity among Isolates of Coccidioides posadasii in Venezuela and Surrounding Regions.

Coccidioides posadasii is a pathogenic fungus that causes coccidioidomycosis in many arid regions of the Americas. One of these regions is bordered by the Caribbean Sea, and the surrounding landscape may play an important role in the dispersion of C. posadasii across South America through southeastern Mexico, Honduras, Guatemala, and Venezuela. Comparative phylogenomic analyses of C. posadasii reveal that clinical strains from Venezuela are genetically distinct from the North American populations found in (i) Arizona and (ii) Texas, Mexico, and the rest of South America (TX/MX/SA). We find evidence for admixture between the Venezuela and the North American populations of C. posadasii in Central America. Additionally, the proportion of Venezuelan alleles in the admixed population decreases as latitude (and distance from Venezuela) increases. Our results indicate that the population in Venezuela may have been subjected to a recent bottleneck and shows a strong population structure. This analysis provides insight into potential for Coccidioides spp. to invade new regions.IMPORTANCE Valley Fever is a fungal disease caused by two species of fungi: Coccidioides immitis and C. posadasii These fungi are found throughout the arid regions of North and South America; however, our understanding of genetic diversity and disease in South America is limited. In this report, we analyze 10 new genomes of Coccidioides posadasii from regions bordering the Caribbean Sea. We show that these populations are distinct and that isolates from Venezuela are likely a result of a recent bottleneck. These data point to patterns that might be observed when investigating recently established populations.

Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples.

At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and sensitivity in fungal DNA-spiked blood and sputum samples, and compare it with previously described molecular markers. Specific amplified fragment length polymorphism (AFLP) amplicons for Coccidioides immitis and Coccidioides posadasii were cloned into the vector pGEM® -T Easy vector and sequenced to develop a SCAR marker. Oligonucleotides were designed to identify Coccidioides spp. by polymerase chain reaction (PCR), and the specificity and sensitivity of these oligonucleotides were tested with the DNA from related pathogens. The specificity and sensitivity of the SCAR marker was evaluated with blood and sputum samples spiked with Coccidioides DNA and compared with other previously described markers (621, GAC2, and Ag2/PRA). In addition, the conditions for its use were established using biological samples. A specific marker named SCAR300 was obtained to identify Coccidioides spp. that exhibited good sensitivity and specificity. The results showed that all of the markers tested in this study can identify Coccidioides spp. However, the SCAR300 and 621 markers were the most sensitive, whereas the SCAR300 marker was the most specific. Thus, the SCAR300 marker is a useful tool to identify Coccidioides spp.

Utility of Whole-Genome Sequencing to Ascertain Locally Acquired Cases of Coccidioidomycosis, Washington, USA.

Coccidioidomycosis is an emerging fungal infection in Washington, USA, and the epidemiology of the disease in this state is poorly understood. We used whole-genome sequencing to differentiate locally acquired cases in Washington on the basis of the previously identified phylogeographic population structure of Coccidioides spp. Clinical isolates from coccidioidomycosis cases involving possible Washington soil exposure were included. Of 17 human infections with epidemiologic evidence of possible local acquisition, 4 were likely locally acquired infections and 13 were likely acquired outside Washington. Isolates from locally acquired cases clustered within the previously established Washington clade of C. immitis. Genetic differences among these strains suggest multiple environmental reservoirs of C. immitis in the state.

Diagnosis of coccidioidomycosis in a non-endemic area: Inference of the probable geographic area of an infection.

BACKGROUND: Coccidioidomycosis is one of the most important endemic mycoses in Northern Mexico. However, diagnosing this disease can be challenging, particularly in patients who do not reside in endemic areas. CASE REPORT: The case of a Mexican HIV+ patient who developed fever, general malaise, a severe cough, and dyspnea during a stay in Acapulco, Guerrero, Mexico, is presented. Since various diseases are endemic to the state of Guerrero, the doctors originally suspected that the patient had contracted influenza A (H1N1), Q fever, or tuberculosis. All the diagnostic tests for those diseases were negative. The patient had received numerous mosquito bites while staying in Acapulco, and a nodule had appeared on his right cheek. Therefore, malaria, cryptococcosis, and histoplasmosis were also suspected, but those infections were also ruled out through diagnostic tests. A direct microscopic examination was performed using KOH on a sample taken from the cheek nodule. The observation of spherules suggested the presence of a species of Coccidioides. The fungus was isolated, and its identity was confirmed by phenotypic and molecular methods. The geographic area in which the infection was likely acquired was identified by random amplified polymorphic DNA (RAPD) analysis. The results suggested a probable endogenous reactivation. CONCLUSIONS: This clinical case illustrates the difficulties associated with diagnosing coccidioidomycosis in non-endemic areas.

Use of Population Genetics to Assess the Ecology, Evolution, and Population Structure of Coccidioides.

During the past 20 years, a general picture of the genetic diversity and population structure of Coccidioides, the causal agent of coccidioidomycosis (Valley fever), has emerged. The genus consists of 2 genetically diverse species, C. immitis and C. posadasii, each of which contains 1 or more distinct populations with limited gene flow. Genotypic data indicate that C. immitis is divided into 2 subpopulations (central and southern California populations) and C. posadasii is divided into 3 subpopulations (Arizona, Mexico, and Texas/South America populations). However, admixture within and among these populations and the current paucity of environmental isolates limit our understanding of the population genetics of Coccidioides. We assessed population structure of Coccidioides in Arizona by analyzing 495 clinical and environmental isolates. Our findings confirm the population structure as previously described and indicate a finer scale population structure in Arizona. Environmental isolates appear to have higher genetic diversity than isolates from human patients.

Molecular detection of airborne Coccidioides in Tucson, Arizona.

Environmental surveillance of the soil-dwelling fungus Coccidioides is essential for the prevention of Valley fever, a disease primarily caused by inhalation of the arthroconidia. Methods for collecting and detecting Coccidioides in soil samples are currently in use by several laboratories; however, a method utilizing current air sampling technologies has not been formally demonstrated for the capture of airborne arthroconidia. In this study, we collected air/dust samples at two sites (Site A and Site B) in the endemic region of Tucson, Arizona, and tested a variety of air samplers and membrane matrices. We then employed a single-tube nested qPCR assay for molecular detection. At both sites, numerous soil samples (n = 10 at Site A and n = 24 at Site B) were collected and Coccidioides was detected in two samples (20%) at Site A and in eight samples (33%) at Site B. Of the 25 air/dust samples collected at both sites using five different air sampling methods, we detected Coccidioides in three samples from site B. All three samples were collected using a high-volume sampler with glass-fiber filters. In this report, we describe these methods and propose the use of these air sampling and molecular detection strategies for environmental surveillance of Coccidioides.

Local Population Structure and Patterns of Western Hemisphere Dispersal for Coccidioides spp., the Fungal Cause of Valley Fever.

UNLABELLED: Coccidioidomycosis (or valley fever) is a fungal disease with high morbidity and mortality that affects tens of thousands of people each year. This infection is caused by two sibling species, Coccidioides immitis and C. posadasii, which are endemic to specific arid locales throughout the Western Hemisphere, particularly the desert southwest of the United States. Recent epidemiological and population genetic data suggest that the geographic range of coccidioidomycosis is expanding, as new endemic clusters have been identified in the state of Washington, well outside the established endemic range. The genetic mechanisms and epidemiological consequences of this expansion are unknown and require better understanding of the population structure and evolutionary history of these pathogens. Here we performed multiple phylogenetic inference and population genomics analyses of 68 new and 18 previously published genomes. The results provide evidence of substantial population structure in C. posadasii and demonstrate the presence of distinct geographic clades in central and southern Arizona as well as dispersed populations in Texas, Mexico, South America, and Central America. Although a smaller number of C. immitis strains were included in the analyses, some evidence of phylogeographic structure was also detected in this species, which has been historically limited to California and Baja, Mexico. Bayesian analyses indicated that C. posadasii is the more ancient of the two species and that Arizona contains the most diverse subpopulations. We propose a southern Arizona-northern Mexico origin for C. posadasii and describe a pathway for dispersal and distribution out of this region. IMPORTANCE: Coccidioidomycosis, or valley fever, is caused by the pathogenic fungi Coccidioides posadasii and C. immitis The fungal species and disease are primarily found in the American desert southwest, with spotted distribution throughout the Western Hemisphere. Initial molecular studies suggested a likely anthropogenic movement of C. posadasii from North America to South America. Here we comparatively analyze eighty-six genomes of the two Coccidioides species and establish local and species-wide population structures to not only clarify the earlier dispersal hypothesis but also provide evidence of likely ancestral populations and patterns of dispersal for the known subpopulations of C. posadasii.

Primary Cutaneous Coccidioidomycosis in an Italian Nun Working in South America and Review of Published Literature.

Coccidioidomycosis is a systemic disease caused by the dimorphic fungus Coccidioides, endemic in parts of the Southwestern USA and Central and South America. Two species, Coccidioides immitis and Coccidioides posadasii, were differentiated. Primary cutaneous coccidioidomycosis (PCC) has been reported rarely. An unusual case of PCC characterized by a persistent solitary lesion diagnosed in Italy in an immunocompetent Italian nun living in Argentina is described. The isolate was identified by sequence analysis as C. posadasii. Antibody screening was negative. A total of 39 cases of PCC have been reported in the literature. Infections occurred as a consequence of traumatic implantation in a natural setting in endemic areas or of accidental inoculation in laboratory workers. Importance of accurate investigation of travel history and of occupational hazards to laboratory workers is outlined.

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.

Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection.

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

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.

Coccidioides immitis identified in soil outside of its known range - Washington, 2013.

Coccidioidomycosis ("valley fever") is caused by inhaling spores of the soil-dwelling fungi Coccidioides immitis or Coccidioides posadasii. Most infections are subclinical. When clinical manifestations do occur (typically 1-4 weeks after exposure), they are similar to those associated with influenza or community-acquired pneumonia. Disseminated disease is rare. Residual pulmonary nodules can lead to chronic lung disease. Fluconazole or other triazoles often are used for treatment, but mild cases often resolve without specific therapy. A total of 17,802 cases were reported in the United States in 2012.

Genetic analysis of the endemic fungal pathogens Coccidioides posadasii and Coccidioides immitis in Mexico.

Coccidioidomycosis (CM) is a mycotic disease that affects mammals, including humans. Official data relative to CM in Mexico has not been collected since 1995, thus its prevalence remains unknown. The objectives of this study were to identify the predominant Coccidioides species in Mexico, infer their current geographical distribution and explore the correlation between species and clinical presentation. We collected 154 strains, which were cultured, inactivated, and processed for DNA extraction. Nine microsatellite loci, the Ag2/PRA gene and Umeyama Region were amplified from each isolate. To infer the current geographical distribution of Coccidioides spp. and to establish a correlation between genotype and clinical presentation, we evaluated genetic population structure under the following grouping criteria: putative origin and clinical presentation records. Microsatellite analysis showed that 82% of the isolates corresponded to C. posadasii and 18% were C. immitis. The species identification results obtained using Umeyama region, Ag2/PRA, and microsatellites of five of the isolates were inconsistent with the data collected for the remaining isolates. C. posadasii strains were found primarily in the northeastern region and C. immitis in the northwestern region. However, there was no relationship between clinical presentation and Coccidioides species. The molecular markers used in this study proved to have a high power of resolution to identify the Coccidioides species recovered in culture. While we found C. posadasii to be the most abundant species in Mexico, more detailed clinical records are needed in order to obtain more accurate information about the infections in specific geographical locations.

Molecular markers in the epidemiology and diagnosis of coccidioidomycosis.

The prevalence of coccidioidomycosis in endemic areas has been observed to increase daily. To understand the causes of the spread of the disease and design strategies for fungal detection in clinical and environmental samples, scientists have resorted to molecular tools that allow fungal detection in a natural environment, reliable identification in clinical cases and the study of biological characteristics, such as reproductive and genetic structure, demographic history and diversification. We conducted a review of the most important molecular markers in the epidemiology of Coccidioides spp. and the diagnosis of coccidioidomycosis. A literature search was performed for scientific publications concerning the application of molecular tools for the epidemiology and diagnosis of coccidioidomycosis. The use of molecular markers in the epidemiological study and diagnosis of coccidioidomycosis has allowed for the typing of Coccidioides spp. isolates, improved understanding of their mode of reproduction, genetic variation and speciation and resulted in the development specific, rapid and sensitive strategies for detecting the fungus in environmental and clinical samples. Molecular markers have revealed genetic variability in Coccidioides spp. This finding influences changes in the epidemiology of coccidioidomycosis, such as the emergence of more virulent or antifungal resistant genotypes. Furthermore, the molecular markers currently used to identify Coccidioides immitis and Coccidioides posadasii are specific and sensitive. However, they must be validated to determine their application in diagnosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Coccidioidomycosis masquerading as skeletal tuberculosis: an imported case and review of coccidioidomycosis in India.

We describe a possible imported case of osteo-articular coccidioidomycosis in India. Culture of the computed tomography-guided aspirate revealed the growth of Coccidioides spp., which was identified as Coccidioides posadasii by sequencing of the internal transcribed spacer (ITS) region of rDNA. He was successfully treated with amphotericin B followed by itraconazole. All the previous published reports of coccidioidomycosis cases diagnosed in India are also reviewed in order to increase the awareness of this disease in non-endemic areas.

Coccidioidomycosis.

Coccidioidomycosis is a common, environmentally acquired, pulmonary fungal infection in arid and semi-arid regions of the West, especially Arizona and California. The infection is frequently associated with striking cutaneous manifestations. Reactive, immunologically mediated eruptions include erythema nodosum, a generalized exanthem, Sweet syndrome, and reactive granulomatous dermatitis. Less commonly, the skin can harbor the actual organisms as a result of dissemination from the lungs. Dermatologists may play a key role in the recognition of coccidioidomycosis