Talaromyces marneffei, Coccidioides species, and Paracoccidioides species-a systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal pathogen priority list. This systematic review aimed to evaluate the epidemiology and impact of infections caused by Talaromyces marneffei, Coccidioides species, and Paracoccidioides species. PubMed and Web of Sciences databases were searched to identify studies published between 1 January 2011 and 23 February 2021 reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 25, 17, and 6 articles were included for T. marneffei, Coccidioides spp. and Paracoccidioides spp., respectively. Mortality rates were high in those with invasive talaromycosis and paracoccidioidomycosis (up to 21% and 22.7%, respectively). Hospitalization was frequent in those with coccidioidomycosis (up to 84%), and while the duration was short (mean/median 3-7 days), readmission was common (38%). Reduced susceptibility to fluconazole and echinocandins was observed for T. marneffei and Coccidioides spp., whereas >88% of T. marneffei isolates had minimum inhibitory concentration values ≤0.015 µg/ml for itraconazole, posaconazole, and voriconazole. Risk factors for mortality in those with talaromycosis included low CD4 counts (odds ratio 2.90 when CD4 count <200 cells/µl compared with 24.26 when CD4 count <50 cells/µl). Outbreaks of coccidioidomycosis and paracoccidioidomycosis were associated with construction work (relative risk 4.4-210.6 and 5.7-times increase, respectively). In the United States of America, cases of coccidioidomycosis increased between 2014 and 2017 (from 8232 to 14 364/year). National and global surveillance as well as more detailed studies to better define sequelae, risk factors, outcomes, global distribution, and trends are required.

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.