First Analysis of Human Coccidioides Isolates from New Mexico and the Southwest Four Corners Region: Implications for the Distributions of C. posadasii and C. immitis and Human Groups at Risk

Abstract

Coccidioidomycosis (Valley Fever) is a disease caused by species of Coccidioides. The disease is endemic to arid regions of the Southwestern US and while most common in CA and AZ is also present in NM. We present the first genetic analysis of clinical isolates from NM. Travel and demographic information was available for a number of patients, which included individuals from NM and the Southwestern US Four Corners region. Multi-gene phylogenetic analyses revealed the presence of both C. posadasii and C. immitis. While NM is predicted to be within the endemic range for C. posadasii, our results expand the known range of C. immitis, often considered to be the "California species". Five of eight infections for which patient ethnicity existed occurred in Native Americans, and two occurred in African Americans. Several isolates came from the northwestern part of NM-outside the predicted "highly-endemic" region. Our study suggests Native Americans represent an unrecognized at-risk group, and it provides a foundation for better defining the geographic distribution of the Coccidioides species and for preventing exposure among populations at risk. In the course of this study, we developed a reliable PCR-based method to distinguish species targeting regions of the mitochondrial genome.

Keywords: American Indian; Coccidioides; New Mexico; Valley Fever; coccidioidomycosis.

Figure 1

Four-gene phylogeny (DNAMLK) for clinical Coccidioides isolates from New Mexico and the Four Corners region. The tree was derived from a concatenated alignment of partial sequences from four gene regions: Serine protease, MCM7, RPB1, and ITS; trees for individual genes are shown in Figure S1 (with all four genes agreeing in terms of species separation). Both C. posadasii and C. immitis isolates were present among those obtained from patients in New Mexico (shown in red). As expected, based on previous analyses of isolates from California, Arizona and Texas, most NM isolates were from C. posadasii (C. immitis being known primarily from CA). One patient infected with C. immitis (isolates NM9443 and NM9737) was a resident of the Four Corners region of Utah, while another (isolate NM3006) was from the Four Corners region of NM with no apparent travel history to California. GenBank accession numbers are given in Table 3. Bootstrap values (percentage of 1000 replicates) greater than 60% are shown for maximum likelihood analysis above the branches and for parsimony analysis below the branches. The tree was rooted with Aspergillus steynii. Maximum likelihood and parsimony analyses performed without sequences from A. steynii, and employing midpoint rooting, separated C. immitis and C. posadasii clades with 100% bootstrap support and placed the root between the two species (results not shown). Arrows indicate branches leading to isolates with identical sequences.

Figure 2

An mtDNA region can be employed to distinguish between C. immitis and C. posadasii using PCR. (A) Summary results from a BLASTN search employing the cox1 mtDNA region from C. posadasii strain C735 delta SOWgp (GenBank: ACFW01000039). Cp = hits against C. posadasii sequences, Ci = hits against C. immitis, and Onyg = hits against other Onygenales (Microsporum canis CBS 113480, Trichophyton interdigitale M8436, Trichophyton mentagrophytes TIMM 2789, and Trichophyton interdigitale H6). Note that the gap in the intron of C. immitis relative to C. posadasii represents a large indel. The intergenic region (intg) between the histidine tRNA and cox1 is conserved between the two Coccidioides species but not between the latter and other Onygenales. The sequence corresponding to primer P1F is present in both Coccidioides species but not in other genera for which sequences are currently available in Genbank. The P1R sequence is contained within the first cox1 exon and is conserved in both species. The P2F primer sequence is entirely absent from C. immitis. The P2R primer sequence is wholly contained within the C. posadasii intron but spans the predicted exon–intron border of C. immitis. (B) Primer pair P1F/P1R amplifies a fragment from both C. immitis and C. posadasii (Aspergillus fumigatus, Af, included as negative control). Primer pair P2F/P2R amplifies a fragment from C. posadasii only (although this fragment was amplified in all 15 C. posadasii strains, results are shown for only three strains.