During 2014–2017, a total of 167 confirmed cases of coccidioidomycosis were reported in Washington. For these cases, 18 isolates (Table) from 17 cases were available. Travel and exposure histories suggested local acquisition in Washington (9 case-patients) or were unknown at the time of specimen collection (8 case-patients).

Figure

Figure. Genetic relationships among Coccidioides immitis isolates. A) Isolates from patients in Washington, USA, compared with isolates from other locations. B) Genetic relationships among C. immitis isolates from the Washington clade; single-nucleotide...

We speciated 14 isolates as C. immitis and 4 as C. posadasii and subjected these isolates to WGS with 25–160 times sequencing coverage. A total of 68,717 SNPs were identified by comparison with the reference isolate. Phylogenetic analysis and comparison with sequenced isolates of C. immitis demonstrated that 5 isolates from 4 patients in Washington were highly genetically related to each other and to isolates from the identified Washington clade that contained human and soil isolates (13). No more than 354 SNPs differentiated any 2 isolates within the Washington cluster, which was also supported by 100% bootstrap values (Figure). In contrast, >10,000 SNPs differentiated the Washington cluster from its nearest neighbor, an isolate from another case-patient in Washington. This case-patient (B12496) reported travel history to disease-endemic regions in California and Mexico. Tens of thousands of SNPs differentiated Washington cluster isolates from those from other states. No more than 3 SNPs were identified among multiple soil isolates collected 4 years apart from a single sampling site, and 4 SNPs were identified between isolates collected 2 years apart from 1 patient in Washington who had a chronic infection. However, 234–324 SNPs were identified in pairwise comparisons between isolates from different patients in the Washington clade (Figure, panel B).

Three of the 4 case-patients infected with Washington clade strains reported no travel history outside Washington. Conversely, 8 of 9 C. immitis isolates that did not cluster with the Washington clade were isolated from patients who reported travel to California.

Washington strains can be distinguished from non-Washington strains in 3 genomic regions on the basis of annotations from C. immitis strain H538-4; locus DS016982: adipocyte-derived leucine aminopeptidase; locus DS01702: hypothetical protein; and locus DS016985: glyoxal oxidase (Appendix). The DNA fragment from locus DS016982 contains SNP GG at positions 94460–94461 for Washington strains and SNP CA for non-Washington strains and can be amplified by using forward primer 5′-GGTACGTCACAAGTCCCCAG-3′ and reverse primer 3′-AAGAGTACTCGCGAAGGAAGC-5′. For locus DS017021, the DNA fragment amplified with forward primer 5′-CTTGACTGTGCAGGGCCTTA-3′ and reverse primer 3′-ACCGGCCTAACTCCATGGTA-5′ encompasses SNPs GGT for Washington strains and TGC for non-Washington strains at positions 105566–105568) The fragment of locus DS016985 can be amplified by using primer pairs 5′-TTCCGCTTGATGGCTGAAGT-3′/3′-TGTGGCCCTCCTATTGCTTG-5′ and contains consecutive SNP CC for Washington strains and SNP GA for non-Washington strains (positions 232442–232443).

We identified 4 case-patients who had C. posadasii infections by using internal transcribed spacer sequencing and confirmed by WGS. Isolates from the 4 case-patients clustered with described C. posadasii populations from Arizona (data not shown). Three of those case-patients had traveled to Arizona, and 1 case-patient had an unknown travel history.

Determining the potential sources of exposure and geographic distribution of Coccidioides in areas where coccidioidomycosis is uncommon is a surveillance challenge because patients often report travel histories to other disease-endemic areas. To address this problem, we implemented WGS to better determine the likely geographic origin of isolates obtained from patients in Washington who had suspected local acquisition or unknown exposure histories. This method is based on the observation that Coccidioides populations have well defined geographic structure, and most isolates can be assigned to specific geographic areas on the basis of their WGS genotypes, such as Arizona, Texas, Mexico, central California, or Washington (20). We demonstrated the utility of this tool for investigating the epidemiology of coccidioidomycosis in Washington.

Of 18 isolates from 17 patients included in this study, 5 (28%) clustered with strains isolated from soil in Washington, which was consistent with local exposure. The remaining 13 (72%) isolates clustered with populations from other areas, such as Arizona, California, or Mexico, which was generally consistent with reported travel histories of patients. The only discordance between WGS and epidemiologic data was observed for isolate B11863, which clustered with C. immitis isolates from California. The patient from whom this isolate originated reported travel out of state but also reported extensive soil exposure in a county in Washington that had previously reported autochthonous cases. Specifically, the only reported travel was to Hawaii 2 months before disease onset and to Costa Rica 1 year before disease onset, with a layover in California during which the patient did not leave the airport. In addition, the patient reported 1 trip to southern California 6 years before disease onset. This discordance was an unexpected finding and might represent more diversity among Coccidioides spp. in Washington than previously understood, reactivated infection because of previous travel, or infection from a fomite or other carrier source of C. immitis. Further investigation and surveillance data are needed to determine which hypothesis is most plausible.

Of the 4 patients infected with the local strains from Washington, 3 patients reported no travel history outside Washington. The fourth patient reported travel to Arizona 1 year before the onset of coccidioidomycosis but also reported extensive exposure to local soils in Washington and dust during hiking and gardening. This result indicates that compatible travel history alone does not exclude the possibility of local exposure and demonstrates the added value of genomic approaches to determining exposure location. Although isolates in the Washington clade were genetically related (Figure, panel A), considerably higher genetic diversity was observed for new strains compared with that detected for the original isolates from Washington identified in 2013 (13). Specifically, 245–299 pairwise SNPs were identified among the newly identified strains from Washington, compared with <5 SNPs detected among previously described isolates from Washington from the same exposure site (Figure, panel B). This small but apparent genetic divergence of the new isolates indicates that new infections were acquired from different local C. immitis populations, likely at different sites, and suggests the presence of multiple environmental loci for Coccidioides spp. in Washington. The divergent isolates were collected from soil in a location ≈70 miles from the suspected exposure location of 1 of the patients. However, in sharp contrast to the populations of Coccidioides spp. in Arizona or California, where isolates are differentiated by thousands of SNPs, the overall low genetic variability in the Washington population is consistent with a relatively small population size, strongly suggesting a recent common ancestry of Washington strains and a relatively recent expansion of C. immitis to Washington, (Figure).

Our study has 2 main limitations. First, there might be additional genetically diverse populations of Coccidioides in Washington that have not yet been discovered or proven; therefore, some genotypes that have been deemed as acquired outside Washington might have been acquired locally. For example, 1 isolate from a patient who was highly suspected of having local acquisition clustered with isolates from California. Likewise, there might be genetically similar populations of Coccidioides spp. in Oregon, California, or other nearby states that have not yet been documented. These genetically similar populations might add complexity to determination of location of exposure. However, to date, no clinical or environmental isolates with strong epidemiologic links to locations outside Washington that cluster with isolates from Washington have been identified. This finding is consistent with those of previous studies that demonstrated that C. immitis and C. posadasii have well-defined geographic structures (15–19).

Second, all patients in our study were given diagnoses of rather atypical cases of coccidioidomycosis that were either asymptomatic (diagnosed during biopsy), cutaneous, chronic, or unusually severe. In general, the acute primary pulmonary form of coccidioidomycosis constitutes the most common manifestation in highly endemic areas and often represents the earliest manifestation of the disease. Conversely, reactivation of latent infections can occur months to years after initial exposure, typically in immunosuppressed persons. Many complicated infections might follow a subacute or chronic disease progression. This lack of acute respiratory cases and preponderance of atypical disease in Washington indicates the likely gap between early identification and delayed diagnosis because of lack of awareness and might also explain the high prevalence of the out-of-state strains of C. immitis in our study. However, even in highly endemic regions, acute pulmonary disease is often missed or delayed in diagnosis for months (31,32), supporting the possibility that an underlying background of typical acute disease might remain unidentified in Washington (33).

Our results indicate that WGS is a useful tool to assist in determining exposure location in surveillance situations in which exposure histories are unclear or unknown. For patients in this study, coccidioidomycosis was more commonly associated with travel to other disease-endemic areas compared with local exposure; however, travel to endemic regions does not preclude local acquisition of the disease. Our results also indicate that coccidioidomycosis is likely to be underdiagnosed and underreported in Washington on the basis of atypical disease manifestations for locally acquired cases. More research is needed to determine the true prevalence of locally acquired coccidioidomycosis in Washington.