Detection of Coccidioides posadasii from xerophytic environments in Venezuela reveals risk of naturally acquired coccidioidomycosis infections

Figures & data

Site identification, location, sampling data, temperature range, rain precipitation, geographic coordinates, soil type, and vegetation-type of samples areas in Lara and Falcon states of Venezuela

Sites	Location	Date of collection	Mean temperature range	Annual precipitation (mm)	Geographic coordinates	Soil type	Vegetation
1 and 2	Urumaco (San José de Bruzual)	04/13/2015	>34 °C	600–800	70°19'48,328"W 11°4'47,769"N	Clay	Shrubs tropophilous deciduous and semi-deciduous
3, 4, and 5	Sucre (Pedregal)	04/15/2015	32–34 °C	500–600	69°51'49,916"W 11°4'27,331"N	Clay	Tropophilous forest basimontane
6, 7, and 8	Democracia (Pecaya)	04/21/2015	32–34 °C	400–500	70°7'13,178"W 11°1'17,091"N	French-sandy	Spiny xerophytic bushes
9, 10, and 11	Urdaneta (Siquisique)	07/20/2015	32–34 °C	<400	69°45'8344"W 10°35'4025"N	Clay-sandy	Agricultural soil
12, 13, and 14	Torres (La Majada)	07/22/2015	30–32 °C	600–800	70°10'14,917"W 10°17'41,557"N	Clay	Thorny xeric shrublands
15	Torres (La Burra)	07/23/2015	28–30 °C	800–1000	70°25'56,35"W 9°58'54,79"N	Clay	Shrubs tropophilous deciduous and semi-deciduous

CocciEnv primers and probe

CocciEnv assay primers	Name	Sequence 3′–5′	Final conc. (μM)
Forward	CocciEnv_F1d1	CGTTGCACRGGGAGCACCT	0.375
	CocciEnv_F2	AAGCTTTGGATCTTTGTGGCTCT	0.375
	CocciEnv_F3	AATTGATCCATTGCAAGCACCT	0.25
	CocciEnv_F4	AATCCAACCTTTGGAACTACACCT	0.25
	CocciEnv_F5	TTTTCCGGTATGGACTAGCACCT	0.375
	CocciEnv_F6d2	TGTTAGGTAATCYAACYAGCACCT	0.125
	CocciEnv_F7d2	TRTTAGGTAATYCAACTAGCACCT	0.125
	CocciEnv_F8d1	TGTTAGATAATCCAACYAGCACCT	0.125
	CocciEnv_F9d2	GKTARGTAATCCAACTAGCACCT	0.125
	CocciEnv_F10d2	TGTTAGGTARTCCAACTAGCAYCT	0.125
	CocciEnv_F11d2	TGTTAGGTAATCCAACTMGCACYT	0.125
Reverse	CocciEnv_R1	GATGGAGGACTCTATATGCTTGT	0.375
	CocciEnv_R2	ATGGAGGACTCGTTATGCCTGT	0.375
	CocciEnv_R3	GGAGGACCCGTATGCTTGTGT	0.375
	CocciEnv_R4	TGCTAAATGATGGAGGGCTTGT	0.375
	CocciEnv_R5	GATGGAGGCTCGTATGCTTGT	0.375
	CocciEnv_R6	AAGGGGTTTGTGGTGAATCCTTA	0.375
	CocciEnv_R7	CAGAAAAATAGCCGTATGCTTGT	0.375
	CocciEnv_R8d2	TRATGGAGRACTTGTATGCTTGT	0.125
	CocciEnv_R9d1	TGATGGAGGACTCGTATGCYTGT	0.125
	CocciEnv_R10d2	TGATGGARRACTCATATGCTTGT	0.125
	CocciEnv_R11d2	TGATAGAGAACTTGTATRCTTRT	0.125
	CocciEnv_R12d2	TGATGAAGAACTTRTATRCTTGT	0.125
	CocciEnv_R13d2	TGATRRAGGACTTGTATGCTTGT	0.125
	CocciEnv_R14	TGATGGAAAACTTGTATGCTTGT	0.125
	CocciEnv_R15d2	TGATGGAGGACTTGTAYAYTTGT	0.125
	CocciEnv_R16d2	TGATGGAGGACTTGTAYGCTTRT	0.125
	CocciEnv_R17d2	TGATGGAGGACTYATATGCTTRT	0.125
	CocciEnv_R18d2	GATGGAGGACTCGTWYGCTTGT	0.125
Taqman probe	CocciEnv_FMGB	6FAM-ACCCACATAGATTAGC-MGBNFQ	0.25

Mean of C_t values obtained for the qPCR-based CocciEnv assay

Site	Replicate 1	Replicate 2	Replicate 3	Replicate 4
1	n.d.	n.d.	39.170457	n.d.	Low positive
2	32.44438067	33.684223	31.253591	32.57464933	High positive
3	38.0875965	n.d.	n.d.	n.d.	Low positive
4	31.043169	29.483968	31.108808	32.5819565	High positive
5	n.d.	37.40752733	37.943037	n.d.	Moderate positive
6	32.039917	30.76345	31.93577333	31.11106433	High positive
7	n.d.	38.681686	n.d.	n.d.	Low positive
8	31.93436033	30.68055833	n.d.	n.d.	Moderate positive
9	40.3225925	n.d.	n.d.	36.743083	Moderate positive
10	n.d.	n.d.	n.d.	39.8178845	Low positive
11	n.d.	n.d.	n.d.	39.017355	Low positive
12	39.985367	n.d.	38.475866	36.31935467	Moderate positive
13	n.d.	37.238985	n.d.	n.d.	Low positive
14	39.1133525	n.d.	n.d.	n.d.	Low positive
15	36.5966605	33.26852667	n.d.	37.1464845	Moderate positive

DNA extractions were performed in quadruplicates for each site analyzed due to the likelihood of variable and potentially rare presence of Coccidioides DNA in the Lara and Falcon states of Venezuela. C_t values are averages of three replicate reactions for each DNA sample. n.d. not detected

Number of C. posadasii ITS2 amplicons sequenced for each of the four replicate DNA extractions representing each analyzed site in Lara and Falcon states of Venezuela

Site	Replicate 1	Replicate 2	Replicate 3	Replicate 4
1	1.1	1.2	1.3	1.4
denovo49	274	246	356	249
denovo8395	0	6	17	4
2	2.1	2.2	2.3	2.4
denovo49	423	235	211	611
denovo8395	7	13	43	0
3	3.1	3.2	3.3	3.4
denovo49	0	0	0	4
denovo8395	0	0	0	0
4	4.1	4.2	4.3	4.4
denovo49	9	290	26	2
denovo8395	455	3516	9	0
6	6.1	6.2	6.3	6.4
denovo49	209	450	4	88
denovo8395	0	145	0	0
8	8.1	8.2	8.3	8.4
denovo49	59	6	3	28
denovo8395	22	49	0	0
10	10.1	10.2	10.3	10.4
denovo49	1	0	0	2
denovo8395	0	0	0	0
12	12.1	12.2	12.3	12.4
denovo49	1	4	1	87
denovo8395	0	0	0	0
15	15.1	15.2	15.3	15.4
denovo49	7	2	3	1
denovo8395	0	0	0	0

Fig. 1 C. posadasii relative abundance in soil samples from Venezuela.

a Overall relative abundance of C. posadasii comparing three different methods: rarefied to the lowest depth sample (Rarified) or normalized by CSS or DESeq2 transformations. b Site-specific relative abundance of C. posadasii comparing the three matrix normalization methods mentioned above

Fig. 2 Alpha and Beta species diversity index calculated for the fungal communities observed in Lara and Falcon states of Venezuela.

Species diversity indexes were calculated based on the amount of C. posadasii DNA in the soil (a, b), individual sites (c, d), and Venezuelan municipalities (e, f)

Fig. 3 Maximum likelihood (ML) tree of the 300 bp fragment of ITS2 locus of environmental, veterinarian, and clinical samples of Coccidioides immitis and C. posadasii.

Branch lengths are proportional to the number of genetic changes and bootstrap values >70% are displayed next to the branches