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.

Introduction

Coccidioidomycosis (Valley fever) is a fungal disease caused by two closely related pathogens: Coccidioides immitis and C. posadasii. Coccidioides immitis is endemic to the San Joaquin Valley of California. Coccidioides posadasii is found in the desert regions of the southwestern United States, including Arizona, Utah, New Mexico, and West Texas, and parts of Mexico, Argentina, Paraguay, and Central America [1]. Coccidioides immitis is likely to present outside the recognized endemic area as evident from human infections and DNA-positive soil samples from Washington state [2,3].

The number of Valley fever cases has increased over the years with 2,271 cases reported in 1998, and 18, 407 cases in 2019 (https://www.cdc.gov/fungal/diseases/coccidioidomycosis/statistics.html). Notably, in 2011, more than 20,000 cases were reported in the US, twice as many cases as tuberculosis [4]. Further documentation of yearly Valley fever cases from Arizona, California, Nevada, New Mexico, Utah, and other jurisdictions are published in the recent updates (https://www.cdc.gov/fungal/diseases/coccidioidomycosis/statistics.html) [5,6]. It is believed that C. immitis and C. posadasii infect ‘tens of thousands’ of people per year and many of whom are sick without knowing the cause or have cases so mild that they are not detected [4]. The rise in Coccidioides infection is attributed to increased travel, relocation to endemic areas, and possible broader distribution of C. immitis and C. posadasii than previously recognized [7,8]. Arthroconidia produced by these fungi are highly infectious, and climate change, including dry and hot weather followed by dust storms makes these conidia easily air borne to cause pulmonary infection [9]. Lung infections typically resolve rapidly leaving the patient with a robust acquired immunity to re-infection [10]. However, in some individuals, the disease may progress to a chronic pulmonary condition or to a systemic disease involving the meninges, bones, joints, subcutaneous, and cutaneous tissues [11]. Identifying Coccidioides to the species level is likely beneficial in the proper treatment of the patients and disease surveillance. Such a laboratory test could also become an important tool in ongoing studies aimed at defining similarities and differences in Valley fever caused by C. immitis and C. posadasii [7,8,12].

Coccidioides immitis and C. posadasii are not easily differentiated in the clinical laboratory on account of similar morphology. Coccidioides posadasii was first differentiated from C. immitis by microsatellite analysis [13,14]. However, this approach was time consuming and technically challenging. Subsequently, several real-time PCR assays, based on LightCycler and TaqMan chemistries, were developed to differentiate C. immitis from C. posadasii [15,16]. Both approaches rely on a single nucleotide polymorphism in different regions of the target gene, and it may have limitations when large number of strains of C. immitis and C. posadasii are tested. Umeyama et al [17] described disparity PCR using species-specific primers designed from the Ci45815 PCR fragment (GenBank AB597180.1). Contiguous deletion of 86-bp nucleotides in corresponding C. posadasii contig Cp45810 PCR fragment (GenBank AB597183.1) resulted in convenient distinction of C. immitis from C. posadasii by conventional PCR. In the present study, we describe a TaqMan duplex real-time PCR assay using PRA2 gene encoding proline-rich antigen specific for the Coccidioides genus and C. immitis PCR fragment Ci45815 specific for C. immitis. The first set of primers and probe targets the PRA2 gene specific for Coccidioides genus. The second set of primers and probe targets C. immitis contig Ci45815 (CiC). The duplex real-time PCR assay was compared against redesigned diagnostic conventional PCR with 100% concordance for culture isolates. We show that duplex real-time PCR assay is highly sensitive (10 gene copies) and identifies C. immitis and C. posadasii from culture and primary specimens. The reliable, and sensitive duplex real-time PCR assay will allow rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens, and further augment the treatment and surveillance of coccidioidomycosis.

Materials and methods

Coccidioides isolates and primary specimens

Two hundred and seventeen suspected isolates of Coccidioides species, 10 formalin-fixed paraffin-embedded tissues, three vials of rhesus monkey kidney (RhMK) cell (Lot # A491216B) [18], six lung tissue, three cerebrospinal fluid (CSF), two bone marrow, one plural fluid, and one whole blood sample, received as a part of reference service from 1995- to 2020, from various diagnostic laboratories of the New York State and neighboring states’ laboratories were part of this investigation. Seventeen isolates of C. immitis were received from the Microbial Diseases Laboratory, California State Department of Public Health. All cultures were stored as sterile water and glycerol stocks at 30°C and -80°C, respectively. Fifteen residual CSF and pleural fluids, which tested positive for coccidioidomycosis by immunodiffusion and complement fixation at the University of California Davis, were also included in the study.

DNA extraction

Extraction of DNA from culture of Coccidioides spp. was carried out in the biological safety cabinet 2 (BSC 2) in the biological safety level 3 (BSL 3) laboratory, and DNA extraction from primary human and animal specimens was carried out in BSC 2 in the BSL 2 laboratory. Qiagen DNA mini kit on the QIAcube automated extractor was used for DNA extraction from all sample types. In brief, for Coccidioides isolates, approximately 5 x 5 mm size of fungal mat grown on SDA slant for 7 to 10 days was removed by sterile loop and suspended in lysis buffer containing approximately 0.2 g of glass beads and incubated for 1 h at 90°C in the BSL 3 laboratory. Heat-killed fungal cells along with the beads was brought to the BSL 2 laboratory, bead-beating in the Precellys homogenizer at 6,500 rpm for 15 sec. for three times using program number 5 (6500-3x60-015). The homogenized fungal suspension was transferred to a 2-ml screw-cap tube, leaving behind the beads. DNA from homogenized samples was extracted using the Qiagen DNA mini kit in the QIAcube semiautomated DNA extractor, resulting in 50 μl of eluted DNA. For DNA extraction from fungal mat grown in the RhMK cell line, approximately 2 ml RhMK medium containing fungal growth was first centrifuged at 12,000 RPM; the supernatant was decanted, and fungal pellet was suspended in lysis buffer containing beads and heat-killed at 90°C for 1 h in the BSL 3 laboratory followed by DNA extraction in the BSL 2 laboratory as described above. Formalin-fixed paraffin-embedded tissue sections were processed by placing 10 sections of 10 μm each into 2-ml tube containing 1 ml of xylene (Sigma Aldrich). The sections were then incubated in xylene for 5 min at room temperature and xylene was removed with fine-tip disposable pipette. A second incubation of 1 ml of xylene was performed followed by centrifugation and removal of xylene. To the sample pallet, 1 ml of 100% ethanol was added, gently vortexed and incubated at room temperature for 5 min, followed by centrifugation and removal of ethanol. The pellet was washed one more time with 1 ml of 100% ethanol. The tissue was dried at room temperature for 15–20 min, followed by addition of lysis buffer, incubation at 70°C for 1 h, bead-beating and DNA extraction using QIAcube as described above. For other primary samples (blood, CSF, pleural fluid, and bone marrow), approximately 400 μl of each sample was directly mixed with lysis buffer containing beads, heated at 56°C for 10 min followed by bead beating and DNA extraction in QIAcube as described above. Lung tissues were minced with sterile blade followed by lysis and DNA extraction as described for other primary samples. All extracted DNAs were stored at −80°C. DNA of fungi (yeasts and molds) other than Coccidioides spp. were procured from the Wadsworth Center Mycology Laboratory (WCML) DNA Collection Repository. DNA from bacterial species including Bacillus megaterium, Escherichia coli, Nocardia farcinica, Pseudomonas aeruginosa, and Streptococcus pneumonia was procured from the Wadsworth Center Bacteriology Laboratory.

Modified conventional PCR

A modification of conventional diagnostic PCR described by Umeyama et. al [17] was used. The primers were designed from the conserved region of Ci45815 (GenBank No. AB597180.1) and Cp45810 (GenBank No. AB597183.1) flanking the deleted region of C. posadasii (S1A Fig). The nucleotide sequences of the diagnostic primers were V2119 5’-CCGGGTACTCCGTACATCAC-3’, and V2120 5’-ATGCGTGAAGCCAATTCTTT-3’ and the PCR conditions were initial denaturation at 95°C for 1 min followed by 30 cycles consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 68°C for 1 min followed by final extension at 68°C for 3 min.

Duplex real-time PCR assay

The PRA2 gene and contig Ci45815/Cp45810 [17] were used to design the duplex real-time PCR assay (S1B and S1C Fig). The nucleotide sequences of primers and probe for PRA2 gene target were forward primer V1753 5’GTGCGAGAAGTTGACCGACTT-3’, reverse primer V1754 5’AGGCGTGATCTTTCCTGGAA-3’, probe V1755 5’-Cy5’-AAGTGCCACTGCGCCAAGCCC-3BHQ and primers and probe for contig Ci45815 target were forward primer V2116 5’-GGTGAAATGCCCGAAAAGAG-3’ reverse primers V2118 5’-CCAATCCTTAGGTAACCGTGAG-3’, and probe V2117 5’/56FAMTTGCACTTT/ZEN/CGTTGACTAGCCGC/3IABkFQ/-3’. Reaction for each real-time PCR contained 1× PerfeCTa multiplex qPCR ToughMix (Quanta Biosciences), a 1,000 nM concentration of primers and a 250 nM concentration of probes, and 2 μl of genomic DNA (approximately 1 to 10 ng) from isolates or 5 μl of tissue DNA extracted from primary clinical specimens in a final volume of 20 μl. Each PCR run also included 2 μl (1 ng) of positive extraction control (C735; C. posadasii), 2 μl (1 ng) of positive amplification controls (C735, C. posadasii and 249, C. immitis), and 2 μl of negative extraction (extraction reagents only) and negative amplification (sterilized nuclease-free water) controls. Parallel to each PCR assay, inhibitory PCR was also performed by incorporating 1 ng of Coccidioides gDNA into each primary clinical DNA sample. The unidirectional workflow kept the reagent preparation, specimen preparation, and amplification and detection areas separate to avoid cross-contamination. Cycling conditions on the ABI 7500 FAST system (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA) were initial denaturation at 95°C for 20 s, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Based on the limit of detection (LOD), a cycle threshold (Ct) value of ≤38 was reported as positive; and >38 was reported as negative. For all primary samples, specimens were reported as inconclusive if PCR inhibition was observed for the primary specimens.

Analytical sensitivity, specificity, and reproducibility of the duplex real-time PCR assay

The 10-fold serial dilutions of genomic DNA from one isolate each of C. posadasii (C-735) and C. immits (249) were used to assess the analytical sensitivity of the duplex real-time PCR assay. The assay specificity was assessed by using an extensive DNA panel comprising various fungi both closely and distantly related to Coccidioides spp. and few bacterial pathogens primarily responsible for causing pulmonary infection (S1 Table). The assay reproducibility was determined by using varying concentration of gDNA from three isolates each of C. posadasii and C. immitis, ran on three different days in duplicate (inter-assay reproducibility), and on the same day in triplicate (intra-assay reproducibility). The assay’s precision was assessed using blinded panel with varying concentration of gDNA from 20 isolates each of C. posadassii and C. immitis, and 10 isolates of fungi other than Coccidioides spp.

Statistical analysis

The GraphPad Prism 8.0 software for macOS was used for calculation of mean, standard deviation, and percent coefficient of variance (CV) for Ct values generated for assay sensitivity and reproducibility. The student t test was used for the analysis of means, and variance, and P value of < 0.05 was considered as statistically significant.

Results

Modified conventional PCR assay

Initially, we modified conventional diagnostic PCR assay using species-specific primers designed from the Ci45815 PCR fragment (GenBank AB597180.1) described by Umeyama et al [17]. The modification reduced the amplicon length down to 200-bp for C. immitis and 114-bp for C. posadasii, yielding better electrophoretic separation (Fig 1). Also, PCR efficiency was higher, with a smaller amplicon size. Of 234 human isolates suspected of Coccidioides were investigated, 172 were C. posadasii, and 43 were C. immitis by conventional PCR, and 19 isolates were negative for Coccidioides spp. (Table 1). This approach was not successful for the analysis of Coccidioides DNA from primary specimens except for three RhMK cell samples (Tables 1 and S2). Overall, our results showed that the conventional diagnostic PCR could be used successfully to identify C. immitis and C. posadasii from culture isolates but not from the primary specimens.

Fig 1

A modified conventional diagnostic PCR assay.

A modification of assay described by Umeyama et al. (2006) allowed generation of smaller amplicons and better electrophoretic separation while maintaining test accuracy. First and last lane 100-bp DNA ladder; lane 1 non-template control (NTC), lanes 2, 4, 6, and 8 C. posadasii; lanes 3, 5, and 7 C. immitis.

Table 1

Results of histopathology, serology, culture, conventional and duplex real-time.

Sample	Type	No	Histopathology Positive/Negative	Serology Positive/Negative (ND)	Culture ID$ Positive/Negative	Conventional PCR		Duplex PCR		Sensitivity (%)	Specificity (%)	PPV	NPV
						114-bp	200-bp	Cp (Cy5 +/FAM-)	Ci (CY5 +/FAM+)
Primary specimens	Paraffin Tissue	10	6/4	NA	NA	0	0	6+3#	0	100	NA	100	NA
	RhMK Cell	3	NA	NA	3/0	3	0	3	0	100	NA	100	NA
	CSF	9	NA	6/0 (2)	0	0	0	0	0	NA	NA	NA	NA
	Pleural Fluid	10	NA	3/6 (1)	0	0	0	2	0	100	86	67	100
	Tissues	10	ND	NA	0	0	0	0	0	0	0	0	0
Culture	California*	17	NA	NA	17/0	4	13	4	13	100	NA	100	NA
	New York@	217	NA	NA	198/19	168	30	168	30	100	100	100	100

NA = Not Applicable; ND = Not Done

The histopathology or serology or culture was used for different specimen types to determine the performance of Coccidioides duplex real-time PCR assay.

$Culture was identified either by GenProbe (Hologic Inc.), or sequencing of the ribosomal gene (ITS), or by Coccidioides immitis/C. posadasii) lab-developed, and Clinical Laboratory Evaluation Program-NYSDOH-approved single plex real-time PCR assay.

#Histopathology was not done for four samples of which three were positive by Coccidioides duplex real-time PCR. Therefore, they were removed from the calculation for PCR accuracy.

* Received as C. immitis

Suspected isolates of Coccidioides spp. received as part of reference testing

Duplex real-time PCR assay sensitivity, specificity, and reproducibility

We designed a duplex real-time PCR assay with the first set of primers and probe targeting PRA2 gene identifying Coccidioides species (S1B Fig), and the second set of primers and probe targeting C. immitis contig Ci45815 (CiC) identifying C. immitis but not C. posadasii due to 86-bp deletion in the corresponding C. posadasii contig 45810 (S1C Fig). The duplex real-time PCR assay was highly sensitive, with the limit of detection was 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii (Fig 2 and Table 2). None of the other fungal or bacterial DNA yielded any Ct value, confirming the high specificity of the duplex real-time PCR assay (S1 Table). The assay was highly reproducible as it yielded correct ID for C. immitis and C. posadasii when varying concentration of gDNA tested within the same day or on different days (S3A and S3B Table), and when samples were blinded by one operator and tested by another operator (S4 Table). Since Coccidioides genus comprises only two species, positive results with both probes identified C. immitis while the positive, negative results with PRA2 and Ci45815 contig (CiC) probes, respectively, identified C. posadasii (Table 3).

Fig 2

Coccidioides duplex real-time PCR assay sensitivity.

Genomic DNA from two control strains of Coccidioides species, C. immitis (249) and C. posadasii (C-735) were serially diluted and tested in triplicate in duplex real-time PCR assay. The assay was linear over 5 orders of magnitude and the limit of detection was 0.001 ng or 1 picogram gDNA/PCR reactions at 45 PCR cycles confirming high sensitivity. As indicated, the assay targeting CiC gene did not yield Ct values against C. posadasii DNA. The test was repeated with similar results.

Table 2

Coccidioides duplex real-time PCR assay sensitivity.

gDNA ng/PCRRxn	Probe	C. posadasii (C-735)			Mean Ct ± SD	%CV	C. immitis (249)			Mean Ct ± SD	% CV
		Ct 1	Ct 2	Ct 3			Ct 1	Ct 2	Ct3
100	PRA2 (Cy5)	18.66	18.86	18.7	18.74 ± 0.106	0.565	19.04	19.14	19.20	19.12 ± 0.08	0.423
10		22.36	22.28	22.25	22.29 ± 0.057	0.255	23.16	23.58	23.18	23.16 ± 0.012	0.050
1		26.34	25.96	26.56	26.28 ± 0.304	1.155	27.00	27.17	26.80	26.85 ± 0.129	0.479
0.1		29.80	30.05	29.89	29.91 ± 0.127	0.423	30.83	30.75	30.60	30.62 ± 0.196	0.640
0.01		33.21	33.47	33.3	33.32 ± 0.132	0.396	33.97	33.69	34.51	34.24 ± 0.266	0.776
0.001		36.38	35.25	35.3	35.64 ± 0.638	1.791	37.37	36.65	37.32	37.10 ± 0.397	1.070
0.0001		0.0	0.0	0.0			0.0	0.0	0.0	0.0
100	CiC (FAM)	0.0	0.0	0.0			19.40	19.47	19.52	19.45 ± 0.051	0.254
10		0.0	0.0	0.0			23.58	23.50	23.55	23.54 ± 0.040	0.172
1		0.0	0.0	0.0			27.17	27.40	27.35	27.30 ± 0.121	0.443
0.1		0.0	0.0	0.0			30.75	31.40	31.00	31.05 ± 0.328	1.056
0.01		0.0	0.0	0.0			33.69	34.78	34.50	34.32 ± 0.566	1.649
0.001		0.0	0.0	0.0			37.83	37.50	37.60	37.64 ± 0.169	0.450
0.0001		0.0	0.0	0.0			0.0	0.0	0.0	0.0

Table 3

Test interpretation of Coccidioides duplex real-time PCR assay.

Test Interpretation	Probe
	Prp2 (Cy5)	CiC (FAM)
Coccidioides immitis	Positive	Positive
Coccidioides posadasii	Positive	Negative
Other Fungi	Negative	Negative
Bacteria	Negative	Negative

Performance of the duplex real-time PCR assay

The duplex real-time PCR assay correctly identified all cultures of Coccidioides species to either C. immitis or C. posadasii, and the results were corroborated with modified diagnostic PCR assay, confirming the high utility of the duplex real-time PCR assay (Table 1). Of 217 human fungal isolates suspected of Coccidioides received for reference testing, 168 were identified as C. posadasii, representing 125 cases, and 30 isolates were identified as C. immitis, representing 23 cases (Fig 3). Of 17 isolates received from California, 13 were confirmed as C. immtits and four were confirmed as C. posadasii (Tables 1 and S5). Next, we assessed the duplex real-time PCR assay’s ability to detect Coccidioides species DNA from 42 primary specimens. Nine of the ten paraffin-embedded tissues including four human and five animal, and three vials of RhMK cells were positive for C. posadassi DNA (S2 Table). All of the three RhMK cell vials positive for C. posadasii DNA also yielded Coccidioides in culture, and subsequently confirmed as C. posadasii (Tables 1 and S2). We also evaluated 15 residual CSF and pleural fluid samples, which were earlier tested positive for coccidoidomycosis by immunodiffusion and complement fixations tests. Of these samples, only two were positive for C. posadasii DNA by duplex real-time PCR assay (Tables 1 and S6).

Fig 3

Human coccidioidomycosis cases from 1995 to 2020 as part of reference diagnostic service.

Isolates received from various facilities from New York and neighboring states were analyzed retrospectively by newly developed duplex real-time PCR assay. Of 152 cases, 129 were confirmed as being caused by C. posadasii, and 23 were caused by C. immitis. The predominance of C. posadasii over C. immitis was observed in the isolates analyzed. Four of the 129 cases were defined based on primary specimens positive for C. posadasii DNA.

Discussion

PCR-based fungal diagnostic methods offer accuracy, sensitivity, and speed of identification, and the use of DNA instead of highly infectious live cultures. Microsatellite analysis has been the primary molecular method used to distinguish the two species of Coccidioides [13,14]. However, this method is both time consuming and technically challenging for clinical and reference laboratories. The light-cycler PCR assay developed by Binnicker et al. [19] was highly sensitive and specific for Coccidioides species, but this assay could not distinguish C. immitis from C. posadasii. Umeyama et al. [17] described disparity PCR using species-specific primers that resulted in different size amplicons for a convenient distinction of C. immitis from C. posadasii by conventional PCR. In the present study, we modified the Umeyama et. al. [17] disparity PCR, which led to the reduction in amplicon size of 200-bp for C. immitis and 114-bp for C. posadasii. The conventional diagnostic PCR assay was excellent for the identification of C. immitis and C. posadasii from culture, but it was not good for the analysis of Coccidioides DNA from primary specimens. One reason could be that the amount of Coccidioides DNA present in the primary samples were below the threshold for detection by conventional PCR, or the template was degraded beyond the capacity of the conventional PCR to yield a positive result. PCR allelic-discrimination assay, referred to as CocciDiff, was developed using TaqMan chemistry [16]. This assay could effectively identify Coccidioides cultures and could accurately distinguish C. immitis from C. posadasii based upon the presence of an individual highly informative canonical SNP [16]. CocciDiff has yet to be validated with clinical specimens. Although, the CocciDiff assay appears to be quite promising, the assay specificity based on canonical SNP can be problematic if such mutations are not consistently present in a given strain of Coccidioides. In the present study, while developing a duplex real-time PCR assay, we used more stringent conditions where the first set of primers and probe targeting PRA2 gene identified Coccidioides to the genus level, and the second set of primers and probe targeting C. immitis contig, Ci45815 (CiC), identified C. immitis but not C. posadasii due to a deletion of 86-bp sequence in the corresponding Cp45810 contig. Since there are only two species within the Coccidioides genus, the positive results with both probes of the duplex assay allowed the identification of C. immitis while positive, negative results with probes PRA2 and Ci45815, respectively, led to the identification of C. posadasii.

Of 168 C. posadasii, 151 isolates were from patients from New York, followed by eight isolates from patients from New Jersey, two isolates each from patients from Pennsylvania and Arizona, one isolate from a patient from Washington DC. All 30 C. immitis isolates were from patients from NY. Since Coccidioides is not found in the soil of New York and other states in the northeast region, the findings of positive isolates suggested that the patients acquired Coccidioides infection while travelling to a known area of endemicity. These findings are consistent with our earlier study where travel history to endemic areas for Coccidioides was linked to positive clinical cases from New York [20]. In the present study, we confirmed over five-fold more isolates as C. posadasii than as C. immitis; this observation supports a previous publication on possible larger population size and more diverse distribution of C. posadasii vis-a-vis C. immitis [21]. Among a smaller sampling of isolates from California, we found four C. posadasii isolates. Also, positive RhMK cell samples from a monkey housed in California were also identified as C. posadasii. These results raised the possibility that a small niche of C. posadasii exists in California or patients might have traveled to areas where C. posadasii is endemic. Further studies are needed to delineate the geographic distribution of C. immitis and C. posadasii and the areas of overlap if any.

Serology tests are the mainstay of coccidioidomycosis diagnosis [22,23]. Our study included a small sample size of serology-positive clinical specimens, but the findings do not permit firm correlation with real-time PCR test. The gold standard for the diagnosis of coccidioidomycosis is the culture of the organism from primary specimens. Culture is highly sensitive, and the DNA probe for confirmatory testing of culture isolates has yielded excellent specificity [24,25]. However, growth in culture may take several days to weeks resulting in a delay in diagnosing and initiating treatment in infected individuals. The highly infectious nature of arthroconidia produced by Coccidioides species presents a safety risk to the laboratory personnel if a culture is not quickly identified and handled appropriately under biosafety level 3 containment. It is important to note that laboratory-acquired infections due to Coccidioides species have been reported in the literature [26]. A rapid Coccidioides DNA test could become an adjunct surveillance tool to study the incidence, prevalence, and overlapping features of Valley fever caused by Coccidioides species [7,8]. In summary, our modified diagnostic conventional PCR assay was excellent for the culture identification of C. immitis and C. posadasii, and the new duplex real-time PCR assay had broader utility for identifying two Coccidioides species from culture and primary specimens. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment coccidioidomycosis treatment and surveillance.

Multiple alignments of contigs and PRA2 gene for the design of primers and probes for conventional and real-time PCR assays.

(A) Nucleotide sequences of PCR amplicons of C. immitis (Ci45815 & Ci45816) and C. posadasii (Cp45809 & Cp45810) contigs [17] were aligned using Geneious R9 9.1.6 software (Biomatters, Inc., San Diego, CA). The gray background defines the consensus sequences between C. immitis and C. posadasii while non-consensus sequences are highlighted in color. Conventional PCR forward (V2119-F) and reverse (V2020-R) primers were designed from the consensus region flanking the deleted region (86-bp) of C. posadasii. As a result, primer set produced 114-bp product for C. posadasii and 200-bp product for C. immitis. (B) PRA2 gene from both C. immitis and C. posadasii were aligned and consensus region of the gene was used for the design of forward (V1753-F), and reverse (V1754-R) primers, and probe (V1755). The resulting primers and probe produced amplicons against both C. immitis and C. posadasii. (C) C. immitis (Ci45815 & Ci45816) and C. posadasii (Cp45809 & Cp45810) contigs were aligned as described in S1A Fig. The forward primer (V2116-F) was designed from C. immits contig region deleted in C. posadasii while reverse primer (V2118-R) and probe (V2117) were designed from consensus region of the contigs. The resulting primers and probe produced amplicon against C. immits, but not against C. posadasii.

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Coccidioides duplex real-time PCR assay specificity.

Genomic DNA from pathogenic fungi both closely and distantly related to Coccidioides species were tested in duplicate. A few bacterial pathogens, usually associated with common respiratory diseases, were also included in specificity panel. DNA from none of the fungal or bacterial isolates yielded detectable fluorescence, and thus, confirming the high specificity of Coccidioides duplex real-time PCR assay.

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Comparison of the diagnostic and Coccidioides duplex real-time PCR assay for detection of Coccidioides DNA from primary human and animal specimens.

DNA was extracted from both fixed and unfixed human and animal primary specimens and subjected to diagnostic PCR and Coccidioides duplex real-time PCR assay. Results revealed the duplex real-time PCR assay was superior to conventional diagnostic PCR for the detection of C. immitis or C. posadasii from various sample types investigated.

(XLSX)

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Assay reproducibility of Coccidioides duplex real-time PCR.

(A) Inter-assay reproducibility: DNA of varying concentrations from three isolates each of C. posadasii and C. immitis were tested in duplicate on 3 separate days. The Ct values obtained were consistent with % CV of <5%, which confirmed an excellent inter-assay reproducibility, (B) Intra-assay reproducibility: DNA samples from three isolates each of C. immitis and C. posadasii with varying concentrations were tested in triplicate on the same day on same plate in one run. The Ct values obtained were consistent with % CV of <5%, which confirmed an excellent intra-assay reproducibility.

(XLSX)

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Blinded panel of Coccidioides duplex real-time PCR.

The assay’s precision was assessed using blinded panel with varying concentration of gDNA from 20 isolates each of C. posadassii and C. immitis, and 10 isolates of fungi other than Coccidioides spp. The duplex assay produced Ct values corresponding to input gDNA of either C. immitis or C. posadasii but not to other fungal species, confirming assay accuracy.

(XLSX)

Click here for additional data file.

Diagnostic PCR and Coccidioides duplex real-time PCR testing of Coccidioides isolates from California.

Genomic DNA from 17 C. immitis isolates, received from California Department of Public Health, were evaluated by conventional diagnostic and duplex Coccidioides real-time PCR assay. Of 17 isolates, 13 were confirmed as C. immitis and four as C. posadasii by both assays, further confirming the utility of these assays for culture identification of both these species.

(XLSX)

Click here for additional data file.

Evaluation of archived CSF and pleural fluid samples by Coccidioides duplex real time PCR assay.

DNA extracted from 15 residual CSF and pleural fluid samples, which were positive by serology tests (immunodiffusion & complement fixation), were tested by Coccidioides duplex real-time PCR assay. Of these samples, only two were positive for C. posadasii DNA by real-time PCR, suggesting a possible low correlation with the serology tests.

(XLSX)

Click here for additional data file.

24 Jun 2021

Dear Dr. Chaturvedi,

Thank you very much for submitting your manuscript "Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Angel Gonzalez, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objective of the study are clearly articulated. But this study is no a hypothesis based study. It is a methods based study. The study design does address the stated objectives.

The samples and populations are clearly described but a hypothesis is not being tested.

The statistical test were not adequately described. On Page 10 in the Statistical analysis section, the authors should state the statistical test performed e.g., t-test, anova etc. including the post-hoc tests.

Yes, the ethical and regulatory requirements seem to be met.

Reviewer #2: While statistical testing was briefly mentioned, it is extremely vague and no specific tests were mentioned. No evidence from statistical tests are given in any of the tables.

Reviewer #3: Chuterverdi et al have provided a very nice article on the development of RT-PCR testing for Coccidioides spp. There methods are sound, all relevant information is provided, and their labs has a history of strong work in similar areas. I do not have any methodologic concerns.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Yes.

Reviewer #2: (No Response)

Reviewer #3: Results are very easy to follow, are justified by the data, and clear.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes.

Reviewer #2: (No Response)

Reviewer #3: Conclusions are sound. Dsicussion should be improved a bit with additional references added, and the clinical usefulness of RT-PCR discussed. The results of Barker et al with regard to spp specific differences and how this may be of clinical significance (not just epi significance) can be added.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: On Page 10 in the Statistical analysis section, the authors should state the statistical test performed e.g., t-test, anova etc. including the post-hoc tests.

Page 11: Lines 1-2 Amplification efficiency is important information to this study because the efficiency can impact the limit of detection. Amplification efficiency will also allow reviewers to access how reliable the assay is. Good quality assay typically have amplification efficiencies of 90 - 110 %. Please, add this data.

Reviewer #2: The methods can be difficult to follow at times. It would benefit from subheadings, especially in the DNA extraction section, to differentiate between protocols for the various sample types. The manufacturers' info is not always complete in this section and the formatting of units is not consistent.

The PCR sections may benefit from diagrams, particularly for the primer and probe design.

Many of the tables contain raw data, rather than summaries for sample types, and are not interpretation-friendly.

The manuscript would benefit if the raw data is kept in the supplement and interpretations presented in the main manuscript.

Reviewer #3: Italicize Coccidioides throughout (page 5 – line 3 etc; why were continuous line numbers not used throughout? Far simpler for reviewers).

Page 15, lines 3-8; this has previously been described by Taylor et al and Barker et al. Appropriate references could be added.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: What are the main claims of the paper and how significant are they for the discipline?

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of two Coccidioides species remains problematic in clinical laboratories as conventional methods are limited by complexity or time constraints. Therefore, the authors developed TaqMan-probes for a duplex qPCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens.

Are these claims novel? If not, which published articles weaken the claims of originality of this one?

Although the techniques used here are not novel per se, the TaqMan-probes developed here overcome short comings of current techniques for diagnosis of Coccidioidomycosis.

Are the claims properly placed in the context of the previous literature? Have the authors treated the literature fairly?

The claims appear to be properly placed in the context of previous literature.

Do the data and analyses fully support the claims? If not, what other evidence is required?

The data partially support the claims made here.

On Page 10 in the Statistical analysis section, the authors should state the statistical test performed e.g., t-test, anova etc. including the post-hoc tests.

Would additional work improve the paper? How much better would the paper be if this work were performed and how difficult would it be to do this work?

I don’t believe that additional work is necessary.

PLOS Neglected Tropical Diseases encourages authors to publish detailed protocols and algorithms as supporting information online. Do any particular methods used in the manuscript warrant such treatment? If a protocol is already provided, for example for a randomized controlled trial, are there any important deviations from it? If so, have the authors explained adequately why the deviations occurred?

No.

Is this paper outstanding in its discipline? If yes, what makes it outstanding? If not, why not?

The authors have developed a technique with advantages over current methodologies.

If the paper is considered unsuitable for publication in its present form, does the study itself show sufficient potential that the authors should be encouraged to resubmit a revised version?

No. With very minor revision the paper is suitable for publication.

Are original data deposited in appropriate repositories and accession/version numbers provided for genes, proteins, mutants, diseases, etc.?

N/A

Are details of the methodology sufficient to allow the experiments to be reproduced?

Page 11: Lines 1-2 Amplification efficiency is important information to this study because the efficiency can impact the limit of detection. Please, add this data instead of stating data not shown.

Is the manuscript well organized and written clearly enough to be accessible to non-specialists?

Yes.

Reviewer #2: Financial disclosure section has not been completed to the specifications of the journal.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

19 Aug 2021

Submitted filename: Response to reviewers comments_Sc081421.docx

Click here for additional data file.

25 Aug 2021

Dear Dr. Chaturvedi,

We are pleased to inform you that your manuscript 'Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Angel Gonzalez, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

10 Sep 2021

Dear Dr. Chaturvedi,

We are delighted to inform you that your manuscript, "Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases