Valley fever, a disease caused by the soilborne fungal pathogen Coccidioides spp., is on the rise in the southwestern United States and is suspected of expanding its habitat in response to climate change. Many people residing in endemic areas are unaware of the risk of contracting coccidioidomycosis by inhaling fugitive dust that may contain dormant arthroconidia of this fungus. In an effort to teach students about the ongoing epidemic of valley fever and reduce incidence of the disease through education, we developed an inquiry-based, multi-focus laboratory exercise that (1) increases awareness of valley fever incidence, disease symptoms, and ongoing efforts of disease prevention; (2) teaches about the pathogen's ecology; and (3) familiarizes students with molecular techniques targeting pathogen identification. This laboratory exercise uses polymerase chain reaction to detect Coccidioides spp. in DNA extracts from soil samples collected by students across different soil environments. Additionally, this exercise will teach students how to use publicly available data to investigate disease incidence over time and characterize soils the pathogen may inhabit.

Introduction

Valley fever, also known as coccidioidomycosis, is a disease that has been documented in California since the early 20th century and is caused by the fungal pathogen Coccidioides (meaning “resembling cocci”) immitis (“not mild”). The pathogen is adapted to semiarid and arid climates, and a close relative of this fungus, C. posadasii (named after the Argentinian physician Alejandro Posadas), occurs in other southwestern states, in Mexico, and in some dry valleys of South America (Brown et al., 2013). When soil is disturbed, arthroconidia of these fungal species can become airborne alongside dust, and the inhalation of these dormant forms of the pathogens can lead to mycosis in the lungs (Galgiani et al., 2005). In more severe instances, infections of skin, bones, joint tissue, and brain and spinal fluid can develop (Ampel, 2011).

In the United States, public health departments have documented a steady increase in valley fever incidence since the late 1990s, mainly due to increased soil disturbance and drought (Colson et al., 2017; Gorris et al., 2018). Coccidioidomycosis is a reportable disease, and in Arizona and California, annual cases number in the thousands. Incidence peaked in 2011 with >16,000 reported cases in humans (Cat et al., 2017; Valley Fever Center for Excellence, 2018). Overall, the endemic area where Coccidioides spp. are established includes ~20% of the United States (Centers for of Disease Control and Prevention [CDC] website). These endemic regions receive hundreds of thousands of visitors annually from all over the world; therefore, valley fever awareness is relevant to both residents and visitors. Furthermore, the endemic area of the pathogen appears to be expanding, and more cases of valley fever are being reported from outside traditional endemic areas in recent years (Marsden-Haug et al., 2014; Turabelidze et al., 2015). Overall, as of 2017, valley fever is affecting ~24% of the population of the United States (~79 million people) who reside in areas of high endemicity such as California, Arizona, western Texas, New Mexico, and Nevada.

Although thousands of people (and animals) are diagnosed with the disease each year, the public is not well informed about the “danger that is lurking in the dust.” Schools traditionally focus on sexually transmitted diseases in the classroom, which are also on the rise in some areas of the United States, but endemic diseases are rarely part of school curricula. Incidence of coccidioidomycosis in California is highest in Kern County, where California State University Bakersfield (CSUB) is located. The area is known for its intensive agriculture, recent increases in construction, and poor air quality and has been subject to the effects of ongoing droughts. The pathogen has frequently been detected in soils collected in Kern County (Smith, 1940; Lauer et al., 2014; Cooksey et al., 2017).

To promote awareness of valley fever and enhance understanding of the ongoing epidemic, we propose a multi-focus exercise that includes an inquiry-based portion followed by an investigatory component. This original exercise will be well suited for a college-level undergraduate microbiology course but can also be adapted for an advanced AP high school biology course. The sections of this multi-focus exercise can be modified to stand-alone lessons. We recommend beginning this exercise with didactic lectures focused on background information regarding the ecology of Coccidioides, how valley fever can be contracted, symptoms and signs of the disease, and methods for detecting the pathogen, and culminating with a discussion about reasons why more cases are being observed in recent times. This will build the foundation for the inquiry part of this exercise.

In addition to raising valley fever awareness, students will be introduced to basic molecular tools to safely detect the pathogen using techniques with real-world applications. We recommend including student access to disease incidence data that can be obtained from the websites of the CDC's National Notifiable Diseases Surveillance System (NNDSS; https://wwwn.cdc.gov/nndss/) and from annual health reports that are released by public health departments. Additionally, students can explore environmental parameters that characterize the habitat of Coccidioides by using the WebSoilSurvey (WSS) database of the U.S. Department of Agriculture (USDA).

Concept Explanation

Today's biology courses rarely include topics relevant to students' interests. Failing to attract students' attention leads to a lack of understanding of why acquiring knowledge about certain topics benefits their education or might even be useful in daily life. Furthermore, concepts of microbiology are rarely included in a high school biology course yet are often an important part of a later undergraduate biology degree (Huppert et al., 2002; Gasper & Gardner, 2013; McKenney et al., 2016). Student engagement, participation, and learning outcomes can be cultivated by interweaving coursework with applied topics that connect with students. One example includes valley fever health risks and prevention. Our exercise proposes using polymerase chain reaction (PCR) to detect Coccidioides spp. in soil samples collected by students and working with real disease incidence data and environmental data accessed from publicly available databases. Databases such as NNDSS and WSS are readily accessible. This exercise is student centered and involves students in all steps (Figure 1).

Figure 1.

Flowchart showing the structure of the exercise, highlighting the different subtopics after project introduction (white box, inquiry part): soil characterization (light gray boxes, left), detection of the pathogen in soil samples collected from diverse types of soils using diagnostic PCR (medium gray boxes, center), and working with disease incidence data (dark gray boxes, right) (investigation part).

Figure 1.

Flowchart showing the structure of the exercise, highlighting the different subtopics after project introduction (white box, inquiry part): soil characterization (light gray boxes, left), detection of the pathogen in soil samples collected from diverse types of soils using diagnostic PCR (medium gray boxes, center), and working with disease incidence data (dark gray boxes, right) (investigation part).

Many laboratory-based biology curricula are dominated by exercises that rely on outdated techniques that are rarely used by modern research institutions. This leads to students being ill-prepared for both the workforce and graduate-level research. Fortunately, an increasing number of funding opportunities combined with the availability of affordable, discounted, or second-hand laboratory equipment has led to better access to modern scientific technology.

Our multi-focus exercise provides students with a deeper understanding of an emerging infectious disease. It combines critical-thinking skills with hands-on research, focusing on discussions, formulation of hypotheses, and data analysis.

Suggested Resources for Learning More about Valley Fever

Materials Needed

The supplies listed below are sufficient for analyzing 50 soil samples.

Soil Sampling

  • Clean zip-lock bags

  • Clean spoon or garden shovel

  • Respirator mask (Fisher Scientific, cat. no. 19-168-175) (needed only when soil is dry)

DNA Extraction, PCR & Agarose Gel Electrophoresis

Equipment

  • Centrifuge (e.g., Accu Spin Micro 17, Fisher Scientific)

  • Vortexer with horizontal adapter (e.g., Vortex-Genie 2, MoBio Laboratories)

  • Micropipettes and sterile pipette tips, different volumes (0–10 μL, 20–200 μL, 100–1000 μL) (Fisher Scientific)

  • Thermocycler (e.g., PTC200, MJ-Research)

  • Gel electrophoresis chamber (Fisher Scientific)

  • UV light transilluminator (e.g., UVP Benchtop UV Transilluminator, Fisher Scientific)

Laboratory supplies

  • Lab coats and nitrile gloves (Fisher Scientific, cat. nos. 17-100-809 and 19-130-1597)

  • Disinfectant (10% bleach solution or 70% ethanol solution, Fisher Scientific, cat. no. AC615095000)

  • DNEasy PowerSoil DNA extraction kit (Qiagen DNeasy PowerSoil Kit, cat. no. 12888-50)

  • 0.6 mL PCR tubes (Fisher Scientific, cat. no. 05-408-121)

  • PCR primers (RDS478/482, ITSC1A/ISTC2, e.g., Invitrogen or IDTDNA)

  • PCR Mastermix (GoTaq Green Mastermix with sterile water, Fisher Scientific, cat. no. PRM7122)

  • Positive control for PCR, C. posadasii NR-4548, nonpathogenic strain (BEI-Resources)

  • PCR marker (e.g., G3161, Fisher Scientific, cat. no. PR-G3161)

  • Low Melting Agarose (Fisher Scientific, cat. no. AC159820100)

  • 10x Tris-borate EDTA (TBE) buffer (Fisher Scientific, cat. no. NC0017762)

  • Nucleic acid stain (SYBRSafe, Fisher Scientific, cat. no. S33102)

  • Agarose gel loading dye (Fisher Scientific, cat. no. BP633-5)

Purification of PCR Products & Sequencing

  • Zymo clean gel DNA recovery kit (Zymo Research, cat. no. D4007)

  • ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix, cat. no. 78200)

  • Sequence data: outsource to a DNA sequencing center (currently ~$6/reaction; e.g., Laragen)

Accessing Online Databases (NNDSS & WSS)

  • Laptops

  • Smart lab (to project websites and progress of this exercise on a large screen)

It is worth noting that high schools are eligible for special educational discounts from vendors such as Fisher Scientific (https://www.fishersci.com/us/en/education-products.html). Furthermore, high school teachers might want to consider collaborating with a university or community college where equipment for molecular analyses is standard. Certain vendors also offer used laboratory equipment for a fraction of the regular price (e.g., https://www.labx.com, http://www.biosurplus.com, https://www.cambridgescientific.com/used-lab-equipment, and even eBay: https://www.ebay.com/b/Lab-Equipment/).

To reduce costs, students can work in pairs or in groups of four. Omitting the purification and sequencing of the PCR products could help in further reducing the costs of this exercise. Students would also obtain many benefits of this exercise by only working with the NNDSS and WSS databases, which can be taught separately from the molecular part of this exercise.

Safety

It is important that students receive instruction about safety measures prior to this experiment. At the beginning of the project, students should be instructed about safety measures during sampling and during work in the laboratory. Any soil sample can potentially contain a pathogenic microorganism. We suggest performing soil sampling during the wet season, so that students are not exposed to dust that may include spores from soilborne pathogens. To avoid any potential danger of exposing students to soilborne microorganisms, soil samples could also be collected by the instructor, who should wear a dust mask (e.g., N95 or similar) when samples are collected during the dry season. Once soil aliquots are transferred to microbead tubes supplied by the DNA extraction kit, students are no longer exposed to the original soil samples, and then even immune-compromised students are not at risk of contracting an infectious disease from soilborne microbes. Regardless, safety procedures should be followed that are essential in all laboratories, meaning students should wear lab coats and nitrile gloves during extraction procedure and PCR preparations and apply aseptic techniques to avoid cross-contamination of samples.

Experimental Details

Following the instructor's presentation of background information on valley fever, the recent increase in reported cases, and techniques used in this exercise, students develop a sampling plan that covers different soil types by using the WSS database (https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm). Students also access disease incidence information, which is included in the inquiry part of this exercise. Step-by-step instructions for accessing these databases are displayed in Figure 2.

Figure 2.

Flowchart showing, step by step, how valley fever incidence data and soil parameter information can be obtained from publicly available online sources. Students can use the data to further explore disease incidence over time by making a line graph or by investigating differences of soil parameters for Coccidioides positive versus negative sites (Microsoft Excel also provides tools to perform correlations between sets of values).

Figure 2.

Flowchart showing, step by step, how valley fever incidence data and soil parameter information can be obtained from publicly available online sources. Students can use the data to further explore disease incidence over time by making a line graph or by investigating differences of soil parameters for Coccidioides positive versus negative sites (Microsoft Excel also provides tools to perform correlations between sets of values).

The investigation portion of this exercise is initiated by collecting soil samples (~25 g, 5–10 cm depth). As noted above, soil sampling should not be performed in the dry season, to avoid putting students at risk of inhaling spores and conidia from dormant soil microbes. Soil samples should be processed immediately or frozen at −20°C to avoid changes in the microbial community. DNA extractions using commercially available kits are widely used in molecular biological laboratories and are very convenient to work with. DNA extractions should follow the manufacturer's protocol. Briefly, soil samples are added to a bead beating tube for rapid and thorough homogenization using a vortexer. Cell lysis occurs by mechanical and chemical methods, using various buffers, and incubations on ice. Supernatants that contain DNA are separated from cell debris via centrifugation, and genomic DNA is captured on a silica membrane in a spin column. The captured DNA is then washed and eluted from the membrane and ready for PCR reactions. To ensure that DNA extractions were successful, aliquots of extracted DNA (e.g., 8 μL) are mixed with a few microliters of agarose loading dye, investigated via 2% agarose gel electrophoresis (185 V, 20 minutes in 1XTBE buffer), and documented using a UV light transilluminator. When 2% agarose gels are prepared, 4 μL of SYBR Safe gel stain are added to 100 mL of molten agarose before the gel is poured. Once the successful extraction of DNA is confirmed, a multiplex PCR (25 μL reaction) is prepared to target the pathogen. The multiplex PCR in this exercise uses two primer pairs: one based on the 18S ribosomal gene to target fungi in general (RDS478/RDS482, ~350 bp), and one Coccidioides specific primer pair (ITSC1A/ITSC2, 220 bp) that selectively amplifies a highly variable area of the ITS2 region of the ribosomal gene (Greene et al., 2000; Lauer et al., 2012). The Green GoTaq Mastermix is used to prepare 25 μL PCRs following recommendations of aliquots of DNA, primer, and sterile PCR water in the manufacturer's protocol. The success of the multiplex PCRs can be investigated via 2% agarose gel electrophoresis viewed on a UV light transilluminator. Details about PCR procedures, including cycling conditions, can be obtained from Greene et al. (2000) and Lauer et al. (2014). Bands that indicated the presence of C. immitis are excised from the agarose gel, and DNA can be extracted with the Zymo clean gel DNA recovery kit following the manufacturer's protocol. Retrieved DNA can then be used for re-amplification with the C. immitis specific primer pair ITSC1A/ITSC2 using 35 cycles instead of 50, followed by a purification step using 2 μL of the ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix) and 5 μL of a PCR product, followed by an incubation step of 15 minutes at 37°C and 15 minutes at 80°C (in a PCR cycler). The purified PCR product is then sequenced and compared to entries in the GenBank nucleotide database provided by the National Center of Bioinformatics and Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) that identifies closest matches to an unknown sequence and thus can confirm that a sequence originates from Coccidioides spp. (see https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). For an overview of methods and techniques used in this exercise, see Figure 1.

Student Instruction

A laboratory group exercise can be successful and generate meaningful data only if teachers and students are interested and engaged, work together as a team, and have acquired the necessary knowledge and basic skills. After the teacher introduces the exercise, a discussion of the topic and the methods should eliminate any misunderstanding on the students' side and encourage critical thinking. Therefore, teachers should guide students to formulate meaningful hypotheses and predictions regarding the outcome of this exercise using the scientific method. This engages students in “inquiry” before they start the “investigation.” Well-developed hypotheses like the following should be formulated:

It is hypothesized that the pathogen distribution is influenced by soil type. The prediction is that multiplex PCRs will result more often in positive amplicons for DNA extracts obtained from natural soils with elevated pH compared to soils with a lower pH.

We suggest that students work in pairs or small groups in the laboratory. This multi-focus exercise includes three lectures (~75 minutes each, covering background information, discussion, theory/application of methods, and discussion of results) and four laboratory parts (~2.5 hours each):

Table 1.
Suggested timeline with lecture and lab content, learning outcome, and student assessment. Quizzes (10 minutes, five questions) should be given at the end of the lecture or at the beginning of the next lecture or lab period.
TopicType of InstructionTimeLearning OutcomeStudent Assessment
Introduction to valley fever, introduction to the USDA WSS database Lecture 1 and homework ~75 min Understanding the underlying causes of increase in valley fever in the U.S., ability to use the USDA WSS to propose a soil sampling plan that includes different types of soil Quiz and homework 
Introduction to DNA extraction, PCR, gel electrophoresis, and sequencing Lecture 2 and homework ~75 min Understanding the principles of molecular tools used in this exercise Quiz 
Introduction to the NNDSS Morbidity Tables, accumulation of incidence data on valley fever, generating graphs with Microsoft Excel that show increased incidence over time or that compare incidence in different counties, completion of soil sampling plan Lab 1 ~2.5 h Acquiring skills to use the NNDSS Morbidity Tables and to work with the program Excel Homework 
Review of introductory material, performing DNA extraction and agarose gel electrophoresis Lab 2 ~2.5 h Acquiring skills to perform DNA extractions, perform gel electrophoresis, and being able to evaluate whether the extraction was successful Discussion and quiz 
Review of introductory material, performing PCR and agarose gel electrophoresis Lab 3 ~2.5 h Acquiring skills to perform PCR and gel electrophoresis and being able to evaluate whether desired amplicons were obtained Discussion and quiz 
Discussion of all results, instructions on how to write a report Lecture 3 ~75 min Acquiring writing skills, scientific writing Lab report 
TopicType of InstructionTimeLearning OutcomeStudent Assessment
Introduction to valley fever, introduction to the USDA WSS database Lecture 1 and homework ~75 min Understanding the underlying causes of increase in valley fever in the U.S., ability to use the USDA WSS to propose a soil sampling plan that includes different types of soil Quiz and homework 
Introduction to DNA extraction, PCR, gel electrophoresis, and sequencing Lecture 2 and homework ~75 min Understanding the principles of molecular tools used in this exercise Quiz 
Introduction to the NNDSS Morbidity Tables, accumulation of incidence data on valley fever, generating graphs with Microsoft Excel that show increased incidence over time or that compare incidence in different counties, completion of soil sampling plan Lab 1 ~2.5 h Acquiring skills to use the NNDSS Morbidity Tables and to work with the program Excel Homework 
Review of introductory material, performing DNA extraction and agarose gel electrophoresis Lab 2 ~2.5 h Acquiring skills to perform DNA extractions, perform gel electrophoresis, and being able to evaluate whether the extraction was successful Discussion and quiz 
Review of introductory material, performing PCR and agarose gel electrophoresis Lab 3 ~2.5 h Acquiring skills to perform PCR and gel electrophoresis and being able to evaluate whether desired amplicons were obtained Discussion and quiz 
Discussion of all results, instructions on how to write a report Lecture 3 ~75 min Acquiring writing skills, scientific writing Lab report 

To enhance their learning outcomes and increase their awareness of the ongoing valley fever epidemic, students should obtain valley fever incidence data from the NNDSS tables for a specified number of years. The data should be entered into a spreadsheet and used to produce a graph that shows a change in incidence over time (Figure 3).

Figure 3.

(A) Total numbers of reported coccidioidomycosis cases, 2010–2017, in Kern County, California, and Maricopa County, Arizona. (B) Incidence of coccidioidomycosis in Kern and Maricopa counties. Incidence can be calculated by dividing the number of cases by the population (population numbers are available from https://www.census.gov) and then multiplying by the factor K (e.g., 100,000). Note that even though the reported cases in Maricopa County are higher, incidence of the disease is higher in Kern County since 2013.

Figure 3.

(A) Total numbers of reported coccidioidomycosis cases, 2010–2017, in Kern County, California, and Maricopa County, Arizona. (B) Incidence of coccidioidomycosis in Kern and Maricopa counties. Incidence can be calculated by dividing the number of cases by the population (population numbers are available from https://www.census.gov) and then multiplying by the factor K (e.g., 100,000). Note that even though the reported cases in Maricopa County are higher, incidence of the disease is higher in Kern County since 2013.

We recommend letting students explore the WSS database to determine where soil samples should be collected, leading to the diversity of soil types included in this exercise. Coccidioides spp. are often detected in soils with an elevated pH, increased conductivity, high percentages of fine particles (silt and clay), and natural vegetation, compared to agricultural soils and garden soils. Based on that knowledge, hypotheses and predictions can be formulated like the ones outlined earlier. Figure 4 shows a map of the CSUB campus (our area of interest, which students used to develop a soil sampling plan) and indicates where the pathogen was detected. Figure 5 shows different pH values in the area of interest, and Figure 6 shows results of the diagnostic PCR for several of the investigated soil samples. We suggest that students use worksheets (Table 2).

Table 2.
Sample student worksheet.
Site Location & Brief DescriptionMolecular Biological TasksEnvironmental Parameters
Sampling SiteCoordinatesSite DescriptionDNA Extraction (Yes/No)Diagnostic PCR (Pos/Neg)Closest Match in GenBank, Accession Number, and Percent SimilaritySoil TypeMap Unit SymbolpHElectrical Conductivity (mS/mm)Percent clayWind Erodibility Index
CSU campus, site 2 35.343475 N, 119.09808 W Eroded field, dry grass, scattered small shrubs, multiple rodent holes, loose soil Yes Pos C. immitis, KY306695, 100% Kimberlina fine sandy loam, 0 to 2 % slopes 174 10 86 
Site Location & Brief DescriptionMolecular Biological TasksEnvironmental Parameters
Sampling SiteCoordinatesSite DescriptionDNA Extraction (Yes/No)Diagnostic PCR (Pos/Neg)Closest Match in GenBank, Accession Number, and Percent SimilaritySoil TypeMap Unit SymbolpHElectrical Conductivity (mS/mm)Percent clayWind Erodibility Index
CSU campus, site 2 35.343475 N, 119.09808 W Eroded field, dry grass, scattered small shrubs, multiple rodent holes, loose soil Yes Pos C. immitis, KY306695, 100% Kimberlina fine sandy loam, 0 to 2 % slopes 174 10 86 
Figure 4.

Soil sampling plan showing different soil types on the CSUB campus. The area of interest lies within the white square. Areas of different soil types are separated by white dashed lines, and their soil unit numbers are in white dashed squares: 125 (Granoso loamy sand), 243 (Wasco sandy loam), 127 (Granoso sandy loam), 174 (Kimberlina fine sandy loam). Two lab sections participated in this exercise. Sites where the pathogen was detected via diagnostic PCR are indicated with black dots within white circles. Coccidioides immitis negative sites are denoted by white dots. Letters next to dots are students' initials. This soil sampling plan was generated using the USDA WSS database and Microsoft PowerPoint.

Figure 4.

Soil sampling plan showing different soil types on the CSUB campus. The area of interest lies within the white square. Areas of different soil types are separated by white dashed lines, and their soil unit numbers are in white dashed squares: 125 (Granoso loamy sand), 243 (Wasco sandy loam), 127 (Granoso sandy loam), 174 (Kimberlina fine sandy loam). Two lab sections participated in this exercise. Sites where the pathogen was detected via diagnostic PCR are indicated with black dots within white circles. Coccidioides immitis negative sites are denoted by white dots. Letters next to dots are students' initials. This soil sampling plan was generated using the USDA WSS database and Microsoft PowerPoint.

Figure 5.

Averaged pH values of soils around the CSUB campus (in the center) obtained from the USDA WSS database: pH 7 (light gray) and pH 7.5–8 (darker gray). Different soil types are indicated by soil map unit numbers in white dashed squares: 125 (Granoso loamy sand), 243 (Wasco sandy loam), 127 (Granoso sandy loam), 174 (Kimberlina fine sandy loam), and others outside our area of interest.

Figure 5.

Averaged pH values of soils around the CSUB campus (in the center) obtained from the USDA WSS database: pH 7 (light gray) and pH 7.5–8 (darker gray). Different soil types are indicated by soil map unit numbers in white dashed squares: 125 (Granoso loamy sand), 243 (Wasco sandy loam), 127 (Granoso sandy loam), 174 (Kimberlina fine sandy loam), and others outside our area of interest.

Figure 6.

Results of multiplex PCRs indicating the presence of fungi in many soil samples (~600 bp). The pathogen was detected in some (~220 bp). (A) Coccidioides immitis was detected in sample JCo. (B) The pathogen was detected in samples JC, PS, EJ, Nora, and KD (PC = positive control, DNA from C. immitis; NC = negative control; not all class data are shown, white arrows point toward PCR amplicons of correct size).

Figure 6.

Results of multiplex PCRs indicating the presence of fungi in many soil samples (~600 bp). The pathogen was detected in some (~220 bp). (A) Coccidioides immitis was detected in sample JCo. (B) The pathogen was detected in samples JC, PS, EJ, Nora, and KD (PC = positive control, DNA from C. immitis; NC = negative control; not all class data are shown, white arrows point toward PCR amplicons of correct size).

Student Assessment

Students are assessed regarding their understanding of the topic via two conceptual quizzes early on in this exercise (see Supplemental Material with the online version of this article). The first quiz includes questions about the life cycle of Coccidioides, symptoms and signs of valley fever, endemic areas, at-risk populations, factors that cause increased disease incidence, and the soil environment as a habitat for Coccidioides. Students take this assessment after the instructor has given two lectures on the topic and students have assessed disease incidence data from the NNDSS website and have suggested a soil sampling plan. The second quiz assesses comprehension of methods, primarily regarding developing a comprehensive sampling plan, performing DNA extractions, PCR procedures, gel electrophoresis, and sequencing principles. Assessment includes questions about the theories and applications behind these methods. These quizzes reveal whether students are ready for the investigation portion of this exercise. An instructor-facilitated discussion focusing on students' experimental results, with reflection on the hypotheses formulated earlier, is also used to further assess student learning outcomes. Methodological issues that are encountered during DNA extractions and PCR are explored during the discussion assessment. Finally, students write individual lab reports with analyses of class results using the format and structure of a research manuscript (Introduction, Material and Methods, Results, Discussion, References).

Teacher Implementation

The exercise proposed here is based on original ongoing research at CSUB. We recommend collecting soil samples in the spring season when the soil is moist and has warmed up, so that microorganisms are present in their vegetative form, which facilitates DNA extractions and increases the chances of detecting Coccidioides. Again, sampling in the dry season is not recommended in Coccidioides endemic areas, to avoid putting students at risk of contracting valley fever, and if samples are collected during the dry season by the instructor, he or she should wear an N95 dust mask in the field. In the dry season, the pathogen and most other microbes are dormant and additional steps should be undertaken to extract DNA from dormant microbes (e.g., three freeze–thaw cycles at 70°C and −20°C, each 15 minutes, each followed by a proteinase K treatment at 56°C for one hour). The sequencing part of the exercise is needed for ultimate confirmation of the presence of the pathogen. However, the primer pair is very specific and only rarely results in false-positive amplicons (slightly longer or shorter than 220 bp) and therefore could be omitted.

We recommend that teachers consider applying for extramural support for laboratory supplies and equipment or collaborate with a university. The National Science Foundation has funded research at high schools through special education grants on STEM in the past (https://search.nsf.gov/search?query=high+school+grant&affiliate= nsf&search=).

Teachers in states where incidence of valley fever is low or absent can adopt the general structure of this exercise with another opportunistic soilborne fungal pathogen in the focus, for example Histoplasma capsulatum (the most common fungal pathogen in the United States), Cryptococcus spp., or Blastomyces dermatitidis. Diagnostic primer pairs for these pathogens can be found in peer-reviewed literature using Google Scholar (e.g., McCarthy & Walsh, 2016, and references within). Soilborne bacterial pathogens such as Clostridium tetani, Clostridium botulinum, or Bacillus anthracis can also be detected via diagnostic PCRs and could be the target of a similar exercise by using the freely available Insignia program (http://insignia.cbcb.umd.edu) to produce DNA signatures for these species (see Phillippy et al., 2009; Nagamine et al., 2015) that can be used in PCRs. Teachers who are concerned about the safety of their students can focus on nonpathogenic fungi (e.g., some Penicillium or Fusarium spp.) or plant pathogens such as Phytophtora spp., by focusing on developing a soil sampling plan and performing DNA extractions and PCR, excluding working with the NNDSS database.

We thank all the students in the General Microbiology course 2013 for their participation and acknowledge Ajaypal Dhillon and Burton Martinez for some of the data analyses. Furthermore, we acknowledge support from the Louis Stokes Alliance for Minorities (LSAMP, NSF-HRD no. 0331537), which generously supported Alex Valenzuela, and we thank Professor Maynard Moe for reviewing our manuscript.

References

References
Ampel, N.M. (
2011
). Coccidioidomycosis. In
Essentials of Clinical Mycology
(pp.
349
366
).
New York, NY
:
Springer
.
Brown, J., Benedict, K., Park, B.J. & Thompson, G.R., III (
2013
).
Coccidioidomycosis: epidemiology
.
Clinical Epidemiology
,
5
,
185
.
Cat, L.A., Gorris, M.E., Randerson, J.T., Riquelme, M. & Treseder, K.K. (
2017
).
Crossing the line: human disease and climate change across borders
. https://cloudfront.escholarship.org/dist/prd/content/qt38t7d87v/qt38t7d87v.pdf.
Centers for Disease Control and Prevention
(
2019
).
Valley fever endemic area
. from https://www.cdc.gov/fungal/diseases/coccidioidomycosis/causes.html.
Colson, A.J., Vredenburgh, L., Guevara, R.E., Rangel, N.P., Kloock, C.T. & Lauer A. (
2017
).
Large-scale land development, fugitive dust, and increased coccidioidomycosis incidence in the Antelope Valley of California, 1999–2014
.
Mycopathologia
,
182
,
439
458
.
Cooksey, G.S., Nguyen, A., Knutson, K., Tabnak, F., Benedict, K., McCotter, O., Jain, S. & Vugia, D. (
2017
).
Notes from the field: increase in coccidioidomycosis—California, 2016. MMWR
.
Morbidity and Mortality Weekly Report
,
66
,
833
.
Galgiani, J.N., Ampel, N.M., Blair, J.E., Catanzaro, A., Johnson, R.H., Stevens, D.A. & Williams, P.L. (
2005
).
Coccidioidomycosis
.
Clinical Infectious Diseases
,
41
,
1217
1223
.
Gasper, B.J. & Gardner, S.M. (
2013
).
Engaging students in authentic microbiology research in an introductory biology laboratory course is correlated with gains in student understanding of the nature of authentic research and critical thinking
.
Journal of Microbiology & Biology Education
,
14
(
1
),
25
.
Gorris, M.E., Cat, L.A., Zender, C.S., Treseder, K.K. & Randerson, J.T. (
2018
).
Coccidioidomycosis dynamics in relation to climate in the southwestern United States
.
GeoHealth
,
2
,
6
24
.
Greene, D.R., Koenig, G., Fisher, M.C. & Taylor, J.W. (
2000
).
Soil isolation and molecular identification of Coccidioides immitis
.
Mycologia
,
1
,
406
410
.
Huppert, J., Lomask, S.M. & Lazarowitz, R. (
2002
).
Computer simulations in the high school: Students' cognitive stages, science process skills and academic achievement in microbiology
.
International Journal of Science Education
,
24
,
803
821
.
Lauer, A., Baal, J.D., Baal, J.C., Verma, M. & Chen, J.M. (
2012
).
Detection of Coccidioides immitis in Kern County, California, by multiplex PCR
.
Mycologia
,
104
,
62
69
.
Lauer, A., Talamantes, J., Olivares, L.R., Medina, L.J., Baal, J.D., Casimiro, K., et al. (
2014
).
Combining forces – the use of landsat TM satellite imagery, soil parameter information, and multiplex PCR to detect Coccidioides immitis growth sites in Kern County, California
.
PLoS One
,
9
,
e111921
.
Marsden-Haug, N., Hill, H., Litvintseva, A.P., Engelthaler, D.M., Driebe, E.M., Roe, C.C., et al. (
2014
).
Coccidioides immitis identified in soil outside of its known range – Washington, 2013
.
Morbidity and Mortality Weekly Report
,
63
,
450
.
McCarthy, M.W. & Walsh, T.J. (
2016
).
PCR methodology and applications for the detection of human fungal pathogens
.
Expert Review of Molecular Diagnostics
,
16
,
1025
1036
.
McKenney, E., Flythe, T., Millis, C., Stalls, J., Urban, J.M., Dunn, R.R. & Stevens, J.L. (
2016
).
Symbiosis in the soil: citizen microbiology in middle and high school classrooms
.
Journal of Microbiology & Biology Education
,
17
(
1
),
60
.
Nagamine, K., Hung, G.C., Li, B. & Lo, S.C. (
2015
).
DNA sequence signatures for rapid detection of six target bacterial pathogens using PCR assays
.
Microbiology Insights
,
8
, MBI-S29736.
Phillippy, A.M., Ayanbule, K., Edwards, N.J. & Salzberg, S.L. (
2009
).
Insignia: a DNA signature search web server for diagnostic assay development
.
Nucleic Acids Research
,
37
,
W229
W234
.
Smith, C.E. (
1940
).
Epidemiology of acute coccidioidomycosis with erythema nodosum (“San Joaquin” or “Valley Fever”)
.
American Journal of Public Health and the Nation's Health
,
30
,
600
611
.
Turabelidze, G., Aggu-Sher, R.K., Jahanpour, E. & Hinkle, C.J. (
2015
).
Coccidioidomycosis in a State where it is not known to be endemic – Missouri, 2004–2013
.
Morbidity and Mortality Weekly Report
,
64
,
636
639
.
Valley Fever Center for Excellence
, University of Arizona at Tucson (
2018
). https://vfce.arizona.edu/sites/vfce/files/tutorial_for_primary_care_ professionals_0.pdf.
Vargas-Gastélum, L., Romero-Olivares, A.L., Escalante, A.E., Rocha-Olivares, A., Brizuela, C. & Riquelme, M. (
2015
).
Impact of seasonal changes on fungal diversity of a semi-arid ecosystem revealed by 454 pyrosequencing
.
FEMS Microbiology Ecology
,
5
,
1
13
.

Supplementary data