An MNP marker site for Histoplasma capsulatum, primer composition, kit and its application

Abstract

This invention discloses an MNP marker site for *Histoplasma capsulatum*, a primer composition, a kit, and their applications. The MNP marker site refers to a genomic region selected from the *Histoplasma capsulatum* genome that distinguishes it from other species and possesses multiple nucleotide polymorphisms within the species, including at least one of the marker sites MNP-1 to MNP-3. The primers are shown in SEQ ID NO.1 to SEQ ID NO.6. The primer pair combination is designed as a multiplex PCR amplification primer that can amplify the MNP marker in a single reaction. The kit integrates multiplex PCR amplification and next-generation sequencing technologies, performing sequence analysis of multiple MNP markers of *Histoplasma capsulatum* in a single reaction. It has the technical advantages of high throughput, multiple targets, high sensitivity, high accuracy, and culture-free operation in applications such as disease prevention and control, scientific research, and mutation monitoring of *Histoplasma capsulatum*.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an MNP marker site for Histoplasma capsulatum, a primer composition, a kit, and its application. Background Technology

[0002] Histoplasma capsulatum causes histoplasmosis, a highly contagious deep fungal infection that primarily affects the reticuloendothelial system or lungs. Identification and genetic variation detection of Histoplasma capsulatum are crucial for monitoring the pathogenicity of the fungus and the performance of industrial microorganisms. Traditional fungal detection methods mainly include microscopic examination after smear staining, pathogen isolation and culture, and nucleic acid detection technologies based on PCR and metagenomic sequencing, which have been developed in recent years. These technologies each have their advantages, but they also have one or more limitations in terms of time, operational complexity, throughput, accuracy and sensitivity of variant detection, and cost. For example, fungal isolation and identification are complex and time-consuming, and cannot detect variants; PCR detection technology targets only one or two markers of a fungus per reaction, resulting in low efficiency and susceptibility to detection failure due to primer region variations, and it cannot detect variants simultaneously. Metagenomic sequencing technology often involves large amounts of host sequencing data; fungal genomes are large, and testing samples with low pathogen loads requires ultra-deep sequencing, leading to high data sequencing costs.

[0003] Therefore, how to provide multiple novel molecular markers with high polymorphism for Histoplasma capsulatum and achieve efficient, accurate, and sensitive detection and variation monitoring of Histoplasma capsulatum in a single assay is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a specific MNP marker site for Histoplasma capsulatum, primer composition, kit, and its application, which can be used for qualitative identification and mutation detection of Histoplasma capsulatum, and has the effects of multi-target, high throughput, and high sensitivity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect of the invention, a combination of MNP markers for Histoplasma capsulatum is provided, the combination of MNP markers including at least one of three marker sites, MNP-1 to MNP-3, which are genomic regions selected on the Histoplasma capsulatum genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, and the nucleotide sequences of the marker sites MNP-1 to MNP-3 are shown in SEQ ID NO.7 to SEQ ID NO.9.

[0007] In the above technical solution, the specific MNP-1 to MNP-3 marker sites are shown in Table 1 of the specification. The start and end positions of the MNP markers marked in Table 1 are determined based on the reference sequence corresponding to the same row of MNPs in Table 1.

[0008] In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition comprising at least one of three pairs of primers, the nucleotide sequences of the three pairs of primers being shown in SEQ ID NO.1–SEQ ID NO.2, SEQ ID NO.3–SEQ ID NO.4, and SEQ ID NO.5–SEQ ID NO.6, respectively.

[0009] In the above technical solution, the primers for each MNP marker site include an upper primer and a lower primer, as shown in Table 1 of the specification.

[0010] In a third aspect of the invention, a detection kit is provided for detecting the MNP marker site of Histoplasma capsulatum, the kit comprising the primer composition described above.

[0011] Furthermore, the kit also includes a multiplex PCR premix.

[0012] In a fourth aspect of the invention, the application of the MNP marker site of Histoplasma capsulatum, the multiplex PCR primer composition, or the detection kit described herein is provided in the identification of Histoplasma capsulatum.

[0013] In a fifth aspect of the invention, the application of the MNP marker site of Histoplasma capsulatum, the multiplex PCR primer composition, or the detection kit described herein is provided in the monitoring of Histoplasma capsulatum variation and the construction of a database.

[0014] The specific operating steps in the above-described application are as follows:

[0015] First, total fungal DNA is obtained from the sample to be tested. Using the kit of this invention, a first round of multiplex PCR amplification is performed on the total DNA and a blank control, with a cycle number not exceeding 25. After purification of the amplification products, sample tags and next-generation sequencing adapters based on a second round of PCR amplification are added. The second-round amplification products are purified and quantified. When detecting multiple strains, equal volumes of the second-round amplification products are mixed and high-throughput sequencing is performed. The sequencing results are aligned to the reference sequence of the fungus to obtain the number of detection sequences and genotype data in the total DNA. Based on the number of sequencing sequences of the fungus obtained from the total DNA and the blank control, and the number of detected MNP sites, data quality control and data analysis are performed on the sequencing data of the total DNA to obtain the number of detected MNP sites, the number of sequencing sequences covering each MNP site, and the genotype data of the MNP sites.

[0016] When used for the identification of *Histoplasma capsulatum*, the presence of *Histoplasma capsulatum* nucleic acid in the test sample is determined after quality control based on the number of *Histoplasma capsulatum* sequencing sequences and the number of detected MNP sites in the test sample and blank control. Specifically, the quality control protocol and determination method use *Histoplasma capsulatum* DNA with a known copy number as the test sample to evaluate the sensitivity, accuracy, and specificity of the kit for detecting *Histoplasma capsulatum*, and to formulate the quality control protocol and determination method for the kit's detection of *Histoplasma capsulatum*.

[0017] When used in the construction of a Histoplasma capsulatum variation monitoring and DNA fingerprint database, the genotype data of the MNP loci of Histoplasma capsulatum identified from the samples are entered into a database file to form a DNA fingerprint database of Histoplasma capsulatum. Each time a different sample is identified, the Histoplasma capsulatum in the sample is compared with the DNA fingerprint database of Histoplasma capsulatum to determine whether the Histoplasma capsulatum in the sample differs from the strains in the database in terms of major genotype (genotype supported by more than 50% sequencing fragments at one MNP locus). Histoplasma capsulatum with major genotype differences at at least one MNP locus is considered a new variant type, and its genotype is included in the DNA fingerprint database.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] This invention provides MNP marker sites, primer compositions, kits, and applications for *Histoplasma capsulatum*. By analyzing the genome sequence of *Histoplasma capsulatum*, this invention screened three MNP marker sites and designed a multiplex primer composition based on the sequence information of these three MNP marker sites. Multiplex PCR amplification was performed using the designed multiplex primer composition, and the amplified products were sequenced using a next-generation sequencing platform. This allows for the simultaneous detection of all three MNP marker sites in *Histoplasma capsulatum*, overcoming the limitations of existing technologies that rely on parallel testing of standard samples for detecting only one marker in one *Histoplasma capsulatum* species. This invention achieves high-throughput, high-efficiency, and high-accuracy detection of *Histoplasma capsulatum*, providing technical support for *Histoplasma capsulatum* identification and variation monitoring. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram illustrating the principle of MNP marker polymorphism provided in an embodiment of this application;

[0022] Figure 2 A flowchart of screening and primer design for MNP marker sites in Histoplasma capsulatum provided in this application embodiment;

[0023] Figure 3 This is a flowchart illustrating the detection process of MNP marker sites provided in an embodiment of this application. Detailed Implementation

[0024] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0027] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0028] This application provides MNP marker sites for Histoplasma capsulatum. The MNP marker sites are species-specific genomic regions selected from the Histoplasma capsulatum genome that have multiple nucleotide polymorphisms within the species. The MNP marker sites include at least one of three marker sites from MNP-1 to MNP-3, and the nucleotide sequences of the marker sites from MNP-1 to MNP-3 are shown in SEQ ID NO.7 to SEQ ID NO.9.

[0029] Based on a general inventive concept, embodiments of this application provide a multiplex PCR primer composition for detecting the MNP marker site, the primer composition comprising at least one of three pairs of primers, the nucleotide sequences of the three pairs of primers being shown in SEQ ID NO.1~SEQ ID NO.2, SEQ ID NO.3~SEQ ID NO.4, and SEQ ID NO.5~SEQ ID NO.6, respectively.

[0030] The primers do not conflict with each other and can be used for efficient amplification via multiplex PCR.

[0031] In this embodiment, the composition of the specific multiplex amplification primer composition for detecting MNP marker sites is refined, enabling comprehensive detection of Histoplasma capsulatum, thereby achieving high-throughput, high-efficiency, high-accuracy, and high-sensitivity detection and variation monitoring of Histoplasma capsulatum.

[0032] The multiplex PCR primer composition is designed based on the above-mentioned MNP marker site. The specific composition and region of the MNP marker site can be referred to the above embodiments. Since the multiplex PCR primer composition adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0033] The multiplex PCR primer composition can be used as a detection kit for the MNP marker site of Histoplasma capsulatum, and the kit provided in this application can accurately identify Histoplasma capsulatum.

[0034] Based on a general inventive concept, embodiments of this application provide a kit for detecting the MNP marker site, the kit comprising the multiplex PCR primer composition.

[0035] This kit is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0036] Based on a general inventive concept, embodiments of this application provide the application of the MNP marker site, the primer composition, or the kit in the preparation of an identification product for Histoplasma capsulatum.

[0037] In this embodiment of the application, when used for the identification of Histoplasma capsulatum, the presence of Histoplasma capsulatum nucleic acid in the test sample is determined after quality control based on the number of Histoplasma capsulatum sequencing sequences and the number of MNP markers detected in the test sample and blank control. The quality control scheme and determination method use known Histoplasma capsulatum DNA as the detection sample to evaluate the sensitivity, accuracy, and specificity of the kit for detecting Histoplasma capsulatum, and to formulate the quality control scheme and determination method for detecting Histoplasma capsulatum using the kit.

[0038] In the reproducibility test of this application, the logarithm of the difference in major MNP marker genotypes between different libraries and between different library construction batches for each sample was 0, the reproducibility r = 100%, and the accuracy a = 100%.

[0039] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0040] The following will provide a detailed description of the MNP marker site, primer composition, kit, and application of Histoplasma capsulatum according to this application, in conjunction with examples, comparative examples, and experimental data.

[0041] Example 1: Screening of MNP marker sites in Histoplasma capsulatum and design of primers for multiplex PCR amplification.

[0042] 1. Screening of MNP marker sites in Histoplasma capsulatum:

[0043] The specific steps for the above screening are as follows:

[0044] The genome sequence of a representative strain of Histoplasma capsulatum was selected as a reference genome, and the other genome sequences of the fungus were compared with the reference genome to obtain the single nucleic acid polymorphism sites of the fungus.

[0045] On the reference genome, a window of 100bp to 300bp is used for window translation with a step size of 1bp to screen and obtain regions of multiple candidate MNP marker sites. The candidate MNP marker site regions are required to contain ≥2 of the single nucleotide variant sites and there are no single nucleotide polymorphism sites on the sequences at both ends of 30bp.

[0046] At least five sites with high DP (discrimination) values ​​in the candidate polynucleotide polymorphism (PNP) regions were selected; the selection criteria included the following:

[0047] DP = d / t,

[0048] In the formula, t is the number of comparisons when all genomes are compared pairwise in the candidate polynucleotide polymorphism site region, and d is the number of genome pairs with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.

[0049] Other step sizes can also be used in the window translation stage, not limited to 1 bp, but the use of 1 bp in this application is beneficial for the comprehensive screening of polymorphic sites on the fungal genome.

[0050] After obtaining the MNP marker sites of the fungus, sequence alignment was performed in a public database, and at least 5 fungus-specific sites were selected as candidate MNP marker sites.

[0051] 2. Design of primers for multiplex PCR amplification:

[0052] like Figure 2 As shown, multiplex PCR amplification primers for the candidate MNP marker were designed using primer design software. The primer design followed the principle that primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.

[0053] 3. Evaluation of the detection efficiency of primer combinations:

[0054] The MNP marker detection kit was used to detect positive samples of Histoplasma capsulatum, with a total of 3 replicates. Sequencing data were analyzed to screen out 3 pairs of highly compatible and product-specific primers for the specific identification of 3 MNP markers of Histoplasma capsulatum.

[0055] like Figure 1 As shown in Table 1, based on the publicly available genome sequence of Histoplasma capsulatum, three MNP marker sites for Histoplasma capsulatum were selected through sequence alignment.

[0056] Table 1 - The starting positions of the MNP marker sites on the reference sequence and the sequences of the detection primers.

[0057]

[0058] Example 2: Performance evaluation and threshold setting of MNP markers and primers for identifying Histoplasma capsulatum

[0059] 1. The detection of Histoplasma capsulatum exhibits technical stability, high specificity, and sensitivity as low as 10 copies / reaction.

[0060] Samples of *Histoplasma capsulatum* were prepared with copy numbers of 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction. An equal volume of sterile water was used as a blank control. Three replicate libraries were analyzed for each sample over four consecutive days, resulting in 12 sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and thresholds for quality control system contamination and detection of the target *Histoplasma capsulatum* were established. The detection procedure for MNP marker sites is as follows: Figure 3 As shown.

[0061] Table 2 shows the sensitivity and stability analysis of the MNP-labeled detection kit for detecting Histoplasma capsulatum.

[0062]

[0063]

[0064] As shown in Table 2, in the detection of positive samples with 10 copies / reaction and 100 copies / reaction, all three MNP marker sites of Histoplasma capsulatum were detected, and the sequence was specifically aligned with the reference sequence of the fungus. This indicates that the primers and detection method have technical stability, high specificity and sensitivity as low as 10 copies / reaction for detecting the fungus.

[0065] 2. Evaluation of the reproducibility and accuracy of the MNP marker detection kit for Histoplasma capsulatum:

[0066] Based on whether the genotypes of the common detection sites in the two replicates can be reproduced, the reproducibility and accuracy of the MNP marker detection method for detecting Histoplasma capsulatum were evaluated. Specifically, the genotypes of each MNP site generated from 12 sets of data from 100 copies / reaction positive samples were compared pairwise, and the results are shown in Table 3.

[0067] Table 3 - Reproducibility and accuracy assessment of MNP marker detection kits for genotype detection

[0068] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a S-1 S-2 3 3 100％ 100％ S-1 S-3 3 3 100％ 100％ S-1 S-4 3 3 100％ 100％ S-1 S-5 3 3 100％ 100％ S-1 S-6 3 3 100％ 100％ S-1 S-7 3 3 100％ 100％ S-1 S-8 3 3 100％ 100％ S-1 S-9 3 3 100％ 100％ S-1 S-10 3 3 100％ 100％ S-1 S-11 3 3 100％ 100％ S-1 S-12 3 3 100％ 100％

[0069] As shown in Table 3, the number of MNP markers with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of the number of reproducible loci to the number of shared loci. In the reproducibility test of this invention, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, i.e., the reproducibility rate r = 100%, and the accuracy a = 100%.

[0070] In both 10-copy / reaction and 100-copy / reaction positive samples, the three MNP markers of Histoplasma capsulatum were reliably detected, while at most one MNP marker was detected in the blank control. Therefore, the criterion for determining Histoplasma capsulatum positivity in this application is as follows: when at least two MNP marker sites of Histoplasma capsulatum are detected in the sample and the abundance of the detected sequences (i.e., the ratio of the number of sequences in the sample that are aligned to the MNP marker sites of Histoplasma capsulatum to the total number of sequences in the sample) is at least 10 times that in the blank control, the nucleic acid of Histoplasma capsulatum is detected in the sample.

[0071] Example 3: Application of MNP marker sites, kits, and methods in the identification of Histoplasma capsulatum

[0072] Six nucleic acid samples provided by Hubei Provincial Center for Disease Control and Prevention were tested using an MNP marker site detection kit. The samples were named S1-S6 (S1 was a Histoplasma capsulatum negative sample, and S2-S6 were Histoplasma capsulatum positive samples). The results are shown in Table 4.

[0073] Table 4 - Analysis of Histoplasma capsulatum detection in samples

[0074] Sample number Fungi detected by sequencing Number of MNP markers detected Judgment Result S1 Histoplasma capsulatum 0 Not detected S2 Histoplasma capsulatum 3 Detected S3 Histoplasma capsulatum 3 Detected S4 Histoplasma capsulatum 3 Detected S5 Histoplasma capsulatum 3 Detected S6 Histoplasma capsulatum 3 Detected

[0075] As shown in Table 4, the kit and method accurately detected all three MNP marker sites in *Histoplasma capsulatum* in each sample in a single reaction. In contrast, the same detection method based on fluorescent PCR, which detects one marker per reaction, requires three tests, demonstrating the accuracy and efficiency of the kit and method in detecting *Histoplasma capsulatum*.

[0076] Example 4: Application of MNP marker sites, kits, and methods in the detection of genetic variations in Histoplasma capsulatum.

[0077] Four copies of a single *Histoplasma capsulatum* strain were tested using the aforementioned kit and MNP marker combination detection method. The samples were named S1-S4. All three MNP markers of *Histoplasma capsulatum* were detected in all samples, with an average sequencing coverage of 4000-fold for each MNP marker (Table 5). Pairwise comparisons of the genotypes of the four strains at the three markers were performed. The results are shown in Table 5. S2 showed major genotypic differences compared to the three new *Histoplasma capsulatum* samples tested in the same batch (Table 5), indicating that S2 exhibits genetic variation.

[0078] Table 5-1 Detection and analysis of four copy samples of a novel Cryptococcus strain.

[0079]

[0080] As shown in Table 5, the kit can identify genetic variations between strains by detecting the sequence of MNP markers. It can be used to detect variations between strains in disease prevention and control monitoring, and to ensure the genetic consistency of the same named strains in different laboratories in scientific research, thereby ensuring the comparability of research results. Therefore, it is of great significance for disease prevention and control monitoring, precision treatment and scientific research.

[0081] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0082] (1) The three MNP marker sites of Histoplasma capsulatum provided in the embodiments of this application have high specificity in the identification of Histoplasma capsulatum;

[0083] (2) The multiplex PCR amplification primer composition for Histoplasma capsulatum provided in this application can amplify three MNP marker sites in one reaction and integrate with a second-generation sequencing platform for sequencing of the amplification products, thereby realizing sequence analysis of three marker sites of Histoplasma capsulatum in one reaction. Compared with the fluorescent PCR method, which only uses one pair of primers to detect one site and detects the fluorescent signal, the multiplex primer pair of this application provides technical support for efficient and accurate sequence-based identification of Histoplasma capsulatum.

[0084] (3) The Histoplasma capsulatum MNP marker site detection kit provided in this application can detect all three MNP marker sites in a single reaction. In the reproducibility test, the three marker sites were stably detected, indicating the high stability of the kit. The logarithm of the difference in major MNP marker genotypes between different libraries and between different library preparation batches detected by the kit was 0, the reproducibility r = 100%, and the accuracy a = 100%, indicating the high accuracy and stability of the kit in detecting Histoplasma capsulatum.

[0085] (4) The method provided in this application uses high-throughput sequencing to detect samples. By adding a unique tag to each sample, it is possible to detect hundreds or thousands of samples at once, which further improves the detection efficiency.

[0086] (5) The method provided in this application uses high-throughput sequencing to detect samples and identifies fungi by detecting the MNP-labeled base sequence, without the need for parallel detection of standard samples. In contrast, the fluorescence PCR-based method detects fluorescence signals and relies on parallel experiments with standard samples.

[0087] (6) The method provided in this application uses high-throughput sequencing to detect samples and obtain the base sequence of the detected MNP marker. In one reaction, not only can fungi be identified, but genetic variations of pathogens between samples can also be detected at the same time.

[0088] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0089] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0090] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A multiplex PCR primer composition for detecting MNP marker combinations in Histoplasma capsulatum, characterized in that, The MNP marker combination includes three marker sites, MNP-1 to MNP-3, with nucleotide sequences as shown in SEQ ID NO.7 to SEQ ID NO.9; the multiplex PCR primer composition includes three pairs of primers, with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.2, SEQ ID NO.3 to SEQ ID NO.4, and SEQ ID NO.5 to SEQ ID NO.6, respectively.

2. A detection kit for detecting MNP marker combinations in Histoplasma capsulatum, characterized in that, The kit includes the primer composition of claim 1.

3. The detection kit according to claim 2, characterized in that, The kit also includes a multiplex PCR premix.

4. The use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in the preparation of an identification product for Histoplasma capsulatum.

5. The application of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in the monitoring of Histoplasma capsulatum variation and database construction for non-diagnostic purposes.

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