Preliminary clinical evaluation of SCAR-type molecular markers for the identification of the section Fumigati, A. flavus, and A. niger in bronchoalveolar lavage samples

The aim of this study was to develop and conduct a preliminary clinical evaluation of SCAR molecular markers for identification of section Fumigati DNA, A. flavus, and A. niger. The RAPD-PCR method was used to obtain specific bands from polymorphic patterns for the section Fumigati, A. flavus, and A. niger. Of the 34 polymorphic patterns obtained, only OPF-01, P54, and 1253 were selected, as they exhibited specific bands for the section Fumigati, A. flavus and A. niger, respectively. SCAR markers were generated from these bands by cloning and sequencing them. Subsequently, the specificity of the designed markers was evaluated by testing them against other species of the genus Aspergillus and other medically important genera. To evaluate sensitivity, serial dilutions (10 ng/µL– 0.001 ng/µL) of genomic DNA from the section Fumigati, A. flavus, and A. niger were used. Finally, the positivity of the SCAR-PCR assay was analyzed in galactomannan-positive bronchoalveolar lavage (BAL) samples from patients evaluated for aspergillosis. The primers OPF-01, P54, and 1253 produced unique bands for the section Fumigati, A. flavus, and A. niger, respectively. Subsequently, three primer pairs were designed: the first amplified a 180 bp band that showed section-level specificity for identifying the section Fumigati DNA; the second amplified a 193 bp band that recognized A. flavus; and the third amplified a 220 bp band in A. niger and A. luchuensis. These SCAR markers were named AfOPF-01 180, AfP54 193.1, and An1253 220. The limit of detection with the SCAR markers was 0.005 ng/µL. SCAR-PCR positivity of the marker OPF-01 180 in BAL samples was 19/51 (37.25%); SCAR-PCR positivity of the marker AfP54 193. 1 was 3/51 (5.88%); There was no SCAR-PCR positivity for the marker An1253 220. Based on the results, the developed SCAR markers show preliminary clinical feasibility, identifying the section Fumigati DNA by the AfOPF-01 180 marker and at the species level by the AfP54 193.1 and An1253 220 markers. However, prospective, multicenter validation is required to evaluate its diagnostic clinical capability.

Species belonging to the genus Aspergillus are widely distributed in the environment, primarily in soil and decaying plant matter, and 453 species are currently recognized (1). Several of these species can cause aspergillosis in humans by inhalation of airborne conidia, which are present in both indoor and outdoor environments. The clinical forms of aspergillosis depend on host factors and the host’s immune response. These include non-invasive forms such as allergic bronchopulmonary aspergillosis (ABPA) and allergic fungal rhinosinusitis, while invasive forms include chronic pulmonary aspergillosis and invasive pulmonary aspergillosis (2, 3). It is important to mention that among the clinically relevant species, due to their ability to infect people with compromised immune systems due to immunosuppression by biologics, solid organ transplantation, or hematopoietic stem cells, neutropenia, chemotherapy and, recently, have been linked to non-traditional risk factors such as SARS-CoV-2 infection and influenza, are mainly Aspergillus fumigatus, A. flavus, A. niger and A. terreus (4–7).

The clinical and environmental importance of species of the section Fumigati, among these are A. fumigatus, as well as A. flavus and A. niger is increasing as global health systems face a growing burden of fungal diseases. These species are among the most common causes of aspergillosis and are associated with high morbidity and mortality, especially in immunocompromised individuals (8–10).

A. fumigatus remains the primary cause of invasive disease in approximately 70–80% of cases, and the World Health Organization recognizes it as a critical priority fungal pathogen due to its high mortality rate and increasing antifungal resistance (11). A. fumigatus could expand its range by up to 77%, potentially exposing millions more people, particularly in temperate regions of Europe, North America, and Asia, to airborne spores. A. flavus, which accounts for approximately 15–20% of infections, is characterized by its aflatoxin production and predominance in warm regions, while A. niger causes fewer invasive infections (approximately 5–10%), which often manifest as respiratory or superficial ear infections, but with increasing clinical relevance (12, 13). A. flavus and A. niger could expand to 16% or more, raising the risks associated with airborne exposure and food chain contamination through aflatoxin-producing strains (14).

The clinical diagnosis of aspergillosis is made by serological detection of biomarkers such as 1,3-β-D-glucan or galactomannan, and by culture. However, these techniques have limitations such as nonspecificity or a lack of positivity (culture) in detecting the etiological agent of this mycosis. Nevertheless, new tools for amplifying Aspergillus DNA using Polymerase Chain Reaction (PCR) have been developed, though only some have been validated (15).

Among the most widely used diagnostic tests for aspergillosis are PCR variants. These include qPCR, Multiplex PCR, Multiplex qPCR, Multiplex real-time PCR, nested PCR, semi-nested PCR, reverse transcriptase PCR, and endpoint PCR. Other tools, such as universal digital high-resolution melt (U-dHRM) and droplet digital PCR (ddPCR), have also been recently integrated. High-throughput sequencing (HTS) techniques—next-generation sequencing (NGS) and third-generation sequencing (TGS)—are used to diagnose aspergillosis from various samples. However, all these options have limitations related to the patient, the presence of fungal DNA in the sample, difficulty distinguishing colonization from infection, and the high cost and inaccessibility in developing countries (16).

Due to these limitations, another alternative for the clinical diagnosis of aspergillosis is the development of specific molecular markers, such as SCAR (Sequence Characterized Amplified Region) markers. Among their advantages is their method of acquisition, as they can be obtained from polymorphic patterns generated by AFLP (Amplified Fragment Length Polymorphism), RFLP (Restriction Fragment Length Polymorphism), RAPD-PCR (Random Amplified Polymorphic DNA), and ISSR (Inter-Simple Sequence Repeat) (17–20). However, the most efficient technique for the development of SCAR markers is RAPD-PCR, since the use of other marker systems (AFLP, ISSR, and RFLP) usually involves higher costs, greater technical complexity, longer execution times, and, in some cases, the need to have prior information of the genomic sequence (18). Furthermore, these markers offer high sensitivity and specificity for the rapid, reliable, and easy-to-implement detection of the microorganism (19–21).

SCAR markers have proven highly useful for diagnosing fungal infections, such as histoplasmosis (21), with high specificity and sensitivity. In the case of the genus Aspergillus, SCAR markers have been developed, from a food-related perspective, for the differential identification of Aspergillus strains that produce and do not produce multiple enzymes of interest to the food industry (22); however, these markers have not yet been developed for the diagnosis of aspergillosis.

Therefore, this study aimed to develop and conduct a preliminary clinical evaluation of SCAR molecular markers for the identification of the section Fumigati DNA, A. flavus, and A. niger.

Fungal isolates

In this study, 80 Aspergillus isolates were used, previously identified phenotypically and genotypically (23). The isolates were obtained from the Molecular Mycology Laboratory collection of the Department of Microbiology and Parasitology, Faculty of Medicine, National Autonomous University of Mexico (UNAM). In addition, five reference strains were included, obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) (Table 1; Figure 1).

Obtaining polymorphic patterns by RAPD-PCR

The RAPD-PCR method was used as described by Kersulyte et al. (24) and Woods et al. (25). The primers OPF-01 (5’-ACGGATTCTG-3’), P54 (5’-GGCGATTTTTGCCG-3’), and 1253 (5’-GTTTCCGCCC-3’) were used, which generate specific patterns for the section Fumigati, A. flavus, and A. niger, respectively, as reported by Valencia et al. (23). The assays were carried out in triplicate as described below. The RAPD-PCR reaction mixture was prepared in a 25 µL volume containing 1X buffer, 2.5 mM MgCl 2, 20 ng of DNA, 200 pmol/µL of each primer (Invitrogen, Carlsbad, CA, USA), and 1 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific Inc.). The PCR conditions were as follows: 1 cycle of 7 min at 94 °C, followed by 45 cycles of 1 min at 92 °C, 1 min at 35 °C, and 1 min at 72 °C, and a final extension of 5 min at 72 °C. The products were subjected to electrophoresis on a 1.5% agarose gel stained with Biotium GelRed™ 10000X (Fremont, CA, USA). Images of the gels were captured using a Synoptics photodocumentation system (Syngene, San Diego, CA, USA). DNA, 200 µM of each dNTP (Applied Biosystems Inc., Waltham, MA, USA), 1 U of Taq DNA polymerase (Figure 1).

Selection, cloning, and sequencing of a selected RAPD-PCR band for the section Fumigati, A. flavus, and A. niger

Based on the polymorphic patterns generated with primers OPF-01, P54, and 1253, specific bands were selected for the section Fumigati, A. flavus, and A. niger. It should be noted that the reproducibility of these bands across triplicate assays of the polymorphic patterns conferred by the three primers was considered when selecting specific bands. Furthermore, the band was present in all isolates of the same species. Finally, bands with a molecular weight greater than 100 bp were considered. Each band of interest was excised from the gel using the QIAquick Gel Extraction Kit (Qiagen, Austin, TX, USA) according to the manufacturer’s protocol. An aliquot of the recovered DNA was re-amplified using the primer from which it originated to verify the presence of a unique band. Subsequently, each purified band was ligated into the pGEM ® -T Easy cloning vector (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The ligated products were then transformed into competent Escherichia coli JM109 cells, as described by Sambrook et al. (26). The plasmid DNA from the recombinants was purified and subsequently digested with the restriction enzyme EcoRI (Invitrogen, Carlsbad, CA, USA) to recover the inserted DNA. The presence of the DNA fragment in the vector was verified by colony PCR (27). The plasmid DNA from the positive clones was purified using the alkaline lysis method described by Sambrook et al. (26). Subsequently, the cloned fragments were sequenced at the Molecular Biology Unit, Institute of Cellular Physiology, UNAM, using an ABI Prism 3100 automated DNA sequencer (Applied Biosystems). Finally, the sequences obtained from each cloned fragment were aligned and compared with all sequences in the GenBank database using the BLAST algorithm (BLAST + 2.15.0) to rule out homology with sequences from related pathogenic fungi (28). Open reading frames (ORFs) were predicted using the ORF Finder tool from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/orffinder, last accessed June 23, 2025) (Figure 1) (29).

SCAR marker design

The sequences obtained from the cloned products were used to design specific primers, called SCAR markers, using the Primer3 Input program (https://primer3.ut.ee, accessed September 9, 2024). The design recommendations were considered, including the ideal oligonucleotide length (18–25 bp), the guanine-cytosine ratio (40–60%), and the annealing temperature (Tm) (55–65 °C). Furthermore, the primers must be 100% complementary to the template DNA, non-self-complementary, and must not form secondary structures that would prevent their alignment with the template DNA. These measures were taken to prevent formation of secondary structures or primer dimers, as well as nonspecific hybridization, and to adjust the melting temperatures to achieve adequate internal stability. The primers were synthesized by Sigma-Genosys (The Woodlands, TX, USA) (Figure 1).

PCR conditions for SCAR markers

The PCR mixtures (25 µL volume) for the AfOPF-01 180 SCAR marker assays consisted of: 1 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific Inc.), 10 mM Tris-Cl, 50 mM KCl, pH 8.3 (Waltham, MA, USA), 200 µM of each dNTP (Boehringer Mannheim), 100 pmol of each primer, 2.0 mM MgCl 2, and 10 ng/µL (2 µL) of fungal DNA. Amplification was performed under the following conditions: 94 °C for 5 min; 30 amplification cycles (30 sec denaturation at 94 °C, 30 sec at 55 °C, 1 min extension at 72 °C), and a final 5-min extension at 72 °C. PCR mixes (25 µL volume) for the SCAR marker assay AfP54 193.1 consisted of 1 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific Inc.), 10 mM Tris-HCl, 50 mM KCl, pH 8.3, 100 µM of each dNTP (Boehringer-Mannheim), 100 pmol of each primer, 2.0 mM MgCl 2, and 20 ng/µL of fungal DNA. Amplification was carried out under the following conditions: 94 °C for 5 min; 30 amplification cycles (30 sec denaturation at 94 °C, 30 sec at 55 °C, 1 min extension at 72 °C); and a final 5-minute extension at 72 °C. PCR mixes (25 µL volume) for the SCAR marker assay An1253 220 consisted of 1 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific Inc.), 10 mM Tris-HCl, 50 mM KCl (pH 8.3), 150 µM of each dNTP (Boehringer-Mannheim), 100 pmol of each primer, 1.0 mM MgCl 2, and 20 ng/µL of fungal DNA. Amplification was performed under the following conditions: 94 °C for 5 min; 30 amplification cycles (30 sec denaturation at 94 °C, 30 sec annealing at 55 °C, 1 min extension at 72 °C); and a final extension of 5 minutes at 72 °C. The amplified products were separated by electrophoresis on 1.5% agarose gels (Figure 1).

Evaluation of the specificity and sensitivity of SCAR markers

The specificity of the SCAR markers was evaluated by PCR using DNA from all Aspergillus isolates included in the study, as well as DNA obtained from isolates of some Aspergillus sections and from species of unrelated genera (Table 1; Figure 1). In this sense, the PCR assays were executed with the PCR conditions previously described for each marker.

The sensitivity of the SCAR markers was determined using PCR; for this purpose, DNA dilutions were prepared (5 ng/μl, 2.5 ng/μl, 0.5 ng/μl, 0.05 ng/μl, 0.025 ng/μl, 0.0125 ng/μl, 0.0025 ng/μl, and 0.0005 ng/μl), and 2 µl of each dilution were used to obtain concentrations of 10 ng/µl, 5 ng/µl, 1 ng/µl, 0.1 ng/µl, 0.05 ng/µl, 0.025 ng/µl, 0.005 ng/µl, and 0.001 ng/µl. Sensitivity assays were performed in triplicate to ensure reproducibility and included both positive and negative controls in every SCAR-PCR run. The limit of detection was determined based on the absence of a visible product (band of interest) following agarose gel electrophoresis.

Preliminary clinical feasibility

To perform a preliminary validation of the SCAR markers, they were tested by endpoint PCR using DNA from 51 galactomannan-positive bronchoalveolar lavage (BAL) samples from patients evaluated for aspergillosis. The samples originated from the biological sample collection of the Laboratorio de Virología y Micología, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER), Mexico City, Mexico.

DNA was obtained from the clinical sample using the DNeasy Tissue kit (Qiagen, Austin, TX, USA). Subsequently, PCR was performed for the SCAR markers AfOPF-01 180, AfP54 193.1, and An1253 220 according to standardized conditions for each marker, along with positive (C+) and negative (C−) controls. A re-amplification of the first PCR was then performed.

The first PCR test was carried out as follows: Reaction mixtures were prepared according to the specific conditions for each marker. For AfOPF-01 180, PCR was performed in a 25 µL reaction mixture containing 1 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA), 10 mM Tris-HCl, 50 mM KCl (pH 8.3), 200 µM of each dNTP (Boehringer Mannheim), 100 pmol of each primer, 2.0 mM MgCl 2, and 20 ng/µL of fungal DNA. For AfP54 193.1, PCR was performed in a 25 µL reaction mixture containing 1 U of AmpliTaq Gold DNA polymerase, 10 mM Tris-HCl, 50 mM KCl (pH 8.3), 100 µM of each dNTP, 100 pmol of each primer, 2.0 mM MgCl 2, and 20 ng/µL of fungal DNA. Finally, for An1253 220, PCR was performed in a 25 µL reaction mixture containing 1 U of AmpliTaq Gold DNA polymerase, 10 mM Tris-HCl, 50 mM KCl (pH 8.3), 150 µM of each dNTP, 100 pmol of each primer, 1.0 mM MgCl 2, and 20 ng/µL of fungal DNA. Amplification of the three SCAR markers was carried out using the same thermal cycling program: an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min.

Subsequently, the products obtained from the first PCR were re-amplified. For this purpose, 2 µL of the first amplification product was used as the template. Re-amplification was performed using the same primers, reaction conditions, and thermal cycling program described for the first PCR. A positive control (C+) and a negative control (C−) were included in each PCR reaction. To prevent contamination, all assays were performed in a NOVATECH PCR-90 laminar flow cabinet. (Centro Integral de Servicio para Laboratorio S de RL de CV, Jalisco, Mexico). PCR products were separated by electrophoresis on 1.5% agarose gels.

Obtaining polymorphic patterns by RAPD-PCR

The polymorphic pattern obtained with the OPF-01 primer revealed specific bands at 1000 bp, 1250 bp, and 1500 bp, useful for identifying the section Fumigati. Meanwhile, the polymorphic patterns generated with the P54 primer showed bands of 350 bp, 400 bp, 550 bp, and 1300 bp, useful for identifying the fungus A. flavus. Likewise, the polymorphic pattern generated with the 1253 primer showed species-specific bands at 1000 bp, 1200 bp, 1300 bp, and 1400 bp, allowing identification of the fungus A. niger.

Selection, cloning, and sequencing of a selected RAPD-PCR band for section Fumigati, A. flavus, and A. niger

The three specific bands for the section Fumigati of 1000 bp, 1250 bp, and 1500 bp were generated with the OPF-01 primer. The four specific bands for A. flavus of 350 bp, 400 bp, 550 bp, and 1300 bp were generated with the P54 primer. The four specific bands for A. niger of 1000 bp, 1200 bp, 1300 bp, and 1400 bp were generated with the 1253 primer. Each set of bands was selected, purified, and re-amplified with the primers that generated them. Of the three bands obtained with the OPF-01 primer, only the 1000 bp band designated AfOPF-01(1000 bp) was successfully cloned. Of the four bands obtained with the P54 primer, only the 400 bp band designated AfP54(400 bp) was successfully cloned. Of the four bands obtained with the 1253 primer, only the 1000 bp band designated An1253(1000 bp) was successfully cloned.

The sequences from AfOPF-01(1000 bp), AfP54(400 bp), and An1253(1000 bp) fragments were analyzed using the BLAST algorithm (28) to identify similarities with all fungal sequences in GenBank. AfOPF-01(1000 bp) was specific, recognizing only the section Fumigati with 100% coverage and similarity. AfP54 (400 bp) from A. flavus was also species-specific, recognizing only A. flavus with 100% coverage and 97.94% similarity. Similarly, An1253(1000 bp) from A. niger recognized only A. niger with 100% coverage and 98.18% similarity. Primers were designed from these sequences, each specific for the section Fumigati, A. flavus, or A. niger. For the fragment used to design the SCAR marker for the section Fumigati, only one open reading frame (ORF) was identified (frame 3, start <178, stop 77; 102 nucleotides, 33 amino acids), corresponding to an RNA-binding subunit complex (XM_077804753.1). For the sequence for A. flavus SCAR markers, only one ORF was identified (frame 3, start 77, stop >3; 75 nucleotides, 24 amino acids), corresponding to a GRAM protein domain (XM_041292823.1). For the A. niger sequence, no ORF was identified. BLAST analysis showed similarity to a genomic contig sequence (AM270030.1).

Design of SCAR markers to identify species of the section Fumigati, A. flavus and A. niger

The Primer 3 Input program (https://primer3.ut.ee) was used to design the primers from the cloned 1000 bp sequences of the section Fumigati, 400 bp of A. flavus, and 1000 bp of A. niger. For the section Fumigati two pairs of primers were chosen: 1) AfOPF-01 180 F (5’- CAACTCCTGCTTCGTAGGGG-3’) with a Tm= 60.11 °C and 60.0% GC and the primer AfOPF-01 180 R (5´-GCTCGCCATCGTTCAAGGAAA-3´) with a Tm= 59.20 °C and 50.0% GC that amplify a region of 180 bp; and 2) AfOPF-01 180.1 F (5’-GCGGTTGCAACTAAAAACCCC-3’) with a Tm= 60.87 °C and 52.38% GC and the first AfOPF-01 180.1 R (5´-CTGTCCCTTCTGATCCCCCT-3´) with a Tm= 60.33 °C and 60.0% GC that amplify a 180 bp region. The primers were synthesized by Sigma-Genosys (The Woodlands, TX). For A. flavus, two primer pairs were chosen: AfP54 193 F (5’- GGAAGGGGAGAAGGATGCAC-3’) with a Tm= 60.11 °C and 60.00% GC and the AfP54 193 R primer (5´-AACTCACCGCTCAGGTTCAG-3´) with a Tm= 59.97 °C and 55% GC that amplify a 193 bp region; and 2) AfP54 193.1 F (5´-CCTCCATCCGCAGCTAACAA-3) with a Tm= 60.11 °C and 55.00% GC and the first AfP54 193.1 R (5´-GCCGATACAGCGCTACATGA-3´) with a Tm= 60.32 °C and 55% GC that amplify a 193 bp region. The primers were synthesized by Sigma-Genosys (The Woodlands, TX). Finally, for A. niger, two pairs of primers were chosen: 1) An1253 238 F (5´- TTGTTCACGCTAGCAGCCG-3´) with a Tm= 61.03 °C and 57.89% GC and the primer An1253 238 R (5´-AATATTCCGCCACGGCACAC-3´) with a Tm= 61.37 °C and 55% GC that amplify a region of 238 bp; and 2) An1253 220 F (5´-TTGGGATGTTCGTGCTTTGC-3´) with a Tm= 59.69 °C and 50.00% GC and the primer An1253 220 R (5´-CCCAGATAATGAATGACGTGCG-3´) with a Tm= 59.78 °C and 50% GC that amplify a 220 bp region. The primers were synthesized by Sigma-Genosys (The Woodlands, TX). The conditions for each PCR with each primer pair were adjusted as described in the Materials and Methods section.

Evaluation of the specificity and sensitivity of SCAR markers for identifying section Fumigati, A. flavus, and A. niger

SCAR marker specificity assays showed that AfOPF-01 180 amplified a 180 bp band in the section Fumigati (Figure 2A), but not in the section Flavi species (A. flavus) or the section Nigri species (A. tamarii, A. niger, A. tubingensis). AfOPF-01 180 also did not amplify the expected band in isolates from other medically important genera (Figure 2B).

To test the specificity of the AfP54 193.1 primer, designed to identify A. flavus, DNAs from other species in the section Flavi (A. flavus: 87A, 370B; A. tamarii: 181), the section Fumigati (A. fumigatus: ATCC-3626, MM-12, MM-16, MM-21; A. lentulus: ATCC-3566, 459; A. hiratsukae: 3IC), and the section Nigri (A. niger: ATCC-WB326, 39A; A. tubingensis: ATCC-1004, 146A, 220A) were used. Figure 3A shows that the SCAR marker only amplified the 193 bp band in A. flavus isolates. In addition, specificity was evaluated with other species from medically important genera, and the AfP54 193.1 marker amplified only the 193 bp band in A. flavus (Figure 3B).

To test the specificity of the An1253 220 marker for identifying A. niger, DNAs from other species in the section Nigri (A. niger: ATCC-WB326, 39A, MM-353, MM-333, MM-309, MM-273, MM-294, MM-274, 219; A. tubingensis: ATCC-1004, 146A, 227, 213; A. luchuensis: 203, 205, 221) were analyzed. The 220 bp band was amplified in A. niger and A. luchuensis isolates (Figure 4A). Additional tests with species from other genera of medical importance also showed that a fragment of the expected 220 bp was amplified only in A. niger (Figure 4B).

For sensitivity testing, the assays showed that each set of SCAR primers (AfOPF-01 180 -F and AfOPF-01 180 -R, AfP54 193.1 -F and AfP54 193.1 -R, An1253 220 -F and An1253 220 R) generated the desired target fragments with the same detection limit of 0.005 ng/μL (Figures 2C, 3C, 4C).

Preliminary clinical feasibility of SCAR marker AfOPF-01 180

To determine the SCAR-PCR positivity of the OPF01 180 marker, 51 galactomannan-positive BAL samples from patients evaluated for aspergillosis were tested (Supplementary Material). Amplification was performed under the previously established conditions for this marker. However, since no amplification of the band of interest was observed, a re-amplification was carried out under the same cycling conditions. After this second amplification, the band of interest (180 bp) was amplified, allowing the identification of the section Fumigati DNA. SCAR-PCR positivity of the marker OPF01 180 in BAL samples was 19/51 (37.25%) (Figure 5).

Preliminary clinical feasibility of SCAR marker AfP54 193.1

To determine the SCAR-PCR positivity of the AfP54 193.1 marker, 51 galactomannan-positive BAL samples from patients evaluated for aspergillosis were analyzed ((Supplementary Material). An initial amplification was performed using the previously established conditions for the marker, but A. flavus was not detected at this stage. Upon re-amplification, SCAR-PCR analysis revealed that 3 out of 51 samples (5.88%) tested positive for marker P54193.1 (Figure 6).

Preliminary clinical feasibility of SCAR marker An1253 220

To determine the SCAR-PCR positivity of the An1253 220 marker, 51 galactomannan-positive BAL samples from patients evaluated for aspergillosis were tested (Supplementary Material). Despite performing an initial amplification and re-amplification, there was no SCAR-PCR positivity for the marker An1253 220.

The rising number of immunocompromised patients has led to an increase in invasive aspergillosis cases. Rapid, reliable identification of Aspergillus species is challenging due to the presence of cryptic species within the genus (1). Species-level identification is necessary because antifungal susceptibility varies, which is vital for appropriate patient treatment (30). Although new culture-independent methods for Aspergillus identification exist, all have limitations (16). SCAR markers from RAPD-PCR are useful diagnostic tools (31–37).

SCAR markers offer several advantages: they reveal higher polymorphism levels due to lower hybridization temperatures and increased primer sequence specificity (38, 39); they enable microorganism identification via simple PCR using primers from RAPD-PCR amplicon sequences (33, 38); and they serve as useful, reliable, rapid tools in resource-limited settings. Given these advantages, we developed SCAR markers from the RAPD-PCR method, using the primers OPF-01, P54, and 1253, which were useful for revealing specific polymorphisms of the section Fumigati, A. flavus, and A. niger, respectively.

The SCAR marker AfOPF-01 180, developed to identify the section Fumigati, showed adequate specificity at the section level. This is because species grouped in the same section, and defined as sister species, are indistinguishable by morphological methods. Therefore, molecular methods, particularly target gene sequencing (40–43), are crucial for correctly identifying and distinguishing the species included in this section, such as A. lentulus, A. udagawae, A. felis, A. hiratsukae, and A. viridinutans. However, other authors have used markers at the section level for diagnosing aspergillosis caused by species of the sections Fumigati, Flavi, Nigri, Terrei, Nidulantes, and Usti (10, 44–46), demonstrating the diagnostic utility of section-level markers over other traditional methods such as the galactomannan test, which only identifies the fungus at the genus level.

It is important to note that Aspergillus DNA in the section Fumigati was detected during the re-amplification step. Consequently, this limitation regarding the markers that tested positive in the SCAR-PCR assay must be considered. This result may be due to the low concentration of Aspergillus DNA available in the sample. Although BAL fluid is the most useful biological specimen for diagnosing aspergillosis, its yield varies depending on the PCR methodology, primers used, and extraction protocol; specifically, the extraction method plays a role, as some methods are less efficient at disrupting the cell wall, which significantly impacts the efficiency and yield of genetic material recovery (47). Furthermore, additional precautions should be taken during PCR assays to prevent contamination. Finally, clinical validation is necessary, as the present study only allowed for the assessment of the marker’s analytical performance and preliminary viability.

Furthermore, no cross-reactivity was observed with the SCAR marker AfOPF-01 180 in the panel of isolates, which included other medically important fungal genera such as C. immitis, C. posadasii, C. albicans, C. glabrata, H. capsulatum, S. schenckii, and Lichtheimia spp. as the expected band was not amplified. However, the SCAR-PCR positivity rate for the AfOPF-01 180 marker in BAL samples was 19/51 (37.25%). However, this marker has limitations regarding cross-reactivity with species from other sections of Aspergillus spp.

The SCAR marker AfP54 193.1 allowed the identification of A. flavus, which was confirmed by tests with other Aspergillus species and other medically important genera. For the preliminary validation of this SCAR marker, 51 BAL samples were used. It was necessary to perform a re-amplification of the first PCR because the markers did not detect the fungus in the first amplification of the analyzed clinical samples. Therefore, a second PCR was performed to confirm the presence of the fungus. Of 51 BAL samples, SCAR-PCR positivity for marker AfP54 193. 1 was 3/51. Although A. flavus is the second most common cause of aspergillosis, few diagnostic tools are available for its identification (48–51).

The SCAR marker An1253 220 showed specificity for A. niger, although it also recognized A. luchuensis. We consider that it is a useful marker for A. niger since clinical cases of aspergillosis caused by A. luchuensis are rare (52, 53). It is worth mentioning the SCAR marker An1253 220, there was no SCAR-PCR positivity in clinical samples. It is worth noting that although A. niger was not detected in the analyzed biological samples, this result does not necessarily rule out its presence in the sample. The absence of amplification could be attributed to various factors, including a low fungal load falling below the technique’s limit of detection; non-homogeneous distribution of the microorganism within the sample; inadequate sample quality due to potential degradation during storage; the presence of PCR inhibitors derived from clinical sample components; or limited sensitivity of the molecular marker used.

In addition, as with A. flavus, few studies have focused on designing PCR-based methodologies to identify species of the section Nigri from clinical and environmental samples (54–56). In particular, the assays developed for the diagnosis of aspergillosis have been very diverse (57); however, none have considered RAPD-PCR. In the present work, markers were developed using RAPD-PCR to identify the section Fumigati DNA, which are the main agents of aspergillosis in humans, as well as A. flavus and A. niger (58).

Although other SCAR markers exist for the identification of A. fumigatus, A. flavus, A. terreus, and A. niger, these showed low specificity when tested in multiplex PCR assays. While promising, the technology has limitations, including low efficiency in some cases and the need for validation in a larger patient cohort and more diverse biological samples (59).

It is worth noting that the SCAR markers obtained in this study are of great epidemiological relevance, as they were designed from autochthonous isolates, which is important given the high genetic variability observed across Aspergillus species, particularly A. fumigatus (60).

To date, no assay using SCAR markers has been developed to identify the section Fumigati DNA, A. flavus, and A. niger. Furthermore, because SCAR markers are specific sequences derived from unique regions of the microorganism’s genome, they allow direct amplification of its DNA by PCR from clinical samples (BAL) without prior culture. It is worth mentioning that, although this marker detects down to 0.005 ng/µL, it was necessary to perform PCR re-amplification with the SCAR markers to reveal the presence of the expected band size.

A notable limitation of this study is the lack of data regarding the classification of aspergillosis in the patients whose BAL samples were used. Furthermore, it was not possible to assess the concordance between laboratory results and those obtained using the developed SCAR markers, as the diagnosis was based exclusively on galactomannan detection rather than the isolation and identification of Aspergillus. The limited number and lack of heterogeneity of the biological samples used to determine the positivity of the SCAR-PCR assays constituted another limitation. This was compounded by the fact that the limit of detection was determined subjectively. Specifically, based on the presence or absence of the band of interest for each sensitivity assay associated with each marker. Regarding the SCAR-PCR assays, it was not possible to include an internal amplification control for the BAL samples. Additionally, the limited number of isolates representing various sections of the Aspergillus genus available for specificity testing posed another limitation, given the potential for cross-reactivity with Aspergillus sections not included in this study. Consequently, further studies are required to evaluate the diagnostic performance of the designed SCAR markers across diverse populations and sample types.

The AfOPF-01 180 marker allows for the identification of the section Fumigati DNA, while the marker AfP54 193.1 proved to be an, excellent candidate for the identification of A. flavus, and marker An1253 220 showed specificity for A. niger and A. luchuensis.

This novel assay, based on SCAR-type markers, is characterized by its low cost, speed of execution, and ease of interpretation.

However, prospective, multicenter validation is required to evaluate its diagnostic clinical capability.

Based on the results obtained in this study, the developed markers are promising candidates for the rapid and specific identification of the section Fumigati DNA, A. flavus, A. niger, and A. luchuensis.