The Use of MALDI-TOF MS as a Diagnostic Tool for Adult Trichuris Species

ORIGINAL RESEARCH published: 11 May 2022 doi: 10.3389/fvets.2022.867919 Frontiers in Veterinary Science | www.frontiersin.org 1May 2022 | Volume 9 | Article 867919 Edited by: Raquel Simões, Universidade Federal Rural do Rio de Janeiro, Brazil Reviewed by: Anders Toftegaard Boysen, Aarhus University, Denmark Jesús Alonso Panti May, Universidad Autónoma de Yucatán, Mexico *Correspondence: Cristina Cutillas [email protected] Specialty section: This article was submitted to Parasitology, a section of the journal Frontiers in Veterinary Science Received: 01 February 2022 Accepted: 13 April 2022 Published: 11 May 2022 Citation: Rivero J, Zurita A, Cutillas C and Callejón R (2022) The Use of MALDI-TOF MS as a Diagnostic Tool for Adult Trichuris Species. Front. Vet. Sci. 9:867919. doi: 10.3389/fvets.2022.867919 The Use of MALDI-TOF MS as a Diagnostic Tool for Adult Trichuris Species Julia Rivero, Antonio Zurita, Cristina Cutillas*and Rocío Callejón Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain Trichuriasis is considered a neglected tropical disease, being the second most common helminthiasis in humans. Detection of Trichuris in routine diagnosis is usually done by microscopic detection of eggs in fecal samples. Other molecular analyses are more reliable and could be used, but these analyses are not routinely available in clinical microbiology laboratories. The use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is increasing since the last decades due to its recent evidence as a potential role for reliable identification of microorganisms and a few nematodes. But, for parasites detection, normalized protocols and the acquisition and introduction of new species to the database are required. We carried out a preliminary study confirming the usefulness of MALDI-TOF MS for the rapid and reliable identification of Trichuris suis used as control and the creation of an internal database. To create main spectra profiles (MSPs), the different parts of five whipworms (esophagus and intestine) were used, developing different tests to verify the repeatability and reproducibility of the spectra. Thus, to validate the new internal database, 20 whipworms, separating the esophagus and intestine, were used, of which 100% were accurately identified as T. suis, but could not distinguish between both parts of the worm. Log score values ranged between 1.84 and 2.36, meaning a high-quality identification. The results confirmed that MALDI-TOF MS was able to identify Trichuris species. Additionally, a MALDI-TOF MS profile of T. suis proteome was carried out to develop the first internal database of spectra for the diagnosis of trichuriasis and other Trichuris spp. Keywords: Trichuris, MALDI-TOF MS, diagnosis, nematode, internal database INTRODUCTION Helminths constitute one of the most common, ecologically diverse, and speciose animal groups in the world. Several species of nematodes are of huge economic importance or medical interest. The most important species of soil-transmitted helminth (STH) infections, infecting humans are Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) and Necator americanus and Ancylostoma duodenale (hookworms). Nearly 24% of the world’s population is infected with soilborne helminths (1). The prevalence is higher in marginalized populations in the tropics and subtropics, where there is a lack of basic sanitation services (2). T. trichiura is a nematode, which is the etiological agent of the parasitic disease known as “trichuriasis.” Trichuriasis is considered as a neglected tropical disease and has a worldwide geographical distribution. T. trichiura is the second most common helminth in humans. Moreover, Rivero et al. MALDI-TOF MS for Trichuris Species whipworms are among the most common intestinal parasites of humans and animals, causing significant diseases and economic losses globally (3,4). Whipworms can be found in a large range of hosts, in addition to humans (T. trichiura), in suids (T. suis), sheep, goats, and bovines (T. ovis and Trichuris discolor), dogs (Trichuris vulpis), non-human primates (NHP) (Trichuris spp.) and several putative new Trichuris species (5–10). Traditionally, the identification of Trichuris species was carried out according to the host where the whipworm was found, and later, by studies based on morphological and biometrical features of adults. But due to the phenotypic plasticity of these parasites (host-induced variation, lack of morphological characteristics, and overlap of morphological characteristics and biometrical data between species), it is highly difficult to distinguish between closely related Trichuris species (11–14). Hence, molecular studies, such as polymerase chain reaction (PCR) and sequencing, are used as a tool to differentiate species (5–7,9,10,15–20). While the morphological identification is a rapid and less costly procedure, the related Trichuris species are hardly morphologically distinguishable. Moreover, the diagnosis of Trichuris is made by observing eggs in fecal samples and requires qualified personnel. Thus, the improvement of an accurate, fast, less expensive, and more accessible diagnostic technique for the identification of parasites would be desirable. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been commonly introduced as a diagnostic method in laboratories, that analyzed complex molecules, such as proteins, by producing protein fingerprint signatures (spectra) from proteins extracts of organisms (21). The creation of reference spectra database by the acquisition of spectra has been used to identify species of parasites (22–25). MALDI-TOF MS has been suggested as a rapid and reliable identification technique of bacteria (26,27). Lately, authors have demonstrated that MALDI Biotyper software can be used, in addition to bacteria (28–31), mycobacteria (28,32), fungi (31), and most recently, in viruses (33), protozoans, arthropods (22,34,35), and a few nematodes (24,36–40). MALDI-TOF MS has revealed many advantages compared with other diagnostic tools (such as PCR assays). When the mass spectrometer and the corresponding databases are available in a laboratory, the identification is inexpensive, and the sample preparation procedure does neither require highly skilled technicians or complex additional laboratory infrastructure (41– 43). Other advantages are that MALDI-TOF MS is significantly less susceptible to contamination, since the samples do not require special collected and preserved conditions, and the results are available within a few minutes. Nevertheless, the constant power supply is a limitation for the suitability of the technique in resource-limited environments. However, MALDI-TOF MS is more and more available in reference laboratories around the world including in developing countries (41–45). Furthermore, recent studies suggested that MALDI-TOF MS technique is of great importance due to its applicability in the discovery of antibiotic resistance in microorganisms, disinfectants, and the production of toxins from pathogens (29,46–48). Recently, a review about the use of MALDI-TOF in human and veterinary helminthology confirms that this technique is reliable and reproducible for nematode parasites. They suggested that it is necessary for many studies for more different species of nematodes, especially in common nematode parasites in the world, and to create a single database with all nematode species. And also, add the necessary spectra to the internal o commercial database to be able to identify larvae and eggs to open the possibility to analyze fecal samples by MALDI-TOF MS and obtain a fast and reliable identification and diagnosis (49). Based on this background, the aim of this study was to evaluate the usefulness of MALDI-TOF MS as an effective diagnostic tool for the specific identification of Trichuris species. For this purpose (i) a standard protocol for the extraction of proteins was developed using Trichuris suis as control, (ii) a preliminary specific reference spectra database was created characterizing this species, T. suis, (iii) the standardized protocol of T. suis was validated for more species of Trichuris using MALDI-TOF MS analysis: Trichuris sp. from Hystrix cristata,T. trichiura from Macaca sylvanus,T. vulpis from Canis lupus familiaris and T. ovis from Capra hircus collected on Spain, (iv) an MS reference spectra in-house database for Trichuris speciesspecific identification was created based on molecular Trichuris identification comparatively with the new species of Trichuris analyzed, and (v) an analysis of the results obtained by MALDITOF MS was performed to verify the usefulness of MALDI-TOF MS in the phylogenetic and taxonomic study of Trichuris species. MATERIALS AND METHODS Ethics Statement This study does not require approval by the ethics committee. Adult Trichuris samples were obtained from their caecum postmortem. The specimens of T. suis and T. ovis were obtained through a slaughterhouse; in a zoo, for adults of T. trichiura from M. sylvanus and Trichuris sp. from H. cristata, and in groups of dogs, for adults of T. vulpis, in strict accordance with good animal practices. Sample Collection For this study, whipworms belonging to the species T. suis from swine (Sus scrofa domestica) and T. ovis from goats (Capra hircus) were collected in slaughterhouses in the provinces of Seville and Huelva, and Seville (Spain), respectively. Specimens identified as Trichuris sp. from H. cristata and T. trichiura from M. sylvanus were collected from Bioparc Fuengirola (Spain), and Zoo Castellar (Spain), respectively. Finally, T. vulpis adults were collected from dogs (Canis lupus familiaris) in the provinces of Seville and Huelva. Separately, whipworm samples were washed several times in saline solution (0.9% w/v), and then frozen at −20◦C until posterior analysis. Sample Preparation and Morphological Identification Adult worms were defrosted and dried at room temperature. The morphological identification of different Trichuris species was carried out according to previous studies (15,18,20, 50–52). Nonetheless, morphologically the different species of Frontiers in Veterinary Science | www.frontiersin.org 2May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species Trichuris are difficult to differentiate. Thus, a molecular study was carried out. Then, two different samples of the T. suis adults (esophagus and intestine) were used separately for MALDI-TOF MS analysis to check which part of the worm gave greater reliability and easier reproducibility. For this reason, the preliminary study with only adults of T. suis was performed using 31 T. suis worms, using the esophagus and the intestine of adults as samples. Further, these two samples were assayed in the rest of the species of Trichuris for proteomic and molecular analysis, respectively. Molecular Analysis DNA Extraction, PCR, and Sequencing To verify the accuracy of the species identification of the samples collected for MALDI-TOF MS, the morphological studies were corroborated with molecular analysis. The total genomic DNA from intestine worms was extracted using DNeasy Blood and Tissue Kit (Qiagen; Hilden, Germany) according to the manufacturer’s instructions. The quality of extractions was assessed using 0.8% agarose gel electrophoresis infused with SYBR R Safe DNA gel stain (Thermo Fisher Scientific, MA, USA). The cytochrome b (cytb) mitochondrial DNA (mtDNA) gene of two to five intestines DNA samples of each Trichuris species (T. suis,Trichuris sp. from H. cristata,T. trichiura,T. vulpis, and T. ovis), were amplified by standard PCR by a thermal cycler (Eppendorf AG; Hamburg, Germany), while the esophagus was reserved for MALDI-TOF MS. The forward primer utilized was D769 (5′-GAGTAATTTTTATAATRCGRGAAGT- 3′) (53) and the reverse primer utilized was D770 (5′- AATTTTCAGGRTCTCTRCTTCAATA-3′) (53), and the following PCR mix: 5 µl each primer (10 µM), 25 µl GoTaq G2 Green Master Mix, 5 µl template DNA, and nuclease-free water to 50 µl. The following conditions were applied: 94◦C at 5 min (denaturing); 36 cycles at 94◦C at 30 s (denaturing), 50◦C at 30 s (annealing), and 72◦C at 30 s (primer extension); followed by 7 min at 72◦C (final extension). The PCR products were visualized on SYBR Safe stained with 2% w/v Tris– Borate–EDTA (TBE) agarose gels. Then, bands were eluted and purified using the Wizard SV Gel and PCR Clean-Up System Kit (Promega, WI, USA). Once purified and concentrated, the PCR products were sequenced in both directions by Stab Vida (Lisbon, Portugal). Sequence Analysis Species Identification The forward and reverse sequences obtained were analyzed using Multiple Sequence Alignment by CLUSTALW, to generate a consensus sequence for each specimen. The sequences were compared with sequences available in the National Center for Biotechnology Information (NCBI) GenBank database using the Basic Local Alignment Search Tool (BLASTn) algorithm for identification (https://blast.ncbi.nlm.nih.gov/ Blast.cgi). Sequences obtained were submitted to the NCBI GenBank database. MALDI-TOF Analysis Preliminary MALDI-TOF Analysis Protein Extraction For the preliminary study with T. suis worms, each worm sample, esophagus, and intestine, separately was placed in a 1.5 ml sterile Eppendorf. Afterward, each sample was pooled with 10 mg zirconia/silica beads (0.5 mm) along with 20–30 µl of a mix of 70% (v/v) formic acid and 50% (v/v) acetonitrile. Then, the samples were homogenized using the TissueLyser II system (Qiagen GmbH) in three cycles of 30 s at a frequency of 30 Hz. The homogenized samples were centrifuged at 10,000 g for 30 s. Target Plate Preparation and Measurements From each sample, 1 µl of the supernatant was carefully dropped on to the MALDI-TOF target in eight different spots for the creation of the main spectrum profiles (MSPs) (25), and four times for the validation test (54). Air dried and each spot was then recovered with 1 µl of CHCA matrix solution composed of saturated α-cyano-4-hydroxycinnamic acid (Sigma–Aldrich, Co., MO, USA), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Thermo Scientific, Rockford, IL, USA), and 47.5% (v/v) high-performance liquid chromatography (HPLC) grade water (34). Bacterial test standard (BTS) (Bruker Daltonics, Bremen, Germany) was used to calibrate the machine (an Escherichia coli extract), which is spiked with two high molecular weight proteins. The matrix solution was loaded in quadruplicate to control the matrix quality (54). Then, at room temperature, having dried for several minutes, the plate was placed into the Microflex LT Mass Spectrometer (Bruker Daltonics) for MALDI-TOF MS. MALDI-TOF Parameters The MALDI-TOF MS measurements were carried out on a range of 2,000–20,000 Da, m/z(mass to charge) and with detection in the linear positive in mode with a laser frequency of 50 Hz, following the calibration with BTS. For each spot, 240 laser shots were performed in four regions, and the measurements were automatically acquired using the AutoXecute method of the flexControl v3.4 software (Bruker Daltonics; Bremen, Germany). The spectrum profiles (protein mass profiles) were generated and visualized by Flex Analysis v3.3 software and were exported to ClinProTools v2.2 and MALDI- Biotyper v3.1.66 (Bruker Daltonics; Bremen, Germany) for data processing (smoothing, baseline subtraction, and peak picking). The acceleration voltage was 20 kV, and the extraction delay time was 200 ns (54). Concisely, the maximum mass error of each individual spectrum was 2,000 Da, the desired peak frequency minimum was 25% and the desired mass error for the MSP was 200 Da. Spectral Analysis and Preliminary Database Creation For the creation of species-specific MSPs, according to Diarra et al. (55), 2–6 specimens of each species is enough. Hence, protein extracts of five esophagus from T. suis worms and three intestines from T. suis worms were spotted on the MALDI-TOF target plate eight times per sample. Then, each spot was measured four times. For each sample worm, this procedure was carried out Frontiers in Veterinary Science | www.frontiersin.org 3May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species on two replicates on different days to demonstrate repeatability and the reproducibility analysis. The combination of the results of the spectra from each specimen was used to create MSP by the automated function of the MALDI-Biotyper using the default parameter set of the “Bio Typer MSP Creation Standard Method” (54). The quality of each raw spectra was assessed with Flex Analysis software version 3.4. (Bruker Daltonics; Bremen, Germany). This program was also used to remove all flatlines and outlier peaks and smooth intensities and edit peak changes within spectra whenever they exceed 500 ppm. Validation Test The recently developed internal database underwent two different validation procedures. Starting with an internal validation, where all the spectra of each group of samples obtained through the MSP creation process were analyzed. And finally, a blind test analysis, in which the samples were measured by MALDI-TOF to evaluate the ability of the database to reliably identify these samples. For the blind test analysis, 20 specimens were analyzed. Protein extract from each sample was spotted on the MALDI-TOF target in quadruplicate. Hence, each sample was associated with four spectra. A total of 72 highquality spectra from T. suis’s esophagus (for two samples the protein extract process failed) and 80 from T. suis’s intestine, were selected to query the database. The results obtained against the reference library were shown as log score values (LSVs) for each spectrum. LSV range from 0 to 3 reflects the results of a comprehensive comparison of peak position and intensity between two spectra (LSV from 0 to 1.699: no reliable identification; 1.7 to 1.999: probable genus identification; 2.0 to 2.299: secure genus identification and probable species identification, and 2.300 to 3.000: highly probable species identification) (25,40). Validation of MALDI-TOF Analysis The analysis was carried out according to the preliminary study to validate and verify the procedures and the parameters used. Spectral Analysis and Database Creation For each Trichuris species (Trichuris sp. from H. cristata,T. trichiura,T. vulpis, and T. ovis), MSPs were created as previously described by the preliminary protocol. To actualize the internal database, five specimens for Trichuris sp. from H. cristata and for T. trichiura from M. sylvanus, two specimens for T. vulpis, and four specimens for T. ovis were used by running the MALDI Biotyper software automatically. Validation Test To assess the ability of the database to reliably identify these samples, a blind test was performed. A total of 63 good quality spectra from the four different Trichuris species analyzed were selected to examine the updated database. For each specimen, the protein extracted was spotted on the MALDI-TOF target four times, generating four spectra associated with the same sample. The results obtained for the query of the internal database are shown as log score values (LSVs) for each spectrum. To determine the distances and similarity among MSPs, a hierarchical clustering of the mass spectra was performed, using the spectra utilized for the MSP creation, and the dendrogram function within MALDI Biotyper software. Phylogenetic Analysis To evaluate the similarity among all cytbTrichuris sequences, the number of nucleotide and amino acids differences per sequence was calculated using Compute Pairwise Distances based on the number of differences method of MEGA X v10.1.8 (56). For phylogenetic analysis, two methods—maximum likelihood (ML) and Bayesian inferences (BI)—were used. PhyML 3.0 was used to generate the ML tree (57), and for BI was used MrBayes v.3.2.6 (58). jModelTest to resolve the best-fit substitution model for the nucleotide data set was employed (59). According to Akaike Information Criterion, models of evolution were determined (57,60). GTR +I+G model, with rate variation along the length of the alignment (+G) and allowing for a proportion of invariant sites (+I), was selected for the nucleotide data set. Topology support was examined using bootstrapping (heuristic option) (61) over 1,000 replications to assess the relative reliability of clades. The Bayesian posterior probabilities (BPPs) comprise the percentage converted. To determine if the number of generations completed was sufficient, was used the standard deviation of split frequencies; every 500 generations the chain was sampled, and each dataset was run for 10 million generations. Based on an assessment of convergence, trees from the first million generations were discarded. Examination of the loglikelihood values of the chains is determined empirically during the burn. RESULTS Preliminary MALDI-TOF Analysis Comparative Analysis of T. suis Samples Used for MSP Database Creation (MALDI-TOF Analysis) As mentioned before, for the creation of different reference spectra, firstly, two distinct body parts (esophagus and intestine) of T. suis worms were used. We included five specimens of which five esophagus and three intestines were used. The cox1 mtDNA partial sequence valid the specific analysis in the reference samples against previously deposited sequences by BLASTn, revealing approximately a 100% identity between T. suis (Supplementary Table S1) and the T. suis sequences from this study (reference accession number: OU756954). From the three T. suis worm sequences obtained, only 1 haplotype was generated. The homogenization and sample preparation protocol described above provided high-quality spectra (Figure 1). Both, results based on esophagus and intestines samples showed similar spectra, with profiles of high intensity and strongest peaks placed in the same range, however, peaks intensity in the esophagus samples was slightly higher. The reference MSPs obtained for each type of sample by MALDI-TOF with high-intensity peaks in the 2–20 kDa range are presented in Figure 1. The highest density peaks were in the region comprised between 2 and 9.5 kDa, with clusters of signals in ranges corresponding to 2.1– 2.3, 3–3.3, 5.2–5.6, and 6.2–6.6 kDa (Figure 2). There were no Frontiers in Veterinary Science | www.frontiersin.org 4May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species FIGURE 1 | Comparison of the spectra created for the internal database between esophagus and intestine samples of T. suis worms by MALDI-TOF MS. a.u., arbitrary units; m/z, mass-to-charge ratio (Da). significant differences between esophagus and intestine samples of T. suis. Internal Database Creation All MSPs obtained were analyzed with the commercially common database using FlexAnalysis software for bacteria and fungi identification and reliable identification was not achieved with all LSVs <1.7. The posterior analysis of the raw spectra obtained during MSP creation, which used a combination of the commercial and internal database, revealed the identification of T. suis in all the samples, but it was not possible to differentiate between esophagus and intestines of the same T. suis species. LSVs of T. suis esophagus sample ranging from 2.43 to 2.82, identified as T. suis esophagus, and from 2.10 to 2.27 identified as T. suis intestine. LSVs of T. suis intestine samples ranged from 2.67 to 2.78 with T. suis intestine, and from 2.24 to 2.36 with T. suis esophagus. The LSVs observed for each sample appeared higher when they were compared with their corresponding body part (esophagus or intestine), but with both could be correctly identified. Analysis of Samples for External Database Validation During the study, a total of 20 Trichuris worms were utilized for blind testing 20 esophagus and 20 intestines for MALDI-TOF analysis. Of the 40 samples, two esophagus samples were not successfully extracted. By using the newly developed internal database and considering a threshold of 1.70, MALDI-TOF MS correctly identified 38/38 (100%) of the samples at the probable genus level. When an LSV threshold of ≥2.0 was obtained, the identification rate was 9/18 (50%) for esophagus T. suis samples while for the intestine samples it was 16/20 (80%) for a probable species identification. Lastly, LSV ≥2.3 was 1/18 (5.55%) for esophagus samples and 1/20 (5%) for intestine samples with a highly probable species identification (see Table 1). Validation of MALDI-TOF Analysis To provide a molecular identification, 19 cytb mtDNA partial sequences were assessed, of which the intestines of the whipworms were used, and were tested against previously deposited sequences by BLASTn, demonstrating high values of identity (98.81–100%) (see Supplementary Table S1). Frontiers in Veterinary Science | www.frontiersin.org 5May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species FIGURE 2 | Pseudo-gel representing the protein profile obtained after MALDI-TOF MS analysis of T. suis specimens’ representative of esophagus and intestine samples. On the x-axis, the mass-to-charge values (m/z, kDa) are reported, at the same time, on the right y-axis, the scale bar reveal the relationship between the color intensity and the peak intensity, expressed by arbitrary units (a.u.). The spectra samples are represented on the left y-axis (Sp.#). TABLE 1 | Identification of 38 T. suis samples by MALDI-TOF MS, using a preliminary developed internal database. Species/body part (sample type) Number of samples correctly extracted Identification LSV range LSV ≥1.70 LSV ≥2.00 LSV ≥2.30 Trichuris suis/Esophagus 18 18 (100%) 9 (50%) 1 (5.55%) 1.84–2.36 Trichuris suis/Intestine 20 20 (100%) 16 (80 %) 1 (5%) 1.90–2.34 LSV, log-score value. The preliminary protocol used for Trichuris samples preparation provided high-quality spectra with elevated reproducibility and intensity of MS spectra. To observe the characteristic high-intensity peaks, a representative protein spectral profile for each Trichuris species (T. suis,Trichuris sp. from H. cristata,T. trichiura,T. vulpis, and T. ovis) is shown in Figure 3. Hence, for each Trichuris species, specific and reproducible MALDI-TOF MS spectra profiles were obtained. Moreover, the internal database was updated with four new Trichuris species, and 16 new reference spectra: Trichuris sp. from H. cristata (n=5), T. vulpis (n=2), T. ovis (n=4), and T. trichiura (n=5). When submitting the new MSPs of Trichuris species to the in-house database (combined with those data commercially available), all the 79 specimens used in the blind test were successfully queried in the extended database. The blind test results yielded 100% correct identification for the specimens evaluated since all samples were LSVs greater than 1.70 (Table 2). For the blind test, the percentage of samples with LSVs ≥2.00 was for T. trichiura from M. sylvanus: 88.89%; for Trichuris sp. from H. cristata: 72.22%; for T. ovis: 100%; and for T. vulpis: 87.5% (Table 2). Finally, the blind test confirmed a complete agreement with the molecular and MALDI-TOF MS identification methods. Furthermore, to confirm the accurate analysis, a dendrogram based on MSPs was added to the internal database with the five Trichuris species (including T. suis), and all species provided highly specific spectra with separated clades for each species (Figure 4). Molecular Study The accession number of all nucleotide sequence data obtained in this work were deposited at the GenBankTM, EMBL, and DDBJ databases, and are available in Table 3. To compare the intraspecific and interspecific similarities obtained among Trichuris species, pairwise nucleotide and amino acid distances for the cytb partial gene sequences were performed (Table 4). The intraspecific similarity between all the samples of the same species studied was similar with values close to Frontiers in Veterinary Science | www.frontiersin.org 6May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species FIGURE 3 | Representative spectral profiles of the esophagus of Trichuris species acquired for the creation of the internal database. a.u., arbitrary units; m/z, mass-to-charge ratio. TABLE 2 | Identification of Trichuris species esophagus samples by MALDI-TOF MS, using an internal database. Species Number of samples correctly extracted Identification LSV range LSV <1.7 LSV 1.7–1.799 LSV 1.8–1.999 LSV 2–2.299 LSV ≥2.3 LSV ≥1.70 LSV ≥2.00 Trichuris trichiura from M. sylvanus 18 0 0 2 (11.11%) 13 (72.22%) 3 (16.67%) 18 (100%) 16 (88.89%) 1.89–2.38 Trichuris sp. from H. cristata 18 0 1 (5.55%) 4 (22.22%) 12 (66.67%) 1 (5.55%) 18 (100%) 13 (72.22%) 1.72–2.42 Trichuris ovis 19 0 0 0 1 (5.26%) 18 (94.74%) 19 (100%) 19 (100%) 2.185–2.577 Trichuris vulpis 8 0 0 1 (12.5%) 5 (62.5%) 2 (25%) 8 (100%) 7 (87.5%) 1.95–2.52 LSV, log-score value. 100% (Table 4), except for the intraspecific similarity among Trichuris sp. from H. cristata, which showed two different groups. The first group consisted of THCM1, THCM2, and THCF3 samples, and the second group of THCF1 and THCF2 samples, with intraspecific nucleotide similarity values of 100 and 99.8%, respectively, and 100% for amino acid distances. The interspecific nucleotide similarity between both groups was 88.2–88.4%, and amino acid similarity was 90.8%. The minimum interspecific similarity among all Trichuris species obtained was 68% (between T. trichiura from M. sylvanus and Trichuris sp. from H. cristata) in nucleotide distances, and the maximum was 73.6% (between T. ovis and Trichuris sp. from H. cristata) and the minimum in amino acid distances was 65.3% (between T. trichiura from M. sylvanus and Trichuris sp. from H. cristata) and the maximum was 76.3% (between T. trichiura from M. sylvanus and T. suis) (Table 4). Phylogenetic tree based on cytb sequences, was rooted including outgroup Trichinella spiralis and Trichinella pseudospiralis. The alignment of 20 sequences of Trichuris species (including outgroups), yielded a dataset of 505 characters. The phylogenetic tree revealed five main clades, corresponding each clade with one different species of Trichuris. All clades separately were highly supported (100% ML and 100% BPP). In addition, the clades T. suis and T. trichiura are more related to each other, but this relation is only supported by BPP, since there is a bootstrap value of <60% between the clades in ML. Both clades are, at the same time, more related to T. vulpis. And all the above is more related to Trichuris sp. from H. cristata, with the clade of T. ovis separated from all of them, although within the group of Trichuris. In addition, two different lineages, highly supported, among Trichuris sp. from H. cristata were revealed (Figure 5). DISCUSSION The genus Trichuris has been the subject of a wide-ranging controversy due to its difficult specific differentiation. Many authors have evidenced synonymies (62), cryptic species (18,63), Frontiers in Veterinary Science | www.frontiersin.org 7May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species FIGURE 4 | Dendrogram analysis constructed using representative spectra of each Trichuris species used to develop the internal database. and new species (6,7,10,64,65). Since Dujardin (66) reviewed the genus of Trichuris for the first time, many studies have been based on morphometric and molecular analyses. The difficulty of relating the species described with exclusively morphological studies and with species described with only molecular studies makes the study of taxonomy even more difficult to solve. In support of this, additional complementary methods might be necessary to elucidate the different species. The aim of this study was to confirm whether MALDITOF MS analysis could be used as a diagnostic tool for the identification of adult Trichuris species. Our results showed high-quality spectra and a high similarity among the samples, regardless of the type of sample analyzed, so we confirm the use of both parts of the body of the nematode for its diagnosis using this technique. To achieve this goal, an internal database has been developed with samples of both, the esophagus and intestine of T. suis, providing similar spectra between them, and, therefore, being adequate to identify this species. All specimens (38/38) were accurately identified. Moreover, the preliminary protocol was validated for more species of Trichuris, including four more species of Trichuris in the internal database. These Trichuris species were also 100% correctly identified. As the values revealed in the preliminary study of samples, both esophagus and intestines, showed similar results, we suggest the preference of using the worm’s esophagus through MALDI-TOF MS technique since it is easier to manage the esophagus and saving the part of the worm’s intestine for molecular studies. MALDI-TOF MS has been used previously as an effective diagnostically tool in microbiology clinical to identify pathogenic microorganisms. Even now, a systematic review of about MALDI-TOF MS in human and veterinary helminthology was carried out, concluding that more studies are needed since there is evidence for the reliable and rapid identification of nematodes using MALDI-TOF MS, and the identification of these nematodes, whether larvae, adults, or eggs in fecal samples (49). Hence, the internal database for helminth identification is being generated with species-specific MSPs. It is necessary the advancement of one standardized approach for protein Frontiers in Veterinary Science | www.frontiersin.org 8May 2022 | Volume 9 | Article 867919 Rivero et al. MALDI-TOF MS for Trichuris Species TABLE 3 | Summary of the Trichuris sequences obtained in this work and available in GenBank database. Species Host Sample ID Accession number Sequence length Content of G +C% Trichuris sp. Hystrix cristata THCM1 OM457227 519 27.2 Trichuris sp. Hystrix cristata THCM2 OM457228 519 27.2 Trichuris sp. Hystrix cristata THCF1 OM457229 519 25.1 Trichuris sp. Hystrix cristata THCF2 OM457230 519 24.9 Trichuris sp. Hystrix cristata THCF3 OM457231 519 27.2 Trichuris vulpis Canis lupus familiaris TVM1 OM457232 519 28.5 Trichuris vulpis Canis lupus familiaris TVF1 OM457233 519 28.3 Trichuris ovis Capra hircus TOF1 OM457234 519 31.4 Trichuris ovis Capra hircus TOF2 OM457235 519 31.6 Trichuris ovis Capra hircus TOM1 OM457236 519 31.4 Trichuris ovis Capra hircus TOM2 OM457237 519 31.4 Trichuris trichiura Macaca sylvanus TMSF1 OM457238 519 30.7 Trichuris trichiura Macaca sylvanus TMSF2 OM457239 519 30.7 Trichuris trichiura Macaca sylvanus TMSF3 OM457240 519 31.1 Trichuris trichiura Macaca sylvanus TMSM1 OM457241 519 31.1 Trichuris trichiura Macaca sylvanus TMSM2 OM457242 519 30.7 Trichuris suis Sus scrofa domestica TSM1 OM457243 519 28.7 Trichuris suis Sus scrofa domestica TSF1 OM457244 519 28.9 Trichuris suis Sus scrofa domestica TSF2 OM457245 519 28.9 TABLE 4 | Pairwise nucleotide and amino acid distances for the cytochrome b partial gene sequences for Trichuris species studied in this work. THCM1 THCM2 THCF1 THCF2 THCF3 TVM1 TVF1 TOF1 TOF2 TOM1 TOM2 TMSF1 TMSF2 TMSF3 TMSM1 TMSM2 TSM1 TSF1 TSF2 THCM1 100 90.8 90.8 100.0 68.2 68.2 68.8 68.8 68.8 68.8 65.3 65.3 65.9 65.3 65.3 69.4 69.4 69.4 THCM2 100.0 90.8 90.8 100.0 68.2 68.2 68.8 68.8 68.8 68.8 65.3 65.3 65.9 65.3 65.3 69.4 69.4 69.4 THCF1 88.4 88.4 100.0 90.8 68.8 68.8 69.4 69.4 69.4 68.8 65.9 65.9 66.5 65.9 65.9 69.9 69.9 69.9 THCF2 88.2 88.2 99.8 90.8 68.8 68.8 69.4 69.4 69.4 68.8 65.9 65.9 66.5 65.9 65.9 69.9 69.9 69.9 THCF3 100.0 100.0 88.4 88.2 68.2 68.2 68.8 68.8 68.8 68.8 65.3 65.3 65.9 65.3 65.3 69.4 69.4 69.4 TVM1 70.7 70.7 73.2 73.2 70.7 99.4 71.7 71.7 71.7 71.7 72.8 72.8 71.7 72.8 72.8 69.9 69.9 69.9 TVF1 70.7 70.7 73.2 73.2 70.7 99.8 71.7 71.7 71.7 71.7 72.8 72.8 71.7 72.8 72.8 69.9 69.9 69.9 TOF1 71.7 71.7 73.6 73.6 71.7 69.9 69.9 100.0 100.0 99.4 72.3 72.3 71.1 72.3 72.3 73.4 73.4 73.4 TOF2 71.5 71.5 73.4 73.4 71.5 69.7 69.7 99.8 100.0 99.4 72.3 72.3 71.1 72.3 72.3 73.4 73.4 73.4 TOM1 71.5 71.5 73.4 73.4 71.5 69.7 69.7 99.2 99.4 99.4 72.3 72.3 71.1 72.3 72.3 73.4 73.4 73.4 TOM2 71.5 71.5 73.2 73.2 71.5 69.7 69.7 99.2 99.4 98.8 72.8 72.8 71.7 72.8 72.8 73.4 73.4 73.4 TMSF1 68.2 68.2 69.4 69.6 68.2 70.3 70.5 70.1 69.9 69.7 70.1 100.0 98.8 100.0 100.0 76.3 76.3 76.3 TMSF2 68.2 68.2 69.4 69.6 68.2 70.3 70.5 70.1 69.9 69.7 70.1 100.0 98.8 100.0 100.0 76.3 76.3 76.3 TMSF3 68.2 68.2 69.4 69.6 68.2 69.9 70.1 69.7 69.6 69.4 69.7 99.6 99.6 98.8 98.8 76.3 76.3 76.3 TMSM1 68.0 68.0 69.2 69.4 68.0 70.1 70.3 70.1 69.9 69.7 70.1 99.6 99.6 99.2 100.0 76.3 76.3 76.3 TMSM2 68.2 68.2 69.4 69.6 68.2 70.3 70.5 70.1 69.9 69.7 70.1 100.0 100.0 99.6 99.6 76.3 76.3 76.3 TSM1 71.1 71.1 72.1 72.1 71.1 72.1 72.1 72.1 71.9 72.1 71.9 71.1 71.1 71.1 70.9 71.1 100.0 100.0 TSF1 70.9 70.9 71.9 71.9 70.9 71.9 71.9 72.1 71.9 72.1 71.9 70.9 70.9 70.9 70.7 70.9 99.8 100.0 TSF2 70.9 70.9 71.9 71.9 70.9 71.9 71.9 72.1 71.9 72.1 71.9 70.9 70.9 70.9 70.7 70.9 99.8 100.0 Nucleotide genetics distances are given below the diagonal and amino acid genetic distance above the diagonal. extraction and MALDI-TOF MS spectra to create accurate databases and specific and reproducible results in different laboratories for further research (25). Nonetheless, these internal databases are created in each reference laboratory, and for that reason, we find ourselves with the added difficulty of not being able to compare the different species recorded in the different databases created, being of huge importance to the creation of a single common database or being able to share the spectra obtained from the analyzes of each research laboratory. Nagorny et al. (67), in their research for the identification of different nematodes (Dirofilaria and Ascaris) by MALDI-TOF MS using tissues from adult worms, suggested that in the range from 8 to 20 kDa, the spectra allowed differentiating between different species of nematodes, and in the range of 2 to 6 kDa, the Frontiers in Veterinary Science | www.frontiersin.org 9May 2022 | Volume 9 | Article 867919