Analysis of 26 STR loci (PowerPlex® Fusion 6C System) in a mestizo population from Mexico city.

BACKGROUND: Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). METHODS AND RESULTS: 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. CONCLUSIONS: The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.

The Missing Person problem through the lens of information theory.

Missing person cases typically require a genetic kinship test to determine the relationship between an unidentified individual and the relatives of the missing person. When not enough genetic evidence has been collected the lack of statistical power of these tests might lead to unreliable results. This is particularly true when just a few distant relatives are available for genotyping. In this contribution, we considered a Bayesian network approach for kinship testing and proposed several information theoretic metrics in order to quantitatively evaluate the information content of pedigrees. We show how these statistics are related to the widely used likelihood ratio values and could be employed to efficiently prioritize family members in order to optimize the statistical power in missing person problems. Our methodology seamlessly integrates with Bayesian modeling approaches, like the GENis platform that we have recently developed for high-throughput missing person identification tasks. Furthermore, our approach can also be easily incorporated into Elston-Stewart forensic frameworks. To facilitate the application of our methodology, we have developed the forensIT package, freely available on CRAN repository, which implements all the methodologies described in our manuscript.

Forensic parameters and population structure based on 21 autosomal STRs with the investigator 24plex QS in mestizos from the Mexico City population.

Allele frequencies and forensic parameters for 21 STR autosomal markers (CSF1PO, D10S1248, D12S391, D13S317,D16S539, D18S51, D19S433, D1S1656,D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX and vWA) were reported in 289 unrelated individuals from Mexico City, Mexico. In addition, an interpopulation analysis was performed including other world populations. In brief, the established population database of 21 autosomal STR markers in the present work is adequate for human identification purposes.

Analysis of 12 X-STRs loci in a population from southeastern Brazil.

Short tandem repeat (STR) markers on the X chromosome have a high potential for solving complex kinship analysis and individual identification cases. To achieve such purposes, allele and haplotype frequencies for the specific population are necessary. Nonetheless, such frequencies are not always available. Therefore, we obtained haplotypes from 520 unrelated males from four different geographic regions of Espírito Santo - Brazil, using the Investigator Argus X-12 kit. Forensic parameters for linked groups of four X-STR loci are reported. Genetic distance analyzes suggest that ES population is genetically closer to the Italian population and farther from the Mexican one, among the populations analyzed in this study.

Identification of the Remains of an Adult Using DNA from Their Deciduous Teeth as a Reference Sample.

In many forensic cases, the identification of human remains is performed by comparing their genetic profile with profiles from reference samples of relatives, usually the parents. Here, we report, for the first time, the identification of the remains of an adult using DNA from the person's deciduous teeth as a reference sample. Fragments of a skeletonized and burned body were found, and a short tandem repeat (STR) profile was obtained. A woman looking for her missing son went to the authorities. When the DNA profile of the woman was compared to a database, a positive match suggested a first-degree kinship with the person to whom the remains belonged. The woman had kept three deciduous molars from her son for more than thirty years. DNA typing of dental pulp was performed. The genetic profiles obtained from the molars and those from the remains coincided in all alleles. The random match probability was 1 in 2.70 × 1021. Thus, the remains were fully identified. In the routine identification of human remains, ambiguous STR results may occur due to the presence of null alleles or other mutational events. In addition, erroneous results can be produced by false matches with close family members or even with people who are completely unrelated to the victim, such that, in some cases, a probability of paternity greater than 99.99% does not necessarily indicate biological paternity. Whenever possible, it is preferable to use reference samples from the putative victim as a source of DNA for identification.

Reference population database for 24 STR loci of selected provinces of Argentina.

BACKGROUND: Argentinean population is the result of admixture between South Amerindians, Europeans and to a lesser degree, Africans. Since the advent of forensic molecular genetics, the construction of local reference databases became mandatory. Aiming to further extend the technical quality reference database of Argentina, we present herein the allele frequencies for 24 autosomal STRs, including D22S1045, and SE33 (not previously reported for Argentina in STRidER). CONCLUSIONS: Genotypes of 6454 unrelated individuals (3761 males and 2694 females) from 13 out of 23 provinces were analysed. Forensic parameters were calculated for each marker. The observed heterozygosity ranged from 0.661 (TPOX) to 0.941 (SE33). The locus SE33 was revealed to be the most informative marker showing the highest values for PIC (0.955), GD (0.952), TPI (8.455) and PE (0.879). On the other hand, TPOX turned out to be the least informative marker: PIC (0.618), GD (0.669), and PE (0.371). The high number of analyzed individuals allowed detecting low frequency alleles and microvariants in CSF1PO; D16S539 and D21S11 D18S51; PENTA D; PENTA E and at locus D6S1043. METHODS AND RESULTS: This study is the most extensive for Argentina and complements the already reported information concerning the autosomal STRs commonly used in forensic identification. The results were submitted passing STRidER quality control standards (QC), receiving the reference number STR000327 v.2.

Three mutations at a Y-STR haplotype defy a paternal half-brothers kinship case analysis.

This work presents the results of a DNA test aimed to determine a possible biological link of paternal half brotherhood of two males. The combined use of biparentally inherited markers (autosomal STRs) and a panel of 27 Y-STRs allowed us to determine the existence of a biological relationship of kinship, even after detecting three mutations at their Y-STR haplotypes along the analyses, constituting an infrequent multiple mutation situation. This case is an example illustrating the importance of having different analytical markers sets and strategies for clarifying complex kinship cases where mutations occur.

Allelic frequencies distribution and forensic parameters of 23 autosomal short tandem repeats in the population of the State of Pernambuco, Brazil.

Located in the Northeast Region, the Pernambuco State is one of the 27 federative units of Brazil. Here, we determined populational data for 23 short tandem repeat (STR) markers - CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, PENTA D, PENTA E and SE33 - of the Pernambuco population. The sample consisted of 767 healthy, adult, unrelated individuals (437 males, 330 females) born and resident in the State of Pernambuco. STRs were amplified using three multiplex kits, according to the availability: PowerPlex® Fusion 6C System (Promega Corporation), PowerPlex® Fusion System (Promega Corporation) and GlobalFiler™ Express (Thermo Fisher Scientific). Allelic frequencies, forensic parameters and Hardy-Weinberg equilibrium determinations were estimated for all the 23 loci. No deviations from the Hardy-Weinberg equilibrium were observed for any of the markers, after Bonferroni correction. We observed that the most and less informative markers were SE33 and TPOX, respectively. The combined power of discrimination (CPD) was 0.99999999999999999999999999999, and the combined power of exclusion (CPE) was 0.99999999997. The cumulative typical paternity index was 37,919,301,869.3021. Interpopulation analyses (Nei's genetic distance) based on the expanded CODIS core loci was performed between the Pernambuco sample and other global populations. Pernambuco was the closest Brazilian population to African group and stayed distant from the Native American group. This work contributed to show that a panel of 23 autosomal STR loci is very informative, being able for forensic applications related in this population.

Characterization of 58 STRs and 94 SNPs with the ForenSeq™ DNA signature prep kit in Mexican-Mestizos from the Monterrey city (Northeast, Mexico).

BACKGROUND: STR allele frequency databases from populations are necessary to take full advantage of the increased power of discrimination offered by massively parallel sequencing (MPS) platforms. MATERIAL AND METHODS: For this reason, we sequenced 58 STRs (aSTRs, X-STRs, and Y-STRs) and 94 identity informative SNPs (iiSNPs) on 105 Mestizo (admixed) individuals from Monterrey City (Northeast, Mexico), with the Primer Set-A of the ForenSeq™ DNA Signature Prep Kit. RESULTS: Most of the STR markers were in Hardy Weinberg equilibrium, with a few exceptions. We found 346 different length-based alleles for these 58 STRs; nevertheless, they became 528 alleles when the sequence was assessed. The combined power of discrimination from autosomal STRs (aSTRs) was -virtually- 100% in both length and sequence-based alleles, while the power of exclusion was 99.9999999976065 and 99.9999999999494%, respectively. Haplotypes based on X-STRs and Y-STRs showed 100% of discriminatory capacity. CONCLUSIONS: These results provide -for the first time- forensic genomic population data from Mexico necessary for interpretation in kinship and criminal analyses.

Genetic study with 21 autosomal STRs in five Peruvian macro regions for human identification purposes.

In this population study 1541 samples in total were collected and analyzed. The samples were collected from five jurisdictions: North macro region (n = 272), Central macro region (n = 404), South macro region (n = 272), East macro region (n = 197), and the Lima macro region (n = 396). The samples were analyzed using the Investigator 24 plex GO and Investigator 24 plex QS kits which enable typing of 21 autosomal STR loci and an amelogenin marker for sex determination. The combined power of discrimination and the combined probability of exclusion for the total population were 0.9999999999 and 0.99999978, respectively. These population geographic subgroupings are similar, supporting that the combined Peruvian data or individual subgroupings could be used for generating statistics in forensic casework.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Analysis and comparison of the STR genotypes called with HipSTR, STRait Razor and toaSTR by using next generation sequencing data in a Brazilian population sample.

Short tandem repeats (STRs) are particularly difficult to genotype with rapid evolving next-generation sequencing (NGS) technology. Long amplicons containing repetitive sequences result in alignment and genotyping errors. Stutters arising from polymerase slippage often result in reads with additional or missing repeat copies. Many tools are available for analysis of STR markers from NGS data. This study has evaluated the concordance of the HipSTR, STRait Razor, and toaSTR tools for STR genotype calling; NGS data obtained from a highly genetically diverse Brazilian population sample have been used. We found that toaSTR can retrieve a larger number of genotypes (93.8%), whereas HipSTR (84.9%) and STRait Razor present much lower genotype calling (75.3%). Accuracy levels for genotype calling are very similar (identical genotypes ~95% and correct alleles ~ 97.5%) across the three methods. All the markers presenting the same genotype through the methods are in Hardy-Weinberg equilibrium. We found that combined match probability and combined exclusion power are 2.90 × 10-28 and 0.99999999982, respectively. Although toaSTR has varying locus-specific differences and better overall performance of toaSTR, the three programs are reliable genotyping tools. Notwithstanding, additional effort is necessary to improve the genotype calling accuracy of next-generation sequencing datasets.

A forensic population database in El Salvador: 58 STRs and 94 SNPs.

We have genotyped the 58 STRs (27 autosomal, 24 Y-STRs and 7 X-STRs) and 94 autosomal SNPs in Illumina ForenSeq™ Primer Mix A in a sample of 248 men and 143 women from El Salvador, Central America. Regional division (Centro, Oriente, Occidente) showed in almost all cases FST values not significantly different from 0, and further analyses were applied only to the undivided, country-wide population. The overall random match probability (RMP) decreased from 6.79 × 10-31 in length-based genotypes in the 27 autosomal STRs to 1.47 × 10-34 in repeat-sequence based genotypes. Combining the autosomal loci in this set, RMP reaches 2.97 × 10-70. In a population genetic analysis, El Salvador showed the lowest FST values with US Hispanics both for autosomal and X-STRs; however, it was much closer to Native Americans for the latter than for the former, in accordance with the well-known gender-biased admixture that created most Latin American populations.

Touch DNA recovery from vehicle surfaces using different swabs.

Several methods of DNA collection are used in places or objects related to crimes, the most common being the use of swabs. However, it is known that the efficacy of touch DNA recovery can be affected by collection devices and surfaces. The aim of this work was to evaluate the efficiency of three different types of swab in recovering touch DNA collected from different parts of a vehicle. The following swabs were tested: PurFlock® swab (Puritan, USA), 4N6FLOQSwabs™ (Copan S.p.A., Italy), and cotton swab (Labor Import). The experiments were carried out in the same vehicle, using the gearshift knob, the parking brake lever, and the steering wheel as support for the collection of touch DNA. Swabs showed significant differences in the amount of DNA recovered (Hc = 53.52; p < 0.05) and in the rate of allele amplification (Hc = 24.3; p < 0.05). The results indicated a greater DNA recovery efficiency by PurFlock® swab, followed by cotton, and then 4N6FLOQSwabs™. However, there was no significant difference among the surfaces analyzed. PurFlock® swab was more efficient for recovering donor alleles than the others (cotton and 4N6FLOQSwabs™), especially for small DNA amounts. This swab was, therefore, suitable for collections in vehicles involved in crime. Furthermore, this study highlights the need to assess different materials and methods of collection of biological samples, considering collection, extraction, and amplification.

Population data for 21 STRs for the Salvadoran population using GlobalFiler Express and GlobalFiler STR Amplification Kits.

With the advent of expanded STR (short tandem repeats) typing kits, it was necessary to determine allele frequencies and other appropriate population data parameters for El Salvador. Samples were collected from the central, east, and west regions of the country and typed for 21 forensically relevant STR loci. The data indicate that all loci are highly polymorphic, the three regions are genetically similar, and the population data are similar to those of US Hispanics. The results of this study support that the allele frequency data described herein can be used for statistical calculations for human identity testing in El Salvador.

DNA fingerprinting reveals varietal composition of Vietnamese cassava germplasm (Manihot esculenta Crantz) from farmers' field and genebank collections.

KEY MESSAGE: A molecular analysis using informative SNP markers in 1570 clones of cassava from Vietnam reveals varietal composition from farmers' field and genebank collections Cassava is the most important smallholder cash crops in Southeast Asia and is especially used in industrial products. Yet, systematic genetic studies on molecular markers from Vietnamese germplasm have not been considered for breeding and conservation programs. We conducted a molecular analysis of 1570 clones of cassava germplasm from farms across six agro-ecological zones using informative SNP markers. We unraveled the genetic diversity and population structure and provided insights into the value of breeding and conservation programs. Duplicated genotypes comprised 98% of the total sample of the Central Highlands region. Ninety-six SNPs were amplified Central Highlands and South East provinces had the highest allelic richness, covering up to 83% of alleles. The average observed heterozygosity (Ho = 0.43) was slightly higher than expected (He = 0.40) across SNP markers, suggesting an excess of heterozygotes plants. Diversity indexes indicated that cassava populations from North West and Eastern Vietnam are genetically diverse (mean He = 0.40). Genetic parentage tests identified 85 unique genetic groups within the varieties KM94, KM419, BRA1305, KM101, KM140, PER262, KM60, KM57 and two unidentified varieties, which accounted for 82% of the frequency distribution. KM94 is the most dominant variety in Vietnamese farms surveyed (38%), reflecting its superior quality and productivity. Discriminant analysis of principal components (DAPC) revealed four main subgroups, which were partially corroborated by neighbor joining (NJ) analyses. After removing duplicates, 31 unique genotypes were distributed across five of the agro-ecological zones. These were well distributed in the subgroups revealed via DAPC and NJ analyses. The genetic groups identified herein could be used to select unique accessions that should ideally conform with ex situ germplasm collections and identify areas where on-farm conservation programs should be targeted. Newly identified genotypes may also contribute as genetic breeding resources that could be used to adapt cassava to future changes and farmers' needs.

Evaluation of Promega PowerSeq™ Auto/Y systems prototype on an admixed sample of Rio de Janeiro, Brazil: Population data, sensitivity, stutter and mixture studies.

Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10-28 to 7.6 × 10-33. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (-1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeqTM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.

Patterns of genetic diversity in Colombia for 38 indels used in human identification.

The current population of Colombia has a genetic heterogeneity resulting from different migrations from other continents and within the country. In addition, there are small groups in their territory that have remained isolated and therefore have a different genetic pool in relation to that of the neighbouring urban populations. This population stratification must be considered in forensic analysis, being more complex for markers with marked intercontinental differentiation. In this study, population differentiation in Colombian admixed, native, and Afro-descendant populations was evaluated for a group of 38 indels described for forensic use. Allelic frequencies and parameters of forensic relevance were determined in each of the groups defined based on population differentiation analyses. In addition to the differences found between population groups, the results show that the set of 38 indels analysed could be useful in studies of individual identification in Colombia. The exclusion power presented by this set of markers suggests the need for joint use with other markers, being able to complement the STRs in paternity cases. High levels of both power of discrimination and exclusion were found when complementing the 38 HID-indels with a second multiplex, for a total of 83 indels.

Autosomal STR and SNP characterization of populations from the Northeastern Peruvian Andes with the ForenSeq™ DNA Signature Prep Kit.

Autosomal DNA data from Peru for human identity testing purposes are scarce in the scientific literature, which hinders obtaining an appropriate portrait of the genetic variation of the resident populations. In this study we genetically characterize five populations from the Northeastern Peruvian Andes (Chachapoyas, Awajún, Wampís, Huancas and Cajamarca). Autosomal short tandem repeat (aSTR) and identity informative single nucleotide polymorphism (iiSNP) data from a total of 233 unrelated individuals are provided, and forensic genetic parameters are calculated for each population and for the combined set Northeastern Peruvian Andes. After correction for multiple testing in the whole dataset of the Northeastern Peruvian Andes, the only departure from Hardy-Weinberg equilibrium was observed in locus rs2111980. Twenty one out of 27 aSTR loci exhibited an increased number of alleles due to sequence variation in the repeat motif and flanking regions. For iiSNPs 33% of the loci displayed flanking region variation. The combined random match probability (RMP), assuming independence of all loci (aSTRs and iiSNPs), in the Chachapoyas, the population with the largest samples size (N = 172), was 8.14 × 10-62 for length-based data while for sequence-based was 4.15 × 10-67. In the merged dataset (Northeastern Peruvian Andes; N = 233), the combined RMP when including all markers were 2.96 × 10-61 (length-based) and 3.21 × 10-66 (sequence-based). These new data help to fill up some of the gaps in the genetic canvas of South America and provide essential length- and sequence-based background information for other forensic genetic studies in Peru.

Molecular Identification of Parasites Causing Cutaneous Leishmaniasis in Panama.

Isolates from 475 cutaneous leishmaniasis (CL) patients from three endemic regions were studied by three typing techniques. The molecular analysis from lesion scrapings based on hsp70 PCR-restriction fragment length polymorphism (RFLP) showed that 78.1% (371/475) restriction patterns corresponded to Leishmania (Viannia) panamensis, 19% (90/475) to Leishmania (Viannia) guyanensis, and 3.0% (14/475) to Leishmania (Viannia) braziliensis. Promastigotes isolated by culture from lesions of 228 patients (48.0%, 228/475) were identified by multi-locus enzyme electrophoresis. Of them, 95.2% (217/228) were typified as L. (V.) panamensis, 1.3% (3/228) as L. (V.) guyanensis, 2.2% (5/228) as L. (V.) braziliensis, and 1.3% (3/228) as hybrids (L. [V.] braziliensis/L. [V.] panamensis). However, a partial sequencing analysis of the hsp70 gene from 77 selected samples showed 16.9% (13/77) typified as L. (V.) panamensis, 68.8% (53/77) as Leishmania (V.) sp., 1, 3.9% (3/77) as L. (V.) guyanensis, 1.3% (1/77) as L. (V.) braziliensis outlier, 2.6% (2/77) as Leishmania (Viannia) naiffi, 2.6% as (2/77) Leishmania (V.) sp., and 2 and 3.9% (3/77) hybrid isolates of L. (V.) braziliensis/L. (V.) guyanensis. These results confirm L. (V.) panamensis as the predominant species and cause of CL lesions in Panama and that L. (V.) guyanensis, L. (V.) braziliensis, and L. (V.) naiffi are circulating to a lower degree. Furthermore, the determination of parasite isolates belonging to atypical clusters and hybrid isolates suggests the circulation of genetic variants with important implications for the epidemiology and clinical follow-up of CL in Panama. No evidence of the existence of parasites of the Leishmania (Leishmania) mexicana complex in Panamanian territory was found in this study.