From Skin to Sequence: Pan-species Detection and Speciation of Leishmania

Abstract

Background

Leishmaniasis remains a significant public health concern. Accurate and efficient diagnostic methods are crucial for timely intervention and management. The limitations of current diagnostic approaches necessitated the exploration of novel targets and tools. This study describes 2 tandemly repeated sequences within the Leishmania genome that allow for sensitive and specific pan-Leishmania detection and speciation via quantitative polymerase chain reaction (qPCR) and recombinase polymerase amplification (RPA) and sequencing.

Methods

Through bioinformatic analysis of highly repeated elements of all Leishmania sp genomes, pan-Leishmania qPCR targets (CL3 and CL179) were identified and validated by qPCR. Amplicons from an RPA-based assay for CL179 qPCR with DNA from clinical samples were sequenced by nanopore sequencing.

Results

The limits of detection for CL3 and CL179 qPCRs were ∼96 and ∼217 fg of genomic DNA across available Leishmania spp known to infect humans. The CL3 and CL179 qPCR could detect a single L donovani–infected macrophage. When tested with DNA extracted from skin biopsies (n = 8) and swabs (n = 7) from patients confirmed positive by 18S qPCR, CL3 and CL179 exhibited 100% sensitivity. Microbiopsies (n = 9) were also positive, with sensitivities of 88% and 67% for CL3 and CL179 qPCRs, respectively. Nanopore sequencing of RPA products resulted in the accurate speciation of Leishmania strains in patients confirmed to have leishmaniasis.

Conclusions

CL3 and CL179 demonstrated high sensitivity and specificity, showing no cross-reactivity with related parasitic intracellular protists. Our data provide an ultrasensitive approach to the rapid diagnosis and subsequent speciation of all Leishmania strains known to infect humans.

This work adds a novel tool adaptable to field use and reference centers alike. Its superior sensitivity to current targets and its cost-effective ability to provide species-level identification of Leishmania spp will enable timely individualized therapy to patients in diverse settings.

Leishmaniasis is a neglected tropical disease (NTD) caused by intracellular protist parasites belonging to the genus Leishmania. Leishmania is transmitted to humans by a diverse array of phlebotomine sand flies in the family Psychodidae, subfamily Phlebotominae [1]. Clinical manifestations of leishmaniasis are diverse, often a reflection of the species of Leishmania. The most common forms of leishmaniasis are cutaneous (CL), mucocutaneous (MCL), and visceral (VL). CL, the most prevalent form of the disease, most often presents as a single cutaneous lesion at the site of the phlebotomine sand fly blood meal [2, 3]. MCL most frequently causes lesions on the oral and nasal mucous membrane lesions that can result in significant facial disfigurement [4] and may manifest as much as a year after resolution of CL lesions [2, 4]. VL is characterized by infection of the liver, spleen, and bone marrow and is the most lethal form of leishmaniasis [1, 2]. The estimated combined burden of CL and VL in terms of disability-adjusted life years is estimated to be 770 000 as of 2017 [5]. Although recent progress has been made toward eliminating leishmaniasis, CL, MCL, and VL remain a large public health problem in 4 ecoepidemiologic regions of the world: the Americas, East Africa, North Africa, and West and Southeast Asia [6]. An estimated 12 million people are infected with Leishmania and an additional 350 million people are at risk for infection [7]. In 2022, the World Health Organization reported the identification of 205 986 new CL cases and 12 842 new VL cases globally [8].

Early diagnosis of leishmaniasis improves prognosis and reduces risk of transmission. Furthermore, as treatments for leishmaniasis can be toxic and treatment regiments vary by the species of infecting Leishmania, clinical manifestations alone are not enough to deliver optimal care [9, 10]. Unfortunately, confirmation of a diagnosis is often not pragmatic in low-resource countries [2]. Thus, there is an urgent need for better diagnostics that are more sensitive, rapid, and less invasive than skin biopsies (the current gold standard) [5]. Toward achieving the goals set forth in the 2021–2030 NTD road map, the Diagnostic Technical Advisory Group to the World Health Organization identified the need for rapid point-of-care tests for (1) post–kala azar dermal leishmaniasis and (2) confirmation of CL at peripheral health facilities [11].

Commonly used and more traditional diagnostic methods for leishmaniasis include microscopy, parasite culture, and antibody detection. Microscopy of Giemsa-stained skin biopsies and lesion swabs in the case of CL and MCL, lymph node biopsies, bone marrow aspirates, and splenic biopsies in the case of VL [3] remains the reference test for diagnosis. While these provide a low-cost approach, sensitivity can be low, potentially yielding numerous false negatives in those with low parasite burdens [12, 13]. Culturing of Leishmania (in vivo) can increase detection sensitivity by allowing the multiplication of the parasites, but this method is time-consuming and labor intensive and requires sophisticated laboratory infrastructure [3]. Although leishmanin (Montenegro) skin test aids in the diagnosis of CL, it is neither a marker of active infection nor is it widely available; moreover, it is not a good tool for VL and is hence of limited value [14]. In comparison, CL Detect (InBios) based on the detection of Leishmania peroxiredoxin has high specificity, though its sensitivity is quite variable across species and endemic regions [15–18]. For VL, the direct agglutination test with freeze-dried antigen and the rK39 immunochromatographic test are the most widely used with varying sensitivities (90%–94%), depending on the geographic locations [19, 20]. Although antibody detection tests can indicate exposure to the parasite, they do not distinguish between current and past infections and can be prone to false positives due to cross-reactivity with Mycobacterium tuberculosis, Toxoplasma gondii, and Trypanosoma spp [3, 12, 13].

Quantitative polymerase chain reaction (qPCR) has emerged as an attractive alternative for the detection of Leishmania due to its high sensitivity and specificity; however, the genetic diversity among Leishmania species poses a challenge for the development of a universal assay [21]. Several pan-Leishmania qPCRs [22–26] have recently been developed to detect Leishmania in sandflies [24] and in clinical settings [26], such as an 18S qPCR assay [23] or the amplification and sequencing of ITS2 [25], which can suffer from cross-reactivity with other kinetoplastids or variable copy number across species, introducing difficulty in quantification of parasite load [22].

The recombinase polymerase amplification (RPA) assay targeting 18S rRNA and kinetoplast minicircle DNA has gained attention as a promising alternative due to its rapidity, isothermal nature, simplicity, and minimal equipment requirements [27, 28]. The present study focuses on the development and validation of a qPCR assay targeting conserved tandemly repeated sequences across Leishmania species with high sensitivity. Additionally, it introduces an RPA-based test for rapid diagnostics with potential point-of-care applications. Furthermore, variations in the repeat region enable species-level identification through sequencing of RPA amplicons.

METHODS

Patients and Sample Collection

All patients were seen as part of a National Institutes of Health institutional review board–approved protocol (NCT00344188) under the auspices of the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases. Eligibility criteria, diagnostic workup, and sample collection methods can be found at www.clinicaltrials.gov/study/NCT00344188. In brief, 2-mm skin punch biopsies collected for clinical care underwent DNA extraction by the clinical microbiology department at the NIH Clinical Center and were subjected to 18S sequencing. Remaining DNA was subjected to additional qPCR testing for this study. Swabs (dry or wet) were obtained from the ulcerative surface of the lesion. Microbiopsies were collected from the edge of the lesions where there was intact skin. Sequential patients enrolled from October 2021 to December 2024 were included in this study. Written informed consent was obtained from all participants to the use of stored specimens (biopsies, swabs, and microbiopsies) obtained as part of the initial evaluation.

Bioinformatic Identification of Target Sequences

The sequence read archives for various Leishmania species were processed to trim, clean, and obtained paired reads (supplementary data). The sequence read archive reads were analyzed according to a previously published RepeatExplorer2 [29] pipeline to obtain highly repeated elements in the genomes of the various Leishmania species and targets identified with little to no homology with closely related organisms.

DNA Extraction, qPCR, and RPA Reactions

DNA extracted from biopsies, swabs, or microbiopsies were stored at −20 °C until use (supplementary data). qPCR assays (3–5 different sets for each target) were designed for the most promising targets by the PrimerQuest Tool (Integrated DNA Technologies), and custom oligos were synthesized. An 18S qPCR assay was performed as described previously [23]. qPCR reactions were run in primer/probe combinations (Supplementary Table 1) and 1 μL of template on the ViiA 7 Real-time PCR System With 384-Well Block (Thermo Fisher Scientific). Reactions that did not demonstrate amplification prior to 40 cycles were listed as having a cycle threshold (Ct) of 40 cycles.

The RPA assays were done according to the manufacturer's instructions (TwistDx) with the TwistAmp basic kit. Primers can be found in Supplementary Table 2. Reactions were briefly vortexed and spun down before being incubated on a heat block at 40 °C for 5 minutes, at which point they were briefly vortexed and spun down once more before completing 25 more minutes of the 40 °C incubation (supplementary data).

Infected Macrophages

Murine RAW 264.7 macrophages were infected with Leishmania donovani stably expressing DsRed [30]. Infected macrophages were serially diluted into 20-μL aliquots of 3 standards: 30, 3, 1, 0.3, and 0.03 macrophages/μL. Replicates (n = 16) were boiled at 95 °C for 10 minutes, cooled, and centrifuged, and 1 μL of lysate was added to a qPCR reaction (supplementary data).

Sequencing and Analyses of Amplicons

The sequencing of amplicons for CL179 were performed by conventional Sanger sequencing and next-generation nanopore sequencing (Oxford Nanopore Technologies). Samples for sequencing on the Oxford Nanopore system were barcoded with the Rapid Barcoding Kit 96 SQK-RBK110.96 and sequenced on the MIN-106 flow cell with a MinION Mk1C. The sequenced reads (Sanger and Nanopore) were mapped to CL179 by Minimap2 (version 2.24) in Geneious Prime (version 2024.0) with the following parameters: k-mer size, 15; strategy, Oxford Nanopore (more sensitive); secondary alignments; minimum secondary to primary alignment score ratio, 0.8. Phylogenetic trees were generated in Geneious per the neighbor-joining method and Tamura-Nei model for genetic distances. Global alignment with free end gaps was used to obtain the distance matrix.

Statistical Considerations

All statistical analyses were performed in Prism (version 9; GraphPad). Due to the small sample sizes for clinical and experimental comparisons, formal hypothesis testing was not conducted. Instead, results are presented descriptively with geometric means as a measurement of central tendency.

RESULTS

CL3 and CL179 Targets Are Found in All Leishmania Species

NGS reads (sequence read archives of reference genome assemblies) for all Leishmania species deposited at NCBI as of October 2022 were analyzed in RepeatExplorer2 [29]. Comparative analyses identified highly repeated elements that were common or selectively present across all available species/strains (Figure 1A). Sequence analyses (blastn) of all contigs against the nonredundant nucleotide database resulted in the identification of 6 contigs (CL3, CL5, CL14, CL17, CL39, and CL179) as promising targets with little to no homology to other organisms. Although CL5, CL14, CL17, and CL39 had detectable reads across all species, only the CL3 and CL179 qPCR assays were able to detect each of the species utilized for screening with genomic DNA (gDNA; Supplementary Figure 1). By nature, all the clusters identified were repetitive elements in the genomes. While the CL3 monomer is only 45 base pairs in length and occurs as multiple copies in tandem in chromosome 35, CL179 with 1652 base pairs happens to be related to snoRNA and is located on chromosome 26. qPCR assays targeting CL3 and CL179 were performed with a wider set of gDNA standards from all clinically relevant species of Leishmania (Figure 1B). CL3 and CL179 were able to detect all species tested. This reactivity of CL3 and CL179 was specific to Leishmania, as gDNA of related protozoan parasites (Neospora caninum, Trypanosoma cruzi [types I–VI], Trypanosoma rangeli, or Toxoplasma gondii; Supplementary Table 4) failed to be amplified at similar concentrations of gDNA (Figure 1C).

Figure 1.

Identification of CL3 and CL179 as specific and sensitive pan-Leishmania qPCR targets. A, The heat map depicts the relative abundance of reads mapped to each target cluster (columns) in each species (rows). The heat map shows only the top 60 clusters. Red to blue indicates high to low abundance qPCR for CL3 and CL179: B, gDNA standards of clinically relevant species of Leishmania; C, 1 ng of gDNA from closely related protozoan parasites. 100 pg of L infantum gDNA was used as positive control. Ct, cycle threshold; gDNA, genomic DNA; qPCR, quantitative polymerase chain reaction.

As shown in Table 1, qPCR assays with gDNA expressed as copies of the haploid genome from multiple Leishmania species demonstrated that CL3 and CL179 were consistently positive for all Leishmania species tested. CL3 exhibited superior analytic sensitivity as compared with CL179, having an average cycle time 5.7 cycles lower than CL179, which translates to a ∼52-fold difference. Despite the differences in Ct values between CL3 and CL179, both targets had high analytic sensitivity with limits of detection for all species tested to be <6 copies of the haploid genome. Based on multiple replicates (n = 12), the observed average limit of detection was 2.6 and 5.9 copies of the haploid genome for CL3 and CL179, respectively, across all species, which translates to ∼96 and ∼217 fg of Leishmania gDNA for CL3 and CL179.

Table 1.

Analytic Limits of Detection of the CL3 and CL179 qPCR on Multiple Species of Leishmania

	CL3							CL179
	Mean Ct Value			Percentage Positive				Mean Ct Value			Percentage Positive
Species	30	3	0.3	30	3	0.3	LOD	30	3	0.3	30	3	0.3	LOD
L aethiopica	22.7	26.0	30.2	100	100	100	—	28.7	32.8	36.7	100	100	67	2.6
L amazonensis	27.0	34.7	39.5	100	75	8	3.8	35.7	39.7	40.0	87	25	0	33.8
L braziliensis	23.0	26.7	35.3	100	100	67	2.6	30.5	34.5	38.6	100	100	50	2.7
L chagasi	22.1	25.5	30.4	100	100	92	1.3	27.7	31.7	37.2	100	100	75	2.5
L donovani	25.0	28.8	34.8	100	100	75	2.5	32.1	36.6	39.2	100	75	25	4.1
L guyanensis	25.5	31.2	38.4	100	83	17	3.5	31.2	35.7	39.8	100	92	8	3.1
L infantum	27.5	30.8	36.8	100	100	50	2.7	32.2	36.9	39.8	100	67	8	4.3
L major	23.7	27.1	32.2	100	100	83	2.2	30.0	35.0	38.7	100	92	33	3.1
L mexicana	22.7	25.9	31.3	100	100	100	—	28.0	32.3	36.4	100	100	75	2.5
L panamensis	25.4	29.3	33.7	100	100	75	2.5	31.8	36.2	39.6	100	83	16	3.5
L tropica	23.8	27.1	33.8	100	100	75	2.5	31.5	35.6	40.0	100	100	0	2.9

CL3 and CL179 qPCR was performed on genomic DNA concentrations listed in terms of copy number (30, 3, 0.3) of the haploid genome of each Leishmania species. The averaged Ct values (n = 12) and percentage of wells that were positive (defined as Ct <40) for each concentration in each species is shown. The LODs were estimated by interpolating the 95% CI via a linear trend from 2 data points: 1 above and 1 below 95% positivity.

Abbreviations: Ct, cycle threshold; LOD, limit of detection; qPCR, quantitative polymerase chain reaction.

CL3 and CL179 Detect Leishmania in Biopsies, Swabs, and Microbiopsies

To evaluate the utility of the CL3 and CL179 qPCR in the clinical setting, DNA extracted from the skin biopsies of patients confirmed to have CL (n = 8) were compared with the currently used 18S qPCR assay. As shown in Figure 2A, CL3 and CL179 targets were detectable by qPCR in all patients. Furthermore, CL3 had Ct values on average 8.0 cycles lower than the currently used 18S qPCR, representing a 256-fold increase in analytic sensitivity. The Ct values of CL179 were on average 0.7 cycles lower than 18S across all samples, representing a 62% increase in analytic sensitivity.

Figure 2.

CL3 and CL179 detect Leishmania in various patient sampling methods. Mean Ct values of the CL3 and CL179 qPCR with extracted DNA: A, skin biopsies of patients confirmed positive for Leishmaniasis by 18S qPCR; B, swabs of patient lesions confirmed positive for Leishmania by 18S qPCR (DNA from skin biopsies); C, microbiopsies of lesions (L1, L2). Except for patient 1, the normal skin microbiopsies on the contralateral side of all other patients were negative for CL3 and CL179. Ct, cycle threshold; gDNA, genomic DNA; qPCR, quantitative polymerase chain reaction.

In a small subset of patients, the efficacy of the CL3 and CL179 qPCRs were evaluated with DNA extracted from swabs and microbiopsies [31, 32] as less invasive sampling methods as compared with skin biopsies. As shown in Figure 2B, qPCR of DNA from swabs (n = 7) was positive for CL3 and CL179. Wet swabs were more efficient as CL3 and CL179 were detected ∼4 and ∼3 cycles earlier, respectively, than DNA from dry swabs. Similar to the swabs, microbiopsies (n = 9) had higher Ct values when compared with the corresponding DNA from skin biopsies (Figure 2C). More important, the Ct values among different microbiopsies of the same lesions varied by several cycles, suggesting variability in the amount of Leishmania sampled among microbiopsies of the same lesion. Moreover, except for 1 patient, the normal skin microbiopsies taken from the contralateral side were negative for CL3 and CL179.

Single Infected Macrophages Can Be Reliably Detected

To further test the CL3 and CL179 qPCRs, we evaluated the efficacy of detecting a single infected macrophage in a sampled tissue. RAW macrophages infected with L donovani at multiplicities of infection (MOIs) of 1:5 and 1:10 (macrophage: Leishmania) were sorted by flow cytometry (Supplementary Figure 2A) and tested at limiting dilutions of 30, 3, 1, 0.3, and 0.03 macrophages/μL by qPCR for CL3 and CL179. As expected, CL3 qPCR had superior sensitivity as compared with CL179, where the delta between CL179 and CL3 Ct values ranged between 6 and 8 cycles (64- to 256-fold) lower for CL3 across all concentrations (30, 3, 1, 0.3, and 0.03) of macrophages (Figure 3A). This analytic sensitivity was similar irrespective of the MOI (Figure 3B, Supplementary Figure 2B). This resulted in determining the limits of detection for CL3 and CL179 as 0.228 and 1.4 infected macrophages, respectively, for an MOI of 1:5.

Figure 3.

CL3 and CL179 detect single Leishmania-infected macrophage. A, Mean Ct values of the CL3 and CL179 qPCRs (n = 12) with DNA from an estimated number of Leishmania donovani–infected macrophages with a multiplicity of infection of 1:5. B, The percentages of wells detected as positive (defined as Ct <40) are plotted for each macrophage standard. The limits of detection were estimated by interpolating the 95% CI via a linear trend from 2 data points: 1 above and 1 below 95% positivity. Ct, cycle threshold; qPCR, quantitative polymerase chain reaction.

Pan-Leishmania–Sensitive RPA

Due to its relatively small size, CL3 was not amenable for design of the RPA assay; hence, RPA assays were focused on CL179 only. Multiple RPA assay sets (Supplementary Table 3) targeting the CL179 repeat were screened with 100 pg of Leishmania gDNA in a basic RPA reaction, of which the combination of F8 and R8 (set 13; Supplementary Tables 2 and 3) detected all species tested (Figure 4A).

Figure 4.

Indels and SNPs in recombinase polymerase amplification targeting CL179 differentiate species of Leishmania. A, A simulated gel (Agilent 2100 Bioanalyzer) featuring the recombinase polymerase amplification products with set 13 of CL179 on various species of Leishmania. The numbers denote the estimated sizes of the amplicons in terms of base pairs. B, The consensus sequences as determined by previous whole genome sequencing of various species were aligned to CL179. Indels and SNPs in the set 13 region are highlighted, which allow speciation of Leishmania. C, The algorithm utilized to speciate patient samples is shown. Reads would be aligned to the consensus of CL179 across all species. Blue boxes describe possible SNPs or inserts present in some species that differ from the CL179 consensus across all species. Starting at step 1, if the insertion and SNPs listed in the blue box are present, the algorithm would be followed to the next blue box to the left in step 2. If the listed insertion and SNPs are not present, the algorithm would be followed to the next blue box to the right in step 2. This process would be repeated until an orange box is reached, indicating the sample to be infected with the species that has been determined by the algorithm. SNP, single-nucleotide polymorphism.

CL179 RPA–Based Speciation

As seen in Figure 4A, the size of amplicons following RPA varied among the species tested, suggesting possible differences in sequence that could allow for speciation. Phylogenetic analyses of the CL179 sequences from each species mimicked the known dendrogram structure of the genus Leishmania (Supplementary Figure 3). Preliminary analyses with existing genomes of various Leishmania species indicated that there would be enough variation in the CL179 region to aid in speciation based on SNPs and indels (Figure 4B). A consensus sequence of the CL179 repeat was generated from the Leishmania species used in Table 1, and species-specific SNPs and indels were identified by aligning individual sequences to this consensus. Based on the sequence variations, a flowchart-based algorithm was generated to enable species identification (Figure 4C). Comparison of amplicons sequenced by Nanopore and Sanger displayed good concordance (Supplementary Figure 4). Examples of the utilization of this algorithm in clinical samples can be found in Supplementary Figure 5.

The ability to speciate patient samples was carried out in a semiblinded manner. While qPCR was performed on biopsies, swabs, and microbiopsies, RPA was evaluated only on biopsies and swabs due to the limited availability of microbiopsy material. CL179 RPA reactions were sequenced with Nanopore using DNA from the skin biopsies (n = 9) and swabs (wet; n = 4) of patients who had previously been confirmed to have active leishmaniasis through 18S and CL3 qPCRs (of which 8 samples had been speciated through sequencing of an 7SL PCR [33]). When aligned with CL179, the resulting sequences confirmed the species identification initially determined by sequencing of the 7SL PCR products in all cases except the swab of patient 10, in which a closely related strain was indicated (Table 2). Notably, sequencing the CL179 RPA amplicons was able to identify previously unspeciated biopsies from patients 3 and 5. Additionally, the CL179 RPA sequencing provided improved species resolution for patients 4, 8, and 15, offering greater specificity than sequencing of 7SL PCR products.

Table 2.

Set 13 CL179 RPA Allows Sensitive Speciation of Leishmania From Patient Samples

Patient	7SL Skin Biopsies	Set 13 Skin Biopsies	Set 13 Swabs	18S/CL3
1	L major	L major	NAa	+/+
3	—b	L guyanensis/panamensis	NA	+/+
4	L guyanensis/panamensis	L panamensis	L guyanensis/panamensis	+/+
5	—	L guyanensis/panamensis	NA	+/+
8	L mexicana/amazonensis	L mexicana	NA	+/+
9	L aethiopica	L aethiopica	NA	+/+
10	L braziliensis	L braziliensis	L guyanensis/panamensis	+/+
12	L aethiopica	L aethiopica	NA	+/+
15	L tropica/aethiopica	L tropica	NA	+/+
20	L guyanensis/panamensis	NA	L guyanensis	+/+
21	NA	NA	L major	NA/+

All patients confirmed positive for leishmaniasis via an 18S or CL3 qPCR are listed. The positivity for the 18S and CL3 qPCRs is listed to the right. Species of Leishmania as determined by sequencing of 7SL PCR are shown when possible.

Abbreviation: qPCR, quantitative polymerase chain reaction.

^a NA (not available) denotes unable to be sequenced due to a lack of sufficient sample.

^bDash denotes that samples were not sequenced due to the 18S qPCR yielding a cycle threshold value >32, indicating a low likelihood of sequencing success.

DISCUSSION

The relative ease of use, sensitivity and specificity of qPCR assays for detection of Leishmania targeting the splice leader RNA region and kinetoplast minicircle DNA, some of which demonstrated high clinical sensitivity for Leishmania in DNA extracted from lesion skin biopsies [21, 25, 27, 34–36]. However, there is currently no consensus on a pan-Leishmania target for the detection of Leishmania. The identification of repeated sequences in the genomes of parasites allowed for development of highly sensitive diagnostics qPCRs targeting tandem repeats [37–40]. Using a similar approach, we describe 2 highly sensitive qPCR assays targeting different tandemly repeated sequences within the Leishmania genomes to detect Leishmania in CL and MCL lesion-sampling strategies such as biopsies, swabs, and microbiopsies.

Although both assays had high analytic sensitivity with limits of detection in the low single-digit copy numbers of haploid genomes, CL3 and CL179 were observed to have slightly lower efficacy for Leishmania guyanensis and Leishmania amazonensis, respectively. The slight variations across species could be due to evolutionary pressure on the repeated regions or the imperfect nature of the tandem repeats that influence qPCR primer/probe binding, or the number of repeats were comparatively less in L amazonensis or L guyanensis than in other Leishmania species, resulting in reduced but still high analytic sensitivity. Although the CL3 and CL179 assays were efficient in the diagnosis and speciation of clinical samples in this study, which originated from multiple geographic locations, further testing with a wider range of clinical isolates and hybrids is needed.

Swabbing CL and MCL lesions through the use of a cotton swab or cytology brush provides a less painful, less invasive, quicker, and easier-to-perform method of obtaining samples from patients as compared with taking a skin biopsy or lesion aspirates [32, 41]. Moreover, microbiopsies of CL and MCL lesions are gaining traction as an additional, even less invasive method of sample collection [42]. Although sampling and subsequent detection of Leishmania in the lesion site (CL or MCL) by microbiopsies can be tricky, microbiopsies provide a unique advantage to lesion swabs. Swabs work best when lesions are wet, such as those found in Latin America; however, they become less effective in regions where lesions are dry, making microbiopsies an attractive option outside of Latin America [31].

When compared with skin biopsies, swabs collect less total tissue; therefore, Leishmania is generally harder to detect in these samples [43, 44]. Given the small amount of tissue obtained in a swab or microbiopsy and the clustered distribution of Leishmania within the lesion, there remains a possibility of not sampling an infected macrophage and hence a false-negative result [31, 43, 45]. The ability to detect a single infected macrophage with either CL3 or CL179 highlights the utility in detecting infections in microbiopsies from noncutaneous leishmaniasis as well. This is probably reflected in the CL3 positivity in the presumed normal contralateral skin microbiopsies, which indicated a potentially disseminated infection (Figure 2C). The observed Ct variability across microbiopsies also highlights the likely need to sample multiple lesion sites to reduce false negatives, which in turn will result in higher costs. It needs to be determined whether this minimally invasive and less painful sampling helps to improve patient compliance to justify the increased resources.

RPA is a promising method for isothermal amplification and potentially for point-of-care testing due to its sensitivity, rapidity, ease of use to reduce technical requirements [46, 47], and independence from electricity, which reduces infrastructure requirements [48–50]. Although highly sensitive pan-Leishmania RPAs have been developed [27], CL179 RPA provides a unique benefit in that it allows subsequent speciation through sequencing of RPA amplicons/products. More recently, an ITS2-based qPCR assay was developed that provides high sensitivity and specificity in distinguishing among Leishmania species [25]. CL179 RPA offers an alternative method of speciating Leishmania with increased clinical sensitivity as compared with previous 7SL PCR sequencing methods. From a clinical perspective, certain species of Leishmania are associated with increased clinical risk of MCL such that determination of the species of Leishmania infecting a patient can be helpful in clinical management, especially in situations where >1 species is coendemic or where the patient has traveled across multiple geographic regions [10]. Interestingly, for patient 10, the sequencing results differed between biopsy and swab samples. This potentially reflects a mixed-species infection, site-specific sampling variability, or a limitation in distinguishing closely related Viannia species. Although this study relied on the generic infrastructure of well-equipped laboratories, implementation in resource-limited settings, though challenging, is not impossible. On average, the cost of RPA and sequencing of pooled barcoded samples amounts to approximately US $14 per sample.

CONCLUSION

The development of highly sensitive qPCR and RPA assays targeting conserved tandemly repeated sequences in Leishmania species represents a significant advance in the diagnostic capabilities for leishmaniasis. The CL3 and CL179 qPCR assays demonstrate superior clinical sensitivity across various species of Leishmania, allowing for reliable detection even in low–parasite burden samples and less invasive sampling methods, such as skin swabs and microbiopsies. Furthermore, the CL179 RPA assay not only provides a rapid and field-deployable diagnostic option but also enables species determination through sequencing of RPA amplicons that can be done at the point of patient contact by using portable sequencing technologies such as MinION (Nanopore). The ability to accurately detect and differentiate Leishmania species holds promise for improving patient outcomes through early diagnosis and tailored treatment strategies. Future validation studies should focus on confirming the effectiveness of these assays across diverse geographic regions and clinical settings.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. T cruzi samples were a kind gift from Michael Lewis (London School of Hygiene & Tropical Medicine). All data produced in the present work are contained in the article.

Financial support. This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.