A total of 21 households were visited during the sampling period, and 23 people were recruited. The sampled households were built similarly as simple huts with wood planks tied together as walls, little to no separation between rooms, a roof of palm leaves, and a latrine built nearby. The distribution of the households in the community is presented in Figure 1.
Sand fly diversity and distribution by collection environment
Ninety-three sand flies were captured at selected houses in the six months of fieldwork, of which 73 (78.5%) were females and 20 (21.5%) were males. Overall, the extradomicile yielded a slightly higher proportion of collected sand flies than the intradomicile (37.6% and 35.4%, respectively), and the peridomicile was less productive (27%).
Of the 93 captured sand flies, 76 were successfully identified to the species level (5 species), 15 were identified only to the genus level (Table 1); two individuals could not be identified by either morphological or molecular methods and were not included in the results. The most abundant species were Nyssomyia ylephiletor , Lutzomyia (Tricolateralis) cruciata and Brumptomyia mesai. The bioinformatic confirmation of species (BIN and neighbor-joining dendrogram) is presented in the Supplementary Materials (Supplementary Fig S1 and Table S1).
Table 1. Sand fly species collected at the study site (March–August 2022) by sex.
Sand fly species
|
Females
|
Males
|
Total
|
Nyssomyia ylephiletor
|
26
|
-
|
26
|
Lutzomyia (Tricholateralis) cruciata
|
20
|
4
|
24
|
Brumptomyia mesai
|
9
|
13
|
22
|
Dampfomyia sp.
|
9
|
2
|
11
|
Lutzomyia sp.
|
1
|
-
|
1
|
Brumptomyia sp.
|
2
|
1
|
3
|
Dampfomyia (Coromyia) deleoni
|
2
|
-
|
2
|
Bichromomyia olmeca
|
1
|
-
|
1
|
Pintomyia sp.
|
1
|
-
|
1
|
Total
|
71
|
20
|
91
|
Of the female sand flies, five species were collected in at least two trapping environments (Figure 2). Seven species were captured in both the extradomicile and peridomicile, representing 35.7% and 21.8% of the females collected in those areas, respectively. In the intradomicile, 42.5% of the females was collected and belonged to four species. More than 70% of the anthropophilic Ny. ylephiletor specimens was collected in the intradomicile, whereas Lu. cruciata was mostly collected in the extradomicile (45%).
Identification of sand fly blood meal sources
Three out of 73 females collected, had blood visible in the abdomen. All three were Ny. ylephiletor. Two were captured in the intradomicile, and one was captured in the peridomicile. Cytochrome b PCR and sequencing successfully identified the blood sources as human (2 samples) and domestic pig (Sus scrofa domestica) (1 sample). The obtained sequences had >98% identity with sequences deposited in GenBank.
Identification of Leishmania in sand flies
The 73 female sand flies were screened for Leishmania DNA by hsp70 PCR-N, and four (5.48%) were positive. Indiscriminate Leishmania DNA was detected in one Ny. ylephiletor captured in the intradomicile. The L. guyanensis complex (L. guyanensis or L. panamensis) was detected in three sand flies captured in the peridomicile or extradomicile (Table 2) but discrimination between the two possible species could not be achieved.
Table 2. Leishmania species identified by sand fly species and environment.
Sand fly species
|
Environment
|
Leishmania species
|
Nyssomyia ylephiletor
|
Intradomicile
|
Leishmania sp.
|
Dampfomyia deleoni
|
Extradomicile
|
L. guyanensis/L. panamensis
|
Brumptomyia sp.
|
Extradomicile
|
L. guyanensis/L. panamensis
|
Dampfomyia sp.
|
Peridomicile
|
L. guyanensis/L. panamensis
|
Demographic characteristics of CL patients
The MoH diagnosed 23 subjects with CL during the study period. Most of the identified patients were adult men, all of whom were farmers. Five adolescents (aged 12 to 17) were also diagnosed with CL, and four of these were girls. The most common lesion location was the lower legs, followed by the hands, neck, arms, and feet. The most recent lesion appeared a month before diagnosis, and one patient reported that their lesion had appeared more than 10 years ago (Table 3).
Table 3. Demographic characteristics of patients diagnosed with CL from March to August 2022 at the study site.
Characteristic
|
|
|
Age, years, mean ± SD
|
34 ± 16
|
Number of lesions per participant, mean (range)
|
1.2 (1–3)
|
|
n
|
%
|
Sex
|
|
|
Male
|
13
|
56%
|
Female
|
10
|
44%
|
Maya Q’eqchi’ ethnic group
|
23
|
100%
|
Previous leishmaniasis
|
2
|
8%
|
Location of lesions
|
|
|
Lower legs
|
10
|
34.5%
|
Hands
|
5
|
17.2%
|
Neck
|
3
|
10.3%
|
Feet
|
3
|
10.3%
|
Forearms
|
2
|
7%
|
Back
|
2
|
7%
|
Upper legs
|
2
|
7%
|
Ear
|
1
|
3.4%
|
Arm
|
1
|
3.4%
|
Identification of Leishmania in clinical samples
Of the 23 patients diagnosed during the study time, only 11 tissue scrapes were obtained from the MoH for this study. The remaining tissue scrapes had a positive diagnosis confirmed by the MoH but were not available as they were send to the reference laboratory for quality control within the MoH.
PCR-F product sequencing successfully typed 10 of 11 clinical samples, identifying four Leishmania species. For one clinical sample, no amplification was obtained from the PCR-F, so PCR-N was performed, followed by sequencing. This sample was identified as part of the L. guyanensis complex (L. guyanensis/L. panamensis), and discrimination between the species was not possible (Table 4).
Table 4. Leishmania species identified in clinical samples (lesion scraping) of CL patients from the study site.
Leishmania species
|
Clinical samples, n (%)
|
L. panamensis
|
5 (46%)
|
L. guyanensis
|
3 (27%)
|
L. braziliensis
|
1 (9%)
|
L. infantum
|
1 (9%)
|
L. guyanensis /L. panamensis
|
1 (9%)
|
Total
|
11
|