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Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia
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Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia
Mabel Moreno Investigationmethodology and writing original draft , ´ Lina Guzman-Rodr´ ıguez Investigationmethodology and writing- review and editing , Carlos Valderrama-Ardila Conceptualizationinvestigationmethodology and writing- review and editing , Neal Alexander Conceptualizationinvestigationmethodology and writing- review and editing , Clara B. Ocampo Conceptualizationinvestigationmethodologywriting- review and editingsupervision and funding ac PII: DOI: Reference:
S0001-706X(19)31062-9 https://doi.org/10.1016/j.actatropica.2019.105315 ACTROP 105315
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
12 August 2019 18 December 2019 18 December 2019
Please cite this article as: Mabel Moreno Investigationmethodology and writing original draft , ´ Lina Guzman-Rodr´ ıguez Investigationmethodology and writing- review and editing , Carlos Valderrama-Ardila Conceptualizationinvestigationmethodology and writing- review and editing , Neal Alexander Conceptualizationinvestigationmethodology and writing- review and editing , Clara B. Ocampo Conceptualizationinvestigationmethodologywriting- review and editingsupervision and funding ac Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105315
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Highlights A rapid method successfully characterized exposure to phlebotomine vectors of cutaneous leishmaniasis.
Monocultures around rural houses favor abundance and dominance of particular phlebotomine species.
Increasing distance between monocultures and houses decreases phlebotomine abundance around the latter.
Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia
Mabel Moreno1 *, Lina Guzmán-Rodríguez1, Carlos Valderrama-Ardila2**, Neal Alexander1, 2, Clara B. Ocampo1,2
1
Centro Internacional de Entrenamiento e Investigaciones Médicas, CIDEIM. Cali, Colombia.
2
Universidad ICESI. Cali, Colombia
* Current affiliation, Coomeva EPS regional suroccidente. Cali, Colombia ** Current affiliation, Universidad del Rosario, Bogotá, Colombia
Corresponding Author: Clara B. Ocampo, Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Carrera 125 # 19-225, Cali, Valle del Cauca 760031, Colombia. email: [email protected].
Abstract American cutaneous leishmaniasis is a public health concern in Colombia, its incidence being sustained or focally increased principally by the emergence of domestic transmission concomitantly with the adaptation of the phlebotomine vectors to habitat transformation around households. The objective of the study is to scale up a rapid characterization methodology for evaluating the relationship of land use around the house to the composition and abundance of phlebotomines. Five sites with a history of domestic leishmaniasis transmission in the Andean area of Colombia were selected. The peri-domestic
habitat was evaluated at 10 meter intervals along eight radial transects, centered on each house, at 45° intervals using a web pattern. Phlebotomines were captured by placing three CDC light traps over two nights both indoors and outdoors (10 m from the house). Blood source and infection were determined by PCR. Spearman rank correlation coefficients and negative binomial regression were used to quantify associations between the phlebotomine abundance and habitat categories. The study demonstrated that the vectors were largely anthropophagic (62% of 79 were human blood) and that a single species in each site was favored by the pertaining agriculture monoculture. Specifically, Pintomyia (Pifanomyia) quasitownsendi was associated with sugar cane in Novillero and La Esmeralda; while Pi. (Pif.) longiflocosa was associated with coffee plantations in Agua Bonita and El Cucal. Honda Alta had a more diverse array of land use and forest coverage with a lower number of specimens but higher species diversity. In terms of distance from the house to an area of a given land use, the abundance of Pi. (Pif.) quasitownsendi was inversely related to the distance to sugar cane plantation (Spearman correlation coefficient, ρ = -0.56, p < 0.001 for outdoor catches, and ρ = -0.50, p < 0.001 indoors). A similar inverse relationship was observed for Pi. (Pif.) longiflocosa with regard to technified coffee (ρ = -0.51, p < 0.001 outdoors, and ρ = -0.48, p < 0.001 indoors). This rapid characterization methodology could guide public heath decision makers in identifying those houses at higher risk of domestic transmission, and also educate farmers to increase the distance between their crops and any neighboring houses.
Keywords Leishmaniasis; land use; phlebotomines; risk factors; Andean region; Colombia.
1.
Introduction
The leishmaniases are a group of neglected tropical diseases caused by parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) transmitted by the bite of infected female phlebotomines (Psychodidae: Phlebotominae) to wild and domestic animals and increasingly to humans (Fitzpatrick and Engels, 2015; Pan American Health Organization, 2018; World Health Organization, 2010).
In America, leishmaniasis transmission in domestic and peri-domestic environments has generated a notable increase of cases, especially of the cutaneous form (Ampuero et al., 2005; Maroli et al., 2013; Valderrama-Ardila et al., 2010). In Colombia, during the 1990s, an average of 6,500 new cases of leishmaniasis were reported per year, however, recently reports has increase to an average of 14,000 cases which shows an unusual increase in cases during the last years, were 97% of leishmaniasis is the cutaneous form (Ministerio de Salud, 2010). This epidemiological change has been attributed mainly to the adaptation of the vectors to new transformed habitats, e.g. by finding human and non-human blood sources in domiciliary areas. Other factors include the ability of the parasites to adapt to other vector species, high rates of human migration from endemic areas, and haphazard and rapid urbanization (Colwell et al., 2011; Ferro et al., 2011; King et al., 2004; Ocampo et al., 2012).
In Colombia, cutaneous leishmaniasis (CL) is the most frequent clinical form, with the Andean region accounting for most reported cases (Zambrano and Mercado, 2014). In terms of the transmission risks already mentioned, a specific form of habitat transformation is the extension of agricultural monoculture (Contreras-Gutiérrez et al., 2014). Knowledge of the interaction of these risk factors is crucial to understanding the transmission dynamics in a specific focus. To identify disease control strategies, a rapid eco-epidemiological assessment technique was developed in a previous study carried
out in Chaparral, Colombia. Characterization of the land use along eight transects radiating from a specific house was able to identify houses with a higher density of sandflies that was associated with a higher risk of CL transmission (Ocampo et al., 2012). This study found that the proximity of dwellings to forest, or to traditional coffee plantations, was associated with abundance of the main vector Pintomyia (Pifanomyia) longiflocosa (Ocampo et al., 2012).
The objective of the current study is to scale up this rapid characterization methodology and apply it to four other foci of domestic transmission of CL in the Andean region of Colombia, and to evaluate the relationship of land use around the houses to the composition and abundance of phlebotomines.
2.
Materials and methods
2.1 Study locations A cross-sectional eco-epidemiological study was conducted in four townships selected from highly endemic municipalities for cutaneous leishmaniasis in the Andean region of Colombia: Novillero- San Benito Municipality (6.0678, -73.5617 – Santander department); La Esmeralda – Villeta (5.0666, -74.5333 – Cundinamarca department); Honda Alta-Rivera, (2.7543, -75.2153 – Huila department) and El Cucal – Rovira (4.1789,-75.3804 -Tolima department) (Figure 1). Collections were carried out during the dry season, in July and September of 2014 (Novillero – La Esmeralda) and February and May of 2015 (El Cucal – Honda Alta). Additionally, we use the data obtained in the ―focal study‖ carried out in Agua Bonita-Tolima (Ocampo et al., 2012) to increase the scope and statistical power of this analysis. The regression analyses described below had not previously been applied to these data.
Figure 1. Study areas in four departments of the Andean region of Colombia with domestic transmission of American cutaneous leishmaniasis.
The climate of the Andean region of Colombia varies with its topography. The study sites lie in the portion of this region which is subject to leishmaniasis transmission: between the eastern and central mountain ranges, and in the inter-Andean valley of the Magdalena River. The main agricultural products are coffee, sugar cane, banana, rice and cotton. The sites were selected in collaboration with the Local Secretary of Health, based on reported cases of cutaneous leishmaniasis during 2000- 2009 (Department of Public Health – Sistema de Vigilancia en Salud Pública - SIVIGILA and municipal hospitals) and accessibility to the Health Services. Reported numbers of cases were: San Benito (98 cases), Villeta (163), Rovira (279) and Rivera (203). At these four new sites, the location of the houses located within an altitudinal range of 1000 to 2000 m.a.s.l, was mapped. A number was assigned to each of the houses and these were placed in a plastic bag and randomly drawn without replacement. Fifteen houses were sampled per site.
2.2 Sample size determination The sample size calculation used data from the above-mentioned previous study in Chaparral (Ocampo et al., 2012). We chose the number of houses (N) based on the putative Spearman rank correlation coefficient (ρ) between density of phlebotomines and land use: √N – 3C(ρ) = Zα + Zβ (Lachin, 1981), where C is Fisher‘s inverse hyperbolic tangent transformation, and Z represents the quantiles of the normal distribution for significance level and power 1-. With 42 houses from Agua Bonita (Chaparral) and 15 from each of the four new sites, to a total of 102, the power is 86% for ρ=0.30.
2.3 Demographic and epidemiological data. A demographic and epidemiological questionnaire was applied to one adult in each selected house. This included information on: the number of residents, their gender and age; whether each person had active or inactive lesions suggestive of leishmaniasis, type of any treatment received, habitual use of
bed nets, house construction, and use of domestic insecticides (mainly pyrethroids). In Colombia, leishmaniasis control is mainly associated to case treatment, sporadic fumigation and bed net distribution; however this last two has low coverture and depends on disease transmission.
2.4 Phlebotomine sampling To evaluate the composition and abundance of phlebotomine sand flies, three Centers for Disease Control and Prevention (CDC) incandescent light traps were located in each house over two consecutive nights. One trap was placed indoors, and another two at different points 10 m from the house. All traps were placed at a height of 1.5 m and were active from 18:00 to 06:00 hours. The catches were recovered from the traps early the following morning and immobilized with triethylamine (TEA: 04885-1; Fisher Scientific, Pittsburgh, PA). The collection mesh of the CDC traps was introduced into a black plastic bag for the immobilization of the insects captured using a piece of cotton moistened with 1 ml of TEA for 15 minutes. The phlebotomines were separated from other insects and stored in plastic tubes with 70% ethanol during transportation to the laboratory (Ferro et al., 2011). Each tube was labeled recording the code of the house, the date of capture and the location of the trap.
2.5 Species identification An initial identification was made with some specimens to associate the species with its external morphological features. This initial identification was made clarifying the specimens with 10% KOH and eugenol using taxonomic keys (Young and Duncan, 1994;Galati, 2003). Given that the most abundant species in this study were subject to future PCR analysis, the taxonomic determination of the remaining specimens was based on the external morphological characteristics recognized during the initial identification. To confirm the identification of the species, a subsample (10%) from each of the
sites was clarified with the same technique, and evaluated using taxonomic keys (Young and Duncan, 1994; Galati, 2003).
2.6 Blood source analysis DNA was isolated using the DNeasy extraction kit (Qiagen) according to the manufacturer‘s recommendations and guidelines. Blood source identification was carried out using cytochrome B (cytB) primers as previously described (Molaei et al., 2008, 2006): (CCATCCAACATCTCAGCATGAAA[f], GCCCCTCAGAATGATATTTGTCCTCA[r]), which generate a fragment of 395 bp. The PCR conditions for blood source identification were those previously described (Ferro et al., 2011). The positive control was Aedes aegypti fed with human blood and the negative control was sterile water. Specimens of Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi were preferentially selected for this analysis, due to their abundance and vectorial role in other foci of domestic transmission.
2.7 Leishmania (Viannia) sp infection in phlebotomines DNA from female phlebotomines was processed for evaluation of Leishmania (V.) sp infection by PCR of the mini-circle kDNA, using LV and B1 primers set (ATTTTTGAACGGGGTTTCTG [f], GGGGTTGGTGTAATA TAGTGG[r]) and Southern blot analysis as previously described (Ferro et al., 2011; Figueroa et al., 2009; Vergel et al., 2005). The positive control was DNA from parasite culture of Leishmania (Viannia) panamensis, and the negative control was sterile water. This was conducted in the most abundant phlebotomine species, Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi. Female specimens (non-blood-fed) were pooled by species in vials of 10 or fewer, and blood-fed females were processed individually. The pools were grouped according to trap location (indoor and outdoor), house code and month of capture.
2.8 Habitat characterization The habitat within 90 meters of the selected houses was characterized at 10 meter intervals along eight radial transects, centered on each house, at 45° intervals using a web pattern as previously described (Ocampo et al., 2012). The houses selected were geo-referenced with a Global Positioning System (GPSmap 76CSx). Habitats were classified according to the following categories: hen house; pigsty; horse stable or cow barn; forest; mixed coffee plantation; unshaded or ‗technified‘ coffee plantation (monoculture); cultivation (annual crops); shrubs; paddock; pasture and other (typically burned or bare areas) as described previously (Ocampo et al., 2012). Around each house, the percentages of points in each category were used to characterize the habitat.
2.9 Data analysis Questionnaire data was entered into a Microsoft Access database and exported to Microsoft Excel. In order to estimate the number of species in unequal sample sizes, species rarefaction curves were calculated for the five sites using EstimateS 9.1.0 (Colwell 2013) and projected to 80 samples (Colwell et al., 2012). Three indexes of vector species diversity were evaluated: the Shannon-Wiener diversity index (H’) which measures species richness, the Simpson index (D) which emphasizes the dominance of species, and the equitability index (J=H’/Hmax) which evaluates the heterogeneity of the sampled species based on the calculated Shannon-Wiener diversity index and the estimated maximum diversity or the species or ln S, where S is the number of species (Magurran 2004). Descriptive measures were calculated for categorical and continuous variables. Spearman rank correlation coefficients were calculated to quantify associations between the phlebotomine abundance and habitat categories. Additionally, the total number of collected phlebotomines and capture rate (phlebotomines /trap/night) at each study site was calculated. Given the over-dispersion of the response variable (number of
phlebotomines), a negative binomial regression model with random effects was estimated for Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi, both indoors and outdoors. For the selection of candidate variables, we used a stepwise forward methodology, with an exclusion p value of greater than 0.20 and an inclusion p value less than 0.05. Models were compared by likelihood ratio test (LRT). Statistical analysis was performed using STATA® version 12.0 and R Version 3.3.2.
2.10 Ethical statement Approval of the project was obtained from the Committee of Ethics in (CIEIH) of CIDEIM. All participants signed informed consent before participating in the study. Phlebotomine specimens were collected in collaboration with the local Secretary of Health and with the permission issued by the National Environmental Licensing Authority (ANLA) to CIDEIM (Resolution 0107 of February 4, 2015).
3.
Results
3.1 Socio-demographic data The demographic and epidemiological characteristics of each locality are shown in Table 1. A total of 102 houses and 448 people were evaluated at the five sites. All the sites had a history of leishmaniasis transmission, and at least one person with an active lesion suggestive of leishmaniasis was observed at each. Most of the cases with parasitological diagnosis had received meglumine antimoniate (MA) treatment.
Regarding the physical structure of the houses, brick was the most common material used (Table 2). Fumigation of the houses by their inhabitants was frequent, except in La Esmeralda and Agua Bonita,
where a high proportion of dwellings were said to have had no fumigation in the years prior to the data collection (Table 2).
3.2 Land coverage There were clear differences between the municipalities in terms of land coverage (Table 2): Novillero and La Esmeralda had a greater coverage of sugar cane (27.1% - 43.5%), shrub (20% - 12.4%) and paddock (15% - 22.6%). On the other hand, the coverage of technified (unshaded) coffee (42.6%) and mixed coffee plantation (27.3%) was greater in El Cucal and Agua Bonita respectively (Table 2). In Honda Alta, the main land coverage was forest (33%) with a variety of other uses totaling less than 15.9%. The predominant land uses at each site were found between 10 and 30 meters from the dwellings.
Table 1. Demographic and epidemiological characteristics of the people surveyed, by locality. Novillero-
Esmeralda-
Honda Alta-
El Cucal-
Agua Bonita-
Santander
Cundinamarca
Huila
Tolima
Tolima
(N. Houses=15)
(N. Houses=15)
(N. Houses=15)
(N. Houses=15)
(N. Houses=42)
Number of people
56
59
49
52
232
Number people/house: mean (SD)
3.73 (2.2)
3.93 (2.3)
3.26 (1.1)
3.46 (1.3)
5.41 (2.8)
Age in years: median (IQR)
37 (13-50)
38 (15-63)
37 (15-47)
30 (12-44)
17 (9-35)
0-15
16 (28.6)
16 (27.1)
13 (26.5)
17 (32.7)
110 (47.4)
16-30
8 (14.3)
9 (15.2)
11 (22.5)
10 (19.2)
51 (22)
34-45
13 (23.2)
10 (17)
12 (24.5)
12 (23.1)
41 (17.7)
>45
19 (33.9)
24 (40.7)
13 (26.5)
13 (25)
30 (12.9)
Male
31 (55.3)
33 (55.9)
26 (53.1)
27 (51.9)
128 (55.2)
Female
25 (44.7)
26 (44.1)
23 (46.9)
25 (48.1)
104 (44.8)
Variable
Age group in years: n (%)
Sex: n (%)
One or more person with active lesion(s): n (%) No
54 (96.4)
58 (98.3)
47 (95.9)
48 (92.3)
231 (99.6)
Yes
2 (3.6)
1 (1.7)
2 (4.1)
4 (7.7)
1 (0.4)
One or more person with inactive lesion(s): n (%) No
47 (83.9)
59 (100)
45 (91.8)
46 (88.5)
169 (72.8)
Yes
9 (16.1)
0
4 (8.2)
6 (11.5)
63 (27.2)
No
1 (1.8)
0
0
3 (5.8)
2 (0.9)
Yes
9 (16.1)
1 (1.7)
4 (8.2)
6 (11.5)
61 (26.3)
Not applicable
46 (82.1)
58 (98.3)
45 (91.8)
43 (82.7)
169 (72.8)
Glucantime
9 (16.1)
1 (1.7)
4 (8.2)
4 (7.7)
61 (26.3)
Other
0
0
0
2 (3.8)
0
Not applicable
47 (83.9)
58 (98.3)
45 (91.8)
46 (88.5)
171 (73.7)
Received treatment: n (%)
Type of treatment: n (%)
Habitual use of bednet: n (%) No
29 (51.8)
49 (83)
10 (20.4)
29 (55.8)
89 (38.3)
Yes
27 (48.2)
10 (17)
39 (79.6)
23 (44.2)
143 (61.7)
SD. Standard deviation. IQR. Interquartile range
Table 2. House construction, insecticide use, and land use coverage.
Novillero
Esmeralda
Honda Alta
El Cucal
Agua Bonita
(N° Houses=15)
(N° Houses=15)
(N° Houses=15)
(N° Houses=15)
(N° Houses=42)
1490 (1361-1670)
1487 (1338-1644)
1325 (1238-1489) 1589 (1526-1680) 1554 (1200-1858)
Wattle & daub (bareque)
2 (13.3)
1 (6.7)
4 (26.7)
4 (26.7)
18 (41.9)
Wood
0
2 (13.3)
5 (33.3)
0
7 (16.2)
Brick
12 (80)
10 (66.7)
6 (40)
8 (53.3)
18 (41.9)
Adobe
1 (6.7)
2 (13.3)
0
3 (20)
0
No
1 (6.7)
10 (66.7)
5 (33.3)
3 (20)
37 (88.1)
Yes
14 (93.3)
5 (33.3)
10 (66.7)
12 (80)
5 (11.9)
Forest
9.4 (17.9)
9.3 (10.2)
33 (18.9)
9.3 (12.1)
15.3 (16)
Sugar cane
27.1 (16.1)
43.5 (26.1)
1.3 (2.7)
0.5 (1.8)
0.8 (1.3)
Technified coffee
0
0.5 (1.5)
8.6 (13.7)
42.6 (22.4)
3.7 (8.9)
Abandoned coffee
0
0.8 (2.8)
4 (6.9)
1.9 (4.1)
2.7 (8.7)
Coffee (with shade tree)
8.8 (10.3)
0
1.5 (3.9)
0.9 (2.4)
3.8 (15.7)
Mixed coffee
1.9 (2.5)
3.8 (13.5)
4.4 (8.6)
16.1 (23)
27.3 (22.4)
Paddock
15 (14.9)
22.6 (13.6)
8.6 (12.9)
5.5 (10.2)
0.2 (0.7)
Shrub
20 (10.3)
12.4 (13.1)
8.8 (9.6)
12.3 (16.3)
15.3 (14.6)
Burnt areas
0
0.4 (0.9)
0.2 (0.7)
0
24.1 (17.8)
Banana
2.1 (2.6)
3.5 (4.1)
4.3 (5.5)
1.2 (2.5)
2.3 (5.6)
Corn (maize)
1.7 (4.2)
0
0.4 (1.4)
3 (5.8)
1.1 (3.3)
Pasture
1.1 (2.2)
0
3.2 (6.1)
1 (2.8)
0.3 (0.8)
Chicken coops
0.2 (0.5)
0.6 (1.5)
0.2 (0.4)
0.1 (0.3)
0
Beans
0.1 (0.3)
0
0
0
1 (3.8)
Others
7.4 (5.2)
0.5 (1.4)
15.9 (12.1)
2.9 (4.7)
0.7 (2.8)
Variable
Altitude (m.a.s.l): median (IQR) House construction n (%)
Insecticide use in the house: n (%)
Land use %: mean (SD)
SD- Standard deviation. % - percentage. IQR- Interquartile range
3.3 Entomological study At three sites, all the phlebotomine specimens were taxonomically identified. In Novillero and El Cucal a random sample of one-third of the total specimens was identified due to the large numbers collected (n=1770 and 4853, respectively). A total of 2937 were taxonomically identified. Of these, 708 were caught indoors and 2229 outdoors (Table 3). A total of 13 species were recorded, of which Pi. (Pif.) quasitownsendi — collected in Novillero and La Esmeralda — and Pi. (Pif.) longiflocosa — collected in Honda Alta, El Cucal and Agua Bonita — were the most abundant. Pi. (Pif.) quasitownsendi was caught mostly indoors, and Pi. (Pif.) longiflocosa mostly in the peridomiciliary area. In Honda Alta, although fewer individuals were caught, a greater diversity of phlebotomine species was observed (Table 3). Estimated species accumulation curves for the 5 sites show that the highest species richness was observed in Honda Alta, and similar curves for the sites Novillero, El Cucal and Agua Bonita, while La Esmeralda presented intermediate values (Figure 2). Rarefaction curves calculated for each site with the 95% confidence interval are presented in the supplementary data (Figure S1). The species richness among the five sampling locations ranged from 2 to 5 indoors, and from 2 to 6 outdoors. The diversity index (H) shows that the highest diversity was observed in the Honda Alta site, followed by La Esmeralda and Agua Bonita; the lowest values were observed in Novillero and El Cucal respectively. Accordingly, the dominance index (D) was highest in El Cucal, Novillero, and Agua Bonita, and the lowest in Honda Alta and La Esmeralda. Likewise, the lowest equitability values were observed in Novillero and El Cucal (Table 4).
2
60 t/n
S/t/n
4.03
4.0
0.03
La
Indoor
n
43
23
16
4
Esmeralda
30 t/n
S/t/n
1.43
0.76
0.53
0.13
Outdoor
n
73
33
29
4
5
2
60 t/n
S/t/n
1.21
0.55
0.48
0.06
0.08
0.03
Honda
Indoor
n
21
13
4
1
1
2
Alta
30 t/n
S/t/n
0.70
0.43
0.13
0.03
0.03
0.06
Outdoor
n
35
7
19
4
3
1
1
60 t/n
S/t/n
0.58
0.11
0.31
0.06
0.05
0.01
0.01
Indoor
n
128
127
1
30 t/n
S/t/n
4.26
4.2
0.03
Outdoor
n
1585
1573
10
2
60 t/n
S/t/n
26.41
26.2
0.16
0.03
Agua
Indoor
n
140
93
4
43
Bonita
84 t/n
S/t/n
1.66
1.1
0.04
0.51
Outdoor
n
294
206
3
7
75
3
168 t/n
S/t/n
1.75
1.22
0.01
0.04
0.44
0.01
Indoor
n
708
398
233
16
1
4
4
1
5
43
3
204 t/n
S/t/n
3.47
1.95
1.14
0.07
0.00
0.01
0.01
0.00
0.02
0.21
0.01
Outdoor
n
2229
273
1786
29
13
19
4
5
4
3
1
11
75
4
2
408 t/n
S/t/n
5.46
0.66
4.37
0.07
0.03
0.04
0.00
0.01
0.00
0.00
0.00
0.02
0.18
0.00
0.00
El Cucal
Total
t/n – traps per night. S/t/n- Sandflies caught per trap per night.
B. beaupertuyi
240
M. trinidadensis
242
Lutzomyia sp
n
L. (Helcocyrtomyia)
Outdoor
L. (H). sanguinaria
0.03
L. (H). ceferinoi
12.5
L. (H). hartmanni
12.53
L. gomezi
S/t/n
L. lychi
30 t/n
L. longipalpis
1
P. columbiana
375
P. ovallesi
376
Novillero
P. longiflocosa
P. quasitownsendi
n
area
Capture rate
Indoor
Study
trap location
Total Phlebotomines
Table 3. Phlebotomine composition and capture rates indoors and outdoors
10
Species
8
6
4
2
0 1
11
21
31
41
51
61
71
Sample Units La Esmeralda
Cucal
Novillero
Honda Alta
Agua Bonita
Figure 2. Estimated species accumulation curves for the five sites. The curves for the sites La Esmeralda, El Cucal, Novillero and Honda Alta were extrapolated by rarefaction from 45 sample units, marked with an arrow, to the sample units of Agua Bonita.
Table 4. Species richness evaluated in each study locality: indexes of alpha diversity (Shannon-Wiener, Simpson, fairness).
Study area
Trap location
S
Shannon-Wiener H’
Simpson D
J= (H’/Hmax)
Indoor
2
0.018
0.995
0.027
Outdoor
2
0.048
0.984
0.069
Indoor
3
0.923
0.433
0.841
Outdoor
5
1.167
0.371
0.725
Indoor
5
1.127
0.433
0.700
Outdoor
6
1.315
0.357
0.734
Indoor
2
0.046
0.984
0.066
Outdoor
3
0.048
0.985
0.044
Indoor
2
0.172
0.921
0.248
Outdoor
4
0.285
0.886
0.206
Novillero
La Esmeralda
Honda Alta
El Cucal
Agua Bonita
S species identified, H the Shannon-Wiener diversity index, D the Simpson dominance index, and J the equitability index
3.4 Blood source analysis Of the 293 blood-fed female sandflies captured, 44 from Pi. (Pif.) quasitownsendi and 35 from Pi. (Pif.) longiflocosa were successfully sequenced and the source of their blood meal identified. The highest proportion contained human blood (n = 49, 62%), followed by avian blood (n = 22, 27.8%) and that of other domestic animals (n = 7, 8.9%) among them dog (Cannis lupus familiaris), cat (Felis catus domesticus) and pig (Sus scrofa domesticus). One blood meal (1.3%) was from a wild mammal, the two-toed sloth (Choloepus hoffmanni) (Table 5).
Table 5. Phlebotomines blood meal sources at each study site
Blood source blood-fed Study area
DNA
DNA
Two-toed
Species females
amplified
Human
Avian
Dog, cat, or pig
n (%)
n (%)
n (%)
sequenced
sloth n (%)
Novillero
Pi. (Pif) quasitownsendi
116
53
41
31 (75.6)
5 (12.2)
5 (12.2)
-
La Esmeralda
Pi. (Pif) quasitownsendi
3
3
3
1 (33.3)
2 (66.7)
-
-
Honda Alta
Pi. (Pif.) longiflocosa
3
3
3
1 (33.3)
1 (33.3)
1 (33.3)
-
El Cucal
Pi. (Pif.) longiflocosa
94
32
32
16 (50)
14 (43.8)
1 (3.1)
1 (3.1)
216
91
79
49(62)
22 (27.8)
7 (8.9)
1 (1.3)
Total
3.5 Natural infection by Leishmania (V.) sp At the four sites studied between 2014 and 2015, only one female of Pi. (Pif.) quasitownsendi, from Novillero, was positive for infection with Leishmania (V.) sp: a prevalence of 0.51% (1/192). This was a non-blood-fed female caught in the peridomestic environment. The previous study in Agua Bonita detected positive females of Pi. (Pif.) longiflocosa indoors (prevalence = 5%, n = 233), and 10 meters from the dwelling (prevalence = 3%, n = 156) (Ocampo et al., 2012).
3.6 Effect of land coverage on phlebotomines sand fly abundance Spearman rank correlations were used to evaluate associations between percentage of land use coverage and phlebotomine abundance in the domestic environment (Table S1). Pi. (Pif.) quasitownsendi showed a considerable positive correlation with the percentage of sugar cane (ρ = 0.72, p < 0.001 for outdoors traps, and ρ=0.70, p < 0.001 indoors). For Pi. (Pif.) longiflocosa, a positive correlation was observed with the percentage of technified coffee (ρ = 0.56 outdoors and 0.50 indoors, both p <0.001) (Table S1). In terms of distance from each land use to the house, the abundance of Pi. (Pif.) quasitownsendi increased as the distance to sugar cane decreased (ρ = -0.56 outdoors and -0.50 indoors, both p < 0.001) (Table S2). In the case of Pi. (Pif.) longiflocosa, a similar tendency was
observed: as the distance of the dwelling to the technified coffee decreased, the abundance of this species increased significantly (ρ = -0.51 outdoors and -0.48 indoors, both p < 0.001) (Table S2).
The negative binomial regression model for outdoor Pi. (Pif.) quasitownsendi is consistent with the above results: greater distance to sugar cane was associated with lower abundance, more specifically 6% per meter (IRR = 0.94, p < 0.001). Again, this was similar for those caught indoors (IRR=0.92; p < 0.001) (Table 6). For Pi (Pif.) longiflocosa, the regression model showed that the percentage of technified coffee and mixed coffee around the house are positively associated with the abundance of this species in the domestic environment, especially outdoors. For each percentage point of coverage of technified and mixed coffee plantations, the abundance of Pi. (Pif.) longiflocosa increased 16% outdoors (IRR: 1.16, p < 0.001) and 14% indoors (IRR: 1.14, p < 0.001) (Table 7). On the other hand, higher pasture cover and greater distance to mixed coffee plantation around the houses were associated with lower abundance of this species.
Table 6. Land uses associated with Pi. (Pif.) quasitownsendi abundance, outdoors and indoors Outdoor
Indoor
Pi (Pif) quasitownsendi
IRR
CI95%
p value
IRR
CI95%
p value
Distance to burnt area
1.08
1.04 – 1.12
<0.0001
1.05
1.01 – 1.08
0.003
Percentage pasture
1.07
1.03 – 1.11
0.001
---
---
---
Distance to technified coffee
1.04
1.01 – 1.06
<0.0001
1.05
1.02 – 1.08
0.001
Distance to cane sugar
0.94
0.92 – 0.97
<0.0001
0.92
0.88 – 0.96 <0.0001
Distance to other crops
---
---
---
0.96
0.94 – 0.98
0.001
Likelihood ratio (LR) tests were done to compare the negative binomial models, as shown, with models using the Poisson distribution. For the outdoor catches, the LR test statistic was 32.15 (p=0.0211), and for indoor catches it was 36.01 (p=0.0029): both favor the negative binomial models shown.
Table 7. Land uses associated with Pi. (Pif.) longiflocosa abundance, outdoor and indoor Outdoor Pi (Pif) longiflocosa
IRR
Technified coffee plantation percentage
Indoor
CI95%
p value
IRR
CI95%
p value
1.16
1.12 – 1.22 <0.0001
1.14
1.07 – 1.21
0.000
Mixed coffee percentage
1.04
1.01 – 1.08
0.011
---
---
---
Pasture percentage
0.86
0.78 – 0.94
0.002
---
---
---
Mixed coffee distance
---
---
---
0.96
0.94 – 0.98
0.003
As for the previous table, LR tests were carried out to compare negative binomial and Poisson models. Again they favor the negative binomial distribution for both outdoor and indoor catches, with LR statistics of 20.74 (p=0.05) and 20.97 (p=0.0072), respectively.
4.
Discussion
This study evaluated five sites of the Andean region in Colombia which, although similar demographically and epidemiologically, were distinct in terms of land use and in the associated phlebotomine diversity and abundance. Overall, dominance of any one species was associated with
monoculture: in Novillero and La Esmeralda, Pi. (Pif.) quasitownsendi was associated with sugar cane; while in El Cucal and Agua Bonita Pi. (Pif.) longiflocosa was associated with coffee plantations. Although the females of the two species Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa are indistinguishable, we were able to identify them due to their allopatric occurrence in the investigated areas. Interestingly, in Honda Alta, the locality with forest coverage and more heterogeneous land use, fewer specimens were caught, but with higher species diversity.
The overall results regarding diversity reflect the values observed for richness and abundance. Some sites, such as Novillero and El Cucal, had a combination of high individual abundance, high species dominance and low diversity, while others, like Honda Alta and La Esmeralda, had a contrasting pattern of low abundance, low dominance and higher equitability. The sites with higher species diversity tended to show lower abundance of individuals while avoiding the dominance of a single species. In Novillero and El Cucal, diversity was very low, and the species Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa were, respectively, almost exclusively dominant, and are recognized vectors of cutaneous leishmaniasis.
Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa play an important role in the transmission of cutaneous leishmaniasis in the Magdalena Valley of Colombia due to their dominant abundance, endophagy, and anthropophagy (Ferro et al., 2011; Ocampo et al., 2012; Pardo et al., 2006; Valderrama-Ardila et al., 2010) . Both species are part of the Townsendi series of the Verrucarum (Cohnstaedt et al., 2011) group which is abundant in mountainous areas and well adapted to traditional and technified coffee crops (Bejarano et al., 2003). In the current study, Pi. (Pif.) quasitownsendi was found to be infected with Leishmania (V.) sp, as was Pi. (Pif.) longiflocosa in the previous study in Agua Bonita (Ferro et al., 2011; Ocampo et al., 2012). The majority of the blood meals had been made
on humans, with smaller proportions from birds and other domestic animals, confirming the anthropophagic behavior.
Correlation analysis showed that Pi. (Pif.) quasitownsendi was associated with highly anthropic sites with sugarcane crops and paddock, and Pi. (Pif.) longiflocosa mainly with technified and mixed coffee. For neither species was forest associated with abundance, possibly because, at the majority of sites, forest was sparse and far from the houses. The regression analysis showed that the abundance of Pi. (Pif.) quasitownsendi decreased significantly with the distance between the dwellings and sugarcane; while the abundance of Pi. (Pif.) longiflocosa increased significantly according to the greater coverage of technified coffee. In Honda Alta, which had the greatest forest coverage, the species diversity was high, as found previously elsewhere (Chaniotis et al., 1972; Jimenez et al., 2009; Memmott, 1992; Thies et al., 2016).
In terms of Pi. (Pif.) longiflocosa, these findings agree with those of other focal studies in the Andean zone of Colombia where this species was the most abundant. In Planadas, Tolima, it was found to be strongly associated with high coverage of coffee (Cárdenas et al., 2014). In the municipality of Chaparral, also in Tolima department, the results of a focal study carried out in the post-epidemic period, showed that high abundance of indoor Pi. (Pif.) longiflocosa was associated mainly with the proximity of houses to technified coffee (Ocampo et al., 2012). Elsewhere in Latin America, studies have also found strong associations between some phlebotomines species and land use, e.g. in Brazil, between banana plantations and the abundance of Nyssomyia whitmani, Nyssomyia intermedia and Migonemyia migonei (Luz et al., 2000; Membrive et al., 2012).The current study is the first to demonstrate the association of Pi. (Pif) quasitownsendi with a monoculture such as sugar cane.
The main objective of this study was to establish a rapid characterization methodology that would allow the identification of houses at higher risk of Leishmania transmission due to the composition and abundance of phlebotomines indoors and outdoors (10 m from the peridomestic environment). The previous analysis (Ocampo et al., 2012) confirmed an association between the abundance of some phlebotomine species and land use. Our radial sampling, based on ecological methodologies, allowed us to evaluate differences between houses due to land use. The negative binomial regression model found that increasing distance from the house to sugar cane monoculture was associated with a reduction of 6% per meter in the abundance of Pi. (Pif.) quasitownsendi. This result suggests that simply increasing the distance of crops from the house could be a protective measure. In the case of Pi (Pif.) longiflocosa, it was the percentage coverage of technified coffee plantation that favored the abundance: 16% increase per percentage point coverage for outdoors catches (IRR: 1.16, p < 0.001) and 14% for indoor captures (IRR: 1.14, p < 0.001) and, to a lesser extent, mixed coffee (IRR 1.04, p = 0.011 outdoor). On the other hand, higher pasture cover and greater distance to mixed coffee plantation around the houses were associated with lower abundance of this species.
Rapid assessment methodologies have been used as a proxy to evaluate species diversity at sites which are remote, and difficult and expensive to access (Álvarez et al., 2004), in order to justify their inclusion in conservation schemes. These methodologies allow the standardization of the biodiversity in many taxonomic groups maximizing the capture efforts in a particular locality. In accordance with this philosophy, we implemented this methodology for a rapid evaluation of the conditions that allowed us to associate the peri-domestic environment with the presence and abundance of phlebotomine vector species of importance in domestic transmission. We sought a methodology that could be easily replicated by state health agencies, such as Departmental and Municipal health secretariats, which
usually have very limited resources for fieldwork, but that allow relevant standardized information on the peri-domestic environment to be obtained in a very short period of time (5 – 6 days for 15 houses), efficiently and at a low cost.
The model presented here could guide public heath decision makers in identifying houses at higher risk of domestic transmission and also educate the farmers to increase the distance of crops from their houses. Most of these rural areas are made up of smallholdings (minifundios) with limited economic resources that force them to increase the density of cultivated areas as close as 1 meter to the house. Interestingly, this methodology also suggests that maintaining biodiversity and forest coverage around the house could help to decrease the profusion of any one species, and maintain a balance between the species as was observed in Honda Alta. However, the small number of sites means that more studies are needed to confirm this finding.
5.
Conclusion
The current study showed that the transformation of forest into agricultural land around the domicile favors the presence and abundance of phlebotomines, both outdoors and indoors. In particular, highly modified sites, such as monocultures, favor particular species that increase the risk of domestic transmission. Although such associations have been observed previously, this is the first study to do so based on a rapid and informative field methodology that can be applied by the local health authorities to prioritize their interventions in those houses with potential risk of transmission in a cutaneous leishmaniasis focus.
Supplementary material, Table S1. Supplementary material, Table S2.
Supplementary material, Figure S1.
Acknowledgments We gratefully acknowledge the technical and logistical support of the Secretary of Health of the Department of Santander, Cundinamarca, Huila and Tolima in providing access to the area and assisting in the conduct of fieldwork. We are also grateful to Luis Ernesto Ramirez for his technical support in the field and laboratory. This study was conducted in partial fulfillment of the Masters of Science degree in Epidemiology of the School of Public Health of the Universidad del Valle.
Financial support This study was partially financed by the National Administrative Department of Science, Technology and Innovation - COLCIENCIAS Contract 673-2013 (569-2012) and NIAID-NIH [Award number U19AI129910]. Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Conflicts of Interest The authors declare that none of them has any conflict of interest. Authors´ addresses Mabel Moreno, Lina Guzmán, Neal Alexander, Clara Ocampo, Centro Internacional de Entrenamiento e Investigaciones Médicas CIDEIM, Cali, Colombia. Carlos Valderrama-Ardila, Universidad Icesi,
Cali Colombia. E-mails: [email protected]; [email protected]; [email protected]; [email protected]; [email protected].
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Graphical abstract
Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia
Mabel Moreno Investigationmethodology and writing original draft , ´ Lina Guzman-Rodr´ ıguez Investigationmethodology and writing- review and editing , Carlos Valderrama-Ardila Conceptualizationinvestigationmethodology and writing- review and editing , Neal Alexander Conceptualizationinvestigationmethodology and writing- review and editing , Clara B. Ocampo Conceptualizationinvestigationmethodologywriting- review and editingsupervision and funding ac PII: DOI: Reference:
S0001-706X(19)31062-9 https://doi.org/10.1016/j.actatropica.2019.105315 ACTROP 105315
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
12 August 2019 18 December 2019 18 December 2019
Please cite this article as: Mabel Moreno Investigationmethodology and writing original draft , ´ Lina Guzman-Rodr´ ıguez Investigationmethodology and writing- review and editing , Carlos Valderrama-Ardila Conceptualizationinvestigationmethodology and writing- review and editing , Neal Alexander Conceptualizationinvestigationmethodology and writing- review and editing , Clara B. Ocampo Conceptualizationinvestigationmethodologywriting- review and editingsupervision and funding ac Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105315
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Highlights A rapid method successfully characterized exposure to phlebotomine vectors of cutaneous leishmaniasis.
Monocultures around rural houses favor abundance and dominance of particular phlebotomine species.
Increasing distance between monocultures and houses decreases phlebotomine abundance around the latter.
Land use in relation to composition and abundance of phlebotomines (Diptera: Psychodidae) in five foci of domiciliary transmission of cutaneous leishmaniasis in the Andean region of Colombia
Mabel Moreno1 *, Lina Guzmán-Rodríguez1, Carlos Valderrama-Ardila2**, Neal Alexander1, 2, Clara B. Ocampo1,2
1
Centro Internacional de Entrenamiento e Investigaciones Médicas, CIDEIM. Cali, Colombia.
2
Universidad ICESI. Cali, Colombia
* Current affiliation, Coomeva EPS regional suroccidente. Cali, Colombia ** Current affiliation, Universidad del Rosario, Bogotá, Colombia
Corresponding Author: Clara B. Ocampo, Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Carrera 125 # 19-225, Cali, Valle del Cauca 760031, Colombia. email: [email protected].
Abstract American cutaneous leishmaniasis is a public health concern in Colombia, its incidence being sustained or focally increased principally by the emergence of domestic transmission concomitantly with the adaptation of the phlebotomine vectors to habitat transformation around households. The objective of the study is to scale up a rapid characterization methodology for evaluating the relationship of land use around the house to the composition and abundance of phlebotomines. Five sites with a history of domestic leishmaniasis transmission in the Andean area of Colombia were selected. The peri-domestic
habitat was evaluated at 10 meter intervals along eight radial transects, centered on each house, at 45° intervals using a web pattern. Phlebotomines were captured by placing three CDC light traps over two nights both indoors and outdoors (10 m from the house). Blood source and infection were determined by PCR. Spearman rank correlation coefficients and negative binomial regression were used to quantify associations between the phlebotomine abundance and habitat categories. The study demonstrated that the vectors were largely anthropophagic (62% of 79 were human blood) and that a single species in each site was favored by the pertaining agriculture monoculture. Specifically, Pintomyia (Pifanomyia) quasitownsendi was associated with sugar cane in Novillero and La Esmeralda; while Pi. (Pif.) longiflocosa was associated with coffee plantations in Agua Bonita and El Cucal. Honda Alta had a more diverse array of land use and forest coverage with a lower number of specimens but higher species diversity. In terms of distance from the house to an area of a given land use, the abundance of Pi. (Pif.) quasitownsendi was inversely related to the distance to sugar cane plantation (Spearman correlation coefficient, ρ = -0.56, p < 0.001 for outdoor catches, and ρ = -0.50, p < 0.001 indoors). A similar inverse relationship was observed for Pi. (Pif.) longiflocosa with regard to technified coffee (ρ = -0.51, p < 0.001 outdoors, and ρ = -0.48, p < 0.001 indoors). This rapid characterization methodology could guide public heath decision makers in identifying those houses at higher risk of domestic transmission, and also educate farmers to increase the distance between their crops and any neighboring houses.
Keywords Leishmaniasis; land use; phlebotomines; risk factors; Andean region; Colombia.
1.
Introduction
The leishmaniases are a group of neglected tropical diseases caused by parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) transmitted by the bite of infected female phlebotomines (Psychodidae: Phlebotominae) to wild and domestic animals and increasingly to humans (Fitzpatrick and Engels, 2015; Pan American Health Organization, 2018; World Health Organization, 2010).
In America, leishmaniasis transmission in domestic and peri-domestic environments has generated a notable increase of cases, especially of the cutaneous form (Ampuero et al., 2005; Maroli et al., 2013; Valderrama-Ardila et al., 2010). In Colombia, during the 1990s, an average of 6,500 new cases of leishmaniasis were reported per year, however, recently reports has increase to an average of 14,000 cases which shows an unusual increase in cases during the last years, were 97% of leishmaniasis is the cutaneous form (Ministerio de Salud, 2010). This epidemiological change has been attributed mainly to the adaptation of the vectors to new transformed habitats, e.g. by finding human and non-human blood sources in domiciliary areas. Other factors include the ability of the parasites to adapt to other vector species, high rates of human migration from endemic areas, and haphazard and rapid urbanization (Colwell et al., 2011; Ferro et al., 2011; King et al., 2004; Ocampo et al., 2012).
In Colombia, cutaneous leishmaniasis (CL) is the most frequent clinical form, with the Andean region accounting for most reported cases (Zambrano and Mercado, 2014). In terms of the transmission risks already mentioned, a specific form of habitat transformation is the extension of agricultural monoculture (Contreras-Gutiérrez et al., 2014). Knowledge of the interaction of these risk factors is crucial to understanding the transmission dynamics in a specific focus. To identify disease control strategies, a rapid eco-epidemiological assessment technique was developed in a previous study carried
out in Chaparral, Colombia. Characterization of the land use along eight transects radiating from a specific house was able to identify houses with a higher density of sandflies that was associated with a higher risk of CL transmission (Ocampo et al., 2012). This study found that the proximity of dwellings to forest, or to traditional coffee plantations, was associated with abundance of the main vector Pintomyia (Pifanomyia) longiflocosa (Ocampo et al., 2012).
The objective of the current study is to scale up this rapid characterization methodology and apply it to four other foci of domestic transmission of CL in the Andean region of Colombia, and to evaluate the relationship of land use around the houses to the composition and abundance of phlebotomines.
2.
Materials and methods
2.1 Study locations A cross-sectional eco-epidemiological study was conducted in four townships selected from highly endemic municipalities for cutaneous leishmaniasis in the Andean region of Colombia: Novillero- San Benito Municipality (6.0678, -73.5617 – Santander department); La Esmeralda – Villeta (5.0666, -74.5333 – Cundinamarca department); Honda Alta-Rivera, (2.7543, -75.2153 – Huila department) and El Cucal – Rovira (4.1789,-75.3804 -Tolima department) (Figure 1). Collections were carried out during the dry season, in July and September of 2014 (Novillero – La Esmeralda) and February and May of 2015 (El Cucal – Honda Alta). Additionally, we use the data obtained in the ―focal study‖ carried out in Agua Bonita-Tolima (Ocampo et al., 2012) to increase the scope and statistical power of this analysis. The regression analyses described below had not previously been applied to these data.
Figure 1. Study areas in four departments of the Andean region of Colombia with domestic transmission of American cutaneous leishmaniasis.
The climate of the Andean region of Colombia varies with its topography. The study sites lie in the portion of this region which is subject to leishmaniasis transmission: between the eastern and central mountain ranges, and in the inter-Andean valley of the Magdalena River. The main agricultural products are coffee, sugar cane, banana, rice and cotton. The sites were selected in collaboration with the Local Secretary of Health, based on reported cases of cutaneous leishmaniasis during 2000- 2009 (Department of Public Health – Sistema de Vigilancia en Salud Pública - SIVIGILA and municipal hospitals) and accessibility to the Health Services. Reported numbers of cases were: San Benito (98 cases), Villeta (163), Rovira (279) and Rivera (203). At these four new sites, the location of the houses located within an altitudinal range of 1000 to 2000 m.a.s.l, was mapped. A number was assigned to each of the houses and these were placed in a plastic bag and randomly drawn without replacement. Fifteen houses were sampled per site.
2.2 Sample size determination The sample size calculation used data from the above-mentioned previous study in Chaparral (Ocampo et al., 2012). We chose the number of houses (N) based on the putative Spearman rank correlation coefficient (ρ) between density of phlebotomines and land use: √N – 3C(ρ) = Zα + Zβ (Lachin, 1981), where C is Fisher‘s inverse hyperbolic tangent transformation, and Z represents the quantiles of the normal distribution for significance level and power 1-. With 42 houses from Agua Bonita (Chaparral) and 15 from each of the four new sites, to a total of 102, the power is 86% for ρ=0.30.
2.3 Demographic and epidemiological data. A demographic and epidemiological questionnaire was applied to one adult in each selected house. This included information on: the number of residents, their gender and age; whether each person had active or inactive lesions suggestive of leishmaniasis, type of any treatment received, habitual use of
bed nets, house construction, and use of domestic insecticides (mainly pyrethroids). In Colombia, leishmaniasis control is mainly associated to case treatment, sporadic fumigation and bed net distribution; however this last two has low coverture and depends on disease transmission.
2.4 Phlebotomine sampling To evaluate the composition and abundance of phlebotomine sand flies, three Centers for Disease Control and Prevention (CDC) incandescent light traps were located in each house over two consecutive nights. One trap was placed indoors, and another two at different points 10 m from the house. All traps were placed at a height of 1.5 m and were active from 18:00 to 06:00 hours. The catches were recovered from the traps early the following morning and immobilized with triethylamine (TEA: 04885-1; Fisher Scientific, Pittsburgh, PA). The collection mesh of the CDC traps was introduced into a black plastic bag for the immobilization of the insects captured using a piece of cotton moistened with 1 ml of TEA for 15 minutes. The phlebotomines were separated from other insects and stored in plastic tubes with 70% ethanol during transportation to the laboratory (Ferro et al., 2011). Each tube was labeled recording the code of the house, the date of capture and the location of the trap.
2.5 Species identification An initial identification was made with some specimens to associate the species with its external morphological features. This initial identification was made clarifying the specimens with 10% KOH and eugenol using taxonomic keys (Young and Duncan, 1994;Galati, 2003). Given that the most abundant species in this study were subject to future PCR analysis, the taxonomic determination of the remaining specimens was based on the external morphological characteristics recognized during the initial identification. To confirm the identification of the species, a subsample (10%) from each of the
sites was clarified with the same technique, and evaluated using taxonomic keys (Young and Duncan, 1994; Galati, 2003).
2.6 Blood source analysis DNA was isolated using the DNeasy extraction kit (Qiagen) according to the manufacturer‘s recommendations and guidelines. Blood source identification was carried out using cytochrome B (cytB) primers as previously described (Molaei et al., 2008, 2006): (CCATCCAACATCTCAGCATGAAA[f], GCCCCTCAGAATGATATTTGTCCTCA[r]), which generate a fragment of 395 bp. The PCR conditions for blood source identification were those previously described (Ferro et al., 2011). The positive control was Aedes aegypti fed with human blood and the negative control was sterile water. Specimens of Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi were preferentially selected for this analysis, due to their abundance and vectorial role in other foci of domestic transmission.
2.7 Leishmania (Viannia) sp infection in phlebotomines DNA from female phlebotomines was processed for evaluation of Leishmania (V.) sp infection by PCR of the mini-circle kDNA, using LV and B1 primers set (ATTTTTGAACGGGGTTTCTG [f], GGGGTTGGTGTAATA TAGTGG[r]) and Southern blot analysis as previously described (Ferro et al., 2011; Figueroa et al., 2009; Vergel et al., 2005). The positive control was DNA from parasite culture of Leishmania (Viannia) panamensis, and the negative control was sterile water. This was conducted in the most abundant phlebotomine species, Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi. Female specimens (non-blood-fed) were pooled by species in vials of 10 or fewer, and blood-fed females were processed individually. The pools were grouped according to trap location (indoor and outdoor), house code and month of capture.
2.8 Habitat characterization The habitat within 90 meters of the selected houses was characterized at 10 meter intervals along eight radial transects, centered on each house, at 45° intervals using a web pattern as previously described (Ocampo et al., 2012). The houses selected were geo-referenced with a Global Positioning System (GPSmap 76CSx). Habitats were classified according to the following categories: hen house; pigsty; horse stable or cow barn; forest; mixed coffee plantation; unshaded or ‗technified‘ coffee plantation (monoculture); cultivation (annual crops); shrubs; paddock; pasture and other (typically burned or bare areas) as described previously (Ocampo et al., 2012). Around each house, the percentages of points in each category were used to characterize the habitat.
2.9 Data analysis Questionnaire data was entered into a Microsoft Access database and exported to Microsoft Excel. In order to estimate the number of species in unequal sample sizes, species rarefaction curves were calculated for the five sites using EstimateS 9.1.0 (Colwell 2013) and projected to 80 samples (Colwell et al., 2012). Three indexes of vector species diversity were evaluated: the Shannon-Wiener diversity index (H’) which measures species richness, the Simpson index (D) which emphasizes the dominance of species, and the equitability index (J=H’/Hmax) which evaluates the heterogeneity of the sampled species based on the calculated Shannon-Wiener diversity index and the estimated maximum diversity or the species or ln S, where S is the number of species (Magurran 2004). Descriptive measures were calculated for categorical and continuous variables. Spearman rank correlation coefficients were calculated to quantify associations between the phlebotomine abundance and habitat categories. Additionally, the total number of collected phlebotomines and capture rate (phlebotomines /trap/night) at each study site was calculated. Given the over-dispersion of the response variable (number of
phlebotomines), a negative binomial regression model with random effects was estimated for Pi. (Pif.) longiflocosa and Pi. (Pif.) quasitownsendi, both indoors and outdoors. For the selection of candidate variables, we used a stepwise forward methodology, with an exclusion p value of greater than 0.20 and an inclusion p value less than 0.05. Models were compared by likelihood ratio test (LRT). Statistical analysis was performed using STATA® version 12.0 and R Version 3.3.2.
2.10 Ethical statement Approval of the project was obtained from the Committee of Ethics in (CIEIH) of CIDEIM. All participants signed informed consent before participating in the study. Phlebotomine specimens were collected in collaboration with the local Secretary of Health and with the permission issued by the National Environmental Licensing Authority (ANLA) to CIDEIM (Resolution 0107 of February 4, 2015).
3.
Results
3.1 Socio-demographic data The demographic and epidemiological characteristics of each locality are shown in Table 1. A total of 102 houses and 448 people were evaluated at the five sites. All the sites had a history of leishmaniasis transmission, and at least one person with an active lesion suggestive of leishmaniasis was observed at each. Most of the cases with parasitological diagnosis had received meglumine antimoniate (MA) treatment.
Regarding the physical structure of the houses, brick was the most common material used (Table 2). Fumigation of the houses by their inhabitants was frequent, except in La Esmeralda and Agua Bonita,
where a high proportion of dwellings were said to have had no fumigation in the years prior to the data collection (Table 2).
3.2 Land coverage There were clear differences between the municipalities in terms of land coverage (Table 2): Novillero and La Esmeralda had a greater coverage of sugar cane (27.1% - 43.5%), shrub (20% - 12.4%) and paddock (15% - 22.6%). On the other hand, the coverage of technified (unshaded) coffee (42.6%) and mixed coffee plantation (27.3%) was greater in El Cucal and Agua Bonita respectively (Table 2). In Honda Alta, the main land coverage was forest (33%) with a variety of other uses totaling less than 15.9%. The predominant land uses at each site were found between 10 and 30 meters from the dwellings.
Table 1. Demographic and epidemiological characteristics of the people surveyed, by locality. Novillero-
Esmeralda-
Honda Alta-
El Cucal-
Agua Bonita-
Santander
Cundinamarca
Huila
Tolima
Tolima
(N. Houses=15)
(N. Houses=15)
(N. Houses=15)
(N. Houses=15)
(N. Houses=42)
Number of people
56
59
49
52
232
Number people/house: mean (SD)
3.73 (2.2)
3.93 (2.3)
3.26 (1.1)
3.46 (1.3)
5.41 (2.8)
Age in years: median (IQR)
37 (13-50)
38 (15-63)
37 (15-47)
30 (12-44)
17 (9-35)
0-15
16 (28.6)
16 (27.1)
13 (26.5)
17 (32.7)
110 (47.4)
16-30
8 (14.3)
9 (15.2)
11 (22.5)
10 (19.2)
51 (22)
34-45
13 (23.2)
10 (17)
12 (24.5)
12 (23.1)
41 (17.7)
>45
19 (33.9)
24 (40.7)
13 (26.5)
13 (25)
30 (12.9)
Male
31 (55.3)
33 (55.9)
26 (53.1)
27 (51.9)
128 (55.2)
Female
25 (44.7)
26 (44.1)
23 (46.9)
25 (48.1)
104 (44.8)
Variable
Age group in years: n (%)
Sex: n (%)
One or more person with active lesion(s): n (%) No
54 (96.4)
58 (98.3)
47 (95.9)
48 (92.3)
231 (99.6)
Yes
2 (3.6)
1 (1.7)
2 (4.1)
4 (7.7)
1 (0.4)
One or more person with inactive lesion(s): n (%) No
47 (83.9)
59 (100)
45 (91.8)
46 (88.5)
169 (72.8)
Yes
9 (16.1)
0
4 (8.2)
6 (11.5)
63 (27.2)
No
1 (1.8)
0
0
3 (5.8)
2 (0.9)
Yes
9 (16.1)
1 (1.7)
4 (8.2)
6 (11.5)
61 (26.3)
Not applicable
46 (82.1)
58 (98.3)
45 (91.8)
43 (82.7)
169 (72.8)
Glucantime
9 (16.1)
1 (1.7)
4 (8.2)
4 (7.7)
61 (26.3)
Other
0
0
0
2 (3.8)
0
Not applicable
47 (83.9)
58 (98.3)
45 (91.8)
46 (88.5)
171 (73.7)
Received treatment: n (%)
Type of treatment: n (%)
Habitual use of bednet: n (%) No
29 (51.8)
49 (83)
10 (20.4)
29 (55.8)
89 (38.3)
Yes
27 (48.2)
10 (17)
39 (79.6)
23 (44.2)
143 (61.7)
SD. Standard deviation. IQR. Interquartile range
Table 2. House construction, insecticide use, and land use coverage.
Novillero
Esmeralda
Honda Alta
El Cucal
Agua Bonita
(N° Houses=15)
(N° Houses=15)
(N° Houses=15)
(N° Houses=15)
(N° Houses=42)
1490 (1361-1670)
1487 (1338-1644)
1325 (1238-1489) 1589 (1526-1680) 1554 (1200-1858)
Wattle & daub (bareque)
2 (13.3)
1 (6.7)
4 (26.7)
4 (26.7)
18 (41.9)
Wood
0
2 (13.3)
5 (33.3)
0
7 (16.2)
Brick
12 (80)
10 (66.7)
6 (40)
8 (53.3)
18 (41.9)
Adobe
1 (6.7)
2 (13.3)
0
3 (20)
0
No
1 (6.7)
10 (66.7)
5 (33.3)
3 (20)
37 (88.1)
Yes
14 (93.3)
5 (33.3)
10 (66.7)
12 (80)
5 (11.9)
Forest
9.4 (17.9)
9.3 (10.2)
33 (18.9)
9.3 (12.1)
15.3 (16)
Sugar cane
27.1 (16.1)
43.5 (26.1)
1.3 (2.7)
0.5 (1.8)
0.8 (1.3)
Technified coffee
0
0.5 (1.5)
8.6 (13.7)
42.6 (22.4)
3.7 (8.9)
Abandoned coffee
0
0.8 (2.8)
4 (6.9)
1.9 (4.1)
2.7 (8.7)
Coffee (with shade tree)
8.8 (10.3)
0
1.5 (3.9)
0.9 (2.4)
3.8 (15.7)
Mixed coffee
1.9 (2.5)
3.8 (13.5)
4.4 (8.6)
16.1 (23)
27.3 (22.4)
Paddock
15 (14.9)
22.6 (13.6)
8.6 (12.9)
5.5 (10.2)
0.2 (0.7)
Shrub
20 (10.3)
12.4 (13.1)
8.8 (9.6)
12.3 (16.3)
15.3 (14.6)
Burnt areas
0
0.4 (0.9)
0.2 (0.7)
0
24.1 (17.8)
Banana
2.1 (2.6)
3.5 (4.1)
4.3 (5.5)
1.2 (2.5)
2.3 (5.6)
Corn (maize)
1.7 (4.2)
0
0.4 (1.4)
3 (5.8)
1.1 (3.3)
Pasture
1.1 (2.2)
0
3.2 (6.1)
1 (2.8)
0.3 (0.8)
Chicken coops
0.2 (0.5)
0.6 (1.5)
0.2 (0.4)
0.1 (0.3)
0
Beans
0.1 (0.3)
0
0
0
1 (3.8)
Others
7.4 (5.2)
0.5 (1.4)
15.9 (12.1)
2.9 (4.7)
0.7 (2.8)
Variable
Altitude (m.a.s.l): median (IQR) House construction n (%)
Insecticide use in the house: n (%)
Land use %: mean (SD)
SD- Standard deviation. % - percentage. IQR- Interquartile range
3.3 Entomological study At three sites, all the phlebotomine specimens were taxonomically identified. In Novillero and El Cucal a random sample of one-third of the total specimens was identified due to the large numbers collected (n=1770 and 4853, respectively). A total of 2937 were taxonomically identified. Of these, 708 were caught indoors and 2229 outdoors (Table 3). A total of 13 species were recorded, of which Pi. (Pif.) quasitownsendi — collected in Novillero and La Esmeralda — and Pi. (Pif.) longiflocosa — collected in Honda Alta, El Cucal and Agua Bonita — were the most abundant. Pi. (Pif.) quasitownsendi was caught mostly indoors, and Pi. (Pif.) longiflocosa mostly in the peridomiciliary area. In Honda Alta, although fewer individuals were caught, a greater diversity of phlebotomine species was observed (Table 3). Estimated species accumulation curves for the 5 sites show that the highest species richness was observed in Honda Alta, and similar curves for the sites Novillero, El Cucal and Agua Bonita, while La Esmeralda presented intermediate values (Figure 2). Rarefaction curves calculated for each site with the 95% confidence interval are presented in the supplementary data (Figure S1). The species richness among the five sampling locations ranged from 2 to 5 indoors, and from 2 to 6 outdoors. The diversity index (H) shows that the highest diversity was observed in the Honda Alta site, followed by La Esmeralda and Agua Bonita; the lowest values were observed in Novillero and El Cucal respectively. Accordingly, the dominance index (D) was highest in El Cucal, Novillero, and Agua Bonita, and the lowest in Honda Alta and La Esmeralda. Likewise, the lowest equitability values were observed in Novillero and El Cucal (Table 4).
2
60 t/n
S/t/n
4.03
4.0
0.03
La
Indoor
n
43
23
16
4
Esmeralda
30 t/n
S/t/n
1.43
0.76
0.53
0.13
Outdoor
n
73
33
29
4
5
2
60 t/n
S/t/n
1.21
0.55
0.48
0.06
0.08
0.03
Honda
Indoor
n
21
13
4
1
1
2
Alta
30 t/n
S/t/n
0.70
0.43
0.13
0.03
0.03
0.06
Outdoor
n
35
7
19
4
3
1
1
60 t/n
S/t/n
0.58
0.11
0.31
0.06
0.05
0.01
0.01
Indoor
n
128
127
1
30 t/n
S/t/n
4.26
4.2
0.03
Outdoor
n
1585
1573
10
2
60 t/n
S/t/n
26.41
26.2
0.16
0.03
Agua
Indoor
n
140
93
4
43
Bonita
84 t/n
S/t/n
1.66
1.1
0.04
0.51
Outdoor
n
294
206
3
7
75
3
168 t/n
S/t/n
1.75
1.22
0.01
0.04
0.44
0.01
Indoor
n
708
398
233
16
1
4
4
1
5
43
3
204 t/n
S/t/n
3.47
1.95
1.14
0.07
0.00
0.01
0.01
0.00
0.02
0.21
0.01
Outdoor
n
2229
273
1786
29
13
19
4
5
4
3
1
11
75
4
2
408 t/n
S/t/n
5.46
0.66
4.37
0.07
0.03
0.04
0.00
0.01
0.00
0.00
0.00
0.02
0.18
0.00
0.00
El Cucal
Total
t/n – traps per night. S/t/n- Sandflies caught per trap per night.
B. beaupertuyi
240
M. trinidadensis
242
Lutzomyia sp
n
L. (Helcocyrtomyia)
Outdoor
L. (H). sanguinaria
0.03
L. (H). ceferinoi
12.5
L. (H). hartmanni
12.53
L. gomezi
S/t/n
L. lychi
30 t/n
L. longipalpis
1
P. columbiana
375
P. ovallesi
376
Novillero
P. longiflocosa
P. quasitownsendi
n
area
Capture rate
Indoor
Study
trap location
Total Phlebotomines
Table 3. Phlebotomine composition and capture rates indoors and outdoors
10
Species
8
6
4
2
0 1
11
21
31
41
51
61
71
Sample Units La Esmeralda
Cucal
Novillero
Honda Alta
Agua Bonita
Figure 2. Estimated species accumulation curves for the five sites. The curves for the sites La Esmeralda, El Cucal, Novillero and Honda Alta were extrapolated by rarefaction from 45 sample units, marked with an arrow, to the sample units of Agua Bonita.
Table 4. Species richness evaluated in each study locality: indexes of alpha diversity (Shannon-Wiener, Simpson, fairness).
Study area
Trap location
S
Shannon-Wiener H’
Simpson D
J= (H’/Hmax)
Indoor
2
0.018
0.995
0.027
Outdoor
2
0.048
0.984
0.069
Indoor
3
0.923
0.433
0.841
Outdoor
5
1.167
0.371
0.725
Indoor
5
1.127
0.433
0.700
Outdoor
6
1.315
0.357
0.734
Indoor
2
0.046
0.984
0.066
Outdoor
3
0.048
0.985
0.044
Indoor
2
0.172
0.921
0.248
Outdoor
4
0.285
0.886
0.206
Novillero
La Esmeralda
Honda Alta
El Cucal
Agua Bonita
S species identified, H the Shannon-Wiener diversity index, D the Simpson dominance index, and J the equitability index
3.4 Blood source analysis Of the 293 blood-fed female sandflies captured, 44 from Pi. (Pif.) quasitownsendi and 35 from Pi. (Pif.) longiflocosa were successfully sequenced and the source of their blood meal identified. The highest proportion contained human blood (n = 49, 62%), followed by avian blood (n = 22, 27.8%) and that of other domestic animals (n = 7, 8.9%) among them dog (Cannis lupus familiaris), cat (Felis catus domesticus) and pig (Sus scrofa domesticus). One blood meal (1.3%) was from a wild mammal, the two-toed sloth (Choloepus hoffmanni) (Table 5).
Table 5. Phlebotomines blood meal sources at each study site
Blood source blood-fed Study area
DNA
DNA
Two-toed
Species females
amplified
Human
Avian
Dog, cat, or pig
n (%)
n (%)
n (%)
sequenced
sloth n (%)
Novillero
Pi. (Pif) quasitownsendi
116
53
41
31 (75.6)
5 (12.2)
5 (12.2)
-
La Esmeralda
Pi. (Pif) quasitownsendi
3
3
3
1 (33.3)
2 (66.7)
-
-
Honda Alta
Pi. (Pif.) longiflocosa
3
3
3
1 (33.3)
1 (33.3)
1 (33.3)
-
El Cucal
Pi. (Pif.) longiflocosa
94
32
32
16 (50)
14 (43.8)
1 (3.1)
1 (3.1)
216
91
79
49(62)
22 (27.8)
7 (8.9)
1 (1.3)
Total
3.5 Natural infection by Leishmania (V.) sp At the four sites studied between 2014 and 2015, only one female of Pi. (Pif.) quasitownsendi, from Novillero, was positive for infection with Leishmania (V.) sp: a prevalence of 0.51% (1/192). This was a non-blood-fed female caught in the peridomestic environment. The previous study in Agua Bonita detected positive females of Pi. (Pif.) longiflocosa indoors (prevalence = 5%, n = 233), and 10 meters from the dwelling (prevalence = 3%, n = 156) (Ocampo et al., 2012).
3.6 Effect of land coverage on phlebotomines sand fly abundance Spearman rank correlations were used to evaluate associations between percentage of land use coverage and phlebotomine abundance in the domestic environment (Table S1). Pi. (Pif.) quasitownsendi showed a considerable positive correlation with the percentage of sugar cane (ρ = 0.72, p < 0.001 for outdoors traps, and ρ=0.70, p < 0.001 indoors). For Pi. (Pif.) longiflocosa, a positive correlation was observed with the percentage of technified coffee (ρ = 0.56 outdoors and 0.50 indoors, both p <0.001) (Table S1). In terms of distance from each land use to the house, the abundance of Pi. (Pif.) quasitownsendi increased as the distance to sugar cane decreased (ρ = -0.56 outdoors and -0.50 indoors, both p < 0.001) (Table S2). In the case of Pi. (Pif.) longiflocosa, a similar tendency was
observed: as the distance of the dwelling to the technified coffee decreased, the abundance of this species increased significantly (ρ = -0.51 outdoors and -0.48 indoors, both p < 0.001) (Table S2).
The negative binomial regression model for outdoor Pi. (Pif.) quasitownsendi is consistent with the above results: greater distance to sugar cane was associated with lower abundance, more specifically 6% per meter (IRR = 0.94, p < 0.001). Again, this was similar for those caught indoors (IRR=0.92; p < 0.001) (Table 6). For Pi (Pif.) longiflocosa, the regression model showed that the percentage of technified coffee and mixed coffee around the house are positively associated with the abundance of this species in the domestic environment, especially outdoors. For each percentage point of coverage of technified and mixed coffee plantations, the abundance of Pi. (Pif.) longiflocosa increased 16% outdoors (IRR: 1.16, p < 0.001) and 14% indoors (IRR: 1.14, p < 0.001) (Table 7). On the other hand, higher pasture cover and greater distance to mixed coffee plantation around the houses were associated with lower abundance of this species.
Table 6. Land uses associated with Pi. (Pif.) quasitownsendi abundance, outdoors and indoors Outdoor
Indoor
Pi (Pif) quasitownsendi
IRR
CI95%
p value
IRR
CI95%
p value
Distance to burnt area
1.08
1.04 – 1.12
<0.0001
1.05
1.01 – 1.08
0.003
Percentage pasture
1.07
1.03 – 1.11
0.001
---
---
---
Distance to technified coffee
1.04
1.01 – 1.06
<0.0001
1.05
1.02 – 1.08
0.001
Distance to cane sugar
0.94
0.92 – 0.97
<0.0001
0.92
0.88 – 0.96 <0.0001
Distance to other crops
---
---
---
0.96
0.94 – 0.98
0.001
Likelihood ratio (LR) tests were done to compare the negative binomial models, as shown, with models using the Poisson distribution. For the outdoor catches, the LR test statistic was 32.15 (p=0.0211), and for indoor catches it was 36.01 (p=0.0029): both favor the negative binomial models shown.
Table 7. Land uses associated with Pi. (Pif.) longiflocosa abundance, outdoor and indoor Outdoor Pi (Pif) longiflocosa
IRR
Technified coffee plantation percentage
Indoor
CI95%
p value
IRR
CI95%
p value
1.16
1.12 – 1.22 <0.0001
1.14
1.07 – 1.21
0.000
Mixed coffee percentage
1.04
1.01 – 1.08
0.011
---
---
---
Pasture percentage
0.86
0.78 – 0.94
0.002
---
---
---
Mixed coffee distance
---
---
---
0.96
0.94 – 0.98
0.003
As for the previous table, LR tests were carried out to compare negative binomial and Poisson models. Again they favor the negative binomial distribution for both outdoor and indoor catches, with LR statistics of 20.74 (p=0.05) and 20.97 (p=0.0072), respectively.
4.
Discussion
This study evaluated five sites of the Andean region in Colombia which, although similar demographically and epidemiologically, were distinct in terms of land use and in the associated phlebotomine diversity and abundance. Overall, dominance of any one species was associated with
monoculture: in Novillero and La Esmeralda, Pi. (Pif.) quasitownsendi was associated with sugar cane; while in El Cucal and Agua Bonita Pi. (Pif.) longiflocosa was associated with coffee plantations. Although the females of the two species Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa are indistinguishable, we were able to identify them due to their allopatric occurrence in the investigated areas. Interestingly, in Honda Alta, the locality with forest coverage and more heterogeneous land use, fewer specimens were caught, but with higher species diversity.
The overall results regarding diversity reflect the values observed for richness and abundance. Some sites, such as Novillero and El Cucal, had a combination of high individual abundance, high species dominance and low diversity, while others, like Honda Alta and La Esmeralda, had a contrasting pattern of low abundance, low dominance and higher equitability. The sites with higher species diversity tended to show lower abundance of individuals while avoiding the dominance of a single species. In Novillero and El Cucal, diversity was very low, and the species Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa were, respectively, almost exclusively dominant, and are recognized vectors of cutaneous leishmaniasis.
Pi. (Pif.) quasitownsendi and Pi. (Pif.) longiflocosa play an important role in the transmission of cutaneous leishmaniasis in the Magdalena Valley of Colombia due to their dominant abundance, endophagy, and anthropophagy (Ferro et al., 2011; Ocampo et al., 2012; Pardo et al., 2006; Valderrama-Ardila et al., 2010) . Both species are part of the Townsendi series of the Verrucarum (Cohnstaedt et al., 2011) group which is abundant in mountainous areas and well adapted to traditional and technified coffee crops (Bejarano et al., 2003). In the current study, Pi. (Pif.) quasitownsendi was found to be infected with Leishmania (V.) sp, as was Pi. (Pif.) longiflocosa in the previous study in Agua Bonita (Ferro et al., 2011; Ocampo et al., 2012). The majority of the blood meals had been made
on humans, with smaller proportions from birds and other domestic animals, confirming the anthropophagic behavior.
Correlation analysis showed that Pi. (Pif.) quasitownsendi was associated with highly anthropic sites with sugarcane crops and paddock, and Pi. (Pif.) longiflocosa mainly with technified and mixed coffee. For neither species was forest associated with abundance, possibly because, at the majority of sites, forest was sparse and far from the houses. The regression analysis showed that the abundance of Pi. (Pif.) quasitownsendi decreased significantly with the distance between the dwellings and sugarcane; while the abundance of Pi. (Pif.) longiflocosa increased significantly according to the greater coverage of technified coffee. In Honda Alta, which had the greatest forest coverage, the species diversity was high, as found previously elsewhere (Chaniotis et al., 1972; Jimenez et al., 2009; Memmott, 1992; Thies et al., 2016).
In terms of Pi. (Pif.) longiflocosa, these findings agree with those of other focal studies in the Andean zone of Colombia where this species was the most abundant. In Planadas, Tolima, it was found to be strongly associated with high coverage of coffee (Cárdenas et al., 2014). In the municipality of Chaparral, also in Tolima department, the results of a focal study carried out in the post-epidemic period, showed that high abundance of indoor Pi. (Pif.) longiflocosa was associated mainly with the proximity of houses to technified coffee (Ocampo et al., 2012). Elsewhere in Latin America, studies have also found strong associations between some phlebotomines species and land use, e.g. in Brazil, between banana plantations and the abundance of Nyssomyia whitmani, Nyssomyia intermedia and Migonemyia migonei (Luz et al., 2000; Membrive et al., 2012).The current study is the first to demonstrate the association of Pi. (Pif) quasitownsendi with a monoculture such as sugar cane.
The main objective of this study was to establish a rapid characterization methodology that would allow the identification of houses at higher risk of Leishmania transmission due to the composition and abundance of phlebotomines indoors and outdoors (10 m from the peridomestic environment). The previous analysis (Ocampo et al., 2012) confirmed an association between the abundance of some phlebotomine species and land use. Our radial sampling, based on ecological methodologies, allowed us to evaluate differences between houses due to land use. The negative binomial regression model found that increasing distance from the house to sugar cane monoculture was associated with a reduction of 6% per meter in the abundance of Pi. (Pif.) quasitownsendi. This result suggests that simply increasing the distance of crops from the house could be a protective measure. In the case of Pi (Pif.) longiflocosa, it was the percentage coverage of technified coffee plantation that favored the abundance: 16% increase per percentage point coverage for outdoors catches (IRR: 1.16, p < 0.001) and 14% for indoor captures (IRR: 1.14, p < 0.001) and, to a lesser extent, mixed coffee (IRR 1.04, p = 0.011 outdoor). On the other hand, higher pasture cover and greater distance to mixed coffee plantation around the houses were associated with lower abundance of this species.
Rapid assessment methodologies have been used as a proxy to evaluate species diversity at sites which are remote, and difficult and expensive to access (Álvarez et al., 2004), in order to justify their inclusion in conservation schemes. These methodologies allow the standardization of the biodiversity in many taxonomic groups maximizing the capture efforts in a particular locality. In accordance with this philosophy, we implemented this methodology for a rapid evaluation of the conditions that allowed us to associate the peri-domestic environment with the presence and abundance of phlebotomine vector species of importance in domestic transmission. We sought a methodology that could be easily replicated by state health agencies, such as Departmental and Municipal health secretariats, which
usually have very limited resources for fieldwork, but that allow relevant standardized information on the peri-domestic environment to be obtained in a very short period of time (5 – 6 days for 15 houses), efficiently and at a low cost.
The model presented here could guide public heath decision makers in identifying houses at higher risk of domestic transmission and also educate the farmers to increase the distance of crops from their houses. Most of these rural areas are made up of smallholdings (minifundios) with limited economic resources that force them to increase the density of cultivated areas as close as 1 meter to the house. Interestingly, this methodology also suggests that maintaining biodiversity and forest coverage around the house could help to decrease the profusion of any one species, and maintain a balance between the species as was observed in Honda Alta. However, the small number of sites means that more studies are needed to confirm this finding.
5.
Conclusion
The current study showed that the transformation of forest into agricultural land around the domicile favors the presence and abundance of phlebotomines, both outdoors and indoors. In particular, highly modified sites, such as monocultures, favor particular species that increase the risk of domestic transmission. Although such associations have been observed previously, this is the first study to do so based on a rapid and informative field methodology that can be applied by the local health authorities to prioritize their interventions in those houses with potential risk of transmission in a cutaneous leishmaniasis focus.
Supplementary material, Table S1. Supplementary material, Table S2.
Supplementary material, Figure S1.
Acknowledgments We gratefully acknowledge the technical and logistical support of the Secretary of Health of the Department of Santander, Cundinamarca, Huila and Tolima in providing access to the area and assisting in the conduct of fieldwork. We are also grateful to Luis Ernesto Ramirez for his technical support in the field and laboratory. This study was conducted in partial fulfillment of the Masters of Science degree in Epidemiology of the School of Public Health of the Universidad del Valle.
Financial support This study was partially financed by the National Administrative Department of Science, Technology and Innovation - COLCIENCIAS Contract 673-2013 (569-2012) and NIAID-NIH [Award number U19AI129910]. Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Conflicts of Interest The authors declare that none of them has any conflict of interest. Authors´ addresses Mabel Moreno, Lina Guzmán, Neal Alexander, Clara Ocampo, Centro Internacional de Entrenamiento e Investigaciones Médicas CIDEIM, Cali, Colombia. Carlos Valderrama-Ardila, Universidad Icesi,
Cali Colombia. E-mails: [email protected]; [email protected]; [email protected]; [email protected]; [email protected].
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Graphical abstract