**2.4. Study of the phlebotomine population**

**2.3. Conceptions, popular attitudes and practices (CAP)**

36 The Epidemiology and Ecology of Leishmaniasis

the disease [19].

of knowledge.

or with a statistically representative sample.

measures that are economical and effective.

Both rural and sylvan domestic endemic foci of leishmaniasis are characterized by poor and remote communities in urban centers with serious social conflict and little governmental health institution presence. In these areas, the conceptions of the cause of disease vary among population groups and are not necessarily associated with a parasite or the bite of sandflies. Communities have their own medical systems that include their conceptions and practices about the origin of the disease, facilities where they receive care when they are sick and procedures that are available for the diagnosis, treatment, cure, and prevention of

Identifying the conceptions, attitudes, and practices relating to leishmaniasis and medical systems operating in communities is an important component of the study of foci. This information is critical to the design and implementation of primary health education, social assistance programs, and control measures. It is therefore important to determine the locations where residents seek care when they have leishmaniasis, including healers, herbalists, pharmacists, physicians, or other healthcare providers. It is also necessary to determine the variety of treatments used, what residents believe causes disease, how residents refer to the vector and leishmaniasis, and what having leishmaniasis or skin ulcers means within their magical-religious conceptions through a respectful dialogue that enables the sharing

This type of research (the social type) is conducted mainly using qualitative methods but can be supplemented with quantitative methods. Within qualitative studies, techniques such as participant observations may be used. These studies require the constant presence of the researcher in the community, enabling a better approach to obtain this knowledge and facilitating the creation of bonds of trust that allow the collection of more credible information. It also allows researchers to construct an overview of the community, thereby knowing, for example, how people interrelate within the community itself and how these relationships (in one way or another) influence the CAP that have developed related to the disease. Other techniques include interviews with key stakeholders in the community, including herbalists and community leaders. Within the quantitative method exists the application of surveys with closed answers, which are most ideally conducted with all of the community members

The use of social science methods allows researchers to characterize the material conditions that, in short, form a simple or complex network of social relations. These conditions influence a large number of people who are consolidated as a community with similar livelihoods and conceptions about disease. These methods also allow for the observance of human behaviors that may predispose individuals towards contact with insect vectors. Hence, there is a need for constant communication throughout the multidisciplinary group to share findings and refocus targeted observations; all together, these processes may help to determine the epidemiological risk of infection, which is the first step to design prevention and control *Leishmania* vectors belong to the family *Psychodidae* and subfamily *Phlebotominae*. There has been no general agreement established as to the classification of phlebotomines into genus and more general taxonomic categories, but according to the most widely accepted categorization scheme, there are six genera: *Lutzomyia*, *Brumptomyia,* and *Warileya* in the New World and *Phlebotomus, Sergentomyia, and Chinius* in the Old World. The vector species of *Leishmania* belong to the genera *Phlebotomus* and *Lutzomyia* [2].

Although the biology of each species of phlebotomine is unique and complex, generally they are small nocturnal insects that range from 2 to 5 mm in length. By day, they remain at rest in burrows, caves, and hollow trees. The life cycle and behavior of phlebotomines are conditioned by abiotic factors (temperature, humidity, photoperiod) and biotic factors. Only the female is hematophagous, needing blood to develop eggs and maintain the gonotrophic cycle [20]. Phlebotomines are very fragile insects, and they are considered sedentary species with a short range of flight. Some studies using the techniques of capture, marking, and recapture with fluorescent powders have determined that the majority of recaptures occur within a range of less than 200 meters from the site of release [21].

The identification of phlebotomines is based on morphological characteristics in both male and female insects, especially the genitalia, alar indices, pharynx, and cibarium. Examination of morphological characteristics may help to solve problems associated with identification (usually cases where males can be distinguished but not females, which have the greatest epidemiological importance). Currently, taxonomic identification based on morphological characters may be corroborated by mitochondrial cytochrome c oxidase (COI) DNA genebased molecular techniques, which can be provided as a barcode‐specific marker for each phlebotomine species [22].

There are more than 800 currently described phlebotomine species. Approximately 465 of these species have been identified in the New World, and 375 of these species have been identified in the Old World; however, not all phlebotomine species are considered vectors [23]. A phlebotomine naturally infected with the promastigote forms of *Leishmania* is not necessarily capable of transmitting the parasite. For a species to be incriminated as a vector, certain criteria are required: an anthropophilic nature; contact with both humans and the disease reservoir; and infection by the same Leishmania strain identified in human cases. Additionally, the transmitted parasite must be able to develop in the vector, the vector must be able to be transmitted through the bite of the parasite, and the geographical distribution of the parasite must be compatible with that of the vector [2]. In America, approximately 400 species of *Lutzomyia* have been identified*,* but only 22 of these species have been implicated as vectors [24]. In Colombia, 153 species of *Lutzomyia* have been identified [25], but only the following species have been found to be naturally infected with *Leishmania* and incriminated as vectors: *Lu. trapidoi* and *Lu. gomezi* with *L. panamensis* [6, 21], *Lu. spinicrassa* and *Lu. gomezi* with *L. braziliensis* [26], *Lu. umbratilis* with *L. guyanensis* [27], *Lu. olmeca* with *L. mexicana* [28] and *Lu. longipalpis a*nd *Lu. evansi* with *L. chagasi* (=*L. infantum*) [29].

Although the incrimination of a species as a vector is a specialized job that requires the support of entomological research laboratories, a list of species incriminated as vectors in different countries has already been developed. Furthermore, by analyzing collected data, it is possible to identify species that potentially transmit the disease, thereby linking different elements of the study of transmission foci.

In the study of foci, one of the objectives is to determine the limits of the macrofocus, and this determination requires a combination of techniques, such as entomological transects, which involve the simultaneous capture of phlebotomines in different geographical locations, for example, along paths and roads and in houses and the extradomicile using sticky traps (which are traps with castor oil-impregnated paper). Five to 10 days later, the traps may be removed and sand flies be recovered from each sticky trap. This information can be used to determine the relative density per square meter of a particular vector species in a given geographical area. It is also possible to establish the area where the phlebotomines are present, thus specifying the limits of the focus.

To determine the risks within a microfocus, simultaneous captures of phlebotomines may be performed inside, around, and outside the home. Presence and relative density of the vector species may be then determined and correlated with other study data, such as Montenegro test positivity and the population group with the highest number of new and old cases of the disease.

To determine high activity hours during which the vector species is most likely to bite, catches may be made overnight from 18:00 in the evening to 6:00 the next day inside the dwelling (nictemeral monitoring). The specimens caught may then be stored for periods of 2 hours using a vial for each capture period (each properly labeled, indicating the hour of the capture and the place and date to be stored until identification). Additionally, captures should be performed during different seasons (rainy–summer) because the species composition and density may vary during different seasons.

To capture phlebotomines, a series of traps have been designed, among which we will mention the most frequently recommended for the study of foci.


lands, it is captured as soon as possible thus avoid being bitten by the sand fly. This procedure has been approved by the Ethics Committee after reviewing the protocols. This technique allows for the identification of anthropophilic behavior in phlebotomine species that attempt to bite humans. During these captures, information regarding landing rates (# of sandflies/hour/man) can be obtained. Capture on protected human bait can be performed in the intra-, peri-, and extradomicile. When performed on animals, it can be used to infer the zoophilic behavior of a phlebotomine.

− Sticky traps: These traps are considered complementary to captures at rest and require less effort. Sticky traps consist of sheets of 20 by 25 cm bond paper that are fixed on a bamboo stick with a side that is castor oil-impregnated [29, 30]. Phlebotomines that settle on these traps can be used determine the density of each species per square meter trap, considering that a trap has an area of 0.01 square meter for capture. These traps are very useful for transects and to determine the spatial distribution of species and limits of foci of transmission. The traps are placed in the intra-, peri-, or extra-domicile and the number of phlebotomines captured at each point or "station" can vary, as can density because it is determined per square meter trap. When placing these traps, the geographical coordinates of the location and relationship to the domicile should be taken into consideration [6, 29–31].

The community can and should actively participate in entomological studies. The community may recognize *Lutzomyia* species using local popular names, which may vary in different regions of the country, and the community can help guide practitioners regarding the times when they see a greater abundance of phlebotomines. In Colombia, the highest phlebotomine density is frequently recorded after the onset of the rainy season. Thus, the community may inform researchers that they are being bitten more frequently inside or outside the house or at certain peak hours; however, this information must be corroborated by capture of specimens.

Entomological specimens captured using light, bait animal, resting, and human traps should be killed by introducing cotton soaked with ethyl acetate or other reagents in capture containers. Ethyl acetate must be handled carefully because of its toxicity. Subsequently, specimens should be deposited on a plate with a white background, and entomological tweezers should be used to separate the phlebotomines from bycatch species (other insects). Finally, phlebotomines should be deposited in vials, plastic bottles, or glasses with screw caps that are dry or contain 70% alcohol, according to the study objectives. The vials containing the phlebotomines should be marked with the capture source, date, method of collection, and person responsible, among other data.

#### **2.5. Study of reservoirs**

Although the incrimination of a species as a vector is a specialized job that requires the support of entomological research laboratories, a list of species incriminated as vectors in different countries has already been developed. Furthermore, by analyzing collected data, it is possible to identify species that potentially transmit the disease, thereby linking different

In the study of foci, one of the objectives is to determine the limits of the macrofocus, and this determination requires a combination of techniques, such as entomological transects, which involve the simultaneous capture of phlebotomines in different geographical locations, for example, along paths and roads and in houses and the extradomicile using sticky traps (which are traps with castor oil-impregnated paper). Five to 10 days later, the traps may be removed and sand flies be recovered from each sticky trap. This information can be used to determine the relative density per square meter of a particular vector species in a given geographical area. It is also possible to establish the area where the phlebotomines are present,

To determine the risks within a microfocus, simultaneous captures of phlebotomines may be performed inside, around, and outside the home. Presence and relative density of the vector species may be then determined and correlated with other study data, such as Montenegro test positivity and the population group with the highest number of new and old cases of the disease.

To determine high activity hours during which the vector species is most likely to bite, catches may be made overnight from 18:00 in the evening to 6:00 the next day inside the dwelling (nictemeral monitoring). The specimens caught may then be stored for periods of 2 hours using a vial for each capture period (each properly labeled, indicating the hour of the capture and the place and date to be stored until identification). Additionally, captures should be performed during different seasons (rainy–summer) because the species composition and

To capture phlebotomines, a series of traps have been designed, among which we will men-

− CDC traps with white light: These traps are installed between 6:00 pm and 6:00 am inside houses and in the peri- and extradomicile. These traps enable the capture of live speci-

− Shannon trap: A Shannon trap is usually installed in the peri- or extradomicile and com-

fabric. Captures are performed using a "mouth aspirator," which consists of a glass or transparent acrylic tube connected to a rubber hose to which mouth suction is applied to suck the insect into the tube. This manual type of capture may be also used to collect insects that are dormant in housing walls, animal pens, rocks, caves, burrows, tree trunk, the but-

− Capture on protected "human bait": During capture, people are dressed properly and leave exposed only a portion of the lower extremities (legs); in their hands they charge a grabber and a flashlight and stay attentive to the sandflies landing. When a sand fly

emitted by people to capture insects that land on the

elements of the study of transmission foci.

38 The Epidemiology and Ecology of Leishmaniasis

thus specifying the limits of the focus.

density may vary during different seasons.

bines attraction to light with CO<sup>2</sup>

tresses of large trees, etc.

tion the most frequently recommended for the study of foci.

mens, which is useful for the isolation of parasites.

The ecology of *Leishmania* spp. is associated with their hosts; therefore, all factors affecting the survival and behavior of a host may affect the transmission cycle of a parasite. Most leishmaniases are zoonoses for which different species of animals may act as reservoirs of the human parasite. The transmission of the disease to humans requires interactions between a human being and the ecological niche of a vector and wild or domestic reservoirs [32].

It is understood that reservoirs provide an ecological system in which the infectious agent survives indefinitely. This system includes hosts, any intermediate host, or vector and any environmental component that is necessary to maintain the agent indefinitely [33]. Depending on the duration that hosts are able to maintain the parasite, they may be classified into primary (maintaining the infection for a long time), secondary, or accidental.

Reservoirs may be domestic, wild, or synanthropic, and for some species of the parasite, humans are the main reservoir. That is, the case of VL caused by *L. donovani donovani* and CL caused by *L. tropica tropica*. In the New World, leishmaniases are zoonoses; however, there is evidence of anthroponotic transmission during outbreaks, previously identified in the Andean region of the country [6, 34].

Generally, within a focus, there is a primary reservoir for each species of *Leishmani*a; however, other mammals in the same area can become infected, thereby becoming secondary or accidental hosts. Domestic, synanthropic, or wild (marsupial carnivores, rodents, Meshed, insectivores, and primates) reservoir species infected with *Leishmania* may or may not show obvious signs of infection. In general, reservoirs do not exhibit symptoms of the disease [2]. To be a source of transmission is not necessary to be a primary reservoir, and it is possible that different populations of mammals can maintain a continuous cycle of infection and become the source of infection or "primary reservoir" [35].

In America, approximately 310 species of mammals have been incriminated as possible reservoirs. More than half of these species have been incriminated just for having been detected as infected, but the majority of species have been studied and considered as wild reservoirs, such as species in the order Didelphimorphia, *Didelphis marsupialis* and *Didelphis albiventris*, which are important hosts of all Leishmania species, especially *L. amazonensis amazonensis* and *L. braziliensis*. Meanwhile the most important hosts of *L. mexicana* are *Peromyscus yucatanicus* and *Ototylomys phyllotis* rodents. For *L. panamensis* and *L. guyanensis,* an important reservoir has been identified to be *Choloepus hoffmanni* sloths [36].

The most important domestic reservoir is the dog; canine species are mainly a reservoir of *L. infantum,* but cases of infection by *L. braziliensis* and *L. panamensis* have also been observed [37, 38]. Donkeys and horses have also been incriminated as secondary reservoirs [2].

The study of reservoirs has been associated with decreased interest because of the difficulty in establishing incrimination. Briefly, to incriminate a host reservoir, the following criteria must be met: chronic maintenance of populations of parasites in each ecosystem, presentation of a parasitic load sufficient to ensure transmissibility, and identification of an appropriate population density (20% or more of studied wildlife) to provide opportunities for host-vector, host-environment, or host-host interactions depending on the type of transmission [32].

The activities used for identifying reservoir hosts include clinical, serological, and parasitological evaluations of domestic and wild mammals within a given locality. Performing these activities requires the participation of professionals specialized in the capture, identification, and sampling of domestic and wild mammals. For CL, it is very important to examine the snout, ears, genitals, appendages, and areas with less body hair (as *Lutzomyia* bites may occur on exposed areas of the skin) of a potential reservoir species and identify early CL lesions, or nodules, from which samples may be taken for direct examination, culture and/or PCR.

Some larger lesions, such as ulcerated or plate‐type lesions, may also be identified [39]. In the case of VL, it is important to examine dogs for the previously defined clinical signs of VL such as peri-orbital waxing, increase in popliteal lymph node size, growing nails (onychogryphosis), emaciation (cachexia), peeling, and, sometimes, keratitis. For serology, dog blood should be drawn from the cephalic vein to facilitate collection; however, other blood vessels, including the jugular and saphenous veins, may also be used. It is important to take samples for culture, which can also be obtained by puncturing the popliteal lymph node; this sampling requires, removal of hair from the sampling area and good skin scraping [40].

Skin biopsies can be used for both pathological study and culture and PCR. It is inadvisable to use aspirate samples because pollution levels are usually very high; however, a high percentage of antibiotic and antimycotic can be used on this media [41].
