**2. Activities to develop in a study of leishmaniasis transmission focus**

When the study of a focus of transmission is initiated, it is necessary to carry out different activities involving all stakeholders involved in the transmission of infection, that is, the human population living in the area, possible vectors, and populations of mammals serving as potential reservoirs. Similarly, knowing the conceptions, attitudes, and practices of the people about the disease is critical to design appropriate control measures for each community. The following sections describe each of these activities.

#### **2.1. Study of human population**

may be zoonotic (from animals to humans) or anthroponotic (from one human to another). In humans, *Leishmania* infections cause a spectrum of illness that depends on the parasite species involved, inoculum size, and host immune response [1, 2], affecting skin, mucous tissues, or organs of the mononuclear phagocyte system and producing clinical symptoms of cutaneous leishmaniasis (CL), mucosal leishmaniasis (ML), and visceral leishmaniasis (VL), respectively.

*Leishmania* spp. infections are acquired when an infected vector bites a mammal to consume its blood. In turn, the vector is infected when it feeds upon the blood of an infected reservoir species and ingests the transmitting parasites (as amastigotes). Transmission cycles of leishmaniasis have been found to have a focal distribution in specific geographic areas. These sites are called the natural foci of infection. The foci of infection are the places where the key elements necessary for transmission are present: vectors and reservoirs. The limits of infection foci are generally determined by the spatial distribution and relative density of the vector species. Hence, conducting entomological surveys and determining the behavior of vectors are important to clarify the epidemiological risk of infection. In turn, the presence of these elements, and especially of vectors, is conditioned by abiotic ecological factors such as climate,

The study of the foci of Leishmaniasis transmission is complex due to the following factors: (1) the diversity of phlebotomine species; (2) the variety of *Leishmania* species; (3) the incrimination of a phlebotomine species requires that the vector meet certain criteria, including exhibiting anthropophilic behavior, being infected with the same species of *Leishmania* isolated from humans infected during the outbreak, and demonstrating geographical distribution consistent with the distribution of reported human cases; (4) the variety of methods required to incriminate potential mammalian reservoirs in an area, which involve capturing and analyzing many samples, isolating and identifying the species of *Leishmania* and determining the parasite prevalence and transmissibility; and (5) the challenge of diagnosing human cases with the clinical form of leishmaniasis, *Leishmania* species causing the disease, and locations

On the other hand, to facilitate the design and implementation of specific control measures, recognition of the transmission mode within a focus and determination of the eco-epidemiological risk of infection are required. Factors that must be determined include the following: (1) the geographical area associated with greatest risk of transmission, or "macrofocus"; (2) the population group at increased risk of becoming infected and developing the disease; (3) the time of year during which increased activity of phlebotomine species in the intradomicile environment occurs (nictemeral behavior); and (4) the place within the dwelling where contact between vectors and the population group at highest risk of infection, or "microfoci," are located [2]. Therefore, understanding the transmission of *Leishmania* infection, identifying the foci of transmission, and designing strategies and control measures requires a combination of different disciplines such as the health sciences, epidemiology, social sciences, entomology, cellular and molecular biology, and ecology, giving rise to eco-epidemiology. In addition to the inclusion of transmission (i.e., the vector), parasite, and reservoir dynamics as objects of eco-epidemiological study, this methodology also includes the study of associated ecological

factors and human behaviors that affect the transmission of the disease.

humidity, altitude, temperature, and vegetation [3, 4].

32 The Epidemiology and Ecology of Leishmaniasis

in dwellings where transmission occurred [5, 6].

Studies of resident populations are conducted by active case finding, conducting epidemiological surveys, and comparing the knowledge, conceptions, attitudes, and practices within a community against the prevalence of the disease and behaviors that facilitate being bitten by vector species.

Active surveillance for cases is performed to develop a detailed report describing the residents of a population, with a special emphasis on the identification of leishmaniasis lesions on both the skin and mucous membranes through clinical examination of the nasal-oropharyngeal region. Additionally, any signs or symptoms compatible with VL, such as fever and/or hepatosplenomegaly, should be identified, especially in children and adolescents. Examination of naso-oropharyngeal mucosa should also be performed on all people with a history of the disease or with compatible scars. During active case finding, it is possible to identify patients who have very advanced mucosal lesions causing destruction of the nose, mouth, pharynx, and lip. Because of their disfigurement, these individuals may not seek treatment at health centers or hospitals within the region, as they have lost all hope of being cured and have been isolated from the social environment due to their facial disfigurement. These people should be actively sought out using information provided by the community in order to obtain the necessary samples to make a diagnosis and initiate treatment. Samples may be then collected from the suspected cases of CL or ML, including scraping for smear and aspiration for culture in the NNN (Novy-MacNeal-Nicolle) medium. All samples should also be analyzed using PCR. In cases of ML, scraping for smear has been found to provide a useful sample [8]. In VL cases, a sample of spleen aspirate or bone marrow (for the smear, culture, and blood tests to detect specific antibodies against *Leishmania* spp.) may be collected; all these procedures have been established by the World Health Organization (WHO) [2].

It is very important to examine the largest possible number of residents in a study site to diagnose active cases and include patients presenting injuries in the healing process due to the use of empirical treatment (such as people with compatible scars and a clinical history that gives reason to suspect that the person had leishmaniasis) and record previous cases. In the analysis of this information, new and old cases may be compared by age, sex, profession, or occupation. Thus, population groups at increased risk of leishmaniasis may be individually identified.

It is also important to diagnose leishmaniasis early because it allows for the initiation of specific treatment as soon as possible. When treatment is initiated early, it is possible to control the progression of the disease, relieve the signs and symptoms of the disease and improve quality of life in patients who are exposed to great social stigma due to the physical "marks" (scars) left by leishmaniasis that make it easy to identify those who had or have the disease.

Since clinical and epidemiological findings are not pathognomonic of the disease, and it is necessary to obtain a laboratory diagnosis to verify clinical suspicion of leishmaniasis. This diagnosis is based on visualization of the parasite in spread (smear) or cultures obtained from lesion material (in cases with CL and ML) or material obtained from aspirate, biopsy of bone marrow, or the spleen (in cases with VL).

However, in some VL or ML cases, it is not always possible to visualize or isolate the parasite, and thus clinical diagnosis may be aided by the presence of specific antibodies against *Leishmania* spp. or molecular tests.

In the case of VL, the use of a clinical and epidemiological history with a positive rK-39 has been accepted as the criteria for diagnosis of the disease. For diagnosis of leishmaniasis, the following clinical laboratory tests may be performed:

**1.** Smear (or spread) is an easy, economical, and rapid procedure; with appropriate sampling and interpretation techniques, the sensitivity of this procedure can reach 90%. The test involves the collection of tissue from the active edge of a lesion and center of an ulcer for CL and ML. If a case has VL, the sample is taken from bone marrow, liver, or the spleen by aspiration, as described later. For cases with skin lesions, it is important to select lesions that are younger and do not display superimposed infection [9].


There are variations of PCR, such as real-time PCR, that employ labeled probes to visualize the amplification reaction. This characteristic makes PCRs more sensitive and enables quantitative analysis of the parasites in a sample [16].

#### **2.2. Epidemiological survey using the Montenegro skin test**

disease or with compatible scars. During active case finding, it is possible to identify patients who have very advanced mucosal lesions causing destruction of the nose, mouth, pharynx, and lip. Because of their disfigurement, these individuals may not seek treatment at health centers or hospitals within the region, as they have lost all hope of being cured and have been isolated from the social environment due to their facial disfigurement. These people should be actively sought out using information provided by the community in order to obtain the necessary samples to make a diagnosis and initiate treatment. Samples may be then collected from the suspected cases of CL or ML, including scraping for smear and aspiration for culture in the NNN (Novy-MacNeal-Nicolle) medium. All samples should also be analyzed using PCR. In cases of ML, scraping for smear has been found to provide a useful sample [8]. In VL cases, a sample of spleen aspirate or bone marrow (for the smear, culture, and blood tests to detect specific antibodies against *Leishmania* spp.) may be collected; all these procedures have

It is very important to examine the largest possible number of residents in a study site to diagnose active cases and include patients presenting injuries in the healing process due to the use of empirical treatment (such as people with compatible scars and a clinical history that gives reason to suspect that the person had leishmaniasis) and record previous cases. In the analysis of this information, new and old cases may be compared by age, sex, profession, or occupation. Thus, population groups at increased risk of leishmaniasis may be individually identified.

It is also important to diagnose leishmaniasis early because it allows for the initiation of specific treatment as soon as possible. When treatment is initiated early, it is possible to control the progression of the disease, relieve the signs and symptoms of the disease and improve quality of life in patients who are exposed to great social stigma due to the physical "marks" (scars) left by leishmaniasis that make it easy to identify those who had or have the disease. Since clinical and epidemiological findings are not pathognomonic of the disease, and it is necessary to obtain a laboratory diagnosis to verify clinical suspicion of leishmaniasis. This diagnosis is based on visualization of the parasite in spread (smear) or cultures obtained from lesion material (in cases with CL and ML) or material obtained from aspirate, biopsy of bone

However, in some VL or ML cases, it is not always possible to visualize or isolate the parasite, and thus clinical diagnosis may be aided by the presence of specific antibodies against

In the case of VL, the use of a clinical and epidemiological history with a positive rK-39 has been accepted as the criteria for diagnosis of the disease. For diagnosis of leishmaniasis, the

**1.** Smear (or spread) is an easy, economical, and rapid procedure; with appropriate sampling and interpretation techniques, the sensitivity of this procedure can reach 90%. The test involves the collection of tissue from the active edge of a lesion and center of an ulcer for CL and ML. If a case has VL, the sample is taken from bone marrow, liver, or the spleen by aspiration, as described later. For cases with skin lesions, it is important to select lesions

been established by the World Health Organization (WHO) [2].

marrow, or the spleen (in cases with VL).

following clinical laboratory tests may be performed:

that are younger and do not display superimposed infection [9].

*Leishmania* spp. or molecular tests.

34 The Epidemiology and Ecology of Leishmaniasis

This activity includes the application (to the general community) of the Montenegro skin test or "leishmanin" antigen test. This test causes an intradermal reaction that measures delayed cellular immunity response, and it has been commonly used in epidemiological studies for the identification of *Leishmania* exposure [17]. This delayed hypersensitivity test should be read 48 hours after being applied, and the pen method should be used to define the area of induration [18]. Although the Montenegro skin test is very sensitive and specific, it does not differentiate between current and past infections. The test is considered positive, or reactive, when the induration is more than 5 mm [2]. It is important to apply the test always on the same forearm (right or left) on each member of the population to facilitate reading, especially when people have negative reactions.

To interpret the results of an epidemiological survey, the common characteristics of people who have a positive test result (which can be a particular age group, profession, or occupation, without discrimination among household members) may be analyzed. For the analysis of these data, it is convenient to divide the population into four age groups: 0 to 4 years (children who usually stay inside the house), 5–14 years (children and adolescents who may have school activities and, in some rural regions, help their parents in agricultural work); 15–60 years (the economically active population); and 61 or more years (the population that most frequently stays at home). Thus, the population group that is at higher risk of infection (the population to which prevention and control measures should be prioritized) may be determined. For analysis purposes, it is helpful to determine the positivity rate, which is the number of positive tests out of the total tests performed in each subpopulation.

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

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 the disease [19].

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 of knowledge.

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 or with a statistically representative sample.

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 measures that are economical and effective.
