**4.2. Methods for detecting naturally infected vectors**

The liposomal form of AmpB is ideal in the treatment of leishmaniasis, since enables the drug to concentrate specifically at the site of infection, reducing the concentration in others organs [43, 44]. More recently, other drugs such as miltefosine, paromomycin and pentamidine have been used in the treatment of VL in some countries of Africa and Asia, but the efficacy and required dosage of several of these medicines have not been demonstrated in all endemic areas and may differ

**Table 1.** Medvications for treatment of VL according to molecular formula, presentation, dose and route of administration

The duration of treatment should be based on clinical outcome, considering the speed of response and the presence

**Medication Molecular formula Presentation Dose/route administration**

(lyophilized)

Sb Pentavalent antimony (Sb+5)

Ampoules 5 mL (300 mg/mL)

Bottle with 50 mg (lyophilized)

20 mg/Sb+5/kg/day, once daily, endovenosa or intramuscular for 30 days. Max dose of 3 ampoules

1 mg/kg/day by infusion for

3 mg/kg/day by infusion for 7 days or 4 mg/kg/day for 5 days

per day.

14–20 days\* .

single dose.

Some criteria need to be observed for the choice of treatment, such as assessment and stabili‐ zation of clinical conditions and comorbidities present at the diagnosis of VL and electrocar‐ diogram. The use of methylglucamine antimoniate has been especially critical in cases where

Unfortunately, the majority of the population affected by VL is of low income, having no access to diagnosis and treatment options, thereby increasing the mortality rate due to the infection. In endemic areas, VL diagnosis is in most cases based only on clinical characteristics and epidemiologic aspects. Despite the urgent needs, research and development on leish‐

According to Killick‐Kendrick [47], four criteria must be fulfilled before incriminating a given specie as a vector for a zoonotic disease: feeding on humans and in the animal reservoir, supporting the parasites after ingestion, displaying indistinguishable parasites from those

*Lutzomyia (Lutzomyia) longipalpis* is the most competent vector for *L. (L.) infantum chagasi* in VL Latin American foci; however, other sandflies species may be acting in the cycle of VL, mainly in areas where *Lu. longipalpis* is absent [48, 49]. In fact, *Pintomyia (Pifanomyia) evansi* has been

resistance against pentavalent antimonials is widely spread [45].

**4. Natural infection of phebotomine with Leishmania**

isolated from patients and transmitting the parasite by biting.

related to VL transmission in Colombia [50–53] and Venezuela [54, 55].

maniasis have been regrettably neglected.

**4.1. Vectors of** *L. (Leishmania) infantum chagasi*

between these areas [20].

Antimonial N‐methylglucamine

\*

of comorbidities.

recommended in Brazil.

Source: Ministério da Saúde [46].

C7 H20NO9

64 The Epidemiology and Ecology of Leishmaniasis

Amphotericin B C47H73NO17 Amphotericin B deoxycholate

Liposomal amphotericin B C47H73NO17 Bottle/ampoule with 50 mg

The report of natural infection by *L. (L.) i. chagasi* in female phlebotomine sandflies is an important tool for epidemiological investigation, being indispensable for appropriate VL con‐ trol strategy. Distinct techniques have been applied to identify parasitic infection in the insect, including classical and molecular methods.

The classical method to detect natural infection is based on the direct observation of parasites under microscopy, after sandfly gut dissection. However, this *in loco* identification is labori‐ ous, time consuming and requires experience. Another limiting factor is the difficulty in pro‐ cessing the large number of samples required in epidemiological studies [70, 71]. In addition, since other flagellated parasites can be found in the digestive tract of the insects, infection needs to be confirmed by in vitro culture of *Leishmania* or by inoculation into laboratory animals [72, 73]. Furthermore, low parasitemia may underestimate the rates of natural sand‐ fly infection, which are usually about 0.2% using the classical approach, often contrasting with the high frequency of VL in endemic areas [64, 74–76]. However, the dissection method has the advantage of allowing to determine the course and location of infection by *Leishmania* in the sandfly digestive tract [77].

Alternatively, molecular approaches represent a more specific and sensitive technique, allow‐ ing the DNA detection of a single *Leishmania* parasite, regardless of its stage and localiza‐ tion in the insect gut [78, 79]. Indeed, PCR‐based technique was eight times more efficient in detecting trypanosomatids than the dissection method and two times more efficient in identifying natural infection by *Leishmania* [80]. However, molecular methods have the dis‐

advantage of not being able to distinguish between viable and dead parasites [81]. To access the genetic material of the parasite, DNA/RNA is extracted generally using a pool of about 10 female phlebotomine sandflies [82, 83].

Multiple molecular markers from nuclear and kinetoplast *Leishmania* DNA have been used to detect naturally infected phlebotomines, including the miniexon‐derived RNA gene, rRNA gene, repeated genomic sequences and the kinetoplast minicircle DNA (kDNA), which is present at thousands of copies per cell [84–87]. These molecular markers are assessed by PCR methods using specific primers to amplify conserved regions, with kDNA amplification having greater reliability as a marker for the parasite when compared to miniexon and 18S rRNA [88]. Currently, PCR assays are able to detect and identify the parasite (*L. (L.) i. chagasi*) and vector (*Lu. longipalpis*) responsible for VL [82, 89–91]. Besides that, qPCR combines the identification of genetic material with the quantification of para‐ sites present in the phlebotomine*,* which is important for VL transmission and the estab‐ lishment of infection [83].

#### **4.3. Disease cases and natural infection rates in Latin America**

The magnitude of VL in Latin America is not completely known, mainly because most coun‐ tries do not have effective surveillance systems [92–94]. VL was reported in at least 12 coun‐ tries in Latin America, with Brazil having the highest number of cases, followed by Paraguay, Argentina and Colombia [21, 25] (**Figure 3**).

**Figure 3.** Visceral leishmaniasis cases in four Latin American countries: Brazil, Paraguay, Colombia and Argentina (2001–2013). Source: PAHO/WHO [21, 25].

The Brazilian Ministry of Health declared a total of 78,444 VL cases in 25 years of notifica‐ tion (1990–2014), with approximately 67% of them in the Northeast region. In this period, the annual mean in the country was 3137 cases and the incidence was two cases/100,000 inhabit‐ ants [22]. In addition, an increase of 3.2–6.6% in mortality rate caused by leishmaniasis was reported in Brazil from 2000 to 2014 [23].

Although resources have been invested in the VL control and establishment of protocols for specific treatment, important territorial expansion of VL in Latin America countries has been registered [21, 25, 95]. In Brazil, it was initially restricted to poor rural areas in the northeast of the country; however, since 1980s, the disease has gradually spread to major cities and peri‐urban areas in North, Southeast, South and Midwest regions [3, 96], occurring in 23 of the 27 Brazilian states [97] (**Figure 4**).

advantage of not being able to distinguish between viable and dead parasites [81]. To access the genetic material of the parasite, DNA/RNA is extracted generally using a pool of about

Multiple molecular markers from nuclear and kinetoplast *Leishmania* DNA have been used to detect naturally infected phlebotomines, including the miniexon‐derived RNA gene, rRNA gene, repeated genomic sequences and the kinetoplast minicircle DNA (kDNA), which is present at thousands of copies per cell [84–87]. These molecular markers are assessed by PCR methods using specific primers to amplify conserved regions, with kDNA amplification having greater reliability as a marker for the parasite when compared to miniexon and 18S rRNA [88]. Currently, PCR assays are able to detect and identify the parasite (*L. (L.) i. chagasi*) and vector (*Lu. longipalpis*) responsible for VL [82, 89–91]. Besides that, qPCR combines the identification of genetic material with the quantification of para‐ sites present in the phlebotomine*,* which is important for VL transmission and the estab‐

The magnitude of VL in Latin America is not completely known, mainly because most coun‐ tries do not have effective surveillance systems [92–94]. VL was reported in at least 12 coun‐ tries in Latin America, with Brazil having the highest number of cases, followed by Paraguay,

The Brazilian Ministry of Health declared a total of 78,444 VL cases in 25 years of notifica‐ tion (1990–2014), with approximately 67% of them in the Northeast region. In this period, the annual mean in the country was 3137 cases and the incidence was two cases/100,000 inhabit‐ ants [22]. In addition, an increase of 3.2–6.6% in mortality rate caused by leishmaniasis was

**Figure 3.** Visceral leishmaniasis cases in four Latin American countries: Brazil, Paraguay, Colombia and Argentina

Although resources have been invested in the VL control and establishment of protocols for specific treatment, important territorial expansion of VL in Latin America countries has been registered [21, 25, 95]. In Brazil, it was initially restricted to poor rural areas in the northeast

10 female phlebotomine sandflies [82, 83].

66 The Epidemiology and Ecology of Leishmaniasis

Argentina and Colombia [21, 25] (**Figure 3**).

reported in Brazil from 2000 to 2014 [23].

(2001–2013). Source: PAHO/WHO [21, 25].

**4.3. Disease cases and natural infection rates in Latin America**

lishment of infection [83].

**Figure 4.** Distribution of visceral leishmaniasis cases in Latin America countries in 2013. Source: PAHO/WHO [97].

Current control strategies to limit the VL expansion are directed against the vector, using insecticides; the canine reservoir by serological screening, by euthanasia in seropositive dogs and by the use of vaccine in asymptomatic animals with negative serological results, in addi‐ tion to the diagnosis and treatment of human cases. Unfortunately, the results of those inter‐ ventions have been shown to be modest [3, 96]. Since VL epidemiological data are generally based only on the prevalence of human infection [98], surveillance strategies based on a better definition of transmission, risk areas and rates of naturally infected sandflies are necessary in order to provide better control of the disease.

Natural infection rates by *L. (L.) i. chagasi* in phlebotomine are still poorly investigated even in VL endemic areas (**Table 2**). Literature has shown that infection ratios are usually low, rang‐ ing around 1–3% in Latin America, often contrasting with the high incidence of the disease in these regions [74, 76, 99].


**Table 2.** Natural infection ratios by *Leishmania (L.) chagasi* in *Lu. longipalpis* females in Latin America.

According to Cimerman and Cimerman [109], transmission depends on the presence of high densities of *Lu. longipalpis*, as observed during outbreaks of the disease. Several factors may be associated with the difference between natural infection rates detected and VL human cases reported. However, it is possible that even low infection rates are sufficient to maintain circulating infection, highlighting the importance of monitoring sandfly vectors in order to prevent the occurrence of VL, as well as for the definition of risk areas.

On the other hand, high rates of natural infection were observed by Freitas‐Lidani et al. [88] and Saraiva et al. [104], with 8.6 and 19%, respectively, in Pará and Minas Gerais states (North and Southeast regions of Brazil). Both rates were determined using molecular approaches by indi‐ vidual vector analysis. The local incidences of VL for the same period were 281 (Pará state, Brazil) and 407 (Minas Gerais state, Brazil) cases [22], respectively. Although the assessment of individual vectors may be more laborious, the great advantage over pooled samples is the achievement of more informative rates of infected sandflies, especially in areas where new cases are beginning to emerge in dogs and humans.
