Entomological Factors Affecting the Low Endemicity of Chagas Disease in Nazca,Southwestern Peru

Introduction

T he American Trypanosomiasis, or Chagas disease, causes more deaths in the Americas than does any other parasitic disease (WHO 2003, Levy et al. 2006). In Peru it is endemic in the coastal desert of the southwestern and the forested eastern regions (Solis Acosta et al. 1997, Cornejo del Carpio 2003). The majority of clinical cases are reported in the southwestern region, particularly in the department of Arequipa, where recent studies have shown that 19.3% Triatoma infestans are infected with Trypanosoma cruzi (Levy et al. 2006). Further, local authorities report that this index is 30% in some areas (Cornejo del Carpio 2003).

Chagas disease is transmitted to humans when insect feces containing T. cruzi trypomastigotes enter the host open skin or mucous membranes. The vectors, some species of the family Reduviidae, become infected by feeding blood from infected hosts containing T. cruzi trypomastigotes (Zeledon and Rabinovich 1981). In the intestinal tract of the bugs the parasites undergo several transformations. It is possible to identify spheromastigotes, epimastigotes, and trypomastigotes as the most representative stages, in addition to the intermediate forms described by Kollien and Schaub (1998a). In Peru, the main reservoirs of the disease in domestic and peridomestic environments are humans, cats, dogs, rodents, and guinea pigs (Mendoza Ticona et al. 2005).

In the southwestern region, Chagas disease has also been reported as endemic in the departments of Ica, Moquegua, and Tacna (Cornejo del Carpio 2003); however, information for these departments is scant. Triatomine infestation has been reported since early studies in Nazca (Ayulo-Robles 1946), but scarce information about the triatomines infection rates appear to be available for this region. Solis Acosta et al. (1997) reported that 20.7% of the population evaluated from Nazca, the southernmost province of the Ica department, was seropositive to Chagas disease. This study had two main objectives: the first is to investigate the natural rate of infection (tripano-triatomine index) in Nazca, and the second is to determine the vector efficiency of the T. infestans populations that exist there. The presence of trypomastigotes in the insect feces and the defecation patterns of the vector were used to evaluate the vector efficiency (Zeledon et al. 1977, Kirk and Schofield 1987, Trumper and Gorla 1991, Crocco and Catala 1996, Rodriguez et al. 2008). We are hoping to contribute to the knowledge of the transmission of the Chagas disease in the province of Nazca, to date largely unknown.

Materials and Methods

Collections

The province of Nazca, or Nasca, is one of the five provinces of the department of Ica. It is located in the southwestern region of Peru, bordering with the Arequipa department in the south, Ica and Palpa in the north, Ayacucho in the east, and the Pacific Ocean in the west (Fig. 1). The area is arid with scarce precipitations. Mining, agriculture, animal farming, and tourism are among the most economically important activities of the Nazca inhabitants (Solis Acosta et al. 1997). Entomological surveys took place on August 2003 and November 2005, in the districts Vista Alegre and Nazca, in the province of Nazca (Fig. 1). We only visited triatomine-infested houses, selected using information from prior surveys carried out by staff from the Ministry of Health in Nazca.

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FIG. 1.

Map of Peru showing collection sites in the province of Nazca, obtained from the Institute of Statistics and Informatics of Peru (inei.gob.pe). Surveys were carried out in the districts of Vista Alegre and Nazca. Notice the geographic proximity between Nazca and Arequipa, a highly endemic area of Chagas disease in Peru.

Domestic and peridomestic environments were explored. We followed Walter et al. (2007) definition for a peridomestic environment: the full area around the house supporting permanent or temporary structures built and used by humans or by their domestic animals. Bugs were collected from chicken coops, corrals of domestic mammals, charcoal piles, pigeon houses, palms, and dog houses. Within the houses, bugs were collected from holes in walls and ceiling. When insects were found in wall crashes and holes, they were pushed with metal sticks and forceps. Specimens collected were placed into metal cages covered with nylon tulle and then transported to the University of San Marcos (UNMSM) laboratories in Lima.

Results

Infective capacity of Trypanosoma cruzi individuals

All triatomines fed on infected mice contained parasites in their feces. The total number of parasites quantified in the five observations was somewhat similar in the two populations evaluated: 1455 parasites were counted in the 30 specimens from the Arequipa colony and 1368 in the 30 individuals from Ica. Results are given in terms of the percentage of parasites observed, since the total numbers depended on the amount of fecal sample or saline examined by the examiner and, therefore, subject to errors.

Seven days after infection (dai), the drop-like intermediate stages (between spheromastigotes and epimastigotes or trypomastigotes) dominated in both populations. These were 70% and 61.9% of the parasites observed in Ica and Arequipa, respectively. Their number decreased 2 weeks after infection (Fig. 2A). The other stages present 7 dai were epimastigotes and parasites with “slender shape.” No trypomastigotes were observed at this point. The spheromastigotes (9.09% and 11.45% of Ica and Arequipa specimens) appeared 14 dai, except a single flagellate observed in the Nazca specimens 21 dai (Fig. 2B). The metacyclic trypomastigotes were observed 14 dai in both set of insects, 7.95% in Ica and 4.11% in Arequipa specimens. Their number increased each time they were observed (Fig. 2C). On the last examination, 90 dai, trypomastigotes become the most abundant stage observed in Ica triatomines (34.77%). In contrast, in the Arequipa specimens, trypomastigotes (29.32%) were outnumbered by the drop-like parasites (33.83%). Epimastigotes were observed from the first to the last observation in both sets of specimens (Fig. 2B). We calculated the linear regression coefficient of the percentage of T. cruzi stages as a function of time (Fig. 2). Trypomastigotes were the only stage that showed a linear trend during the five observations. Regression coefficients were close to unity (0.98 and 0.94 for Ica and Arequipa specimens, respectively). We used the slope of the linear regression to define the growth rate of trypomastigotes. The differences between Ica and Arequipa trypomastigotes growth rates (Ica, 0.37 ± 0.05; Arequipa, 0.33 ± 0.03) were negligible (Fig. 2).

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FIG. 2.

Percentage of Trypanosoma cruzi stages observed in Triatoma infestans from Ica and Arequipa. (A) Intermediate stages: drop-like stage (lines) and slender-shape parasites (bars); (B) epimastigotes (lines) and spheromastigotes (bars), and (C) trypomastigotes.

Discussion

For decades, the Peruvian department of Ica was considered to be having a high risk of becoming a Chagas disease–endemic area (Ayulo-Robles 1946). However, there is a lack of confirmed Chagasic patients and external evidence of the disease (such as the Romaña's sign) in the last years, except for introduced cases from Arequipa (Cabrera et al. 2002). Further, scant information on T. cruzi infection rates in the triatomines vector is also difficult to obtain from this province (Cornejo del Carpio 2003).

We found no T. infestans bugs infected by T. cruzi in the 624 specimens examined from Nazca, even though houses in this area show high infestation by triatomines. Low T. cruzi infection rates in triatomines were previously reported by Solis et al. (2003), where a single triatomine infected with T. cruzi was identified out of specimens in 494 houses surveyed in Nazca during 1997–1998. In contrast, in the neighboring department of Arequipa, Levy et al. (2006) reported that 19.3% of the 397 houses surveyed in this region harbored infected triatomines. Additionally, infection rates as high as 30% in Arequipa have been reported (Cornejo del Carpio 2003).

Our results are consistent with serological surveys carried out by the local authorities (Direccion de Salud de Ica, Health Direction of Ica, unpublished), and with the Panamerican Health Organization, which report 0% of seropositivity to Chagas in Ica inhabitants in 2000 (PAHO 2001). Nevertheless, these conflict with those of the 20.6% of seropositivity found by Solis Acosta et al. (1997) in Nazca. It is important to note that the main limitation of Solis Acosta et al. study was that they used the indirect fluorescent antibody test only, whereas current diagnosis of Chagas disease requires positive results on two or more serological tests (WHO 2000). Since serological tests are based on the detection of antibodies, they could not easily discriminate between patients with an acute or chronic phase of the disease. The reconstruction of Nazca houses with masonry material after the earthquake of 1996 may have limited the transmission of Chagas in some areas and positive results probably detected mainly chronic infections. Large epidemiological studies are required, including serological and entomological surveys, to evaluate whether reconstructed areas have lower incidence rates in Nazca. It would also be important to investigate if there are any transmission hotspots in Nazca, as reported in Arequipa (Levy et al. 2007).

T. infestans appears to be an efficient vector in all regions where it is found, and no conclusive evidence demonstrating vector refractoriness to parasite infection has been found (Zeledon and Rabinovich 1981). However, future research on the molecular interactions between strains of within the vector is required (Araujo et al. 2007). There is some evidence of co-adaptation between this parasite and its vectors, being triatomines exhibiting a higher susceptibility to their local parasites (Vallejo et al. 2003, Araujo et al. 2008). Some triatomines species have an hemolytic factor that can affect the survival of certain T. cruzi serotypes (Kollien and Schaub 2000). Taking these lines of evidence into consideration, an experimental infection of T. infestans specimens from Nazca (Ica) and Arequipa was undertaken to investigate the vectorial capacity of this species. Unexpectedly, both insect sets were capable of producing metacyclic trypomastigotes. Similarly, the Arequipa specimens showed less epimastigotes. It is likely that specimens from Arequipa may have diminished their vectorial capacity for being kept in a colony for several generations. Regardless of these differences, both insect groups were capable of completing T. cruzi cycle.

In both insect groups, a peak in total parasite numbers was observed 2 weeks after infection. These results were consistent with those obtained by De Stephani Marquez et al. (2006), who found a higher number of T. rangeli parasites 10 dai. Trypomastigotes were detected from the second observation date, 14 dai. Their number increased linearly (regression coefficients close to unity) through the days until the last examination, 2 months after infection. Trypomastigotes from both populations grew at the same rate (slope values 0.37 ± 0.05 and 0.33 ± 0.03 for Nazca and Arequipa, respectively), indicating that they had a similar grow rate (Fig. 2C). The rest of the stages showed low regression coefficients, indicating a lack of trend. These results differ from the natural infection in T. brasiliensis, which showed trypomastigotes 3 dai (Araujo et al. 2008), but are more similar to those found by Piesman and Sherlock (1985), who detected trypomastigotes of T. cruzi between 2 and 4 weeks after infection in Panstrongylus megistus. The development of spheromastigotes within the intestine of the vector has been described as an alternative route to metacyclogenesis and has been related to stress conditions, such as starvation (Kollien and Schaub 2000). However, spheromastigotes were found 2 weeks after infection in both groups of bugs, which had not been subject to starvation. Similarly, prolonged starvation has been linked to the increase in the number of drop-like intermediate stages (Kollien and Schaub 1998b, 2000), but we found them as the dominant stage in both groups during the insect fecal examinations. It is then likely that the specimens were undergoing certain form of stress as a result of manipulation during experiments. It is also possible that these stages are observed under normal circumstances. The limited information in this respect did not allow us to arrive to further conclusions.

Zeledon et al. (1977) maintained that triatomines that defecate before 10 min postfeeding are potentially effective transmitters of T. cruzi. In this study we showed that specimens from Nazca defecate immediately after feeding when completing their blood intake, demonstrating that they are efficient vectors. However, a proportion of the insects did not accepted the blood meal on mice immediately. This may be explained by the stress caused on insects during experiments. We considered the possibility that insects developed preference for bird blood as they had been previously fed on chickens in the insectary. However, this seems unlikely according to recent studies (Gurtler et al. 2009).

Feeding preferences may have affected the role of T. infestans populations as vectors of Chagas disease in Nazca. Solis et al. (2003) showed that triatomines in this province had strong preference for avian blood. Using the precipitin test, authors observed that 62.8% of bugs collected from Nazca (reactive to any type of blood) fed on birds only. Houses surveyed in Nazca had indoor environments reconstructed with masonry material after the earthquake in 1996. However, peridomestic environments where chicken dwellings are located are still constructed with adobe materials, favoring triatomines colonization. Since host proximity may be the main reason for the eclectic host-feeding patterns in T. infestans (Minter 1976), birds accessibility in Nazca may explain the results of Solis et al. (2003). Taking into account that birds are refractory to T. cruzi infection (Teixeira et al. 2006), this may also explain the low infection rates found in this area. Conversely, Arequipa guinea pigs, which are important reservoir of T. cruzi, are consumed as typical food in the Andes area. Levy et al. (2006) showed that enclosures with guinea pigs had an average of 33.9 triatomines, which is 1.69 times more likely to keep triatomines than other animal dwellings. We suggest that host availability is the most plausible explanation for the differences observed in T. infestans infection rates in the Peruvian regions of Nazca and Arequipa.

Particular care must be taken on winter seasons, as triatomines may prefer to colonize warmer indoor environments. As vector control for Chagas disease requires effective measures that reduce expenses and increase efficiency, budgetary limitations currently restrict insect control and monitoring programs, and thus disease control is considered insufficient in Peru (Cornejo del Carpio 2003). This can only be remedied by encouraging research that improves our understanding of vector biology, including feeding preferences, dwelling preferences, and seasonal variability. We recommend that insecticide spraying to control T. infestans in Nazca should be carried out before the winter season, when the infected triatomines have a higher risk of transmitting the disease to humans, and while insects may prefer to stay in the peridomestic environments instead of seeking shelter and warmth in human dwellings.