Follow-up of experimental chronic Chagas' disease in dogs: use of polymerase chain reaction (PCR) compared with parasitological and serological methods.

In this study, the polymerase chain reaction (PCR) was compared with parasitological and serological methods to detect the infection in dogs, 5-12 years after experimental infection with Trypanosoma cruzi. The ability of parasitological methods to identify a positive animal was 22 and 11% by hemoculture and xenodiagnosis/xenoculture, respectively. On the other hand, the serological tests, including conventional serology and anti-live trypomastigote antibodies (ALTA) were positive in all infected dogs. Despite its low sensitivity, if considering only one reaction, the PCR analysis showed 100% of positivity, demonstrating the presence of parasite kDNA in all infected dogs. To identify a positive dog required at least two blood samples and up to nine repeated reactions using the same sample. Serial blood sample collection, ranging from 1 to 9, revealed that the percentage of dogs with positive PCR ranged from 67 to 100%. These findings suggested that, although the PCR is useful to detect the parasite in infected hosts, it should not be used isolated for the diagnosis of Chagas' disease and warn for the necessity of serial blood collection and re-tests. Moreover, these data validate once more the dog as a model for Chagas' disease since they demonstrate the permanence of infection by PCR, parasitological and serological methods, reaching relevant requisites for an ideal model to study this disease.     

Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas' disease

Trypanosoma cruzi isolates from 23 acute chagasic patients from localities of Western Venezuela (state of Barinas) where Chagas' disease is endemic were typed using ribosomal and mini-exon gene markers. Results showed that isolates of the two major phylogenetic lineages, T. cruzi I and T. cruzi II, were isolated from these patients. Six isolates (26%) were typed as T. cruzi II and 17 (74%) as belonging to T. cruzi lineage I. Analysis of random amplified polymorphic DNA (RAPD) patterns confirmed these two groups of isolates, but did not disclose significant genetic intra-lineage polymorphism. Patients infected by both T. cruzi I or T. cruzi II showed different clinical profiles presenting highly variable signs and symptoms of acute phase of Chagas' disease ranging from totally asymptomatic to severe heart failure. The predominance of T. cruzi I human isolates in Venezuela allied to the higher prevalence of severe symptoms of Chagas' disease (heart failure) in patients infected by this lineage do not corroborate an innocuousness of T. cruzi I infection to humans. To our knowledge, this is the first study describing predominance of T. cruzi lineage I in a large number of acute chagasic patients with distinct and well-characterized clinical profiles.     

Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina

Long-term variations in the dynamics and intensity of sylvatic transmission of Trypanosoma cruzi were investigated around eight rural villages in the semiarid Argentine Chaco in 2002-2004 and compared to data collected locally in 1984-1991. Of 501 wild mammals from 13 identified species examined by xenodiagnosis, only 3 (7.9%) of 38 Didelphis albiventris opossums and 1 (1.1%) of 91 Conepatus chinga skunks were infected by T. cruzi. The period prevalence in opossums was four-fold lower in 2002-2004 than in 1984-1991 (32-36%). The infection prevalence of skunks also decreased five-fold from 4.1-5.6% in 1984-1991 to 1.1% in 2002-2004. Infection in opossums increased with age and from summer to spring in both study periods. The force of infection per 100 opossum-months after weaning declined more than six-fold from 8.2 in 1988-1991 to 1.2 in 2002-2004. Opossums were mainly infected by T. cruzi lineage I and secondarily by lineage IId in 1984-1991, and only by T. cruzi I in 2002-2004; skunks were infected by T. cruzi IId in 1984-1991 and by IIc in 2002-2004. The striking decline of T. cruzi infection in opossums and skunks occurred in parallel to community-wide insecticide spraying followed by selective sprays leading to very low densities of infected Triatoma infestans in domestic and peridomestic habitats since 1992; to massive deforestation around one of the villages or selective extraction of older trees, and apparent reductions in opossum abundance jointly with increases in foxes and skunks. These factors may underlie the dramatic decrease of T. cruzi infection in wild reservoir hosts.     

Variables that modulate the spatial distribution of Trypanosoma cruzi and Trypanosoma evansi in the Brazilian Pantanal

An evaluation was made on how the landscape and cattle ranching affect the transmission cycles and the patterns of tripanosomatid infection (Trypanosoma cruzi and Trypanosoma evansi) of small wild mammals in the Pantanal. This region comprises a large natural environment with a multiplicity of habitats, wide variety of biodiversity besides the presence of livestock. T. cruzi and T. evansi infections were evaluated by parasitological and serological methods in one preserved and one cattle ranching area. The diversity of the small mammal fauna showed to be the same in the two studied areas, however, their relative abundance was different. Distinct enzootiological scenarios of both trypanosomatids could be observed. Transmission of T. cruzi occurred mainly in forested areas, in the two study areas, while T. evansi occurred dispersed among all habitats studied in the unpreserved area. The arboreal rodent Oecomys mamorae, the most abundant species in both areas, displayed high T. cruzi and T. evansi serum prevalence and parasitemias. Also, the caviomorph rodent Thrichomys pachyurus was shown to be an important host due to its expressive relative abundance, prevalence of infection by both trypanosomatid species and a broad range use of habitats. The role of the small mammal fauna in the transmission cycle of both trypanosomes species seems to be distinct according to land use since we found a broad range of T. evansi infected hosts in the preserved area in contrast to cattle ranching area and a half number of the rodents species infected with T. cruzi in unpreserved in comparison to protect area. The present study showed that cattle ranching in this study area did not enhance overall prevalence of T. cruzi infection among small wild mammals. Together with the observation that small mammals diversity in FA is similar to RN area suggest that ranching activity may also not necessarily conduct to biodiversity loss or risk of Chagas disease.     

Higher seroprevalence of Trypanosoma cruzi infection in dogs than in humans in an urban area of Campeche, Mexico

The reservoir capacity of dogs for Trypanosoma cruzi infection was analyzed in the city of Campeche, an urban town located in the Yucatan peninsula in Mexico. The city is inhabited by ~96,000 dogs and ~168,000 humans; Triatoma dimidiata is the only recognized vector. In the present study, we sampled 262 dogs (148 stray dogs and 114 pet dogs) and 2800 young people (ranging in age between 15 and 20 years old) and tested for T. cruzi antibodies by enzyme-linked immunosorbent assay, Indirect Immunofluorescence, and Western blotting serological assays. Seroprevalence in stray dogs was twice higher than in pet dogs (9.5% vs. 5.3%) with general seroprevalence of 7.6%. In humans, the observed seroprevalence was 76 times lower than in dogs (0.1% vs. 7.6%, respectively). Western blotting analysis showed that dogs' antibodies recognized different T. cruzi antigenic patterns than those for humans. In conclusion, T. cruzi infection in Campeche, Mexico, represents a low potential risk to inhabitants but deserves vigilance.     

Differential impact of metacyclic and blood trypomastigotes on parasitological, serological and phenotypic features triggered during acute Trypanosoma cruzi infection in dogs

A detailed follow-up investigation of the major parasitological, serological and phenotypic features in dogs experimentally infected with metacyclic (MT) and blood (BT) trypomastigotes of Trypanosoma cruzi strain Berenice-78, typifying vectorial and transfusional transmission of human Chagas disease, has been conducted. Although there were no changes with respect to the window of patent-parasitaemia, significant differences between MT- and BT-infected dogs in both the prepatent period (days 23 and 19, respectively) and the day of maximum parasitaemia (days 26 and 22, respectively) were recorded. A progressive enhancement in the level of T. cruzi-specific antibodies accompanied infection by both MT and BT forms, although higher IgG titres developed on days 14 and 21 following infection with MT forms. Higher Thy-1(+)/CD21(+) and lower CD4(+)/CD8(+) cell ratios, occasioned by increased levels of Thy-1(+) and CD8(+) T-cells and reduced frequencies of CD4(+) T-cells and CD21(+) B-lymphocytes, were observed in both MT- and BT-infected animals. The reduced frequency of CD14(+) leukocytes was revealed as the most relevant phenotypic feature intrinsic to T. cruzi infection independent of inoculum source. BT-specific phenotypic features included an early reduction in the percentage of circulating CD21(+) and CD14(+) leukocytes, together with a higher Thy-1(+)/CD21(+) cell ratio on day 42. On the other hand, higher levels of CD8(+) T-cells, together with a lower CD4(+)/CD8(+) cell ratio on day 28, were characteristic of MT infection. These findings emphasise the importance of inoculum source and suggest that vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during acute Chagas disease.     

Congenital transmission of Trypanosoma cruzi: an operational outline for detecting and treating infected infants in north-western Argentina

We designed a set of procedures for first-line local health services to detect and treat the congenital transmission of Trypanosoma cruzi at a province-wide scale, and field-tested the programme in the province of Tucumán, northwestern Argentina, from 1992 to 1994. The programme consists of routine screening of pregnant women for seroreactivity to T. cruzi, serological and parasitological follow-up of the newborn at least twice during the first year of age, treatment of the infected infants, and evaluation of the outcome. 927 (5.5%) of 16 842 pregnant women were seroreactive to T. cruzi by indirect haemagglutination assay and ELISA. Twenty-one (6.7%) of 315 newborns to seroreactive mothers were diagnosed as infected with T. cruzi parasites microhaematocrit concentration before 30 days of age. Five newborns who initially tested negative had a T. cruzi infection detected by microhaematocrit and/or serological techniques at 3 or 6 months of age. Thus, congenital infection was diagnosed in 26 (7.1%) infants born to seroreactive women and residing in houses free of triatomine bugs. Four of 6 infants born to seroreactive mothers died during the first year of age and had some evidence of T. cruzi infection; one of the deaths was attributed to T. cruzi based on clinical evidence. After specific treatment with nifurtimox or benznidazole, 30 of 32 infants remained parasitologically and serologically negative. This study shows the feasibility of controlling the incidence of congenitally acquired T. cruzi infections at a province-wide scale by means of a specific screening programme at first-line health services level.     

Detection and significance of inapparent infection in Chagas disease in western Venezuela

Inapparent infections of Trypanosoma cruzi were detected in symptomless seropositive people living in close proximity, and under the same conditions of risk, to patients with acute Chagas disease. Similar infections were also detected in sera samples of people from 25 villages of western Venezuela where Chagas disease is endemic. Seropositivity in all the 1,251 studied samples was established by use of 3 serological methods (direct agglutination test, indirect immunofluorescence antibody test, and enzyme-linked immunosorbent assay). Each seropositive sample was tested for detection of anti-T. cruzi-specific immunoglobulin (Ig) M and IgG levels and specific T. cruzi infection by molecular methodology (polymerase chain reaction assay). The combined analysis of the serologic (IgM and IgG levels), molecular (specific T. cruzi DNA), and statistical findings demonstrated the existence of a different stage of T. cruzi infection in asymptomatic patients, which is suggested to be recognized as inapparent infection. Its definition, significance, and comparison with typical Chagas disease phases are presented, and its potential epidemiological importance is discussed.     

Short report: Trypanosoma cruzi infection in wild mammals from a chagasic area of Chile

We report results of PCR-DNA based detection of Trypanosoma cruzi in wild mammal reservoirs from a chagasic area of Chile. We analyzed 157 blood samples from wild mammals including the marsupial Thylamis elegans and the rodents Octodon degus, Phyllotis darwini, and Abrothrix olivaceus. In addition, 42 blood samples from goats (i.e., a peridomestic mammal) were analyzed. Blood samples were used to extract DNA, and PCR was performed using the amplification of minicircle DNA sequences. Southern analysis was used to confirm diagnosis with a universal probe of P(32)-labeled kinetoplast DNA. Altogether, 51% of the wild and 36% of the peridomestic mammals were infected with T. cruzi. These findings suggest that the real T. cruzi infection levels in wild and peridomiciliary reservoirs are higher than those previously determined with serological and parasitological conventional methods. The relevance of our results is discussed in term of the risk factor of infection in human.     

A prospective study of the effects of sustained vector surveillance following community-wide insecticide application on Trypanosoma cruzi infection of dogs and cats in rural Northwestern Argentina

Domestic dogs were used as natural sentinels to assess prospectively the long-term impact of selective, community-based spraying with pyrethroid insecticides after community-wide spraying on transmission of Trypanosoma cruzi in rural villages under surveillance between 1992 and 2002. In 2000 and 2002 light infestations by Triatoma infestans were recorded, and 523 dogs and cats were examined serologically or by xenodiagnosis. The prevalence of T. cruzi infection in dogs decreased from 65% at baseline to 8.9% and 4.7% at 7.5 and 10 years after sustained vector surveillance, respectively. The average annual force of infection dropped 260-fold from 72.7 per 100 dog-years at baseline to <0.3% in 2002, as determined prospectively and retrospectively from the age-prevalence curve of native dogs born during surveillance. Multiple logistic regression analysis showed that prevalent cases in dogs in 2000 and 2002 were associated positively and significantly with the peak number of T. infestans caught in domestic areas at the dog's compound during its lifetime. The sustained decline in T. cruzi infections in dogs and cats is the result of selective, community-based insecticide spraying that kept the abundance of infected T. infestans at marginal levels, fast host population turnover, and low immigration rates from areas with active transmission.     

Impact of community-based vector control on house infestation and Trypanosoma cruzi infection in Triatoma infestans, dogs and cats in the Argentine Chaco

The relative impact of two community-based vector control strategies on house infestation by Triatoma infestans and Trypanosoma cruzi infection in bugs, domestic dogs and cats was assessed in two neighboring rural areas comprising 40 small villages and 323 houses in one of the regions most endemic for Chagas disease in northern Argentina. The prevalence and abundance of domestic infestation were 1.5- and 6.5-fold higher, respectively, in the area under pulsed, non-supervised control actions operating under the guidelines of the National Vector Control Program (NCVP) than in the area under sustained, supervised surveillance carried out jointly by the UBA research team and NCVP. The prevalence of infestation and infection varied widely among village groups within each area. In the pulsed control area, the prevalence of infection in bugs, dogs and cats was two- to three-fold higher than in the area under sustained surveillance, most of the infected animals qualified as autochthonous cases, and evidence of recent transmission was observed. Infection was highly aggregated at the household level and fell close to the 80/20 rule. Using multiple logistic regression analysis clustered by household, infection in dogs was associated positively and significantly with variables reflecting local exposure to infected T. infestans, thus demonstrating weak performance of the vector surveillance system. For high-risk areas in the Gran Chaco region, interruption of vector-mediated domestic transmission of T. cruzi requires residual insecticide spraying that is more intense, of a higher quality and sustained in time, combined with community participation and environmental management measures.     

Trypanosoma cruzi congenital transmission in wild bats

Trypanosoma cruzi congenital transmission in wild bats (Molossus molossus), associated with infected Rhodnius prolixus in a natural habitat from a rural locality in western Venezuela, is reported. T. cruzi blood circulating trypomastigotes in a pregnant bat were detected by parasitological methods. Polymerase chain reaction (PCR) assays carried out in samples from the heart and the fetus of the same infected female, revealed the presence of T. cruzi-specific DNA in both of the tissues, demonstrating transmission of the infection from the mother to the offspring. Eighty percent of the captured bats and 100% of the examined fetuses from pregnant specimens were shown to be infected by T. cruzi, indicating that M. molossus is a very susceptible species for this parasite, and that T. cruzi congenital transmission is a common phenomenon in nature. To our knowledge, this seems to be the first report on congenital T. cruzi transmission in wild bats in Venezuela. The circulation of T. cruzi lineage I in the study area was demonstrated by typing the isolates from bats and triatomine bugs captured in the same habitat. The potential epidemiological implication of these findings in areas where Chagas disease is endemic is discussed.     

Incidence of trypanosoma cruzi infection among children following domestic reinfestation after insecticide spraying in rural northwestern Argentina

Following increasing reinfestation with Triatoma infestans after insecticide spraying, the household incidence of infection with Trypanosoma cruzi in children was positively related to the domestic abundance of infected T. infestans and the presence or proportion of infected dogs or cats in Amamá, a rural village in northwestern Argentina. Seven (12.1%) children seronegative for antibodies to T. cruzi at baseline, with no history of travel or blood transfusion, seroconverted after three years. Six incident cases lived in houses heavily infested with T. infestans, with high proportions of bugs infected with T. cruzi and having fed on humans or dogs. The remaining incident case occurred under a very light domestic infestation detected only at the endpoint, and most bugs had fed on humans. Dogs had a 17 times greater force of infection than children (4.3% per year). Sustained vector surveillance is crucially needed in high-risk areas for Chagas disease such as the Gran Chaco.     

Polymerase chain reaction reveals Trypanosoma cruzi infection suspected by serology in cutaneous and mucocutaneous leishmaniasis patients

The existence of patients suffering a double infection caused by Trypanosoma cruzi and Leishmania spp. has been suggested by several authors. Since the conventional serological tests now available for the diagnosis of Chagas' disease lack specificity due to the cross-reactivity between these two parasites, a serological confirmation of a T. cruzi infection cannot be made unless specific antigens are used. An enzyme linked immunosorbent assay (ELISA) to detect antibodies against a specific T. cruzi antigen, named Ag163B6, and immunoblotting using T. cruzi epimastigotes, are non-conventional serological techniques that could be employed for specific diagnosis of Chagas' disease. Using these two methods 34 cutaneous or mucocutaneous leishmaniasis patients were classified into two groups: (A) patients with serological evidence of T. cruzi infection, i.e. those who tested positive in at least one assay (18/34); and (B) patients with no serological evidence of T. cruzi infection, i.e. those who were negative for both assays (16/34). Taking into account the difficulties of xenodiagnosis and its low sensitivity (less than 50%) for a direct diagnosis in the chronic period of the disease, we used polymerase chain reaction (PCR) to confirm a T. cruzi infection in those leishmaniasis patients who presented positive results with the non-conventional serological techniques. Of the 18 patients with serological evidence of T. cruzi infection, 17 gave positive results when genomic DNA primers were used. Using minicircle primers, 15/18 of that group were positive. Nevertheless, all the patients suspected of being double infected were positive in at least one PCR test. Just one patient with no serological evidence of T. cruzi infection gave a positive PCR result when amplifying the minicircle sequence. The proof of the existence of a T. cruzi infection by PCR in leishmaniasis patients suspected to be chagasic when non-conventional serology was used, strongly supports the use of the specific Ag163B6 and immunoblotting with epimastigotes as specific serological diagnostic tools to determine a T. cruzi infection.     

Isozyme profiles of Trypanosoma cruzi stocks from Colombia and Ecuador

A total of 74 of 82 domestic Rhodnius prolixus from the same locality in eastern Colombia were found to be infected with Trypanosoma cruzi or T. rangeli. One of three domestic Triatoma dimidiata from Ecuador also showed T. cruzi infection. A total of 59 T. cruzi stocks from these and five other localities in Colombia were isolated from man, marsupials and triatomine bugs. Cellulose-acetate electrophoresis of nine or ten enzymes characterized all T. cruzi stocks as zymodeme 1 (reference clone Silvio X10/1). Differences in electrophoretic patterns between the newly isolated stocks and the zymodeme 1 standard were seen with the enzymes G6PD and HK. These results are in agreement with the previously described geographical distribution of T. cruzi zymodemes. Stocks were isolated from both low and high altitudes and there was no evidence of adaptative significance of T. cruzi enzyme polymorphism.     

Lutzomyia migonei as putative vector of visceral leishmaniasis in La Banda, Argentina

Four autochthonous cases of human visceral leishmaniasis (VL) were reported in La Banda, Santiago del Estero from June 2007 to May 2008. In the vicinity of these cases there were 3/47 rK39 sero-positive dogs, and another 4 dogs with VL were reported by passive surveillance. The sero-positive dogs and infected humans lived within a 3.1 km radius. Phebotomine sand fly captures were performed twice during November/December 2007 and April 2008. In 20 of the 59 sampled sites in the areas of the human and canine cases (220 night/traps) 151 phlebotomine sand flies were collected and consisted of: Lutzomyia migonei 93%, Lutzomyia cortelezzii 5.6% and Lutzomyia neivai 1.4%. We propose that there was an enzootic cycle of VL with accidental human transmission due to L. migonei and suggest that there be a surveillance of human isolated cases of VL within the L. migonei dispersion area.     

Dynamics and distribution of house infestation by Triatoma dimidiata in central and southern Belize

Chagas disease is a major public health problem from South America to Mexico, with approximately 10 million infected people. Chagas disease is known to occur in Belize, but little is known about the prevalence of Trypansoma cruzi infection in the Belizean population or the Chagas vector in this region. An entomologic survey of triatomines in the central and southern region of Belize was thus performed. Triatomines were collected by community participation in 37 villages of the Cayo (central) and Toledo (southern) districts and analyzed for infection with T. cruzi by microscopic examination and polymerase chain reaction. Two hundred fifty-six triatomines were collected in 34/37 villages, indicating a wide distribution, and all were identified as T. dimidiata. The majority (87%) were adults (42% males, 58% females), and 13% were larval stages. The infection rate with T. cruzi was 28%. Triatomines were more abundant during the hot season (March-July) compared with the cooler season (September-February). These results confirm that there is a significant risk for autochthonous Chagas disease transmission in central and southern Belize and suggest a pattern of seasonal infestation by nondomiciliated adult triatomines, which are likely to be closely related to T. dimidiata from Yucatan, Mexico. Further entomologic and epidemiologic studies should be performed to precisely determine T. cruzi transmission risk to humans, as well as the seroprevalence of T. cruzi infection and incidence of Chagasic cardiomyopathy in the Belizean population.     

High Incidence Cold Autoagglutinins of Anti-IT Specificity in Yanomama Indians of Venezuela

Summary. A cold auto-agglutinin that reacts with human group O red cells from both adult and cord bloods was found in high incidence (84%) among 90 group O Yanomama Indians from several villages on the upper Orinoco River of Venezuela. The serological characteristics of the cold antibody revealed anti-I^T specificity. This type of cold agglutinin was absent among 30 Warao Indians who inhabit the Orinoco Delta of Venezuela, and among 50 donors residents from the city of Caracas. In preliminary tests, no correlation was found between the cold agglutinins and antibodies against Trypanosoma cruzi or several ARBO virus antibodies.     

Trypanosoma cruzi infection in dogs and cats and household seroreactivity to T. cruzi in a rural community in northeast Brazil.

The prevalence of Trypanosoma cruzi parasitemia as determined by xenodiagnosis on domestic dogs and cats was correlated with household rates of seroreactivity to T. cruzi and household Panstrongylus megistus infestation in a rural area in northeast Brazil where P. megistus was the only domiciliary triatomine vector. T. cruzi infection was present in about 18% of domestic dogs and cats. Two-thirds of seroreactive children below age 10 resided in houses with T. cruzi-infected animals. In houses with a T. cruzi-infected dog or cat, as well as at least one infected P. megistus, the household rate of seroreactivity to T. cruzi was five times greater than in houses with non-infected domestic animals and no infected triatomine vectors. Domestic dogs and cats are important reservoirs of T. cruzi in an endemic area where P. megistus is the only domiciliary triatomine vector.     

Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST)

Leishmaniasis caused by Leishmania infantum is a prevalent disease in dogs and humans. A serological survey of Leishmania infection in dogs was carried out in the endemic region of Alto Douro (north Portugal). Two hundred and ninety-four dogs from the municipality of Peso da Régua were examined for clinical signs of canine leishmaniasis (CanL), and sera samples were evaluated by the direct agglutination test (DAT) and the fast agglutination screening test (FAST). The sero-prevalence of infection was 20.4%, after screening the study population by FAST and subsequent confirmation by DAT. The overall prevalence of disease was 3.1%. Only 15.0% of the sero-positive dogs had clinical signs of CanL. A high degree of agreement (88.4%; kappa value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test for canine Leishmania infection in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of CanL.