A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study

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1 International Journal of Clinical Medicine Research 2016; 3(1): Published online March 4, 2016 ( ISSN: A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study Nestor Anez 1, *, Gladys Crisante 1, Agustina Rojas 1, Rafael O. Rojas 2, Javier Bastidas 3 1 Center for Parasitological Research J. F. Torrealba, Department of Biology, Faculty of Sciences, University of Los Andes, Merida, Venezuela 2 Regional Epidemiology Office, Health Corporation, Ministry of Health, Merida, Venezuela 3 Rural Endemic Division, Ministry of Health, Merida, Venezuela Keywords Chagas Disease, Trypanosoma cruzi, Oral Transmission, Venezuela Received: February 19, 2016 Revised: February 27, 2016 Accepted: February 29, address nanes@ula.ve (N. Anez) Citation Nestor Anez, Gladys Crisante, Agustina Rojas, Rafael O. Rojas, Javier Bastidas. A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study. International Journal of Clinical Medicine Research. Vol. 3, No. 1, 2016, pp Abstract A massive, simultaneous and severe acute Chagas disease micro-epidemic outbreak associated with Trypanosoma cruzi oral transmission, detected in a rural locality of Merida, Venezuela, is reported. The presence of similar severe clinical profiles, massive active parasitemia, specific anti-t. cruzi IgM and infection by the same genetic T. cruzi genotype (DTU I) detected in nine patients involved in the outbreak, together with the absence of Romaña s sign, strongly indicate simultaneous infections by ingesting T. cruzi-contaminated food. The analysis of the clinical profiles detected in the acute chagasic patients revealed a total of 18 symptoms with an average per patient of 9±4 (range: 5-16) symptoms with 44% lethality. In the four fatal cases hepatomegaly was present (100%) and myocarditis, pericardial effusion and cardiomegaly were respectively detected in 75%, 50% and 25% of them. In all cases, similar values of anti-t. cruzi antibodies, amount of blood circulating trypomastigotes and same DNA bands obtained by combination of sero-parasitological and molecular (Polymerase Chain Reaction-PCR) assays revealed patients with a similar clinical history, which support the oral mode of infection. Histopathological observations revealed the presence of large number of T. cruzi-tissue forms and massive lymphomonocytic infiltration in samples from heart, liver, spleen, skeletal muscle and tongue of a fatal case, which demonstrated the severity of the parasite circulating in the studied area. The epidemiological significance of the findings is discussed, and the alert to clinicians and population on the orally transmitted Chagas disease is provided. 1. Introduction The nowadays ample knowledge on the typical vectorial Trypanosoma cruzi transmission by triatomine bugs seems to have contributed in underestimating the oral route of Chagas disease infection, which is perhaps the most ancient and efficient mode of infection in nature amongst wild animals. This fact, among others, appears to be supported by the cannibalism frequently occurring between animals acting as T. cruzi reservoirs at enzootic foci, a behavior revealing this primitive mode of transmission which could have occurred prior to the association of the parasite with the blood sucking triatomine-bugs [1, 2, 3, 4]. The oral route of transmission has been experimentally demonstrated infecting specimens of opossums after been fed with rodents bearing

2 30 Nestor Anez et al.: A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study T. cruzi-infection [5]. Regarding efficiency, oral transmission provides a better explanation for the infection of predators than that of skin contamination with bugs feces of the often dense animal furs [6]. In addition, ingestion of infected insects, a common practice observed in wild and domestic animals, increases the possibility of getting T. cruzi oral infection after ingesting triatomine-bugs with full parasitic charge instead of an aliquot of parasites deposited on the skin [7, 8, 9, 10]. Contamination of food with T. cruzi metacyclic forms from the anal glands of the opossum Didelphis marsupialis or from the rectal ampoule of Rhodnius prolixus have provided experimental evidence on the efficacy of the oral route to infect laboratory mice [11, 12]. In relation to orally acquired human infection, first Chagas disease cases were described in Argentina by Mazza [13] who reported acute symptoms in persons after ingesting either a local medicine prepared by witch-doctors, consisting of beverages made up of herbs mixed with opossum s blood, or uncooked meat from wild animals. This mode of transmission was later confirmed in a higher number of patients not acquainted with triatomine-bugs or that did not receive blood transfusions, but referred frequent ingestion of meat from hunted wild animals prepared according to regional costumes [14]. Despite the elapsed time from the recognition of Chagas disease oral transmission in the Latin- American subcontinent, it is only during the last decade when scientific information has increased showing results demonstrating the appearance of new cases of T. cruziinfection due to contamination of food with infective forms from triatomine-bugs or fluids from reservoirs of the parasite [15, 16]. This includes the most recent outbreaks from Brazil, Colombia and Venezuela, being the former of more frequent appearance and the latter where the larger number of patients has been detected [15, 17]. The present work deals with the detection of an outbreak occurred in a rural village at Merida, Western Venezuela, affecting a family group resulting in four fatal cases. Considering the intense outbreak producing a Chagas disease familiar micro-epidemic associated with oral ingestion of contaminated food, a comprehensive study was carried out using sero-parasitological, histopathological, molecular (Polymerase Chain Reaction-PCR) and epidemiological tools, looking for an explanation for the aggressive behavior of the circulating T. cruzi local genotype and its link with the observed severe clinical conditions. 2. Patients and Methods 2.1. Characteristic of the Study Area and Origin of Chagasic Patients The study patients came from the village La Macarena, located at the Nueva Bolivia parish, municipality Tulio Febres Cordero, Merida state, Western Venezuela. The locality is a relatively new small rural town made up of farm workers, transport workers and other people doing different agricultural activities in and outside the town. It is located besides the so called Pan-American road, one of the main western motor ways connecting with most part of the country. The village is located at an altitude of 71 m.a.s.l, with an average temperature of 28 C, a relative humidity >80% and at N and W. A total of nine people were involved in the outbreak, seven from a family group and two neighbors. They were two children, 2 and 6 year old sisters, and 7 adults (5 males and 2 females). The group showed an average age of 25.7±15 years (range 2-58 year old). The relation of the family members was as follows: case coded mother of case (fatal), grandmother of sisters and (fatal cases) and mother in law of cases and The case was a relative of (cousin) and and (fatal) neighbors living in close proximity. Seven out of the 9 patients involved in the outbreak were evaluated, for clinical diagnosis, at the emergency unit of Los Andes University General Hospital, Merida, Venezuela after they were referred from the rural medical service located near the locality where the outbreak occurred Parasitological Diagnosis Blood samples from 5 of the 9 studied patients were received in our laboratory from the above referred General Hospital. Samples were microscopically examined to detect blood forms of Trypanosoma cruzi. When fresh samples showed flagellates, 5µL of blood were placed on a glass slide, smeared, fixed with methanol and stained with 10% Giemsa in phosphate buffer. Both fresh and stained samples were examined under an Axioscop microscope (Zeiss- Germany) linked to a 480 Noticam camera connected to a Laptop-HP computer to record activity and morphology of the parasites, respectively. When parasites were seen in each of the identified sample, 0.5 ml of infected blood was placed into culture tubes containing NNN biphasic medium. The hemocultures were observed every 5 days until observing flagellates in enough amounts to be transferred into new culture media in order to amplify the parasite population and stabilize the isolate under laboratory conditions for further work. To assess parasite infectivity, an aliquot of blood from each of the infected patients was intradermically inoculated into healthy mice, which were periodically examined to detect circulating T. cruzi blood forms Serological Diagnosis Serological tests used to detect anti-t.cruzi circulating antibodies (Ab) in sera samples from seven of the nine patients involved in the acute Chagas disease outbreak, included a direct agglutination test (DAT) and an indirect immunofluorescence antibody test (IFAT), for both polyvalent and specific IgM/IgG subtypes as described previously [18, 19]. As serological criterion to consider a sample as seropositive, reactivity in both DAT and IFAT serological methods must be coincident.

3 International Journal of Clinical Medicine Research 2016; 3(1): Histopathological Diagnosis In one of the fatal cases (01-15) necropsy was carried out at the pathological anatomy unit of the hospital referred above and samples from heart, skeletal muscle, liver, spleen and tongue were sent to our laboratory to be prepared for histopathological diagnosis. The tissue samples were fixed with 10% formaldehyde before embedding in paraffin wax. Serial sections of 7 µm thickness were cut and then stained by Giemsa stain looking for T. cruzi tissue forms following protocols previously reported [18, 19, 20] Molecular Diagnosis To corroborate the morphological identification of the flagellates obtained from active parasitemia of the infected patients, blood samples were used to carry out a specific PCR assay to demonstrate the presence of T. cruzi-dna. The assay was performed using primers S35 (5 - AAA TAA TGT ACG GGT GAG ATG CAT GA-3 ) and S36 (5 -GGG TTC GAT TGG GGT TGG TGT-3 ) following protocols previously established [21]. Samples of T. cruzi-dna previously identified were typed through PCR amplification using primers D71 (5 -AAG GTG CGT CGA CAG TGT GG-3 ) and D72 (5 - TTT TCA GAA TGG CCG AAC AGT- 3 ) designed from the divergent domain of the 24SαRNA gene, and primers TC (5 - CCC CCC TCC CAG GCC ACA CTG-3 ), TC1 (5 - GTG TCC GCC ACC TCC TTC GGG CC-3 ) and TC2 (5 - CCT GCA GGC ACA CGT GTG TGT G-3 ) for amplification of an intergenic region of the miniexon gene described by Souto et al [22]. In addition, part of the tissue samples referred above was processed for PCR to demonstrate the presence of T. cruzi- DNA as indicated above. In all cases, the T. cruzi-dna both from the blood samples and from the tissue biopsies, obtained after necropsy of the patient 01-15, was extracted by the classical phenol-chloroform method, and the PCR amplified product was separated by electrophoresis in 3% agarose gels, and stained with ethidium bromide [19] Epidemiological Survey After confirming the acute oral Chagas disease outbreak, a serological study was carried out in 110 individuals to check out seroprevalence in inhabitants living under the same risk conditions of the infected patients. The study group was made up of 58 females (53%) and 52 males (47%) with a female: male ratio of 1.1 and an average age of 28±22 years (range years). In the included individuals, samples consisted of 5 ml peripheral blood obtained by venipuncture for routine serological testing. The serological tests used to detect circulating anti-t.cruzi antibodies were the same indicated above for serological diagnosis of the patients involved in the acute outbreak. In this case, diagnostic criteria to consider a sampled individual as seropositive were the same used in previous diagnosis, i.e. both DAT and IFAT tests showing Ab-titers 64 [19, 23]. In general, epidemiological characteristics observed in the locality included rural houses in good conditions and inhabitants having good standard of living. Domestic animals were present, predominating dogs which freely circulate all around. Triatomines bugs were present in some domestic environments. As cultural behavior families use to celebrate frequent week end parties sharing food and drinks prepared in mass to enjoy together. This part of the study was under the supervision of the Rural Endemic Division and the Regional epidemiology Office, both of the Venezuelan Ministry of Health, which were in charge of the epidemiological conditions and risk factors evaluation in the study locality, assuming all responsibility for ethical procedures Ethical Considerations The study involving blood and tissue samples from chagasic patients followed the conditions by the ethical committee established at the infectology and cardiological units of Los Andes University General Hospital, Merida, Venezuela. 3. Results 3.1. Clinical Findings According to the housewife testimony as head of the family involved in the acute Chagas disease outbreak, most clinical signs and symptoms appeared almost simultaneously in the members of the family, being 5 days the difference between the first and last showing initial symptoms. All of them began with fever not responsive to antibiotic or antipyretic treatment, and headache not responsive to analgesic therapy. After consulting at the local rural medical service, they were clinically examined and informed on a possible virus infection (dengue) or suffering Leptospirosis. However, they were kept without specific diagnosis and/or treatment for more than a week. At day 19 after general symptoms started, patient 00-15, a 2 year old girl, was referred to the Valera General Hospital, Trujillo state, where she suddenly died. The rest of symptomatic patients were interned at Los Andes University General Hospital, Merida for diagnosis and treatment. Due to the severity of the infection, patients and died soon after they were diagnosed. Similarly, patient died at home because he refused to be sampled for diagnosis or be referred to the hospital. In summary, 4 out of 9 (44%) patients died as consequence of the acute infection. The analysis of the clinical profiles of the 9 acute chagasic patients, revealed a total of 18 signs or symptoms with an average per patient of 9±4 (range: 5-16) symptoms, with 3 of them present in all the patients, including fever, headache and myalgia. The other symptoms were detected in 11% to 78% of patients. It is interesting to note that hepatomegaly was present in all the fatal cases and in a survival patient (06-15) showing severe clinical condition. Similarly, myocarditis was revealed in 3 out of 4 fatal cases (75%) and pericardial effusion detected by echocardiography in 2 of them (50%). The major number of symptoms was 16 in a fatal case (02-

4 32 Nestor Anez et al.: A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study 15), and 14 in a patient who survived after suffering severe clinical condition (06-15). In no cases neither Romaña s sign (bipalpebral edema) nor chagoma, an edema at the point of parasite penetration, frequently observed after vectorial transmission, was detected. The five survivor patients after receiving treatment and recovering physical conditions left Table 1. Signs and symptoms detected in orally infected acute chagasic patients. the hospital and went home, and soon after they went back to normal life. Complete information on clinical profiles, including specific signs and symptoms detected in each of the acute patients orally infected with T. cruzi, is shown in Table 1. Signs and Symptoms PATIENTS CODE Total Nº (%) * Fever (100) Feverish cold (shivering) (78) Convulsion + 1 (11) Headache (100) Myalgia (100) Artralgia (78) Cough (33) Vomit (33) Diarrhea (22) Edema (44) Disnea (33) Hepatomegaly (56) Abdominal pain (56) Abdominal inflammation (33) Tachycardia (33) Myocarditis (33) Pericardial effusion (22) Cardiomegaly + 1 (11) Total Signs/symptoms Final condition Fatal case *: Papule in tongue border and stiffen neck Fatal case Fatal case Treated Treated Treated Treated Treated Table 2. Sero-parasitological and molecular (PCR) findings and genetic typing of Trypanosoma cruzi in acute chagasic patients from an oral outbreak in Merida, Venezuela. CODE Parasitological AGE Serological Diagnosis PCR Assay Clinical Gender Diagnosis (Years) Condition DAT IFAT (poly*) IFAT (IgM) IFAT (IgG) BCP HC ILA Blood DTU F ND ND Fatal case** F 1:512 1:1024 1:512 - ND ND ND ND ND Fatal case M 1:4096 1:1024 1:512 - ND ND ND ND ND Fatal case M 1:64 1:256-1: ND Mild symptoms F 1:128 1:512-1: DTU I Mild symptoms F 1:256 1:1024 1:64 1: DTU I Mild symptoms M 1:64 1:512 1:512 1: DTU I Severe Sympt M 1:64 1:512 1:512 1: DTU I Mild symptoms M ND ND Fatal case*** *: Polyvalent; BCP: Blood circulating parasites; HC: Hemoculture; ILA: Inoculation in laboratory animals. **: Died at Valera General Hospital Trujillo State; ***: Dead at home, refused to be examined for diagnosis DAT, IFAT, PCR, DTU: Explanation in the text; ND: not done Fatal case 3.2. Sero-Parasitological and Molecular Findings Serological results obtained in samples from seven referred patients revealed in all of them the presence of circulating anti-t. cruzi antibodies. The coincidence of reactivity, detected in both DAT and IFAT as the used serological methods, fulfilled established criterion for seropositivity. Detection of specific anti-t.cruzi antibody titers revealed levels from 1: 64 to 1: 4096 for DAT and between 1: 256 and 1: 1024 for IFAT. In addition, high levels of IgM (1: 512) were detected in 4 out of the 7 patients, which indicate an evident acute profile. Details on the serological results obtained in each of the studied patients are presented in Table 2. Fresh blood samples microscopically examined from five patients involved in the acute Chagas disease outbreak, revealed the presence of circulating trypomastigotes in 4 of them (80%). The morphological identification of T. cruzi blood circulating forms was corroborated by detecting them in Giemsa stained blood smears (Fig. 1). Parasitological diagnosis was verified by observing parasite development growing in mass in NNN culture medium, and its infectivity in laboratory mice previously inoculated. Details of the

5 International Journal of Clinical Medicine Research 2016; 3(1): results recorded on the positive patients (04-15, 05-15, and 07-15) including presence of blood circulating parasites (BCP), detection of flagellates in hemoculture (HC) and infection in inoculated laboratory animals (ILA) are shown in Table The genetic typing of T. cruzi isolates from acute patients (04-07/15), carried out by PCR amplification of 24 α Sribosomal locus, showing bands of 110bp, and mini-exon genes (SL), showing bands of 350 bp, revealed the presence of DTU I (T. cruzi I genotype). Table 2 shows identification of DTU in each of the infected patients, giving details on its relation with sero-parasitological characteristics and clinical conditions of the patients suffering chagasic infection. In Fig. 3 characteristics of the amplification are given, comparing the present results with the reference strains for DTU I and DTU II. Fig. 1. Trypanosoma cruzi blood circulating trypomastigote detected in a sample stained by Giemsa (X 1000). In addition, the use of a PCR assay allowed the detection of specific T. cruzi-dna in blood samples from infected patients (04-15, 05-15, and 07-15) by the amplification of 330 bp bands from the kdna minicircle variable region of the parasite, which matched with T.cruzi-DNA used as reaction control. Details of the recorded results obtained with blood samples using a PCR assay to corroborate Chagas disease diagnosis are presented in Table 2 and Fig. 2. Fig. 2. PCR-assay in blood samples from an oral Chagas disease outbreak. Lane1: T.cruzi DNA from a control acute case. Lanes2-5: Patients , respectively. C+: T.cruzi DNA controls. C-: Negative control. MW: Molecular weight ladder. Fig. 3. Trypanosoma cruzi typing through 3% agarose gel electrophoresis of PCR products generated by amplification of mini-exon and ribosomal 24 Sα gene sequences of DNA from acute patients detected in an oral Chagas disease outbreak. DTU I Lanes 1-5 (2,3,4,5=patients 06, 04, 05, 07; 1=DNA control from an acute patient). G and Y: DNA from reference DTU I and DTU II, respectively as controls. MW: Molecular weight Histopathological and Molecular Findings Microscopic examination carried out in sectioned Giemsa stained samples from heart, skeletal muscle, liver, spleen and tongue obtained from a fatal case (01-15), revealed intracellular T. cruzi amastigotes pseudocysts in most of them. The size of pseudocysts and the number of intracellular amastigotes varied from a tissue to another, being the heart in which the biggest cysts were detected. In most tissues inflammatory infiltrate was detected with mononuclear predominance and lymphocyte abundance. Fig. 4 shows some selected samples including a heart s section with an evident T. cruzi amastigote pseudocyst (A) and inflammatory infiltrate (B), which is also observed in the liver (C). The molecular evaluation using PCR assay confirmed the presence of specific T. cruzi-dna in all the organs investigated for parasite invasion, supporting the results obtained with the histopathological methodology. The detection of T. cruzi-dna in all the studied organs was evidenced by the amplification of similar strong bands of 330 bp in each corresponding tissue as shown in Fig. 5.

6 34 Nestor Anez et al.: A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study Fig. 4. Giemsa stained sections of heart (A-B) and liver (C) from a Trypanosoma cruzi infected fatal case (01-15). Note the presence of a nest of parasite in A (arrowed) and severe lymphomonocytic infiltration (B-C). Magnification: 1000X evaluated members of the study community including age grouping and distribution of T. cruzi seropositivity is shown in Table 3. Although most people did not recognize clinical symptoms attributable to Chagas disease, 51% of them were able to identify triatomine bugs as risk factor for the infection transmission. Indeed, after searching around the houses specimens of triatomine bugs were found. This consisted of three specimens of Rhodnius prolixus, which after microscopically examined for parasite, showed T. cruzi metacyclic forms. 4. Discussion Fig. 5. Trypanosoma cruzi specific PCR assay from tissue samples of the patient a fatal case occurred in an acute Chagas disease outbreak in a rural locality of Merida, Venezuela. Lanes: H (heart), L (Liver), S (spleen), SM (skeletal muscle), T (tongue). C+: T. cruzi DNA controls. C-: Control of reaction. MW: Molecular weight. Table 3. Serological response to Trypanosoma cruzi in individuals living in close proximity of patients from an acute Chagas disease outbreak associated to oral transmission. Age Nº (%) Nº (%) Serological diagnosis group Individuals Infected DAT IFAT IgM IgG (25.4) 0 (00) (23.6) 1 (20) 1:2048 1:256 1: (13.6) 1 (20) 1:2048 1: (10.0) 0 (00) (8.2) 1 (20) 1:2048 1:128 1: (8.2) 0 (00) (5.5) 1 (20) 1:2048 1:1024 1:64 >70 6 (5.5) 1 (20) 1:2048 1:1024 1:16 1:32 Total 110 (100) 5 (4.5) 3.4. Epidemiologic Findings Serological analysis carried out in 110 individuals living in the village where the acute Chagas disease outbreak was detected, revealed positive results for anti-t.cruzi antibodies in 5 (4.5%) of them. This result was observed in healthy asymptomatic individuals with an average age of 48±25 year old (range: years). In all cases seropositives showed high titers, corresponding to dilution 1:2048 for direct agglutination test (DAT), and between 1:128 and 1:1024 for immunofluorescence antibody test (IFAT) when a polyvalent conjugate was used. However, detected values of anti-t. cruzi specific IgM and IgG were relatively low showing titers corresponding to dilutions from 1:16 to 1:64, which allowed considering them as bearing inapparent infections. Details on Acute Chagas disease outbreaks due to ingestion of T.cruzi-contaminated food have been reported with increasing frequency in some Latin-American countries. This includes recorded cases mainly from Brazil, Bolivia, Colombia and Venezuela where massive and severe fatal infections, affecting people from rural and urban, endemic and non-endemic areas, have been detected during the last decade [10, 15, 16, 17, 24, 25, 26, 27, 28, 29, 30]. The general characteristics of the outbreak reported here matched with those established by the Pan American Health Organization guidelines to define a confirmed acute orally transmitted Chagas disease case, following parasitological, serological, epidemiological and clinical criteria [27, 31]. Among these indicators, the absence of Romaña s sign, the presence of active parasitemia with the same T.cruzi genetic type, the specific anti-t. cruzi IgM in patients from the same cluster, besides the detection of simultaneous onset of acute symptoms in a family group with a common history, together with the observed severity of symptoms/high lethality and the findings of other risk factors at the village where the event occurred, seem to indicate that the here reported Chagas disease outbreak happened as a consequence of an oral ingestion of T. cruzi-infected food. In relation to the recorded clinical pattern detected in chagasic patients involved in the present outbreak, a total of 18 symptoms were recognized. These ranged from mild symptoms, as those commonly observed in infection caused by other etiologies, to severe manifestation which produced prostration and death in 44% (4/9) of the infected patients. The analysis revealed an average of 9 ± 4 symptoms per patient, ranging from 5 to 16 symptoms and showing 3 simultaneous symptoms in 100% of the studied patients including fever, headache and myalgia. In addition, two more symptoms were detected in 78% of the acute chagasic patients, including artralgia and shiver. Hepatomegaly and abdominal pain were recorded in more than half of the T.cruzi-infected patients (56%) who showed the more severe clinical patterns responsible for 4 deaths and one extremely severe case gravely affected and recovered after a long convalescent post treatment period. Regarding the unfortunate end patients, their course of infection revealed several heart failures including severe myocarditis in 3 of them (75%), pericardial effusion in two (50%) and

7 International Journal of Clinical Medicine Research 2016; 3(1): cardiomegaly in one (25%). The echocardiographic study also revealed left ventricular ejection fraction of 30% and concentric hypertrophy without signs of cardial plug up. Tachycardia was detected in 3 patients (33%), including a fatal case and two survival patients showing one of them severe symptoms and the other mild symptoms. Other symptoms as leg edema, disnea, diarrhea, vomit, cough and convulsion were also detected between 11 to 44% of the infected patients. The described clinical profiles for patients involved in the present outbreak where 18 symptoms were detected, resemble those previously reported for a microepidemic oral acute Chagas disease also occurred in Merida, Venezuela [10]. Apart from the presence of an internal facial swollen with paresthesia in the tongue detected in the mentioned previous outbreak, which were not recorded in the present one, most symptoms as well as the course of infection and epidemiological testimonies resulted quite similar in the two events. Studies on orally transmitted T. cruzi producing acute outbreaks of Chagas disease have been reported in other Latin American countries, showing a frequency of appearance and severity of symptoms similar as those described in the present report. In fact, characteristics of clinical patterns reported from outbreaks detected in Brazil [25, 27, 32, 33, 34], Colombia [16, 29, 35], Bolivia [28] and Venezuela [10, 17] showed great similarity among them, especially considering that contamination probably occurred through food and/or drinks inadequately stored, which became contaminated by feces of T. cruzi-infected bugs or fluids from wild animals. To corroborate the origin of the clinical features observed in the study patients, the use of a multifarious methodology unquestionably demonstrated the presence of T. cruzi as the etiological agent responsible for the observed damage. Indeed, the accomplished parasitological, serological, molecular (PCR assay) and histopathological methods allowed demonstrating that the massive and simultaneous infection detected in the patients involved in the outbreak had the same etiological origin. In addition, the similar clinical characteristics observed in the patients, same time of appearance of symptoms, similar aggressive behaviour of the parasite and almost same time when deaths occurred, reinforce that infection was due to ingestion of food/drink contaminated with infective metacyclic forms of T. cruzi. Similarly, the observed response with coincident seropositivity, detecting among patients almost the same values of anti-t. cruzi antibodies both using DAT and IFAT serological techniques and the similar titers of anti-igm in the examined samples, including those belonging to fatal cases, support the possibility of a simultaneous infection as claimed above. Besides the serological findings, the use of parasitological methods allowed the demonstration of T. cruzi blood circulating trypomastigotes in 4 out of 5 samples examined from symptomatic patients involved in the outbreak. The amount of BCP detected by microscopic examination including fresh samples and Giemsa s stained blood smears allowed the establishment of isolates in culture medium, whose infectivity was also demonstrated after inoculating healthy mice. This fact unequivocally corroborated that the acute outbreak was caused by T. cruzi and that the isolates obtained from different infected patients showed a similar behaviour both in vitro and in inoculated laboratory animals. Indeed, the way how the parasites were easily microscopically detected, together with its successful adaptation to artificial condition in culture medium and the rapid infection produced in the experimental hosts, suggest the presence of massive infections in the observed samples and should explain the number of symptoms and its severity/lethality to the patients during the course of the acute infection. In order to add more scientific evidence on the identification of the parasite isolated from patients suffering the acute outbreak, PCR assays were performed in all the obtained isolates. These findings not only corroborated the microscopic results identifying T.cruzi, but also showed concordance in all the isolates which matched with the T. cruzi-dna used as control. In addition, the genetic typing performed in all the isolates from the patients revealed the circulation of T. cruzi-dtu I in the locality where the outbreak occurred. This fact, also allowed recognizing this T. cruzi-dtu associated with the appearance of severe acute Chagas disease familiar micro-epidemic causing death in 44% of the infected people. These findings support previous reports on the detection of T. cruzi-dtu I in a large number of acute chagasic patients infected by vectorial transmission in other localities of western Venezuela where this genotype is predominant in the parasite population [36]. Moreover, the detection of T. cruzi-dtu-i allied to severe symptoms and death of patients infected by this genotype, as reported here, did not agree with its innocuousness as previously claimed by various authors [37, 38, 39]. Although the aim of the present paper is not going into deeply considering the here recorded T. cruzi DTU I-clinical profile association, it may serve to alert clinicians on the possibility to find out Chagas disease lethal cases in any latitude where this parasite circulates, supporting previous opinions [36, 40]. Other considerations regarding the T. cruzi DTUs and its relationship with defined patterns will be discussed elsewhere (Crisante, personal communication). To accomplish observations concerning the effect of T. cruzi-infection on patients involved in the present outbreak, the combination of histopathological and molecular (PCR) methods in samples from a fatal case, showed the massive invasion of the parasite to different tissues, which undoubtedly contributed to produce prostration and death in 4 of the 9 infected patients during the familiar microepidemic. The enormous amount of parasites detected in all the examined organs appear to be the cause of the sudden deaths occurred in just 19 days after the first symptoms were recognized. The aggressive effect of the T. cruzi circulating in the area was demonstrated not only by the capacity of invasion to organs, but also by the severe lymphomonocytic infiltration and the strong T. cruzi-dna bands observed in heart, liver, skeletal muscle, spleen and tongue examined during the study.

8 36 Nestor Anez et al.: A New Acute Oral Chagas Disease Outbreak in Merida, Venezuela: A Comprehensive Study The present epidemiological findings appear to validate the occurrence of the here reported acute oral Chagas disease outbreak due to the presence of risk factors in the study area. Indeed, the finding of T. cruzi-infected triatomine bugs in environments around the houses, besides the large number of dogs wandering all around the village and the forest surrounding the locality, together with the detection of seropositivity to anti-t. cruzi antibodies in 5% of the study population, seem to be reasons enough to corroborate the above statement to consider the locality as endemic for Chagas disease where several mode of transmission should be acting to infect the human population. Finally, taking into consideration the outcome showed in this comprehensive study together with the increasing frequency of the orally transmitted acute Chagas disease outbreaks, the efficacy of the mode of transmission through ingestion of T. cruzi-contaminated food and the severe clinical features responsible for high lethality rate, make as mandatory to: i. Draw attention to rural population and clinicians, at local medical services, to keep aware on the potential risk for cluster T. cruzi-infection (micro-epidemic) appearance, at any time, in Chagas disease endemic areas; ii. Establish the need for a quick sero-parasitological diagnosis to detect T. cruzi-infection complemented with clinical evaluation of patients with persistent fever; iii. Refer patients to general hospitals for cardiovascular evaluation and perform differential or complemented diagnosis; iv. Establish a health education program tending to consider the orally acquired Chagas disease as a potential risk in rural and suburban areas. 5. Conclusion The combination of clinical, sero-parasitological, histopathological, molecular (PCR assay) and epidemiological methodologies performed during the present comprehensive study led to reach the conclusion of the occurrence of an orally transmitted acute Chagas disease micro-epidemic based on the following observations: i. Absence of Romaña s sign, common in vectorial transmission, or any other external point of T. cruzi entrance in the total studied patients; ii. Presence of simultaneous onset of Chagas disease acute symptoms in members of the same family with similar frequency, history and severity; iii. Massive detection of blood circulating parasites and similar T. cruzi genotype (DTU I) found in all the examined samples; iv. Detection of specific anti-t. cruzi IgM in all patients affected during the micro-epidemic event; v. High lethality (44%) with death occurring almost simultaneously. Acknowledgements We are indebted to Prof. G. Fermín for carefully review of the manuscript. We thank technical assistance of Miss Sonia Araujo. We also thank personnel from The Rural Endemic Division and the Regional Epidemiology Office, Ministry of Health, Merida, Venezuela for help received during field work. This work was supported by CDCHTA-ULA (Grants C AA (NA) and C B (GC). Support from the Administrative Vice-Chancellor of University of Los Andes, Merida, Venezuela, is grateful acknowledged. References [1] Pipkin AC. Transmission of Trypanosoma cruzi by arthropod vectors: anterior versus posterior route infection. Intern Rev Trop Med 1969; 3: [2] Diotaiuti L, Pereira AS, Loiola CF, Fernandes AJ, Schofield CJ, Dujardin JP, et al. Interrelation of sylvatic and domestic transmission of Trypanosoma cruzi in areas with and without domestic vectorial transmission in Minas Gerais, Brazil. 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