Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Update on oral Chagas disease outbreaks in Venezuela: epidemiological, clinical and diagnostic approaches

Orally transmitted Chagas disease has become a matter of concern due to outbreaks reported in four Latin American countries. Although several mechanisms for orally transmitted Chagas disease transmission have been proposed, food and beverages contaminated with whole infected triatomines or their faeces, which contain metacyclic trypomastigotes of Trypanosoma cruzi, seems to be the primary vehicle. In 2007, the first recognised outbreak of orally transmitted Chagas disease occurred in Venezuela and largest recorded outbreak at that time. Since then, 10 outbreaks (four in Caracas) with 249 cases (73.5% children) and 4% mortality have occurred. The absence of contact with the vector and of traditional cutaneous and Romana’s signs, together with a florid spectrum of clinical manifestations during the acute phase, confuse the diagnosis of orally transmitted Chagas disease with other infectious diseases. The simultaneous detection of IgG and IgM by ELISA and the search for parasites in all individuals at risk have been valuable diagnostic tools for detecting acute cases. Follow-up studies regarding the microepidemics primarily affecting children has resulted in 70% infection persistence six years after anti-parasitic treatment. Panstrongylus geniculatushas been the incriminating vector in most cases. As a food-borne disease, this entity requires epidemiological, clinical, diagnostic and therapeutic approaches that differ from those approaches used for traditional direct or cutaneous vector transmission.

Connections: can the 20th century coronary heart disease epidemic reveal something about the 1918 influenza lethality?

This essay proposes that the ecologic association shown between the 20th century coronary heart disease epidemic and the 1918 influenza pandemic could shed light on the mechanism associated with the high lethality of the latter. It suggests that an autoimmune interference at the apoB-LDL interface could explain both hypercholesterolemia and inflammation (through interference with the cellular metabolism of arachidonic acid). Autoimmune inflammation, then, would explain the 1950s-60s acute coronary events (coronary thrombosis upon influenza re-infection) and the respiratory failure seen among young adults in 1918. This hypothesis also argues that the lethality of the 1918 pandemic may have not depended so much on the 1918 virus as on an immune vulnerability to it, possibly resulting from an earlier priming of cohorts born around 1890 by the 1890 influenza pandemic virus.

Aedes albopictus may not be vector of dengue virus in human epidemics in Brazil

Over 60,500 dengue cases were reported in the state of Espírito Santo (ES), Brazil, between 1995 and 1998. The study's purpose was to identify whether Aedes albopictus was transmitting the dengue virus during an epidemic in the locality of Vila Bethânia (Viana County),Vitória, ES. From April 3 to 9, 1998, blood and serum samples were collected daily for virus isolation and serological testing. Four autochthonous cases were confirmed through DEN 1 virus isolation and two autochthonous cases through MAC ELISA testing. Of 37 Ae. aegypti and 200 Ae. albopictus adult mosquitoes collected and inoculated, DEN1 virus was isolated only from a pool of two Ae. aegypti female mosquitoes. The study results suggest that Ae. albopictus still cannot be considered an inter-human vector in dengue epidemics in Brazil.

Dengue and the risk of urban yellow fever reintroduction in São Paulo State, Brazil

OBJECTIVE: To propose a mathematical method for the estimation of the Basic Reproduction Number, R0, of urban yellow fever in a dengue-infested area. METHODS: The method is based on the assumption that, as the same vector (Aedes aegypti) causes both infections, all the quantities related to the mosquito, estimated from the initial phase of dengue epidemic, could be applied to yellow fever dynamics. It is demonstrated that R0 for yellow fever is, on average, 43% lower than that for dengue. This difference is due to the longer dengue viremia and its shorter extrinsic incubation period. RESULTS: In this study the analysis was expanded to the epidemiological situation of dengue in São Paulo in the year 2001. The total number of dengue cases increased from 3,582 in 2000 to 51,348 in 2001. It was then calculated R0 for yellow fever for every city which have shown R0 of dengue greater than 1. It was also estimated the total number of unprotected people living in highly risky areas for urban yellow fever. CONCLUSIONS: Currently there is a great number of non-vaccinated people living in Aedes aegypti infested area in the state of São Paulo.

Outbreak of persistent cutaneous abscesses due to Mycobacterium chelonae after mesotherapy sessions, Lima, Peru

Outbreaks of rapidly growing mycobacteria have been occasionally described. The article reports an outbreak of cutaneous abscesses due to Mycobacterium chelonae following mesotherapy in Lima, Peru. From December 2004 through January 2005, 35 subjects who had participated in mesotherapy training sessions presented with persistent cutaneous abscesses. Thirteen (37%) of these suspected cases consented to underwent clinical examination. Skin punch-biopsies were collected from suspicious lesions and substances injected during mesotherapy were analyzed. Suspected cases were mainly young women and lesions included subcutaneous nodules, abscesses and ulcers. Mycobacterium chelonae was isolated from four patients and from a procaine vial. In conclusion, it is important to consider mesotherapy as a potential source of rapidly growing mycobacteria infections.

Imported and autochthonous cases in the dynamics of dengue epidemics in Brazil

OBJECTIVE: To estimate the basic reproduction number (R0) of dengue fever including both imported and autochthonous cases. METHODS: The study was conducted based on epidemiological data of the 2003 dengue epidemic in Brasília, Brazil. The basic reproduction number is estimated from the epidemic curve, fitting linearly the increase of initial cases. Aiming at simulating an epidemic with both autochthonous and imported cases, a "susceptible-infectious-resistant" compartmental model was designed, in which the imported cases were considered as an external forcing. The ratio between R0 of imported versus autochthonous cases was used as an estimator of real R0. RESULTS: The comparison of both reproduction numbers (only autochthonous versus all cases) showed that considering all cases as autochthonous yielded a R0 above one, although the real R0 was below one. The same results were seen when the method was applied on simulated epidemics with fixed R0. This method was also compared to some previous proposed methods by other authors and showed that the latter underestimated R0 values. CONCLUSIONS: It was shown that the inclusion of both imported and autochthonous cases is crucial for the modeling of the epidemic dynamics, and thus provides critical information for decision makers in charge of prevention and control of this disease.

An outbreak of bat-transmitted human rabies in a village in the Brazilian Amazon

During 45 days without electrical power, 57 individuals (8.7% of the population) from the village of Antônio Dino (municipality of Turiaçu, Northeastern Brazil) were attacked by bats and 16 died from human rabies. The aim of the study was to analyze the factors associated with bat attacks and the development of human rabies. Of the 46 individuals, who suffered bat attacks, 36 (78.3%) were under 17 years of age. The risk factors associated with bat attacks were age under 17 years, having observed bats inside the bedroom and having been without electrical power in the house. Age under 17 years and having been without electrical power in the house were factors associated with human rabies.

Yellow fever in Brazil: thoughts and hypotheses on the emergence in previously free areas

This article describes and discusses factors associated to the reemergence of yellow fever and its transmission dynamics in the states of São Paulo (Southeastern Brazil) and Rio Grande do Sul (Southern) during 2008 and 2009. The following factors have played a pivotal role for the reemergence of yellow fever in these areas: large susceptible human population; high prevalence of vectors and primary hosts (non-human primates); favorable climate conditions, especially increased rainfall; emergence of a new genetic lineage; and circulation of people and/or monkeys infected by virus. There is a need for an effective surveillance program to prevent the reemergence of yellow fever in other Brazilian states.

Chikungunya risk for Brazil

This study aimed to show, based on the literature on the subject, the potential for dispersal and establishment of the chikungunya virus in Brazil. The chikungunya virus, a Togaviridae member of the genusAlphavirus, reached the Americas in 2013 and, the following year, more than a million cases were reported. In Brazil, indigenous transmission was registered in Amapa and Bahia States, even during the period of low rainfall, exposing the whole country to the risk of virus spreading. Brazil is historically infested by Ae. aegypti and Ae. albopictus, also dengue vectors. Chikungunya may spread, and it is important to take measures to prevent the virus from becoming endemic in the country. Adequate care for patients with chikungunya fever requires training general practitioners, rheumatologists, nurses, and experts in laboratory diagnosis. Up to November 2014, more than 1,000 cases of the virus were reported in Brazil. There is a need for experimental studies in animal models to understand the dynamics of infection and the pathogenesis as well as to identify pathophysiological mechanisms that may contribute to identifying effective drugs against the virus. Clinical trials are needed to identify the causal relationship between the virus and serious injuries observed in different organs and joints. In the absence of vaccines or effective drugs against the virus, currently the only way to prevent the disease is vector control, which will also reduce the number of cases of dengue fever.

Successful prevention of the transmission of vancomycin-resistant enterococci in a Brazilian public teaching hospital

INTRODUCTION: Vancomycin-resistant enterococci (VRE) can colonize or cause infections in high-risk patients and contaminate the environment. Our objective was to describe theepidemiological investigation of an outbreak of VRE, the interventions made, and their impact on its control. METHODS: We conducted a retrospective, descriptive, non-comparative study by reviewing the charts of patients with a VRE-positive culture in the University Hospital of Campinas State University, comprising 380 beds, 40 of which were in intensive care units (ICUs), who were admitted from February 2008-January 2009. Interventions were divided into educational activity, reviewing the workflow processes, engineering measures, and administrative procedures. RESULTS: There were 150 patients, 139 (92.7%) colonized and 11 (7.3%) infected. Seventy-three percent were cared for in non-ICUs (p = 0.028). Infection was more frequent in patients with a central-line (p = 0.043), mechanical ventilation (p = 0.013), urinary catheter (p = 0.049), or surgical drain (p = 0.049). Vancomycin, metronidazole, ciprofloxacin, and third-generation cephalosporin were previously used by 47 (31.3%), 31 (20.7%), 24 (16%), and 24 (16%) patients, respectively. Death was more frequent in infected (73%) than in colonized (17%) patients (p < 0.001). After the interventions, the attack rate fell from 1.49 to 0.33 (p < 0.001). CONCLUSIONS: Classical risk factors for VRE colonization or infection, e.g., being cared for in an ICU and previous use of vancomycin, were not found in this study. The conjunction of an educational program, strict adhesion to contact precautions, and reinforcement of environmental cleaning were able to prevent the dissemination of VRE.

Bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses?

An increasingly asked question is 'can we confidently link bats with emerging viruses?'. No, or not yet, is the qualified answer based on the evidence available. Although more than 200 viruses - some of them deadly zoonotic viruses - have been isolated from or otherwise detected in bats, the supposed connections between bats, bat viruses and human diseases have been raised more on speculation than on evidence supporting their direct or indirect roles in the epidemiology of diseases (except for rabies). However, we are convinced that the evidence points in that direction and that at some point it will be proved that bats are competent hosts for at least a few zoonotic viruses. In this review, we cover aspects of bat biology, ecology and evolution that might be relevant in medical investigations and we provide a historical synthesis of some disease outbreaks causally linked to bats. We provide evolutionary-based hypotheses to tentatively explain the viral transmission route through mammalian intermediate hosts and to explain the geographic concentration of most outbreaks, but both are no more than speculations that still require formal assessment.

Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines

Psittaciformes are one of the most endangered groups of birds, and several Brazilian species are classified between vulnerable and critically endangered. It is thus necessary to identify agents that cause infections in captive wild animals and to assess the risks posed thereof and to design interventions to minimize the possibility of disease outbreaks, leading to the conservation of endangered species. The purpose of this study was to identify enteropathogenic Escherichia coli (EPEC) cloacal isolates from asymptomatic psittacines in captivity and evaluate the distribution of the EPEC pathotype. Cloacal swabs were obtained from 46 asymptomatic birds, and resulting isolates were tested by polymerase chain reaction (PCR) for the presence of the attaching and effacing gene (eae) and bundle-forming pilus structural gene (bfpA) of EPEC. Samples from several species were tested, and three samples were found to be positive for the eae and bfpA genes and characterized as typical EPEC. This is the first report of this pathotype in asymptomatic psittacines. Although certain E. coli strains are more pathogenic than others, various factors should be considered when determining the potential of E. coli isolates to cause disease in captive psittacines. Birds that are positive for the EPEC (typical) strain could be zoonotic sources of infection, and may have acquired these strains through contact with humans or domestic animals. These findings may also be valuable for the long-term management of endangered species ex situ as one EPEC sample was isolated from a Red-tailed Amazon (Amazona brasiliensis).

Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012)

Cases of vesicular and exanthematic disease by Vaccinia virus (VACV) have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goiás state, Midwestern Brazil (2010-2012), involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old) were affected in all cases and calves (2 to 9 months old) were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm), painful and coalescent papules (3-8 mm), which resulted in ulcers (5-25mm) and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to vgf gene of Orthopoxvirus was positive in samples collected from all outbreaks, and in some cases, genomic VACV sequences were identified by nucleotide sequencing of the PCR amplicons. Infectious virus was isolated in cell culture from scabs from one outbreak. Antibodies to Orthopoxvirus were detected in at least 3 or 4 animals in most outbreaks, by ELISA (outbreaks 1, 2, 3, 4, 5 and 7) or virus-neutralization (outbreak 6). Neutralizing titers ranging from 8 to 64 in outbreak 6. In all outbreaks, VACV infection was suspected based on the clinical and pathological findings and it was confirmed by laboratory tests. Upon the etiological confirmation, other agents associated with vesicular disease were discarded. In all outbreaks, at least one milker who handled the affected cows developed malaise, headache, fever, painful vesico-pustular lesions mainly in the hands, but also in the neck and nose. These results confirm the circulation of VACV in the region and call attention for a correct diagnosis and the adoption of prophylactic and control measures.

Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil

Abstract: Canid herpesvirus 1 (CHV-1) is a widespread pathogen of dogs and produces infertility, abortions and severe systemic disease in young puppies. Clinical data indicate the circulation of CHV-1 among Brazilian dogs yet definitive diagnosis has rarely been accomplished. This article describes the clinicopathological findings of four independent cases/outbreaks of neonatal disease by CHV-1 in Bulldog puppies followed by virus identification and genetic characterization. Three events occurred in a kennel holding dogs of different breeds at reproductive age (March 2013, October 2013 and April 2014). Puppies from three French or English Bulldog litters, aging 9 to 30 days were affected, presenting dyspnea, agonic breathing, pale mucous, abdominal pain and tension, evolving to death within about 24 hours. At necropsy, the puppies presented necrohemorrhagic hepatitis, multifocal and moderate necrohemorrhagic nephritis and fibrinonecrotic interstitial pneumonia. Virus isolation was positive in clinical specimens from one litter and CHV-1 DNA was detected by PCR in tissues from all four cases. Virus-neutralizing assays with samples of the affected kennel revealed 9/12 adult animals with high antibody titers to CHV-1. Nucleotide sequencing of glycoprotein B, C and D genes revealed 99-100% of identity among the viruses and with CHV-1 sequences available in GenBank. Phylogenetic analyses of gC sequences showed a segregation of the samples, even among three isolates from the same kennel. These findings support CHV-1 infection as the cause of disease and death in these dog litters, reinforcing the need for correct etiologic diagnosis, prevention and immunization against CHV-1 in dogs from Southern Brazil.