RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

The practical application of the agglutination test for the diagnosis of glanders.

Thesis (D.V.M.) - Cornell University, June 1907.

Comparative assessment of the gelatin particle agglutination test and an enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis

The performances of the gelatin particle agglutination test (GPAT) and enzyme-linked immunosorbent assay (ELISA) for the diagnosis of strongyloidiasis with reference to the results of the agar plate culture technique (APCT) were evaluated with samples from 459 individuals from communities in northeast Thailand where strongyloidiasis is endemic. The prevalence of strongyloidiasis in five sample groups determined by GPAT varied between 29.3 and 61.5% (mean, 38.8%). ELISA and APCT, employed concurrently, gave lower prevalence rates of 27.5% (range, 21.6 to 42.1%) and 22.7% (range, 12.7 to 53.8%), respectively. By using APCT as the standard method, the sensitivity of GPAT was generally higher than that of ELISA (81 versus 73%). The specificity of GPAT was slightly lower than that of ELISA (74 versus 86%). The resulting GPAT titers exhibited positive linear relationships with the ELISA values (optical density at 490 nm) (P < 0.05), which suggests that the GPAT titer also reflects the levels of specific antibody comparable to those reflected by the ELISA values. Based on the relative ease and simplicity of use of the technique as well as the acceptable rates of sensitivity and specificity of the test, GPAT is more practical for screening for strongyloidiasis than the conventional ELISA.

Serological Survey for Antibodies to Encephalitozoon cuniculi in Ownerless Dogs From Urban Areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and IS (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.

Diagnosing visceral leishmaniasis and HIV/AIDS co-infection: a case series study in Pernambuco, Brazil

HIV/AIDS-associated visceral leishmaniasis may display the characteristics of an aggressive disease or without specific symptoms at all, thus making diagnosis difficult. The present study describes the results of diagnostic tests applied to a series of suspected VL cases in HIV-infected/AIDS patients admitted in referral hospitals in Pernambuco, Brazil. From a total of 14 eligible patients with cytopenias and/or fever of an unknown etiology, and indication of bone marrow aspirate, 10 patients were selected for inclusion in the study. Diagnosis was confirmed by the following examinations: Leishmania detection in bone marrow aspirate, direct agglutination test, indirect immunofluorescence, rK39 dipstick test, polymerase chain reaction and latex agglutination test. Five out of the ten patients were diagnosed with co-infection. A positive direct agglutination test was recorded for all five co-infected patients, the Leishmania detection and latex agglutination tests were positive in four patients, the rK39 dipstick test in three, the indirect immunofluorescence in two and a positive polymerase chain reaction was recorded for one patient. This series of cases was the first to be conducted in Brazil using this set of tests in order to detect co-infection. However, no consensus has thus far been reached regarding the most appropriate examination for the screening and monitoring of this group of patients.

Acceptance and potential barriers to effective use of diagnostic tests for visceral leishmaniasis in an urban area in Brazil

Abstract: INTRODUCTION: Acceptance of the IT LEISH(r) and direct agglutination test- made in the Laboratório de Pesquisas Clínicas (DAT-LPC) by healthcare professionals and patients suspected of visceral leishmaniasis (VL) in Ribeirão das Neves was evaluated. METHODS: Ninety-two patients and 47 professionals completed three questionnaires. RESULTS: Eighty-eight (96%) patients considered fingertip blood collection a positive test feature, and 86% (37) and 91% of professionals considered the IT LEISH(r) easy to perform and interpret, respectively. All professionals classified the DAT-LPC as simple and easy. CONCLUSIONS: Patients and healthcare professionals in Ribeirão das Neves demonstrated a high degree of acceptance of the IT LEISH(r) and DAT-LPC.

Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT) and hemagglutination test (IHAT) were standardized, and complement fixation test (CFT) and direct agglutination test (DAT) were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1) parasitemic dogs of two localities of endemic area (EA) of Santiago del Estero province in Argentina (n = 134); (2) non-parasitemic dogs of the same area (n = 285); (3) dogs experimentally infected with T. cruzi in the patent period (n = 6); (4) non-infected dogs (n = 56) which were born in the city of Buenos Aires (BA), one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.

Reliability of serological methods for detection of leishmaniasis in portuguese domestic and wild reservoirs

A direct agglutination test (DAT) and an immunofluorescence technique (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrábida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres >1:320 and >1:128). Of 34 samples collected from apparently healthy dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25%. Considering these and previous studies in the adjacent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be of high magnitude.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Prevalence and risk factors for human toxoplasmosis in a rural community

Toxoplasma gondii infection may lead to important pathological questions, especially in rural areas, where several sources of infection exist. Therefore, it is important to determine risk factors in order to establish adequate prophylactic measures. The present study aimed to assess the prevalence and risk factors involved in human toxoplasmosis infection in a rural community, in Eldorado, Mato Grosso do Sul State, Brazil. This community was composed of 185 farms - with 671 inhabitants - from which 20 were randomly chosen. In these farms, blood samples were collected from rural workers, who also answered a risk factor questionnaire. Serum samples were analyzed by means of direct agglutination test for the detection of anti-Toxoplasma gondii antibodies. From 73 samples collected, 79.45% were positive. None of the studied variables was significantly associated with the prevalence of the infection. However, among the individuals who reported eyesight impairments, 94.4% had anti-T. gondii antibodies, compared with 74.0% who did not report eyesight changes (p = 0.0594). Moreover, most individuals in the study (68.20%) were older than 18 years and presented 84.44% positivity, compared with 66.67% of positive individuals younger than 18 years old. We were able to conclude that a high prevalence of antibodies did not imply significant associations with the risk factors studied.

Detection of Toxoplasma gondii DNA in the milk of naturally infected ewes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diferenciação entre os estágios agudo e crônico na infecção toxoplásmica pelo método de aglutinação direta modificado e pesquisa do agente no leite de ovelhas naturalmente infectadas por Toxoplasma gondii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transmissão galactogênica de toxoplasma gondii na infecção experimental de ratas Wistar

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção de antígeno de Toxoplasma gondii em células de sarcoma murino TG180

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta imune de tilápias do Nilo (Oreochromis niloticus) vacinadas contra Streptococcus agalactiae

Pós-graduação em Medicina Veterinária - FCAV