Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

MONTENEGRO SKIN TEST AND AGE OF SKIN LESION AS PREDICTORS OF TREATMENT FAILURE IN CUTANEOUS LEISHMANIASIS

A case-control study was conducted to examine the association among the Montenegro skin test (MST), age of skin lesion and therapeutic response in patients with cutaneous leishmaniasis (CL) treated at Evandro Chagas National Institute of Infectious Diseases (INI), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil. For each treatment failure (case), two controls showing skin lesion healing following treatment, paired by sex and age, were randomly selected. All patients were treated with 5 mg Sb5+/kg/day of intramuscular meglumine antimoniate (Sb5+) for 30 successive days. Patients with CL were approximately five times more likely to fail when lesions were less than two months old at the first appointment. Patients with treatment failure showed less intense MST reactions than patients progressing to clinical cure. For each 10 mm of increase in MST response, there was a 26% reduction in the chance of treatment failure. An early treatment - defined as a treatment applied for skin lesions, which starts when they are less than two months old at the first appointment -, as well as a poor cellular immune response, reflected by lower reactivity in MST, were associated with treatment failure in cutaneous leishmaniasis.

USE OF MUELLER-HINTON BROTH AND AGAR IN THE GERM TUBE TEST

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

DENGUE OUTBREAK IN MATO GROSSO STATE, MIDWESTERN BRAZIL

Dengue virus (DENV) is the most frequent arbovirus worldwide. In this study, we report a large outbreak in Mato Grosso State (MT). Serum samples from 604 patients with acute febrile illness for less than five days were inoculated in C6/36 cells, then infected cells were subjected to an indirect immunofluorescence test for DENV serotypes and yellow fever virus. Serum samples were submitted to a multiplex-semi-nested-RT-PCR for 11 flaviviruses. DENV-4 was isolated in 150/604 (24.8%) and DENV-1 in 19/604 (3.1%) specimens. By RT-PCR, 331 (54.8%) samples tested positive for DENV; 321 had single infections (DENV-4 n = 305; DENV-1 n = 15; DENV-3 n = 1), nine had co-infections of DENV-1/DENV-4, and one of DENV-2/DENV-4. DENV-4 was detected in 315/331 (95.2%) positive patients from 17 municipalities, and DENV-1 in 24/331 (7.2%) patients from five cities in north-central MT and the city of Cuiaba. The incidence of infection was higher in patients aged 20-39 (142/331; 42.9%). The NS5 partial nucleotide sequence of DENV-1 was most similar to that of genotype V, DENV-2 to Southeast Asian/American, DENV-3 to genotype III, and DENV-4 to genotype II strains, considered the most frequent strains in Brazil. This outbreak coincided with the introduction of DENV-4 in the state. Cuiaba was hyperendemic for the four DENV serotypes, highlighting the necessity for arbovirus surveillance in MT.

SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY

SUMMARYDuring recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

A modification of the indirect fluorescence test (IFT) for Paracoccidioidomycosis

A modification of the indirect fluorescence test (IFT) for serological diagnosis of Paracoccidioidomycosis is described in which formolized budding forms (yeast cells) of the fungus Paracoccidioides brasiliensis are used as antigen. A further modification introduced is a less eiaborate technical procedure of the test without lowering the sensitivity of the reaction. The test may be considered as adequateiy accurate and easy to perform in any laboratory with immunofluorescence facilities.

Evaluation of a direct immunofluorescent antibody (difma) test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

A direct immunofluorescent antibody (DIFMA) test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL) in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in Northern Brazil

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

The usefulness of Umelosa hepatitis C vírus qualitative kit as supplemental test for confirmation of hepatitis C virus infection

Forty voluntary blood donors from two different blood banks in Havana, Cuba, who were repeatedly reactive on the routine screening of antibodies to hepatitis C virus, by Umelisa HCV test, were analyzed for the presence of HCV RNA using a nested PCR assay of the HCV 5' untranslated region, Umelosa HCV qualitative. Sera from 45 patients of a specialized gastroenterology consultation, positive to Umelisa HCV, were also assayed with the Umelosa HCV qualitative, to establish their condition related to the presence of HCV RNA previously to the indication of a treatment or after three, six or twelve months of antiviral therapy. Serum HCV-RNA was detected in 21/40 (52.5%) donors who had repeatedly positive ELISA results, confirming the HCV infection for them. In specialized consultation HCV-RNA was detected by PCR analysis in 30/45 (66%) analyzed sera.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

Evaluation of an immunochromatography test for malaria diagnosis under different storage conditions

This study aimed to evaluate the second-generation OptiMal test for malaria diagnosis under various storage conditions. It detected all the positive samples, except for two Plasmodium malariae samples. Further research evaluating diverse environmental conditions are important for ICT test applicability in Brazilian malaria areas.

Detection of Mycobacterium leprae infection in wild nine-banded armadillos (Dasypus novemcinctus) using the rapid ML Flow test

Mycobaterium leprae infection was investigated in armadillos from the State of Espírito Santo, Brazil. The ML Flow test was performed on 37 nine-banded armadillos and positive results were found in 11 (29.7%). The ML Flow test may be used to identify possible sources of Mycobaterium leprae among wild armadillos.