Swine foetal myogenesis in different gestation periods.

This experiment was conducted to evaluate the effects of sex and uterus position on swine foetal myogenesis at different gestational ages. Fifteen primiparous sows were divided into three groups according to gestational age: 50, 80, and 106 days. The experiment was a block randomized factorial design with two sexes (male and female) and three uterine regions (apex, middle, and base). After slaughter, each uterus horn was divided into three segments of equal length: apex region near the ovary; base region near the uterine body; and the middle region, lying between the apex and base regions. The foetuses were weighed, identified, and longitudinally opened to harvest the semitendinosus muscle for later morphological analysis. After 50 days of pregnancy, male foetuses had greater (p < .05) weight than females. The number of primary fibres at 50 days of gestation was negatively correlated (r = -.29, p = .04) with the number of foetuses in utero. After 80 days, foetuses in the base region had less (p < .05) secondary area of muscle fibres compared to the apex region, which was accompanied by differences in the weight of the foetuses, the lowest weight were for foetuses located in the base region (p < .05). In the same period, the ratio of secondary to primary fibres had a positive correlation with weight. In conclusion, sex did not influence myogenesis in the gestational ages studied and the development of secondary muscle fibres of the foetuses at 80 days of gestation was affected by their uterine position with foetuses at the base of the uterine horn being less developed.

Padronização da técnica de plasmaférese automatizada em equinos / Standardization of automated plasmapheresis in horses

O presente artigo objetivou estudar a viabilidade da técnica de plasmaférese automatizada e padronizá-la em cinco equinos hígidos, bem como apresentar as complicações durante o procedimento, as adequações em relação aos procedimentos em humanos e avaliar a recuperação de volume globular e proteínas plasmáticas totais nos doadores. Os procedimentos foram realizados com o equipamento Fresenius AS104, com duração média de 1h46min, processamento de 5758mL de sangue total e colheita média de 3133mL de plasma. Não foram observadas alterações significativas do volume globular após a plasmaférese automatizada. A recuperação dos níveis plasmáticos de proteínas foi de 91,4% em 96 horas após o procedimento. A plasmaférese automatizada apresentou-se viável para a espécie equina, diminuindo o tempo de recuperação hematimétrica nos doadores.

This paper aimed to study feasibility and standardize the automated plasmapheresis in five healthy horses, showing the complications during the procedure, adjustments in relation to the procedures in humans and assessing the recovery of globular volume and plasma total proteins in donors. The procedures were performed with the Fresenius AS104 equipment, with an average duration of one hour and forty six minutes, processing 5758mL of whole blood and harvest average of 3133mL of plasma. There were no significant variations in globular volume after the automated plasmapheresis. The recovery of plasma total proteins was 91.4% at 96 hours after the procedure. The automated plasmapheresis appeared viable for the equine species, decreasing the time of hematimetric level recovery in donors.

Hepatite granulomatosa em bovino causada por Mycobacterium avium subsp. paratuberculosis / Granulomatous hepatitis in cattle caused by Mycobacterium avium subsp. paratuberculosis

Samples from intestines, liver, and lymph nodes were collected from a dairy steer with clinical suspicion of paratuberculosis. The samples were processed for histologic examination with hematoxylin-eosin and Zihel-Neelsen (ZN) staining for the detection of acid-fast bacilli (AFB), and submitted to immunohistochemistry (IHC). Macroscopic changes were observed in the small intestines, with thickening and corrugation of the mucosa. The main microscopic changes were found in small intestines, lymph vessels in the mesentery, and mesenteric lymph nodes characterized by enteritis, lymphangiectasia, and lymphadenitis. Liver presented with granulomatous hepatitis, an uncommon histopathological feature for paratuberculosis. The clinical features associated with positive culture of Mycobacterium avium subsp. paratuberculosis and detection of AFB by ZN and IHC in the cytoplasm of macrophages (epithelioid) in the intestinal mucosa and submucosa, lymph nodes, and liver were important to confirm the diagnosis of paratuberculosis.

Occurrence of false-positive results in three paratuberculosis - ELISAs performed in a tuberculous herd.

The interference of bovine tuberculosis (TB) on the efficacy of paratuberculosis (PTB) diagnostic tests has been evaluated. A group of 32 tuberculous cows identified by both intradermal tests and gamma-interferon assay, 16 of them confirmed by the recovery of M.bovis from tissues, was tested by three different PTB- ELISAs, being two commercials and one in-house. The rest of the adult animals of the herds, totalizing 216 TB-negative animals, were also tested as a control group. Fecal culture for PTB was negative in all animals, but seven (21.8%) tuberculous cows produced false-positive reactions when tested by various PTB-ELISAs, leading to a misdiagnosis. Tuberculosis impairs the specificity of serological tests for paratuberculosis diagnosis and should be considered for the reliability of PTB control programs.

Prevalence of pulmonary tuberculosis among respiratory symptomatic subjects in an out-patient primary health unit.

SETTING: Out-patient primary health unit (OPHU) in Rio de Janeiro City, Brazil. OBJECTIVE: To evaluate the impact on the detection of tuberculosis (TB) cases of reducing the time of respiratory symptoms from 'cough > or = 3 weeks' to 'cough > or = 1 week' as a criteria for TB case finding among individuals visiting an OPHU for any other reason. DESIGN: Cross-sectional study. RESULTS: During the period of the study, 10.7% (765/ 7174) of subjects reported cough > or = 1 week. Among 542 subjects enrolled in the study with cough > or = 1 week, 15 (2.7%) cases were diagnosed with pulmonary tuberculosis (PTB, 2767/100000). The probability of detecting TB in the OPHU setting among subjects seeking care for respiratory symptoms was significantly higher than among those presenting to the OPHU for other reasons (OR 31.5, 95% CI 4.1-241.9; P < 0.0001). The probability of identifying TB among patients seeking care due to respiratory symptoms was not influenced by the duration of cough (P = 0.7). CONCLUSION: These findings suggest that the screening criteria for TB case finding of cough for less than the usual 3 weeks among patients who attend a health facility due to respiratory symptoms in settings with a high prevalence of TB may significantly improve the proportion of TB cases diagnosed.

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres.

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.

Oxacillin- and mupirocin-resistant Staphylococcus aureus: in vitro activity of silver sulphadiazine and cerium nitrate in hospital strains.

Nasal carriage is an important reservoir of oxacillin-resistant Staphylococcus aureus (ORSA). Mupirocin is a topical drug used to remove S. aureus from nares. However, isolates resistant to mupirocin have been reported all over the world. Silver sulphadiazine (SSD) is a topical agent, which when associated with cerium nitrate (CN), has been shown to be useful in the treatment of burn infections and could be an alternative drug for patient decolonization. Susceptibility to oxacillin in 203 S. aureus isolates was evaluated by the agar diffusion test, while the agar diffusion and agar dilution methods were used for mupirocin. A PCR-multiplex method was performed to detect the mecA and ileS-2 genes. Minimum inhibitory concentration (MICs) to SSD and CN, used alone or in association, were determined by the agar dilution method. One hundred and sixty-three (80.3%) strains were oxacillin-resistant, and 37 (18.2%) were mupirocin resistant. The MIC of SSD alone or in association with CN was 64 microg/mL, while for CN alone was 2048 microg/mL for all isolates. SSD presented anti-staphylococcal activity at concentrations (64 microg/mL) much lower than those commonly used in commercial preparations (10 mg/g) and had good activity against mupirocin-resistant strains, showing that this drug could be used for nasal decolonization in ORSA carries.

Agar gel immunodiffusion test (AGID) evaluation for detection of bovine paratuberculosis in Rio de Janeiro, Brazil.

AIMS: The aim of this study is to evaluate the AGID serological test for detection of antibodies anti-Mycobacterium paratuberculosis and its possible adoption as diagnostic method in our field conditions. METHODS: Bovine serum samples from dairy herds in Rio de Janeiro State, Brazil, were screened for the presence of antibodies against Myco. paratuberculosis using three different ELISA tests. A panel of 48 randomly selected sera were evaluated by an Agarose Gel Immunodiffusion (AGID) test using Protoplasmatic Paratuberculosis Antigen (PPA). AGID results were compared to the standards--the results of the three ELISA tests, and the specificity and sensitivity were calculated. RESULTS: From 48 sera tested for AGID, 14 (29.17%) were positive and 34 (70.83%) were negative. AGID sensitivity was 57% with two false-positive reactions, and specificity was 92.5% with nine false-negative results. The positive predictive value was calculated in 85.7% for a confidence interval of 95%. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its low sensitivity and specificity rates, AGID test has shown to be unsatisfactory as a screening diagnostic method for subclinical herd infection, but it can be useful as a confirmatory test for clinical suspect animals.

Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis.

In this study, our objective was to evaluate Etest strips containing exponential gradients of isoniazid (INH), rifampin (RIF), and streptomycin (STR) for susceptibility testing of Mycobacterium tuberculosis. M. tuberculosis isolates were tested for antimicrobial susceptibilities by the standard proportion method using Löwenstein-Jensen (LJ) medium and by the Etest. The MICs determined by the Etest were obtained at 5, 7, or 10 days. In some strains with Etest-discrepant results, radiometric susceptibility testing (BACTEC) was performed to determine a consensus result. M. tuberculosis concordance between the two methods was 97% (86 of 89 isolates) for RIF, 96% for INH (84 of 87 isolates), and 80% (61 of 76 isolates) for STR. Most of the MICs determined by the Etest were easy to interpret and readable within 5 days. Results correlated well with those obtained by the LJ proportion and BACTEC methods for INH and RIF. However, a high proportion of false-sensitive and false-resistant results were observed, most often for STR. We also observed that variations in the inoculum size of M. tuberculosis isolates affected the MICs to a substantial degree. These discrepancies, along with the expense of the media, the Etest strips, and the specialized equipment required (CO2 incubator), make this method less useful in developing countries.

Mycobacterium tuberculosis typing: usefulness of DRE-PCR to confirm cross-contamination in the mycobacteriology laboratory of a general reference hospital for AIDS.

In this study two molecular typing methods, a simple double repetitive element PCR-based assay and the standardized restriction fragment length polymorphism (RFLP), were used to confirm cross-contamination in the mycobacteriology laboratory. Clinical specimens from 12 patients, submitted for acid-fast bacilli stain smear and processed for culture in Lowenstein-Jensen on the same day, resulted in positive bacterioscopy (+++) and confluent growth only for one of the patients. The specimens from all the other patients but two were smear-negative and culture-positive, with one or two colonies. None of them had clinical symptoms and radiological findings for active tuberculosis (TB). The suspicion of false-positive cultures arose when a health care worker who had had a PPD skin test conversion, claimed to be healthy and had no TB symptoms, was found to have a positive sputum culture. DRE-PCR demonstrated that all nine cultures typed belonged to one cluster, further confirmed by RFLP. Although DRE-PCR has been found to be poorly reproducible, it has enough discriminatory power to be useful for rapid epidemiological investigation in selected settings.

The use of Ag85 complex as antigen in ELISA for the diagnosis of bovine tuberculosis in dairy cows in Brazil.

Several enzyme-linked immunosorbent assay (ELISA) methods have been investigated to evaluate their performance in the diagnosis of tuberculosis in cattle. Increased production of antibodies to the proteins of antigen 85 complex (Ag85) after experimental and natural infection in cattle shows that they are strongly immunogenic in vivo. The purpose of this study was to evaluate the use of Ag85 as an antigen in ELISA for the diagnosis of bovine tuberculosis in dairy cows in Brazil. The test groups consisted of 46 serum samples from intradermal tuberculin test (ITT)-positive animals (Group A) and 46 samples from ITT-negative animals (Group B). Group C comprised 12 samples from a tuberculosis-free herd and was the control group of the study. Samples were tested in an ELISA using Ag85 as antigen. Differences between the mean ODs of groups A and B and A and C were significant (P < 0.01), but no significant difference (P > 0.05) was observed between groups B and C. The sensitivity of the ELISA using Ag85 was 91.3% (42/46) and its specificity was 94.8% (55/58). These results were not significantly different (P > 0.05) from those observed in a previous study of an ELISA using purified protein derivative (PPD). We concluded that, although Ag85 can be used as antigen for ELISA tests in the diagnosis of bovine tuberculosis with good sensitivity and specificity rates, no significant advantages were observed in relation to the ELISA using PPD that could justify the purification and utilization of Ag85 as a single antigen in routine methods of diagnosis.

[Relationship between serological diagnosis (ELISA) and severity of pulmonary tuberculosis in children]. / Relação entre o diagnóstico sorológico (ELISA) e a gravidade da tuberculose pulmonar na infância.

Forty eight children from 0 to 13 years old were submitted to the enzyme-linked immunosorbent assay (ELISA) serological test with a view to detect anti PPD IgG antibodies, for diagnosis of pulmonary tuberculosis and to establish the relationship between immune response and radiological gravity of pulmonary tuberculosis (mild, moderate and severe). There were 29 children with pulmonary tuberculosis and 19 children without tuberculosis. The median ELISA optical density were: 0.098 in children with primary complex (mild); 0.092 in children with pneumonic pattern (moderate) and 0.134 in children with miliary tuberculosis (severe). These data show higher positive serological test results in severe forms of pulmonary tuberculosis (p = 0.0007).

RFLP patterns and risk factors for recent tuberculosis transmission among hospitalized tuberculosis patients in Rio de Janeiro, Brazil.

Isolates of Mycobacterium tuberculosis from 120 tuberculosis patients seen in the 12 months ending September 1994 at 2 tertiary-care centres in Rio de Janeiro were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis. Ninety-seven patients (81%) had isolates with unique RFLP patterns, while 23 patients (19%) had isolates that belonged to 11 different RFLP cluster patterns. The strains from the latter patients were distributed among 1 group of 3 patients and 10 groups of 2 patients each. The cluster-pattern strains were not associated with gender, age, HIV infection, type of residence, living in shelter, homelessness or previous history of tuberculosis. However, clustering was strongly associated with multidrug resistance (P = 0.006). These data suggest that recent exogenous transmission may be important for the development of new cases of multidrug-resistant disease in patients attending tertiary-care centres in Rio de Janeiro, Brazil.

Genotypic patterns of multiple isolates of M. tuberculosis from tuberculous HIV patients.

We investigated whether the recurrence of tuberculosis in HIV-infected patients is due to an exogenous reinfection or relapses after antituberculosis chemotherapy. We reviewed clinical information on 32 patients at a Rio de Janeiro hospital from whom multiple Mycobacterium tuberculosis isolates were taken. All isolates were analysed by DRE-PCR fingerprinting technique, and those with identical DRE-PCR patterns were analysed by the RFLP method. Twenty patients had M. tuberculosis simultaneously isolated from different organs. These patients and nine others with sequential positive cultures after 2 months of therapy showed stable DRE-PCR and RFLP patterns. One patient's isolate became resistant to isoniazid, but the molecular pattern remained unchanged despite the development of drug resistance. In three patients, the DRE-PCR patterns of the isolates changed dramatically. Clinical and microbiological evidence was consistent with active tuberculosis caused by a new strain of M. tuberculosis. The exogenous reinfection of the three patients was not due to an outbreak, but the isolates from each patient showed unique patterns.

Mycobacterium tuberculosis lipid antigens: use of multi-antigen based enzyme immunoassay for free and complex dissociated antibodies.

OBJECTIVE: To test the sensitivity and specificity of four lipid antigens of Mycobacterium tuberculosis: BDA-TDA, DAT, SL-I, and PIMs, adsorbed in the same microplate well, to detect reactive IgG by enzyme-immunoassay (EIA) from plain serum (MA-EIA) and dissociated immune complexes (ICMA-EIA). DESIGN: IgG antibodies against four antigens, placed in the same microplate well, were evaluated in serum from 155 tuberculous (TB) cases non-infected with the human immunodeficiency virus (HIV): 78 patients with positive bacilloscopy and culture, 33 patients with positive culture and 44 patients diagnosed by clinical and radiological criteria; and from 211 HIV negative control subjects: 32 patients with other pulmonary diseases, 100 healthy people and 79 close contacts. RESULTS: MA-EIA had an overall sensitivity and specificity of 61% (94/155) and 95% (200/211), respectively. We further examined whether the dissociation of immune complexes increases the number of positive reactions in those initially found to be seronegative (SN). The subset of 112 (76 controls and 36 TB) MA-EIA SN samples tested using ICMA-EIA yielded an overall sensitivity and specificity of 83% and 100%. The ICMA-EIA results improved the overall sensitivity from 61 to 80% without changing specificity. CONCLUSION: These preliminary results suggest that MA-EIA followed by ICMA-EIA, for SN samples, might serve as a fast, cheap, and easy method for the diagnosis of TB in less than 48 hours.

Predictive value of the acid fast smear for detection of Mycobacterium tuberculosis in respiratory specimens in a reference center of HIV/AIDS in Rio de Janeiro, Brazil.

In order to evaluate the predictive value of acid fast bacilii (AFB) smear for the diagnosis of Mycobacterium tuberculosis in respiratory specimens in a setting with a high prevalence of AIDS and an unknown prevalence of nontuberculous mycobacteria (NTM), we retrospectively examined specimens cultured for mycobacteria between 1 September 1993 and 30 September 1994 and medical records of patients with positive culture in a General Hospital, AIDS reference in Rio de Janeiro, Brazil. Seventy three per cent (1517/2077) of samples were respiratory specimens and mycobacteria were recovered from 20.6% (313/1517) of these. M. tuberculosis was identified in 94.2% (295/313) and NTM in 5.8% (18/313). The yield of positive AFB smear and of positive culture was 6.1% (93/1517) and 20.6% (313/1517), respectively. The positive predictive value (PPV) of AFB for M. tuberculosis was 98.4% in expectorated sputum and 96.4% in bronchoalveolar lavage. Forty four percent (130/295) of specimens with positive culture for M. tuberculosis and 66.7% (12/18) for NTM were from patients HIV positive. The conclusion was that in our study population, the PPV of AFB for M. tuberculosis in respiratory specimens was high and the prevalence of NTM was low despite the high prevalence of HIV positive.

Drug Resistance Patterns among Hospitalized Tuberculous Patients in Rio de Janeiro, Brazil, 1993-1994

The purpose of this study was to analyze the prevalence and risk factors for drug resistance among hospitalized patients in two tertiary care centers, an acquired immunodeficiency syndrome (AIDS) reference center and a sanatorium, in Rio de Janeiro, Brazil. From 1993-1994, 389 patients were diagnosed as having tuberculosis (TB). Isolates from 265 patients were tested for in vitro susceptibility to rifampin and isoniazid. Resistance to one or more drugs was detected in 44 patients (16.6 percent) and was significantly more common among recurrent cases in both hospitals (p=0.03 in the AIDS center and p=0.001 in the sanatorium). Twenty seven patients (10.2 percent) had isolates resistant to both isoniazid and rifampin. Multi-drug resistance was associated with human immunodeficiency virus (HIV) infection among patients who had never been treated for TB. In conclusion, drug-resistant TB is high in hospitalized patients in Rio de Janeiro, especially among HIV infected patients. Therefore, measures to control TB and prevent nosocomial transmission need urgently to be set up in the Brazilian hospitals

Comparison between a gamma-IFN assay and intradermal tuberculin test for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major problem in Brazil. The intradermal tuberculin test is the standard test for detection of bovine tuberculosis in Brazil but can lack both sensitivity and specificity. The purpose of this study was to compare a bovine gamma-IFN assay with the tuberculin test under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested using the single cervical tuberculin test (SCTT). Among those animals, about 15% of each herd, 220 in total, represented a high-risk group and were selected to be tested using the gamma-IFN test. Of the 1632 animals tested, 207 presented significant reactions representing 12.7% of the cattle studied. In the selected group the number of animals positive by the gamma-IFN assay was 126/220 (57.3%) and the total number of reactive cows on SCTT was 106/220 (48.2%). The real number of infected cattle, following standards, was 120/220 (54.5%). From these results the relative sensitivity rate of gamma-IFN test was 100% including the false-positive results and 88.3% for the SCTT--a significant (P < 0.01) difference in favour of the gamma-IFN test of 11.7%. The gamma-IFN assay also identified some positive animals 60-120 days earlier than the SCTT. In conclusion, we believe that the gamma-IFN assay can be used alone or in combination with the SCTT, as a valuable tool for the control of bovine tuberculosis in the Brazilian national herd.