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.

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.

Mechanisms of immunologic eradication of a syngeneic guinea pig tumor. I. Quantitative analysis of adoptive immunity.

Adoptive immunity against a syngeneic hepatoma (line-10) of Sewall-Wright inbred strain 2 guinea pigs was analyzed by a two-dimensional titration of iv transferred immune lymphoid cells versus intradermal tumor challenges. Tumor resistance increased exponentially as a function of the number of immune lymphoid cells transferred. Within the tumor challenge doses analyzed, suppression of tumor growth mediated by the transferred immune lymphoid cells appeared to be independent of the primary immune response in the recipient. Quantitatively, rejection of a given number of tumor cells reflected the number of transferred immune cells and was independent of the presence of the same tumor at other skin sites. There was no evidence indicating that transferred immune cells were attracted specifically to the tumor inoculation site. The number of tumor cells that could be rejected at a skin site by adoptive immunity was greater than the estimated number of immune lymphoid cells present at the challenge site.

Mechanisms of immunological eradication of a syngeneic guinea pig tumor: participation of a component(s) of recipient origin in the expression of systemic adoptive immunity.

The effects of carrageenan and trypan blue on the expression of adoptive immunity to the syngeneic guinea pig line 10 hepatoma were investigated. Adoptive immunity was assessed by observing dermal tumor growth in recipients of immune cells and by bioassays in which tumor challenge sites were transplanted into secondary hosts. Carrageenan abrogated transferred immunity in treated animals as evidenced by dermal tumor growth and by development of fatal ascites tumors in peritoneal cavities of the secondary hosts. Trypan blue, on the other hand, did not abrogate transferred immunity in treated animals. However, the i.p. bioassay revealed the presence of line 10 cells in the tumor challenge sites 10 days after adoptive transfer. In vitro and in vivo exposure of immune spleen cells to carrageenan or trypan blue had no significant effect on the subsequent adoptive transfer, indicating that the inhibitory activity of these agents cannot be attributed to direct toxicity to immune lymphoid cells. Tumor challenge sites taken from carrageenan or trypan blue-treated animals 5 days after adoptive transfer failed to grow progressively when transplanted s.c. into secondary hosts. This observation suggests the presence of immune cells at tumor challenge sites. Thus, the inhibitory effects were unlikely due to interference with recirculation of the i.v.-transferred immune cells. Adoptive immunity was not influenced in guinea pigs that received a lethal dose of irradiation (500 rads). These results demonstrate that a recipient component(s) sensitive to carrageenan and trypan blue but resistant to radiation is essential to the expression of adoptive immunity.

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.

Mycobacteremia in patients with the acquired immunodeficiency syndrome.

BACKGROUND: Bacillemia is a key event in the pathogenesis of tuberculosis. Although current evidence indicates that Mycobacterium tuberculosis bacteremia is rare in patients seronegative for the human immunodeficiency virus, it has been increasingly reported in patients with the acquired immunodeficiency syndrome (AIDS). OBJECTIVE: To determine clinical and laboratory characteristics of patients with AIDS and tuberculosis with and without bacillemia. METHODS: Fifty patients with AIDS with clinical suspicion of disseminated mycobacterial disease were prospectively selected. Three consecutive blood samples were collected for culture using a standardized protocol. RESULTS: Mycobacterium was isolated from any body site in 42 patients (84%). Bacillemia was detected in 30 (71.4%) of these 42 patients: 11 (28.2%) caused by Mycobacterium avium-intracellulare complex and 19 (71.8%) caused by M tuberculosis. Blood culture was the only method used to confirm the diagnosis in 5 (15%) of the 33 tuberculosis cases. Tuberculosis in patients with AIDS developed with nonspecific insidious symptoms, a remarkable elevated alkaline phosphatase level, and without the classic miliary radiological pattern. We could demonstrate 2 previously unrevealed clinical characteristics of bacteremic tuberculosis in patients with AIDS: a shift to the left in the white blood cell count and abdominal lymph node enlargement. In patients with tuberculosis, the in-hospital mortality rate was higher among patients with bacillemia, although the posttreatment survival rate was comparable. CONCLUSIONS: Blood culture is a valuable tool to confirm the clinical diagnosis of disseminated tuberculosis in patients with AIDS and can distinguish patients with characteristic clinical findings and outcome. Abdominal ultrasonography may be an additional helpful tool to identify these patients.

Mechanisms of immunological eradication of a syngeneic guinea pig tumor. II. Effect of methotrexate treatment and T cell depletion of the recipient on adoptive immunity.

The influence of methotrexate on the development of immunity to the line 10 hepatoma was studied in guinea pigs. Chronic methotrexate treatment had no apparent effect on the ability of immune guinea pigs to suppress the growth of inoculated tumor cells. In contrast, the same methotrexate regimen inhibited the development of tumor immunity if started before the 8th day after immunization with a vaccine containing viable line 10 cells admixed with Bacillus Calmette-Guérin (BCG) cell walls. Thus, methotrexate selectively inhibited the afferent limb of the immune response. In adoptive transfer experiments, methotrexate-treated recipient guinea pigs were capable of being passively sensitized with immune spleen cells, indicating that the primary cell-mediated immune response of the recipient was not required for adoptive immunity. The contribution of recipient T cells in adoptive immunity was further investigated in guinea pigs deleted of T cells by thymectomy, irradiation, and bone marrow reconstitution. Despite demonstrable deficiency in T lymphocyte reactions, "B" animals were fully capable of rejecting tumors after transfer of immune cells. These results suggest that the expression of adoptive immunity was independent of recipient T cell participation. In addition, sublethal irradiation of immune spleen cells prior to adoptive transfer abolished their efficacy. Proliferation of transferred immune cells in the recipient may be essential for expression of adoptive immunity.

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.

Association between an early humoral response to Mycobacterium tuberculosis antigens and later development of tuberculosis in human immunodeficiency virus-infected individuals.

OBJECTIVE: To evaluate the association between an early humoral response to Mycobacterium tuberculosis antigens and the later development of tuberculosis (TB) disease in human immunodeficiency virus (HIV)-infected individuals. METHODS: Using an ELISA test, IgG antibodies against 4 M. tuberculosis antigens--purified protein derivative (PPD); 2,3 diacyl trehalose (DAT); a lipooligosaccharide (LOS) and a trehalose dicarboxylic acid bis N,N-dioctadecylamide (BDA.TDA)--were measured in sera from 25 HIV-infected tuberculous patients and 52 HIV-infected persons without TB. RESULTS: With the DAT and LOS antigens, a positive result in sera obtained in the 12 months preceding the onset of TB was significantly associated with later development of TB. Using the BDA.TDA antigen, the same association was observed in sera collected during the 6 months before the diagnosis of TB. No significant association was found with the PPD antigen. CONCLUSIONS: These results suggest that specific antibody markers may be useful to evaluate the risk of active TB in HIV-infected individuals, and a helpful indicator for preventive treatment.

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.

Incidence surveillance of wound infection in hernia surgery during hospitalization and after discharge in a university hospital.

A six-month prospective incidence surveillance of wound infection was conducted in the department of general surgery of the Rio de Janeiro University Hospital. Postoperative infections were classified according to Centers for Disease Control criteria. This study reports a rate of 14.04% in surgical infections limited to herniorrhaphy and detected by surveillance. The majority (87.50%) of them were only apparent after hospital discharge. Fourteen out of 16 patients (88.60%) were not deemed to be at risk for surgical infections. Staphylococcus aureus was the most important pathogen associated with infection. This report shows that community surveillance is necessary to determine accurate rates of hospital-acquired infection and will help establish prevention and control policies in Brazil.

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.

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.

Use of the anti-PGL-I antibody ELISA and the Mitsuda reaction in early diagnosis of leprosy.

Cutaneous reaction to the Mitsuda antigen and anti-PGL-I IgM antibodies (ELISA) were determined in 134 leprosy patients, 290 household contacts, 52 healthy controls and 43 tuberculous individuals. The multibacillary patients did not develop cell-mediated immunity (CMI), although they presented high levels of IgM (absorbance at 492 nm greater than 0.5). The paucibacillary patients presented CMI, although in varying degrees, and IgM levels did not exceed 0.5 absorbance units. Most of the contacts (107) showed a Mitsuda-positive test, and 25 of them were anti-PGL-I IgM seropositive (absorbance less than 0.5 but greater than 0.22), although none became ill during the two-year follow-up. Of the 17 Mitsuda-negative contacts, two exhibited an immunological status of lepromatous leprosy (negative Mitsuda test and positive serology; absorbance greater than 0.5) and became ill (one borderline lepromatous and one indeterminate leprosy). These results show that the immunological status of lepromatous leprosy can appear prior to clinical symptoms, and thus serology for anti-PGL-I IgM together with the Mitsuda test can be useful in an active search for new preclinical cases among high risk populations.

The dot-ELISA test for the detection of anti-PGL-1 IgM in leprosy patients and their contacts.

1. Since dot-ELISA has recently been reported to be a sensitive, simple and fast method, we have compared it with the conventional microplate ELISA method. Sera of 124 leprosy patients, 136 household and professional contacts, and 92 controls were tested for antibodies against a Mycobacterium leprae antigen using dot-ELISA on nitrocellulose membrane filters and microplate ELISA. 2. The sensitivity of the two techniques was similar for multibacillary patients, but dot-ELISA was less sensitive for paucibacillary patients although it was more specific (100%) than ELISA (93.4%). 3. Of 21 household contacts that gave a response by ELISA, 3 were also positive by dot-ELISA; one of these 3 developed indeterminate leprosy 12 months later and the other was diagnosed as borderline lepromatous after 28 months. 4. These data indicate that dot-ELISA has a high specificity and can be a useful tool in field evaluation.

Relationship between immune response and clinical manifestations in patients with tuberculosis.

Cutaneous reactivity to purified protein derivative (PPD) and antibody levels were investigated in 122 adults and 28 children with tuberculosis. IgG anti-PPD levels (measured by ELISA and reported as absorbance at 405 nm) were higher in adult patients with the disease for more than one year (0.533 +/- 0.391, N = 38 vs 0.224 +/- 0.256, N = 50 in patients with the disease for less than one year) as well as in children with disseminated tuberculosis (0.138 +/- 0.137, N = 11, vs 0.072 +/- 0.055, N = 15 in children with localized disease). The cut-off values (mean + 2SD) for healthy children and adults were 0.09 and 0.22 absorbance at 405 nm. In both adult and pediatric patients, cutaneous reactivity to PPD was inversely correlated with antibody levels. The present data provide additional evidence for the existence of an unstable spectrum of immune response in tuberculosis patients in whom changes in clinical situation are dynamic.

Evaluation of an ELISA-PPD for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major health problem in Brazil. The intradermal tuberculin test is the standard test for its detection, but it can lack both sensitivity and specificity. The purpose of this study was to evaluate a bovine enzyme-linked immunosorbent assay- (ELISA - PPD) under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested with the intradermal tuberculin test (ITT). Two hundred and seven cows gave a positive reaction, which represents 12.7 per cent of the cattle studied. The sensitivity and specificity rates to ITT were 87.7 per cent and 95.2 per cent, respectively. From the 1632 animals 15 per cent of each herd (220 in total) were selected to be tested by the ELISA. Differences between mean optical density (OD) of the control group, ITT -positive and ITT -negative groups were all significant (P<0.01). The sensitivity rates to ELISA - PPD were 86.7 per cent, while specificity was 90.6 per cent. The use of ELISA - PPD is suggested for situations where the investigation of the whole herd is more important than the individual testing of each cow. In addition, the ELISA - PPD can also be helpful when a collective diagnosis is desired to elucidate clinical suspicions of disease, or in the first steps of a control program, for identification of foci.

[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).