Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.     

Sources of GABAergic input to the inferior colliculus of the rat

We have studied the GABAergic projections to the inferior colliculus (IC) of the rat by combining the retrograde transport of horseradish peroxidase (HRP) and immunohistochemistry for gamma-amino butyric acid (GABA). Medium-sized (0.06-0.14 microliter) HRP injections were made in the ventral part of the central nucleus (CNIC), in the dorsal part of the CNIC, in the dorsal cortex (DCIC), and in the external cortex (ECIC) of the IC. Single HRP-labeled and double (HRP-GABA)-labeled neurons were systematically counted in all brainstem auditory nuclei. Our results revealed that the IC receives GABAergic afferent connections from ipsi- and contralateral brainstem auditory nuclei. Most of the contralateral GABAergic input originates in the IC and the dorsal nucleus of the lateral lemniscus (DNLL). The dorsal region of the IC (DCIC and dorsal part of the CNIC) receives connections mostly from its homonimous contralateral region, and the ventral region from the contralateral DNLL. The commissural GABAergic projections originate in a morphologically heterogeneous neuronal population that includes small to medium-sized round and fusiform neurons as well as large and giant neurons. Quantitatively, the ipsilateral ventral nucleus of the lateral lemniscus is the most important source of GABAergic input to the CNIC. In the superior olivary complex, a smaller number of neurons, which lie mainly in the periolivary nuclei, display double labeling. In the contralateral cochlear nuclei, only a few of the retrogradely labeled neurons were GABA immunoreactive. These findings give us more information about the role of GABA in the auditory system, indicating that inhibitory inputs from different ipsi- and contralateral, mono- and binaural auditory brainstem centers converge in the IC.     

Detection of antibiotic resistance in Enterococcus sp. Comparison of GPS-TA (BioMérieux-Vitek), Uniscept MIC-3 (bioMérieux-Vitek) and conventional methods

BACKGROUND: The treatment of severe enterococcus infections requires synergism of a beta-lactamic or glycopeptide and a aminoglycoside, but when resistance to first one or high-level resistance to aminoglycosides are present, synergism would be lost. We compared the adequacy of two commercially available systems to detect antibiotic resistance. METHODS: We studied 158 isolates of Enterococcus sp., with high-level resistance to gentamicin (40 isolates) and streptomycin (89 isolates), resistance to ciprofloxacin (34 isolates), resistance to ampicillin (7 isolates) and with intermediate susceptibility to vancomycin (3 isolates). No one was beta-lactamase producer by Cefinase disk method. We use disk diffusion as reference technique to detect high-level streptomycin resistance. The susceptibility to the remainder antibiotics was studied by agar dilution method, according to NCCLS. We studied the accuracy of GPS-TA cards and Uniscept MIC-3 in relation to the degree of agreement with conventional means, following FDA criteria. RESULTS: Essential agreement for MIC was less than 90 with MIC-3 for ampicillin (81.5%) and ciprofloxacin (71.3%). Categorical agreement rate was less than 90% (76.4%) and major error rate was higher than 3% (10.9%) with the use of MIC-3 for ciprofloxacin. Very major errors for ampicillin, vancomycin and ciprofloxacin were not produced by any system. The very major error rates for high level resistance to gentamicin and streptomycin with GPS-TA card were 5 and 15.7%, respectively. CONCLUSIONS: We do not recommend the use of the Uniscept MIC-3 panel with visual reading to detect susceptibility to ciprofloxacin. Detection of high levels of aminoglucoside resistance by GPS-TA card should be supplemented with conventional techniques because of the high rate of major error. Due to the low number of strains that have been studied, we can not assure the suitability of these systems to detect ampicillin or vancomycin resistance.     

Liver fibrosis

Surfactant abnormalities may contribute to the impairment of gas exchange observed in Pneumocystis carinii pneumonia. Analysis of rat bronchoalveolar lavage (BAL) lipid extracts from normal controls, steroid controls, trimethaprim-sulfamethoxazole (TMP-SMX) controls, TMP-SMX/P. carinii pneumonia controls, and P. carinii pneumonia animals reveal similar total phospholipid and total protein levels. However, there was a marked reduction in phosphatidylglycerol (PG) from the BAL of P. carinii pneumonia rats as compared with control animals, with a decrease from 4.91 +/- 1.29 nmol/mg protein to 0.46 +/- 0.57 nmol/mg protein (p<0.05) and a decrease, as a percent of total phospholipids, from 7.7% +/- 0.88% to 0.91% +/- 0.59% (p<0.001). Furthermore, in vitro surface activities of BAL lipid extracts from control and P. carinii pneumonia rats revealed minimum surface tension increases from 9.38 +/- 1.71 mN/m in controls to 16.36 +/- 0.83 mN/m in P. carinii pneumonia rats (p<0.05) and likewise maximum surface tension increases from 22.14 +/- 4.34 mN/m to 38.57 +/- 2.07 mN/m (p<0.01). Of interest, the surface activity of PG-deficient P. carinii pneumonia BAL lipid extracts is completely restored to that of normal controls by the addition of exogenous PG. These findings suggest that a functionally abnormal surfactant occurs in P. carinii pneumonia and that this may account, in part, for the impairment of gas exchange observed in this disorder.     

Dyslipemia in a population with a high rate of cardiovascular mortality

The activity of N-acetyltransferase (NAT) and catalase was measured in the tissues of placenta, full venous blood and full umbilical cord blood in 141 complicated pregnancies. The control group consisted of 34 physiological pregnancies. The enzymes activity was marked by use of biochemical methods. It has been shown that NAT and catalase is the most active in the group of physiological pregnancies. The decrease of NAT and catalase in venous and umbilical cord blood is correlated with the decrease of the activity these enzymes in placenta.     

Cardiovascular effects produced by R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin in the preoptic area of conscious rats

The effect of ancrod-induced defibrinogenation on thrombosis and bleeding time was determined in anesthetized rats. Functional plasma fibrinogen levels were reduced 42, 71, 94 and 93% by ancrod doses of 5, 10, 20 and 30 U/kg, respectively, while a 2.5 U/kg dose was without significant effect. Ancrod inhibited vena cava thrombosis induced by partial stasis of blood flow combined with mild vascular injury. Thrombus weight was decreased 85 and 93% by the 10 and 20 U/kg doses, but was unaffected at lower doses. In contrast, ancrod doses of up to 30 U/kg did not significantly decrease carotid artery thrombi formed in response to oxidative transmural vessel injury. Ancrod caused a dose-dependent increase in bleeding time measured by puncturing small mesenteric arteries with a hypodermic needle. The bleeding time increase was approximately 38% in response to the 2.5 and 5 U/kg doses, and 182% in response to the 10 U/kg dose. These studies demonstrate that ancrod-induced reductions in plasma fibrinogen more effectively inhibit venous compared to arterial thrombosis, although these activities require doses that also increase bleeding time in small arteries.     

Development of rapid tolerance to pentobarbital and cross-tolerance to ethanol on a motor performance test with intoxicated practice

Male Wistar rats given a single moderate dose (1.7 mg/kg, IP) of pentobarbital (PB), followed by six trials on the moving belt apparatus during the next hour, showed tolerance to the motor-impairing effects of a second dose of 17 mg/kg given 24 h later. A control group that received saline before the first test showed the usual initial sensitivity when tested with PB 24 h later. Three weeks later, the first group showed cross-tolerance to the effects of ethanol (1.7 g/kg, IP) on the same test, while the second group did not. These findings support the suggestion that rapid tolerance is closely similar to chronic tolerance and that the contribution of intoxicated practice results in a long-lasting component that applies to cross-tolerance to ethanol on the same test.