Occurrence and sensitivity profile of extended spectrum beta-lactamase-producing Enterobacteriaceae at a tertiary hospital in Southern Brazil

Abstract: INTRODUCTION: Nosocomial infections are closely associated with antimicrobial drug resistance. One of the most important mechanisms of resistance to β-lactam antibiotics is the production of extended spectrum β-lactamases (ESBLs). The objective of the present study was to evaluate the prevalence and antimicrobial susceptibility profile of ESBL-producing strains and to assess the evolution of antimicrobial drug resistance between 2007 and 2013 at the Hospital São Vicente de Paulo, Passo Fundo, State of Rio Grande do Sul, Brazil. METHODS: We conducted a descriptive, observational, cross-sectional study. Bacterial culture was performed from January to December 2013. The antimicrobial susceptibility profile of these cultures was determined using the disk diffusion method. Phenotypic screening for ESBL production was performed using the disk approximation method. RESULTS : We analyzed a total of 19,112 cultures, 11.5% of which were positive for Enterobacteriaceae. Of these, 30.3% of the isolates were positive for ESBL production, and the most prevalent species was Klebsiella sp. (37.5%). Over 95% of these isolates showed reduced susceptibility to all cephalosporins, aztreonam, and amoxicillin/clavulanic acid. The isolates also showed high sensitivity to the following antimicrobials: amikacin, meropenem, and piperacillin/tazobactam. Overall, the resistance rates among ESBL-producing Enterobacteriaceae decreased from 2007 to 2013. CONCLUSIONS : In our hospital, the increased sensitivity to certain antimicrobial agents seems to be directly related to the implementation of improvements in the methods to prevent and control nosocomial infections in addition to the natural development of other resistance mechanisms.

Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Features to validate cerebral toxoplasmosis

Introduction Neurotoxoplasmosis (NT) sometimes manifests unusual characteristics. Methods We analyzed 85 patients with NT and AIDS according to clinical, cerebrospinal fluid, cranial magnetic resonance, and polymerase chain reaction (PCR) characteristics. Results In 8.5%, focal neurological deficits were absent and 16.4% had single cerebral lesions. Increased sensitivity of PCR for Toxoplasma gondii DNA in the central nervous system was associated with pleocytosis and presence of >4 encephalic lesions. Conclusions Patients with NT may present without focal neurological deficit and NT may occur with presence of a single cerebral lesion. Greater numbers of lesions and greater cellularity in cerebrospinal fluid improve the sensitivity of PCR to T gondii.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

An analysis of electrocardiographic criteria for determining left ventricular hypertrophy

OBJECTIVE: To determine the most sensitive criterion for the detection of left ventricular hypertrophy according to echocardiographically defined left ventricular mass. METHODS: The Sokolow-Lyon voltage, Sokolow-Lyon-Rappaport, Cornell voltage duration product, White-Bock, and Romhilt-Estes point scoring criteria were compared with left ventricular mass index, corrected for body surface, obtained from the echocardiograms of 306 outpatients (176 females, 130 males), of all age groups. RESULTS: The Cornell voltage duration product criteria index had the greatest sensitivity in women (54.90%), and the Sokolow-Lyon-Rappaport index was most sensitive in men (73.53%). When applied to men at the same voltage amplitude (20mm) as that in women, the Cornell index showed increased sensitivity relative to the conventional index (28mm) of 67.65% (P<=0.01) and a sensitivity similar to that of the Sokolow-Lyon-Rappaport index, with higher specificity (P<=0.01). The White-Bock and Romhilt-Estes criteria were the least sensitive in men and women, despite their high specificity. The electrocardiographic criteria were more efficient when dilatation predominated over left ventricular hypertrophy. CONCLUSION: The Cornell index had greater sensitivity in women, and the Sokolow-Lyon-Rappaport index was more sensitive in men. When applied to men at the same voltage amplitude as that of women, the Cornell index had an increase in sensitivity similar to that of the Sokolow-Lyon-Rappaport index.

Parasitological diagnosis of schistosomiasis mansoni: fecal examination and rectal biopsy

Even with all progress in the search of sensitive and methods for the immunological diagnosis of schistosomiasis, the microscopic detection of eggs of the parasite in the stool still remains the most widely used tool for the actual diagnosis of active infection. Among the coproscopic methods, Kato's technic modified by Katz et al (Kato/Katz) has the advantages of higher sensitivity, the possibility of egg quantification, its low operational cost and its feasibility in areas with minimal infra-structure. The oorgram of the rectal mucosa is valuable in initial clinical trials of schistosomicides, when it is needed to observe egg morphology in tissue. It could be an alternative method for individual diagnosis, being more sensitive than a single stool exam in low intensity infection. However, the increased sensitivity of a higher number of fecal exams makes that invasiveprocedure unnecessary. In the assessment of cure of schistosomiasis, Kato/Katz method (three fecal samples in one, three and six months after treatment) and the rectal biopsy four months after treatment, are equally reliable.

Effect of electrolytic lesion of the dorsal raphe nucleus on water intake and sodium appetite

The present study determined the effect of an electrolytic lesion of the dorsal raphe nucleus (DRN) on water intake and sodium appetite. Male Wistar rats weighing 290-320 g with a lesion of the DRN (L-DRN), performed two days before experiments and confirmed by histology at the end of the experiments, presented increased sensitivity to the dehydration induced by fluid deprivation. The cumulative water intake of L-DRN rats reached 23.3 ± 1.9 ml (a 79% increase, N = 9) while sham-lesioned rats (SL-DRN) did not exceed 13.0 ± 1.0 ml (N = 11, P < 0.0001) after 5 h. The L-DRN rats treated with isoproterenol (300 µg kg-1 ml-1, sc) exhibited an increase in water intake that persisted throughout the experimental period (L-DRN, 15.7 ± 1.47 ml, N = 9 vs SL-DRN, 9.3 ± 1.8 ml, N = 11, P < 0.05). The L-DRN rats also showed an increased spontaneous sodium appetite during the entire period of assessment. The intake of 0.3 M NaCl after 12, 24, 36 and 72 h by the L-DRN rats was always higher than 20.2 ± 4.45 ml (N = 10), while the intake by SL-DRN was always lower than 2.45 ± 0.86 ml (N = 10, P < 0.00001). Sodium- and water-depleted L-DRN rats also exhibited an increased sodium appetite (13.9 ± 2.0 ml, N = 11) compared to SL-DRN (4.6 ± 0.64 ml, N = 11) after 120 min of observation (P < 0.02). The sodium preference of L-DRN rats in both conditions was always higher than that of SL-DRN rats. These results suggest that electrolytic lesion of the DRN overcomes a tonic inhibitory component of sodium appetite.

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 ± 0.7% in 2-month-old Cx (N = 5) vs 13.4 ± 0.3% (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 ± 0.9% in 2-month-old Cx (N = 5) vs 15.5 ± 0.3% (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 ± 1.2% in 10-month-old vs 9.8 ± 0.9% in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 ± 0.2% in 10-month-old vs 18.6 ± 0.7% in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Increased osmotic sensitivity for antidiuretic response in chronic chagas' disease

The osmotic threshold for attaining the antidiuretic response to hypertonic saline infusion and Progressive dehydration was studied in 31 patients with the chronic form of Chagas' disease and 16 control patients. The chagasic patients exhibited enhanced osmoticsensitivity to the antidiuretic response. This was demonstrated by lower values of the increments in plasma osmolarity sufficient to induce a significant fall in water clearance, without alterations in the osmolar clearance or creatinine excretion. The time needed to attain the antidiuretic response was shorterfor chagasics in relation to normal subjects. The results suggest the existence of a disturbance in the fine control of osmoregulation in the chagasic patients. They are interpreted to be a consequence of the denervation in hypothalamic or extrahypothalamic areas that regulate the secretion of vasopressin in chronic Chagas' disease.

RC-IAL cell line: sensitivity of rubella virus grow

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL² with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.

Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

Echocardiographic Assessment of the Pulmonary Venous Flow: An Indicator of Increased Pulmonary Flow in Congenital Cardiac Malformations

OBJECTIVE: To identify the left inferior pulmonary vein as an indirect marker of increased pulmonary flow in congenital heart diseases.METHODS: We carried out a prospective consecutive study on 40 patients divided into 2 groups as follows: G1 - 20 patients diagnosed with congenital heart disease and increased pulmonary flow; G2 (control group) - 20 patients who were either healthy or had congenital heart disease with decreased or normal pulmonary flow. We obtained the velocity-time integral of the left inferior pulmonary vein flow, excluding the "reverse A" wave, with pulsed Doppler echocardiography.RESULTS: In G1, 19 out of the 20 patients had well-identified dilation of the left inferior pulmonary vein. No G2 patient had dilation of the left inferior pulmonary vein. Dilation of the left inferior pulmonary vein in conditions of increased pulmonary flow had sensitivity of 95%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 95% (1 false-negative case). The integral of time and velocity of the pulmonary venous flow obtained with pulsed Doppler echocardiography was greater in the G1 patients (G1=25.0±4.6 cm versus G2=14.8±2.1 cm, p=0.0001).CONCLUSION: The identification of dilation of the left inferior pulmonary vein suggests the presence of congenital heart disease with increased pulmonary flow. This may be used as an indirect sign of increased flow, mainly in malformations of difficult diagnosis, such as atrial septal defects of the venous sinus or coronary sinus type.