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INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

Background Influenza virus infection is an important cause of under-five mortality. Maternal vaccination protects children younger than 3 months of age from influenza infection. However, it is unknown to what extent paediatric influenza-related mortality may be prevented by a maternal vaccine since global age-stratified mortality data are lacking. Methods We invited clinicians and researchers to share clinical and demographic characteristics from children younger than 5 years who died with laboratory-confirmed influenza infection between January 1, 1995 and March 31, 2020. We evaluated the potential impact of maternal vaccination by estimating the number of children younger than 3 months with in-hospital influenza-related death using published global mortality estimates. Findings We included 314 children from 31 countries. Comorbidities were present in 166 (53%) children and 41 (13%) children were born prematurely. Median age at death was 8·6 (IQR 4·5–16·6), 11·5 (IQR 4·3–24·0), and 15·5 (IQR 7·4–27·0) months for children from low- and lower-middle-income countries (LMICs), upper-middle-income countries (UMICs), and high-income countries (HICs), respectively. The proportion of children younger than 3 months at time of death was 17% in LMICs, 12% in UMICs, and 7% in HICs. We estimated that 3339 annual influenza-related in-hospital deaths occur in the first 3 months of life globally. Interpretation In our study, less than 20% of children is younger than 3 months at time of influenza-related death. Although maternal influenza vaccination may impact maternal and infant influenza disease burden, additional immunisation strategies are needed to prevent global influenza-related childhood mortality. The missing data, global coverage, and data quality in this study should be taken into consideration for further interpretation of the results. Funding Bill & Melinda Gates Foundation.

PURPOSE Adenocarcinoma is the most common histologic subtype of non–small-cell lung cancer, representing 40% of all diagnoses. Several biomarkers are currently used to determine patient eligibility for targeted treatments, including analysis of molecular alterations in EGFR and ALK, as well as programmed death-ligand 1 (PD-L1) protein expression. Epidemiologic data reporting the frequency of these biomarkers in Brazilian patients with lung adenocarcinoma (LUAD) are limited, and existing studies predominantly included patients from the southeast region of the country. MATERIALS AND METHODS The goal of this study was to investigate the frequency of somatic mutations in the EGFR, KRAS, NRAS, and BRAF genes, ALK, and PD-L1 expression in a series of Brazilian patients diagnosed with LUAD predominantly recruited from centers in southern Brazil. Molecular analysis of the EGFR, KRAS, NRAS, and BRAF genes was performed by next-generation sequencing using DNA extracted from tumor tissue. Immunohistochemistry was used to detect ALK and PD-L1 expression. RESULTS Analysis of 619 tumors identified KRAS mutations in 189 (30.2%), EGFR mutations in 120 (19.16%), and BRAF mutations in 19 (3%). Immunohistochemistry demonstrated ALK and PD-L1 expression in 4% and 35.1% of patients, respectively. CONCLUSION To our knowledge, this is the first study investigating the molecular epidemiology of patients with LUAD from southern Brazil and the largest assessing the frequency of multiple predictive biomarkers for this tumor in the country. The study also reveals a distinct mutation profile compared with data originating from other regions of Brazil.

The emergence of SARS-CoV-2 P.1 lineage coincided with a surge in hospitalisations in the North region of Brazil. In the South region’s Rio Grande do Sul state, severe COVID-19 case numbers rose 3.8 fold in February 2021. During that month, at a COVID-19 referral hospital in this state, whole-genome sequencing of a subset of cases’ specimens (n = 27) revealed P.1 lineage SARS-CoV-2 in most (n = 24). Findings raise concerns regarding a possible association between lineage P.1 and rapid case and hospitalisation increases.

The emergence SARS-CoV-2 P.1 lineage has been coincidental with a rapid growth in hospitalization in the northern region of Brazil. An exponential growth of severe COVID-19 occurred in Rio Grande do Sul state, Southern Brazil in February-2021. Whole-genome sequencing revealed that the previously undetected P.1 lineage accounted for 88.9% of specimens collected from patients at a referral COVID-19 hospital. These findings raise concerns regarding a possible association between lineage P.1 and rapid growth in cases and hospitalizations.

RT-qPCR for SARS-CoV-2 is the main diagnostic test used to identify the novel coronavirus. Several countries have used large scale SARS-CoV-2 RT-qPCR testing as one of the important strategies for combating the pandemic. In order to process the massive needs for coronavirus testing, the usual throughput of routine clinical laboratories has reached and often surpassed its limits and new approaches to cope with this challenge must be developed. This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR in a general population. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. In the first protocol (Protocol A); 10 clinical samples were pooled before RNA extraction. The second protocol (Protocol B) consisted of pooling the already extracted RNAs from 10 individual samples. Results from Protocol A were identical (100% agreement) with the individual results. However, for results from Protocol B, reduced agreement (91%) was observed in relation to results obtained by individual testing. Inconsistencies observed were related to RT-qPCR results with higher Cycle Thresholds (Ct > 32.73). Furthermore, in pools containing more than one positive individual, the Ct of the pool was equivalent to the lowest Ct among the individual results. These results provide additional evidence in favor of the clinical use of pooled samples for SARS-CoV-2 diagnosis by RT-qPCR and suggest that pooling of samples before RNA extraction is preferrable in terms of diagnostic yield.

Introduction: Tuberculosis (TB) persists as a severe global public health issue. The aim of the present study was to evaluate the performance of an in-house TB PCR (polymerase chain reaction) in sputum. Methods: DNA from sputum specimens were submitted to a nested-PCR protocol for the IS6110 region detection. PCR results were compared to those of the traditional methods for TB diagnosis, i.e., acid-fast bacilli (AFB) smear microscopy and culture. We analyzed sputum samples obtained from 133 patients. Results: A total of 48 (36%) cultures yielded indeterminate results due to contamination. This high contamination rate may be explained by the fact that samples from fibrocystic patients were included in this study. Additionally, other five samples were positive for nontuberculous mycobacteria (NTM). Therefore, it was possible to compare 80 patients for M. tuberculosis detection. We found 14 positive samples: five presented positive results in the three methods (5/14; 35.7%), two were positive in culture and PCR (2/14; 14.3%), one was positive in AFB and PCR (1/14; 7.1%), five were positive only in PCR (5/14; 35.7%) and 1 was positive only in culture (1/14; 7.1%). Thus, positivity rates for each technique were: 7.5% for AFB (6/80), 10% for culture (8/80) and 16.25% for PCR (13/80). Among the 48 patients who had indeterminate results in sputum culture, two samples were positive in PCR. Conclusion: Considering the limitations of the traditional methods, the use of PCR as a molecular technique could be advantageous for TB diagnosis.

The objective of this study was to compare the positive detection rates obtained using the Oxoid IMAGEN® direct immunofluorescence assay (designated as IF) with those obtained using the CLART® PneumoVir multiplex RT-PCR DNA microarray assay (designated as RT-PCR) in the diagnosis of respiratory viruses in hospitalized children. This was a retrospective study of 62 individuals18 years old who had nasopharyngeal aspirates collected for virus identification in a tertiary university hospital in south Brazil between January 1st, 2014 and December 31st, 2014. All 62 nasopharingeal aspirates were analyzed using both assay methods. The main outcome to be measured was the difference in the proportion of test samples returning a positive virus detection result between the IF and the RT-PCR. The McNemar test was used for data analysis and the results showed that the RT-PCR and the IF methods produced 55 (88.7 %) and 17 (27.4 %) virus-positive samples, respectively (p 0.001). The most prevalent virus was rhinovirus (45.5 % of the RT-PCR positive samples). The RT-PCR method increased the detection rates of human respiratory syncytial virus, influenza A virus and parainfluenza 3 virus. The RT-PCR and IF had concordant results in 19 samples (30.6 %) and discordant results in 43 samples (69.4 %). It is concluded that in comparison to the Oxoid IMAGEN® IF method, the CLART® PneumoVir multiplex RT-PCR method had a greater potential to contribute to the clinical management of hospitalized children due its greater ability in detecting respiratory viruses than the IF method.

Introdução: A infecção pelo vírus BK (BKV) em pacientes de transplante renal pode levar a disfunção do aloenxerto renal e perda do enxerto. A determinação precisa da carga viral do BKV é fundamental para prevenir a nefropatia associada ao BKV (BKVAN), mas o ponto de corte de melhor valor preditivo para BKVAN ainda é foco de debates. Objetivo: Avaliar o desempenho de um teste de qPCR comercial e outro desenvolvido internamente para detecção quantitativa de vírus BK em receptores de transplante renal. Métodos: O presente estudo prospectivo incluiu receptores de transplante renal de dois grandes hospitais universitários no Brasil. Os pacientes foram testados para infecção por BKV a cada três meses no primeiro ano pós-transplante com um teste comercial de reação em cadeia de polimerase quantitativa em tempo real (qPCR) e outro desenvolvido internamente. A presença de BKVAN foi confirmada com base na histopatologia. A área sob a curva para o qPCR plasmático foi determinada a partir da análise da característica de operação do receptor. Resultados: Um total de 200 pacientes foram incluídos. Cinquenta e oito por cento eram do sexo masculino, 19,5% tinham diabetes mellitus e 82% tiveram seus rins transplantados de doadores falecidos. Viremia de BKV foi detectada em 32,5% dos pacientes e oito (4%) foram diagnosticados com BKVAN. BKVAN foi associada a viremia de 4,1 log cópias/mL usando o kit comercial. O corte para o ensaio interno foi de 6,1 log cópias/ mL. A linearidade entre o kit comercial e o ensaio interno foi R2 = 0,83. Conclusão: Nosso estudo demonstrou uma acentuada variabilidade na carga viral de BKV quando diferentes metodologias de qPCR foram utilizadas. O ensaio interno de qPCR mostrou- -se clinicamente útil, além de ser uma opção menos onerosa em relação aos kits comerciais de qPCR. Há uma necessidade urgente de se definir padrões de BKV para a comunidade internacional. Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/ mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/ mL. The linearity between the commercial kit and the in-house assay was R2 =0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Outbreaks associated with rapidly growing mycobacteria (RGM) have been increasingly reported worldwide, including in Brazil. Among the RGM, theMycobacterium abscessuscomplex is the most pathogenic and related to multidrug resistance. The aim of this study was to evaluate the antimicrobial susceptibility and molecular profile of RGM isolates involved in new postsurgical infection outbreaks in Brazil since 2007. Of the 109 cases reported in the state of Rio Grande do Sul between 2007 and 2011, 43 (39 %) had confirmed mycobacterial growth in culture. Clinical isolates were obtained from biopsy specimens or abscess aspirates. PRA-hsp65restriction pattern identified the isolates asM. abscessustype 2, and partialrpoBsequencing confirmed the identification asM. abscessussubsp.bolletii. All isolates were susceptible to amikacin and resistant to ciprofloxacin, doxycycline, sulfamethoxazole, moxifloxacin and tobramycin. Most isolates (72 %) were fully susceptible to cefoxitin but six isolates (14 %) were fully resistant to clarithromycin. The latter differed from the susceptibility profiles of the previously described BRA100 clone from other Brazilian regions. Nevertheless, pulsed-field gel electrophoresis analysis revealed that these isolates belonged to a single BRA100 clone. In conclusion, our study reports the persistence of an emergent single and highly resistant clone ofM. abscessussubsp.bolletiifor several years even after national implementation of infection control measures.

Emerging reports have associated chronic pulmonary obstructive disease (COPD) with invasive aspergillosis (IA), particularly in patients treated with mechanical ventilation and/or corticosteroids. This is a multicentre study in which COPD patients demonstrating a new lung infiltrate while being mechanically ventilated were prospectively evaluated for the presence of IA. From the 47 patients studied, Aspergillus fumigatus was recovered in culture in two patients (4.2%). While serum galactomannan (GM) was negative for 94% of patients, GM levels in respiratory samples were >0.5, >1.0 and >1.5 for 74.5, 40.5, and 21.3% of patients, respectively. PCR was positive for 10 patients in the study but did not differentiate Aspergillus colonization from infection. The combination of PCR and GM in respiratory samples may be an interesting alternative to diagnose IA in COPD patients.

Introdução: O citomegalovírus humano (HCMV), causador de infecção latente, reativa com frequência em pacientes imunossuprimidos. Portanto, o HCMV permanece uma das infecções mais comuns após transplantes de órgãos sólidos e de células hematopoiéticas resultando em significativa morbidade, perda do enxerto e ocasional mortalidade. Assim, o diagnóstico precoce para uma terapia preventiva é de grande importância. Este estudo visa comparar o desempenho dos métodos PCR qualitativo in-house e antigenemia pp65 para o diagnóstico de infecção por CMV em pacientes imunossuprimidos do Hospital de Clínicas de Porto Alegre. Métodos: O estudo foi realizado em 216 amostras de sangue total (EDTA) coletadas de 85 pacientes, entre agosto de 2006 e janeiro de 2007. Resultados: Dentre as 216 amostras analisadas, 81 (37,5%) amostras apresentaram resultados positivos na PCR, enquanto 48 (22,2%) apresentaram resultados positivos na antigenemia. A sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo para a PCR, considerando antigenemia como padrão foram 87,5%, 76,8%, 51,8% e 95,5%, respectivamente. Conclusões: Estes resultados demonstraram que a PCR tem alta sensibilidade e valor preditivo negativo. Consequentemente PCR é especialmente indicada para o diagnóstico inicial de infecção por HCMV. No caso da estratégia de terapia preventiva, a identificação de pacientes com alto risco para a doença por HCMV é fundamental e a PCR pode ser uma ferramenta útil. Introduction: Human cytomegalovirus (HCMV) is often reactive in latently infected immunosuppressed patients. Accordingly, HCMV remains one of the most common infections following solid organ and hemopoietic stem cell transplantations, resulting in significant morbidity, graft loss and occasional mortality. The early diagnosis of HCMV disease is important in immunosuppressed patients, since in these individuals, preemptive treatment is useful. The objective of this study was to compare the performance of the in-house qualitative polymerase chain reaction (PCR) and pp65 antigenemia to HCMV infection in immunosuppressed patients in the Hospital de Clínicas of Porto Alegre (HCPA). Methods: A total of 216 blood samples collected between August 2006 and January 2007 were investigated. Results: Among the samples analyzed, 81 (37.5%) were HCMV-positive by PCR, while 48 (22.2%) were positive for antigenemia. Considering antigenemia as the gold standard, sensitivity, specificity, positive predictive values and negative predictive values for PCR were 87.5%, 76.8%, 51.8% and 95.5% respectively. Conclusions: These results demonstrated that qualitative PCR has high sensitivity and negative predictive value (NPV). Consequently PCR is especially indicated for the initial diagnosis of HCMV infection. In the case of preemptive treatment strategy, identification of patients at high-risk for HCMV disease is fundamental and PCR can be useful tool.

Although many studies have investigated the function of cellular prion protein (PrPc), its physiologic role remains elusive. PrPc null mice have been reported to develop normally and to show normal performance in most behavioural tests. In the present study we investigated whether this also holds true after episodes of acute stress. PrPc gene ablated (Prnp0/0) and wild-type mice were subjected to restraint stress, electric foot shock, or swimming and compared with non-stressed animals. Immediately after the stressful situation, the anxiety levels and locomotion of the animals were measured using plus-maze and open-field tests. Among non-stressed animals, there was no significant difference in performance between Prnp0/0 and wild type animals in either test. However, after acute stress provoked by a foot shock or a swimming trial, Prnp0/0 animals showed a significant decrease in anxiety levels when compared with control animals. Moreover, after the swimming test, knockout mice presented decreased locomotion when compared to wild-type mice. Because of this observation, we also assessed both types of mice in a forced swimming test with the objective of better evaluating muscle function and found that Prnp0/0 animals presented reduced forced swimming capacity when compared to controls. As far as we know, this is the first report suggesting that cellular prion protein is involved in modulation of anxiety or muscular activity after acute psychic or physical stress.

The effects of gabapentin, 400 mg and 800 mg, on anxiety induced by simulated public speaking (SPS) were investigated. Thirty-two normal male volunteers (aged 17-30 years) had their anxiety and mood evaluated by self-scales [Visual Analogue Mood Scale (VAMS) and Profile of Mood State (POMS)] during the SPS procedure. Physiological measures (heart rate and blood pressure) were taken. Treatment with gabapentin at 800 mg attenuated the anxiety of subjects that had a decrease on the VAMS item calm-excite. In addition, volunteers that received gabapentin at 400 mg and 800 mg showed a decrease in the hostility score in POMS. Our results suggest, in agreement with other studies, an anxiolytic potential to gabapentin.

Recent studies demonstrated important differences between short- and long-term memory mechanisms. Besides, the emotional component has a crucial role in memory formation. This study was carried out to answer whether there is a differential influence of emotional arousal in short- and long-term memory in healthy adults. Thirty-one healthy volunteers were divided into two major groups. In the first group long-term memory (LTM) was evaluated, with the testing session one week after training. The second group was tested 1 h after training, where short-term memory (STM) was evaluated. Each group was divided in to two subgroups. One half of the volunteers was exposed to an emotionally neutral story, and the other half of each group was exposed to a closely matched but more emotionally arousing story. The testing session consisted of a questionary containing 80 questions of multiple choices. The results were evaluated through percentage of correct answers. Results showed that correct answers were increased, in LTM measures, in the subjects that were given the emotional version of the test. In STM measures, no differences were found between the emotional and neutral version. However, the presentation of emotional story caused an emotional reaction in both groups. The lack of effect of emotional arousal in STM suggests that amygdala is not related to STM mechanisms. Further studies using different approaches are needed to elucidate if STM processes are influenced by emotional arousal.

Background: Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract illness in children worldwide. Molecular analyses show two distinct RSV groups (A and B) that comprise different genotypes. This variability contributes to the capacity of RSV to cause yearly outbreaks. These RSV genotypes circulate within the community and within hospital wards. RSV is currently the leading cause of nosocomial respiratory tract infections in pediatric populations. The aim of this study was to evaluate the G protein gene diversity of RSV amplicons. Methods: Nasopharyngeal aspirate samples were collected from children with nosocomial or community-acquired infections. Sixty-three RSV samples (21 nosocomial and 42 community-acquired) were evaluated and classified as RSV-A or RSV-B by real-time PCR. Sequencing of the second variable region of the G protein gene was performed to establish RSV phylogenetics. Results: We observed co-circulation of RSV-A and RSV-B, with RSV-A as the predominant group. All nosocomial and community-acquired RSV-A samples were from the same phylogenetic group, comprising the NA1 genotype, and all RSV-B samples (nosocomial and community-acquired) were of the BA4 genotype. Therefore, in both RSV groups (nosocomial and community-acquired), the isolates belonged to only one genotype in circulation. Conclusions: This is the first study to describe circulation of the NA1 RSV genotype in Brazil. Furthermore, this study showed that the BA4 genotype remains in circulation. Deciphering worldwide RSV genetic variability will aid vaccine design and development.

Extensive evidence suggests that N-methyl- d -aspartate (NMDA) glutamate receptor channels in the amygdala are involved in fear-motivated learning, and infusion of NMDA receptor antagonists into the amygdala blocks memory of fear-motivated tasks. Recent studies have shown that previous training can prevent the amnestic effects of NMDA receptor antagonists on spatial learning. In the present study, we evaluated whether infusion of the NMDA antagonist d,l -2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) impairs reinforcement of inhibitory avoidance learning in rats given previous training. Adult male Wistar rats (220–310 g) were bilaterally implanted under thionembutal anesthesia (30 mg/kg, i.p.) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Infusion of AP5 (5.0 μg) 10 min prior to training in a step-down inhibitory avoidance task (0.4 mA footshock) blocked retention measured 24 h after training. When infused 10 min prior to a second training session in animals given previous training (0.2 mA footshock), AP5 blocked the enhancement of retention induced by the second training. Control experiments showed that the effects were not due to alterations in motor activity or footshock sensitivity. The results suggest that NMDA receptors in the basolateral amygdala are involved in both formation of memory for inhibitory avoidance and enhancement of retention in rats given previous training.

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P

Adult male Wistar rats were bilaterally implanted with indwelling cannulae in four different coordinates of the cingulate cortex: (1) the anterior cingulate (AC), (2) the rostral region of the posterior cingulate (RC), (3) the upper portion of the caudal region of the posterior cingulate (UC), and (4) the lower portion of the caudal region of the posterior cingulate (LC). After recovery, animals were trained in a step-down inhibitory avoidance task (3.0-s, 0.4-mA foot shock). Either immediately, or 90 or 180 min after training, animals received a 0.5-microl infusion of vehicle (phosphate buffer, pH 7.4), of muscimol (0.5 microg), or of AP5 (5.0 microg). Retention testing was carried out 24 h after training. Muscimol was amnestic when given into any of the three coordinates of the posterior cingulate cortex 90 min after training, and when given into LC immediately post-training. In addition, AP5 was amnestic when given into UC 90 min post-training, but not when given into any other region and/or at any other time. None of the treatments had any effect when given into AC. The results suggest that memory processing of the inhibitory avoidance task is regulated by the posterior but not by the anterior cingulate cortex, through muscimol-sensitive synapses, relatively late after training. AP5-sensitive synapses appear to play a very limited role in these processes, restricted to UC.

Prions are the causative agents of transmissible spongiform encephalopathies. The transmissible agent (PrP Sc ) is an abnormal form of PrP C , a normal neuronal protein. The physiological role of PrP C remains unclear. In the present report, we evaluated behavioral parameters in Prnp 0/0 mice devoid of PrP C . Prnp 0/0 mice showed normal short- and long-term retention of a step-down inhibitory avoidance task and normal behavior in an elevated plus maze test of anxiety. During a 5-min exploration of an open field, Prnp 0/0 mice showed normal number of rearings, defecation, and latency to initiate locomotion, but a significant increase in the number of crossings. The results suggest that Prnp 0/0 mice show normal fear-motivated memory, anxiety and exploratory behavior, and a slight increase in locomotor activity during exploration of a novel environment.

Meningitis is a severe and potentially fatal form of tuberculosis. The diagnostic workup involves detection of acid-fast bacilli (AFB) in the cerebrospinal fluid (CSF) by microscopy or culture, however, the difficulty in detecting the organism poses a challenge to diagnosis. The use of the polymerase chain reaction (PCR) in the diagnostic approach to Mycobacterium tuberculosis (MTB) meningitis has been reported as a fast and accurate method, with several commercial kits available. As an alternative, some institutions have been developing inexpensive in house assays. In our institution, we use an in house PCR for tuberculosis. We analyzed the performance of our PCR for the diagnosis of MTB meningitis in 148 consecutive patients, using MTB culture as gold standard. The sensitivity and specificity of CSF PCR for the diagnosis of MTB meningitis was 50% and 98.6% respectively with a concordance with CSF mycobacterial culture of 96% (Kappa=0.52). In contrast to CSF cultures for MTB, our PCR test is a fast, simple and inexpensive tool to diagnose tuberculous meningitis with a performance similar to that obtained with the available commercial kits.

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia. Keywords: Burkholderia cenocepacia, Cystic fibrosis, PFGE, Genetic similarity

Introduction: Resistance to macrolides, lincosamides and streptogramins B (MLSB antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLSB resistance lead to three phenotypes, namely constitutive resistance (cMLSB), inducible resistance (iMLSB), and resistance only to macrolides and streptogramins B (MSB). The iMLSB resistance is the most difficult to detect in the clinical laboratory. Objective: This study investigated the expression of MLSB resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. Methods: Primary MLSB resistance was detected by the disk diffusion method. Isolates with iMLSB phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. Results: A total of 46.7% of staphylococci were positive for cMLSB; 3.3% for iMLSB and 3.3% for MSB. One or more erm genes were present in 50.1% of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. Conclusion: The prevalence of the ermA, ermB and ermC genes were 29.6%, 17.1% and 0.66% respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes. Keywords: Staphylococcus, resistance, erm genes, macrolides

Introdução: A infecção por citomegalovírus (CMV) é freqüente e acomete com significativa morbimortalidade indivíduos i-munossuprimidos, especialmente transplantados e pacientes HIV positivos com doença avançada. Objetivo: Este estudo visou a comparar o desempenho dos métodos reação em cadeia da polimerase qualitativo (PCR) e antigenemia pp65 para o diagnóstico de infecção por CMV em pacientes imunossuprimidos. Métodos: O estudo foi realizado em 216 amostras de sangue total coletadas de 85 pacientes, entre agosto de 2006 e janeiro de 2007, provenientes da internação ou ambulatório do Hospital de Clínicas de Porto Alegre (HCPA). Resultados: Entre as 216 amostras analisadas, 35 amostras apresentaram resultados positivos e 181 amostras apresentaram resultados negativos em uma e/ou outra técnica. Destas 35 amostras positivas, 16 foram positivas em ambas as técnicas; 12 apresentaram-se positivas pela PCR e negativas pela antigenemia; e sete amostras apresentaram-se positivas pela antigenemia e negativas pela PCR. Considerando a antigenemia como padrão-ouro, a técnica de PCR mostrou sensibilidade de 69,6%, especificidade de 93,8%, valor preditivo positivo de 57,1% e valor preditivo negativo de 96,3%. O coeficiente de correlação kappa foi de 0,578. Discussão: Este resultado demonstra que a PCR qualitativa apresenta moderada sensibilidade e alta especifi-cidade para o diagnóstico de infecção para CMV. Apesar de suas limitações, pode ser utilizada para exclusão do diagnóstico em pacientes com suspeita de infecção por CMV, por ser um método mais simples, de baixo custo e de fácil execução., Introduction: Cytomegalovirus (CMV) infection is frequent and has significant morbidity and mortality in immunosuppressed individuals, especially in transplanted and HIV-positive patients with advanced disease. Objective: This study compared the performance of qualitative polymerase chain reaction (PCR) and pp65 antigenemia methods for the diagnosis of CMV infection in immunosuppressed patients. Methods: A total of 216 of peripheral blood samples were collected from 85 inpatients or outpatients, from August 2006 through January 2007 from Hospital de Clínicas de Porto Alegre (HCPA). Results: Of the 216 samples, 35 had positive results and 181 were negative with regard to one and/or another technique. Of the 35 positive samples, 16 were positive in both techniques; 12 were positive for PCR and negative for antigenemia, and seven samples were positive for antigenemia but negative for PCR. Considering antigenemia as the gold standard, the PCR technique showed sensitivity of 69.6%, specificity of 93.8%, positive predictive value of 57.1%, and negative predictive value of 96.3%. The kappa correlation coefficient was 0.578. Discussion: These results demonstrate that the qualitative PCR has moderate sensitivity and high specificity for the diagnosis of CMV infection. Despite its limitations, it can be used for diagnosis exclusion in patients under CMV infection suspicion because of its simplicity, low cost and of easy execution.

The Passiflora extracts have been used in folk medicine because of its reputed sedative and anxiolytic properties. The present study aimed to compare the potential anxiolytic activity of two Passiflora spray-dried powders obtained from P. alata and P. edulis, known in Brazil as 'maracuja'. Male adult Swiss rats were treated with 200, 400 and 800 mg/kg of spray-dried powders p.o. and anxiolytic activity was evaluated using the elevated plus-maze test. The spray-dried powders showed anxiolytic activity in doses of 400 and 800 mg/kg. Our results support the potential anxiolytic effect of Passiflora spray-dried powders (P. alata and P. edulis).