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Introduction Syphilis is a globally occurring sexually transmitted disease caused by Treponema pallidum, a non-cultured in vitro bacterium. Molecular typing of Treponema pallidum strains isolated from patients are useful for investigating the molecular epidemiologic patterns, diversity of strains and antimicrobial resistance patterns.To date, there was no data on the circulating or prevalent subtype in Brazil. In this study we aimed to determine T. pallidum strain diversity and analyse for the mutation associated with macrolide resistance from patients with primary syphilis attended at CSEGPS. Methods We analysed 24 samples of primary lesion collected from patients attended at CSEGPS between 2013 and 2015. DNA was extracted with DNeasy kit (Qiagen). Standard PCR targeting tpp47 and polA genes was used for screening. Molecular typing was performed by CDC established methods, by determination of the 60 bp repeats within the arp gene, and RFLP analysis of tpr subfamily II genes (E, G and J). Completed by sequence analysis of a variable region of the tp0548 gene. The 23S rDNA mutation was analysed by DNA sequencing of PCR product. Results: T. pallidum DNA was detected in samples from 15 patients. Among 12 specimes typed, subtype found were 14d/g (6), 14d/d (5) and 12b/d (1). From 10 samples analysed for 23 rDNA mutation, all showed A2058-G, no mutation was detected at A2059. One case presented a different subtype in re-infection. The first was 14d/g and the second was 14d/d. Conclusion: T. pallidum detected in the samples of patients with primary syphilis are of subtypes 14d/g, 14d/d and 12b/d. The macrolide resistance mutation A2058-G was detected in all samples analysed. T. pallidum subtyping discriminated re-infection.

A neurocisticercose é causada por Cysticercus cellulose, a forma larval de Taenia solium, quando este se aloja no sistema nervoso central. O seu diagnóstico é realizado com base em dados clínicos, epidemiológicos, demonstração do agente etiológico pelas técnicas de imagem e testes laboratoriais. No presente estudo, apresentamos uma revisão do diagnóstico laboratorial, com ênfase no desempenho dos testes para pesquisa de anticorpos específicos e detecção de antígenos circulantes, utilização de antígeno homólogo ou heterólogo, nativo e recombinante, bem como a aplicação de métodos moleculares.

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(Psi)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process.

Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7%) and a specificity of 94.7% (95% CI, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities. O desempenho do teste rápido imunocromatográfico de procedência comercial Determine™ Syphilis TP (Abbott Lab.) para detectar anticorpos específicos contra antígenos treponêmicos foi avaliado com soros de pacientes tendo diagnóstico epidemiológico, clínico e laboratorial de sífilis, de pacientes com outras doenças sexualmente transmissíveis e de indivíduos clinicamente sadios ou com resultados negativos nos testes sorológicos para sífilis. Os valores de sensibilidade e de especificidade do teste Determine™ foram respectivamente de 93,6% e 95,2%, o valor preditivo positivo foi de 95,2% e o valor preditivo negativo 93,6%. O fenômeno de prozona foi observado em uma amostra de paciente com sífilis recente latente. O teste é de fácil e rápida execução, sem necessidade de equipamentos e o resultado pode ser obtido em 15 minutos, que de acordo com suas características diagnósticas poderá ser utilizado como teste alternativo de triagem em determinadas situações de emergência ou em pacientes que vivem em locais remotos.

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays. Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST) em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A especificidade verificada na análise destes antígenos recombinantes, indicam a sua utilidade na padronização de novos testes para o imunodiagnóstico da sífilis.

HIV-1, human T cell lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) and hepatitis C virus (HCV) are common among intravenous drug users (IDUs) and can cause chronic infections in the host. Usually, the diagnosis of such viruses employs serological assays; however, some difficulties in confirming HTLV-2 infection have been reported in high-risk populations in Brazil. We present data of an unusual case of coinfection with HIV-1, HTLV-1, HTLV-2, and HCV in a male IDU in which HTLV-2 was detected only by molecular assays. Comparative analysis of retroviruses from 2002 and 2012 showed identical HTLV-1 and HTLV-2 sequences (LTR, env, and tax), and a change in HIV-1 tropism from CXCR4 to CCR5. No mutation was detected in the hot points of the env region of the HTLV-2 isolate that justified the lack of rgp46-II-specific antibodies. These data emphasize the need for molecular assays to diagnose HTLV-2 in high-risk populations in Brazil.

INTRODUCAO: A utilidade da deteccao de anticorpos da imunoglobulina da classe M (IgM) no diagnostico da sifilis tem sido discutida ha tempos. OBJETIVO: No presente estudo foi analisada a ocorrencia de anticorpo IgM anti-T. pallidum (Tp-IgMAc) nas amostras de pacientes com sifilis recente, na fase de soroconversao e no monitoramento da resposta sorologica pos-tratamento. METODOS: Amostras sericas de 11 individuos. RESULTADOS: Na soroconversao, o Tp-IgMAc foi detectado nas amostras de 10 individuos, e em um paciente a reatividade IgM ocorreu anteriormente ao Venereal Disease Research Laboratory (VDRL). A sororreversao foi evidenciada nas amostras de tres pacientes com sifilis secundaria tratada, e em um individuo com reinfeccao. CONCLUSAO: A deteccao de Tp-IgMAc mostrou ser um potencial marcador diagnostico de sifilis ativa e o desempenho do ensaio imunoenzimatico de captura de IgM (ELISA-IgM) para o monitoramento pos-tratamento foi similar ao da VDRL.

Syphilis is a curable sexually transmitted infection caused by the bacterium Treponema pallidum; the infection can also be passed from mother to her fetus during pregnancy. Diagnosis of syphilis is based on clinical evaluation, detection of the causative organism, and confirmation of the disease by serodiagnosis. T. pallidum cannot be cultured in the laboratory, but can be identified in lesions using dark-field or fluorescence microscopy or by molecular techniques. Most infected individuals have no symptoms or have transient lesions and therefore a serological test must be used to screen for infection.

Serologic monitoring is the way to determine adequate treatment response, which is typically defined as a fourfold decline in Rapid Plasma Reagin (RPR) or Veneral Disease Research Laboratory (VDRL) titer within 6 months of treatment for patients with primary or secondary syphilis and within 12 months of treatment for patients with early latent syphilis. Studies documented close to 100% rate seroreversion 1 -2 years after penicillin treatment, depending on the stage of syphilis and duration of symptoms. HIV co-infection has several effects on the presentation, diagnosis, disease progression, and therapy of syphilis. Unusual syphilis serologic titers have been reported in HIV-infected patients, with either unexpected high or low titer, as well as delayed serologic response at the time of diagnosis. There are several studies concerned to syphilis serological treatment response comparing HIV-infected and HIV-negative patients. Some studies reported a slower serological response in HIV infected patients. A recent study showed that the use of high active antiretroviral therapy (HAART) and the routine use of macrolides for the prevention of opportunistic infections may reduce syphilis serologic failure rates among HIV-infected patients who have syphilis. Penicillin G, administered parenterally, is the preferred drug for treating all stages of syphilis (French et al., 2009; Workowski & Berman, 2010). Treatment failure can occur with any regimen. Assessing response to treatment frequently is difficult, and definitive criteria for cure or failure have not been established. According to the CDC guidelines recommendations, clinical and serologic evaluation should be performed 6 months and 12 months after treatment of patients with primary or secondary syphilis; more frequent evaluation might be prudent if follow-up is uncertain. Patients who have signs or symptoms that persists or recurs or who have sustained fourfold increase in nontreponemal test titer probably failed treatment or were reinfected. The latent syphilis is not transmitted sexually; the objective of treating patients with this stage of disease is to prevent complication. Quantitative nontreponemal serologic tests should be repeated at 6, 12, and 24 months for followup. Limited information is available concerning clinical and serological follow-up of patients who have tertiary syphilis. HIV-infected persons should be evaluated clinically and serologically for treatment failure at 3, 6, 9, 12, and 24 months after therapy (Workowski & Berman, 2010). Usually, treatment failure cannot be distinguished from reinfection with T. pallidum; in these cases a CSF analysis is recommended.

In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Ribosomal RNAs (rRNAs) form a fundamental structure of ribosome, and play crucial roles in protein biosynthesis. To investigate functional importance of RNA helices in both 16S and 23S rRNAs, we devised an E. coli genetic system that allowed us to identify and select functional deletions in rRNAs. We here describe many RNA helices in rRNAs to be shortened or completely truncated. These results suggest architectural flexibility of rRNAs in ribosome assembly and function.

Ribosomal RNAs (rRNAs) play crucial roles in protein biosynthesis. The decoding center of 16S rRNA in 30S subunit affords a place for interaction between mRNA and tRNA, and contributes to the fidelity of the decoding by monitoring the codon-anticodon base pairing. The helices 18 and 44 in 16S rRNA are known to be major components of the decoding center. To investigate functional role of the conserved sequence in rRNAs, we employed a new genetic method that allows us to identify and select from randomized E. coli rRNA libraries those rRNA sequences absolutely required for the ribosome function. Functional consensus sequences were identified in both helices, providing us with a new insight into the decoding mechanism.

Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis