Soroepidemiologia de Chlamydia trachomatis, Chlamydia pneumoniae e Treponema pallidum em portadores do Vírus da imunodeficiência humana (HIV), no Estado do Pará

A Chlamydia trachomatis e o Treponema pallidum compartilham com o HIV uma importante forma de transmissão: a via sexual. Por conta do comprometimento imunológico dos portadores de HIV, a C. pneumoniae pode apresentar um papel potencial em infecções respiratórias. Este trabalho objetivou a descrição da soroprevalência destes três agentes em portadores de HIV do Estado do Pará, Brasil. Entre setembro de 2007 a junho de 2008, foram coletadas 430 amostras de portadores de HIV em Belém, Pará. Estas foram submetidas a um ELISA para detecção de anticorpo IgG e IgM anti-Chlamydia e, dentre os positivos, uma amostragem aleatória foi escolhida e submetida à microimunofluorescência para sorotipagem. Para a detecção de anticorpos anti-Treponema pallidum foi feito um teste não treponêmico (RPR) e um teste treponêmico (ELISA). Os resultados obtidos foram analisados pelo teste do χ². A prevalência geral de anticorpos anti-Chlamydia foi 64,2% (51,6% para IgG e 4% para IgM). A sorotipagem mostrou uma alta prevalência de C. trachomatis (100% tanto para IgG como IgM), e C. pneumoniae (73,5% IgG e 70,5% IgM), sendo que houve uma larga disseminação dos sorotipos que causam infecções genitais da Chlamydia trachomatis. A prevalência geral de anticorpos contra o Treponema pallidum foi de 34,9%, sendo que 7,3% apresentaram resultado laboratorial indicativo de sífilis. As variáveis que apresentaram associação com a infecção por Chlamydia e Treponema pallidum foram: o gênero masculino, maior idade, baixa escolaridade, número de parceiros por semana, a prática de sexo anal, homossexualismo/bissexualismo, uso de droga não-endovenosa, histórico de IST. Faz-se necessário tanto a conscientização como o monitoramento da população, para impedir a transmissão destes agentes e para a melhoria da qualidade de vida dos indivíduos portadores de HIV.

Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazi

INTRODUÇÃO: Apesar das medidas de controle da sífilis materna e congênita estarem disponíveis no Brasil, existem dificuldades da rede em prover o diagnóstico laboratorial da infecção durante o pré-natal. O objetivo deste estudo foi confirmar a presença do Treponema pallidum pela PCR em mulheres com sorologia positiva ao VDRL e com resultado letal da gravidez, isto é, aborto, natimorto e neomorto. MÉTODOS: Estudo retrospectivo realizado em mulheres VDRL-sororeativas com resultado negativo da gravidez, admitidas na Fundação Santa Casa de Misericórdia do Pará FSCM-PA entre janeiro e julho de 2004. As amostras de soro e DNA de sangue total foram obtidas no mesmo período da triagem pelo VDRL. Estas amostras foram analisadas pelo ELISA IgG, FTA-Abs IgM e PCR simples (polA). RESULTADOS: Durante o período de estudo, 0,7% (36/4.912) das mulheres com resultado letal da gravidez apresentaram VDRL positivo. O genepolA foi amplificado em 72,7% (24/33) destas mulheres,com 55,6% (20/36) e 94,4% (34/36) apresentando anticorpos tipo IgG e IgM contra o T. pallidum, respectivamente. A comparação destes resultados mostrou uma diferença estatística significativa, sendo que os resultados da PCR versus FTA-Abs Ig Mmostraram-se concordantes, sugerindo que a sífilis materna era uma infecção ativa. A causa básica de morte dos conceptos não foi relatada em 97,2% (35/36) dos casos. Entre as mulheres que foram submetidas ao VDRL no pré-natal, somente quatro das nove soropositivas receberam tratamento. CONCLUSÕES: A elevada frequência de sífilis no grupo de estudo indica a fragilidade do serviço no diagnóstico, tratamento e monitoramento da infecção, comprometendo o controle epidemiológico.

Prevalence of Treponema Species Detected in Endodontic Infections: Systematic Review and Meta-regression Analysis

Introduction: This systematic review and meta-regression analysis aimed to calculate a combined prevalence estimate and evaluate the prevalence of different Treponema species in primary and secondary endodontic infections, including symptomatic and asymptomatic eases. Methods: The MEDLINE/PubMed, Embase, Scielo, Web of Knowledge, and Scopus data-bases were searched without starting date restriction up to and including March 2014. Only reports in English were included. The selected literature was reviewed by 2 authors and classified as suitable or not to be included in this review. Lists were compared, and, in case of disagreements, decisions were made after a discussion based on inclusion and exclusion criteria. A pooled prevalence of Treponema species in endodontic infections was estimated. Additionally, a meta-regression analysis was performed. Results: Among the 265 articles identified in the initial search, only 51 were included in the final analysis. The studies were classified into 2 different groups according to the type of endodontic infection and whether it was an exclusively primary/secondary study (n = 36) or a primary/secondary comparison (n = 15). The pooled prevalence of Treponema species was 41.5% (95% confidence interval, 35.9-47.0). In the multivariate model of meta-regression analysis, primary endodontic infections (P < .001), acute apical abscess, symptomatic apical periodontitis (P < .001), and concomitant presence of 2 or more species (P = .028) explained the heterogeneity regarding the prevalence rates of Treponema species. Conclusions: Our findings suggest that Treponema species are important pathogens involved in endodontic infections, particularly in cases of primary and acute infections.

Complete Genome Sequence of Treponema paraluiscuniculi, Strain Cuniculi A: The Loss of Infectivity to Humans Is Associated with Genome Decay.

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.

Characterization of humoral immune responses against Treponema pallidum antigens

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with an estimated 12 million new cases per year worldwide. There is no vaccine currently available for the prevention of syphilis. In the present study, the T. pallidum hypothetical protein TP0693 was examined to determine its cellular location, and its potential for use as a vaccine candidate and immunodiagnostic for syphilis. TP0693 was demonstrated to be strongly reactive with sera from rabbits infected experimentally with T. pallidum for >25 days. Results from proteinase K digestion, immunofluorescence and immunoelectron microscopy were consistent with outer surface localization of TP0693. Serum reactivity against TP0693 was detected in only 68% of syphilis patients, which does not support its use as an immunodiagnostic for syphilis. Immunization of rabbits with TP0693 or three other outer membrane candidates did not alter the course of lesion development atter T. pallidum inoculation. We also examined the T. pallidum proteome by two-dimensional gel electrophoresis coupled with mass spectrometry analysis and immunoblotting. This approach resulted in the identification of 95 unique polypeptides, several of which were reactive with sera from infected rabbits and syphilis patients. The analyses described here enabled us to identify antigens potentially useful as vaccine candidates or diagnostic markers, and may provide insight into host-pathogen interactions during T. pallidum infection. ^

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Spherical body formation in the spirochaete Brachyspira hyodysenteriae

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 mu m in diameter, some much larger spherical bodies (6-12 mu m diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mu m in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.

Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

Syphilis at the Crossroad of Phylogenetics and Paleopathology

The origin of syphilis is still controversial. Different research avenues explore its fascinating history. Here we employed a new integrative approach, where paleopathology and molecular analyses are combined. As an exercise to test the validity of this approach we examined different hypotheses on the origin of syphilis and other human diseases caused by treponemes (treponematoses). Initially, we constructed a worldwide map containing all accessible reports on palaeopathological evidences of treponematoses before Columbus's return to Europe. Then, we selected the oldest ones to calibrate the time of the most recent common ancestor of Treponema pallidum subsp. pallidum, T. pallidum subsp. endemicum and T. pallidum subsp. pertenue in phylogenetic analyses with 21 genetic regions of different T. pallidum strains previously reported. Finally, we estimated the treponemes' evolutionary rate to test three scenarios: A) if treponematoses accompanied human evolution since Homo erectus; B) if venereal syphilis arose very recently from less virulent strains caught in the New World about 500 years ago, and C) if it emerged in the Americas between 16,500 and 5,000 years ago. Two of the resulting evolutionary rates were unlikely and do not explain the existent osseous evidence. Thus, treponematoses, as we know them today, did not emerge with H. erectus, nor did venereal syphilis appear only five centuries ago. However, considering 16,500 years before present (yBP) as the time of the first colonization of the Americas, and approximately 5,000 yBP as the oldest probable evidence of venereal syphilis in the world, we could not entirely reject hypothesis C. We confirm that syphilis seems to have emerged in this time span, since the resulting evolutionary rate is compatible with those observed in other bacteria. In contrast, if the claims of precolumbian venereal syphilis outside the Americas are taken into account, the place of origin remains unsolved. Finally, the endeavor of joining paleopathology and phylogenetics proved to be a fruitful and promising approach for the study of infectious diseases.

Association between HIV-1 infection, the etiology of genital ulcer disease, and response to syndromic management

Background: Reports on the effect of HIV-1 infection on healing rates of ulcers are conflicting. Goal: The goal was to determine the etiology and response to treatment of genital ulcer disease (GUD) in relation to HIV-1 infection. Study Design: This was a cohort study of patients with GUD treated with local syndromic management protocols. Results: Among the 587 recruited, the prevalences of infections due to HSV, Treponema pallidum, Chlamydia trachomatis (lymphogranuloma venereum [LGV]), Haemophilus ducreyi, Calymmatobacterium granulomatis, and HIV-1 were 48%, 14%, 11%, 10%, 1%, and 75%, respectively. The prevalence of T pallidum was higher among men (P = 0.03), and an association was seen among HIV-1-seronegatives on univariate and multivariate analyses (P < 0.001; P = 0.01). The prevalence of C trachomatis (LGV) was higher among females (P = 0.004), and an association was seen among HIV-1-seropositives on univariate analysis (P = 0.04). At follow-up, 40/407 (10%) showed a decreased healing tendency, not associated with ulcer etiology or HIV-1 seropositivity. Conclusion: Response to syndromic management of GUD was acceptable and not associated with HIV-1 coinfection.

Advantages and Pitfalls of the Polymerase Chain Reaction in the Diagnosis of Esophageal Ulcers in AIDS Patients

HIV-1-infected patients frequently have opportunistic esophageal infections which, when associated with severe immunodeficiency, can be attributed to unusual pathogens. The clinical presentation of several esophageal diseases is similar and the best method for a specific diagnosis of these patients has not been well defined. To evaluate the role of the polymerase chain reaction (PCR) in the etiologic definition of esophageal ulcers in HIV-1-infected patients, 96 esophageal biopsies from 79 HIV-1-infected patients were processed by PCR using specific primers for cytomegalovirus (CMV), herpes virus (HSV), human papilloma virus (HPV), HIV-1, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Treponema pallidum, and Haemophilus ducreyi. The PCR results were compared to the histopathologic results. Seventy-nine patients were studied (mean age: 34 years; 62% men; median CD4 + T cell = 103.59 cells/mu l (range 1-795.2 cells/mu l). The most common endoscopic findings were as follows: esophageal candidiasis (37.1%), esophageal ulcers (24.7%), esophagitis (11.2%), and lugol-negative areas (10.1%). The histopathologic findings in the esophageal ulcers (22 biopsies) were non-specific inflammation (31.8%), HSV (36.4%), Candida (13.6%), CMV (13.6%), or HPV disease (4.5%). In the esophageal ulcer biopsies, the PCR results were negative in 27.6% of cases, and positive for HIV (65.5%), CMV (31%), HPV (20.7%), HSV (10.3%), and H. ducreyi (6.9%). The histopathologic examination did not identify a pathogen or identified only Candida in 15 biopsies of esophageal ulcers. PCR was positive in ten (66.7%) and negative in five (33.3%) of these biopsies (idiopathic ulcers). PCR detected: HIV (53.3%), CMV (20%), HPV (13.3%), and H. ducreyi (6,7%). PCR detected more etiologic agents in esophageal ulcers than histopathology and was able to detect unusual pathogens. On the other hand, sometimes more than one pathogen was detected in the esophageal ulcers, making it difficult to reach an accurate diagnosis. This finding indicates the need for more studies to evaluate the benefit of this method in the routine evaluation of esophageal ulcer biopsies in HIV-1-infected patients.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63 kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Avaliação do desempenho dos Laboratórios Portugueses nos últimos 10 anos quanto ao diagnóstico de Sífilis

O Programa Nacional de Avaliação Externa da Qualidade (PNAEQ) disponibiliza desde 1994 um programa de Avaliação Externa da Qualidade para avaliação do desempenho dos laboratórios em Sífilis. Neste programa são enviadas amostras de matriz humana para o diagnóstico da sífilis, para pesquisa de anticorpos de Treponema pallidum através dos testes disponíveis nos laboratórios (testes treponémicos e/ou não treponémicos). O objetivo principal deste estudo é avaliar o desempenho dos laboratórios participantes de 2005 a 2015.