Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

ABSTRACT The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p < 0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.

Infection by Streptococcus pneumoniae in children with or without radiologically confirmed pneumonia / Infecção por Streptococcus pneumoniae em crianças com ou sem pneumonia radiologicamente confirmada

Abstract Objective: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. Methods: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n = 249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. Results: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. Conclusions: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.

Resumo Objetivo: Avaliar o papel do raios X de tórax na identificação de casos de pneumonia adquirida na comunidade (PAC) causada por agentes bacterianos. Métodos: A frequência de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com PAC não hospitalizadas foi comparada com a presença de confirmação radiológica da pneumonia (n = 249 crianças com pneumonia radiologicamente confirmada e 366 crianças com raios X de tórax normal). Infecção por S. pneumoniae foi diagnosticada com base na resposta sorológica a pelo menos uma dentre oito proteínas pneumocócicas investigadas (aumento ≥ 2 vezes nos níveis de IgG em relação a Ply, CbpA, PspA1 e 2, PhtD, StkP-C e PcsB-N ou aumento≥ 1,5 vez em relação aPcpA). Infecção por H. influenzae e M. catarrhalis foi definida por aumento ≥ 2 vezes nos níveis de IgG específica a antígenos de cada agente. Resultados: Crianças com pneumonia radiologicamente confirmada apresentaram maior taxa de infecção pelo pneumococo. Além disso, a presença de infecção pneumocócica foi um fator preditor de pneumonia radiologicamente confirmada, o que aumenta sua chance de detecção em 2,8 vezes (IC 95%: 1,8-4,3). O valor preditivo negativo do raios X normal para a infecção por S. pneumoniae foi 86,3% (IC95%: 82,4%-89,7%). Não houve diferença nas frequências de infecção por H. influenzae e M. catarrhalis entre crianças com PAC com ou sem confirmação radiológica. Conclusão: Crianças com diagnóstico clínico de PAC submetidas a um raios X de tórax que apresentam confirmação radiológica têm maior taxa de infecção por S. pneumoniae comparadas com as crianças com raios X normal.

Ehrlichia canis and Rickettsia spp. in dogs from urban areas in Paraiba state, northeastern Brazil / Ehrlichia canis e Rickettsia spp. em cães de áreas urbanas da Paraíba, nordeste do Brasil

Abstract The aims of our study was to identify Ehrlichia canis and antibodies against Rickettsia spp. belonging to the spotted fever group (SFG) in dogs sampled from Paraiba state, northeastern Brazil. Blood and serum samples collected by convenience from dogs in urban areas of five municipalities were analyzed by real-time PCR for the detection of E. canis DNA and by immunofluorescence assay test (IFAT) for the identification of antibodies against Rickettsia rickettsii, R. felis, R. parkeri, R. amblyommii and R. rhipicephali antigens. E. canis DNA was detected in 8.9% (64/719) of the blood samples, whereas 5.63% (43/763) of the serum samples were positive for at least one of the Rickettsia antigens tested by IFAT. This study showed for the first time the occurrence of E. canis and suggested the circulation of SFG Rickettsia in dogs in the study region of Paraiba state, northeastern Brazil.

Resumo Os objetivos do nosso estudo foram identificar Ehrlichia canis e anticorpos contra Rickettsia spp. pertencentes ao Grupo da Febre Maculosa (GFM) em cães amostrados no estado da Paraíba, nordeste do Brasil. As amostras de sangue e soro, coletados por conveniência, de cães em áreas urbanas de cinco municípios foram analisadas por PCR em tempo real para a detecção de DNA de E. canis e pela Reação de Imunofluorescência Indireta (RIFI) para identificação de anticorpos contra Rickettsia rickettsii, R. felis, R. parkeri, R. amblyommii e R. rhipicephali. O DNA de E. canis foi detectado em 8,9% (64/719) das amostras de sangue, enquanto que 5,63% (43/763) das amostras de soro foram positivas para pelo menos um dos antígenos de Rickettsia testados por RIFI. Este estudo mostrou pela primeira vez a ocorrência de E. canis e sugere a circulação de Rickettsia do GFM em cães na região em estudo do estado da Paraíba, Nordeste do Brasil.

The role of environmental tobacco exposure and Helicobacter pylori infection in the risk of chronic tonsillitis in children / O papel da exposição ambiental do tabaco e infecção pelo Helicobacter pylori no risco de amigdalite crônica em crianças

ABSTRACT CONTEXT AND OBJECTIVE: Helicobacter pylori (H. pylori) is a chronic infectious pathogen with high prevalence. This study investigated the interaction between environmental tobacco exposure and H. pylori infection on the incidence of chronic tonsillitis in Chinese children. DESIGN AND SETTING: Cross-sectional study performed in an outpatient clinic in China. METHODS: Pediatric patients with chronic tonsillitis were enrolled. H. pylori infection was determined according to the presence of H. pylori CagA IgG antibodies. Serum cotinine levels and environmental tobacco smoke (ETS) exposure were determined for all participants. RESULTS: There was no significant difference in H. pylori infection between the children with chronic tonsillitis and children free of disease, but there was a significant difference in ETS between the two groups (P = 0.011). We next studied the association between ETS and chronic tonsillitis based on H. pylori infection status. In the patients with H. pylori infection, there was a significant difference in ETS distribution between the chronic tonsillitis and control groups (P = 0.022). Taking the participants without ETS as the reference, multivariate logistic regression analysis showed that those with high ETS had higher susceptibility to chronic tonsillitis (adjusted OR = 2.33; 95% CI: 1.67-3.25; adjusted P < 0.001). However, among those without H. pylori infection, ETS did not predispose towards chronic tonsillitis. CONCLUSION: Our findings suggest that tobacco exposure should be a putative mediator risk factor to chronic tonsillitis among children with H. pylori infection.

RESUMO CONTEXTO E OBJETIVO: Helicobacter pylori (H. pylori) é um patógeno infeccioso crônico com alta prevalência. Este estudo investigou a interação entre exposição à fumaça ambiental do tabaco (FAT) e infecção pelo H. pylori sobre a incidência de amigdalite crônica em crianças chinesas. TIPO DE ESTUDO E LOCAL: Estudo transversal desenvolvido num ambulatório na China. MÉTODOS: Pacientes pediátricos com amigdalite crônica foram recrutados. A infecção por H. pylori foi determinada segundo a presença de anticorpos H. pylori CagA IgG. Foi determinado o nível de cotinina sérica e exposição à FAT de todos os participantes. RESULTADOS: Não houve diferença significativa entre crianças com amigdalite crônica na infecção por H. pylori e sem amidalite, mas existia diferença significativa na FAT entre os dois grupos (P = 0,011). Em seguida, estudamos a associação entre FAT e amigdalite crônica com base no status de infecção por H. pylori. Nos pacientes com infecção por H. pylori, houve diferença significativa na distribuição de FAT entre os grupos de amigdalite crônica e controle (P = 0,022). Tomando os participantes sem FAT como referência, a análise de regressão logística multivariada mostrou que aqueles com alta FAT tinha maior susceptibilidade à amigdalite crônica (OR ajustado IC = 2,33, 95%: 1,67-3,25, ajustado P < 0,001). No entanto, naqueles sem infecção por H. pylori, a FAT não predispôs a amigdalite crônica. CONCLUSÃO: Nossos achados sugerem que a exposição ao tabaco é um fator de risco para amigdalite crônica em crianças com infecção por H. pylori.

Number of leprosy reactions during treatment: clinical correlations and laboratory diagnosis

Abstract INTRODUCTION: The occurrence of leprosy reactions, a common event during treatment, may be mostly related to the action of multidrug therapy on Mycobacterium leprae. The clinical and laboratory monitoring of patients with reactions is important, since collecting data that assists in predicting the risk of reactions may help to prevent disability. METHODS: This was a sectional study, in order to correlate clinical and laboratory diagnosis with the number of reactions during treatment. Spearman's correlation was used to verify the degree of association between the assessed variables. RESULTS: This study was conducted with 211 patients with leprosy reactions during treatment of M. leprae. The borderline tuberculoid group was the most prevalent clinical form (74/211; 35.1%) and the type one reaction showed the highest frequency (136/211; 64.5%). It was observed that 73.5% (155/211) of reactions occurred within 3 months of the initiation of multidrug therapy. The diagnostic values, including the bacterial indices (BIs) of dermal smears (r = 0.21, p < 0.05) and skin biopsies (r = 0.20; p < 0.05), showed a positive correlation with the number of reactions during treatment. CONCLUSIONS: This research showed a positive correlation between bacillary load markers and the number of leprosy reactions. This study provided scientific support to future research aiming to elucidate the influence of antigenic load on the number of leprosy reactions during treatment.

Integrative literature review of the reported uses of serological tests in leprosy management

Abstract: An integrative literature review was conducted to synthesize available publications regarding the potential use of serological tests in leprosy programs. We searched the databases Literatura Latino-Americana e do Caribe em Ciências da Saúde, Índice Bibliográfico Espanhol em Ciências da Saúde, Acervo da Biblioteca da Organização Pan-Americana da Saúde, Medical Literature Analysis and Retrieval System Online, Hanseníase, National Library of Medicine, Scopus, Ovid, Cinahl, and Web of Science for articles investigating the use of serological tests for antibodies against phenolic glycolipid-I (PGL-I), ML0405, ML2331, leprosy IDRI diagnostic-1 (LID-1), and natural disaccharide octyl-leprosy IDRI diagnostic-1 (NDO-LID). From an initial pool of 3.514 articles, 40 full-length articles fulfilled our inclusion criteria. Based on these papers, we concluded that these antibodies can be used to assist in diagnosing leprosy, detecting neuritis, monitoring therapeutic efficacy, and monitoring household contacts or at-risk populations in leprosy-endemic areas. Thus, available data suggest that serological tests could contribute substantially to leprosy management.

Lack of association between helicobacter pylori's virulence and increased serum c-reactive protein levels in functional dyspeptic patients

ABSTRACT Background Recently, a great variety of studies aimed to investigate and even suggestHelicobacter pylori as an important key factor in gastrointestinal and non-gastrointestinal events development. The well-established relationship between bacterial virulence and increased risk for peptic ulcer or gastric carcinoma is not so clear when comparing inflammation markers alterations, such C-reactive protein, with the pathogen. Objective The objective of this study was to evaluate the presence of H. pylori, bacterial virulence and C-reactive protein serum levels in individuals diagnosed with functional dyspepsia. Methods Were prospectively included in this study 489 dyspeptic individuals. They fulfill Rome III clinical criteria for the diagnosis of functional dyspepsia with no organic disease at endoscopy. The bacterial infection was established by histology and urease rapid test. The levels of serum C-reactive protein were obtained by immunonefelometry and CagA status ofH. pylori positive individuals was determined through an imunoenzimatic assay. Results Prevalence rate of H. pylori was 66.3% and virulence factor CagA was detected in nearly 43% of positive samples. In addition, it has been noticed an association between Ilex paraguariensis(yerba maté) consumption and pathogen's prevalence. An important effect of bacterial infection on inflammation was only observed in gastric epithelium. Conclusion No systemic response to the pathogen, measured through C-reactive protein levels, was observed, regardless of CagA status. Otherwise, the intake of yerba maté should be considered as a cultural factor possibly related toH. pylori's transmission.

RESUMO Contexto Recentemente, uma grande variedade de estudos tem investigado e até mesmo sugerido a presença de Helicobacter pylori como um importante fator no desenvolvimento de eventos restritos ou não ao trato gastrointestinal. A relação já bem estabelecida entre virulência bacteriana e risco aumentado para úlcera péptica ou adenocarcinoma gástrico não parece estar tão elucidada quando se comparam alterações de marcadores inflamatórios, como a proteína C-reativa, com a presença do patógeno. Objetivo O objetivo deste estudo foi avaliar a presença da infecção por H. pylori, a virulência bacteriana e os níveis séricos de proteína C-reativa em indivíduos diagnosticados com dispepsia funcional. Métodos Foram incluídos neste estudo, prospectivamente, 489 indivíduos dispépticos. Os pacientes deveriam preencher os critérios clínicos de Roma III para o diagnóstico de dispepsia funcional sem apresentar doença orgânica evidenciada a partir da endoscopia. A infecção bacteriana foi estabelecida por histologia e pelo teste rápido da urease. Os níveis de proteína C-reativa foram quantificados através de imunonefelometria e o status para a presença da CagA dos indivíduos infectados por H. pylorifoi determinado por ensaio imunoenzimático. Resultados A taxa de prevalência de H. pylori foi de 66.3% e o fator de virulência CagA foi detectado em aproximandamente 43% das amostras positivas. Adicionalmente, denotou-se uma associação entre o consumo deIlex paraguariensis (chimarrão) e a prevalência do patógeno. Um importante efeito da infecção bacteriana na inflamação apenas foi observado localmente, no epitélio gástrico. Conclusão Não foi evidenciada resposta sistêmica ao patógeno aferido através dos níveis de proteína C-reativa, independentemente do status para CagA. Por outro lado, o consumo de chimarrão pode ser sugerido como um fator cultural possivelmente relacionado à transmissão de H. pylori.

Factors associated with seropositivity for APGL-Iamong household contacts of leprosy patients

Abstract: INTRODUCTION: Leprosy is mainly transmitted among family members who share genetic and ambient factors. The clinical form of leprosy in the index case and kinship could be risk factors for leprosy transmission. High antibody levels in household contacts (HC) in the absence of neural or skin lesions may characterize latent infection. This study aimed to evaluate the association between seropositivity for anti-phenolic glycolipid-I immunoglobulin M antibodies (APGL-I) in HC and the clinical classification of the index case and to analyze the association between APGL-I positivity with other factors such as age, kinship, and gender. METHODS: We performed a survey among 320 HC of 120 leprosy patients who were evaluated and followed-up in a leprosy outpatient clinic of a university hospital. All HC underwent complete skin examination, peripheral nerve palpation, skin sensory tests, and serologic tests for the detection and quantification of APGL-I. RESULTS: The overall seropositivity rate was 20%, and was greatly affected by kinship. APGL-I seropositivity was higher in siblings (41%), followed by parents (28%), spouses (26%), other (19%), and offspring (14%). Independent risk factors for seropositivity were being siblings (OR 3.3) and being a HC of an index case with indeterminate leprosy (OR 5.3). APGL-I seropositivity was associated with index cases with a bacillary index of 4 (88%; p<.001). Seropositivity among HC was not significantly associated with their gender and age. There was no statistical difference in the seropositivity rates of HC of index patients with paucibacillary and multibacillary leprosy. CONCLUSIONS: Strict evaluation and follow-up of HC with positive results for APGL-I is recommended. Special attention should be paid during the screening of siblings of the index cases, HC of patients with a high bacillary index, and HC of patients with indeterminate leprosy.

Evaluation of the social, clinical and laboratorial profile of patients diagnosed with leprosy in a reference center in São Paulo

BACKGROUND: Leprosy is the main infectious cause of disability. It is said to be eradicated in São Paulo since 2005, but diagnosis is still late. OBJECTIVES: To investigate the social, clinical and laboratorial profile of leprosy patients diagnosed between 01/2007 and 12/2011, in a reference center in São Paulo. METHODS: Retrospective descriptive study. Data of all new leprosy cases diagnosed between 01/2007 and 12/2011 were raised in São Paulo. RESULTS: 103 men and 71 women were diagnosed, most of them were multibacillary. Mean age at diagnosis was 49 yrs; 2,2% were children; 70% had incomplete primary education; 50% were referred without diagnostic suspicion of leprosy. Mean time since first symptoms/signs and diagnosis was 2 years; 64% of patients had some degree of disability, and 26% had grade 2. 23 cases were diagnosed only after being summoned, and 80% of these had no disability. Agreement between the Ridley and Jopling and the WHO classification was 75% (kappa index = 0.44). Serology for IgM anti-PGL1 (87 patients) showed a mean value of 0.25, and an association between MB classification and test positivity (p <0.001). CONCLUSIONS: Leprosy diagnosis in São Paulo is late. The disease mainly affected the socially disadvantaged and economically active population. Failure to detect the disease (41% in the last 10 years) could be due to the lack of suspicion and to decentralization. For the classification of patients with advanced leprosy, both the WHO and R&J classifications proved to be helpful tools. .

Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.

Cloning and expression of a recombinant CagA -gene fragment of Helicobacter pylori and its preliminary evaluation in serodiagnosis / Clonación y expresión de un fragmento recombinante del gen cagA de Helicobacter pylori y su evaluación preliminar en el serodiagnóstico

Introduction: Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations. Objective: To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection Methods: A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test. Results: The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively. Conclusion: The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.

Introducción. Las cepas de Helicobacter pylori que expresan la citotoxina CagA, se asocian más frecuentemente con úlcera péptica, gastritis atrófica y adenocarcinoma gástrico que las que carecen de esta citotoxina. Por lo anterior, el determinar la presencia de anticuerpos anti-CagA adquiere gran importancia clínica en la detección serológica de la infección por H. pylori y la predicción del riesgo de enfermedades. Sin embargo, los métodos serológicos que emplean CagA han sido cuestionados debido a las diferencias encontradas en las evaluaciones de su desempeño en diversas poblaciones. Objetivo. Obtener un fragmento recombinante de la proteína CagA para el serodiagnóstico de la infección por H. pylori . Materiales y métodos. Un fragmento del gen cagA fue clonado en un vector de expresión procariota que contenía el promotor de la T7 ARN polimerasa. El fragmento de la proteína CagA con seis histidinas en la región C-terminal, se expresó, se solubilizó con urea y se purificó por cromatografía de afinidad con iones metálicos inmovilizados. El desempeño de la proteína recombinante se evaluó empleando un método in house de Western Blot y 180 sueros humanos. Los resultados se compararon con la prueba de referencia Helicoblot 2.1. Resultados. La proteína CagA expresada mostró una banda inmunorreactiva de 80 kDa en el Western Blot al emplear dos anticuerpos policlonales anti-CagA específicos. La proteína recombinante fue purificada hasta un 93 % de pureza y el análisis de desempeño del antígeno recombinante purificado mostró buena inmunorreacción y exhibió valores de sensibilidad, especificidad y exactitud de 88,1 %, 100 % y 92,7 %, respectivamente. Conclusiones. El fragmento de la proteína CagA del estudio puede constituir una herramienta útil para el diagnóstico serológico de la infección por cepas de H. pylori positivas para CagA.

Seroprevalence and risk factors for cattle anaplasmosis, babesiosis, and trypanosomiasis in a Brazilian semiarid region / Soroprevalencia e fatores de risco para anaplasmose, babesiose e tripanosomiase bovina em uma região semiárida do Brasil

The seroprevalence of Anaplasma marginale, Babesia bigemina, Babesia bovis and Trypanosoma vivax and the risk factors for these infections were investigated in 509 cows on 37 farms in the semiarid region of Paraíba, northeastern Brazil. Cow serum samples were tested by means of immunofluorescence assay (IFA) against each specific antigen. The mean seroprevalence values per farm were 15.0% (range: 0-75%) for A. marginale, 9.5% (range: 0-40%) for B. bigemina and 26.9% (range: 0-73.7%) for B. bovis. All cows tested negative for T. vivax. Higher prevalence for A. marginale was significantly associated with less frequent acaricide spraying per year and with higher use of injectable antihelminthics. Presence of cows positive for B. bigemina was significantly associated with acaricide use and with presence of horse flies on the farm. Both occurrence and higher prevalence of B. bovis were significantly associated with recent observations of ticks on cattle. Overall, the present results indicate that the region investigated is an enzootically unstable area for A. marginale, B. bigemina and B. bovis, since most animals were seronegative to at least one agent.

A soroprevalência de Anaplasma marginale, Babesia bigemina, Babesia bovis e Trypanosoma vivax, assim como os fatores de risco para estas infecções, foram investigadas em 37 fazendas (total de 509 vacas) da região semiárida da Paraíba, nordeste do Brasil. A presença de anticorpos nos soros dos animais foi detectada pela técnica de imunofluorescência indireta, utilizando antígenos específicos. Os valores médios de soroprevalência por fazenda foram 15,0% (0-75%) para A. marginale, 9,5% (0-40%) para B. bigemina, e 26,9% (0-73,7%) para B. bovis. Todas as vacas foram soronegativas para T. vivax. As maiores prevalências de A. marginale foram significativamente associadas com menor uso de carrapaticidas por ano e com uso mais frequente de antihelmínticos injetáveis. A soroprevalência de B. bigemina foi significativamente associada com o uso de carrapaticidas, e com a presença de mutucas na fazenda. Tanto a ocorrência como a maior soroprevalência para B. bovis nas fazendas foram significativamente associadas com a presença recente de carrapatos nos bovinos. No geral, os resultados indicam que as fazendas amostradas estão situadas em área de instabilidade enzoótica para A. marginale, B. bigemina, e B. bovis, uma vez que a maioria dos animais foi soronegativa para pelo menos um dos agentes.

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil / Pesquisa sorológica por doenças transmitidas por carrapatos em cães da Amazônia oriental, Brasil

Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.

Ehrliquiose canina e babesiose canina são as doenças parasitárias transmitidas por carrapatos de maior prevalência em cães do Brasil. Poucos estudos pesquisaram doenças transmitidas por carrapatos na região da Amazônia brasileira. Um total de 129 amostras de sangue foram colhidas de cães da Amazônia oriental brasileira. Setenta e dois cães eram de áreas rurais de 19 municípios do Estado do Pará, e 57 amostras foram colhidas de cães errantes vadios da área urbana do município de Santarém-PA. As amostras de soro foram submetidas ao ensaio de imunofluorescência indireta, com antígenos de Babesia canis vogeli, Ehrlichia canis, e seis espécies de Rickettsia. A frequência de cães com anticorpos anti-B. canis vogeli, anti-E. canis, e anti-Rickettsia spp. foi de 42,6%, 16,2% e 31,7%, respectivamente. Anticorpos anti-B. canis vogeli foram detectados em 59,6% dos cães urbanos, e em 29,1% dos cães rurais (P < 0.05). Para E. canis, a soroprevalência foi parecida entre os cães urbanos (15,7%) e rurais (16,6%). Para Rickettsia spp., cães rurais apresentaram prevalência (P < 0.05) significativamente maior (40,3%) do que os cães urbanos (21,1%). Esse primeiro estudo sobre agentes transmitidos por carrapatos entre cães da Amazônia oriental brasileira indica que estes animais estão expostos a vários agentes. Estes incluem Babesia principalmente na área urbana, Riquétsias do grupo da Febre Maculosa principalmente nas áreas rurais, e Erliquia em cães de ambas as áreas, rural e urbana.

High rates of undiagnosed leprosy and subclinical infection amongst school children in the Amazon Region

Leprosy in children is correlated with community-level factors, including the recent presence of disease and active foci of transmission in the community. We performed clinical and serological examinations of 1,592 randomly selected school children (SC) in a cross-sectional study of eight hyperendemic municipalities in the Brazilian Amazon Region. Sixty-three (4%) SC, with a mean age of 13.3 years (standard deviation = 2.6), were diagnosed with leprosy and 777 (48.8%) were seropositive for anti-phenolic glycolipid-I (PGL-I). Additionally, we evaluated 256 house-hold contacts (HHCs) of the students diagnosed with leprosy; 24 (9.4%) HHC were also diagnosed with leprosy and 107 (41.8%) were seropositive. The seroprevalence of anti-PGL-I was significantly higher amongst girls, students from urban areas and students from public schools (p < 0.0001). Forty-five (71.4%) new cases detected amongst SC were classified as paucibacillary and 59 (93.6%) patients did not demonstrate any degree of physical disability at diagnosis. The results of this study suggest that there is a high rate of undiagnosed leprosy and subclinical infection amongst children in the Amazon Region. The advantages of school surveys in hyperendemic areas include identifying leprosy patients at an early stage when they show no physical disabilities, preventing the spread of the infection in the community and breaking the chain of transmission.

Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission

Leprosy transmission still occurs despite the availability of highly effective treatment. The next step towards successfully eliminating leprosy is interrupting the chain of transmission of the aetiological agent, Mycobacterium leprae. In this investigation, we provide evidence that household contacts (HHCs) of leprosy patients might not only have subclinical infections, but may also be actively involved in bacilli transmission. We studied 444 patients and 1,352 contacts using anti-phenolic glycolipid-I (PGL-I) serology and quantitative polymerase chain reaction (qPCR) to test for M. leprae DNA in nasal swabs. We classified the patients according to the clinical form of their disease and the contacts according to the characteristics of their index case. Overall, 63.3% and 34.2% of patients tested positive by ELISA and PCR, respectively. For HHCs, 13.3% had a positive ELISA test result and 4.7% had a positive PCR test result. The presence of circulating anti-PGL-I among healthy contacts (with or without a positive PCR test result from nasal swabs) was considered to indicate a subclinical infection. DNA detected in nasal swabs also indicates the presence of bacilli at the site of transmission and bacterial entrance. We suggest that the concomitant use of both assays may allow us to detect subclinical infection in HHCs and to identify possible bacilli carriers who may transmit and disseminate disease in endemic regions. Chemoprophylaxis of these contacts is suggested.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids

Non-bilayer phospholipid arrangements are three-dimensional structures that form when anionic phospholipids with an intermediate structure of the tubular hexagonal phase II are present in a bilayer of lipids. Antibodies that recognise these arrangements have been described in patients with antiphospholipid syndrome and/or systemic lupus erythematosus and in those with preeclampsia; these antibodies have also been documented in an experimental murine model of lupus, in which they are associated with immunopathology. Here, we demonstrate the presence of antibodies against non-bilayer phospholipid arrangements containing mycolic acids in the sera of lepromatous leprosy (LL) patients, but not those of healthy volunteers. The presence of antibodies that recognise these non-bilayer lipid arrangements may contribute to the hypergammaglobulinaemia observed in LL patients. We also found IgM and IgG anti-cardiolipin antibodies in 77% of the patients. This positive correlation between the anti-mycolic-non-bilayer arrangements and anti-cardiolipin antibodies suggests that both types of antibodies are produced by a common mechanism, as was demonstrated in the experimental murine model of lupus, in which there was a correlation between the anti-non-bilayer phospholipid arrangements and anti-cardiolipin antibodies. Antibodies to non-bilayer lipid arrangements may represent a previously unrecognised pathogenic mechanism in LL and the detection of these antibodies may be a tool for the early diagnosis of LL patients.

Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil

New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil

New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil