Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Ecoepidemiología de Chlamydophila psittaci, pecorum, pneumoniae, abortus en Córdoba

Las Clamidias son bacterias patógenas de los animales de producción, de vida silvestre y de compañía. Además de las pérdidas económicas que producen las infecciones en los planteles de producción bovina, ovina, caprina, porcina y aves de corral, la mayoría de las especies tienen importancia zoonótica, pudiendo dar origen a infecciones graves, potencialmente letales en el ser humano. El orden Chlamydiales está integrado por bacterias que actúan como parásitos intracelulares obligados que desarrollan su ciclo de vida únicamente dentro de inclusiones citoplasmáticas. En este orden se encuentra la familia Chlamydiaceae que comprende dos géneros, Chlamydia y Chlamydophila; y las especies, Chlamydia trachomatis, C. suis, C. muridarum, Chlamydophila psittaci, C. abortus, C. felis, C. caviae, C. pecorum, y C. pneumoniae. C. psittaci causa psitacosis o clamidiosis aviar. En Argentina, los primeros casos clínicos de psitacosis fueron reportados en 1929. Los criadores de aves y quienes las poseen como mascotas, representan el grupo de mayor riesgo; pero también las personas que trabajan en pajarerías y aquellas que por su empleo se ven expuestas a contraer la enfermedad (empleados en peladeros donde se carnean y procesan pollos y otras aves para consumo, veterinarios, empleados de zoológicos, etc.). La infección en humanos se presenta como una neumonía severa; con fiebre alta, escalofríos, dolor de cabeza, mialgia y dificultad respiratoria. Ocasionalmente puede presentarse vómitos, dolor abdominal, diarrea y complicaciones como miocarditis, endocarditis, encefalitis, ictericia y fallas multiorgánicas, que pueden ser fatales sino se le administra el tratamiento adecuado. La infección en las mujeres embarazadas puede producir neumonía, hepatitis, insuficiencia renal, sepsis, parto prematuro y muerte fetal. Existen más de 465 especies de aves en las que se registró C. psittaci, incluyendo ornamentales, de corral, silvestres, acuáticas y palomas. Las patologías que pueden producir en estos animales son neumonitis, conjuntivitis, encefalomielitis, placentopatías, fetopatías, anorexia, diarrea e infecciones persistentes asintomáticas u oligosintomáticas. En bovinos, C. pecorum, C. abortus y C. psittaci producen infecciones respiratorias y genitales; que se presentan como cuadros de enteritis, artritis, encefalomielitis, endometritis e hipofertilidad. En Argentina, la infección clamidial en el ganado caprino fue asociada a daños en el tejido uterino, abortos, partos prematuros y crías débiles. En equinos, C. psittaci y C. pneumoniae producen abortos y desórdenes respiratorios, con un gran impacto en ganadería que redunda en pérdidas económicas. Considerando que existen escasos estudios eco-epidemiológicos y clínicos que reporten el estado de situación de estas infecciones en nuestro medio, es que el presente trabajo propone actualizar y profundizar el conocimiento de las especies de Clamidias de importancia médico-veterinaria presentes en la provincia de Córdoba, Argentina. El desarrollo de este proyecto aportará la implementación de técnicas que mejorarán el diagnóstico microbiológico, confirmarán los cuadros clínicos; y por lo tanto contribuirá al conocimiento de estos agentes infecciosos en nuestra región. Esta información es indispensable para los organismos responsables de la Salud Pública (Ministerios de Salud y Educación, Municipios, etc.) para que puedan obrar en consecuencia y generar sistemas de alerta temprana, tomar medidas de prevención y medidas de control frente a la presencia de un brote epidémico por alguna cepa clamidial.

Nucleic acid amplification testing for Neisseria gonorrhoeae: An ongoing challenge

Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.

Rastreio de Infeções Sexualmente Transmissíveis não víricas nos adolescentes: qual o estado da arte

As infeções sexualmente transmissíveis (IST) constituem um problema persistente de saúde pública, sendo os adolescentes e adultos jovens os que apresentam as taxas de prevalência mais elevadas para algumas IST. As IST não víricas nos países desenvolvidos incluem a Chlamydia Trachomatis, a Neisseria gonorrhoeae, o Treponema pallidume a Trichomonas vaginalis. A deteção precoce das IST não víricas tem impacto positivo a nível individual e na saúde pública: permite instituição atem- pada de tratamento adequado, a redução de transmissão entre parceiros, bem como reduzir as complicações a longo prazo, nomeadamente doença inflamatória pélvica, dor pélvica crónica, gravidez ectópica e infertilidade. Várias sociedades médicas internacionais publicaram recomendações para o rastreio de algumas IST não víricas em de- terminados grupos. Em Portugal, a Direção Geral de Saúde (DGS) atualizou em 2014 a norma sobre a notificação obrigatória de doenças transmissíveis, que inclui a gonorreia, a sífilis e a infeção por Chlamydia Trachomatis. Não obstante, os estudos sobre a epidemiologia de IST são parcos em Portugal e apenas recentemente foi contemplado o rastreio oportunístico de infeção genital por Chlamydia Trachomatis no Plano Nacional de Saúde 2011-2016. O médico de família através da sua abordagem holística centrada na pessoa, no seu contexto familiar e social (focando antecedentes pessoais / comportamentos de risco) tem necessariamente um papel determinante na prevenção primária e no rastreio das IST.

Prevalência da infecção por HPV e perfil das mulheres frente ao exame de papanicolau no município de São José de Mipibú/RN.

The Human Papillomavirus (HPV) infection is the major sexually transmitted disease all over the world. There are many factors associated to infection and the virus persistency in the organism. This study aims to evaluate the women's knowledge, attitudes and practice about the Papanicolaou test (Pap), as well as analyze the HPV and Chlamydia trachomatis infections prevalences in sexually active women from the city of São José do Mipibu/RN/Brazil. This research was divided in two steps (step I and step II), using different methodologies and samples each. The samples collected in each step, even socio-demographic or from uterus cervix, are from different patients e were analyzed separated. In step I was evaluated 267 rural and urban zone women s knowledge, attitudes and practices about the Pap by home interview. In the step II were included 605 women with age ranged from 15 to 71 years old, with mean of 33,5 years old and from each one were collected two cervical samples, one for Pap and other for molecular biology, beside the epidemiological interview to investigate the correlation between prevalence of HPV infection and risk factors. To molecular analyses, the samples were processed using a mammal rapid DNA extraction technique protocol. For C. trachomatis DNA detection were used the CP24/27 primers, and GP5+/GP6+ to HPV. PCR products were analyzed by electrophoresis on 8% polyacrylamide gels, followed by silver staining. The results of the step I showed that, in spite of only 46,1% of the interviewed women they have demonstrated to possess appropriate knowledge on the Pap test, the attitude and practice proportions were significantly larger, 63,3% and 64,4% respectively. The largest education degree presented association with adaptation of the knowledge, attitudes and practice, while neglect, lack of solicitation of the exam for the doctor and shame, came as main barriers for the accomplishment of the exam. In the stage II the HPV general prevalence was 28,9%, being 26,7% in the women with normal cytology or benign alterations, 26,7% in the ones that had atypical squamous cells of undetermined significance (ASC-US) and 80% in those with Low grade squamous intraepithelial lesion (LSIL). the HPV infection prevalence was larger in the patients with up to 30 years of age and in the unmarried women, and those that had more than one sexual partner presented larger infection risk. The results show that the sexual relationship with multiple partners increased the infection risk for HPV and consequently the possibility of the occurrence of lesions uterine cervix

Modelo predictivo para uso del condón y consumo de alcohol como conductas de riesgo relacionadas con el contagio de VIH/SIDA en trabajadoras sexuales de Bogotá- 2015

Objetivo: Determinar un modelo predictivo para uso del condón y consumo de alcohol como conductas de riesgo relacionadas el contagio de VIH/Sida en mujeres trabajadoras sexuales de la ciudad de Bogotá en el año 2015. Métodos Estudio de tipo transversal con diseño observacional, se tomaron 255 mujeres trabajadoras sexuales de la ciudad de Bogotá; La información analizada fue tomada del estudio realizado en cinco ciudades de Colombia en el año 2015, las hipótesis planteadas se soportaron en la asociación entre las condiciones sociodemográficas, de conocimiento, practicas, hábitos, apoyo social y de ocupación propia de las mujeres trabajadoras sexuales que podían explicar y predecir la adopción de conductas riesgosas para VIH/sida como son el uso del condón y el consumo de alcohol en ejercicio de su ocupación. Resultados El promedio de edad de inicio en el trabajo sexual fue 22,1±7,1 años, tres cuartas partes son solteras y residen en estrato dos y tres; el 96,5% dijo usar el condón con el último cliente y el 27,8% de ellas consumió alcohol durante su último servicio. En la conducta de riesgo uso del condón, se encontraron asociados entre otras, la edad [OR=1,10(1,03-1,17)], vivir en estrato dos [OR=7,7(1,5-39,5)], el ingreso por trabajo sexual [OR=1,0(1,0-1,0)], la disponibilidad del condón para el servicio [OR=0,03(0,008-0,16)] y contar con otro método de planificación (ligadura de trompas) [OR=4,47(1,0-18,3)]. En la conducta de riesgo consumo de alcohol, se encontró asociado ente otros: estrato socioeconómico dos [OR=5,8(1,54-22,3)], nivel de escolaridad secundaria [OR=0,12(0,16-0,96)], vivir con otros familiares [OR=3,45(1,7-7,02)], ingreso por trabajo sexual [OR=1,0(1,0-1,0)] y el sitio donde se ofrece el servicio [OR=0,07(0,04-0,15)]. Después de ajustar, se encontró que las variables que mejor explican el uso del condón fueron edad [OR=1,1(1,02-1,17)] y disponibilidad del condón [OR=0,04(0,008-0,024)], el modelo tuvo poca sensibilidad 33,3% y buena capacidad predictiva (84,6%). Las variables que mejor explicaron el consumo de alcohol durante el servicio fueron edad [OR= 0,95(0,91-0,98)], Número de clientes por semana [OR=0,9(0,90-0,98)], sitio donde ofrece el servicio [OR=7,1(3,45-14,8)], y estrato socioeconómico [OR=1,8 (0,90-3,83)], resultando un modelo con buena sensibilidad (71,8%) y buena capacidad predictiva (86,4%). Conclusiones Aspectos como la edad, el estrato socioeconómico, escolaridad, estado civil, ingreso económico por trabajo sexual, edad de inicio en el trabajo sexual, número de clientes antiguos en la última semana, disponibilidad del condón para prestar el servicio y ligadura de trompas como método diferente de planificación, se asociaron estadísticamente con el uso del condón. Sin embargo al ajustar las variables solo la edad y la disponibilidad del condón se mantuvieron como variables explicativas. Cabe anotar, que aunque el modelo mostró buena capacidad predictiva (84,6%), la precisión en sus estimaciones fue baja debido a la poca frecuencia del no uso del condón con el ultimo cliente (3,5%), y la sensibilidad del modelo apenas fue del 33,3%. Por otro lado, factores como la edad, el estrato socioeconómico, nivel educativo, ingreso económico, sitio de oferta del servicio, composición familiar, número de hijos, número de clientes atendidos en la última semana y número de clientes antiguos mostraron asociación estadística con el consumo de alcohol. Sin embargo, al ajustar las variables solo edad, estrato socioeconómico, sitio donde se ofrece el servicio y número de clientes por semana mantuvieron asociación estadística; observándose además que el estrato socioeconómico (uno y dos) y sitio donde se ofrece el servicio (establecimiento), son factores de riesgo para el consumo de alcohol en ejercicio de la ocupación y la poca edad y un número reducido de clientes por semana se comportan como factores de protección para el consumo de alcohol. El modelo predictivo que se desarrolló para la conducta de riesgo de consumo de alcohol, con una sensibilidad del 71,8% y un poder predictivo del 86,4%. .

Linfogranuloma venéreo

Material utilizado na unidade 3 do Curso Autoinstrucional de Capacitação em Atenção Integral à Saúde Sexual e Reprodutiva II, produzido pela UNA-SUS/UFMA e voltado para os médicos que atuam na Atenção Básica. Este material aborda o linfogranuloma venéreo, trazendo informações sobre suas especificidades como manifestações, incubação, transmissão e diagnóstico.

Capacitação para identificação, notificação e tratamento de casos de tracoma em escolares do ensino público fundamental do bairro Morumbi

O tracoma é afecção inflamatória ocular crônica, cujo agente etiológico é a Chlamydia trachomatis (sorotipos A, B, Ba e C), uma bactéria gram negativa, que provoca uma ceratoconjuntivite crônica recidivante e transmissível. A única fonte de infecção é o homem com infecção ativa na conjuntiva ou em outras mucosas. As formas de transmissão são a direta, de olho a olho, ou a indireta, através de objetos contaminados. Alguns insetos, como a mosca doméstica (Musca domestica) podem atuar como vetores. Em decorrência de infecções repetidas, o tracoma produz cicatrizes na conjuntiva palpebral, podendo levar à formação de entrópio e triquíase. Estas podem levar a alterações irreversíveis da córnea, causando cegueira. Com a prevalência do tracoma nas crianças de ensino público fundamental, em decorrência das más condições habitacionais e de saúde, além do desconhecimento dos profissionais de saúde acerca da identificação, notificação e tratamento do tracoma, esse trabalho tem como objetivo levar conhecimento, capacitar e informar sobre a notificação e encaminhamento dos casos suspeitos de tracoma

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.

Seroprevalence of Antibodies to Chlamydophila psittaci in Zoo Workers in Brazil

P>To evaluate the prevalence of antibodies to Chlamydophila psittaci 364 serum samples were collected from veterinarians, biologists, animal scientists, veterinary students, animal keepers and others employees in 20 zoos, and from veterinary practitioners in 10 Brazilian states. Subjects ranged from 15 to 64 years of age, with 268 (74%) males and 96 (26%) females. Chlamydial antibodies were determined by the complement fixation test (CFT) and specific anti-C. psittaci IgG antibodies were determined by the microimmunoflurescence (MIF) test. Complement fixation test showed 23.9% (87/364) and MIF test showed 4.7% (17/364) positive serum samples. Titres ranged from 16 to 256 in both assays, demonstrating evidence of recent or current infection. Although chlamydial antibodies were detected in workers of seventeen zoos, MIF test only detected specific C. psittaci antibodies in seven of them. Previous psittacosis infection was suspected in eight workers of two zoos, five of whom reported having pneumonia, while employed at the zoos. However, diagnosis was not established in any of these cases in the past. Results indicated the occurrence of infection and previous contact of Brazilian zoo workers with C. psittaci, as well as the zoonotic potential of psittacosis in this risk population. Other studies are necessary to evaluate the risk factors of infection in this population. This seroepidemiological survey confirmed the need to adopt preventive measures to control avian chlamydiosis and protect the health of zoo workers in the country.

Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

Mycoplasma pneumoniae and Chlamydia pneumoniae in calcified nodules of aortic stenotic valves

Aortic Valve Stenosis (AVS) has been explained as an atherosclerotic process of the valve as they often exhibit inflammatory changes with infiltration of macrophages, T lymphocytes and lipid infiltration. The present study investigated whether the bacteria Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP), detected previously in atherosclerotic plaques, are also present in AVS. Ten valves surgically removed from patients with AVS were analyzed by immunohistochemistry, in situ hybridization, and electron microscopy. The mean and standard deviation of the percentage areas occupied by CP antigens and MP - DNA were respectively 6.21 +/- 5.41 and 2.27 +/- 2.06 in calcified foci; 2.8 +/- 3.33 and 1.78+/- 3.63 in surrounding fibrotic areas, and 0.21 +/- 0.17 and 0.12 +/- 0.13 in less injured parts of the valve. There was higher amount of CP and MP in the calcified foci and in the surrounded fibrosis than in more preserved valvular regions. In conclusion, the fact that there were greater amounts of CP and MP in calcification foci of AVS favors the hypothesis that AS is not an inevitable degenerative process due to aging, but rather that it may be a response to the presence of these bacteria, similarly to the morphology detected in atherosclerosis damage.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Coinfection with Mycoplasma Pneumoniae and Chlamydia Pneumoniae in ruptured plaques associated with acute myocardial infarction

OBJECTIVE: To study atheromas, Mycoplasma pneumoniae (M. pneumoniae), and Chlamydia pneumoniae (C. pneumoniae). METHODS: C. pneumoniae was studied with immunohistochemistry and M. pneumoniae with in situ hybridization (ISH), in segments of coronary arteries (SCA) as follows: group A - thrombosed ruptured plaques (TRP) of 23 patients who died due to acute myocardial infarction (AMI); group B - 23 nonruptured plaques (NRP) of group A patients; group C - NRP of 11 coronary patients who did not die due to AMI; and group D - 11 SCA from patients with dilated cardiomyopathy or Chagas' disease without atherosclerosis. RESULTS: The mean number of C. pneumoniae+ cells/400x in groups A, B, C, and D was, respectively, 3.3±3.6; 1.0±1.3; 1.2±2.4; and 0.4±0.3; and the percentage of M. pneumoniae area was, respectively, 3.9±3.5; 1.5± 1.6; 0.9±0.9; and 0.4±0.2. More M. pneumoniae and C. pneumoniae were found in of group A than in group B (P<0.01). Good correlation was seen between the area of the vessel and the M. pneumoniae area in the plaque (r = 0.46; P=0.001) and between C. pneumoniae+ cells and CD4+ T lymphocytes (r = 0.42; P<0.01). The number of C. pneumoniae+ cells correlated with CD20+ B cells (r=0.48; P<0.01). CONCLUSION: M. pneumoniae and C. pneumoniae are more frequently found in TRP correlate with the intensity of the inflammation and diameter of the vessel (positive remodeling).