Análisis de muestras de orina para la detección molecular de enfermedades infecciosas. Aplicación en la identificación de citomegalovirus humano

La implementación de metodologías de biología molecular como la reacción en cadena de la polimerasa (PCR), ha permitido la realización de diagnósticos sensibles y específicos para múltiples enfermedades, dentro de las cuales son de gran interés las infecciosas. Hasta hoy, los métodos de identificación se basan principalmente en cultivos y serología por su sensibilidad y especificidad, pero consumen tiempo y dinero. Las muestras de orina se han constituido en una alternativa no invasiva de obtención de ADN para la realización de análisis de biología molecular. Metodología: Implementación de una estrategia para la obtención de ADN a partir de muestras de orina. Las muestras fueron tomadas de niños de guardería, para documentar la presencia o no de inhibidores de PCR a través de la amplificación de genes de Citomegalovirus humano (CMVH). Resultados: En el 27,1% de las muestras analizadas se evidenció amplificación específica para CMVH, no se encontraron diferencias significativas en la presencia del virus en los tres estratos, pero sí en la intensidad de las bandas. Conclusión: Se verificó la ausencia de inhibidores de PCR mediante la amplificación del gen de la B-globina. Se estandarizó una metodología molecular para la identificación de CMVH, la cual puede ser aplicada

Neuromielitis óptica. Reporte de caso

La neuromielitis óptica, también conocida como síndrome de Devic, es una enfermedad que combina la neuritis óptica y la mielitis transversa. Hace unos años era considerada una forma de esclerosis múltiple; sin embargo, realmente es una enfermedad diferente, con características clínicas, imágenes, serología e inmunopatología propias. El presente artículo presenta el caso de una mujer de 29 años con un cuadro clínico que inicia en el quinto mes de postparto, con pérdida progresiva de la fuerza en miembros inferiores, asociada a pérdida de la agudeza visual bilateral. El objetivo es hacer, mediante el estudio de caso, una revisión amplia de esta enfermedad, poco común, mediada por un proceso inmune y desmielinizante. Para ello, se resumen los parámetros epidemiológicos más importantes y se presentan las posibilidades diagnósticas y terapéuticas disponibles actualmente.

Prevalencia de enfermedad celiaca en Latinoamérica: revisión sistemática de la literatura y meta-análisis

Introducción: La enfermedad celiaca (EC) es una enfermedad autoinmune (EA) intestinal desencadenada por la ingesta de gluten. Por la falta de información de la presencia de EC en Latinoamérica (LA), nosotros investigamos la prevalencia de la enfermedad en esta región utilizando una revisión sistemática de la literatura y un meta-análisis. Métodos y resultados: Este trabajo fue realizado en dos fases: La primera, fue un estudio de corte transversal de 300 individuos Colombianos. La segunda, fue una revisión sistemática y una meta-regresión siguiendo las guías PRSIMA. Nuestros resultados ponen de manifiesto una falta de anti-transglutaminasa tisular (tTG) e IgA anti-endomisio (EMA) en la población Colombiana. En la revisión sistemática, 72 artículos cumplían con los criterios de selección, la prevalencia estimada de EC en LA fue de 0,46% a 0,64%, mientras que la prevalencia en familiares de primer grado fue de 5,5 a 5,6%, y en los pacientes con diabetes mellitus tipo 1 fue de 4,6% a 8,7% Conclusión: Nuestro estudio muestra que la prevalencia de EC en pacientes sanos de LA es similar a la notificada en la población europea.

Cross-sectional estimation of Babesia bovis antibody prevalence in cattle in two contrasting dairying areas in Tanzania

The crude prevalence of antibodies to Babesia bovis infection in cattle was estimated by serology using indirect ELISA during the period January to April, 1999. Sera were obtained from 1395 dairy cattle (of all ages, sexes and breeds) on smallholder farms, the majority being kept under a zero grazing regime. The crude prevalence of antibodies to Babesia bovis was 6 % for Tanga and 12 % for Iringa. The forces of infection based on the age sero-prevalence profile, were estimated at six for Iringa and four for Tanga per 100 cattle years-risk, respectively. Using random effect logistic regression as the analytical method, the factors (variables) of age, source of animals and geographic location were hypothesised to be associated with sero-positivity of Babesia bovis in the two regions.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

Seroprevalence of Human Herpesvirus 8 Infection in Individuals From Health Care Centers in Mozambique: Potential for Endemic and Epidemic Kaposi`s Sarcoma

Human herpesvirus 8 (HHV-8) infection is common in sub-Saharan Africa, but its prevalence in Mozambique is unknown. The seroprevalence of HHV-8 in a cohort of individuals seen at public health centers in Northern (n = 208), Central (n = 226), or Southern (n = 318) Mozambique was examined. All individuals were interviewed to obtain socioeconomic, demographic and clinical data and were tested for serum anti-HHV-8 antibodies using an immunofluorescence assay. The overall frequency of HHV-8 antibodies was 21.4% and, in spite of the diversity of epidemiological characteristics of the tested individuals, did not differ significantly among regions: 18.7%, 24.3% and 21.4% in the North, Center, and South, respectively (chi(2), 2.37; P = 0.305). The variables that were associated significantly with the presence of HHV-8 antibodies were gender, age, level of education, number of siblings and HIV serostatus, but these differed across the regions. In the North, although tested individuals lived under poor socioeconomic conditions, no association between HHV-8 infection and household variables was detected, with the exception of the number of siblings (P = 0.042). In the Central region, HHV-8 infection was associated with gender (P = 0.010), the number of household members (P = 0.031), and the place of attendance (P = 0.021). In the South, HHV-8 infection was associated with the number of siblings (P = 0.023) and HIV status (P = 0.002). The overall prevalence of HHV-8 seropositivity increased with age. These results demonstrate that Mozambique is another country in Africa with endemic HHV-8 infection, and, because of the AIDS epidemic, continued access to antiretroviral treatment is necessary to avert an outbreak of AIDS-Kaposi`s sarcoma. J. Med. Virol. 82:1216-1223, 2010. (C) 2010 Wiley-Liss, Inc.

Inconclusive results in conventional serological screening for Chagas` disease in blood banks: evaluation of cellular and humoral response

To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted-secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non-chagasic individuals and chronic chagasic patients. TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas` disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. TESA blot is a good confirmatory test for Chagas` disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas` disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas` disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time-consuming) do not support its use as a confirmatory test in blood-bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.

Markers and risk factors for HCV, HBV and HIV in a network of injecting drug users in Melbourne

Background and aims: Current injecting drug users (IDU) in major street drug markets within greater Melbourne were recruited to a longitudinal study on blood borne viruses. Here we investigated risk factors for hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV infection in these IDU at the time of their recruitment.

Methods : Three hundred and eighty-two IDU completed detailed questionnaires on their drug use and risk behaviours, and provided blood samples for serology testing. These data were analysed using univariate and multivariate techniques.

Results : The overall prevalence of exposure to HCV, HBV and HIV was estimated at 70%, 34% and <1%, respectively. Independent predictors of HCV exposure were history of imprisonment (RR 1.34, 95% CI 1.19–1.52), use of someone else's needle or syringe (RR 1.23, 95% CI 1.07–1.42), >7.6 years length of time injecting (RR 1.21, 95% CI 1.07–1.37), and originating from Vietnam (RR 1.12, 95% CI 1.07–1.18). Independent predictors of HBV exposure were HCV exposure (RR 2.15, 95% CI 1.35–3.43), >7.6 years length of time injecting (RR 1.57, 95% CI 1.17–2.13) and originating from outside Australia (RR 1.60, 95% CI 1.22–2.10). Neither prison- nor community-applied tattoos predicted HCV or HBV exposure. Up to 31% of IDU who injected for 1 year or less were HCV antibody positive, as were 53% of those who injected for 2 years or less.

Conclusions : Ongoing engagement with young IDU, through the provision of harm reduction education and resources, is critical if we are to address blood borne viral infections and other health and social harms associated with injecting drug use.

Undiagnosed and potentially lethal parasite infections among immigrants and refugees in Australia

Background: Intestinal parasite infections are a major cause of ill health in many resource-poor countries. This study compares the types and rates of these infections and their risk factors in recently arrived and long-term immigrants in Australia.

Method: Cross-sectional surveys of 127 East African and 234 Cambodian immigrants and refugees were undertaken in 2000 and 2002, respectively, to assess the burden of intestinal parasites and collect demographic information. Serum samples were assessed for eosinophilia and Strongyloides stercoralis and Schistosoma antibodies, and feces examined for ova, cysts, and parasites.

Results: Intestinal parasites were identified in 77/117 fecal samples from East African and in 25/204 samples collected from Cambodian participants. Eleven percent (14/124) of East Africans and 42% (97/230) of Cambodians had positive or equivocal serology for S stercoralis. Schistosoma serology was positive or equivocal in 15% (19/124) of East African participants.

Conclusion: Potentially serious intestinal parasite infections are common among recent and longer term immigrants despite multiple visits to health care providers. Immigrants and refugees from high-risk countries would benefit from comprehensive health checks soon after resettlement.

A boarding school outbreak of pertussis in adolescents : value of laboratory diagnostic methods

Culture for Bordetella pertussis (B. pertussis) is the traditional gold standard for laboratory diagnosis of pertussis but is insensitive, especially later in the course of illness and in vaccinated persons. Interpretation of serology is limited by the lack of an appropriate reference standard. An outbreak of pertussis in a crowded boarding-school dormitory allowed evaluation of laboratory correlates of infection. Questionnaires, serum samples and throat swabs were collected from members of the exposed group. Serum samples from unexposed controls of a similar age group were used for comparison. B. pertussis PCR was performed on throat swabs, and sera were tested for IgA antibodies against whole-cell (WC) B. pertussis antigen and IgG antibodies to pertussis toxin (PT). The Centers for Disease Control and Prevention definition for pertussis was used to define clinical cases. We evaluated the use of a previously published cut-off for PT IgG of 125 EIA units (EU)/ml. Completed questionnaires were obtained from 115 students, of whom 85 (74%) reported coughing symptoms, including 32 (28%) who met the clinical case definition for pertussis. B. pertussis was detected by PCR in 17 (15%) and WC IgA in 22 (19%) students; neither correlated with symptoms, but dormitory of residence strongly predicted PCR status. The mean PT IgG geometric mean concentration, in this situation of high pertussis exposure, correlated with severity of symptoms and was significantly higher in both symptomatic and asymptomatic children exposed during the outbreak (P<0·001) than in control children. A cut-off for PT IgG of 125 EU/ml was too high in an outbreak situation to be sensitive enough to identify pertussis cases. A case of pertussis in a crowded boarding-school dormitory resulted rapidly in an outbreak. Serology and PCR were useful in identifying the outbreak and commencing disease control measures. The use of serology has mostly been evaluated in community serosurveys, where it is not possible to determine if immunity reflects vaccination, asymptomatic disease or symptomatic disease. This outbreak gave us the opportunity to evaluate the value of serology and PCR in the presence of confirmed exposure to pertussis.

Bird biometrics condition and disease database

Contains biometric measurements (tarsus length, bill length, total head length, body mass) of birds caught at various locations in Australia (though mainly South Australia and Victoria), including data on the viral (mainly Avian Influenza Virus) analysis of cloacal and oropharyngeal swabs, serology of sera samples, and additional data on metabolites and parasite prevalence in swabs, blood cells and sera. Approximately 1500 birds (mainly waterbirds) are sampled annually.

Epizootiology of brucella infection in Australian fur seals

Novel members of the bacterial genus Brucella have recently emerged as pathogens of various marine mammal species and as potential zoonotic agents. We investigated the epizootiology of Brucella infection in Australian fur seals (Arctocephalus pusillus doriferus) by establishing demographic and temporal variations in antibody prevalence, attempting isolation of the causative agent, and determining whether this potential pathogen is involved in frequent abortions observed in this pinniped species. Two competitive enzyme-linked immunosorbent assays (cELISAs), an indirect ELISA, and a fluorescence polarization assay (FPA) were used to test sera for Brucella antibodies. The FPA and cELISA proved suitable for use in this species. Significant differences in antibody prevalence were found between age classes of seals sampled between 2007 and 2009 at one colony. Pups sampled at this site (n5134) were negative for Brucella antibodies by all serologic tests but 17 of 45 (38%) of juveniles were antibody-positive. Antibody prevalence in adult females was significantly higher than in juveniles (P50.044). Antibody prevalence for adult females between 2003 and 2009 varied significantly over time (P50.011), and for individuals sampled between 2003 and 2005, the likelihood of pregnancy was greater in individuals positive for Brucella antibodies (P50.034). Inflammatory lesions suggestive of infectious agents were found in 14 of 39 aborted Australian fur seal pups, but pathologic changes were not uniformly consistent for Brucella infection. Culture and PCR investigations on fetal tissues were negative for Brucella. Culture and PCR on selected fresh or frozen tissues from 36 juvenile and adult animals were also negative. We suspect that the prevalence of active infection with Brucella in Australian fur seals is low relative to antibody prevalence. © Wildlife Disease Association 2011.

Serological prevalence study of exposure of cats and dogs in Launceston, Tasmania, Australia to spotted fever group rickettsiae

A sero-epidemiological study of cats and dogs in the Launceston area of Tasmania, Australia was undertaken to determine the prevalence of antibodies to spotted fever group (SFG) rickettsiae. Results showed that 59% of cats and 57% of dogs were positive for antibodies, but there was no correlation between the animal’s health and seropositivity at the time of testing, suggesting that rickettsial exposure is unrelated to ill-health in these two species of domestic animals.