Vaccination Strategies in a Potential Use of the Vaccine against Bovine Tuberculosis in Infected Herds.

Bovine tuberculosis (bTB) is a disease of cattle that represents a risk to public health and causes severe economic losses to the livestock industry. Recently, one of the strategies recommended for reducing the prevalence of the disease in animals is the use of the BCG vaccine, alone or in combination with proteins. It has been shown that the vaccine elicits a strong immune response, downsizes the number of animals with visible lesions, and reduces the rate of infection as well as the bacillary count. This paper, based on scientific evidence, makes suggestions about some practical vaccination alternatives that can be used in infected herds to reduce bTB prevalence, considering BCG strains, vaccine doses, routes of application, and age of the animals. Our conclusion is that vaccination is a promising alternative to be included in current control programs in underdeveloped countries to reduce the disease burden.

Changes in lactate dehydrogenase on admission throughout the COVID-19 pandemic and possible impacts on prognostic capability.

Introduction: The enzyme lactate dehydrogenase (LDH) is a good marker of general hyperinflammation correlated with mortality for COVID-19, and is therefore used in prognosis tools. In a current COVID-19 clinical randomized trial (CRT), the blood level of LDH was selected as an inclusion criterion. However, LDH decreased during the pandemic; hence, the impact of this decrease on the prognostic value of LDH for mortality was evaluated. Methods: Data on LDH levels in 843 patients were obtained and analyzed. Relative risk, standard error and receiver operating characteristic curves were calculated for two cutoff values. Results: Relative risk lost validity and the area under the curve narrowed by trimester during the pandemic. Conclusion: The progressive decrease in LDH impacted the capacity to predict mortality in COVID-19. More studies are needed to validate this finding and its implications.

Effects of humic acids on the recovery of different bacterial strains in an in vitro chicken digestive model.

Humic acids (HA) have been evaluated as growth promoters in poultry, but their effects on the gut microbiota remains controversial using in vitro and in vivo models. The objective of this study was to determine the effect of HA extracted from a wormcompost on the recovery of bacteria: Salmonella Enteritidis (S. Enteritidis), Escherichia coli (E. coli), Clostridium perfringens (C. perfringens), Bacillus subtilis (B. subtilis) and Lactobacillus salivarius (L. salivarius) using an in vitro chicken digestive system. Independent in vitro trials were run for each bacteria using six treatments: 1) Negative control with no bacteria added (Control-), 2) Positive control added with bacteria (Control+), 3) 0.1% HA + bacteria, 4) 0.2% HA + bacteria, 5) 0.5% HA + bacteria and 6) 1% HA + bacteria. Data was subjected to analysis of variance and linear regression. In the crop, S. Enteritidis was lower, C. perfringes and B. subtilis were not affected by HA, while E. coli and L. salivarius were higher at 0.5 and 1% HA inclusion (P ≤ 0.0001). In the proventriculus, S. Enteritidis, E. coli and B. subtilis were higher at 0.5 and 1% HA inclusion (P ≤ 0.0001); C. perfringens and L. salivarius were not affected by HA. In intestine, significant increases of all bacteria strains were observed (P ≤ 0.0001). In conclusion, the results suggests that HA can be used as prebiotic, but their mechanisms of action to stimulate the growth of gut bacteria remains to be elucidated.

A.L.L. Y.O.U. N.E.E.D. I.S. L.O.V.E. Manual on health self-management and patient-reported outcomes among low-income young adult Mexicans on chronic dialysis: Feasibility study.

PURPOSE: We evaluated disease knowledge/self-management skills among low-income Mexican young adults maintained on dialysis and to test the effectiveness of the A.L.L. Y.O.U. N.E.E.D. I.S. L.O.V.E (AYNIL) Manual - Spanish Version on patient-reported outcomes. This is a low literacy teaching tool designed with patients and educators' input. DESIGN AND METHODS: A quasi-experimental study was conducted in 17 chronic dialysis patients at Mexico City's Hospital General de México, Dr. Eduardo Liceaga. Ages 18-30-year-old completed disease knowledge/self-management and quality of life measures before the intervention and 6 weeks later. RESULTS: Significant increases were observed on disease knowledge/self-management scores in the STARx questionnaire from 47 (IQ: 40,51) to 50 (IQ: 48,54) p = 0.04. The UNC-TRxANSITION Index increased significantly from 4.8 (IQ: 3.9,5.7) to 7.7 (IQ: 7.5,8.2) p ≤0.001. Significant increases in scores were detected in the "Burden of kidney disease" (p = 0.008), "Effects of kidney disease" " (p = 0.03) and " Dialysis staff encouragement" (p = 0.027) based on the KDQoL survey. CONCLUSIONS: In this vulnerable population, the Spanish version of the A.L.L. Y.O.U. N.E.E.D. I.S. L.O.V.E. - AYNIL Manual improved CKD/ESRD disease knowledge/self-management skills and HRQoL. This study highlighted the need for low-literacy educational tools to improve patient-reported outcomes. PRACTICE IMPLICATIONS: Young adults with CKD/ESRD can benefit from patient-centered educational interventions to enhance their autonomy and the development of self-management behaviors that improve patient-reported outcomes and potential complications of the disease. Special attention is needed in low-income patients with low rates of adherence to treatments and poor self-management skills.

Prime Vaccination with Chitosan-Coated Phipps BCG and Boosting with CFP-PLGA against Tuberculosis in a Goat Model.

Attempts to improve the immune response and efficacy of vaccines against tuberculosis in cattle, goats, and other animal species have been the focus of research in this field during the last two decades. Improving the vaccine efficacy is essential prior to running long-lasting and expensive field trials. Studies have shown that vaccine protocols utilizing boosting with proteins improve the vaccine efficacy. The use of polymers such as chitosan and PolyLactic-co-Glycolic Acid (PLGA) improves the immune response against different diseases by improving the interaction of antigens with the cellular immune system and modulating the host immune response. This study shows that the prime BCG vaccination, boosted with a culture filtrate protein (CFP), alone or in combination with chitosan and PolyLactic-co-Glycolic Acid (PLGA), have the potential to reduce tuberculosis (TB) dissemination by reducing the number of animals with lesions, the number of lesions per animal, and the size of the lesions in vaccinated animals, compared with those not vaccinated or those vaccinated with BCG alone. The vaccinated groups showed significantly higher Interferon-γ levels in the blood compared to the control, nonvaccinated group after vaccination, after boosting, and after the challenge with the wild-type Mycobacterium bovis strain.

Application of antigenic biomarkers for Mycobacterium tuberculosis.

The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease. Promising new biomarkers for diagnosis and immunotherapy have emerged recently. Mycobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment, likely based on the complexity of the network of interactions between the molecules involved in infection. Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis. Among the most widely investigated antigens are CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secretory antigenic target), Ag85A, Ag85B, CFP-7, and PPE18. Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host. The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide. Presently, the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals. Therefore, these tests depend on the capacity of the host to develop an immune response, which usually is heterogeneous. In the last 20 years, special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity. In this review, we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies.

A repeat dose of misoprostol 800 mcg following mifepristone for outpatient medical abortion at 64-70 and 71-77 days of gestation: A retrospective chart review.

OBJECTIVE: To compare the effectiveness of outpatient medical abortion with mifepristone 200 mg and two misoprostol 800 mcg doses at 64-70 and 71-77 days of gestation. STUDY DESIGN: We conducted a retrospective chart review of medical abortion outcomes among clients with 64-77 day gestations at a Mexico City public clinic between February 2014 and November 2016 who took mifepristone 200 mg followed 24-48 h later by two doses of misoprostol 800 mcg four hours apart (first dose buccally, second dose sublingually). The primary outcome was successful medical abortion, defined as pregnancy expulsion without surgical intervention. We also assessed additional management and visits to other facilities. We compared outcomes by gestational age (64-70 vs 71-77 days). RESULTS: Of 602 charts reviewed, we analyzed 232 and 218 in the respective groups for effectiveness; nearly 25% of clients were lost to follow up. Treatment success occurred in 231 (99.6%, 95% CI 97.6-100%) clients at 64-70 days and 213 (97.7%, 95% CI 94.7-99.3%) clients at 71-77 days (p = 0.11). Ongoing pregnancy occurred in 1 (0.4%, 95% CI 0-2.4%) and 3 (1.4%, 95% CI 0.3-4.0%) clients, respectively (p = 0.36). Two charts from the 71-77 days group documented visits to other facilities: one bleeding concern prior to scheduled follow up and a hemorrhage during an aspiration intervention. CONCLUSIONS: Regimen effectiveness was high at 64-70 and 71-77 days among clients who attended follow up. However, with 25% attrition, it is difficult to draw definitive conclusions about effectiveness and associated safety. IMPLICATIONS: Mifepristone 200 mg followed by two doses of misoprostol 800 mcg four hours apart is a promising medical abortion regimen to improve efficacy in pregnancies from 64-77 days of gestation as compared to regimens with an initial single misoprostol dose. Prospective research is recommended to achieve more robust efficacy estimates.

A non-inferiority study of outpatient mifepristone-misoprostol medical abortion at 64-70 days and 71-77 days of gestation.

OBJECTIVES: This open-label non-inferiority study assessed efficacy of a common outpatient medical abortion regimen among people with pregnancies 64-70 days and 71-77 days of gestation. STUDY DESIGN: We defined non-inferiority by a 6% margin of method success. People with intrauterine pregnancies 64-77 days' gestational age by abdominal ultrasound seeking medical abortion at one of eight clinics and met eligibility criteria were offered participation. Consenting participants took mifepristone 200 mg followed 24-48 h later by misoprostol 800 mcg buccally, and returned after one week for provider evaluation and abdominal ultrasound to determine abortion status. Participants recorded medication use, pregnancy expulsion, daily bleeding and pain scores until the one-week follow up. Clinic staff interviewed participants prior to study discharge to assess acceptability. RESULTS: Seven hundred and nineteen participants were enrolled, 393 and 326 in the respective groups. Successful expulsion without surgical intervention was achieved in 92.3% of the earlier gestational age group and 86.7% of the later group (difference in proportions 5.6%, 1-sided 95% CI 9.6). Ongoing pregnancy accounted for 3.6% and 8.7% (p = 0.007) of outcomes, respectively. Participants in the 71-77 day group reported nausea and weakness more frequently. Pain, bleeding and acceptability measures between groups were similar. CONCLUSION: Although the success rate at 71-77 days of gestation was within the non-inferiority margin, we cannot rule out that it is statistically worse than in the previous gestational week. Significantly more ongoing pregnancies in the later group raise concerns about using the regimen at 71-77 days.

Molecular epidemiology of cattle tuberculosis in Mexico through whole-genome sequencing and spoligotyping.

Mycobacterium bovis infection in cattle persists in Mexico, posing a threat to human health. Control of bovine tuberculosis, through the National Program Against Bovine Tuberculosis, has led to the decrease of disease prevalence in most of the country, except for high dairy production regions. Genotyping of M. bovis has been performed mainly by spoligotyping and variable number tandem repeats (VNTR), but higher resolution power can be useful for a finer definition of the spread of the disease. Whole genome sequencing and spoligotyping was performed for a set of 322 M. bovis isolates from different sources in Mexico: Baja California, Coahuila, Estado de Mexico, Guanajuato, Hidalgo, Jalisco, Queretaro and Veracruz, from dairy and beef cattle, as well as humans. Twelve main genetic clades were obtained through WGS and genetic diversity analysis. A clear differentiation of the Baja California isolates was seen as they clustered together exclusively. However, isolates from the central states showed no specific clustering whatsoever. Although WGS proves to have higher resolving power than spoligotyping, and since there was concordance between WGS and spoligotyping results, we consider that the latter is still an efficient and practical method for monitoring bovine tuberculosis in developing countries, where resources for higher technology are scarce.

Characterization of Leptospira isolates from humans and the environment in Uruguay.

Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

Ultrafast Dischargeable LiMn2O4 Thin-Film Electrodes with Pseudocapacitive Properties for Microbatteries.

LiMn2O4 (LMO) thin films are deposited on Si-based substrates with Pt current collector via multi-layer pulsed-laser-deposition technique. The LMO thin films feature unique kinetics that yield outstanding electrochemical cycling performance in an aqueous environment. At extremely high current densities of up to 1880 µA cm-2 (≈ 348 C), a reversible capacity of 2.6 µAh cm-2 is reached. Furthermore, the electrochemical cycling remains very stable for over 3500 cycles with a remarkable capacity retention of 99.996% per cycle. We provide evidence of significant nondiffusion-controlled, pseudocapacitive-like storage contribution of the LMO electrode.

Effects of Depot Medroxyprogesterone Acetate Injection Timing on Medical Abortion Efficacy and Repeat Pregnancy: A Randomized Controlled Trial.

OBJECTIVE: To evaluate the effects of timing of depot medroxyprogesterone acetate injection on medical abortion outcome and risk of repeat pregnancy within the subsequent 6 months. METHODS: In a multinational randomized trial, we assigned women undergoing medical abortion who wanted depot medroxyprogesterone acetate to administration either with mifepristone (Quickstart group) or after the abortion (Afterstart group). We ascertained abortion outcome, pregnancies, and contraception use over 7 months. RESULTS: From August 2013 to March 2015, we enrolled 461 participants with pregnancy durations of 75 days or less. Of participants included in the abortion outcome analyses, 14 of 220 (6.4%) and 12 of 226 (5.3%) in the Quickstart and Afterstart groups, respectively, had surgery to complete the abortion; the upper 90% confidence limit on this difference was 4.9%, within our prestipulated 5% noninferiority margin. Ongoing pregnancy after initial abortion treatment was significantly more common in the Quickstart group (8/220 [3.6%]) than in the Afterstart group (2/226 [0.9%]); the difference was 2.7% (90% confidence interval 0.4-5.6%). By 6 months, 5 of 213 (2.3%) and 7 of 217 (3.2%) in the Quickstart and Afterstart groups, respectively, became pregnant (exact log-rank test, P=.64). Use of highly effective contraceptives was significantly more common in the Quickstart group at 31 days (P<.001), but no difference was apparent at 6 months. The Quickstart group was significantly more satisfied with group assignment. CONCLUSION: Depot medroxyprogesterone acetate administration with mifepristone did not appreciably increase the risk of surgery after medical abortion but did increase the risk of ongoing pregnancy. It enhanced patient satisfaction, but we found no evidence that it decreased 6-month risk of repeat pregnancy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT01902485.

Genetic diversity based on MIRU-VNTR profile of isolates of Mycobacterium bovis from Mexican cattle.

Bovine tuberculosis (bTB) is a disease caused by Mycobacterium bovis (M. bovis), which affects cattle, animal species and humans. To determinate the genetic structure of strains of M. bovis in mexican cattle, 467 isolates obtained from 2009 to 2010 from different regions of Mexico with known spoligotype were included in the study. The isolates were genotyped by interspersed repeated mycobacterial units-variable number tandem repeats (MIRU-VNTR) obtaining 13 MIRU-VNTR groups. When combining MIRU-VNTR patterns with its spolygotypes, the Hunter genetic discrimination index (HGDI), we obtained 421 genetic patterns distributed in 17 groups. The HGDI for the total loci was 0.99. The locus that presented the higher HGDI was 2461 (0.857), while the locus with the lowest HGDI was 2686 (0.239). When we analyzed our results, using just 6 or 8 MIRU-VNTR we obtained an discriminatory power of 0.8499 and 0.8875 respectively indicating lower HGDI than 12 MIRU-VNTR locus.

Molecular Relationship between Strains of M. bovis from Mexico and Those from Countries with Free Trade of Cattle with Mexico.

The purpose of this study was to identify relationships between spoligotypes of M. bovis from cattle in Mexico and those reported in countries with free trade of cattle with Mexico: Australia, Canada, New Zealand and the United States of America. Mexican spoligotypes were obtained from isolates collected from cattle in different parts of the country. Spoligotypes from Canada and New Zealand were obtained from different reports in the literature. Those from the United States were obtained from the database of the National Veterinary Services Laboratory in APHIS-USDA. In order to perform the analysis in a single data set, spoligotypes were all converted to binary data and classified according to www.mbovis.org or www.pasteur-guadeloupe.fr:8081. Epidemiologic information included country and species infected. From 3,198 isolates, 174 different spoligotypes were obtained, 95 were orphans. Ninety one percent of the isolates came from the Unites States (n = 1,609) and Mexico (n = 1,323). Spoligotype SB0265 is shared between Canada and the United States in cattle and wildlife. Six spoligotypes, SB0673, SB0121, SB0145, SB0971, SB0140 and SB1165, were frequent in cattle and wildlife in the United States and cattle in Mexico, suggesting wide exchange of strains. Spoligotype SB0669 was found only in Mexico. Spoligotype SB0140 was the most common in Australia and the sixth in the United States and Mexico. In a phylogenetic analysis, spoligotype SB0140 appears as the oldest spoligotype in the data set, suggesting this as the ancestral spoligotype for all spoligotypes in the five countries. Some spoligotypes are shared by animals and humans, corroborating the zoonotic importance of M. bovis.

Effect of Immediate Compared With Delayed Insertion of Etonogestrel Implants on Medical Abortion Efficacy and Repeat Pregnancy: A Randomized Controlled Trial.

OBJECTIVE: To evaluate the effect of insertion of etonogestrel implants with mifepristone compared with after the abortion on the risks of medical abortion failure and repeat pregnancy over the subsequent 6 months. METHODS: In a randomized trial, we assigned patients undergoing medical abortion to receive etonogestrel implants either with the mifepristone (Quickstart group) or after the abortion (Afterstart group). We followed them for 7 months to ascertain abortion outcome, pregnancies, and contraception use. RESULTS: Between September 2013 and August 2014, we enrolled 236 participants in the Quickstart group and 240 in the Afterstart group. To examine abortion failure, we conducted a noninferiority analysis from which we excluded nine participants who had missing outcome data and four with specified protocol violations. Of the rest, 9 of 229 (3.9%) and 9 of 234 (3.8%) in the Quickstart and Afterstart groups, respectively, had surgery to complete the abortion; the difference of 0.08% (90% confidence interval -3.1% to 3.3%) excluded our prestipulated noninferiority margin of 5 percentage points. Among participants with pregnancy follow-up through 6 months, 1 of 213 (0.5%) and 3 of 208 (1.4%) in the Quickstart and Afterstart groups, respectively, became pregnant within that time; 6-month pregnancy rates did not differ significantly by group (exact log-rank test, P=.28). At enrollment, significantly more participants in the Quickstart group than in the Afterstart group were satisfied with their group assignments (187/236 [79%] compared with 129/240 [54%], respectively; P<.001). CONCLUSION: Insertion of etonogestrel implants with mifepristone did not appreciably increase medical abortion failure risk and it enhanced patient satisfaction, but we found no evidence that it decreased repeat pregnancy rates. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01902485.

BmVDAC upregulation in the midgut of Rhipicephalus microplus, during infection with Babesia bigemina.

The molecular mechanisms involved during the infection of Rhipicephalus microplus midgut cells by Babesia bigemina are of great relevance and currently unknown. In a previous study, we found a voltage-dependent anion channel (VDAC)-like protein (BmVDAC) that may participate during parasite invasion of midgut cells. In this work, we investigated BmVDAC expression at both mRNA and protein levels and examined BmVDAC localization in midgut cells of ticks infected with B. bigemina at different times post-repletion. Based on the RT-PCR results, Bmvdac expression levels were significantly higher in infected ticks compared to uninfected ones, reaching their highest values at 24h post-repletion (p<0.0001). Similar results were obtained at the protein level (p<0.0001). Interestingly, BmVDAC immunolocalization showed that there was an important differential expression and redistribution of BmVDAC protein between the midgut cells of infected and uninfected ticks, which was more evident 24h post-repletion of infected ticks. This is the first report of BmVDAC upregulation and immunolocalization in R. microplus midgut cells during B. bigemina infection. Further studies regarding the function of BmVDAC during the infection may provide new insights into the molecular mechanisms between B. bigemina and its tick vector and could result in its use as an anti-tick and transmission-blocking vaccine candidate.

What Can Proteomics Tell Us about Tuberculosis?

Tuberculosis (TB) is an infectious disease transmitted by aerosol droplets and characterized by forming granulomatous lesions. Although the number of people infected in the population is high, the vast majority does not exhibit symptoms of active disease and only 5-10% develop the disease after a latent period that can vary from weeks to years. The bases of the immune response for this resistance are unknown, but it depends on a complex interaction between the environment, the agent, and the host. The analysis of cellular components of M. tuberculosis shows important host-pathogen interactions, metabolic pathways, virulence mechanisms, and mechanisms of adaptation to the environment. However, the M. tuberculosis proteome still remains largely uncharacterized in terms of virulence and pathogenesis. Here, we summarize some of the major proteomic studies performed to scrutinize all the mycobacterial components.

Valoración de un procedimiento de inmunofluorescencia indirecta para la detección de anticuerpos tipo IgM (IF-IgM) utilizado en el diagnóstico temprano de leptospirosis / Evaluation of an indirect immunofluorescence assay for detection of IgM (IF-IgM) type antibodies in the early diagnosis of leptospirosis

Introducción: la leptospirosis es una enfermedad febril, aguda, que presenta manifestaciones clínicas variadas. Esto dificulta o retarda el diagnóstico clínico, por lo cual es útil disponer de métodos de laboratorio adecuados para orientar el manejo inicial de estos pacientes. Objetivo: evaluar un procedimiento de inmunofluorescencia para detectar IgM (IF-IgM) de elaboración propia aplicado al diagnóstico temprano de leptospirosis. Material y método: se analizaron por IF-IgM y microaglutinación (MAT) (tomada como estándar de referencia) sueros obtenidos de pacientes con sospecha clínica de leptospirosis. La sensibilidad y especificidad de la IF-IgM versus MAT se establecieron utilizando una tabla de doble entrada. La concordancia entre dos observadores se determinó por el test Kappa. Resultados: de 161 muestras precoces analizadas, 97 sueros correspondieron a pacientes con infección aguda confirmada por MAT y 64 sin infección. La sensibilidad y especificidad de la IF-IgM con sueros de fase aguda fueron 79% y 100%, respectivamente. El índice Kappa fue 1. Conclusiones: la IF-IgM aparece como una herramienta útil para el diagnóstico temprano de pacientes con leptospirosis. No requiere el manejo de bacterias viables, puede realizarse en laboratorios que cuenten con microscopio de luz ultravioleta, se necesita una sola muestra de suero y el resultado está listo en tres a cuatro horas. En cuanto a las desventajas, no identifica los serovares involucrados y un resultado negativo no descarta la infección. Teniendo en cuenta esto último es obligatorio analizar por MAT una segunda muestra de suero obtenida a los 10-20 días de la primera para descartarla o confirmarla...

Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay.

INTRODUCTION: Assays based on DNA amplification can provide information that contributes to the initial management of patients with leptospirosis. However, these have not been adopted in Uruguay. Our aim was to evaluate the performance of the lipL32 real-time PCR (qPCR) for diagnosis of leptospirosis. METHODOLOGY: We analyzed by microscopic agglutination test (MAT) and lipL32 qPCR serum samples from 183 patients with suspected leptospirosis. To establish the analytical sensitivity of the qPCR, experimentally spiked samples with known amounts of Leptospira interrogans were analyzed. RESULTS: The analytical sensitivity of the qPCR was 102 leptospires/mL. In 98 patients MAT results were negative meanwhile 85 showed positive reactions, revealing acute infections. Twenty six acute-phase sera of these 85 patients showed a positive signal by qPCR (diagnostic sensitivity 30%). In these patients the average time between onset of symptoms and collection of the first sample was 8 days. In patients with negative results for qPCR and positive MAT results (n=59) the average interval between onset of symptoms and collection of the first sample was 13 days. The qPCR did not yield false positive results. CONCLUSIONS: The qPCR had a lower diagnostic sensitivity than MAT and a higher cost. However, it allowed to make an early diagnosis in 26 patients. In patients with confirmed acute infections and negative results by qPCR, more than 8 days had elapsed between the onset of the illness and extraction of the first serum sample. Our data support that the qPCR from sera have clinical utility within the first week of illness.

A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem.

Leptospira spp. are delicate bacteria that cannot be studied by usual microbiological methods. They cause leptospirosis, a zoonotic disease transmitted to humans through infected urine of wild or domestic animals. We studied the incidence of this disease in the Uruguayan population, its epidemiologic and clinical features, and compared diagnostic techniques. After examining 6,778 suspect cases, we estimated that about 15 infections/100,000 inhabitants occurred yearly, affecting mainly young male rural workers. Awareness about leptospirosis has grown among health professionals, and its lethality has consequently decreased. Bovine infections were probably the principal source of human disease. Rainfall volumes and floods were major factors of varying incidence. Most patients had fever, asthenia, myalgias or cephalalgia, with at least one additional abnormal clinical feature. 30-40% of confirmed cases presented abdominal signs and symptoms, conjunctival suffusion and altered renal or urinary function. Jaundice was more frequent in patients aged > 40 years. Clinical infections followed an acute pattern and their usual outcome was complete recovery. Laboratory diagnosis was based on indirect micro-agglutination standard technique (MAT). Second serum samples were difficult to obtain, often impairing completion of diagnosis. Immunofluorescence was useful as a screening test and for early detection of probable infections.