SummaryAccurate, timely diagnosis is essential for the control, monitoring and eradication of foot‐and‐mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple‐to‐use technologies, including molecular‐based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)‐specific reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) and real‐time RT‐PCR (rRT‐PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory‐based rRT‐PCR. However, the lack of robust ‘ready‐to‐use kits’ that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT‐PCR and RT‐LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real‐time, and for the RT‐LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

SummaryCapripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real‐time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory‐based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory‐based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory‐based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings.

SummaryHighly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT‐PCR diagnostic system (Enigma MiniLab®) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real‐time RT‐PCR (rRT‐PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

The aim of present study was calculate the Minimum inhibitory concentrations (MICs) of silver nanoparticles and clotrimazole for Candida species and their interaction by the adaptation of standarized methods. The MICs values of clotrimazole were 9 E -04 -3 E -03 ug/ml, 0.1-0.6 ug/ml, 3 E -03 -0.1 ug/ml and 3 E -03 -0.3 ug/ml for Candida albicans susceptible to fluconazole, Candida albicans resistance to fluconazole, Candida krusei and Candida parapsilosis respectively. The MICs values of silver nanoparticles were 26.50-53 ug/ml; 26.50-106 ug/ml; 106-212 ug/ ml and 26.50-53 ug/ml for Candida albicans susceptible to fluconazole, Candida albicans resistance to fluconazole, Candida krusei and Candida parapsilosis respectively. Synergism between clotrimazole and silver nanoparticles was measured by checkerboard BMD (broth microdilution) test and shown only for C. albicans susceptible to fluconazole because the fractional inhibitory concentrations (FICs) values were 0.07 -0.15 ug/ml. Indifference was shown for the other species tested because the FICs values were between 0.5 -2-3.06 ug/ml. The results suggest synergistic activity depending on the fungus species analysed, however we recommend the incorporation of others measurement methodologies to confirm our results. As for measurement methodologies of MICs of silver nanoparticles and clotrimazole international normative were respected to guarantee reproducible and comparable results.

Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the bacterium Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato, a tick with worldwide distribution. When not diagnosed and treated early, disease can be severe. Currently, the disease is confirmed by serological or molecular assays. The objective of this study was to compare a serological assay based on immunochromatography (SPEED® EHRLI immunochromatographic test; BVT, France) and a molecular assay (a screening PCR followed by a nested PCR specific for E. canis) for the diagnosis of E. canis in suspected dogs from Buenos Aires city and southern Greater Buenos Aires, Argentina. Blood samples from 20 clinically healthy dogs (Control Group) and from 80 sick dogs suspected of having CME (Groups 1 to 4) were tested in parallel. Neither the immunochromatographic test nor the PCR assay was able to detect the presence of E. canis in the Control Group. In the group which had been previously tested by serology, the agreement between the tests was low (kappa: 0.200), whereas in the group which had been previously tested by PCR, the concordance between the tests was adequate (kappa: 0.650). The concordance between the tests evaluated in the total population studied was moderate (kappa: 0.496). The results of our study suggest that the use of rapid serological tests as a first approach, together with subsequent confirmation by PCR, will improve the diagnosis of CME.

<p>La hepatozoonosis canina (HC) es una enfermedad transmitida por la ingestión de garrapatas infectadas con Hepatozoon canis que puede causar signos clínicos graves dependiendo de la inmunidad del hospedador. El objetivo de este trabajo fue detectar la presencia de H. canis en muestras de caninos sin signología provenientes del Gran Buenos Aires a través de la observación microscópica en extendidos de sangre periférica y capa flogística. Posteriormente, se determinó la casuística de la HC en los diferentes barrios, la asociación entre las variables raza, sexo, edad y la presencia de garrapatas con el diagnóstico hematológico positivo de HC y la asociación entre las alteraciones del hemograma y la presencia de HC. De las 207 muestras 10 (4,83%) fueron positivas mediante la observación en frotis de sangre periférica y 15 (7,24%) en frotis de sangre periférica y extendidos de la capa flogística. Con respecto a las variables evaluadas, la HC fue más frecuente en adultos, hembras, de raza mestiza y con presencia de garrapatas, no obstante las diferencias no fueron significativas. El mayor porcentaje de muestras positivas se observó en aquellos pacientes provenientes de los barrios de Solano y Quilmes. En relación a las alteraciones hematológicas en el grupo de muestras positivas a HC se observó anemia (20%), leucocitosis (40%), linfocitosis (26,6%) y eosinofilia (80%) en un mayor porcentaje con respecto al grupo de muestras negativas, no obstante las diferencias entre ambos grupos no fueron significativas (p&gt;0,05). Este estudio corrobora la endemicidad de la HC en el Gran Buenos Aires y demuestra que la prevalencia varía con la procedencia de las muestras y del momento en que se realiza el estudio. Asimismo, refleja la utilidad de la concentración leucocitaria para aumentar las probabilidades de observación positiva de gamontes cuando no se disponga de métodos moleculares.</p>

La leptospirosis es una enfermedad zoonótica de distribución mundial causada por bacterias del género Leptospira que afecta a numerosas especies de animales domésticos y silvestres. El diagnóstico en caninos se realiza demostrando seroconversión en muestras del paciente en el periodo agudo y convaleciente de la enfermedad mediante la prueba de microaglutinación (MAT) no obstante, la aplicación de la técnica de reacción en cadena de la polimerasa (PCR) puede proporcionar una confirmación temprana de los casos sospechosos. El objetivo de este trabajo fue validar en una etapa intralaboratorio una técnica de PCR en tiempo real (qPCR) que utiliza cebadores dirigidos a un fragmento del gen lipL32 (lipL32-qPCR). Posteriormente se evaluó su utilidad en el diagnóstico, a partir de muestras clínicas de caninos con sospecha de la enfermedad tomando, como referencia la prueba de MAT. La sensibilidad analítica a partir del ADN de cepas de referencia fue de 1 x 100 equivalente genoma/reacción. Al evaluar la especificidad analítica, todas las cepas patógenas fueron correctamente amplificadas mientras que con el ADN de 2 cepas saprófitas y otros microorganismos se obtuvieron resultados negativos. En muestras biológicas contaminadas experimentalmente la sensibilidad analítica fue de 1 x 102 bacterias/ml en sangre, 1 x 103 bacterias/ml en suero y 1 x 101 bacterias/ml en orina. La técnica lipL32- qPCR demostró mayor sensibilidad analítica a partir del ADN de cepas de referencia y de las muestras de sangre y orina contaminadas experimentalmente que una técnica de PCR convencional dirigida a un fragmento del gen rrs (rrs-PCR 2 convencional), excepto para las muestras de suero en las cuales se obtuvo el mismo valor. En el estudio a campo, se incluyeron un total de 51 caninos con sospecha clínica de la enfermedad. Con la prueba de MAT, 7 de 51 pacientes fueron considerados como casos confirmados. La técnica lipL32-qPCR fue positiva en 6 de los siete casos confirmados por MAT y permitió realizar el diagnóstico en dos pacientes seronegativos. Mientras que la técnica rrs-PCR convencional sólo detectó 4 de 7 casos confirmados. Al comparar la prueba de MAT y la lipL32-qPCR se observó una fuerte concordancia entre ambas (Kappa=0.76, 95 % IC 0.492-1.039). De acuerdo a los resultados obtenidos se concluye que la prueba lipL32-qPCR resulta un excelente complemento de la serología en la confirmación de casos sospechosos y permite diagnosticar un mayor porcentaje de caninos verdaderos positivos.