Irene Teresa Molina Martínez

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniasis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.

Assessment of genetic diversity of zoonotic Brucella spp. Recovered from livestock in Egypt using multiple locus VNTR analysis

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples

DNA-based methods have emerged as an additional tool for Brucella infection-confirmation at a herd level. However, their implementation may require the use of specialized equipment. In this context the recently developed loop-mediated isothermal amplification (LAMP) technique may constitute an additional and cost-effective tool for rapid and specific DNA detection, especially in low income areas. In the present study the usefulness of a newly developed LAMP assay aiming at the multicopy-IS711 sequence was assessed on a variety of clinical samples (n=81 from abortions and ewes; cattle, n=3; swine, n=4) that were analyzed in parallel using real-time PCR and bacteriology. Although overall sensitivities obtained with the three methods were comparable (p>0.05), our results highlighted the complementarity between bacteriology and molecular-based methods for increased sensitivity. Significant differences (p<0.05) were observed with all techniques depending on the nature of the sample. Our results demonstrate the potential of the IS711-LAMP technique for direct Brucella detection.

Optimising the controlled release of dexamethasone from a new generation of PLGA-based microspheres intended for intravitreal administration.

Successful therapy for chronic diseases affecting the posterior segment of the eye requires sustained drug concentrations at the site of action for extended periods of time. To achieve this, it is necessary to use high systemic doses or frequent intraocular injections, both associated with serious adverse effects. In order to avoid these complications and improve patients quality of life, an experimental study has been conducted on the preparation of a new generation of biodegradable poly(D,L-lactide-co-glycolide) (50:50) (PLGA) polymer microspheres (MSs) loaded with Dxm, vitamin E and/or human serum albumin (HSA). Particles were prepared according to a S/O/W encapsulation method and the 20-40μm fraction was selected. This narrow size distribution is suitable for minimally invasive intravitreal injection by small calibre needles. Characterisation of the MSs showed high Dxm loading and encapsulation efficiency (> 90%) without a strong interaction with the polymer matrix, as revealed by DSC analysis. MSs drug release studies indicated a small burst effect (lower than 5%) during the first five hours and subsequently, drug release was sustained for at least 30days, led by diffusion and erosion mechanisms. Dxm release rate was modulated when solid state HSA was incorporated into MSs formulation. SDS-PAGE analysis showed that the protein maintained its integrity during the encapsulation process, as well as for the release study. MSs presented good tolerance and lack of cytotoxicity in macrophages and HeLa cultured cells. After 12months of storage under standard refrigerated conditions (4±1°C), MSs retained appropriate physical and chemical properties and analogous drug release kinetics. Therefore, we conclude that these microspheres are promising pharmaceutical systems for intraocular administration, allowing controlled release of the drug.

Interferon-gamma responses in sheep exposed to virulent and attenuated Brucella melitensis strains

Antibody detection is the basis of large-scale sheep brucellosis diagnosis because of its sensitivity and specificity. In contrast, information on the cellular mediated immune (CMI) response triggered after Brucella melitensis infection, a cornerstone in the protection against this pathogen, is more limited, particularly regarding the effect of the virulence of the infecting strain in the induced CMI reaction. Here, the interferon-gamma (IFN-γ) profiles evoked after exposure by different routes to virulent (H38) and attenuated (Rev.1) B. melitensis strains in 14 pregnant sheep and 87 ewe lambs, respectively, were characterized accounting for different host-related factors, and compared with their serological response and with the basal IFN-γ responses observed in 155 animals non exposed to Brucella. No significant differences in the IFN-γ response of Rev.1 vaccinated animals depending on the inoculation route was observed, in contrast with their serological results. Response in H38-challenged followed a similar trend although peaked later, and an effect of the abortion on the IFN-γ response was detected. This information could help to understand the interaction bacteria-host that leads to its intracellular survival and could be useful for the design of new diagnostic approaches.

Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct-PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach

Abstract Leishmaniasis, caused by Leishmania infantum, is a vector‐borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent‐class model was used here to estimate the sensitivity and specificity of the Immuno‐fluorescence antibody test (IFAT) in serum and a Leishmania‐nested PCR (Ln‐PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two‐population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut‐off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70–80%, whereas the Ln‐PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.

FRI0533 Primary Pyomyositis in Children: A Retrospective Review of 25 Cases

Background Primary pyomyositis is a rare condition in children that should be included in the differential diagnosis of musculoskeletal infections. Objectives To describe the clinical features of pyomyositis as well as its diagnostic and therapeutic approach. Methods A descriptive and retrospective study was performed by reviewing medical records of patients admitted to our hospital from January 1996 to July 2013 with a diagnosis of primary pyomyositis. Results A total of 25 patients (16 men, 9 women) aged between one month and 14 years (median age 2 years) were registered. Two peaks of incidence have been found in the months of July and October. The most frequent clinical manifestations were the presence of local pain (24 patients), fever (18 patients), local swelling (9 patients) and skin rash (7 patients). Predisposing factors in 8 patients (1 strenuous exercise, 4 trauma, 3 intramuscular injections) were found. The primary site was pelvic muscles or lower extremities (20 patients). In laboratory results, we found the presence of leukocytosis, with an average value of 12,936±5.753/uL leukocytes, total neutrophils 4.659/uL ±7.350), ESR of 55±28 mm) and PCR 8±6 mg/dL). CPK was normal in 5 out of 6 patients who were requested. Laboratory results were positive for Staphylococcus aureus (7 patients), Salmonella no tiphy (1 patient) and Bacteroides fragillis (1 patient). Ultrasound suggested the diagnosis in 11 cases and magnetic resonance imaging support the diagnosis in all cases. All patients received intravenous antibiotic therapy (mean duration 11 days) followed by oral antibiotic therapy (mean duration 23 days). 9 patients suffered complications as abscess formation, 3 need abscess aspiration and one of them required surgical drainage. None of our patients had residual functional limitations. Conclusions It is important to have a high index of suspicion for pyomyositis in patients with fever and musculoskeletal pain at an early stage in order to initiate start appropriate antibiotic treatment against Staphylococcus aureus. Although ESR and ultrasound may be useful, it is necessary to complement with magnetic resonance imaging in order to support the diagnosis in all cases. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4467