How is post-mortem microbiology appraised by pathologists? Results from a practice survey conducted by ESGFOR.

Post-mortem microbiology (PMM) is an important tool in forensic pathology, assisting to determine the cause and manner of death. However, there is a lack of standardisation of PMM sampling. In order to get a better insight into the methods used, the available technical options and developmental needs, ESCMID Study Group for Forensic and Postmortem Microbiology (ESGFOR) members designed a survey aimed at pathologists regarding common practices of PMM in clinical and forensic autopsies. Multiple choice questions were developed based on Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech). The questionnaire was sent to pathologists mainly across Europe and Turkey using SurveyMonkey. The survey had 147 respondents. Although all pathologists were aware of the existence of PMM, 39% (19/49) of the participants were not using it. The three main indications for PMM were: (i) clinical suspicion of an infection not confirmed antemortem (83%), (ii) infectious signs at autopsy (83%) and (iii) as part of a standard protocol for foetal/perinatal or paediatric death (67%). Almost 80% of the participants using PMM stated taking 1-10 samples per case. Of the requested examinations, a general bacteriological culture (96%) and a specific polymerase chain reaction (PCR) assay for a particular infectious agent (34%) were most popular. The most frequent samples were: heart blood (66%), peripheral femoral blood (49%), spleen (64%) and lung (56%). Eighty-nine percent of the participants considered PMM a useful resource when investigating the cause of death. Although there are some common uses, this survey indicates that there is a need for improvement towards standardising sampling procedures in PMM.

[Pediatric Extracorporeal Shock Wave Lithotripsy]. / Litotricia extracorpórea en pacientes pediátricos.

INTRODUCTION: Extracorporeal Shock Wave lithotripsy (ESWL) is the cornerstone of pediatric urolitiasis management. We evaluated its efficacy and complications in a series of children. MATERIAL AND METHODS: Children who were managed with ESWL between 2003 and 2012 were retrospectively reviewed. We studied etiology, clinical presentation, stonefree ratio and relevant complications. SPSS 17.0 software was used. RESULTS: 90 children aged 0 to 10 years (median 2.9 years) were included in the study; that accounted for 122 stones and 162 ESWL sessions. Mean follow up was 20 months. Mean stone diameter was 12.2 mm. (range 4-25). Most prevalent clinical sign was urinary infection (55.6%, 50 patients). 75.4% of the lithiasis were found in renal pelvis or calices. A mean of 1.42 ESWL sessions per stone was performed. Stonefree status was reached in 80.3% (98) of the lithiasis. This rate was higher in patients below 5 years of age (86.1% vs., 69.8%, p=0.03), and worse in staghorn calculi (66% vs. 87.2%) and cystine ones (30% vs. 84.8%, p<0.001). We observed 17 complications (10.4% among 162 sessions), 6 UTIs, 6 episodes of fever and 6 episodes of UTI associated with steinstrasse. Almost all complications were associated with bigger size, staghorn calculi and struvite. DISCUSSION: Best results are found in younger patients and small calculi. ESWL is a safe and efficient procedure in pediatric patients.

INTRODUCCION: La Litotricia Extracorpórea por Ondas de Choque (LEOC) constituye el pilar fundamental de la urolitiasis infantil. En este trabajo pretendemos objetivar la tasa de fragmentación y expulsión de cálculos mediante LEOC. MATERIAL Y METODOS: Revisión retrospectiva de procedimientos de LEOC pediátricos, analizando etiología, clínica, tasa de éxito, factores asociados al mismo y complicaciones. Análisis: SPSS 17.0. RESULTADOS: Se revisaron 90 niños (edad: 8 meses-10 años -mediana 2,9 años) que habían presentado 122 litiasis y precisaron 162 procedimientos de LEOC entre 2003 y 2012. Mediana de seguimiento: 20 meses. Diámetro medio del cálculo: 12,2 mm (rango 4-25 mm). La clínica más habitual fue Infección del Tracto Urinario (ITU) (55,6%, 50 niños). El 3,3% de los cálculos se localizaron en vejiga, el 21,3% en uréteres y el 75,4% en riñón. Se realizó una media de 1,41 LEOC por episodio litiásico. Se consiguió fragmentación y expulsión en el 80,3% (98) de los cálculos. Esta tasa fue mayor en niños menores de 5 años (86,1% vs. 69,8%, p=0,03) y en cálculos asociados a prematuridad y estancia prolongada en UCI (100% vs. 78,4%, p=0,19); y peor en cálculos coraliformes (66% vs. 87,2%, p=0,021), y en los de cistina (30% vs. 84,8%, p<0,001). Entre los 162 procedimientos, hubo 17 complicaciones (10,4%): (6 ITUs ­3,7%­, y 5 ITUs asociadas a calle litiásica ­3%­), todas relacionadas con cálculos grandes, coraliformes y/o de estruvita. CONCLUSION: Los mejores resultados en LEOC se objetivan en los pacientes de menor edad. La LEOC pediátrica es eficaz y segura.

Evaluation of canine leishmaniosis vaccine CaniLeish® under field conditions in native dog populations from an endemic Mediterranean area-A randomized controlled trial.

Dog vaccination is considered an effective way of reducing Leishmania infantum infection incidence in the canine population, as well as its transmission to humans. However, the use of partially effective vaccines can have the detrimental effect of "masking" vaccinated asymptomatic carriers, capable of harbouring the parasite and transmitting it to naïve individuals. After eight years on the European market, few studies have been released on CaniLeish® vaccine safety and efficacy. The present study, a one-year randomized CaniLeish® vaccine field trial, was performed in a canine leishmaniosis endemic area and included animals selected from a native dog population (n = 168). No severe adverse reactions were observed in vaccinated dogs (n = 85). Cases of active L. infantum infection were detected by serological, molecular and clinical follow-up of dogs. One-year post-vaccination, no differences in number or severity of L. infantum active infections were observed between study groups (n = 4 in each group). Vaccine-induced cellular immunity, assessed through interferon-γ quantification, showed significantly higher levels of this cytokine one-month post-vaccination in the vaccine group (p < 0.001), but no differences were observed after nine months between trial groups (p = 0.078). These results fail to support the reported CaniLeish® efficacy in the prevention of active L. infantum infection in dogs from endemic areas and naturally exposed to the parasite.

Post-mortem microbiology in sudden death: sampling protocols proposed in different clinical settings.

BACKGROUND: Autopsies, including minimally invasive autopsies, are a powerful tool for determination of the cause of death. When a patient dies from an infection, microbiology is crucial to identify the causative organism. Post-mortem microbiology (PMM) aims to detect unexpected infections causing sudden deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical errors. Additionally, the analysis of the thanatomicrobiome may help to estimate the post-mortem interval. AIMS: The aim was to provide advice in the collection of PMM samples and to propose sampling guidelines for microbiologists advising autopsy pathologists facing different sudden death scenarios. SOURCES: A multidisciplinary team with experts in various fields of microbiology and autopsies on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - study group of forensic and post-mortem microbiology and in collaboration with the European Society of Pathology) developed this narrative review based on a literature search using MedLine and Scopus electronic databases supplemented with their own expertise. CONTENT: These guidelines address measures to prevent sample contamination in autopsy microbiology; general PMM sampling technique; protocols for PMM sampling in different scenarios and using minimally invasive autopsy; and potential use of the evolving post-mortem microbiome to estimate the post-mortem interval. IMPLICATIONS: Adequate sampling is paramount to identify the causative organism. Meaningful interpretation of PMM results requires careful evaluation in the context of clinical history, macroscopic and histological findings. Networking and closer collaboration among microbiologists and autopsy pathologists is vital to maximize the yield of PMM.

Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs.

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.

Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain.

Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.

Canine Leishmaniasis Progression is Associated with Vitamin D Deficiency.

The relationship between vitamin D deficiency and the risk of suffering from a plethora of health disorders, ranging from autoimmune processes to infectious diseases has been widely described. Nonetheless, the potential role of vitamin D in visceral leishmaniasis remains uncharacterized. In the Mediterranean basin, where the dog is leishmania's main peri-domestic reservoir, control measures against the canine disease have shown beneficial effects on the incidence of human leishmaniasis. In this study, we measured the vitamin D levels in serum samples from a cohort of 68 healthy and disease dogs from a highly endemic area and we have also studied the relationship of these levels with parasitological and immunological parameters. The sick dogs presented significantly lower (P < 0.001) vitamin D levels (19.6 ng/mL) than their non-infected (31.8 ng/mL) and the asymptomatic counterparts (29.6 ng/mL). In addition, vitamin D deficiency correlated with several parameters linked to leishmaniasis progression. However, there was no correlation between vitamin D levels and the Leishmania-specific cellular immune response. Moreover, both the leishmanin skin test and the IFN-γ levels displayed negative correlations with serological, parasitological and clinical signs. Further studies to determine the functional role of vitamin D on the progression and control of canine leishmaniasis are needed.

Effect of highly active anti-retroviral therapy and hepatitis C virus co-infection on serum levels of pro-inflammatory and immunoregulatory cytokines in human immunodeficiency virus-1-infected individuals.

This study aimed to determine the effect of highly active anti-retroviral therapy (HAART) and hepatitis C virus (HCV) co-infection on peripheral levels of interleukin (IL)-2, IL-10, IL-12 (p70), IL-18 and soluble tumour necrosis factor receptor type II (sTNFRII). Serum levels were monitored for a 1-year period in 25 patients infected with human immunodeficiency virus-1 (HIV-1) who were naive for HAART at the initiation of the study, and in four HIV-1-infected long-term non-progressors. Serum levels of both IL-18 and sTNFRII at baseline were significantly higher in HIV-1-infected patients than in controls. Baseline levels of IL-18 and sTNFRII were not significantly different in long-term non-progressors compared with the other patients. HCV co-infected patients had significantly higher levels of IL-18 and sTNFRII at each time-point compared with patients who were not co-infected with HCV. Irrespective of HCV status, response to HAART resulted in a significant decrease in the levels of both IL-18 and sTNFRII, particularly among patients who achieved HIV viral suppression, but the net decrease observed at the end of follow-up was lower in patients co-infected with HCV. No information was obtained from IL-2, IL-10 and IL-12 (p70) measurements. The data suggest that analysis of serum levels of IL-18 and sTNFRII may be a valuable tool for evaluating the response to HAART, and perhaps for assessing the degree of immune restoration achieved by HAART responders. The results also highlight the relevance of considering the HCV infection status of HIV-1-infected patients in order to avoid misinterpretation of IL-18 and sTNFRII measurements.

Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis.

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.

Early neutralizing and glycoprotein B (gB)-specific antibody responses to human cytomegalovirus (HCMV) in immunocompetent individuals with distinct clinical presentations of primary HCMV infection.

BACKGROUND: Antibodies with functional anti-Human Cytomegalovirus (HCMV) activity are likely to be involved in preventing virus dissemination and thus may contribute to minimize the clinical manifestations of infection. OBJECTIVES: To investigate the role of humoral immunity in modulating the clinical expression of primary Human Cytomegalovirus (HCMV) infection in immunocompetent persons. STUDY DESIGN: Neutralizing (NA) and glycoprotein B (gB)-specific antibodies were quantitated in acute-phase and late-convalescence phase sera from 19 individuals who developed either HCMV mononucleosis (12) or oligosymptomatic hepatitis (seven). RESULTS: The levels of NA in sera drawn early after infection were significantly lower in the former patients than in the latter (P=0. 032). This difference was not related to either the total serum IgG levels and anti-HCMV IgGs avidity or to the presence of higher viral loads in blood, as assessed by detecting serum HCMV DNA by PCR, in patients experiencing mononucleosis. Increased NA titers were seen in all available late-convalescence sera. In these sera, median NA levels were not significantly different among the study groups. Antibodies to HCMV gB of both IgG and IgM classes were detected in all acute-phase sera analyzed. Median anti-gB IgG and IgM titers did not differ significantly between study groups. Likewise, the IgG subclass reactivity pattern against gB was found to be similar for both groups. CONCLUSIONS: The data revealed that an intense and early antibody response to gB developed in patients undergoing primary HCMV infection irrespective of the clinical manifestation of the disease. In contrast, a deficient NA response was observed in patients with HCMV mononucleosis versus that observed in patients displaying a milder form of disease-suggesting that the strength of NA response to HCMV generated early after infection might determine the severity of primary HCMV infection.

Pharmacokinetics of liposome-encapsulated meglumine antimonate after intramuscular and subcutaneous administration in dogs.

Controlling canine leishmaniasis may reduce the incidence of human leishmaniasis, which affect immunocompromised persons, especially those with human immunodeficiency virus infection. Thus, the pharmacokinetics of liposome-encapsulated meglumine antimonate (LMA) in dogs was studied after intramuscular (I.M.) and subcutaneous (S.C.) administration. Serum concentration-time data for both forms of administration were best described by a triexponential open model. The absorption phase showed statistically significant differences between I.M. and S.C. administrations (K01(I.M.) = 0.046/min, K01(S.C.) = 0.025/min). The first phase of decrease of plasma concentrations showed a longer half-life for S.C. than for I.M. administration, with the delay being caused by the slow absorption process after S.C. injection. Mean terminal phase half-lives after administration of I.M. and S.C. were 904.1 min and 637.4 min, respectively. Peak plasma concentrations after administration of I.M. (Cmax = 43.8 microg/ml) and S.C. (Cmax = 24.9 microg/ml) were detected at 42.8 min and 79.8 min, respectively. Urinary excretion of antimony for both routes surpassed 80% during the first 6 hr, with the rest of the drug being excreted slowly over the following 18 hr. The results obtained with this formulation suggest that for treating canine leishmaniasis, it would be more advisable to inject LMA intramuscularly if we assume that the significantly higher Cmax observed after I.M. administration is more relevant to dog's clinical outcome than is maintenance of concentrations over longer periods.

Functional antibody response to human cytomegalovirus in immunocompetent and HIV-1 infected individuals with antibodies that inhibit virus penetration into cells and intercellular transmission of viral infection.

Antibodies mediating post-attachment virus neutralisation (PN), inhibition of human cytomegalovirus (HCMV)-induced cell fusion in the glioblastoma cell line U373 (IF) and global neutralising activity (NA) were quantified in sera from healthy immunocompetent individuals, asymptomatic HIV-1-infected subjects and AIDS patients to further characterise the neutralising antibody response to HCMV in these population groups and to assess whether HIV-1-infected individuals exhibited an abnormal functional antibody profile. PN and IF antibodies accounted for a minor fraction of the NA activity of sera from all population groups. Sera from HIV-1-infected individuals (particularly AIDS patients) displayed higher levels of PN and IF antibodies than those from the healthy control group; however, the relative contribution of these antibodies to the global serum NA activity appeared to be lower in the former individuals than in immunocompetent controls. Serum antibodies preventing HCMV cell-to-cell spread (IP) were then measured to determine whether a specific deficiency could be detected in the HIV-1-infected group population. Serum IP antibody titres were significantly higher in HIV-1-infected individuals (particularly in AIDS patients) than in controls. The potential implications of the data for explaining the pathogenesis of HCMV infection are discussed.

Evaluation of the efficacy of two leishmanins in asymptomatic dogs.

There are few studies in dogs concerning leishmanin skin test. We evaluated and compared the efficacy of two leishmanin preparations for the detection of dog Leishmania cellular-mediated immunity. Clinically healthy dogs living in an endemic area were studied. A leishmanin preparation 1 (3 x 10(8) promastigotes/ml) was superior to a leishmanin preparation 2 (5 x 10(6) promastigotes/ml), measured as the percentage of positive reactions and the diameter of the induced induration. The leishmanin skin test is a valuable tool, although the results show that the degree of response, as it has been shown in human beings, depends on the preparation used.

The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection.

Veterinarians working in the Balearic Islands (Mallorca), an endemic region of canine leishmaniosis, have reported very few cases of leishmaniosis in Ibizian hounds while concurrently observing that dogs of other breeds had a high incidence of clinical canine leishmaniosis. To further investigate this observation, two populations of dogs from the Balearic Islands were examined for the presence of Leishmania-specific cellular immunity using a delayed type hypersensitivity test (DTH) to leishmanin and for the presence of Leishmania-specific humoral immunity using an ELISA. Fifty-six asymptomatic dogs, 31 Ibizian hounds and 25 dogs belonging to other breeds were examined. Seventy-seven percent of the dogs demonstrated a specific immune response against Leishmania, either humoral or cellular. This finding suggests that the infection rate (77%) was higher than previously considered. For Ibizian hounds 81% were DTH positive while only 48% of the other dogs were DTH positive. A statistical association between Ibizian hounds and positive DTH response was found. A specific humoral response was found in 48% of Ibizian hounds and in 56% of the other dogs. No statistical association relative to the Leishmania-specific IgG1 and IgG2 levels were found between the two groups. The Ibizian hound has been reported to be more resistant to Leishmania infection and we found that the Ibizian hound mounts a significant cellular response to infection. Thus, the Ibizian hound may be an interesting canine model for the investigation of protective anti-Leishmania immune response.

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.

Pharmacokinetics of meglumine antimoniate after administration of a multiple dose in dogs experimentally infected with Leishmania infantum.

Pharmacokinetics of meglumine antimoniate in dogs with experimentally induced leishmaniosis has been investigated. After infection, dogs received a dose of 75 mg kg-1 of meglumine antimoniate twice daily by subcutaneous injection for 10 days. Blood samples were collected throughout the treatment. No statistical differences were found in the kinetic behaviour of the drug administered as a single dose to healthy dogs and that administered as a multiple dose to infected animals. However, peak plasma concentrations (Cmax) of 30.8 +/- 12.8 micrograms ml-1 found after this dosage regimen were higher than those observed after the single dose administration of 100 mg kg-1 24 h-1. Furthermore, sustained antimony concentrations of 1.14 +/- 0.52 micrograms Sb ml-1 were detected throughout the treatment. No signs of toxicity were found in the animals treated indicating that this regimen would be very appropriate to treat canine leishmaniosis.

Long term improvement in the treatment of canine leishmaniosis using an antimony liposomal formulation.

Pharmacokinetic and clinical effectiveness of liposome-encapsulated N-methylglucamine antimoniate (LMA) was performed in dogs suffering from experimental leishmaniosis. LMA was compared with N-methylglucamine antimoniate (MGA), the same drug in its free form. Sb plasma concentrations for LMA were always higher than those for MGA. Mean residence time (MRT), half-life time (t(1/2)) and clearance (Cl) showed that Sb was eliminated slower after liposome administration. The high volume of distribution (Vd) obtained with LMA suggests that Sb could achieve therapeutic concentrations in parasite-infected tissues. Average plasma concentration at steady state (Css(ave)) shows that Sb body concentrations after LMA treatment (9.8 mg/kg Sb, each 24h) would be effective in Leishmania infantum canine infection. Comparing LMA with MGA in a 1-year follow-up we observed no relapses for LMA and total protein and gammaglobulin concentrations were within normal range, while for MGA both began to rise 3 months after treatment. Use of antimonial liposomal formulations may restore effectiveness to an existing drug and reduce toxicity.

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment.

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.

Histological and immunohistochemical study of clinically normal skin of Leishmania infantum-infected dogs.

Skin lesions are the most usual manifestation of canine leishmaniosis. The aim of this study was to investigate the histological pattern and parasite load in clinically normal skin of Leishmania-infected dogs. Two groups of Leishmania-infected dogs were studied. Group A consisted of 15 symptomless animals which, although seronegative or only mildly seropositive, gave a positive polymerase chain reaction (PCR) for Leishmania in the skin. Group B consisted of 20 clinically affected dogs which were highly seropositive and PCR-positive. Biopsies of normal skin from all dogs were processed for routine histology and Leishmania immunohistochemistry. The study demonstrated microscopical lesions and the presence of parasites in the skin from dogs of group B, but not group A. The results cast doubt on the relevance of infected but symptomless dogs in the epidemiology of canine leishmaniosis. In contrast, however, the clinically normal skin of sick dogs harbours the parasite and probably plays a role in the transmission of leishmaniosis.

Study of haemostatic disorders in experimentally induced leishmaniasis in Beagle dogs.

Haemostatic alterations in dogs experimentally infected with Leishmania infantum were studied before and after therapy with meglumine antimonate. Haemostatic function tests including platelet count, collagen-induced platelet aggregation, prothrombin time, activated partial thromboplastin time, thrombin time, plasma fibrinogen determination, and serum fibrinogen/fibrin degradation products concentration were performed. In the course of infection and before treatment, moderate thrombocytopenia (P<0.00001), decreased collagen induced platelet aggregation (P=0.0003), prolonged thrombin time (P=0.0117) and increased fibrinogen/fibrin degradation products were observed. Statistically significant differences of plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time were not encountered. Haemostatic parameters returned to normal values after therapy. The results indicate that Leishmania infection may impair haemostasis suggesting induction of disseminated intravascular coagulation (DIC), and that treating dogs in an early stage of infection may potentially avoid the possibility of developing an uncompensated DIC.