Survival time of Leptospira kirschneri on strawberries.

In the past decade, two leptospirosis outbreaks occurred among strawberry harvesters in Germany, with 13, and 45 reported cases respectively. In both outbreaks, common voles (Microtus arvalis) infected with Leptospira kischneri serovar Grippotyphosa were identified as the most likely outbreak source. In an univariate analysis, eating unwashed strawberries was identified as one of the risk factors associated with Leptospira infection. The aim of this study was to evaluate the survival time of L. kirschneri serovar Grippotyphosa on strawberries under varying conditions. Strawberries were spiked with 5x109 of both a laboratory reference strain (strain Moskva V) and an outbreak field strain (94-6/2007) of L. kirschneri serovar Grippotyphosa sequence type 110. Survival times were investigated in a fully crossed design with three incubation times (2h, 4h, 6h and 8h) and three temperatures (15°C, 21°C and 25°C) with three replicated for each condition. A wash protocol was developed and recovered Leptospira were determined by qPCR, dark field microscopy and culturing. Viable L. kirschneri of both the reference strain and the field strain were identified in all samples at 25°C and an incubation time of 2h, but only 1/9 (11%) and 4/9 (44%) of the samples incubated at 15°C were positive, respectively. Both reference and field strain were viable only in 2/9 (22%) at 25° after 6h. After an 8h incubation, viable Leptospira could not be identified on the surface of the strawberries or within the fruit for any of the tested conditions. Based on these results, the exposure risk of consumers to viable Leptospira spp. through the consumption of strawberries bought at the retail level is most likely very low. However, there is a potential risk of Leptospira infection by consumption of strawberries on pick-your-own farms.

Leptospira spp. in Rodents and Shrews from Afghanistan.

Leptospirosis is an occupational risk for military personnel and many cases have been reported worldwide. Rodents are the most important maintenance hosts for Leptospira spp. and may infect both animals and humans. To determine the occurrence and identity of pathogenic Leptospira spp. in rodent and shrew populations in German military camps in Afghanistan, we examined 751 animals ( Mus musculus, Cricetulus migratorius, Meriones libycus, Rattus tanezumi, Crocidura cf. suaveolens, and Suncus etruscus) from four military camps in Northern Afghanistan from 2009-12. Leptospiral DNA was found in 1.1% of the animals and only in Mus musculus. Partial secY sequencing identified Leptospira borgpetersenii and Leptospira kirschneri as infecting genomospecies. Multilocus sequence typing was successful in the L. borgpetersenii samples, which were identified as sequence type 155. The low prevalence we observed suggested that the exposure risk of military personnel to infectious Leptospira spp. in the region is low.

Specific CD4+ T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis.

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4(+) T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4(+) T cells. The production of inflammatory cytokines such as TNF-α by CD4(+) T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease.

Estimation of the sensitivity and specificity of a Leptospira spp. in-house ELISA through Bayesian modelling.

The microscopic agglutination test (MAT) is still considered the gold standard for the diagnosis of leptospirosis, although studies have shown that the test is an imperfect gold standard for clinical samples and unsuitable for epidemiological studies. Here, test characteristics of an in-house ELISA were identified for both subclinical and clinical populations by Bayesian latent class models. A conditional dependence model for two diagnostic tests and two populations was adapted to analyse a clinical and a subclinical scenario, respectively. These Bayesian models were used to estimate the sensitivity and specificity of the in-house ELISA and the MAT as well as the prevalences. The Bayesian estimates of the in-house ELISA were: clinical sensitivity=83.0%, clinical specificity=98.5%, subclinical sensitivity=85.7% and subclinical specificity=99.1%. In contrast, the estimates of the MAT were: clinical sensitivity=65.6%, clinical specificity=97.7%, subclinical sensitivity 54.9% and subclinical specificity=97.3%. The results show the suitability of the in-house ELISA for both clinical investigations and epidemiological studies in mildly endemic areas.

Severe leptospirosis complicated by Epstein-Barr Virus reactivation.

INTRODUCTION: Weil's disease is a severe, potentially fatal illness following Leptospira interrogans infection. The reported case of a patient suffering from acute renal failure, jaundice, thrombocytopenia, rhabdomyolysis and encephalitis syndrome highlights the clinical challenge in reference to Weil syndrome complicated by Epstein-Barr Virus (EBV) reactivation. MATERIALS AND METHODS: The diagnosis of leptospirosis was performed using four different diagnostic methods. Sera were analyzed with an in-house IgM and IgG enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination assay (IHA). Microscopic agglutination test (MAT) was done using 17 reference strains comprising 14 serogroups and 17 serovars. Polyvalent EBV-IgG analysis, EBV-IgG/IgM/IgA western blot analysis as well as quantitative EBV polymerase chain reaction (PCR) were performed. RESULTS: Leptospira IHA showed an initial titer of 1:640 (cut-off 1:320), leptospiral IgG was negative, but IgM was positive. MAT was negative at that time for all 17 strains analyzed. One week later, leptospirosis IHA titer increased to 1:20,480. Leptospiral IgG was now positive, -IgM remained positive and urine was tested negative for leptospiral DNA. The MAT showed positive results for L. interrogans serovar Bataviae, serovar Copenhageni, serovar Pyrogenes and L. borgpetersenii serovar Serjoe. During follow-up examinations, both the leptospiral IgM and IgG remained positive and MAT showed positive results for L. interrogans of different serovars. EBV IgA immunoblot taken at admission was positive for VCA-p18, quantitative EBV-PCR showed an EBV viral load of 2.8E3 copies/ml indicating acute EBV-reactivation. CONCLUSION: Leptospirosis represents a neglected and re-emerging disease which is difficult to diagnose since Leptospira-PCR from whole blood or urine is frequently negative in the case of early empiric antibiotic treatment. EBV-reactivation might represent a severe complication in Weil's disease which potentially aggravates clinical manifestations of leptospirosis including hepatitis, nephritis, and rhabdomyolysis. Thus, there might be a need for peripheral blood EBV-PCR and EBV blotting in patients suffering from complicated Weil syndrome, also in terms of the choice of antibiotic treatment.

Distribution of Leptospira serogroups in dogs from Berlin, Germany.

Leptospirosis is a bacterial zoonosis in which dogs can act as a reservoir for human infection. The annual vaccination of dogs can prevent leptospirosis caused by serovars included in the vaccine. To date, all available vaccines in Germany include only the serovars Icterohaemorrhagiae and Canicola, the most commonly found serovars prior to the introduction of the leptospirosis vaccines. Yet, the involvement of additional serovars in the clinical presentation of leptospirosis in dogs has been described. The objective of this sero-epidemiological study was to examine the different Leptospira serovars currently circulating in a population of dogs suspicious for leptospirosis from Berlin. In 329 dogs presenting at the Small Animal Clinic in Berlin, the predominant serogroup was Australis (24%), followed by Grippotyphosa (20%) and Pomona (9%). A total of 18% of the dogs were diagnosed with clinical leptospirosis; here the most prevalent serogroups were also Australis (28%), Grippotyphosa (18%), and Pomona (14%). The serovar prevalence data presented here confirm that a change of pattern of infecting Leptospira serovars in dogs has taken place in Berlin. This data corresponds to further sero-epidemiological studies from other regions in Germany. To ensure human and canine health, available vaccines should be adapted to include the most important circulating serovars.

Comparison of two PCR systems for the rapid detection of Leptospira spp. from kidney tissue.

In this study we compared two routine PCR systems for the detection of Leptospira spp. and assessed their performance when directly applied to kidney samples from small mammals. Although the kappa value of 0.9 indicated a high level of agreement between the tests, the outer membrane lipoprotein gene lipl32 based PCR was more robust and showed a higher number of positive kidney samples.

Outbreak of leptospirosis among triathlon participants in Germany, 2006.

BACKGROUND: In August 2006, a case of leptospirosis occurred in an athlete after a triathlon held around Heidelberg and in the Neckar river. In order to study a possible outbreak and to determine risk factors for infection an epidemiological investigation was performed. METHODS: Participants of the triathlon were contacted by e-mail and were asked to fill out a standardized questionnaire. In addition, they were asked to supply a serum sample for laboratory diagnosis of leptospirosis. A confirmed case patient was defined as a clinical case (i.e. fever and at least one additional symptom suggestive for leptospirosis) with at least two of the following tests positive: ELISA IgM, latex agglutination testing, or microscopic agglutination testing. Rainfall and temperature records were obtained. RESULTS: A total of 142 of 507 triathletes were contacted; among these, five confirmed leptospirosis cases were found. Open wounds were identified as the only significant risk factor for illness (p = 0.02). Heavy rains that preceded the swimming event likely increased leptospiral contamination of the Neckar River. DISCUSSION: This is the first outbreak of leptospirosis related to a competitive sports event in Germany. Among people with contact to freshwater, the risk of contracting leptospirosis should be considered by health care providers also in temperate countries, particularly in the summer after heavy rains.

Resurgence of field fever in a temperate country: an epidemic of leptospirosis among seasonal strawberry harvesters in Germany in 2007.

BACKGROUND: Although leptospirosis is a reemerging zoonosis of global importance, outbreaks related to agricultural exposures are primarily situated in tropical countries. In July 2007, a suspected leptospirosis outbreak was recognized among strawberry harvesters from Eastern Europe who were working in Germany. An investigation was initiated to identify the outbreak source and the risk factors for infection. METHODS: We conducted a retrospective cohort study with use of a questionnaire administered to harvesters by health authorities in Romania, Slovakia, and Poland. Collected serum samples were tested by microscopic agglutination test and immunoglobulin M enzyme-linked immunosorbent assay. A case patient was defined as a person who worked in the strawberry field during the period 5 June-8 September 2007 and had leptospirosis-compatible symptoms and either an antibody titer 1:800 and a positive immunoglobulin M enzyme-linked immunosorbent assay result (for a confirmed case) or no serological confirmation (for a suspected case). Local rodents were examined for leptospirosis. RESULTS: Among 153 strawberry harvesters, we detected 13 confirmed case patients who had test results positive for antibodies against Leptospira species serogroup Grippotyphosa and 11 suspected case patients (attack rate, 16%). Risk of disease increased with each day that an individual worked in the rain with hand wounds (odds ratio, 1.1; 95% confidence interval, 1.04-1.14) and accidental rodent contact (odds ratio, 4.8; 95% confidence interval, 1.5-15.9). Leptospires of the serogroup Grippotyphosa were isolated from the kidneys of 7 (64%) of 11 voles. CONCLUSIONS: This is, to our knowledge, the largest leptospirosis epidemic to occur in Germany since the 1960s. Contact between hand lesions and contaminated water or soil and infected voles was the most likely outbreak source. The unusually warm winter of 2006-2007 supported vole population growth and contributed to this resurgence of leptospirosis in Germany. Because of ongoing climate change, heightened awareness of leptospirosis in temperate regions is warranted.

Leptospirosis in urban wild boars, Berlin, Germany.

We found antibodies to leptospires in 25 (18%) of 141 wild boars from Berlin (95% confidence interval 12-25). Seropositivity was associated with chronic interstitial nephritis (odds ratio 10.5; p=0.01), and leptospires were detected in kidney tissues. Wild boars represent a potential source for human leptospirosis in urban environments.