Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-µPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-µPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-µPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-µPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Diagnostic efficacy of urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 for early detection of acute kidney injury in dogs with leptospirosis or babesiosis.

This study evaluates the diagnostic efficacy of urinary biomarkers, Neutrophil Gelatinase-Associated Lipocalin (uNGAL), and Kidney Injury Molecule-1 (uKIM-1), in identifying Acute Kidney Injury (AKI) in dogs affected with leptospirosis or babesiosis. Acute kidney injury was diagnosed based on the increase in serum creatinine levels above 0.3 mg/dL within 48 h and dogs were categorized according to AKI grades based on International Renal Interest Society guidelines. Traditional biomarkers (serum creatinine and blood urea nitrogen) and novel biomarkers like urinary NGAL and urinary KIM-1 levels were measured and compared to concentrations obtained in control dogs. Statistical analysis assessed significant differences (P < 0.01) across AKI grades, specifically noting elevated urinary NGAL and KIM-1 in IRIS grade I AKI (P < 0.001). The study highlights the diagnostic significance of urinary NGAL and KIM-1 as early indicators of renal damage, particularly valuable in non-azotemic AKI cases, offering promising markers for early AKI diagnosis in veterinary clinical settings. These biomarkers demonstrate clinical utility and underscore their potential for improving AKI management in veterinary medicine. Further validation studies involving larger cohorts and diverse etiologies of AKI are needed to confirm the diagnostic accuracy and clinical utility of urinary NGAL and KIM-1 in veterinary practice.

Leptospiral infection in domestic cats: Systematic review with meta-analysis.

The role of domestic cats in the transmission of Leptospira is controversial, being considered either as a protective factor or a potential source of infection for humans. The aims of this study were to obtain an integrated estimate of the prevalence of leptospiral infection in domestic cats, and to characterize those individuals most susceptible to infection. For this systematic review and meta-analysis, five electronic databases, as well as the reference lists of eligible reports were screened for observational studies published during 1979-2022. The overall prevalence of leptospiral antibodies and of pathogenic Leptospira in kidney/urine was estimated using three-level meta-analysis models. To investigate potential sources of heterogeneity, moderator analyses was performed using the sampling year, health status, living environment, and origin of the cats. The odds-ratio (OR) of potential risk factors was estimated using random-effects meta-analysis models for binary outcomes. The literature search identified 61 eligible publications, containing 114 effect estimates. The overall seroprevalence was 11.7 % (95 % CI: 9.3-14.6 %), the prevalence of leptospires in urine was 3.7 % (95 % CI: 1.7-8.1 %), and the prevalence of leptospires in kidney tissue was 12.8 % (95 % CI: 3.2-39.9 %). There were no significant differences in the estimated prevalence after removing articles with medium risk of bias. Seroprevalence was higher in Europe when compared to Latin America and the Caribbean (P = 0.047) and showed a mild decrease over time (P = 0.023). Outdoor cats had almost three times greater probabilities of infection with Leptospira (OR: 2.74, 95 % CI: 1.10-6.84). No other significant effect was detected among the tested moderators or potential risk factors (P > 0.05). Results suggest that feline leptospirosis should receive particular attention in veterinary care, as exposure to leptospires can occur in approximately 1 in every 10 cats, and more frequently in cats with street access. Domestic cats should be considered as potential Leptospira carriers when designing public health strategies for the control and prevention of leptospirosis. Further investigation is required to improve knowledge of the role of these animals in the environmental transmission cycle.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

Comparison of clinicopathological patterns of renal tubular damage in dogs with acute kidney injury caused by leptospirosis and other aetiologies.

In humans, leptospiral acute kidney injury (AKI) is characterised by tubulointerstitial involvement and renal electrolyte losses, impacting clinical presentation and case management. The aim of this study was to evaluate urine chemistry findings in dogs with leptospirosis in order to identify characteristic patterns of tubular damage associated with this disease. Dogs with intrinsic AKI caused by leptospirosis and by other aetiologies were prospectively enrolled. Clinical and clinicopathological variables, including serum and urine chemistry, fractional excretion (FE%) of electrolytes, and urinary neutrophil gelatinase-associated lipocalin (NGAL), were evaluated in both groups and compared statistically. Dogs with leptospirosis (n = 38) had significantly higher serum creatinine concentration than dogs with AKI caused by other aetiologies (n = 37). Serum potassium and glucose concentrations were comparable between groups. Dogs with leptospiral AKI had significantly higher FE of potassium (median 100%, range 20-480 vs. median 68%, range 5-300; P = 0.048), as well as higher magnitude of glucosuria (urine glucose to creatinine ratio, median 0.64, range 0-26 vs. median 0.22, range 0-13; P = 0.023) and frequency of positive glucose dipstick reaction (59% vs. 18%; P = 0.002), than dogs with AKI of other aetiologies. Additional markers of tubular damage considered in this study, including FE of other electrolytes and urinary NGAL, did not differ between groups. In conclusion, when compared to other aetiologies of intrinsic AKI, canine leptospirosis was characterised by increased glucosuria and kaliuresis.

Development of lipL32 real-time PCR combined with an internal and extraction control for pathogenic Leptospira detection.

At least two real-time PCRs for the early diagnosis of leptospirosis have been described, evaluated and validated. However, at least one other report suggested adaptation and modification of primers and probes used in these assays since additional Leptospira species have been described and the primers and probe in use possess a serious mismatch to corresponding target sequence. In this study we developed a real-time PCR for detection of pathogenic Leptospira based on the lipL32 gene. The present method consists of generic primers and probes based on target sequence of 10 pathogenic Leptospira species including Leptospira interrogans. The hybridization, annealing and extension temperature (60°C) were optimized as the optimal temperature of the DNA polymerase enzyme which is used in the amplification reaction. The present assay has a high analytical sensitivity and specificity; the calculated diagnostic sensitivity and specificity were 93.0% and 98.3% respectively. Moreover, the present method includes an internal control which enables easy detection of false negative results and an optional extraction control which enables the estimation of the DNA extraction efficiency.

On-farm risk factors associated with Leptospira shedding in New Zealand dairy cattle.

This study aimed to evaluate risk factors associated with shedding of pathogenic Leptospira species in urine at animal and herd levels. In total, 200 dairy farms were randomly selected from the DairyNZ database. Urine samples were taken from 20 lactating, clinically normal cows in each herd between January and April 2016 and tested by real-time polymerase chain reaction (PCR) using gyrB as the target gene. Overall, 26.5% of 200 farms had at least one PCR positive cow and 2.4% of 4000 cows were shedding Leptospira in the urine. Using a questionnaire, information about risk factors at cow and farm level was collected via face-to-face interviews with farm owners and managers. Animals on all but one farm had been vaccinated against Hardjo and Pomona and cows on 54 of 200 (27%) farms had also been vaccinated against Copenhageni in at least one age group (calves, heifers and cows). Associations found to be statistically significant in univariate analysis (at P < 0.2) were assessed by multivariable logistic regression. Factors associated with shedding included cattle age (Odds ratio (OR) 0.82, 95% CI 0.71-0.95), keeping sheep (OR 5.57, 95% confidence interval (CI) 1.46-21.25) or dogs (OR 1.45, 95% CI 1.07-1.97) and managing milking cows in a single as opposed to multiple groups (OR 0.45, 95% CI 0.20-0.99). We conclude that younger cattle were more likely to be shedding Leptospira than older cattle and that the presence of sheep and dogs was associated with an increased risk of shedding in cows. Larger herds were at higher risk of having Leptospira shedders. However, none of the environmental risk factors that were assessed (e.g. access to standing water, drinking-water source), or wildlife abundance on-farm, or pasture were associated with shedding, possibly due to low statistical power, given the low overall shedding rate.

First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population.

Evaluation of real-time PCR targeting the lipL32 gene for diagnosis of Leptospira infection.

BACKGROUND: Different diagnostic methods have been used for the laboratory confirmation of leptospirosis. Molecular diagnostic techniques are not only faster and more sensitive than culture analysis, but can also detect a Leptospira infection before the appearance of antibodies. The aim of the present study was to analyze and compare two different PCR approaches applied to blood and urine specimens obtained from patients with clinical manifestations that were suggestive of leptospirosis. Furthermore, the results of these different PCR approaches were compared with the results of culture and serology analyses. RESULTS: A total of 400 samples (234 blood or 58.5% and 166 urine of 41.5%) from 310 Slovenian patients with clinical manifestations suggestive of leptospirosis were tested using conventional PCR assays targeting the rrs gene and RT-PCR targeting the lipL32 gene. Additionally, culture, serology and sequence analysis were performed for the majority of these samples. The PCR and RT-PCR results were concordant in 376 out of 400 of these samples (94.0%). Conventional PCR was positive for 27 out of 400 samples (6.8%) and RT-PCR was positive for 47 out of 400 samples (11.8%). Culture and microscopic agglutination tests supported these diagnoses. CONCLUSIONS: A comparison of the two PCR methods indicated that the RT-PCR targeting of the lipL32 gene was faster, more sensitive and more specific for the determination of Leptospira DNA in these clinical samples.

Leptospira interrogans and Leptospira kirschneri are the dominant Leptospira species causing human leptospirosis in Central Malaysia.

BACKGROUND: Leptospirosis, commonly known as rat-urine disease, is a global but endemic zoonotic disease in the tropics. Despite the historical report of leptospirosis in Malaysia, the information on human-infecting species is limited. Determining the circulating species is important to understand its epidemiology, thereby to strategize appropriate control measures through public health interventions, diagnostics, therapeutics and vaccine development. METHODOLOGY/PRINCIPLE FINDINGS: We investigated the human-infecting Leptospira species in blood and serum samples collected from clinically suspected leptospirosis patients admitted to three tertiary care hospitals in Malaysia. From a total of 165 patients, 92 (56%) were confirmed cases of leptospirosis through Microscopic Agglutination Test (MAT) (n = 43; 47%), Polymerase Chain Reaction (PCR) (n = 63; 68%) or both MAT and PCR (n = 14; 15%). The infecting Leptospira spp., determined by partial 16S rDNA (rrs) gene sequencing revealed two pathogenic species namely Leptospira interrogans (n = 44, 70%) and Leptospira kirschneri (n = 17, 27%) and one intermediate species Leptospira wolffii (n = 2, 3%). Multilocus sequence typing (MLST) identified an isolate of L. interrogans as a novel sequence type (ST 265), suggesting that this human-infecting strain has a unique genetic profile different from similar species isolated from rodents so far. CONCLUSIONS/SIGNIFICANCE: Leptospira interrogans and Leptospira kirschneri were identified as the dominant Leptospira species causing human leptospirosis in Central Malaysia. The existence of novel clinically important ST 265 (infecting human), that is different from rodent L. interrogans strains cautions reservoir(s) of these Leptospira lineages are yet to be identified.

Leptospira infection and shedding in dogs in Thailand.

BACKGROUND: Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection. In Northern, Northeastern, and Central Thailand, 273 stray (n = 119) or client-owned (n = 154) dogs from rural (n = 139) or urban (n = 134) areas were randomly included. Dogs that had received antibiotics within 4 weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6 months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald. RESULTS: Twelve of 273 (4.4%; 95% confidence interval (CI): 2.0-6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01-1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2-16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9-50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p = 0.023). CONCLUSIONS: Leptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.

Leptospiral shedding and seropositivity in shelter dogs in the Cumberland Gap Region of Southeastern Appalachia.

BACKGROUND: Leptospirosis, caused by pathogenic Leptospira spp., is a zoonotic infection that affects humans, dogs and many other mammalian species. Virtually any mammalian species can act as asymptomatic reservoir, characterized by chronic renal carriage and shedding of a host-adapted leptospiral serovar. Environmental contamination by chronic shedders results in acquisition of infection by humans and susceptible animals. METHODS: In this study, we investigated if clinically normal shelter dogs and cats harbor leptospires in their kidneys by screening urine samples for the presence of leptospiral DNA by a TaqMan based-quantitative PCR (qPCR) that targets pathogen-associated lipl32 gene. To identify the infecting leptospiral species, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. Additionally, we measured Leptospira-specific serum antibodies using the microscopic agglutination test (MAT), a gold standard in leptospiral serology. RESULTS: A total of 269 shelter animals (219 dogs and 50 cats) from seven shelters located in the tri-state area of western Virginia, eastern Tennessee, and southeastern Kentucky were included in this study. All cats tested negative by both qPCR and MAT. Of the 219 dogs tested in the study, 26/198 (13.1%, 95% CI: 8.4-17.8%) were positive for leptospiral DNA in urine by qPCR and 38/211 (18.0%, 95% CI: 12.8-23.2%) were seropositive by MAT. Twelve dogs were positive for both qPCR and MAT. Fourteen dogs were positive by qPCR but not by MAT. Additionally, leptospiral rpoB gene sequencing from a sub-set of qPCR-positive urine samples (n = 21) revealed L. interrogans to be the leptospiral species shed by dogs. CONCLUSIONS: These findings have significant implications regarding animal and public health in the Cumberland Gap Region and possibly outside where these animals may be adopted.

Study protocol: characterising the clinical, epidemiological and aetiological aspects of leptospirosis in Sri Lanka: a hospital based clinico-epidemiological study.

INTRODUCTION: Sri Lanka has one of the highest incidences of leptospirosis worldwide. We hypothesised that different geographical locations and patient context will have a distinct molecular epidemiology of leptospirosis, based on microgeographical characteristics related to regiona-specific Leptospira predominance. Our objective is to characterise the clinical, epidemiological and molecular aspects of leptospirosis in Sri Lanka to understand disease progression, risk factors and obtain isolates of Leptospira. METHODS AND ANALYSIS: We designed a multicentre prospective study in Sri Lanka to recruit undifferentiated febrile patients and conduct follow-ups during hospital stays. Patients will be recruited from outpatient departments and medical wards. This study will be conducted at two main sites (Anuradhapura and Peradeniya) and several additional sites (Awissawella, Ratnapura and Polonnaruwa). Blood and urine will be collected from patients on the day of admission to the ward or presentation to the outpatient department. Bedside inoculation of 2-4 drops of venous blood will be performed with Ellinghausen-McCullough-Johnson-Harris (EMJH) semisolid media supplemented with antibiotics. Regionally optimised microscopic agglutination test, culture and qPCR-evidence will be performed to confirm the presence of Leptospira in blood which in turn will confirm the presence of disease. Whole genome sequencing will be carried out for all isolates recovered from patients. Multilocus sequence typing (MLST) will be used for the genotyping of new isolates. Sri Lankan isolates will be identified using three published MLST schemes for Leptospira. ETHICS AND DISSEMINATION: Ethical clearance for the study was obtained from Ethics Review Committees (ERC), Medicine and Allied Sciences (FMAS), Rajarata University of Sri Lanka (RUSL) and University of Peradeniya. All genomic data generated through this project will be available at GenBank. Anonymised data will be deposited at the ERC, FMAS, RUSL.

Emerging Leptospira strain poses public health risk for dairy farmers in New Zealand.

Leptospira infection in dairy cattle and leptospirosis in dairy farm workers were common in New Zealand prior to the introduction of dairy cattle vaccination in the 1980s. Despite widespread vaccination within the dairy industry, the long-term effectiveness of vaccination and current Leptospira exposure status remained unknown. A cross-sectional study was conducted from January-April 2016 to investigate the prevalence of pathogenic Leptospira spp. DNA in urine at cow and herd level, and its relationship to five Leptospira serovars known to be endemic. Two hundred dairy farms were randomly selected from the national database. Twenty paired blood and urine samples were collected on each farm from adult cows (n = 4000). Sera were tested using the Microscopic Agglutination Test against serovars Hardjobovis (termed Hardjo), Pomona, Copenhageni, Ballum and Tarassovi with titres ≥48 being considered positive. Urine was tested using quantitative real-time PCR (qPCR) that amplifies the gryB gene. All but one herd had been vaccinated with a bivalent Hardjo/Pomona or trivalent vaccine incorporating Copenhageni. In total, 2.4% of cows were urine qPCR positive and 27% of farms had at least one urine qPCR positive cow. Overall 63% of cows were seropositive to one or more serovars: 44% for Hardjo, 28% for Pomona, 15% for Copenhageni (in vaccinated herds), and for unvaccinated cows: 1% for Copenhageni, and 3% for Ballum and 17% for Tarassovi. Of the 94 qPCR urine-positive cows, 51 were seropositive to Tarassovi, 3 to Ballum, 3 to Copenhageni, 24 to Hardjo, and 17 to Pomona, the latter two presumably reflecting vaccination titres. A strong association was found between shedding and serology for Tarassovi. While there was no evidence that current vaccination programmes were ineffective in protecting against their target serovars, serovar Tarassovi has apparently emerged in NZ dairy cattle. As Tarassovi is currently not included in vaccines and is prevalent in notified leptospirosis cases in dairy workers, we concluded that this serovar poses a public health risk.

Blanding's turtles (Emydoidea blandingii) as a reservoir for Leptospira spp.

Leptospira spp. are re-emerging zoonotic pathogens. Previous research has found that Blanding's turtles (Emydoidea blandingii) experimentally infected with Leptospira interrogans shed leptospires in their urine, suggesting that they could play a role in transmitting pathogen within an aquatic ecosystem. This study investigated whether a population of wild Blanding's turtles known to be exposed to Leptospira spp. actively shed the pathogen under natural conditions. Blood samples were collected for serologic testing and to assess the health of the turtles. Free catch urine was collected for polymerase chain reaction (PCR) testing. All turtles were seropositive for Leptospira spp. and 73.5% (25/34) of the urine samples were PCR positive. All animals appeared clinically healthy and showed no apparent signs of disease. This study confirms that wild Blanding's turtles can actively shed Leptospira spp. in their urine and suggests that they may play a role in the epidemiology of this disease in habitats in which they reside.

Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test / Diagnóstico de leptospirose canina: avaliação de dois ensaios de PCR em comparação com o teste de microaglutinação

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.(AU)

O diagnóstico definitivo da leptospirose canina é geralmente realizado demonstrando a seroconversão em amostras do paciente no período agudo e de convalescença por serologia. No entanto, a aplicação de técnicas de PCR pode contribuir para a confirmação de casos suspeitos num período de tempo mais curto. O objetivo deste estudo foi avaliar dois ensaios de PCR publicados em humanos (PCR-lipL32 em tempo real e PCR-rrs convencional) em culturas puras e em amostras de sangue com anticoagulante, soro e urina experimentalmente contaminados. Posteriormente, investigamos a utilidade de ambos os ensaios de PCR em amostras clínicas de cães com suspeita de leptospirose tomando a técnica de microaglutinação (MAT) como referência. A sensibilidade analítica foi de 1 e 10 genoma equivalente por reação para PCR-lipL32 em tempo real e para PCR-rrs convencional, respectivamente. Ambos os ensaios amplificaram corretamente as 14 estirpes patogênicas, mas foram negativos para avaliar o ADN de outros microrganismos que poderiam estar presentes em amostras clinicas. Em nas amostras experimentalmente contaminadas PCR-LipL32 em tempo real detectou 100 bactérias/mL em sangue total, 1000 bactérias/mL em soro e 10 bactérias/mL em urina. Enquanto o PCR-rrs convencional detectou 1000 bactérias/mL em sangue total e soro e 100 bactérias/mL na urina. Dos 51 cães suspeitos, sete foram considerados casos confirmados pela MAT. O PCR-lipL 32 em tempo real detectou seis dos sete casos confirmados, enquanto o PCR-rrs convencional foi positivo em quatro deles. As técnicas de PCR avaliadas provaram ser uma ferramenta de diagnóstico útil na confirmação de casos clínicos caninos quando utilizados em conjunto com a técnica MAT.(AU)

Persistent Leptospiruria in Five Dogs Despite Antimicrobial Treatment (2000-2017).

In dogs with leptospirosis, doxycycline therapy is recommended as the preferred therapy for its ability to eliminate the organism from all tissues, including the renal tubules. Elimination of organisms from the renal tubules terminates leptospiruria and prevents transmission of the organism. This report describes the discovery of persistent leptospiruria in the face of therapy with doxycycline in four dogs and enrofloxacin in one dog. Leptospiruria was confirmed by polymerase chain reaction testing for pathogenic leptospires in all five dogs. In two dogs, leptospiruria resolved after a change in therapy to enrofloxacin. In three dogs, doxycycline and/or enrofloxacin were ineffective at eliminating leptospiruria, which then resolved after therapy with clarithromycin. Pet owners could be at risk as persistent leptospiruria poses a potential zoonotic risk. The potential reasons for persistent leptospiruria as demonstrated by polymerase chain reaction testing are discussed.

A remotely sensed flooding indicator associated with cattle and buffalo leptospirosis cases in Thailand 2011-2013.

BACKGROUND: Leptospirosis is an important zoonotic disease worldwide, caused by spirochetes bacteria of the genus Leptospira. In Thailand, cattle and buffalo used in agriculture are in close contact with human beings. During flooding, bacteria can quickly spread throughout an environment, increasing the risk of leptospirosis infection. The aim of this study was to investigate the association of several environmental factors with cattle and buffalo leptospirosis cases in Thailand, with a focus on flooding. METHOD: A total of 3571 urine samples were collected from cattle and buffalo in 107 districts by field veterinarians from January 2011 to February 2013. All samples were examined for the presence of leptospirosis infection by loop-mediated isothermal amplification (LAMP). Environmental data, including rainfall, percentage of flooded area (estimated by remote sensing), average elevation, and human and livestock population density were used to build a generalized linear mixed model. RESULTS: A total of 311 out of 3571 (8.43%) urine samples tested positive by the LAMP technique. Positive samples were recorded in 51 out of 107 districts (47.66%). Results showed a significant association between the percentage of the area flooded at district level and leptospirosis infection in cattle and buffalo (p = 0.023). Using this data, a map with a predicted risk of leptospirosis can be developed to help forecast leptospirosis cases in the field. CONCLUSIONS: Our model allows the identification of areas and periods when the risk of leptospirosis infection is higher in cattle and buffalo, mainly due to a seasonal flooding. The increased risk of leptospirosis infection can also be higher in humans too. These areas and periods should be targeted for leptospirosis surveillance and control in both humans and animals.

Seroprevalence, frequency of leptospiuria, and associated risk factors in horses in Kansas, Missouri, and Nebraska from 2016-2017.

Leptospirosis is a worldwide veterinary and public health concern, and well recognized infectious disease of horses. Seroprevalence rates vary with geography, but many studies have confirmed a high exposure rate. The correlation between seropositivity and shedding status has not been made in horses, however. The aims of this study were to use semi-nested PCR on urine from apparently healthy horses to determine period prevalence of leptospiral shedding and to correlate these findings with MAT results to establish associations with client based survey data regarding horse management and environment. Serum and free-catch urine were collected from 204 healthy horses between May 2016-December 2017. Serum was used to determine GGT, creatinine concentrations, and six serovar MAT. Urine samples were submitted for PCR testing of leptospiral 23S rRNA. Client consent and survey data were collected for all subjects. Potential risk factors included drinking water source, exposure to livestock and dogs, geographical location, season, and precipitation. Two horses were positive on urine PCR for leptospirosis (shedding prevalence 1%), yet only one had a high reciprocal MAT titer of ≥ 800. Both horses were negative on urine PCR one month later without treatment. Approximately 77% of horses (157/204) were seroreactive (MAT reciprocal titer ≥ 100) with titers to serogroup Australis detected more frequently than others (47.5%; (97/204)). Apparently healthy horses infrequently shed Leptospira spp. in urine, yet seroreactivity in clinically normal horses is high (77%), confirming high exposure rates to Leptospira spp. in the Central Midwest.

Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples

Abstract A modified TaqMan real-time polymerase chain reaction targeting a 138 bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy dogs were tested for presence of leptospiral DNA using the TaqMan-based assay. The results were compared with those from a well-established conventional PCR targeting the 16S RNA encoding gene associated with nucleotide sequencing analysis. The overall agreement between the assays was 94.8% (confidence interval [CI] 95% 88-100%). The newly developed assay presented 91.6% (CI 95% 71.5-98.5%) relative sensitivity (22[+] lipl32 PCR/24[+] 16S RNA and sequencing), 100% (CI 95% 96.3-100%) relative specificity and 98.7% accuracy (CI 95% 94.8-100%). The lipl32 assay was able to detect and quantify at least 10 genome equivalents/reaction. DNA extracted from 17 pathogenic Leptospira spp., 8 intermediate/saprophytic strains and 21 different pathogenic microorganisms were also tested using the lipl32 assay, resulting in amplification exclusively for pathogenic leptospiral strains. The results also demonstrated high intra and inter-assay reproducibility (coefficient of variation 1.50 and 1.12, respectively), thereby qualifying the newly developed assay as a highly sensitive, specific and reliable diagnostic tool for leptospiral infection in dogs using urine specimens.