Why do Doctors Miss the Diagnosis of Leptospiral Uveitis? Emergence of New Serovars and Challenges in Diagnosis.

PURPOSE: Leptospirosis is an endemic disease in India and uveitis is its late complication. Several Indian reports showed diversity of serovars, changing patterns and existence of new serovars. Failure to add new serovars in testing panel result in increased false-negativity in serology. AIM: To analyse seroprevalence, changing patterns and to discuss the resulting challenges in diagnosis. METHODS: In this retrospective study covering the period from 1994 to 2020, we analysed data from laboratory records of patients diagnosed with leptospiral uveitis in South India. Microscopic agglutination Test (MAT) and/or Enzyme-Linked Immunosorbent Assay (ELISA) were performed on clinically diagnosed leptospiral uveitis cases from our hospital, as well as on systemic leptospirosis patients from government and private hospitals. RESULTS: Out of a total of 87 216 new uveitis cases with varying causes over 27 years, 3,658 (4.1%) were clinically diagnosed as leptospiral uveitis. Among them, 1,268 (34.7%) patients were seropositive. In 1994, 92% of clinically diagnosed leptospirosis patients were seropositive in the MAT performed at the Centers for Disease Control and Prevention in Atlanta. However, the positivity rate gradually declined to 35% over the years. The predominant serovars identified were L. autumnalis, L. icterohaemorrhagiae, and L. australis. There were notable variations in the distribution of serovars over the years. CONCLUSIONS: The data suggest a declining sensitivity of MAT and ELISA, possibly due to the emergence of new serovars. Customizing the panel based on local isolates could enhance the performance of MAT. Critical need is the addition of advanced molecular techniques to improve the diagnosis.

Leptospiral uveitis- "Transition 'from epidemic to endemic form" difficulties in laboratory confirmations.

Purpose: Leptospirosis is a waterborne zoonotic disease that primarily causes systemic illness, followed by uveitis. After heavy flooding in Madurai district, an epidemic outbreak of systemic and ocular leptospirosis occurred in 1994. Our data shows a transition to endemicity after each epidemic. Aim: The aim of this study is to report the clinical signs, epidemic outbreaks, and persistent endemicity of leptospiral uveitis, as well as the diagnostic dilemmas associated with it. Methods: A retrospective analysis of clinical signs was conducted using medical records of leptospiral uveitis patients over a period of 27 years (1994-2020) in a tertiary care eye hospital. The clinical workup of uveitis included a detailed clinical history, systemic, and ophthalmic examination. Microagglutination tests (MATs) was done at the Centers for Disease Control and Prevention (CDC) in Atlanta and later in our regional laboratory. Serum samples were collected from human systemic leptospirosis cases and a small group of animals in and around Madurai. Results: The first epidemic outbreak resulted in 200 seropositive patients. Subsequent epidemic outbreaks occurred in 1997, 1998, 2001, 2005, and 2012, with Madurai experiencing multiple outbreaks. However, the disease remained endemic, with 25-50 patients being observed per year in between the peaks. Ocular examination revealed acute non-granulomatous uveitis (94.9%), pan uveitis (59.8%), vitreous inflammatory reaction (55.4%), retinal vasculitis (29.5%), disc hyperemia (20.9%), and hypopyon. (16.2%). New serovars emerged every year, resulting in decreased sensitivity of the MAT. Over time, the MAT started to miss diagnoses. Conclusion: The persistent endemicity of leptospiral uveitis emphasizes the need for accessible diagnostic tests. The low performance of the MAT can be attributable to the use of an older panel. The incorporation of new isolates in the MAT by a national laboratory will improve the accuracy of diagnosis.

Leptospira seroprevalence and associated risk factors in healthy Swedish dogs.

BACKGROUND: Leptospirosis is an emerging zoonotic infection worldwide and a cause of life-threatening disease in dogs. Seroprevalence in Swedish dogs is unknown. The aims of the present study were to estimate seroprevalence of pathogenic Leptospira in healthy dogs in Sweden using the microagglutination test (MAT) and a rapid point-of-care enzyme-linked immunosorbent assay (ELISA), and to evaluate risk factors of Leptospira exposure in Swedish dogs. RESULTS: Positive MAT titres (≥ 1:50) were detected in 27/369 (7.3%) of included dogs. Five different serovars were represented of which the Saxkoebing serovar was the most common (64.3%), followed by Copenhagi (14.3%), Bratislava (10.7%), Icterohaemorrhagiae (7.1%), and Canicola (3.6%). The ELISA test (SNAP® Lepto) was positive in 3/316 (0.9%) dogs. Living in urban areas and contact with stagnant water were found to be risk factors for Leptospira seropositivity (p < 0.05) in a multivariable logistic regression model. CONCLUSION: In this first seroprevalence study of Leptospira in Swedish dogs, it was shown that healthy dogs without recent (24 months) travel history and antileptospira vaccination had been exposed to pathogenic Leptospira interrogans serovars. Contact with stagnant water and living in urban areas were independent risk factors for seropositivity.

Circulating serogroups of Leptospira in swine from a 7-year study in France (2011-2017).

BACKGROUND: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT) between 2011 and 2017. We used weighted averages to determine serogroup distributions according to MAT results and considering cross-reactions. RESULTS: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippotyphosa, Bataviae, and Pomona, were less common. CONCLUSIONS: Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the serogroups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveillance and prophylactic strategy support.

Are Small Animal Practitioners Occupationally Exposed to Leptospirosis? Results of a Serological Survey.

Leptospirosis is a worldwide zoonosis frequently responsible for clinical disease in dogs and rarely reported in human people. The risk of human exposure to Leptospira has been investigated in a sample population working in the northeast of Italy, a geographical area with high endemicity of canine leptospirosis. Two-hundred twenty-one human serum samples were analyzed for Leptospira microagglutination test (MAT): 112 clinical freelance small animal practitioners (exposed subjects) and 109 people not occupationally exposed to Leptospira-infected animals (unexposed subjects) were voluntarily enrolled. Despite the previously reported serological detection of antibodies vs. Leptospira in people in different Italian regions, this study did not detect any reactivity in the investigated population. This study shows that veterinarians do not appear to be at a greater risk of leptospirosis than the reference population. This may be due to both veterinarian awareness of the Leptospira zoonotic risk and the efficiency of the preventive measures and management of patients. Moreover, it could be the result of the relatively low excretion of Leptospira in symptomatic dogs, which can be considered as an environmental sentinel for Leptospira presence rather than a vehicle of transmission.

Application of PCR for Specific Diagnosis of Leptospirosis in Humans in Ukraine.

Leptospirosis remains one of the most widespread zoonotic diseases in the world and Ukraine, in particular. Ukrainian clinicians have been faced with early detection of the disease due to the availability of only a serological method for routine diagnostics in Ukraine, namely the microscopic agglutination test (MAT). This paper demonstrates the first results of the complex application of MAT and polymerase chain reaction (PCR) for routine verification of leptospirosis, which were first applied simultaneously in Lviv Oblast of Ukraine in 2016. We examined the sera of 150 patients clinically suspected of leptospirosis, 31 of whom were treated at the Lviv Oblast Clinical Hospital for Infectious Diseases (LOCHID). The application of PCR during the first seven days of the disease allowed increasing the share of confirmed leptospirosis cases by 16,1% in patients that were treated in LOCHID during 2016-2017.

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)

Leptospirosis in Ukraine (Lviv Oblast): Clinical and Epidemiological Features.

The article describes the results of a retrospective analysis of medical records of 395 patients with a clinical diagnosis of leptospirosis treated at the Lviv Oblast Infectious Disease Clinical Hospital (Ukraine) between 2002 and 2016. The main risk factors for leptospirosis were contact with rodents or their excrements (26.84%) and bathing in ponds, small lakes, and reservoirs (10.63%). Among 276 patients in whom the anti-leptospira antibodies were detected by the microscopic agglutination test (MAT), the most common serotypes were Leptospira icterohaemorrhagiae (33.33%) and Leptospira grippotyphosa (25.0%). The mortality rate was significantly higher in patients where leptospirosis diagnosis was established based on clinical symptoms without confirmation by MAT (15.13% vs. 5.43%, p < 0.01).

[Diagnostic procedure after abortions in sows after simultaneous infection with leptospira and chlamydia]. / Diagnostische Abklärung von Aborten bei Zuchtsauen nach Leptospiren- und Chlamydieninfektion.

INTRODUCTION: In a farrowing farm 2 first parity sows aborted on day 95 and day 110 of gestation due to an infection with leptospira and chlamydia. The double infection was diagnosed by PCR examination of abortion material. Serum samples of both sows and additional 8 sows taken three weeks after abortions were sent to two different labs for serological examination for antibodies against leptospira and chlamydia using a microagglutination test and a complement fixation test, respectively. In both labs the tests for antibodies against chlamydia were negative. Titers against diverse leptospira serovars varied between both labs and were low, so that they were not indicative for the involvement of the two pathogens regarding abortion. This case report indicates the diagnostic difficulties of direct and indirect detection methods for leptospira and chlamydia to assess the impact of these pathogens on observed reproductive failure.

Determination of O:4 antigen-antibody affinity level in O:5 antigen positive and negative variants of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has two serological variants: one that expresses the O:5 antigen (1,4,5,12:i:1,2) and one that lacks O:5 antigen (1,4,12:i:1,2). For serotyping, S. Typhimurium is agglutinated by diagnostic O:4 antigen serum. This study was carried out to compare the antigen-antibody affinity of O:4 antigen in S. Typhimurium χ3306 O:5-positive and S. Typhimurium χ3306 O:5-negative strains. The affinity of O:4 antigen with O:4 antigen serum was found to be stronger in the O:5-negative strains compared to O:5-positive strains. Next, we investigated the antigen-antibody affinity of O:4 antigen with O:4 antigen serum in field strains of S. Typhimurium, which showed the same tendency in affinity as seen with S. Typhimurium χ3306 O:5-positive and negative strains. This study suggests that the presence or absence of O:5 antigen causes differences in O:4 agglutination reactions with different field strains of S. Typhimurium.

ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied.

Evaluation of the immunochromatographic (Leptocheck) test for detection of specific antibodies against leptospires.

BACKGROUND: Leptospirosis is a febrile worldwide zoonosis. Routine diagnosis of leptospiral infection is based on demonstration of specific antibodies with serological tests. Performance of the reference serological test, the microscopic agglutination test (MAT), requires significant expertise. The aim of our study was to find out if leptospiral infection can be proven with simple, rapid, commercially available immunochromatographic Leptocheck test in order to introduce it for the first level diagnosis in emergency cases with less specialized laboratory staff. METHODS: In all, 590 serum samples of patients with clinical manifestations suggestive of leptospirosis were collected and tested with MAT and Leptocheck test. For confirmation of the results some other diagnostic methods such as polymerase chain reaction (PCR) and Leptospira isolation were performed. RESULTS: Results of both serological tests were consistent in 576/590 (97.63%) cases but Leptocheck gave more positive results in comparison to MAT (36 and 12, respectively) at first patient's testing. Following up the patient, MAT became positive in majority of Leptocheck positive patients at first visit. Leptospiral DNA was detected in nine blood and six urine samples belonging to thirteen different patients while only two samples were culture positive. CONCLUSION: In comparison with serological tests, PCR and culture have low sensitivity. According to our findings we conclude that Leptocheck test can prove leptospiral infection and could be used for rapid diagnosis of leptospirosis, later the sample should be confirmed with MAT.

Seroprevalencia de leptospirosis humana en un asentamiento del área urbana de la ciudad de Guatemala / Seroprevalence of human leptospirosis in a settlement of the urban area in Guatemala city

Introducción: aunque la leptospirosis es considerada una enfermedad de ambientes rurales, la reciente aparición de epidemias urbanas la hace emerger como un problema en salud pública. En Guatemala (2008), se demostró una seroprevalencia de 51,8 % en áreas rurales, por lo que es importante llevar a cabo estudios en áreas urbanas que permitan establecer el impacto que pudiera tener en la población guatemalteca. Objetivos: determinar la seroprevalencia de leptospirosis humana en un asentamiento ubicado en la ciudad de Guatemala, así como los serovares de Leptospira interrogans circulantes y los factores de riesgo asociados a la exposición con esta bacteria. Métodos: participaron 119 habitantes con 6 años y más de los 2 sexos, que aceptaron, previo consentimiento informado. Con una entrevista estructurada se recolectaron los datos sociodemográficos y las muestras de sangre venosa. La técnica de microaglutinación y ELISA IgG se utilizaron para la detección de anticuerpos. Los sueros se enfrentaron a 20 serovariedades de Leptospira interrogans sensu lato. La prevalencia se determinó con un IC95% y las variables sociodemográficas con chi cuadrado, la razón de prevalencia con Epi Info 3.5.1. Resultados: la seroprevalencia de leptospirosis en la población estudiada resultó de 30,3 %, (IC95%). Los serovares más frecuentes fueron Australis y Lanka (11,1 % ambos). El título más frecuente fue de 1:80 por microaglutinación. En la población se encontraron distintos factores de riesgo, pero ninguno mostró una asociación significativa con la presencia de anticuerpos anti-Leptospira (p> 0,05). Conclusiones: la seroprevalencia de leptospirosis detectada fue de 30,3 % en los habitantes del asentamiento ubicado en la ciudad de Guatemala, la cual es comparable a las áreas urbanas de países en donde esta enfermedad es hiperendémica, por lo que es importante implementar medidas de prevención y de control de la enfermedad en la comunidad, en forma conjunta con la municipalidad del distrito.

Introduction: although leptospirosis is considered a rural environment disease, recent urban epidemics make it emerge as a public health problem. A seroprevalence of 51.8 % has been found in rural areas of Guatemala; therefore it is important to establish the impact that this disease may have on the Guatemalan urban population. Objectives: to determine the seroprevalence of human leptospirosis in a settlement of Guatemala city and also to identify urban Leptospira interrogans serovars and risk factors associated with exposure to the bacteria. Methods: there were selected 119 people aged 6 years old and over from both sexes, who agreed to participate after giving their informed consent. Sociodemographic data were collected and venous blood samples were taken. The microagglutination test (MAT) and ELISA IgG were used to detect antibodies. Sera were tested against 20 serovars of Leptospira interrogans sensu lato. The seroprevalence was determined with a 95% confidence interval, and sociodemographic variables were evaluated with Chi square and Odds Ratio using Epi Info 3.5.1. Results: a 30.3 % seroprevalence of leptospirosis was found in the study population (CI, 95%). The more frequently serovars founded were Australis and Lanka (11.1 % each). The most common titer was 1:80 (MAT). Different risk factors were found, but none showed a significant association with the presence of Leptospira IgG antibodies (p> 0.05). Conclusion: the prevalence of IgG anti-Leptospira antibodies detected in the residents of the settlement is comparable to that of the urban areas of other countries where leptospirosis is hyperendemic. For avoiding outbreaks in these areas, it is important to prevent and control the infection in the community.