Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon

Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates

Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars

Genetic Affinities within a Large Global Collection of Pathogenic Leptospira: Implications for Strain Identification and Molecular Epidemiology

Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland

Evaluation of surveillance case definition in the diagnosis of leptospirosis, using the Microscopic Agglutination Test: a validation study

<p>Abstract</p> <p>Background</p> <p>Leptospirosis is endemic in both urban and rural areas of Sri Lanka and there had been many out breaks in the recent past. This study was aimed at validating the leptospirosis surveillance case definition, using the Microscopic Agglutination Test (MAT).</p> <p>Methods</p> <p>The study population consisted of patients with undiagnosed acute febrile illness who were admitted to the medical wards of the Teaching Hospital Kandy, from 1^stJuly 2007 to 31^stJuly 2008. The subjects were screened to diagnose leptospirosis according to the leptospirosis case definition. MAT was performed on blood samples taken from each patient on the 7^thday of fever. Leptospirosis case definition was evaluated in regard to sensitivity, specificity and predictive values, using a MAT titre ≥ 1:800 for confirming leptospirosis.</p> <p>Results</p> <p>A total of 123 patients were initially recruited of which 73 had clinical features compatible with the surveillance case definition. Out of the 73 only 57 had a positive MAT result (true positives) leaving 16 as false positives. Out of the 50 who didn't have clinical features compatible with the case definition 45 had a negative MAT as well (true negatives), therefore 5 were false negatives. Total number of MAT positives was 62 out of 123. According to these results the test sensitivity was 91.94%, specificity 73.77%, positive predictive value and negative predictive values were 78.08% and 90% respectively. Diagnostic accuracy of the test was 82.93%.</p> <p>Conclusion</p> <p>This study confirms that the surveillance case definition has a very high sensitivity and negative predictive value with an average specificity in diagnosing leptospirosis, based on a MAT titre of ≥ 1: 800.</p

Fulminant Leptospirosis (Weil's disease) in an urban setting as an overlooked cause of multiorgan failure: a case report

<p>Abstract</p> <p>Introduction</p> <p>Leptospirosis has recently come to international attention as a globally important re-emerging infectious disease. Our case is unusual given the season, location and setting in which leptospirosis occurred. According to the New York City Board of Health, there were only two other cases of leptospirosis in New York City in the year that our patient was diagnosed.</p> <p>Case presentation</p> <p>A 49-year-old healthy Chinese man presented to our hospital with sepsis and multiorgan failure. The patient did not respond to antibiotics and his multiorgan failure worsened. His workup did not show any significant findings except for a positive nasopharyngeal swab result for influenza A. Later the patient developed hemoptysis with evidence of bilateral infiltrates on radiography. His status mildly improved after he was started on steroids. Eventually, a microagglutination test confirmed the presence of antibodies against <it>Leptospira icterohaemorrhagiae. </it>The patient subsequently recovered after a course of intravenous antibiotics.</p> <p>Conclusion</p> <p>The case of fulminant leptospirosis presented here should serve to alert health care providers and the general public to the clinical importance of this severe, sometimes fatal, disease. Leptospirosis should be considered early in the diagnosis of any patient with acute, non-specific febrile illness with multiorgan system involvement or high fever in a returning traveler. In addition, not only should it be considered in tropical and rural areas between late summer to early fall, but also in any location or time if the risk factors are present.</p

A Comparative Study of Leptospirosis and Dengue in Thai Children

Two of the most common causes of acute febrile illnesses among children in the tropics are leptospirosis and dengue. Early in illness, these two conditions are often indistinguishable and rapid laboratory confirmation of the infecting pathogen is generally not available. An enhanced ability to distinguish leptospirosis from dengue in children would guide clinicians and public health personnel in the appropriate use of limited healthcare resources

Expanding the Diagnostic Use of PCR in Leptospirosis: Improved Method for DNA Extraction from Blood Cultures

Background: Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNAextraction methods, primarily sodium polyanetholesulfonate (SPS). Methodology/Principal Findings: In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested. Conclusions/Significance: This study suggests that a specific and early diagnosis can be obtained in most cases of sever

Detection and Quantification of Leptospira interrogans in Hamster and Rat Kidney Samples: Immunofluorescent Imprints versus Real-time PCR

A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible