The emergence of Leptospira borgpetersenii serovar Arborea in Queensland, Australia, 2001 to 2013.

BACKGROUND: Leptospirosis is an emerging infectious disease, with increasing frequency and severity of outbreaks, changing epidemiology of populations at risk, and the emergence of new serovars. Environmental drivers of disease transmission include flooding, urbanisation, poor sanitation, changes in land use and agricultural practices, and socioeconomic factors. In Queensland, human infection with Leptosira borgpetersenii serovar Arborea was first reported in 2001. This study aims to report the emergence of serovar Arborea in Queensland from 2001 to 2013, and investigate potential risk factors for infection and drivers of emergence. METHODS: Data on laboratory-confirmed cases of human leptospirosis in Queensland were obtained from the enhanced surveillance system at the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis in Brisbane, Australia. The changing epidemiology of serovar Arborea from 2001 to 2003 was described with respect to case numbers, proportion of leptospirosis cases attributed to the serovar, and geographic distribution. Differences in risk factors for the most common serovars were compared. RESULTS: During this period, 1289 cases of leptospirosis were reported, including 233 cases attributed to serovar Arborea. Risk factors for infection include male gender (91 % of cases), occupation, and recreational exposure. Most common occupations recorded were banana workers (28.4 %), meat workers (7.2 %), dairy farmers (5.8 %), graziers/stockmen (5.5 %), 'other agricultural/rural workers' (16.4 %), and tourists or tourism operators (4.6 %). Time trend analysis showed that while non-Arborea cases decreased over the study period, Arborea cases increased by 3.4 cases per year. The proportion of annual cases attributed to Arborea peaked at 49 % in 2011 after unprecedented flooding in Queensland. Mapping of cases by residential location showed expansion of the geographic range of serovar Arborea, concentrating mostly around Brisbane, Cairns and Innisfail. Serovars varied significantly between ages and occupational groups, and serovar Arborea was most strongly associated with 'other agricultural/rural workers'. CONCLUSIONS: Leptospira borgpetersenii serovar Arborea has been emerging in Queensland since 2001, with increase in case numbers, the proportion of leptospirosis infections attributed to the serovar, as well as expansion of its geographic distribution. Reasons for this emergence are unknown, but climatic factors and environmental change are likely to have played important roles.

Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospira-specific IgM enzyme-linked immunosorbent assay in a clinical environment.

A Taqman assay for the detection of pathogenic Leptospira was modified to suit the LightCycler instrument. The modified assay was found to have an analytical sensitivity of 10 copies/reaction. The assay was then compared to the current gold standard for acute phase detection, culture, and to a commercially available Leptospira-specific IgM enzyme-linked immunosorbent assay (ELISA). Of the 236 samples including serum and ethylenediaminetetraacetic acid anticoagulated blood sample submitted for testing, polymerase chain reaction (PCR) was able to detect Leptospira DNA in 27 of the 28 culture positives and in 1 negative culture. Discrepant results were resolved by using the microscopic agglutination test on convalescent sera. The PCR was found to have a clinical specificity and sensitivity of 99.5% and 96.4%, respectively, when compared to the culture. Comparisons between culture and ELISA showed that the ELISA lack sensitivity (4.2%) and positive predictability (3.6%) for the detection of acute phase Leptospira infections. These results show that the modified LightCycler Taqman assay could be used as a replacement of culture for the detection of pathogenic Leptospira in a clinical setting.

Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia.

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.

Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene.

BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.

An improved multiple-locus variable number of tandem repeats analysis for Leptospira interrogans serovar Australis: a comparison with fluorescent amplified fragment length polymorphism analysis and its use to redefine the molecular epidemiology of this serovar in Queensland, Australia.

In this study, an improved multiple-locus variable number of tandem repeats analysis (MLVA) method based upon a previously published method is described. Improvements to the method included redesigned primers and PCR conditions, combined with pooled capillary electrophoresis using multicolored dyes. Allele sizes were converted into an allele string, and each unique allele string was assigned a numerical MLVA type (MVT). The improved MLVA method was then applied to 96 previously characterized Leptospira interrogans serovar Australis isolates from human and animal sources. The improved MLVA was found to have between six and 13 alleles at each locus, compared with three to eight in the original. The mean Hunter-Gaston diversity index (HGDI) for the improved MLVA method was 0.654, compared with 0.599 in the original; this increase in diversity was largely due to changes in the analysis of the variable number of tandem repeat (VNTR) data. When the improved MLVA method was compared with the fluorescent amplified fragment length polymorphism (FAFLP) method, there was a high level of concordance between the profiles; however, the MLVA method produced an additional four unique profiles amongst the subset of 30 isolates tested. Given that the improved MLVA method was found to be superior to the original MLVA method, it was subsequently used to redefine the molecular epidemiology of L. interrogans serovar Australis in Queensland, Australia. Using cluster analysis, the authors were able to demonstrate clonal links amongst rodent isolates, rodent and human isolates, and rodent and canine isolates. These results highlight the role of rodents in the disease, and also the potential role of MLVA in defining the molecular epidemiology of L. interrogans.

Development of a Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia.

BACKGROUND: Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. METHODS: In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. RESULTS: The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. CONCLUSION: The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.

Antibodies to Leptospira among blood donors in higher-risk areas of Australia: possible implications for transfusion safety.

BACKGROUND: Leptospirosis is one of the most common bacterial zoonoses worldwide, and clinical manifestations range from asymptomatic infection to acute febrile illness, multi-organ failure and death. Asymptomatic, acute bacteraemia in a blood donor provides a potential for transfusion-transmission, although only a single such case from India has been recorded. Human leptospirosis is uncommon in developed countries; however, the state of Queensland in Australia has one of the highest rates among developed countries, especially after increased rainfall. This study examined the prevalence of antibodies to Leptospira spp. in blood donors residing in higher-risk areas of Australia, to evaluate the appropriateness of current blood safety guidelines. MATERIALS AND METHODS: Plasma samples collected from blood donors residing in higher-risk areas of Australia during 2009 and 2011 were included in the study. All samples were tested for the presence of antibodies to 22 leptospiral serovars using the microscopic agglutination test. RESULT: No sample had antibody titres suggestive of a current or recent infection, however, seven samples (1.44%, 95% CI: 0.38-2.50%) had titres suggestive of a past infection. DISCUSSION: This study provides data that may support the appropriateness of current relevant donor selection policies in Australia. Given that the risk profile for leptospirosis is expanding and that the infection is likely to become more prevalent with climate change, this disease may become more of a concern for transfusion safety in the future.

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.

BACKGROUND: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

Haemoglobin and red cell counts in leptospirosis patients infected with different serovars.

INTRODUCTION: The aim of the study was to compare haemoglobin and red cell counts between patients known to be infected with a range of leptospiral serovars. METHODS: The study retrospectively compared the haemoglobin and red cell count results from the first blood samples taken from 207 patients at presentation to a Queensland Health hospital. RESULTS: Significant differences were observed in haemoglobin and red cell counts in those infected with Leptospira interrogans serovars Szwajizak and Canicola when compared with most of the other serovars. CONCLUSIONS: These findings suggest that haemoglobin and red cell counts may be useful in differentiating leptospiral serovars in leptospirosis patients.

Causes of non-malarial fever in Laos: a prospective study.

BACKGROUND: Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. METHODS: For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5-49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. FINDINGS: With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. INTERPRETATION: Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. FUNDING: Wellcome Trust, WHO-Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention

Evaluation of the Standard Diagnostics Leptospira IgM ELISA for diagnosis of acute leptospirosis in Lao PDR.

The diagnostic utility of the Standard Diagnostics Leptospira IgM ELISA for detection of acute leptospirosis was assessed in febrile adults admitted in Vientiane, Laos. Using the cut-off suggested by the manufacturer [optical density (OD) ≥0.75], the assay demonstrated limited diagnostic capacity with a sensitivity of 95% and a specificity of 41% compared with the Leptospira microscopic agglutination test, which is the serological gold standard. However, re-evaluation of the diagnostic cut-off to an OD of 1.7 demonstrated improved diagnostic accuracy overall (sensitivity 70%; specificity 78%).

Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease.

Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions.

Serologic-based investigation of leptospirosis in a population of free-ranging eastern grey kangaroos (Macropus giganteus) indicating the presence of Leptospira weilii serovar Topaz.

Eastern grey kangaroos (Macropus giganteus) are one of the most abundant large macropodids sharing the landscape with humans. Despite this, little is known about the prevalence of Leptospira carriage within this species and the role that they may partake in the transmission of this disease in Australia. The sera of 87 free-ranging eastern grey kangaroos, captured in the Warragamba Catchment Area, Sydney, Australia, from June 2004 to November 2006, were screened against a reference panel of 22 Leptospira serovars using the microscopic agglutination test (MAT). Leptospiral antibodies were detected in 47% (41 of 87) of serum samples collected. Leptospira weilii Topaz, a newly emergent serovar in Australia, was detected in all seropositive kangaroos (41 of 41; 100%). The sex and tail-fat body condition index of kangaroos appeared to have no significant effect on the exposure to the disease. This serologic-based study is the first reported for L. weilii serovar Topaz in New South Wales, to our knowledge, having previously been isolated only in humans and two other animal species (bovine and long-nosed bandicoot [Perameles nasuta]) in Western Australia and Queensland. The potential role of eastern grey kangaroos in the maintenance and zoonotic spread of the disease to livestock and humans is discussed.

Molecular epidemiology of Leptospira borgpetersenii serovar Arborea, Queensland, Australia, 1998-2005.

Leptospira borgpetersenii serovar Arborea is an emerging cause of leptospirosis in Australia. It was not previously recognized as an endemic serovar before the 1990s, but at that point, human infections with the serovar increased significantly. Using fluorescent-amplified fragment-length polymorphism (FAFLP) molecular typing, human and rodent isolates were compared genetically. Typing revealed 11 unique profiles among the 23 isolates examined; however, there was no clonality revealed between the human and rodent isolates. There was clonality among rodent isolates from geographically related areas. This study highlights the utility of Leptospira culture combined with FAFLP for the examination of the epidemiology of this disease.

Lymphopenia is observed regularly in the acute (leptospiraemic) phase but not the immune phase of leptospirosis.

Lymphocyte counts in patients with leptospirosis have been shown to be variable. This study retrospectively compared lymphocyte counts from the first blood samples taken following hospital presentation in patients with leptospirosis who were either (i) IgM non-reactive, (ii) IgM reactive and microscopic agglutination test (MAT) non-reactive or (iii) IgM and MAT reactive in an effort to determine whether differences in lymphocyte counts are observed in the acute and immune phase of leptospirosis. Statistical differences in lymphocyte counts were observed between the three groups. In conclusion, this study has shown that the phase of leptospiral infection may affect patient lymphocyte counts.

Reclassification of Leptospira meyeri serovar Perameles to Leptospira interrogans serovar Perameles through serological and molecular analysis: evidence of a need for changes to current procedures in Leptospira taxonomy.

It has been recognized that there is heterogeneity among Leptospira isolates in culture collections worldwide, causing confounding results for researchers utilizing these organisms; one such culture is Leptospira meyeri serovar Perameles. The serovar reference strain Bandicoot 343 was previously identified to the species level by DNA-DNA hybridization; however, subsequent published studies demonstrated results that contradicted the initial speciation. In this study, initial serological testing was performed with isolates from the culture collections of the Centers for Disease Control (CDC), Atlanta, USA (strain Lepto0214), and the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Brisbane, Australia (strain Bandicoot 343), and the original serovar Perameles hyperimmune antiserum produced in 1964. The results indicated that strain Lepto0214 was not serologically reactive to the antiserum. However, further investigations revealed an alternative serovar Perameles strain held in the CDC collection (Lepto0213) that yielded titres against the antiserum. 16S rRNA gene sequencing of the three strains revealed that Lepto0214 had significant sequence similarity with previously sequenced L. meyeri strains; however, strains Lepto0213 and Bandicoot 343 had significant similarity with Leptospira interrogans strains. 16S rRNA gene sequencing results were confirmed by pulsed-field gel electrophoresis; Lepto0214 had a pattern similar to that of L. meyeri serovar Hardjo strain Went 5, and the pattern differed significantly from those of Lepto0213 and Bandicoot 343. This research provides evidence for the reclassification of serovar Perameles from L. meyeri to L. interrogans. This reclassification highlights a need for changes to how reference Leptospira serovars are identified, disseminated and stored, with the aim of reducing heterogeneity of reference strains between culture collections.

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia.

A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).

The microscopic agglutination test (MAT) is an unreliable predictor of infecting Leptospira serovar in Thailand.

A prospective study in Thailand identified 106 patients with culture-proven leptospirosis. The accuracy of the microscopic agglutination test (MAT) in predicting the infecting serovar was evaluated in 78/106 (74%) patients with a diagnostic titer. MAT correctly determined the infecting serovar in 26 cases (33%), indicating that this assay is a poor predictor of infecting serovar in our setting.

Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand.

A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of Nakornrachasima, Thailand. The isolate showed typical Leptospira motility and morphology under dark-field microscopy. Cells were 10-13 mum long and 0.2 mum in diameter, with a wavelength of 0.5 mum and an amplitude of approximately 0.3 mum. Phenotypically, strain Khorat-H2(T) did not grow at 13 degrees C but grew at 30 and 37 degrees C and in the presence of 8-azaguanine. Serological identification using the microscopic agglutination test revealed that strain Khorat-H2(T) had no cross-reaction with any recognized Leptospira serogroups. Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain within the radiation of the genus Leptospira, with sequence similarities of 88.1-97.7 % to recognized Leptospira species. DNA-DNA hybridization against the type strains of the three most closely related Leptospira species was used to confirm the results of the 16S rRNA sequence analysis. The G+C content of strain Khorat-H2(T) was 41.8 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Khorat-H2(T) represents a novel species of the genus Leptospira, for which the name Leptospira wolffii sp. nov. is proposed. The type strain is Khorat-H2(T) (=WHO LT1686(T) =KIT Khorat-H2(T)).

Haemoglobin and red cell counts in leptospirosis patients infected with different serovars

Introduction The aim of the study was to compare haemoglobin and red cell counts between patients known to be infected with a range of leptospiral serovars. Methods The study retrospectively compared the haemoglobin and red cell count results from the first blood samples taken from 207 patients at presentation to a Queensland Health hospital. Results Significant differences were observed in haemoglobin and red cell counts in those infected with Leptospira interrogans serovars Szwajizak and Canicola when compared with most of the other serovars. Conclusions These findings suggest that haemoglobin and red cell counts may be useful in differentiating leptospiral serovars in leptospirosis patients. .