[Show abstract][Hide abstract] ABSTRACT: Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.
Diseases of Aquatic Organisms 08/2014; 110(3):165-72. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract During rehabilitation, acute renal failure due to leptospirosis occurred in eight male northern elephant seals (Mirounga angustirostris) that stranded along the central California coast in 2011. Characteristic histologic lesions including renal tubular degeneration, necrosis, and mineralization, and mild lymphoplasmacytic interstitial nephritis were noted in the six animals examined. Immunohistochemistry, bacterial culture, and PCR were positive in 2/3, 2/3, and 3/4 seals, respectively, and 6/8 had high serum antibody titers to Leptospira interrogans serovar pomona. Pulsed-field gel electrophoresis confirmed one isolate as serovar pomona. Variable number tandem repeat (VNTR) analysis showed both elephant seal isolates were identical to each other but distinct from those isolated from California sea lions (Zalophus californianus). The time from stranding to onset of azotemia was 1 to 38 (median = 24) days, suggesting some seals were infected at the rehabilitation facility. Based on temporal and spatial incidence of infection, transmission among elephant seals likely occurred during rehabilitation. Molecular (VNTR) analysis of the two isolates indicates there is a unique L. interrogans serovar pomona genotype in elephant seals, and sea lions were not the source of infection prior to or during rehabilitation. This study confirms the susceptibility of northern elephant seals to leptospirosis, indicates intraspecies transmission during rehabilitation, and reports the first isolation and preliminary characterization of leptospires from elephant seals.
Journal of wildlife diseases 05/2014; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this survey we investigated a population of small mammals in Eastern Croatia in order to determine Leptospira carriage rates and identify circulating serovars. Out of 67 trapped animals, 20 (29.9%) isolates were obtained. Identification of isolates using microscopic agglutination test, pulsed field gel electrophoresis and multi locus sequence typing revealed that 10 (50.0%) isolates belong to serogroup Pomona, serovar Mozdok, 6 (30.0%) isolates to serogroup Australis, serovar Jalna, 2 (10.0%) isolates to serogroup Sejroe, serovar Saxkoebing, and 1 (5.0%) isolate to serogroup Grippotyphosa, serovar Grippotyphosa. One isolate from serogroup Bataviae was unable to be identified to the serovar level. Amplification of a 331-bp region of the locus LA0322 using real-time polymerase chain reaction determined that 12 (60.0%) isolates belong to L. kirschneri, 6 (30.0%) isolates to L. interrogans, and 2 (10.0%) isolates to L. borgpetersenii. Leptospira carriage rate was high (29.9%), which corresponds to a high incidence of human and domestic animal leptospirosis in Eastern Croatia. Furthermore, 90.0% of the isolates belong to serogroups Pomona, Australis and Sejroe which are also the most prevalent serogroups in humans in this area. These findings suggest that small mammals might be an important source of Leptospira spp. infection in Eastern Croatia.
[Show abstract][Hide abstract] ABSTRACT: Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories.
The American journal of tropical medicine and hygiene 07/2013; · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The syndrome of fever is a commonly presenting complaint among persons seeking healthcare in low-resource areas, yet the public health community has not approached fever in a comprehensive manner. In many areas, malaria is over-diagnosed, and patients without malaria have poor outcomes.
We prospectively studied a cohort of 870 pediatric and adult febrile admissions to two hospitals in northern Tanzania over the period of one year using conventional standard diagnostic tests to establish fever etiology. Malaria was the clinical diagnosis for 528 (60.7%), but was the actual cause of fever in only 14 (1.6%). By contrast, bacterial, mycobacterial, and fungal bloodstream infections accounted for 85 (9.8%), 14 (1.6%), and 25 (2.9%) febrile admissions, respectively. Acute bacterial zoonoses were identified among 118 (26.2%) of febrile admissions; 16 (13.6%) had brucellosis, 40 (33.9%) leptospirosis, 24 (20.3%) had Q fever, 36 (30.5%) had spotted fever group rickettsioses, and 2 (1.8%) had typhus group rickettsioses. In addition, 55 (7.9%) participants had a confirmed acute arbovirus infection, all due to chikungunya. No patient had a bacterial zoonosis or an arbovirus infection included in the admission differential diagnosis.
Malaria was uncommon and over-diagnosed, whereas invasive infections were underappreciated. Bacterial zoonoses and arbovirus infections were highly prevalent yet overlooked. An integrated approach to the syndrome of fever in resource-limited areas is needed to improve patient outcomes and to rationally target disease control efforts.
[Show abstract][Hide abstract] ABSTRACT: Leptospira is a global pathogen of emerging public health importance in both developing and industrialized nations and can infect almost all mammalian species, including humans. As suburbanization and the popularity of outdoor recreational activities increases, so do human-wildlife and companion animal-wildlife interfaces. Florida offers a tropical climate favorable for outdoor activities and a semirural landscape that sustains an abundant feral hog population. Because no survey ofleptospirosis in feral hogs (Sus scrofa) in Florida has been published to our knowledge, we sought to establish preliminary seroprevalence ofleptospirosis exposure in feral hogs in Florida. Blood samples were collected opportunistically from 158 male and 166 female feral hogs taken at managed hunts and by permitted trappers in the northern, central, and southern regions of Florida. Samples were then analyzed using the microscopic agglutination test (MAT) for antibody titers to 20 Leptospira serovars representing 17 serogroups. A titer of > 1:100 was considered positive; 33% (107/324 total samples) were positive to at least one serovar, and 46% of those were positive to multiple serovars. Antibodies to L. interrogans serovar Bratislava strain Jez Bratislava (serogroup Australis) was the most common, with 18% (58/324) testing positive for antibodies. These initial data indicate that there is a significant possibility of feral hogs having a larger role in the complex etiology of leptospirosis in Florida than historically estimated and that further investigation is warranted.
Journal of Zoo and Wildlife Medicine 06/2013; 44(2):404-7. · 0.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Background: Leptospirosis and human immunodeficiency virus (HIV) infection are prevalent in many areas, including northern Tanzania, yet little is known about their interaction. Methods: We enrolled febrile inpatients at two hospitals in Moshi, Tanzania, over 1 year and performed HIV antibody testing and the microscopic agglutination test (MAT) for leptospirosis. Confirmed leptospirosis was defined as ≥four-fold rise in MAT titer between acute and convalescent serum samples, and probable leptospirosis was defined as any reciprocal MAT titer ≥800. Results: Confirmed or probable leptospirosis was found in 70 (8.4%) of 831 participants with at least one serum sample tested. At total of 823 (99.0%) of 831 participants had HIV testing performed, and 203 (24.7%) were HIV infected. Among HIV-infected participants, 9 (4.4%) of 203 had confirmed or probable leptospirosis, whereas among HIV-uninfected participants 61 (9.8%) of 620 had leptospirosis. Leptospirosis was less prevalent among HIV-infected as compared to HIV-uninfected participants [odds ratio (OR) 0.43, p=0.019]. Among those with leptospirosis, HIV-infected patients more commonly presented with features of severe sepsis syndrome than HIV-uninfected patients, but differences were not statistically significant. Among HIV-infected patients, severe immunosuppression was not significantly different between those with and without leptospirosis (p=0.476). Among HIV-infected adolescents and adults, median CD4 percent and median CD4 count were higher among those with leptospirosis as compared to those with other etiologies of febrile illness, but differences in CD4 count did not reach statistical significance (p=0.015 and p=0.089, respectively). Conclusions: Among febrile inpatients in northern Tanzania, leptospirosis was not more prevalent among HIV-infected patients. Although some indicators of leptospirosis severity were more common among HIV-infected patients, a statistically significant difference was not demonstrated. Among HIV-infected patients, those with leptospirosis were not more immunosuppressed relative to those with other etiologies of febrile illness.
[Show abstract][Hide abstract] ABSTRACT: Since 1970, periodic outbreaks of leptospirosis, caused by pathogenic spirochetes in the genus Leptospira, have caused morbidity and mortality of California sea lions (Zalophus californianus) along the Pacific coast of North America. Yearly seasonal epizootics of varying magnitude occur between the months of July and December, with major epizootics occurring every 3-5 years. Genetic and serological data suggest that Leptospira interrogans serovar Pomona is the infecting serovar and is enzootic in the California sea lion population, although the mechanism of persistence is unknown. We report asymptomatic carriage of Leptospira in 39% (33/85) of wild, free-ranging sea lions sampled during the epizootic season, and asymptomatic seroconversion with chronic asymptomatic carriage in a rehabilitated sea lion. This is the first report of asymptomatic carriage in wild, free-ranging California sea lions and the first example of seroconversion and asymptomatic chronic carriage in a sea lion. Detection of asymptomatic chronic carriage of Leptospira in California sea lions, a species known to suffer significant disease and mortality from the same Leptospira strain, goes against widely-held notions regarding leptospirosis in accidental versus maintenance host species. Further, chronic carriage could provide a mechanism for persistent circulation of Leptospira in the California sea lion population, particularly if these animals shed infectious leptospires for months to years.
[Show abstract][Hide abstract] ABSTRACT: Rats are considered the principle maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.
The American journal of tropical medicine and hygiene 01/2013; 88(4):704-709. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species.
We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated.
The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.
[Show abstract][Hide abstract] ABSTRACT: The incidence of leptospirosis, a neglected zoonotic disease, is uncertain in Tanzania and much of sub-Saharan Africa, resulting in scarce data on which to prioritize resources for public health interventions and disease control. In this study, we estimate the incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania.
We conducted a population-based household health care utilization survey in two districts in the Kilimanjaro Region of Tanzania and identified leptospirosis cases at two hospital-based fever sentinel surveillance sites in the Kilimanjaro Region. We used multipliers derived from the health care utilization survey and case numbers from hospital-based surveillance to calculate the incidence of leptospirosis. A total of 810 households were enrolled in the health care utilization survey and multipliers were derived based on responses to questions about health care seeking in the event of febrile illness. Of patients enrolled in fever surveillance over a 1 year period and residing in the 2 districts, 42 (7.14%) of 588 met the case definition for confirmed or probable leptospirosis. After applying multipliers to account for hospital selection, test sensitivity, and study enrollment, we estimated the overall incidence of leptospirosis ranges from 75-102 cases per 100,000 persons annually.
We calculated a high incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania, where leptospirosis incidence was previously unknown. Multiplier methods, such as used in this study, may be a feasible method of improving availability of incidence estimates for neglected diseases, such as leptospirosis, in resource constrained settings.
[Show abstract][Hide abstract] ABSTRACT: The genus Leptospira currently comprises 17 named species. In addition, four un-named hybridization groups were designated Leptospira genomospecies 1, 3, 4 and 5. These groups represent valid species level taxa, but were not assigned names in the original description by Brenner et al. (1999). To rectify this situation, it is proposed that Leptospira genomospecies 1, genomospecies 3, genomospecies 4 and genomospecies 5 should be classified as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov., Leptospira yanagawae sp. nov., respectively with strains L. alstonii 79601T (=ATCC BAA-2439T), L. vanthielii WaZ HollandT (=ATCC 700522T), L. terpstrae LT 11-33T (=ATCC 700639T) and L. yanagawae Sao PauloT (=ATCC 700523T) as type strains.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 09/2012; · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A collection of 101 Leptospira isolates was tested by multilocus sequence typing (MLST) and by traditional serotyping. MLST divided the isolates into 4 sequence types (STs), while serotyping classified them into 6 serogroups. Two isolates failed to generate products for some genes by MLST. MLST was less discriminatory than serotyping for uncommonly occurring isolates from humans in Brazil.
Journal of clinical microbiology 08/2011; 49(11):3940-2. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We enrolled consecutive febrile admissions to two hospitals in Moshi, Tanzania. Confirmed leptospirosis was defined as a ≥ 4-fold increase in microscopic agglutination test (MAT) titer; probable leptospirosis as reciprocal MAT titer ≥ 800; and exposure to pathogenic leptospires as titer ≥ 100. Among 870 patients enrolled in the study, 453 (52.1%) had paired sera available, and 40 (8.8%) of these met the definition for confirmed leptospirosis. Of 832 patients with ≥ 1 serum sample available, 30 (3.6%) had probable leptospirosis and an additional 277 (33.3%) had evidence of exposure to pathogenic leptospires. Among those with leptospirosis the most common clinical diagnoses were malaria in 31 (44.3%) and pneumonia in 18 (25.7%). Leptospirosis was associated with living in a rural area (odds ratio [OR] 3.4, P < 0.001). Among those with confirmed leptospirosis, the predominant reactive serogroups were Mini and Australis. Leptospirosis is a major yet underdiagnosed cause of febrile illness in northern Tanzania, where it appears to be endemic.
The American journal of tropical medicine and hygiene 08/2011; 85(2):275-81. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested paired sera from 584 febrile persons in an low-income urban community in Bangladesh for evidence of Leptospira infection. A total of 8.4% of the persons met criteria for definite or probable infection. Persons with leptospirosis were older than those with undifferentiated fever in this population. The dominant infecting serogroups in Bangladesh differed from serogroups commonly reported in nearby regions.
The American journal of tropical medicine and hygiene 06/2010; 82(6):1127-30. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: On 21 November 2005, a 32-year-old male resident of New York was hospitalized with suspected leptospirosis. He had participated in an endurance-length swamp race on 4-5 November 2005 outside of Tampa, Florida.
We interviewed racers to assess illness, medical care, and race activities. A suspected case was defined as fever plus > or = 2 signs or symptoms of leptospirosis occurring in a racer after 4 November 2005. Individuals with suspected cases were referred for treatment as needed and were asked to submit serum samples for microscopic agglutination testing (MAT) and for rapid testing by the dot enzyme-linked immunosorbent assay dipstick immunoglobulin M immunoassay.
The Centers for Disease Control and Prevention and participating state health departments interviewed 192 (96%) of 200 racers from 32 states and Canada. Forty-four (23%) of 192 racers met the definition for a suspected case. The median age of the patients was 37 years (range, 19-66 years), and 128 (66.7%) were male. Fourteen (45%) of the 31 patients with suspected cases who were tested had their cases confirmed by serological testing (a single sample with MAT titer > or = 400), including the index case patient. Organisms of a potential novel serovar (species Leptospira noguchii) were isolated in culture from 1 case patient. Factors associated with increased risk of leptospirosis included swallowing river water (odds ratio [OR], 3.4; 95% confidence interval [CI], 1.6-7.0), swallowing swamp water (OR, 2.4; 95% CI, 1.1-5.2), and being submerged in any water (OR, 2.3; 95% CI, 1.1-4.7).
This report describes a leptospirosis outbreak that resulted in a high rate of symptomatic infection among adventure racers in Florida. The growing popularity of adventure sports may put more people at risk for leptospirosis, even in areas that have not previously been considered areas of leptospirosis endemicity.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal of systematic and evolutionary microbiology 06/2009; 59(Pt 5):1199-203. · 2.11 Impact Factor