Intermediate Leptospira Research Articles

Advances and challenges in barcoding pathogenic and environmental Leptospira.

Leptospirosis is a zoonotic bacterial disease of global importance. A large spectrum of asymptomatic animal hosts can carry the infection and contribute to the burden of human disease. Environmental sources of human contamination also point to the importance of a hydrotelluric reservoir. Leptospirosis can be caused by as many as 15 different pathogenic or intermediate Leptospira species. However, classification of these bacteria remains complicated through the use of both serological and genetic classification systems that show poor correlation. With the advent of molecular techniques, DNA-based barcoding offers a conceptual framework that can be used for leptospirosis surveillance as well as source tracking. In this review, we summarize some of the current techniques, highlight significant successes and weaknesses and point to the future opportunities and challenges to successfully establish a widely applicable barcoding scheme for Leptospira.

Imported Leptospira licerasiae Infection in Traveler Returning to Japan from Brazil.

We describe a case of intermediate leptospirosis resulting from Leptospira licerasiae infection in a traveler returning to Japan from Brazil. Intermediate leptospirosis should be included in the differential diagnosis for travelers with fever returning from South America. This case highlights the need for strategies that detect pathogenic and intermediate Leptospira species.

Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia.

Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. Intermediate Leptospira was present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. Saprophytic Leptospira was present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii, intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10−1 genomic equivalent ml−1. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.

Leptospira species in floodwater during the 2011 floods in the Bangkok Metropolitan Region, Thailand.

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.

Evaluation of an in-house ELISA using the intermediate species Leptospira fainei for diagnosis of leptospirosis

Leptospirosis is recognized as an emerging zoonotic disease generally affecting urban slums in developing countries and tropical regions. A combination of non-specific symptoms, low awareness among the medical community and a lack of readily available diagnostic tests have made leptospirosis an underdiagnosed disease. In this study, we tested an in-house ELISA with formalin-treated and boiled bacteria from the intermediate species Leptospira fainei as an antigen to detect Leptospira-specific IgM antibodies. A total of 819 serum samples, tested by a microscopic agglutination test (MAT) as a reference test, were used to evaluate the ELISA. Compared with positive and negative sera, the ELISA showed 94 % sensitivity and 99 % specificity. Positive and negative likelihood ratios were 94 and 0.06, respectively. No cross-reactivity was observed in sera from subjects with dengue and syphilis infections. The kappa value was 0.92 (95 % confidence interval 0.88-0.96), which indicated excellent agreement between the MAT and ELISA. The overall performance of this in-house ELISA suggests applicability as a rapid screening test for the diagnosis of leptospirosis in resource-limited settings and in hospitals and laboratories where a MAT is not available.

Accuracy of Loop-Mediated Isothermal Amplification for Diagnosis of Human Leptospirosis in Thailand

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case–control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0–52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0–89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5–94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.

Asymptomatic Renal Colonization of Humans in the Peruvian Amazon by Leptospira

BackgroundRenal carriage and shedding of leptospires is characteristic of carrier or maintenance animal hosts. Sporadic reports indicate that after infection, humans may excrete leptospires for extended periods. We hypothesized that, like mammalian reservoir hosts, humans develop asymptomatic leptospiruria in settings of high disease transmission such as the Peruvian Amazon.Methodology/Principal FindingsUsing a cross-sectional study design, we used a combination of epidemiological data, serology and molecular detection of the leptospiral 16S rRNA gene to identify asymptomatic urinary shedders of Leptospira. Approximately one-third of the 314 asymptomatic participants had circulating anti-leptospiral antibodies. Among enrolled participants, 189/314 (59%) had evidence of recent infection (microscopic agglutination test (MAT0 ≥1∶800 or ELISA IgM-positive or both). The proportion of MAT-positive and high MAT-titer (≥1∶800) persons was higher in men than women (p = 0.006). Among these people, 13/314 (4.1%) had Leptospira DNA-positive urine samples. Of these, the 16S rRNA gene from 10 samples was able to be sequenced. The urine-derived species clustered within both pathogenic (n = 6) and intermediate clades of Leptospira (n = 4). All of the thirteen participants with leptospiral DNA in urine were women. The median age of the DNA-positive group was older compared to the negative group (p≤0.05). A group of asymptomatic participants (“long-term asymptomatic individuals,” 102/341 (32.5%) of enrolled individuals) without serological evidence of recent infection was identified; within this group, 6/102 (5.9%) excreted pathogenic and intermediate-pathogenic Leptospira (75–229 bacteria/mL of urine).Conclusions/SignificanceAsymptomatic renal colonization of leptospires in a region of high disease transmission is common, including among people without serological or clinical evidence of recent infection. Both pathogenic and intermediate Leptospira can persist as renal colonization in humans. The pathogenic significance of this finding remains to be explored but is of fundamental biological significance.

Leptospira wolffii, a potential new pathogenic Leptospira species detected in human, sheep and dog

Leptospirosis is the most common zoonotic disease, which is transmitted to humans through contaminated water or direct exposure to the urine of infected animals. In this study, the presence and prevalence of Leptospira species in the infected samples of human (n=369) and sheep (n=75) sera and also dogs' urine (n=150), collected from four provinces of Iran, were investigated by using nested-PCR/RFLP assay followed by sequencing analysis. Nested-PCR assay detected that 98/369 (26.5%) human, 13/75 (17.33%) of sheep's sera and 33/150 (22%) dogs' urine samples were positive for Leptospira DNA. RFLP assay detected that all positive cases had either pathogenic or intermediate Leptospira species. By sequence analysis, Leptospira interrogans was the most prevalent species among the examined samples of human (53/82, 64.6%) and sheep (11/13, 84.6%). However, in dog samples, Leptospira wolffii (27/29, 93.1%) was detected for the first time and was the dominant species. The presence of L. wolffii with 100% identity in clinical human samples and animals suspected with Leptospira may provide evidence for circulation of L. wolffii and its role in transmission cycle within human and animal hosts. In addition, this species can be potentially pathogenic to human and probably animal hosts. A large epidemiology survey would be needed to define the presence and the prevalence of this species in global endemic regions.

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.

Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.

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.

Prevalence of Leptospira Species in Environmental Soil and Water from National Parks in Sarawak, Malaysia

In Malaysia, there is an increasing number of reported leptospirosis cases which led to mortality. The infection is usually spread through the urine of infected animals and may contaminate the environmental soil and water. This study was conducted to determine the prevalence of Leptospira spp. in two national parks in Sarawak. A total of 50 soil and 60 water samples were collected from Tanjung Datu National Park (TDNP) and Bako National Park (BNP). The samples were filtered through sterile membrane filter prior to inoculation into modified semisolid Ellinghausen-McCullough-Johnson-Harris (EMJH) media added with 5-fluorouracil. The cultures were incubated at room temperature (28-30 °C) for 30 days before specific polymerase chain reaction (PCR) was conducted. PCR-positive samples were subjected to DNA sequencing. Out of the 110 environmental samples from two national parks in Sarawak, the prevalence of pathogenic, intermediate and saprophytic Leptospira was 0.9%, 5.5% and 0%, respectively. Results indicated that Leptospira borgpetersenii serovar Mini and Leptospira wolffii serovar Khorat were the pathogenic and intermediate Leptospira circulating in these study areas, respectively. Due to the ability of Leptospira to survive for months in environment, there is a risk of exposure to the public and tourists who visit these national parks. Increased awareness, continuous monitoring and effective preventive measures should be taken by local authorities to control leptospirosis outbreak.