Optimization of Culture of
            <i>Leptospira</i>
            from Humans with Leptospirosis

Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Limmathurotsakul, Direk; Smythe, Lee D.; Symonds, Meegan L.; Dohnt, Michael F.; Slack, Andrew T.; Limpaiboon, Roongreung; Suputtamongkol, Yupin; White, Nicholas J.; Day, Nicholas P. J.; Peacock, Sharon J.

doi:10.1128/jcm.02430-06

Cited by 66 publications

(69 citation statements)

References 7 publications

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“…blood, cerebrospinal fluid) but this requires prolonged periods of incubation and a set of absorbed antisera to establish the serovar of the recovered strains [7].…”

Section: Introductionmentioning

confidence: 99%

Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

González

Geymonat

Rodríguez-Hernández

et al. 2013

J Infect Dev Ctries

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Introduction: Assays based on DNA amplification can provide information that contributes to the initial management of patients with leptospirosis. However, these have not been adopted in Uruguay. Our aim was to evaluate the performance of the lipL32 real-time PCR (qPCR) for diagnosis of leptospirosis. Methodology: We analyzed by microscopic agglutination test (MAT) and lipL32 qPCR serum samples from 183 patients with suspected leptospirosis. To establish the analytical sensitivity of the qPCR, experimentally spiked samples with known amounts of Leptospira interrogans were analyzed. Results: The analytical sensitivity of the qPCR was 10 2 leptospires/mL. In 98 patients MAT results were negative meanwhile 85 showed positive reactions, revealing acute infections. Twenty six acute-phase sera of these 85 patients showed a positive signal by qPCR (diagnostic sensitivity 30%). In these patients the average time between onset of symptoms and collection of the first sample was 8 days. In patients with negative results for qPCR and positive MAT results (n=59) the average interval between onset of symptoms and collection of the first sample was 13 days. The qPCR did not yield false positive results. Conclusions: The qPCR had a lower diagnostic sensitivity than MAT and a higher cost. However, it allowed to make an early diagnosis in 26 patients. In patients with confirmed acute infections and negative results by qPCR, more than 8 days had elapsed between the onset of the illness and extraction of the first serum sample. Our data support that the qPCR from sera have clinical utility within the first week of illness.

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“…blood, cerebrospinal fluid) but this requires prolonged periods of incubation and a set of absorbed antisera to establish the serovar of the recovered strains [7].…”

Section: Introductionmentioning

confidence: 99%

Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

González

Geymonat

Rodríguez-Hernández

et al. 2013

J Infect Dev Ctries

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“…It is also a relatively insensitive diagnostic method. However, isolation of pathogenic leptospires is proof of an infection and culture is golden standard method (28,29). Also, isolated leptospires can be typed to identify serovars and it is useful in the epidemiological studies of local pathogenic serovars (30).…”

Section: Culturementioning

confidence: 99%

Clinical Laboratory Diagnosis of Human Leptospirosis

Khaki

2016

Int J Enteric Pathog

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Context: Leptospirosis is a worldwide zoonotic infection which appears to be a re-emerging health problem. The clinical features of the disease are broad ranging, but are often similar to those of other infections. As a result, the accuracy of a clinical diagnosis of leptospirosis is low and confirmation requires the use of laboratory tests. Evidence Acquisition: The disease is usually diagnosed in the laboratory by different methods such as direct microscopy, culture, serological methods and molecular methods. The microscopic agglutination test (MAT) is considered the reference test among the several serological methods for leptospirosis diagnosis. However, isolation and identification of the microorganism allows for definitive diagnosis, and provides for epidemiological and prophylactic studies of this disease. Therefore, culture is a golden standard method. Polymerase chain reaction is a rapid, sensitive and specific means of detecting leptospiral infection, in contrast to serology tests. Further benefit is the ability to identify early infection especially during the first few days of the disease even before antibodies are detectable. Conclusions: Choice of test for diagnosis of leptospirosis depends on the stage of the disease. An ideal test will need to discriminate between leptospirosis and a broad spectrum of diseases that cause acute febrile illness and have overlapping clinical presentations. Although detection of antibodies is by itself no proof of a current infection, serological methods (such as MAT and ELISA) are often the most appropriate diagnostic methods.

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“…First, all agar plates were tested for time to growth (visible colonies) of Leptospira interrogans serovar Pyrogenes strain NR-20157, a clinical isolate cultured from a febrile rice farmer presenting to a hospital in northeast Thailand in 2001 (6,26). The inoculum was prepared by adding 1 ml of stock culture to 4 ml of liquid culture medium (EMJH supplemented with 10% RS) and incubating the mixture at 30°C in air for 1 week.…”

Section: Bacterial Isolatesmentioning

confidence: 99%

“…This is not the case for leptospirosis. Although Leptospira cells are present in blood during the first week of illness (6), these fastidious microorganisms do not grow readily in standard laboratory media. Culture can be performed but requires specific semisolid or liquid media and considerable expertise and may take several weeks or months before the organism is isolated (6).…”

mentioning

confidence: 99%

“…Although Leptospira cells are present in blood during the first week of illness (6), these fastidious microorganisms do not grow readily in standard laboratory media. Culture can be performed but requires specific semisolid or liquid media and considerable expertise and may take several weeks or months before the organism is isolated (6). Furthermore, antimicrobial therapy is invariably empirical because of a lack of simple antimicrobial susceptibility testing methods for Leptospira (7).…”

mentioning

confidence: 99%

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Rapid Isolation and Susceptibility Testing of Leptospira spp. Using a New Solid Medium, LVW Agar

Wuthiekanun

Amornchai

Paris

et al. 2013

Antimicrob Agents Chemother

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g Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO 2 incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO 2 for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC 90 values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 g/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

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Optimization of Culture of Leptospira from Humans with Leptospirosis

Cited by 66 publications

References 7 publications

Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

Clinical Laboratory Diagnosis of Human Leptospirosis

Rapid Isolation and Susceptibility Testing of Leptospira spp. Using a New Solid Medium, LVW Agar

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