A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema paUlidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative. Genomic DNA-DNA hybridization helped to define nine species (26) of the genus Leptospira, which is composed of more than 200 serovars. The nonpathogenic Leptospira spp.

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.

Purpose We investigated the effect of a 31-d ketogenic diet (KD) on submaximal exercise capacity and efficiency. Methods A randomized, repeated-measures, crossover study was conducted in eight trained male endurance athletes (V˙O2max, 59.4 ± 5.2 mL⋅kg−1⋅min−1). Participants ingested their habitual diet (HD) (13.1 MJ, 43% [4.6 g⋅kg−1⋅d−1] carbohydrate and 38% [1.8 g⋅kg−1⋅d−1] fat) or an isoenergetic KD (13.7 MJ, 4% [0.5 g·kg−1⋅d−1] carbohydrate and 78% [4 g⋅kg−1⋅d−1] fat) from days 0 to 31 (P < 0.001). Participants performed a fasted metabolic test on days −2 and 29 (~25 min) and a run-to-exhaustion trial at 70% V˙O2max on days 0 and 31 following the ingestion of a high-carbohydrate meal (2 g⋅kg−1) or an isoenergetic low-carbohydrate, high-fat meal (<10 g CHO), with carbohydrate (~55 g⋅h−1) or isoenergetic fat (0 g CHO⋅h−1) supplementation during exercise. Results Training loads were similar between trials and V˙O2max was unchanged (all, P > 0.05). The KD impaired exercise efficiency, particularly at >70% V˙O2max, as evidenced by increased energy expenditure and oxygen uptake that could not be explained by shifts in respiratory exchange ratio (RER) (all, P < 0.05). However, exercise efficiency was maintained on a KD when exercising at <60% V˙O2max (all, P > 0.05). Time-to-exhaustion (TTE) was similar for each dietary adaptation (pre-HD, 237 ± 44 vs post-HD, 231 ± 35 min; P = 0.44 and pre-KD, 239 ± 27 vs post-KD, 219 ± 53 min; P = 0.36). Following keto-adaptation, RER >1.0 vs <1.0 at V˙O2max coincided with the preservation and reduction in TTE, respectively. Conclusion A 31-d KD preserved mean submaximal exercise capacity in trained endurance athletes without necessitating acute carbohydrate fuelling strategies. However, there was a greater risk of an endurance decrement at an individual level.

Leptospirosis is a worldwide-distributed zoonosis, endemic in tropical areas. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests. Recently, molecular typing systems based on variable-number tandem-repeat (VNTR) analysis have been described and have been used to identify Leptospira interrogans strains. Although L. interrogans is the most common Leptospira species encountered in human infections around the world, other pathogenic species, such as Leptospira kirschneri and Leptospira borgpetersenii, are also frequently associated with human leptospirosis. In this study, we aimed to extend multilocus VNTR analysis (MLVA) identification of strains to species other than L. interrogans. We designed primers for VNTR loci found in L. interrogans, L. kirschneri, and L. borgpetersenii. The discriminatory power of the redefined primers was evaluated on collection strains and then on clinical strains. We also carried out a retrospective study on 156 strains isolated from patients and animals from New Caledonia, an area of high endemicity in the South Pacific. Our results show that this simple PCR-based MLVA typing technique is a powerful methodology for the epidemiology of leptospirosis.

Polymerase chain reaction assay (PCR) amplifying a fragment of the Leptospira rrs gene was compared with culture and microagglutination test (MAT) for the diagnosis of leptospirosis in a study of 200 patients with various clinical syndromes compatible with leptospirosis. For the first group of samples tested, PCR identified the 14 cases that later were unequivocally confirmed to be leptospirosis. Thirteen other systemic cases presenting decreasing leptospiral antibody titers were also detected by PCR. The average persistence of leptospiral DNA in serum was estimated at 12 days, with a maximum of 56 days in a culture-confirmed case. The possibility of detecting leptospires in aqueous humor during the ocular complications of the disease was confirmed. The results suggest that PCR is an efficient tool for early diagnosis of leptospirosis during the first 10 days of the disease, especially when the clinical expression of the disease is confusing.