Conventional diagnosis of leptospirosis mainly relies on serological techniques. These methods reach only suitable levels of sensitivity at a late acute phase of the disease when antibiotic treatment may be less effective. Leptospires are present in the blood during the first 5 to 10 days after onset of the disease. Direct detection of them would provide an excellent means to give early confirmation of clinical suspicion. Direct observation of leptospires in blood samples by darkfield microscopy is notoriously unreliable and not recommended 
. Isolation of leptospires can take up to months and does not contribute to early diagnosis. Detection of leptospires through specific PCR amplification of its DNA has been championed as a promising alternative for two decades. Several conventional PCR tests for the specific detection of leptospiral DNA from body fluids and tissues have been described 
. Unfortunately evaluation of the clinical applicability has only been done for two of such PCRs on a limited scale 
, leaving the value of the conventional PCR for the laboratory diagnosis unclear. A further drawback of the conventional PCR is that the technique is particularly prone to contamination resulting to false positive outcomes 
. It is not always clear whether serologically or culture negative but PCR positive samples reflect a higher sensitivity of the PCR or are due to contamination 
Real-time PCR, either using molecular beacons or SYBR Green technology has the advantage that it gives a result much quicker than conventional PCR and is less prone to contamination. By now, several real-time PCRs have been developed for the detection of leptospires 
but solid evaluations are lacking and therefore their usability remains uncertain. An auspicious exception is the recently described evaluation of a Taq-man real-time PCR 
. This test showed an analytical sensitivity of 10 homologous genome copies and when compared with culture proven leptospirosis patients had a DSe and DSp of 100% and 93%, respectively.
Here we describe the development and evaluation of a real-time PCR based on the SYBR Green technology targeting the secY
gene. The primer pair we selected showed high specificity for detection of pathogenic leptospires, excluding all saprophytic strains tested and the vast majority of intermediate ones. The lack of amplification of DNA from most intermediate strains is not unexpected because their Leptospira
species form a separate intermediate clade situated between pathogenic and saprophytic species 
. This signifies a difference in DNA composition compared to pathogenic species apparently resulting in a too low annealing capacity of the primers hampering the amplification. In our opinion, this is of little relevance for the diagnostic potential of the test. Infection of patients with intermediate leptospires is a relatively rare event and their pathogenic status is as yet doubtful. Even though some of the intermediate Leptospira
spp. have been described as clinical isolates, virulence cannot convincingly be demonstrated 
. No cross-reaction was found with other micro-organisms, which is an important feature because secY
is a house-keeping gene that has been demonstrated in many prokaryotic species.
A major advantage of using the secY
gene is its great phylogenetic potential 
. We recently demonstrated that a small 245 bp segment of secY
, flanked by the primer pair G1/G2 
, had a high phylogenic power almost equaling that of the whole gene thus making it a feasible and interesting target for speciation by less sophisticated laboratories 
. We found that the 201 bp fragment of the gene amplified in this real-time PCR assay had a similar phylogenetic potential as the 245 bp G1/G2 restricted fragment, making this target an attractive alternative for sequencing and phylogeny following amplification in a conventional format (Fig. S1
The real-time PCR was validated using the specific instructions from OIE 
. We found an analytical sensitivity of 1 to 50 copies, depending on the degree of homology between strains from different species and the type of sample used for extracting Leptospira
DNA. Taking into account the DNA extraction and PCR protocols used in this study, detection of 1 genome copy per reaction equals a concentration of 100 leptospires per ml culture medium. This implicates a detection range from 100 to 5000 leptospires per ml or tissue equivalent, provided that the DNA extraction is efficient. Hence the high analytical sensitivity cannot be translated in a high practical efficiency, due to the sample processing step in which DNA is not concentrated. Our main future focus is therefore on developing a more adequate extraction procedure.
Notably DNA extracted from urine and kidney samples contained inhibitors. Both sample types are not essential for early diagnosis of leptospirosis but have value in other situations such as post-mortem investigations. We addressed the residual inhibition in two ways. For urine we introduced an extra washing step in the extraction procedure as most optimal tactic. For kidney samples preparing 1
10 dilutions of the extracted DNA appeared the best approach. Both methods have the disadvantage of losing or diluting target DNA but overall the approaches led to markedly higher success rates. Inhibition is a real problem as this leads to false-negative results. To provide a tool to check on inhibitory effects in the PCR we introduced an IAC.
For early diagnosis, blood and serum are ideal samples. The immune system of the human body clears the bacteria from the blood after approximately 5–7 days after appearance of clinical manifestations. From one hand, the real-time PCR had a DSe of 100% when performed within the first four days of illness, which statistically represents a bias, as leptospires are still present at high concentrations in the patients' blood. On the other hand early confirmation of leptospirosis is of utmost importance for initiating adequate treatment. Therefore, from a clinical point of view, the high DSe at the early stage of illness signifies a great value for clinical decision making.
It should be noted that the very promising results of clinical evaluation in this study have been achieved with samples from Dutch patients. Currently half of the infections are acquired in The Netherlands where the serovars Copenhageni, Icterohaemorrhagiae and Grippotyphosa are dominant. This might induce a bias of the performance of the test. For this reason, the last stages of the OIE validation scheme include field studies at other laboratories to assess clinical sensitivity and specificity under different circumstances. We are currently aiming at the implementation and evaluation of the test in endemic areas with a variety of causative serovars.
In this study, culture and serology were considered as gold standard to estimate the clinical sensitivity and specificity in order to measure eventual bias of the results of high bacterial loads in culture and PCR positive samples alone. The overall sensitivity and specificity in this study were estimated as 93% and 100%, respectively. The assay showed complete reproducibility and repeatability as well as high level of robustness since changing in critical PCR parameters has no or slight influence on overall results.
Testing kidney, lung and liver from two early deceased patients as well as some rodent kidneys proved clearly the usefulness of the real-time PCR as an effective tool for the detection of Leptospira in the distinct tissues. This shows the applicability of real-time PCR as a suitable diagnostic tool on post-mortem samples, overcoming the failure to confirm leptospirosis of early deceased patients by serology.