gave a high yield [12]. Leptospires can survive in heparinized blood
stored at room temperature, from which they can be recovered at
the longest of 109 days after blood collection [12]. It has been
shown that some in vitro-cultured Leptospira strains can survive in
commercially available blood culture media for up to 2 weeks
[13]. Girault et al. successfully isolated L. borgpetersenii and
L. interrogans from commercial aerobic blood culture bottles.
They transferred culture bottle samples that were Leptospira realtime polymerase chain reaction (PCR)-positive to EMJH medium
within 2.5 days after blood collection and obtained isolates in
one-third of the patients investigated [14].
Leptospires may be isolated by inoculating 0.5 ml CSF into
5 ml semi-solid culture medium on the first week of illness
[9]. Urine is the most suitable specimen for the isolation during
the leptospiruria phase (on the second week after onset). As the
viability of leptospires is limited in acidic urine, urine needs to be
inoculated into the culture medium within 2 h after voiding. Alternatively, the urine sample is neutralized or centrifuged to obtain
pellets, which are inoculated into the culture medium [9]. 5Fluorouracil and/or some antimicrobial agents may be used to
reduce the risk of contamination [9, 10].
3 NAATs
NAATs have demonstrated superior sensitivity in the acute phase of
leptospirosis to culture and serological diagnostics. Leptospiral
DNA has been amplified from blood, urine, CSF, aqueous
humor, and tissues. A number of conventional PCRs including
nested PCR and real-time PCR, as well as isothermal DNA amplification techniques, have been developed for the early diagnosis of
leptospirosis in the last three decades [15–19]. NAAT using realtime PCR has been evaluated in large populations in clinical settings
and becomes the standard for early diagnosis [20–22]. However,
the instruments used in real-time PCR are expensive and may be
unavailable in resource-limited settings. Isothermal DNA amplification techniques such as loop-mediated isothermal amplification
and recombinase polymerase amplification can amplify the target
DNA sequence without thermal cyclers and can be integrated into
“lab-on-a-chip,” which enables NAATs at the point of care
[23]. Extensive evaluations are needed for the utility of isothermal
DNA amplification techniques for leptospirosis diagnosis in clinical
settings [11, 24].
As with culture, blood samples are most suitable in the acute
phase for NAATs. However, in clinical settings, there are controversial reports: one study showed that serum is a better specimen
than whole blood [21], whereas another study demonstrated that
sensitivity is much higher for whole blood than for serum
Laboratory Diagnosis of Leptospirosis
279
stored at room temperature, from which they can be recovered at
the longest of 109 days after blood collection [12]. It has been
shown that some in vitro-cultured Leptospira strains can survive in
commercially available blood culture media for up to 2 weeks
[13]. Girault et al. successfully isolated L. borgpetersenii and
L. interrogans from commercial aerobic blood culture bottles.
They transferred culture bottle samples that were Leptospira realtime polymerase chain reaction (PCR)-positive to EMJH medium
within 2.5 days after blood collection and obtained isolates in
one-third of the patients investigated [14].
Leptospires may be isolated by inoculating 0.5 ml CSF into
5 ml semi-solid culture medium on the first week of illness
[9]. Urine is the most suitable specimen for the isolation during
the leptospiruria phase (on the second week after onset). As the
viability of leptospires is limited in acidic urine, urine needs to be
inoculated into the culture medium within 2 h after voiding. Alternatively, the urine sample is neutralized or centrifuged to obtain
pellets, which are inoculated into the culture medium [9]. 5Fluorouracil and/or some antimicrobial agents may be used to
reduce the risk of contamination [9, 10].
3 NAATs
NAATs have demonstrated superior sensitivity in the acute phase of
leptospirosis to culture and serological diagnostics. Leptospiral
DNA has been amplified from blood, urine, CSF, aqueous
humor, and tissues. A number of conventional PCRs including
nested PCR and real-time PCR, as well as isothermal DNA amplification techniques, have been developed for the early diagnosis of
leptospirosis in the last three decades [15–19]. NAAT using realtime PCR has been evaluated in large populations in clinical settings
and becomes the standard for early diagnosis [20–22]. However,
the instruments used in real-time PCR are expensive and may be
unavailable in resource-limited settings. Isothermal DNA amplification techniques such as loop-mediated isothermal amplification
and recombinase polymerase amplification can amplify the target
DNA sequence without thermal cyclers and can be integrated into
“lab-on-a-chip,” which enables NAATs at the point of care
[23]. Extensive evaluations are needed for the utility of isothermal
DNA amplification techniques for leptospirosis diagnosis in clinical
settings [11, 24].
As with culture, blood samples are most suitable in the acute
phase for NAATs. However, in clinical settings, there are controversial reports: one study showed that serum is a better specimen
than whole blood [21], whereas another study demonstrated that
sensitivity is much higher for whole blood than for serum
Laboratory Diagnosis of Leptospirosis
279