[22]. Although the sensitivity is low, leptospiral DNA was detected
in urine samples as early as in plasma collected from patients in the
acute phase [11]. As mentioned above, blood culture bottles could
be used for Leptospira detection, but special DNA extraction methods must be employed [25]. Leptospiral DNA detection in blood
culture bottles was conducted in a clinical setting but turned out to
be a limited utility [26]. DNA detection can be performed using
filter paper-dried serum samples stored at room temperature for up
to 30 days, the sensitivity of which was 78.6% compared with that
using fresh serum samples [27].
Some NAAT target genes are specific for pathogenic Leptospira
spp., such as lipL32. There is increasing evidence that the intermediate subclade of Leptospira spp. is responsible for human leptospirosis [28–32]. Therefore, pathogenic species-specific NAATs
potentially fail to diagnose leptospirosis caused by intermediate
Leptospira spp. In addition, new Leptospira spp. in the pathogenic
subclade have been identified from the environment, and a new
degenerate primer/probe set for real-time PCR detecting lipL32
has been described [33]. In contrast, NAATs targeting rrs detect all
subclades, pathogens, intermediates, and saprophytes, but it has
been reported that these primer (and probe) sets could detect
DNAs from other bacteria, especially when they are applied to
urine samples [34–36].
To improve the sensitivity of real-time PCR, RNA-based
[reverse transcription (RT)] PCR has been employed because
RNA transcripts are present in clinical samples much more than
DNA targets which usually present one copy in a bacterial cell
[37, 38]. A new real-time RT-PCR developed by Backstedt et al.
demonstrated significantly higher sensitivity than DNA-based realtime PCR using clinical samples [37].
Clinical manifestations of leptospirosis are similar to other
infectious diseases such as dengue fever, malaria, and scrub typhus
in the tropics, and rapid differential diagnosis could result in appropriate management and improve the outcome. Multiplex real-time
PCR assays, one of which detects Leptospira spp., dengue virus, and
Plasmodium spp., and the other detects Leptospira spp., chikungunya virus, and dengue virus, have been developed [39, 40]. Their
diagnostic accuracy should be evaluated.
4 Serological Diagnosis
As defined by the WHO, microscopic agglutination test (MAT)
using paired serum samples is the definitive serological diagnosis:
seroconversion or at least fourfold increase in titers must be
observed between acute and convalescent serum samples because
antibodies detected by MAT are present for months to years after
infection. WHO also defines 400 MAT titers in a single sample as
280
Nobuo Koizumi
in urine samples as early as in plasma collected from patients in the
acute phase [11]. As mentioned above, blood culture bottles could
be used for Leptospira detection, but special DNA extraction methods must be employed [25]. Leptospiral DNA detection in blood
culture bottles was conducted in a clinical setting but turned out to
be a limited utility [26]. DNA detection can be performed using
filter paper-dried serum samples stored at room temperature for up
to 30 days, the sensitivity of which was 78.6% compared with that
using fresh serum samples [27].
Some NAAT target genes are specific for pathogenic Leptospira
spp., such as lipL32. There is increasing evidence that the intermediate subclade of Leptospira spp. is responsible for human leptospirosis [28–32]. Therefore, pathogenic species-specific NAATs
potentially fail to diagnose leptospirosis caused by intermediate
Leptospira spp. In addition, new Leptospira spp. in the pathogenic
subclade have been identified from the environment, and a new
degenerate primer/probe set for real-time PCR detecting lipL32
has been described [33]. In contrast, NAATs targeting rrs detect all
subclades, pathogens, intermediates, and saprophytes, but it has
been reported that these primer (and probe) sets could detect
DNAs from other bacteria, especially when they are applied to
urine samples [34–36].
To improve the sensitivity of real-time PCR, RNA-based
[reverse transcription (RT)] PCR has been employed because
RNA transcripts are present in clinical samples much more than
DNA targets which usually present one copy in a bacterial cell
[37, 38]. A new real-time RT-PCR developed by Backstedt et al.
demonstrated significantly higher sensitivity than DNA-based realtime PCR using clinical samples [37].
Clinical manifestations of leptospirosis are similar to other
infectious diseases such as dengue fever, malaria, and scrub typhus
in the tropics, and rapid differential diagnosis could result in appropriate management and improve the outcome. Multiplex real-time
PCR assays, one of which detects Leptospira spp., dengue virus, and
Plasmodium spp., and the other detects Leptospira spp., chikungunya virus, and dengue virus, have been developed [39, 40]. Their
diagnostic accuracy should be evaluated.
4 Serological Diagnosis
As defined by the WHO, microscopic agglutination test (MAT)
using paired serum samples is the definitive serological diagnosis:
seroconversion or at least fourfold increase in titers must be
observed between acute and convalescent serum samples because
antibodies detected by MAT are present for months to years after
infection. WHO also defines 400 MAT titers in a single sample as
280
Nobuo Koizumi