The purpose of this study was to examine whether Leptospira spp. associated with culture-negative leptospirosis included a distinct subset at the species level compared with Leptospira spp. associated with culture-positive infection. The driver for this was to explain why the majority of patients with PCR-positive leptospirosis in our setting are culture-negative. Demonstration of a specific culture-negative sub-set would have opened the way to develop alternative culture media and potentially increase the sensitivity of culture, but we found no such differences.
There are several alternative explanations for PCR-positive but culture-negative leptospirosis. The patient may have received an effective antimicrobial drug but may not have cleared non-viable organism by the time that the blood sample was taken. We do not have information on antimicrobial consumption by our study patients prior to hospital admission, and this remains an important possibility. Leptospira spp. might also perish in the blood collection tube prior to laboratory culture due to fluctuations in ambient temperature or other factors, and are fastidious bacteria with highly defined growth requirements. Timing of the sample after the onset of symptoms is also important; we have reported previously for the same group of patients that those who were PCR-positive and culture-positive had a shorter duration of illness prior to sampling than those who were PCR-positive but culture-negative . Two patients were culture-positive but PCR-negative. A possible explanation is that this resulted from a stochastic effect associated with a sample containing a low bacterial concentration; i.e., by chance, the aliquot taken from the sample for culture contained organisms but the sample used for DNA extraction did not. An alternative explanation is laboratory error or contamination.
This study has several limitations. The patient group studied were those people who were sufficiently unwell to require hospitalization, and so the findings cannot be generalized to cases with milder clinical manifestations. Furthermore, the characteristics of Leptospira spp. and the study population in Thailand may be significantly different to that in other settings.
The genetic diversity of rrs described here has been noted previously during a study in which a 452-nucleotide region of the 16S ribosomal RNA gene (defined with reference to rrs2 of L. interrogans serovar Copenhageni strain Fiocruz L1-130) was used as one of six loci of a genotyping scheme devised by Ahmed et al. . This region maps to positions 103 to 554 of the rrs gene of L. interrogans serovar Lai strain 56601, and includes the 443-nucleotide rrs region used in our study (position 89 to 531). The number of alleles and polymorphic sites in 9 pathogenic and 5 intermediate Leptospira spp. were identical for the 443-nucleotide and 452-nucleotide regions. Ahmed et al. evaluated 120 strains belonging to 6 pathogenic species (L. interrogans, L. noguchii, L. kirschneri, L. santarosai, L. alexanderi, and L. borgpetersenii), which were separated by rrs2 into 29 different alleles containing 20 polymorphic sites. This suggests that alleles of Leptospira
rrs arise frequently and could occur through homologous recombination within or between closely related species rather than by mutation [16, 17].
Gene sequence analysis represents an important tool for the delineation of Leptospira species. The genes used previously in such analyses have included 16S rRNA (rrs) [18–29], DNA gyrase subunit B (gyrB) [26, 30–32] and RNA polymerase subunit B (rpoB) [33, 34]. An important question is what constitutes a new Leptospira species based on genetic differences at one or a small number of genetic loci. At one end of the spectrum, a Leptospira species may be clearly genetically distinct from other members of the genus based on a large degree of genetic diversity. Nucleotide distances between pathogenic and intermediate species evaluated in this study were more that 4% when compared using the nearly entire rrs gene (> 3% based on the 443-nucleotide rrs region). However, defining a species based on genetic differences at a small number of genetic loci is problematic. For example, two closely related Leptospira species (L. interrogans and L. kirschneri) have just 1 to 4 nucleotide differences when compared using the nearly entire gene, and 1 to 3 nucleotide differences based on the 443-nucleotide rrs region. Similarly, the sequence difference between the closely related L. inadai and L. broomii is 3 nucleotides based on the nearly entire gene and a single nucleotide based on the 443-nucleotide rrs region. This is equivalent to the number of differences observed within L. noguchii alone, which using our 443-nucleotide rrs region defined 5 polymorphic sites and 3 alleles. The unidentified species in this study (L498) was most closely related to L. borgpetersenii and had 3 nucleotide differences based on the 443-nucleotide rrs region. It remains to be seen whether this is a new species or a variant of an existing one. The lack of a live bacterial culture in this case precludes further characterization using additional tools such as DNA hybridization.