Interlaboratory Agreement of Pulsed-Field Gel Electrophoresis Identification of Leptospira Serovars

Abstract

Abstract. Leptospirosis may be caused by 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: 800 strain patterns; SAMMC database: 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories.

First Report on Leptospira Species Isolated from Patients in Slovenia

“…According to NotI-RFLP, eight clinical isolates were assigned to the serogroups Grippotyphosa (FF 291/02, SS 8049/14, KB 185/02, VV 206/02, and PZ 9474/06) or Icterohaemorrhagiae (OD 8720/11, ČP 226/02, and SS 166/02) (Table 3, Figure 1). The serovars identified for our clinical isolates are comparable to those described in other studies, with minor variations likely due to different electrophoresis conditions [12][13][14]34].…”

“…The serovar of the reference strain Semaranga Patoc Patoc 1 could not be determined because this strain tends to self-degrade, making the DNA unsuitable for NotI-RFLP analysis. Most other studies focused on pathogenic Leptospira strains, and NotI-RFLP was not performed for saprophytic Leptospira strains [13,35], although Naigowit et al [36] described a characteristic NotI-RFLP of a Patoc 1 strain. The NotI-RFLP patterns for two of the reference strains (Tarassovi Tarassovi Mitis Johnson and Ballum Castellonis Castelon 3) were identical, suggesting identical serovars.…”

“…These strains both belong to the species L. borgpetersenii, but we expected them to have different NotI-RFLP patterns because they are reference strains from the European Leptospira reference laboratory (Figure 1). It is possible that the strains were mixed in the original cultures and that multiple passages resulted in one strain overgrowing the other, or that multiple reinoculations resulted in the strains mixing during laboratory work [13].…”

“…Gels were stained with ethidium bromide solution for 30 min and imaged using the Bio-Rad Gel Doc™ XR system (Bio-Rad Technologies, Hercules, CA, USA). The NotI-RFLP patterns of the isolated Leptospira strains were compared to the restriction patterns of our reference strain of a known serovar [11][12][13]. Fingerprint patterns were analyzed by pairwise comparisons using the Dice coefficient.…”