Molecular Subtyping in Cholera Outbreak, Laos, 2010

A cholera outbreak in Laos in July 2010 involved 237 cases, including 4 deaths. Molecular subtyping indicated relatedness between the Vibrio cholerae isolates in this and in a 2007 outbreak, uncovering a clonal group of V. cholerae circulating in the Mekong basin. Our finding suggests the subtyping methods will affect this relatedness.

A cholera outbreak in Laos in July 2010 involved 237 cases, including 4 deaths. Molecular subtyping indicated relatedness between the Vibrio cholerae isolates in this and in a 2007 outbreak, uncovering a clonal group of V. cholerae circulating in the Mekong basin. Our fi nding suggests the subtyping methods will affect this relatedness. C holera is a major public health concern in countries where access to safe water and adequate sanitation cannot be guaranteed for all residents. Vibrio cholerae serogroups O1 and O139 are the causative agents of cholera (1). A major virulence factor is cholera toxin (Ctx) encoded by the ctxAB gene and located on the Ctx prophage. V. cholerae O1 is classifi ed into 2 biotypes, classical and El Tor. The El Tor biotype is responsible for the ongoing seventh pandemic of cholera (2). Since the early 1990s, the El Tor variant strains, which are biotypes of El Tor but carry the classical type of ctxB, have emerged and prevail in multiple regions where cholera is endemic (1,(3)(4)(5)(6).

The Study
In July 2010, a cholera outbreak began in Attapeu Province in southern Laos along the Cambodian border. Onset dates were July 5-September 16. The outbreak spread to 17 villages of the province and involved 237 persons, including 4 who died. To isolate the suspected V. cholera colonies, we screened specimens on thiosulfate citrate bile salt sucrose agar with or without enrichment in alkaline peptone water. Suspected colonies were examined by conventional biochemical tests and PCR amplifi cation of ctx (7,8). Of the 42 fecal specimens tested, 9 were culture positive. The isolates were toxigenic V. cholerae O1 serotype Ogawa with features of the El Tor variant, according to the ctxB-typing method of Morita et al. (9).
We analyzed the 9 V. cholerae isolates from the Attapeu outbreak. We performed pulsed-fi eld gel electrophoresis (PFGE) according to the PulseNet protocol (10) and multilocus variable number tandem repeat analysis (MLVA) using the 7 loci, as described (5,11).
The isolates of the Attapeu outbreak had almost indistinguishable PFGE profi les and MLVA repeat copy numbers. In PFGE analysis, 8 of the 9 isolates showed indistinguishable profi les (PFGE-A). The profi le of the remaining isolate differed from the dominant isolates by 2 bands (PFGE-B) ( Figure). In MLVA, 8 isolates showed the same MLVA type (MLVA-I), and 1 isolate showed another MLVA type that differed from the major MLVA type by being a single-locus variant of MLVA-I with only 1 locus and 1 repeat copy number (MLVA-II) (Table). Seven of the MLVA-I and 1 of the MLVA-II isolates showed the PFGE-A profi le, and 1 of the MLVA-I isolates showed the PFGE-B profi le. Although the source of contamination remains unknown, these results indicate that all isolates were indistinguishable from or similar to each other and that the outbreak could have been caused by a single source of contamination.
For comparison, we also examined 19 isolates from an outbreak that occurred in Xekong Province in 2007. These isolates also were toxigenic V. cholerae O1 serotype Ogawa of the El Tor variant (12). MLVA results clearly indicate that the isolates of the Attapeu outbreak in 2010 differed from those of the Xekong outbreak in 2007. The isolates from the Xekong outbreak comprised 3 MLVA types; 17 isolates were MLVA-III, 1 was MLVA-IV, and 1 was MLVA-V. MLVA-IV and MLVA-V were singlelocus variants of MLVA-III (Table). Of the 7 loci tested, 3 or 4 displayed different repeat copy numbers than did those of the Attapeu and Xekong outbreaks. In PFGE analysis, however, the profi les were similar to each other; the isolates from the Xekong outbreak showed a PFGE-B profi le (Figure).
These results suggest that strains with a specifi c PFGE type and the related strains have been circulating in the area for at least 3 years. Nguyen (15). Representatives of the Xekong and Attapeu isolates also were subjected to sequence analysis of ctxB. The results showed that their ctxB sequences were identical with that of the original classical type, which suggests that the clonal group in the Mekong basin differs from the new Orissa type of V. cholerae in India.

Conclusions
Our study clearly indicates that the 2010 cholera outbreak at Attapeu was caused by 1 source of contamination. Furthermore, isolates from the Attapeu outbreak and the 2007 Xekong outbreak showed similar PFGE profi les, but they were differentiated by MLVA, consistent with their origin. This study suggests that PFGE analysis is useful for identifying the kinds of V. cholerae clones circulating in a specifi c geographic region and might be useful for determining a long-term framework of the region-specifi c V. cholerae because PFGE profi les are probably more stable than the MLVA types. By contrast, MLVA is useful for investigating and discriminating shortterm individual outbreaks in a region. Another cholera outbreak in Cambodia in 2010 also might be related to the Attapeu outbreak. Combined use of both molecular subtyping methods would indicate the relatedness of cholera in the 2010 Cambodian outbreak and the others in the Mekong basin.