To the Editor

In the article ‘Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996–2004 possessing the unique VSP-II region of El Tor variant’ Bhuiyan and colleagues [Reference Bhuiyan1], reported a Zambian variant of El Tor biotype harbouring El Tor-type rstR but producing classical ctxB. They also showed that these recent variants lack VC0493 and VC0498 in the VSP-II island. The authors characterized the ctxB gene by mismatch amplification mutation PCR assay (MAMA)-PCR analysis [Reference Morita2]. Although this method is useful for epidemiological surveillance, it is specific for the ctxB allele and misses polymorphisms outside the target position. Based on the classification of Olsvik et al. [Reference Olsvik3], the authors concluded that the Zambian V. cholerae O1 strains isolated in 1996–1997 were identical to the ctxB genotype 3 of El Tor reference strain N16961, while the strains from 2003–2004 that harboured ctxB genotype 1 corresponded to the classical biotype.

In 2010, Safa and co-workers [Reference Safa, Nair and Kong4] characterized nine ctxB genotypes and showed polymorphism throughout the ctxB coding sequence as well as in five additional positions, not previously considered by Olsvik et al. [Reference Olsvik3]. We analysed the ctxB sequences from Bhuiyan et al. [Reference Bhuiyan1] (GenBank: EU932878-EU932884) and found that the Zambian sequences represent three distinct ctxB alleles and not two as originally stated by the authors. According to Safa's characterization, EU932883 corresponds to genotype 1 and is identical to the classical type. The sequences EU932878-80 and EU932881-82, EU932884 are similar to genotype 6 with mutations at Thr⁶⁸Ile and Tyr³⁹His, respectively. We designated the new variants genotypes 10 and 11 (Table 1).

Table 1. Distribution and comparison of ctxB genotypes in V. cholerae strainsFootnote *

* Adapted from Safa et al. [Reference Safa, Nair and Kong4].

Bhuiyan's paper established a relationship between the ctxB genotypes and the two major clusters of the V. cholerae strains from Zambia. However, it was not possible to determine here the distribution of the ctxB genotypes 1, 10 and 11 among Zambian strains, due to lack of information, regarding the year of isolation and the strain identification of the sequences deposited at GenBank. The exception was the 237/02 strain (1996) (EU932878), classified as genotype 10 (an El Tor prototype according to the authors), belonging to cluster A. Concerning the 2003–2004 isolates, it is possible to state that they are unique due to the characteristics of their VSP-II region and to the new ctxB allele.

We agree with the authors on the implication of the heterogeneity in the B subunit of cholera toxin for vaccine development and diagnosis, as well as concerning the impact of this variation in cholera severity [Reference Ryan5]. The two new emerging ctxB genotypes from Zambia are labels that could be considered in the epidemiological surveillance of cholera epidemics.