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.

To the Editor: Cholera is a major public health problem in countries where access to safe water and adequate sanitation cannot be guaranteed for all. Vibrio cholerae serogroups O1 and O139 are the causative agents of cholera (1). One of the most powerful virulence factors in this organism is cholera toxin encoded by the ctxAB gene, located on the CTX prophage. V. cholerae O1 is classified into 2 biotypes, classical and El Tor. The El Tor type of V. cholerae O1 is responsible for the ongoing seventh worldwide pandemic of cholera (2). The sequence of ctxB of a certain strain has been believed to correspond to its biotype; that is, a biotype classical strain has classical type ctxB, and a biotype El Tor strain has El Tor type ctxB. However, recent research studies suggest that novel types of V. cholerae O1, hybrid strains, and altered El Tor or El Tor variant strains (1,3) are emerging. Altered El Tor or El Tor variant strains are biotype El Tor but produce classical cholera toxin (3,4). Recent reports suggest that this type of V. cholerae O1 is spreading to many areas of the world (5). In December 2007–January 2008, a cholera outbreak occurred in Xekong Province in southeastern Laos, in the Mekong Basin. The first case of the outbreak was detected on December 23, 2007. The outbreak spread to 10 villages and lasted through January 2008. Specifically, in the Thateng District, 117 cases occurred and 2 deaths were reported. The sources of the outbreak were suspected to be regularly used water. In October 2007, 2 months before the outbreak, 3 sporadic cases of V. cholerae infection had been identified in Vientiane (the capital city) and Xaignabouri Province in north-central and northwestern Laos, respectively. The outbreak investigation in the Xekong Province identified no linkage between these sporadic cases and the outbreak cases. In this study, we analyzed 18 V. cholerae isolates obtained in 2007: 3 were from patients with sporadic cases, and 15 were from the Xekong outbreak (13 from patients and 2 from water samples). All the isolates were serotype O1 Ogawa and biotype El Tor, but their ctxB types were classical, according to the method previously described (6). This finding indicates that they were the type of altered El Tor. We used pulsed-field gel electrophoresis (PFGE) to investigate relationships between the isolates according to the PulseNet protocol (7). All 18 isolates from the sporadic cases and the outbreak in 2007 displayed profiles indistinguishable from each other (Figure). We also compared 2 additional V. cholerae O1 isolates, 1 from a patient in Vientiane in 1998 and another from a patient in Louangphabang in 2000 (Figure). The profiles of the isolates obtained in 1998 and 2000 clearly differed from those obtained in 2007. These results indicate that all isolates from sporadic and outbreak cases in 2007 were likely from the same source of contamination, although extensive epidemiologic investigation did not identify any common source. Figure Dendrogram for NotI-digested pulsed-field gel electrophoresis profiles of Vibrio cholerae isolates, Laos, December 2007–January 2008. Origin of each isolate is shown on the right. *Water sample. Nguyen et al. characterized the isolates from a cholera outbreak in Vietnam from late 2007 to early 2008 (8). Their report suggests that the isolates from the outbreaks in Vietnam and Laos shared the same elements of the CTX prophage. Our study suggests a common source for the strains of sporadic cases in Vientiane and Xaignabouri Province in October 2007 and those of the outbreak in Xekong Province in December 2007. Molecular typing suggests that a novel clone of V. cholerae O1 is being disseminated along the Mekong Basin. However, no epidemiologic association has been identified so far. Thus, a more extensive regionwide surveillance system is needed to identify and control V. cholerae infection in Laos and neighboring countries.

The detection rate of rotaviruses from diarrheal stools in Lao People's Democratic Republic (Lao PDR) was studied in the period from 1994 to 2003. Rotavirus antigen was detected using latex agglutination kit. The average detection rate was 2.4%, or 18 of 738 cases examined in total. Rotavirus was not detected from 175 cases examined in 1995, 1998, 2000 and 2003, but 8 of 85 cases (9.4%) examined in 1997 were positive for rotavirus. The detection rate was 6.0% in the age group younger than 2 years and 0.6% in the age group older than 2 years. These detection rates were markedly lower than those in neighboring countries such as Vietnam and Thailand.

Drug susceptibility of Neisseria gonorrhoeae in the past 3 years in Lao People's Democratic Republic (Lao PDR) was examined. The organisms were collected from the patients visiting Center for Laboratory and Epidemiology, Ministry of Health, Vientiane, Lao PDR. A total of 131 isolates of N. gonorrhoeae (45 in 2000. 27 in 2001, and 59 in 2002) were collected, and the drug susceptibilities of the isolates were examined by disc method using 5 anti-microbial agents (ciprofloxacin, ceftriaxone, penicillin, spectinomycin, and tetracycline). The susceptibility was expressed as sensitive (S), intermediate (M) and resistant (R) depending on the Zone Diameter Interpretive Chart attached to the disc preparation. As the results, almost all isolates were resistant to penicillin and tetracycline, but sensitive to ceftriaxone and spectinomycin. Most of the penicillin resistant isolates were regarded as penicillinase producing N. gonorrhoeae (PPNG) depending on the effect of clavulanic acid. N. gonorrhoeae is getting resistant to ciprofloxacin in the 3-year examination period. About 10% of the isolates were resistant to ciprofloxacin in 2000, but the rate of resistant strains went up to 70% in 2002. There were very few isolates showing intermediate susceptibility.

Drug susceptibility of Staphylococcus aureus has been examined in the past 9 years to monitor the appearance of drug resistant strains especially methicillin resistant S. aureus (MRSA). The pattern of drug susceptibility has been relatively constant, but S. aureus is gradually becoming resistant to ampicillin. One MRSA was isolated in 1996 from the nasal vestibulum of a non-infected patient at Mahosot Hospital but not isolated thereafter. In 2001, 2 strains of MRSA which belonged to different clones were first isolated from the infection foci of patients. One was isolated from Setthathirath Hospital, where many Japanese staff have been dispatched since 2000. Careful monitoring and epidemiological studies of MRSA are recommended.

By examining 99 Vibrio cholerae O1 isolates from cholera epidemics in a variety of areas in Laos in 1998, we found two unusual characteristics of the organisms. All except 4 isolates were moderately resistant to tetracycline without having plasmids, and susceptibilities to the other drugs were as expected. Eleven isolates showed the same level of susceptibility to polymyxin B as classical V. cholerae O1. With the exception of 4 tetracycline-susceptible strains, all the isolates were resistant to or poorly sensitive to the vibriostatic agent O/129 (2, 4-diamino-6, 7-di-iso-propyl pteridine phosphate). All isolates, including those susceptible to polymyxin B, produced El Tor hemolysin which was neutralized by anti-El Tor hemolysin and were resistant to cholera phage IV, indicating that they were of the El Tor biotype.

Staphylococcus aureus isolated in Laos (People's Democratic Republic of Lao =Lao PDR) was examined for the drug sensitivities and coagulase type. A total of 114 strains examined consisted of 32 isolated in 1995 and 47 isolated in 1996 from the infection focus, and 35 isolated from nasal mucosa of noninfected patients and nurses working in the wards. One strain with coagulase type IV was regarded as methicillin-resistant S. aureus (MRSA) depending on the minimum inhibitory concentration of the drugs as > 100, 100, 100, > 100, and 25 μg/ml of methicillin, ampicillin, tetracycline, erythromycin, and cefdinir, respectively. The mecA gene was detected in this strain but not in the others. This is the first report of MRSA from Lao PDR.

Thirty-four strains of Vibrio cholerae O1 serotype Ogawa isolated in Lao People's Democratic Republic in 1995 were examined for their biological behaviour and cholera toxin productivity. Biotyping indicated that the 34 strains belonged to the El Tor biotype. All strains were hemolysin positive and resistant to polymyxin B and cholera phage IV. However, 4 strains were negative for the hemolysin activity as determined by the test tube method, although they were positive for the antigenic activity. Prophage typing identified 31 strains of the Celebes type, including 3 cured Celebes type, and 3 Ubol type. All the strains produced detectable levels of cholera toxin; 15 isolates produced more than 100 ng/ml, 10 strains between 10 and 100 ng/ml and 9 strains less than 10 ng/ml, determined by using AKI medium in a stationary test tube. The maximum production was 200 ng/ml.

Etiologic bacteria of 135 cases with diarrheal diseases and the healthy carrier rate in 211 individuals were studied in People's Democratic Republic of Lao. Diarrheagenic Escherichia coli, Shigella and Campylobacter were the predominant pathogens isolated. This country does not face the sea, but Vibrio Parahaermolyticus was isolated from 2 cases. Healthy carrier rate of enteropathogenic E. coli as diagnosed by serogroup Class I was 3 times higher than the detection rate in diarrheal patients (10.9% and 3.7%). Healthy carrier rate of Shigella was 4.3%, whereas the detection rate of Shigella in diarrheal patients was 15.4%. The isolation frequencies of enterotoxigenic E. coli and Campylobacter jejuni in the patients and healthy individuals were almost the same. Verotoxin producing E. coli was found in one healthy individual (Toxin type VT1, O-antigen O111). Rotavirus was not detected from the 29 infant diarrhea examined, but detected from 2 adult cases out of 32 examined.