Use Of Molecular Typing Research Articles

Increased Incidence of Legionellosis after Improved Diagnostic Methods, New Zealand, 2000–2020

Legionellosis, notably Legionnaires' disease, is recognized globally and in New Zealand (Aotearoa) as a major cause of community-acquired pneumonia. We analyzed the temporal, geographic, and demographic epidemiology and microbiology of Legionnaires' disease in New Zealand by using notification and laboratory-based surveillance data for 2000‒2020. We used Poisson regression models to estimate incidence rate ratios and 95% CIs to compare demographic and organism trends over 2 time periods (2000-2009 and 2010-2020). The mean annual incidence rate increased from 1.6 cases/100,000 population for 2000-2009 to 3.9 cases/100,000 population for 2010-2020. This increase corresponded with a change in diagnostic testing from predominantly serology with some culture to almost entirely molecular methods using PCR. There was also a marked shift in the identified dominant causative organism, from Legionella pneumophila to L. longbeachae. Surveillance for legionellosis could be further enhanced by greater use of molecular typing of isolates.

Systematic Analysis of Molecular Subtypes Based on the Expression Profile of Immune-Related Genes in Pancreatic Cancer.

Immunotherapy has a good therapeutic effect and provides a new approach for cancer treatment. However, only limited studies have focused on the use of molecular typing to construct an immune characteristic index for gene expression in pancreatic adenocarcinoma (PAAD) and to assess the effectiveness of immunotherapy in patients with PAAD. Clinical follow-up data and gene expression profile of PAAD patients were retrieved from The Cancer Genome Atlas (TCGA) database. Based on 184 immune features, molecular subtypes of pancreatic cancer were found by the "ConsensusClusterPlus" package, and the association between clinical features and immune cell subtype distribution was analysed. In addition, the relationship between the proportion of immune subtypes and the expression of immune checkpoints was analysed. The CIBERSORT algorithm was introduced to evaluate the immune scores of different molecular subtypes. We used the tumor immune dysfunction and exclusion (TIDE) algorithm to assess the potential clinical effect of immunotherapy interventions on single-molecule subtypes. In addition, the oxidative stress index was constructed by linear discriminant analysis DNA (LDA), and weighted correlation network analysis was performed to identify the core module of the index and its characteristic genes. Expression of hub genes was verified by immunohistochemical analysis in an independent PAAD cohort. Pancreatic cancer is divided into three molecular subtypes (IS1, IS2, and IS3), with significant differences in prognosis between multiple cohorts. Expression of immune checkpoint-associated genes was significantly reduced in IS3 and higher in IS1 and IS2, suggesting that the three subgroups have different responsiveness to immunotherapy interventions. The results of the CIBERSORT analysis showed that IS1 exhibited the highest levels of immune infiltration, whereas the results of the TIDE analysis showed that the T-cell dysfunction score of IS1 was higher than that of IS2 and IS3. Furthermore, IS3 was found to be more sensitive to 5-FU and to have a higher immune signature index than IS1 and IS2. Based on WGCNA analysis, 10 potential gene markers were identified, and their expression at the protein level was verified by immunohistochemical analysis. Specific molecular expression patterns in pancreatic cancer can predict the efficacy of immunotherapy and influence the prognosis of patients.

Hepatitis A outbreak associated with a food handler in Bizkaia, 2017

IntroductionThe reporting of one case of hepatitis A in a food handler at a bakery and five cases in employees of a company after consuming products from the same bakery prompted an outbreak investigation. MethodsOutbreak cases were defined as individuals with laboratory-confirmed hepatitis A (HAV) infection, with symptoms which started in June and who, during the incubation period, worked with the food handler and/or had close contact with him and/or consumed products from the bakery. Epidemiologic questionnaires were performed and blood samples were obtained to be tested for the presence of anti-hepatitis A antibodies. Molecular characterisation was carried out by PCR, sequencing of the VP1/2A region and phylogenetic analysis with the maximum likelihood estimation method, bootstrap 1000 (MEGA 7.0 software). ResultsA total of 14 primary hepatitis A cases were identified: eleven cases related to the consumption of products from the bakery, two cases among co-workers of the food handler, and one case was a household contact. All 12 sequenced viruses were genotype IA, matching one of the strains (RIVM-HAV16-090) responsible for the outbreaks occurring at that time in Europe, mostly affecting men who have sex with men. ConclusionsHAV vaccination of at-risk groups should be reinforced in order to prevent future outbreaks. Increasing the use of molecular typing in hepatitis A cases could improve the investigation of outbreaks, which can be expected to increase in the future because of decreasing immunity in the population.

Assessment of the use and need for an integrated molecular surveillance of tuberculosis: an online survey in Germany

BackgroundThe implementation of an integrated molecular surveillance (IMS) of tuberculosis (TB) is of high priority for TB control. IMS is defined as the systematic inclusion of molecular typing results in the national TB surveillance system. Although not standardized, an IMS of TB is already implemented in several low TB incidence countries. Germany is in the process of implementing a nationwide IMS of TB. This requires close collaboration between national and local health authorities. We conducted an online survey to understand the current use of molecular typing results for TB surveillance among the local public health offices (PHO)s in Germany, and to collect their perception and expectations towards the implementation of a nationwide IMS of TB.MethodsThe online survey was developed using the software Voxco and included 31 questions. The survey was sent to all the 377 local PHOs in Germany in April 2017. Responses were collected until June 2017.ResultsA total of 174/377 (46.2%) local PHOs participated in our survey, and 88/377 (23.3%) used molecular typing results in their routine TB surveillance work. The PHOs used molecular typing results especially as support for epidemiological contact tracing (62/88, 70.4%). We found statistically significant differences between answers of PHOs that did not use molecular typing results (n = 86) vs. PHOs that did use molecular typing results (n = 88): the latter perceived the use of molecular typing results as more beneficial for their work compared to the former (65.9% vs. 34.9%, p < 0.05). Moreover, the PHOs using molecular typing results expect for the future more support and coordination from regional and national public health institutes, especially regarding the identification and analysis of molecular clusters.ConclusionsOur study is a step forward in the broader goal of implementing an IMS of TB in Germany. The local PHOs currently using the molecular typing results highlighted their positive attitude towards the implementation of an IMS, but also their needs of more support. Similar assessments might serve as an example for other countries which are on the way to implement a nationwide IMS of TB.

Brote de hepatitis A asociado a un manipulador de alimentos en Bizkaia, 2017

IntroductionThe reporting of one case of hepatitis A in a food handler at a bakery and five cases in employees of a company after consuming products from the same bakery prompted an outbreak investigation. MethodsOutbreak cases were defined as individuals with laboratory-confirmed hepatitis A (HAV) infection, with symptoms which started in June and who, during the incubation period, worked with the food handler and/or had close contact with him and/or consumed products from the bakery. Epidemiologic questionnaires were performed and blood samples were obtained to be tested for the presence of anti-hepatitis A antibodies. Molecular characterisation was carried out by PCR, sequencing of the VP1/2A region and phylogenetic analysis with the maximum likelihood estimation method, bootstrap 1000 (MEGA 7.0 software). ResultsA total of 14 primary hepatitis A cases were identified: eleven cases related to the consumption of products from the bakery, two cases among co-workers of the food handler, and one case was a household contact. All 12 sequenced viruses were genotype IA, matching one of the strains (RIVM-HAV16-090) responsible for the outbreaks occurring at that time in Europe, mostly affecting men who have sex with men. ConclusionsHAV vaccination of at-risk groups should be reinforced in order to prevent future outbreaks. Increasing the use of molecular typing in hepatitis A cases could improve the investigation of outbreaks, which can be expected to increase in the future because of decreasing immunity in the population.

Outbreak of Salmonella enteritidis PT14b gastroenteritis at a restaurant in England: the use of molecular typing to achieve a successful prosecution

ObjectivesTo describe an outbreak of Salmonella enteritidis phage type (PT) 14b in people who had eaten at a restaurant, and the investigation and subsequent prosecution of the food business operator (FBO). Study designThe local health protection team and environmental health department formed an outbreak control team to investigate the outbreak. MethodsEpidemiological, microbiological, and environmental investigations were undertaken. Epidemiological investigations involved case finding and interviews. Microbiological investigation: stool samples from the suspected cases and environmental samples from the implicated food business were investigated. Salmonella isolates obtained were subjected to multiple locus variable-number tandem repeat analysis (MLVA) profiling and whole genome sequencing. In addition, adenosine triphosphate (ATP) hygiene swab tests were used to verify the quality of cleaning procedures and data loggers were used to determine the water temperature of the mechanical dishwasher. ResultsFifteen cases of illness where the causative agent was shown to be S. enteritidis PT14b were identified, all of whom had eaten at the same restaurant. S. enteritidis PT14b was also identified from three of the 11 food and environmental samples taken at the restaurant and found to have the same MLVA profile as the cases. A case for prosecution was built and the FBO was successfully prosecuted in July 2015. ConclusionsThis investigation highlighted that the use of molecular typing as part of thorough epidemiological, microbiological, and environmental investigations can present a robust case for prosecution against restaurants which pose a risk to public health.

Use of molecular typing to investigate bacterial translocation from the intestinal tract of chlorpyrifos-exposed rats.

BackgroundHuman are confronted on a daily basis with contaminant pesticide residues in food, water and other components of the environment. Although the digestive system is the first organ to come into contact with food contaminants, very few data are available on the impact of low-dose pesticide exposure during the in utero and postnatal periods on intestinal bacterial translocation (BT). Previous studies have revealed that chlorpyrifos (CPF) exposure is associated with intestinal dysbiosis and the contamination of sterile organs. Here, molecular typing was used to investigate intestinal bacterial translocation in rats exposed to chlorpyrifos in utero and during lactation. The translocated bacteria were profiled, and CPF tolerance and antibiotic resistance traits were determined.MethodsA total of 72 intestinal segments and extra-intestinal organs were obtained from 14 CPF-exposed rats. The samples were cultured to isolate bacterial strains that had tolerated treatment with 1 or 5 mg CPF/kg bodyweight/day in vivo. Strains were identified using matrix-assisted laser desorption/ionization (MALDI) Biotyper. The disk diffusion method was used to determine the antibiotic susceptibility. The isolates were genotyped with PCR assays for the enterobacterial repetitive intergenic consensus sequence and random amplification polymorphic DNA.ResultsBacterial translocation was confirmed for 7 of the 31 strains (22.6 %) isolated from extra-intestinal sites. Overall, the most prevalent bacteria were Staphylococcus aureus (55.5 % of the 72 intestinal and extra-intestinal isolates), Enterococcus faecalis (27.7 %) and Bacillus cereus (9.8 %). 5 % of the S. aureus isolates displayed methicillin resistance. Seventy two strains were identified phenotypically, and seven translocated strains (mainly S. aureus) were identified by genotyping. Genotypically confirmed translocation was mainly observed found in pesticide-exposed groups (6 out of 7).ConclusionBT from the intestinal tract colonized normally sterile extra-intestinal organs in CPF-exposed rats. Our findings validate the use of molecular typing for the assessment of intestinal BT in CPF-exposed rats during critical periods of development.

Molecular Epidemiology of Brucella abortus in Northern Ireland—1991 to 2012

BackgroundBrucellosis is the most common bacterial zoonoses worldwide. Bovine brucellosis caused by Brucella abortus has far reaching animal health and economic impacts at both the local and national levels. Alongside traditional veterinary epidemiology, the use of molecular typing has recently been applied to inform on bacterial population structure and identify epidemiologically-linked cases of infection. Multi-locus variable number tandem repeat VNTR analysis (MLVA) was used to investigate the molecular epidemiology of a well-characterised Brucella abortus epidemic in Northern Ireland involving 387 herds between 1991 and 2012.ResultsMLVA identified 98 unique B. abortus genotypes from disclosing isolates in the 387 herds involved in the epidemic. Clustering algorithms revealed the relatedness of many of these genotypes. Combined with epidemiological information on chronology of infection and geographic location, these genotype data helped to identify 7 clonal complexes which underpinned the outbreak over the defined period. Hyper-variability of some VNTR loci both within herds and individual animals led to detection of multiple genotypes associated with single outbreaks. However with dense sampling, these genotypes could still be associated with specific clonal complexes thereby permitting inference of epidemiological links. MLVA- based epidemiological monitoring data were congruent with an independent classical veterinary epidemiology study carried out in the same territory.ConclusionsMLVA is a useful tool in ongoing disease surveillance of B. abortus outbreaks, especially when combined with accurate epidemiological information on disease tracings, geographical clustering of cases and chronology of infection.

Intraspecies classification of rhamnose-positive Yersinia pestis strains from natural plague foci of Mongolia

Comparative analysis of rhamnose-positive Y. pestis strains, which are traditionally recognized as the biovar altaica, was performed with the use of molecular typing. The strains were isolated from the natural plague foci of the Changay mountainous system and the Sailugem natural plague focus in Mongolia. The Russian part of the latter is known as the Altai Mountains focus (focus 36). All isolates were compared with the other rhamnose-positive vole strains from the natural plague foci located in the grassland of Xilingol (focus L, China), the plateau of Qinghai-Tibet (focus M, China), the Gissarian Ridge (focus 34, Tadjikistan and Uzbekistan), and the Talassian Ridge (focus 40, Kyrgyzstan). The strains studied formed a single cluster with two branches, of which the first branch included the talassica (0.PE4?), qinghaiensis (0.PE4ab), and xilingolensis (0.PE4cd) phylogenetic groups, while the second included the hissarica (0.PE9) and altaica (0.PE1) phylogenetic groups. The results of molecular typing suggest that several strains from the Qinghai group of the Mongolian foci belong to the qinghaiensis and xilingolensis biovars but not to the altaica biovar.

Use of Molecular Typing to Elucidate Mycobacterium bovis Transmission in South Korean Beef Cattle

Use of Molecular Typing to Elucidate Mycobacterium bovis Transmission in South Korean Beef Cattle

Mycobacterium bovis in California dairies: A case series of 2002–2013 outbreaks

From 2002 to 2013, bovine tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) has been detected on numerous dairies in California. In total, twelve herds had bTB detected and are included in the case series which describes these recent outbreaks and discusses potential pathways of introduction. Epidemiological investigations to determine the initial source of bTB in each herd included obtaining data on likely pathways of pathogen introduction. Pathways included purchasing cattle, use of heifer-raising operations, commingling of cattle at greater risk of exposure to infected cattle with cattle destined for California dairies, contact with infected wildlife, exposure to humans with bTB infections, community and neighboring herds and others. Epidemiologic and molecular typing data confirmed the source of infection in 3 herds and probable sources of infection in 2 herds. In the 7 remaining herds described in this case series an epidemiologic link to a source could not be determined and molecular typing results did not associate M. bovis isolates acquired from these herds with another specific U.S. herd or U.S.-born animal. Preventing new introductions of M. bovis onto California dairies will require rigorous epidemiologic investigation of all the potential pathways of introduction discussed here. The root cause(s) of bTB on California dairies is certainly multifactorial with complex interactions of herd management practices, importation of cattle at greater risk of exposure to infected cattle, and the potential of human M. bovis exposure. The extensive use of molecular typing has improved epidemiologists’ ability to narrow the scope of potential sources.

Epidemiology of Methicillin-Resistant Staphylococcus aureus in a University Medical Center Day Care Facility

Few data are available on methicillin-resistant Staphylococcus aureus (MRSA) colonization in day care. We performed a study in a medical university child care center to study the epidemiology of MRSA in this population. Survey. A child care center on the campus of a university medical center. One hundred four children who attended the child care center and 32 employees gave samples that were cultured for MRSA. Seventeen household members of the children and employee found to be colonized with MRSA also gave samples that were cultured. Parents and employees completed questionnaires about demographic characteristics, medical conditions and treatments, and possible exposure risks outside the child care center. In addition, 195 environmental samples were taken from sites at the childcare center. Isolates were analyzed for relatedness by use of molecular typing, and statistical analysis was performed. The prevalence of MRSA in the children was 6.7%. One employee (3.1%) was colonized with MRSA. Cultures of samples given by 6 of 17 (35.3%) family members of these children and the employee yielded MRSA. MRSA was recovered from 4 of 195 environmental samples. Molecular typing revealed that many of the MRSA isolates were indistinguishable, and 18 of the 21 isolates were community-associated MRSA. Multivariable analysis revealed that receipt of macrolide antibiotics (P = .002; odds ratio, 39.6 [95% confidence interval, 3.4-651.4]) and receipt of asthma medications (P = .024; odds ratio, 26.9 [95% confidence interval, 1.5-500.7]) were related to MRSA colonization. There was a low prevalence of MRSA colonization in children and employees in the child care center but a higher prevalence of colonization in their families. Molecular typing showed that transmission of MRSA likely occurred in the child care center. The use of macrolide antibiotics and asthma medications may increase the risk of MRSA colonization in this population.

MDR-TB AND HIV: THE PERFECT STORM?

MDR-TB AND HIV: THE PERFECT STORM?

Novel weak D types 31 and 32: adsorption‐elution–supported D antigen analysis and comparison to prevalent weak D types

Weak D types are thought to express rather quantitative than qualitative D antigen variants. Distinct type-specific phenotypes and weak D cases with anti-D alloimmunization, however, suggest a variable degree of D antigen alteration. Variant D types were investigated by use of molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen density determination. Two novel weak D types were discovered, termed weak D type 31 and 32 with single RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches, with antigen densities of approximately 130 and 50 D sites per red blood cell, respectively. Adsorption-elution technique-supported D epitope mapping of these two weak D types, the recently described weak D type 26, and of the most common Central European weak D types (weak D types 1, 2, 3, 4.0, and 4.1) demonstrated the expression of all tested D epitopes. In contrast, a distinct D epitope loss was detected in weak D type 15 and partial D control samples. All novel and prevalent weak D types expressed all tested D epitopes. Our results indicate that adsorption-elution techniques may be of advantage whenever D epitope loss is suspected in extremely weak D variants.

The use of molecular typing for epidemiological surveillance and investigation of endemic nosocomial infections

While molecular typing methods are widely used to help the epidemiologist in the investigation of outbreaks, their use for the investigation of endemic infections has been limited. For the investigation of a micro-epidemiological setting such as the understanding of endemic nosocomial infections, discriminant methods are needed, such as pulsed field gel electrophoresis (PFGE), or PCR related typing methods. However, for long-term surveillance, preference should be given to typing methods that give definite results. The identification of the source or reservoir is only possible with comprehensive screening of the environment and the endogenous flora of patients and staff, and therefore, requires significant resources. However, cross-contamination or the existence of a common exogenous source might be investigated by typing clinical isolates during non-epidemic periods. Cross-contamination is suspected when isolates from different patients belong to the same type and when an epidemiological relation can be established. Thus, molecular typing makes it possible to track the dissemination of specific clones, it may facilitate the breaking down of endemic transmission to the level of micro-epidemics. This is illustrated by the example of one investigation of Pseudomonas aeruginosa colonization/infection in patients hospitalized in intensive care units.

The use of molecular typing to evaluate the dissemination of antimicrobial resistance among Gram-negative rods in Brazilian hospitals.

Antimicrobial resistance has increased rapidly in Brazil and worldwide during the past few years, giving rise to a growing necessity for antimicrobial resistance surveillance programs. These programs have been instituted in order to monitor bacterial resistance in various regions, and to guide empirical antimicrobial therapy. We evaluated the use of molecular typing in multicenter surveillance programs. We also studied the dissemination modes of selected resistance profiles. Antimicrobial susceptibility to various antimicrobial agents was evaluated by the reference broth microdilution method. Bacterial isolates with selected susceptibility patterns were characterized by pulsed field-gel electrophoresis (PFGE). A total of 119 Gram-negative bacteria were molecularly typed, including 22 imipenem-resistant Pseudomonas aeruginosa, 26 ESBL-producing Escherichia coli, 27 cefoxitin-resistant-ESBL-producing Klebsiella pneumoniae, 33 Enterobacter spp., 8 Citrobacter spp., and 3 S. marcescens isolates resistant to ceftazidime. The isolates were from clinically apparent bacteremia of patients hospitalized in medical centers located in 13 cities of 11 Brazilian states. Our molecular typing results revealed a great genetic diversity among isolates of the same species. However, some major PFGE patterns were found in more than one isolate. All repeated PFGE patterns were detected in only 2 isolates, which were isolated within the same institutions or in different medical centers. We conclude that the ability to characterize organisms phenotypically and genotypically is a powerful epidemiologic tool and it provides unique information that is very important for multicenter surveillance programs.

Prospective use of molecular typing of Mycobacterium tuberculosis by use of restriction fragment-length polymorphism in a public tuberculosis-control program.

We performed a prospective, community-based evaluation of molecular typing of Mycobacterium tuberculosis isolates as a method for tuberculosis (TB) control. We performed restriction fragment-length polymorphism (RFLP) analysis of the insertion sequences IS6110 and pTBN12 for isolates recovered from 61 of 62 patients with culture-positive TB in St. Louis during 12 months. Twenty-four (39%) of the 61 patients were infected with an isolate with an RFLP pattern that was shared with >/=1 other isolate, and 11 (46%) also had epidemiologic links with patients in their cluster of cases. One case each of laboratory cross-contamination and occupational transmission were discovered. The patients in clusters were more likely to be younger, black, United States-born, to have substance abuse problems, and to live in poorer areas. A predictive algorithm for molecular identification of clusters had a sensitivity and a specificity of 75%. This study allowed the TB-control program in St. Louis to be redirected toward the affected subpopulations.

Bacterial and epidemiologic study of the resistance to oxyiminocephalosporins in Escherichia coli in a French hospital

OBJECTIVE: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital. METHODS: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of beta-lactam antibiotics, and characterization of the beta-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum beta-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis. RESULTS: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum beta-lactamase. For the other strains, a beta-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a beta-lactam agent was significantly associated with case patients. CONCLUSIONS: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and beta-lactam use in their treatment.

Use of molecular typing to investigate bacterial translocation from the intestinal tract in malnourished children with Gram-negative bacteremia.

OBJECTIVE: To investigate whether translocation of intestinal Enterobacteriaceae was a likely source of bacteremia in children of 2 years or less hospitalized for fever and moderate malnutrition. METHODS: Blood and fecal cultures were obtained from 198 such children admitted in the Princess Basma Hospital (Irbid, Jordan). Enterobacteriaceae isolated from blood cultures were compared with those present in fecal cultures by genomic typing using pulsed-field gel electrophoresis. The characteristics of children whose blood and fecal strain typing patterns were not distinguishable, i.e. with documented translocation, were compared to those of children without documented translocation. RESULTS: Bacterial translocation was documented in 12 of 19 children (63%) with Enterobacteriaceae bacteremia for whom paired blood and fecal strains were available for genomic typing, and was thus the major source of these bacteremias. The clinical or biological signs of malnutrition were not significantly more pronounced in the bacteremic children with documented translocation than in those without. CONCLUSIONS: We demonstrated that bacterial translocation from the intestinal tract was the most common source of Gram-negative bacteremia in the population studied.

Does Hospitalizing Patients with Tuberculosis Ensure Compliance with Drug Regimens?

Bangladesh, the strain has rapidly spread to many parts of the world. The spread of the India strain of V. cholerae 0139 to southern China was confirmed here by both serological and molecular means. This is not unexpected, given the increasing cross-border trading in southwestern China and poor hygienic conditions made worse by the influx of millions of workers from northern China every year. The India and the Bangladesh strains of V. cholerae 0139 were distinguished with use of molecular typing and biotyping methods. The Guangdong strain described herein was identical to the India strain with respect to biotype, serotype, antibiogram, enterotoxin production, and the hybridization pattern of the ct.A cholera toxin gene. Nevertheless, the cholera toxin gene pattern in V. cholerae is known to be an unstable trait. Ribotyping with a broad-spectrum rRNA probe revealed no difference so far in hybridization pattern among all the 0139 strains (unpublished data), despite the presence of different ctxA gene patterns. It is therefore possible that all of the 0139 strains belonged to the same clonal linage, with subsequent divergence in terms of cholera toxin gene arrangement. The presence of two copies of cholera toxin gene in the 0139 strains was also documented in this case. Ou results agree with those of previous investigators who identified tan em repeats of the cholera toxi gene in V. cholerae 0139. This characteristic probably accounts or the huge quantities of toxin produced by this organism that result in infections of the same severity as V. cholerae 01.