https://ojs.wpro.who.int/ 1WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887 Original Research B otulism is a life-threatening condition caused by botulinal neurotoxins (BoNTs). The typical symptoms are neurological, including blurred vision, slurred speech, difficulty swallowing and muscle weakness.1 The mortality rate may be up to 60% without adequate medical intervention.1 BoNTs are produced by Clostridium botulinum, a Gram-positive, rod- shaped, anaerobic, spore-forming, motile bacterium.2 Of the eight types of BoNT (A–H), A, B, E and F are associated with human botulism.1 C. botulinum spores grow and produce toxins in foods in an anaerobic, non- acidic environment with low sugar and salt. The spores are highly resistant to heat (several hours at 100 °C), desiccation, ultraviolet light and alcohol.1 C. botulinum is present in the environment, with type A or B spores being found primarily in terrestrial vegetables and type E commonly found in fish and aquatic products.3 Several outbreaks of botulism have been reported globally, due to consumption of a wide range of foods. In Egypt, a type E botulism outbreak was reported in 1991 in 91 patients, with 19 fatalities, related to consumption of a fermented grey mullet fish (faseikh).4 In Finland, an outbreak of BoNT type E in 1997 was linked to consump- tion of hot-smoked Canadian whitefish.5 Tinned bamboo shoots were found to be the cause of three outbreaks of botulism in Thailand, comprising nine cases in 1998,6 19 cases in 1997–19987 and up to 209 cases in 2006.8 In Taiwan (China), two outbreaks have been recorded, one caused by type A botulism in nine patients who con- sumed preserved peanuts in 19869 and another caused by type B botulism in five cases related to consumption of fermented food in 2006.10 In China, two type A BoNT outbreaks were caused by consumption of smoked ribs by two patients in 201311 and of vacuum-packaged salted fish and ham in four cases in 2021.12 Liquid herbal tea was found to be the main source of a type A botulism outbreak in two elderly people in the United States in 2017.13 No outbreaks of botulism had been reported in Viet Nam before the recent outbreak in the southern provinces in July 2020.14 An initial case series from this outbreak, which comprised the first six cases in a hospital in Ho Chi Minh City, linked cases to consumption of a tinned vegetarian pâté. In addition, the Institute of Public Health a Institute of Public Health, Ministry of Health, Ho Chi Minh City, Viet Nam. Published: 6 January 2022 doi: 10.5365/wpsar.2022.13.1.887 Objective: To investigate the cause of a botulism outbreak in several provinces in Viet Nam in 2020. Methods: An initial investigation was conducted to confirm the outbreak and to form hypotheses about the potential causes, followed by a case–control assessment of the plausible causative food item. Collected food samples were tested to identify the pathogen, and mouse bioassays were performed. Control measures were introduced to stop the outbreak and to prevent similar events in the future. Results: Twelve people in six southern provinces of Viet Nam were identified as having symptoms of botulism, of whom 11 were in critical condition requiring breathing support. A history of foods eaten in the 4 days before illness onset indicated that all the cases had eaten a tinned vegetarian pâté, and a case–control assessment showed that this was significantly associated with the outbreak, with an odds ratio of 35.2 (95% confidence interval: 3.4–∞). Clostridium botulinum type B was detected in three of eight pâté samples collected from the houses of cases. In the mouse bioassay for the toxicity of the pâté samples, all the mice died with clinical symptoms of botulism. Discussion: A tinned vegetarian pâté was the plausible cause of a botulism outbreak in Viet Nam in 2020. Revision of food safety regulations to improve quality control of tinned foods to prevent future outbreaks is recommended. An outbreak of type B botulism in southern Viet Nam, 2020 Tinh Huu Ho,a Ha Phan Ai Nguyen,a Nhan Dinh Trong Le,a Phuong Hoai Hoang,a Ninh Thi Haa and Chinh Van Danga Correspondence to Tinh Huu Ho (email: hohuutinh@iph.org.vn) WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887 https://ojs.wpro.who.int/2 Ho et alAn outbreak of type B botulism in southern Viet Nam, 2020 vision, during the same period who was not admitted to hospital. Age, sex, symptoms, date of onset and history of foods consumed during the 4 days before the date of onset were recorded for each patient. The incubation period was calculated as the time between consumption of the pâté and the onset of symp- toms. For patients who ate the pâté more than once dur- ing the 4 days before onset of symptoms, the incubation period was calculated as the time between the first and last consumption of the pâté and the onset of symptoms. Case–control assessment After the initial investigation, a case–control assessment was conducted to confirm epidemiologically that the implicated food item was the source of the outbreak. The cases were those from the initial investigation, and controls were defined as people who had shared at least one meal with a case in the 4 days before the onset of illness in that case, who showed none of the above symptoms of botulism intoxication. A structured questionnaire was used to interview the cases and controls, which included age, sex and history of food consumption (including names of foods and quantity ingested) within 4 days of the onset of any symptom. Controls were asked only about their exposure to the implicated food. Odds ratios (ORs) were calculated by exact logistic regression, and P values were calculated for exposure to the implicated food only with Fisher’s exact test. Laboratory investigation Vegetarian pâté samples from opened tins that had been consumed by patients within 4 days of symptom onset and samples from three unopened tins were col- lected from household members and sent to the IPH for examination. Patient specimens (faeces and stomach fluid) collected in hospital at admission were also ex- amined. Testing for C. botulinum and botulinal toxin was conducted according to standard methods.15 Briefly, 1 g of pâté sample was added to 15 mL of cooked meat broth in a tube (Becton Dickinson, Sparks, MD, USA). After 5 days of incubation at 35 °C, enrichment cultures were examined for turbidity, gas production, digestion (IPH) within the Ministry of Health, located in Ho Chi Minh City, received notification of several suspected cases from this and two other hospitals via a hotline. The aim of this study was therefore to investigate the source of the botulism outbreak by examining food consumption histories and pathogens from collected samples to provide information for preventing future foodborne outbreaks. METHODS The investigation was conducted in three stages: 1) an initial investigation to confirm the outbreak; 2) an epidemiological investigation to identify foods possibly implicated in the outbreak, including a case–control as- sessment of the implicated food product; and 3) a labora- tory investigation to identify the pathogen. Environmental studies and investigations at food facilities related to the outbreak were conducted by a different institution, and the results were not shared with our investigation team. Initial investigation At the end of July 2020, seven patients with severe neu- rological symptoms were transferred from four provincial hospitals to two central hospitals in Ho Chi Minh City. Ini- tial diagnosis of the six initial cases suggested a potential outbreak of botulism intoxication.14 A full investigation of all cases in the southern provinces was conducted by the rapid outbreak response team of the IPH. Although cases were also recorded in northern provinces, they were not included in this investigation because of limited resources. Details of the cases in all hospitals in the southern provinces were collected to confirm whether an outbreak of food poisoning had occurred. The team examined hos- pital case reports, consulted physicians who treated the patients and interviewed the patients or their caregivers. To avoid missing new cases, the IPH also notified all cen- tral hospitals in Ho Chi Minh City to report all suspected cases to IPH between 1 August and 30 September 2020. The case definition used for the outbreak was any individual admitted to any central hospital in Ho Chi Minh City between July and September 2020 who was diagnosed with botulism intoxication or any individual with three or more of the following symptoms: limb weak- ness, bilateral ptosis, dysphagia, difficulty breathing, dysarthria, descending paralysis and double and blurred WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887https://ojs.wpro.who.int/ 3 An outbreak of type B botulism in southern Viet Nam, 2020Ho et al onset. All but one case reported having eaten at least 1.5 spoonfuls. The ingredients of the pâté were mush- rooms (such as shiitake, wood ear, chicken drumstick, oatmeal and straw mushrooms), soya bean and nuts (cashew, almond and walnut). All other foods reported in the food histories were consumed by 17–42% of cases (Table 2). The incubation period ranged from 11 to 222 hours (median, 73 hours) (Fig. 1). Case–control assessment All 12 cases and nine controls were included in the case–control assessment of the implicated food prod- uct. As most of the cases were vegetarians, their meals were usually different from those of other household members; thus, it was difficult to find controls who had shared meals with the cases. The nine controls identified had all shared a meal with the cases in the 4 days before symptom onset and were relatives (chil- dren, spouses, parents or grandparents) or roommates (Table 3). Controls were not obtained for six cases who ate meals different from those of other household members. As the only food item consumed by all cases was a tinned vegetarian pâté, the case–control analysis in- cluded only the pâté. Only two controls reported having eaten the pâté, giving an OR of 35.2 (95% confidence interval [CI]: 3.4–∞). The OR for consuming more than one spoonful of pâté was 12.5 (95% CI: 0.84–∞); how- ever, this association was not statistically significant. Laboratory investigation Eight pâté samples, three from unopened tins and five from opened tins and consumed by the cases, and specimens from four patients were collected (Table 4). Three of five opened pâté samples were positive for BoNT type B C. botulinum by PCR test. None of the unopened tinned pâté samples was positive for BoNT. In the mouse bioassay, all mice died with clinical symptoms of botulism after exposure to the three pâté samples positive for C. botulinum. The four specimens from cases were all negative for C. botulinum. Six cases had consumed pâté from the three opened tins that were positive for C. botulinum: three in Dong Nai province, one in Ho Chi Minh City and two in Khanh Hoa province. of meat particles, odour and Gram stain. The enrich- ment culture was also inoculated onto anaerobic egg yolk agar (HiMedia, India) and incubated at 35 °C for 48 hours. A single pearly zone colony was selected and inoculated into trypticase peptone glucose yeast extract broth (HiMedia). After incubation for 5 days at 26 °C, the culture in broth was used for further detection of toxin in a mouse bioassay. The culture was diluted 1:5, 1:10 and 1:100 in gelatin phosphate buffer (HiMedia), and mice weighing 15–20 g were injected intraperitoneally with 0.5 mL of each dilution of test sample. All the mice were observed periodically for symptoms of botulism for 48 hours. Typically, signs of botulism in mice begin within the first 24 hours with ruffling of fur, followed in sequence by laboured breathing, weakness of limbs and total paralysis with gasping for breath, followed by death due to respiratory failure. C. botulinum isolates carrying botulinum neurotoxin A, B, E and F genes were identified in a polymerase chain reaction (PCR) assay, as reported previously.16 RESULTS Initial investigation Twelve cases of botulism were linked to the outbreak between 24 July and 15 September 2020, 11 of which were treated in intensive care units in three central hospitals; the other case was not admitted to hospital. The cases ranged in age from 20 to 64 years (median, 38 years). Eight of the 12 were female and reported eat- ing a vegetarian diet. The cases were from six southern provinces, with five cases from two families (two cases in Khanh Hoa province and three in Long An province), three cases among roommates in Dong Nai province and four unlinked cases from Vung Tau (n = 2), Ho Chi Minh City and Binh Duong (Table 1). The three most common symptoms were limb weakness (10/12), bilateral ptosis (9/12) and dysarthria (8/12). All 11 hospitalized patients required ventila- tor support. No deaths were reported (Table 1). The four most serious cases were treated with botulinum antitoxins provided by the World Health Organization; however, we were unable to evaluate the effectiveness of botulinum antitoxin treatment. All 12 cases reported having eaten the same brand of tinned vegetarian pâté in the 4 days before symptom WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887 https://ojs.wpro.who.int/4 Ho et alAn outbreak of type B botulism in southern Viet Nam, 2020 Table 1. Characteristics of cases and controls in the botulism outbreak investigation, southern Viet Nam, 2020 Characteristic Cases (n = 12) Controls (n = 9) n % n % Age (years), median (min–max) 38 (20–64) 27 (18–56) Sex Female 8 67 8 89 Male 4 33 1 11 Vegetarian diet 8 67 3 33 Home province Long An 3 25 0 0 Dong Nai 3 25 2 22 Khanh Hoa 2 17 0 0 Vung Tau 2 17 5 56 Ho Chi Minh City 1 8 2 22 Binh Duong 1 8 0 0 Symptoms Limb weakness 10 83 - - Bilateral ptosis 9 75 - - Dysarthria 8 67 - - Vomiting 6 50 - - Difficulty breathing 5 42 - - Hospitalized 11 92 - - Required ventilator 11 92 - - Relationship to case patients Roommate - - 2 22 Child - - 3 33 Spouse - - 2 22 Parent or grandparent - - 2 22 Fig. 1. Fig. 1. Epidemic curve and incubation period after consumption of the implicated food of 12 cases of botulism, southern Viet Nam, 2020 * Range for incubation period of cases that consumed the vegetarian pâté more than once during the 4 days before symptom onset. 0 1 2 3 4 9-Jul 11-Jul 13-Jul 15-Jul 17-Jul 19-Jul 21-Jul 23-Jul 25-Jul 27-Jul 29-Jul 31-Jul 2-Aug 4-Aug 6-Aug 8-Aug 10-Aug 12-Aug 14-Aug 16-Aug 18-Aug 20-Aug 22-Aug 24-Aug 26-Aug N o. o f b ot ul is m c as es Onset date 90 h 11 h 56 h 24 h 33 h 50 h 24–168 h* 12–222 h* 12–216 h* WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887https://ojs.wpro.who.int/ 5 An outbreak of type B botulism in southern Viet Nam, 2020Ho et al Table 2. Food consumption of 12 cases of botulism in the 4 days before onset of symptoms – southern Viet Nam, 2020 Table 3. Odds ratios (ORs) and 95% confidence intervals (CIs) for case and control consumption of vegetarian pâté – southern Viet Nam, 2020 The most plausible source of this outbreak was a tinned vegetarian pâté. All the cases reported having eaten the same brand of the pâté, and the case–con- trol assessment found that consumption of the pâté was significantly associated with the outbreak, with an OR of 35.2. The incubation period was estimated to be 11–222 hours (median, 73 hours), consistent with foodborne botulism, which can occur between 2 hours to 8 days after exposure (usually 12–72 hours).1 In the laboratory investigation, positive results for DISCUSSION This outbreak of botulism comprised 12 cases in six southern provinces of Viet Nam in 2020. No deaths were recorded, probably due to timely treatment of all patients and to administration of botulinum antitoxins provided by the World Health Organization to the four most serious cases. In addition, the strain identified was BoNT type B, which is less likely to be fatal than other types, such as BoNT types A and E.1 Food n % Vegetarian pâté 12 100 Bread 5 42 Steamed rice 5 42 Pickled vegetables 5 42 Vegetable soup 3 25 Steamed vegetables 3 25 Tofu 3 25 Sweet taro 3 25 Potato 3 25 Bamboo shoot 3 25 Noodle 2 17 Snail 2 17 Cumulative intake of vegetarian pâté (spoonfuls) ≤ 1 1 8 1.5 6 50 2 3 25 > 2 2 17 Pâté consumption Case (n = 12) Control (n = 9) OR (95% CI) Pâté eaten Yes 12 2 35.2 (3.4–∞) No 0 7 Cumulative amount of pâté eaten > 1 coffee spoon 11 0 12.5 (0.84–∞) ≤ 1 coffee spoon 1 2 WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887 https://ojs.wpro.who.int/6 Ho et alAn outbreak of type B botulism in southern Viet Nam, 2020 Table 4. Results of testing of food and patient samples – southern Viet Nam, 2020 C. botulinum spores were found per kg of soil and 2100 type B spores per kg of mushrooms.17 As C. botulinum is ubiquitous in the environment, viable C. botulinum or C. botulinum spores can occur in food. Botulism occurs only when C. botulinum in foods has enough nutritional requirements and anaerobic conditions for growth and production of toxins, such as in tinned foods. Although the optimum temperature for the growth and production of toxins is 35–40 °C, BoNTs can be produced at 3 °C.1,18 The normal temperature in a refrigerator compartment, which is usually set at 4.4 °C (40 °F) or less, may not be low enough to inhibit the growth of C. botulinum. The growth of C. botulinum in tinned foods can nevertheless be controlled by several methods, such as low pH, low water activity, high salt concentration and other food preservatives.16 The limitations of this outbreak investigation are that it was restricted to the southern provinces of Viet Nam and did not include cases linked to consumption of the vegetarian pâté in the northern provinces Lack of access to the results of the environmental investiga- tion also limited our study. The availability of reagent kits for detection of BoNTs has been a challenge for all laboratories in Viet Nam. In this study, we attempted to cultivate C. botulinum from samples and then used PCR to determine BoNT type, in addition to performing mouse bioassays. Another limitation of the response to this outbreak is that botulism is not covered in the national surveillance system, and no outbreaks of botulism had been seen previously. C. botulinum type B were found in three opened pâté tins. The finding that all four patient specimens were negative for C. botulinum may be explained by the fact that the adult gastrointestinal tract is not a natural habitat for C. botulinum. Detection of BoNTs in patient specimens was also hindered by lack of reagents for additional testing. After identification of the contaminated pâté as the cause of this outbreak, the information was widely spread through all media channels, and the population was advised not to eat the product; furthermore, all stocks of the product were recalled. The outbreak was successfully controlled, as no additional cases were identified at the national level. As the pâté implicated in this outbreak was distrib- uted nationally, cases occurred throughout the country. The ingredients of the pâté – mushrooms, soya beans and nuts – are common environments for C. botulism types A and B.1 A limitation of this investigation was the inability to determine at which step(s) of the pâté produc- tion process the contamination was introduced, as the environmental investigation was conducted by another team, and our investigation team could not inspect the canning company. Foods may be contaminated with C. botulinum at all steps of the production process, including cultivation, harvesting, processing and after processing. Contamination often occurs during growth in an environ- ment with a high incidence of spores.17 C. botulinum is common in soil and organic fertilizers, usually at low concentrations.1 In a survey in China, up to 25 000 Substrate Type of sample Culture PCR Toxicity testing in mice Vegetarian pâté 1 2 jars, 450 g (unopened) - N/A N/A Vegetarian pâté 2 1 jar, 200 g (unopened) - N/A N/A Vegetarian pâté 3 1 jar, 450 g (unopened) - N/A N/A Vegetarian pâté 4 1 jar, 200 g (used) + Type B Died, typical symptoms Vegetarian pâté 5 1 jar, 200 g (used) + Type B Died, typical symptoms Vegetarian pâté 6 1 jar (used) - N/A N/A Vegetarian pâté 7 1 jar (used) - N/A N/A Vegetarian pâté 8 1 jar (used) + Type B Died, typical symptoms Gastric fluid - N/A N/A Gastric fluid - N/A N/A Gastric fluid - N/A N/A Faeces - N/A N/A WPSAR Vol 13, No 1, 2022 | doi: 10.5365/wpsar.2022.13.1.887https://ojs.wpro.who.int/ 7 An outbreak of type B botulism in southern Viet Nam, 2020Ho et al 6. Wongwatcharapaiboon P, Thaikrea L, Ungchusak K, Wattanasri S, Sriprasert P, Nanthavas S, et al. Foodborne botulism associated with home-canned bamboo shoots – Thailand, 1998. MMWR Morb Mortal Wkly Rep. 1999;48(21):437–9. pmid:11075968 7. Swaddiwudhipong W, Wongwatcharapaiboon P. Foodborne botu- lism outbreaks following consumption of home-canned bamboo shoots in northern Thailand. J Med Assoc Thai. 2000;83(9):1021– 5. pmid:11075968 8. Kongsaengdao S, Samintarapanya K, Rusmeechan S, Wongsa A, Pothirat C, Permpikul C, et al. An outbreak of botulism in Thailand: clinical manifestations and management of severe respiratory fail- ure. Clin Infect Dis. 2006;43(10):1247–56. doi:10.1086/508176 pmid:17051488 9. Chou JH, Hwang PH, Malison MD. An outbreak of type A food- borne botulism in Taiwan due to commercially preserved peanuts. Int J Epidemiol. 1988;17(4):899–902. doi:10.1093/ije/17.4.899 pmid:3225100 10. Tseng CK, Tsai CH, Tseng CH, Tseng YC, Lee FY, Huang WS. An outbreak of foodborne botulism in Taiwan. Int J Hyg Environ Health. 2009;212(1):82–6. doi:10.1016/j.ijheh.2008.01.002 pmid:18396100 11. Feng L, Chen X, Liu S, Zhou Z, Yang R. Two-family outbreak of bot- ulism associated with the consumption of smoked ribs in Sichuan Province, China. Int J Infect Dis. 2015;30:74–7. doi:10.1016/j. ijid.2014.10.008 pmid:25448333 12. Min M, Bai L, Peng X, Guo L, Wan K, Qiu Z. An outbreak of botu- linum types A, B, and E associated with vacuum-packaged salted fish and ham. J Emerg Med. 2021;60(6):760–3. doi:10.1016/j. jemermed.2020.12.006 pmid:33518376 13. Kim M, Zahn M, Reporter R, Askar Z, Green N, Needham M, et al. Outbreak of foodborne botulism associated with prepackaged pouch- es of liquid herbal tea. Open Forum Infect Dis. 2019;6(2):ofz014. doi:10.1093/ofid/ofz014 pmid:30793007 14. Ngan NTT, Tho VNA, Khanh DTN, Hien VTT, Day JN, Sang NN, et al. Botulism outbreak after the consumption of vegetarian pâté in the south of Viet Nam. Wellcome Open Res. 2020;5:257. doi:10.12688.welcomeopenres.16372.1 15. 17.7.1 AOAC Official Method AOAC 977.26 Clostridium botuli- num and its toxins in foods. Microbiological method. Gaithersburg (MD): AOAC International; 2020. Available from: https://www. edgeanalytical.com/wp-content/uploads/Food_AOAC-977.26.pdf, accessed 1 October 2021. 16. Solomon HM, Lilly T Jr. BAM Chapter 17: Clostridium botulinum. In: Bacteriological analytical manual (BAM). Rockville (MD): Food and Drug Adminitstration; 2001. Available from: https://www.fda. gov/food/laboratory-methods-food/bam-chapter-17-clostridium- botulinum, accessed 1 October 2021. 17. Austin JW. Clostridium: Occurrence and detection of Clostridium botulinum and botulinum neurotoxin. In: Caballero B, Finglas PM, Toldrá F, editors. Encyclopedia of food and health. Oxford: Aca- demic Press; 2016:155–9. 18. Austin JW. Clostridium: Occurrence of Clostridium botulinum. In: Caballero B, editor. Encyclopedia of food sciences and nutrition, second edition. Oxford: Academic Press; 2003:1407–13. In conclusion, this outbreak highlights the risk of botulism from tinned foods, especially once they are opened. Strengthening of regulation of the production of processed foods and public education on food safety at home are recommended to prevent future foodborne out- breaks. Although botulism is rare, preparation of reagent kits for early detection of BoNTs and a standard response protocol to ensure prompt investigation and implementa- tion of control measures should be considered. Acknowledgements We would like to acknowledge Cho Ray Hospital, the National Hospital of Tropical Diseases and 115 People’s Hospitals for collaboration in this investigation. We also acknowledge our colleagues in the Department of Nutri- tion and Food Safety and the Southern Regional Testing Centre for Food Safety at the Institute of Public Health for their time and effort. Conflicts of interest None Funding None References 1. Harris A. Clostridium botulinum. In: Caballero B, Finglas PM, Toldrá F, editors. Encyclopedia of Food and Health. Oxford: Aca- demic Press; 2016:141–5. 2. Pirazzini M, Rossetto O, Eleopra R, Montecucco C. Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharma- col Rev. 2017;69(2):200–35. doi:10.1124/pr.116.012658 pmid:28356439 3. Glass K, Marshall K. Clostridium botulinum. In: Morris JG, Potter ME, editors. Foodborne infections and intoxications, fourth edition, chapter 27. San Diego (CA): Academic Press; 2013:371–87. 4. Weber JT, Hibbs RG Jr, Darwish A, Mishu B, Corwin AL, Rakha M, et al. A massive outbreak of type E botulism associated with traditional salted fish in Cairo. J Infect Dis. 1993;167(2):451–4. doi:10.1093/infdis/167.2.451 pmid:8421179 5. Korkeala H, Stengel G, Hyytiä E, Vogelsang B, Bohl A, Wihlman H, et al. Type E botulism associated with vacuum-pack- aged hot-smoked whitefish. Int J Food Microbiol. 1998;43(1–2):1– 5. doi:10.1016/s0168-1605(98)00080-4 pmid:9761332