, Ahmed Saif Seed Moharem2
, Khaled Abdul-Karim A Al-Moyed1
, Hassan Abdulwahab Al-Shamahy1,5
, Sami Ahmed Al-Haidari1,3
, CHOLERA IN SANA'A, YEMEN: CLINICAL FEATURES, RISK FACTORS AND ANTIBIOTIC SENSITIVITY OF VIBRIO CHOLERAE
Eman Mohmmed Ahmed Al-Mohanadi1, Ahmed Saif Seed Moharem2, Khaled Abdul-Karim A Al-Moyed1, Hassan Abdulwahab Al-Shamahy1,5, Sami Ahmed Al-Haidari1,3,
Ahmed Mohamed Al-Hadad4
1Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University, Republic of Yemen.
2Medical Microbiology department, Faculty of Medicine, Dhamar University, Dhamar city, Republic of Yemen.
3Diseases Control & Surveillance, NTDs, Ministry of Health and population, Al-Husabaa Street, MoPHP - Sana'a.
4Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Hadhramout University, Republic of Yemen.
5Medical Microbiology department, Faculty of Medicine, Genius University for Sciences & Technology, Dhamar city, Republic of Yemen.
Background: Cholera, caused by Vibrio cholerae O1 or O139 serotypes, is one of the most important healthcare associated infections (HAI) among patients in Yemen.
Aim: This study aimed to determine the risk factors associated with cholera outbreaks, clinical presentations, and antibiotic susceptibility of the V. cholerae strains isolated among inpatient in Diarrheal Treatment Centers (DTCs) in Sana’a City.
Methods: This is a matched case-control study carried out on 134 DTC inpatients (cases) aged from 2 to 85 years who had a mean age of 26.8 years; and 134 community healthy individuals (control), ranged in age from 2 to 85 years with a mean age of 27.1 years in Sana'a. The identified isolates were tested for antibiotics susceptibility using disc diffusion technique. Data were analyzed using Epi Info 7.2. Express the quantitative data as mean values, standard deviation (SD), when the data are normally distributed.
Results: Among the cases, females are more susceptible to cholera than males (62.7% vs. 37.3%), and there is an increase in the incidence of cholera with age as 32.8% of cases were in the ≥35-year age group. There were significant risk factors for cholera with unwashed fruits (OR=33, p<0.001), unwashed vegetables (OR=5.3, p=0.001), outside foods (OR=129, p<0.001), and leftovers un-cooled food (OR=2, p=0.04). Also, collapsed drinking water and sanitation system (OR=4.5, p<0.001), sewage running in street (OR=8.7, p<0.001), and contact with diarrhea persons in the home (OR=33.3, p<0.001) were factors associated with cholera, while being vaccinated with cholera vaccines (OR=0.11, p<0.001) and use of pipes for sewage disposal (OR=0.5, p=0.02) were factors protectively associated with cholera.
Conclusion: Cholera affects all age groups in Sana’a, with females and persons of the age group greater than 35 years mostly affected. The most common clinical presentations were watery diarrhea and abdominal pain. Consumption of unwashed fruits, vegetables, outside food, unrefrigerated food, use of breakdown sewage system, dilapidated sewage near the home, running of street sewage, and contact with diarrhea in the home were risk factors, while cholera vaccination was protective against it. Therefore there is need to ensure that proper hygiene and sanitation to prevent infection.
Keywords: antibiotics susceptibility, Case- control study, cholera, clinical manifestations, DTCs, epidemic, risk factors, Sana’a city, V. Cholerae.
INTRODUCTION
Vibrio cholerae is the causative agent of cholera, a Gram-negative, rod-shaped pathogen that can be transmitted between two different environments persisting in saltwater ponds and transmission from the human intestine, transmitted from environmental reservoirs to the human host through contamination of food or water. V. cholerae is hypersensitive to low gastric pH, and thus the infective dose of this bacterium is high at more than x108 organisms1. Those cells that escape stomach acid eventually colonize the intestine. The toxin co-regulated pilus (TCP) promotes in colonization by encouraging the formation of bacterial micro-colonies. V. cholerae produces cholera toxin (CT), which disturbs the ordinary ion transport of the intestinal epithelium, resulting in a large influx of water into the intestine resulting in devastating vomiting and diarrhea2. The transition between biofilm formation and movement during infection is a key component of the colonization of V. cholerae3. The main risk factor connected with cholera is restricted approach to clean drinking water and adequate sanitation. As a result, the specific situations in which people live in such suboptimal sanitary conditions were also considered relevant risk factors, such as severe overcrowding, urban slums with limited health infrastructure, and temporary refugee or IDP camps, whether humanitarian or environmental crises and civil unrest4. Even with current managements, it is expected that there are more than 3 million cholera cases as well as more than 100,000 deaths annually5.
In Yemen, there are new published reports indicating the increased prevalence of communicable and non-communicable diseases that are closely related to war, poverty and the collapse of health systems6-25. The most recent outbreak of cholera in Yemen began in October 2016626, and continues until April 201927. In February and March 2017, the outbreak appeared to be declining during the cold weather wave, but the number of cholera cases re-emerged in April 201728. As of October 2018, more than 1.2 million cases have been reported, and more than 2,500 people have died (58% of whom are children in Yemen)29. The first cases were mostly in the capital, Sana'a, and some occurred in Aden. By the end of October, cases were reported in the governorates of Aden, Al Hudaydah, Hajah, Ibb, Lahj and Taiz, and in late November also in Al Dhale’ and Amran28,29.
Antimicrobial treatment is optional for critically ill patients and hospitalized patients. It is especially recommended for patients who are severe or moderately dehydrated and continue to pass a large amount of stool during rehydration therapy. Antimicrobial drugs can shorten the period of diarrhea, decrease the volume of rehydration solutions requisite, and shorten the extent of Vibrio excretion. Data of the sensitivity of regional strains to antibiotics, if any, be supposed to be used to direct antimicrobial therapy, so antimicrobial susceptibility testing must be performed30.
The cholera outbreak in Yemen is the largest in the recent history of this disease. The scale of this outbreak can likely be explained by the overall breakdown of public services, including hygiene and sanitation, linked to the war in Yemen. However, evidence of the clinical features, risk factors and antibiotic sensitivity of isolates of V. cholerae is needed to guide an appropriate public health response. Therefore this study aimed to identify risk factors for cholera epidemic in inpatients in DTCs, clinical features and antibiotic susceptibility to V. cholerae isolated.
SUBJECTS, MATERIAL, AND METHODS
Study design: This a matched case-control study to determine the potential risk factors for cholera infection. Also conducted a laboratory analysis of stool samples to identify the bacterial isolates, and determine the antimicrobial susceptibility pattern of the V. Cholerae isolates.
Study area: This study was conducted in cholera treatment centers (DTCs) in Sana'a city. Sana’a is located on a plain of the same name, the Haql Sana’a, which is over 2,300 meters (7,500 ft) above sea level. The plain is roughly 50–60 km long north–south and about 25 km wide, east–west, in the area north of Sana’a, and somewhat narrower further south. To the east and west, the Sana’a plain is bordered by cliffs and mountains, with wadis coming down from them. Sana’a has a population of approximately 3,937,500, making it Yemen's largest city.
Study population: This consist of 134 cholera cases admitted in DTC and 134 community controls identified in Sana’a. The cases and controls were selected and recruited into the study employing the following definitions:
Case: A case was defined as any patient, aged five years and over admitted in DTC from April 2020 to June 2020 with clinical presentations such as vomiting, diarrhea and abdominal pain, and laboratory-confirmed with V. Cholerae infection.
Controls: A control was defined as any person living in Sana’a, age, gender and neighborhood matched with cases without cholera clinical presentations or V. Cholerae isolated.
Sample size determination: The estimated sample size of 134 case patients and 134 controls was calculated with the assumption of a projected incidence of potential risk factors of V. cholera infection of 25% among case patients and 5% among individual controls from an earlier survey by Sheiban et al.,25 in Yemen, with a confidence level of 99.9% and power of 90% using Epi Info 7.2.
Data collection method: Respondents demographic data (age, sex) clinical presentations (diarrhea, nausea, vomiting, abdominal pain etc.) and risk factors (food, water source, waste disposal system, vaccination status) were collected using a pre-designed structured questionnaire administered by the interviewer.
Laboratory testing: After swab samples were collected from the rectum or stool, the samples were immediately placed in Cary-Blair transfer medium as transport media. All samples were cultured in the Bacteriological Department of the National Center for Public Health Laboratories (NCPHL) Sana'a, using standard bacteriological methods30,31. In the laboratory, rectal swabs or stool samples were incubated in alkaline peptone water (APW) at 37 °C for 4 h. Rectal swabs or stool samples were inoculated, and further enriched with broth for 4 h by placing streaks on taurocholate-telluride-gelatin agar (TTGA). Colonies resembling V. Cholerae were agglutinated with antigens specific to V. Cholerae O1 and V. Cholerae O1392131.
Antimicrobial susceptibility testing: The isolated and identified V. Cholerae were tested for suscepti-bility toamoxicillin (AM) 10 mcg, tetracycline (TE 30 mcg), doxycycline (30 mcg), cefuroxime (CXM 30 mcg), and co-trimoxazole (SXT, 1, 25/23, 75 mcg), ciprofloxacin (CI 5 mcg), nalidixic acid (NA 30 mcg), minocycline (MN 30 mcg), furazolidone (FR 50 mcg), and erythromycin (ER 15 mcg) (Oxoid Ltd) using the disc diffusion method32.
Statistical analysis: By using Epi Info statistical program version 6 (CDC, Atlanta, USA) the analysis of data was performed. Expressing the quantitative data as mean values, standard deviation (SD), when the data was normally distributed. Expressing the qualitative data as percentages; Chi square test was used for comparison of two variables to determine the P value. Odd ratio (OR) was used with 99% confidence interval to determine risk factors of cholera, p value <0.05 was considered statistically significant.
Ethical consideration: From all participants, consents were taken and participants were informed that participation is voluntary and that they can reject this exclusive of stating any reason.
RESULTS
The cases were 37.3% males and 62.7 females. The ages of the cases ranged from 2 to 85 years with a mean age±SD equal to 26.8±16.3 years. Most of the cases were in the age groups 35 years (32.8%) followed by the age group 15-24 years (23.9%) and 5-14 years (22.3%), while only 6 cases (4.5%) were recorded in less than 5 years group (Table 1). Ages in the control group ranged from 2 to 85 years with a mean age±SD equal to 27.1±16.1 years.
Most of the controls were in the ≥35 years (35.8%) age-group followed by the 15-24-year-old (25.3%), 5-14-year (20.9%), and 4.5% under-5-year-old group (Table 1). Females at higher risk of contracting cholera than male (62.7% versus 37.3%), also an increase in cholera incidence with age in which 32.8% of the cases were in age group ≥35 years (Table 1). The most common symptoms among cholera cases were watery diarrhea (98.5%) and abdominal pain (81.3%), followed by nausea (55.2%), vomiting (53.7%) and headache (46.3%), while bloody diarrhea was very low (1.5%), and mucosal diarrhea was 0%; and the mortality rate was zero (Table 2). There were significant risk factors for developing cholera with unwashed fruits (OR=33, p<0.001), unwashed vegetables (OR=5.3, p=0.001), outdoor foods (OR=129 p<0.001), and un-cooled food residues (OR=2, p=0.04) (Table 3). When assessing drinking water as a risk factor for cholera; there was no association between all sources of water with cholera infection (Table 3). Collapse of the drinking and sanitation system, dilapidated sanitation near the home, street running for wastewater and contact with diarrhea at home were cholera risk factors (OR=4.5, p <0.001; OR=1.6, p=0.046; and OR=8.7, p <0.001; OR=33.3, p<0.001; respectively). Positive cholera vaccine was a preventive factor against cholera infection (OR=0.11 average protection, P <0.001) (Table3). V. cholera isolates were 100% resistant to nalidixic acid; amoxicillin; 95.5% erythromycin and 98% for tetracycline, while the rates of resistance were low with cefotaxime (2.2%) and furazolidone (3.8%) (Table 4). The higher incidence of cholera in older patients may be associated with exposure to risk factors associated with outdoor activities. Our result also contradicts the finding of Sack et al., children were more likely to be infected, and children aged two to four years had the highest rates of cholera36. Females were more likely to have cholera in our study as females had 62.7% of cholera cases versus 37.3% for males (Table 1). These findings differ from those reported in cholera pandemics in the past 20 years, in Africa, Iraq, India and Vietnam37-39 where the incidence of cholera is approximately equal in both sexes. In the current study, abdominal pain and watery diarrhea occurred significantly (81.2% and 98.5%; p < 0.01, respectively) (Table 2). However, other typical cholera symptoms like vomiting etc. were less frequent. In severe cases of cholera, cholera patients may experience lethargy and may have sunken eyes, dry mouth, cold or wet skin, or hands and feet. Kussmaul's breathing, a deep and exhausting breathing pattern can also be caused by acidosis from faecal bicarbonate loss and lactic acidosis associated with poor perfusion. Blood pressure declines as a result of dehydration, peripheral pulse is fast and strenuous, and urine production reduces eventually. Muscle cramps and weakness, altered consciousness, seizures, or even coma due to an electrolyte imbalance are frequent, particularly in particular in children36. But all of our cases did not develop these symptoms due to prompt, appropriate DTC treatment. The approximation of cholera prevalence is on the whole important to take effective control measures, comprising the provision of clean water, enhanced hygiene and sanitation, and presentation of cholera vaccines. Oral cholera vaccines have been found to be safe and effective40-42. In our study there was protection role for vaccination in which the vaccinated individual had OR equal to 0.1, p<0.01(Table 3). Nevertheless, representation studies have revealed that sanitation measures and water may afford an equally viable solution, particularly in the long term, because the immunization approved by vaccines declines over time43-45. Two inactivated types of cholera vaccines are presently offered: one have killed cholera whole cells (rBS-WC) and recombinant cholera toxin B subunit; and the other have only killed cholera whole cells (WC)46,47. Field trials confirmed that the two vaccines afforded >50% protection for 3 yrs47,48. In spite of this, the WC vaccine is cheaper, (US$1.85 per dose) in the public division, with a protective effectiveness of 66% all through the third year of follow-up, as described in a study from Kolkata, India49. Realistic data concerning incidence of cholera is at present unavailable in Yemen, which limits the validity of any cost effectiveness evaluation of a potential intervention programme. When the sources of drinking water versus V. Cholerae infection were considered, there was a highly significant increasing in the rate of V. Cholerae infection with sabil water (OR=4.1) (Table 3). Higher rate of cholera with this type of water might be related to faecal contamination of this source. There was a highly significant increasing in the rate of V. Cholerae infection with breakdown of potable and sanitation system (OR=4.5), dilapidate sanitation near the house (OR=1.6) and swage run in the street (OR=8.7) (table 3). These risks might be related to faecal contamination of drinking water from sanitation system. Madbah area (the center study) is a densely populated area and has one of the largest concentrations of slums in Sana'a city. Slum settlements often have unhygienic latrines, poor garbage management systems, and sewers that overflow into houses. In most cases, latrines are linked with sewerage lines and municipal water pipes are commonly exposed to sewerage lines which may lead to faecal contamination of the supply water source. The main objective of this study was to analyze trends in multiple antibiotic resistances among clinical strains of V. cholerae isolated in 2019-2020 in Sana'a city, Yemen. Even though the therapy for cholera is principally supportive, antimicrobial therapy can be useful in decreasing the volume of stools and length of illness50,51. While tetracycline has been the mainstay of therapy, erythromycin, furazolidone and co-trimox-azole are the other reported alternatives52,53. Multidrug resistant classical V. cholerae strains and simultaneous epidemic outbreaks of classical of V. cholerae has been reported in Yemen. Majority of the classical 01-0139 strains in this study was resistant to ampicillin and erythromycin and a similar trend of resistance was seen in Bangladesh and India54. All strains of V. cholerae in our study were sensitive to co-trimoxazole; since cholera is a non-invasive disease, drugs such as co-trimoxazole, which is not absorbed from the gastrointestinal tract, must be good choice for treatment of cholera in Yemen. Most strains isolated in our study were sensitive to tetracycline (98.5%) (Table 4). This result is on contrast with that of India, Latin America, Tanzania, Bangladesh and Zaire; in which high incidence of V. cholerae non-O1, non-O139 strains resistant to tetracycline were reported25,55-58. In the current study ciprofloxacin was 100% effective against isolated strains of V. Cholerae. Reservation about promotion of ciprofloxacin as a first line drug for the treatment of cholera in developing countries has been expressed59, since it is an important substitute drug for treatment of multidrug resistant enteric and other pathogens. Extensive use of this drug and empirical therapy for treating diarrheal infection might have promoted incidence of ciprofloxacin resistant V. cholerae, which has emerged for the first time in India during 1992 among V. cholerae non-O1 non-O139 and during 1995 among V. cholerae O1 and O139 strains60. Sundaram & Murthy61 reported that only 2±7% non-O1 isolates were multi drug resistant in Madras; an area endemic for cholera in south India, but none of the strains was resistant to nalidixic acid. In the current study, it was observed that all -O139 isolates exhibited resistance to nalidixic acid. It is amply clear that long-term surveillance programmers are essential to identify changes in the spectrum of microbial pathogens causing serious infection and to monitor trends in antimicrobial resistance patterns62-64. Limitations This research suffers from specific limitations ranging from common flaws (not all results and answers to all research questions were justified) due to the lack of previous studies to compare with it in Yemen even in cholera-endemic areas in recent times. There were methodological problems as the study time was short (3 months), and the molecular methods for typing V. cholerae isolates and for antibiotic sensitivity testing was not performed, so further studies in a larger sample size with the use of the molecular technique is recommended in further studies.
CONCLUSION
Cholera affects all age groups in Sana’a, with females and persons of the age group greater than 35 years mostly affected. The most common clinical presentations were watery diarrhea and abdominal pain. Consumption of unwashed fruits, vegetables, outside food, unrefrigerated food, use of breakdown sewage system, dilapidated sewage near the home, running of street sewage, and contact with diarrhea in the home were risk factors, while cholera vaccination was protective against it. So, there is the need to ensure that proper hygiene and sanitation to prevent infection.
ACKNOWLEDGEMENTS
The authors would like to thank the Bacteriological Department of the National Center for Public Health Laboratories (NCPHL) Sana'a, Yemen for the support as well as the Ministry of Health and Population, Sana'a for allowing working in DTC and providing laboratory materials for collection, isolation, identification and antibiotic sensitivity testing.
CONFLICT OF INTEREST
No conflict of interest associated with this work.
AUTHOR’S CONTRIBUTIONS
This research is part of a master's degree in the Department of Medical Microbiology, first author Eman Mohmmed Ahmed Al-Mohanadi, who conducted field work in hospitals, and who did laboratory work and other authors contributed to data analysis, drafting and review of the paper, and gave final approval to the research.
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