EPIDEMICITY OF VIBRIO CHOLERA IN SANA'A CITY, YEMEN: PREVALENCE AND POTENTIAL DETERMINANTS

In 2017, a total of 889854 suspected cholera cases with 2578 deaths were reported from Yemen, thus WHO considered these figures to be the worst epidemic of cholera in recent history of humanity. The aims of the study were to determine the prevalence of Vibrio cholera and protozoa causes in severe diarrhea patients and the potential risk factors of the contracting Vibrio cholera. Hospital-based diarrhoeal disease surveillance has been done for 12 days in Bany-alharth district of Sana'a city, where all patients admitted with severe diarrhoea in all health centers in the area were enrolled and tested for Vibrio cholerae, and others causes. The study was conducted on 345 patients and demographic, clinical, and potential risk factors were collected, then stool specimens were collected and processed by standard methods. The prevalence of V. cholerae was 8.1%, intestinal Entamoeba histolytica was 50.7%, and Giardia lamblia was 6.7% and one case of EPEC while 42% of diarrheal cases were undiagnosed. There was slightly increasing in the rate of V. cholerae infection with increasing age (15%). Also there were significant risk factors of dispose sewages to surround environment (OR=3.4 times, PV=0.02) and reused Jerry can bottles for drinking water (OR=3.1, PV= 0.03) with V.cholerae infection Vibrio cholera infection rate and intestinal protozoa infection rates were significantly high. The findings emphasize that there is cholera epidemic in Sana' city and diarrheal epidemic due to various diagnosed and non diagnosed pathogenic microorganisms which may predispose population of the study to significant health risks.


INTRODUCTION
Cholera occurs following infection of the intestine by the O1 or O139 sero-groups of the bacterium Vibrio cholerae 1,2,3,4 . About 20% of infected individuals develop acute, watery diarrhoea and 10-20% of these progress severe watery diarrhoea 5 . Even though casefatality rates have dropped due to oral and intravenous rehydration therapy, cholera can cause severe disease because of its rapid onset; residents in low-income locations as Yemen are at particularly high risk of infection in areas where public health systems cannot cope with outbreaks as in Yemen in which about 60% of public health system have been destroyed by the Saudi aggression on Yemen for 3 years and still continue 6 .
In 2017, a total of 889854 suspected cholera cases were reported from Yemen, including 2578 deaths, to the World Health Organization (WHO) 6,7 . So WHO considered these figures to be worst epidemic of cholera in recent history of humanity. As the fact that the WHO considered reported figures from endemic areas of cholera are underestimates, as poor surveillance systems and fear of negative impact on trade and tourism in many countries likely led to underreporting 7,8 . WHO estimates that officially reported cases represent only 5-10% of the actual number occurring worldwide annually 6 . Cholera is an endemic in Yemen 9 . In Yemen, cholera occurs year-round with seasonal peaks typically before and after rainy seasons with limited number of cases 9 . The true burden of cholera is unknown in Yemen due to the lack of a population-based surveillance system. The estimation of cholera prevalence is particularly important to take effective control measures, including the provision of clean water, improved hygiene and sanitation, and introduction of cholera vaccines. Oral cholera vaccines have been found to be safe and effective 10,11,12 . However, modeling studies have shown that water and sanitation measures may provide an equally viable solution, especially in the long term, since the immunization granted by vaccines wanes over time 13,14,15 . Two types of inactivated cholera vaccines are currently available: one containing recombinant cholera toxin B subunit and killed cholera whole cells (rBS-WC) and the other containing only killed cholera whole cells (WC) 16,17 . Field trials demonstrated that both vaccines provided >50% protection for 3 years 16,18 . However, the WC vaccine is cheaper, at US$1.85 per dose in the public sector, with a protective efficacy of 66% during the third year of follow-up, as reported in a recent study from Kolkata, India 19 . Credible data regarding incidence of cholera is currently unavailable in Yemen, which limits the validity of any costeffectiveness evaluation of a potential intervention programme. The aims of the study were to determine the prevalence of Vibrio cholera and protozoa causes among Yemeni patients suffering from severe diarrhea and the potential risk factors of the contracting Vibrio cholera.

Case definition
We defined severe diarrhoea as frequent loose or liquid stools for which a person had to be admitted to a healthcare facility, or had to receive intravenous rehydration, or had died as a result of the diarrhoeal illness.

Data collection
Data including demographic data of the patients, clinical information, and potential risk factors as water sources, food ingestion, sewage discarding, etc. time of disease, time of collection the specimen, etc. The findings were recorded in a form with laboratory results.

Laboratory testing
Following rectal swab or stool specimens collection, samples were immediately placed in Cary-Blair transport media. All samples were cultured in the Al-Thorah hospital microbiology laboratory using standard bacteriological methods 20

Ethical approval
The field team obtained written consent from the identified severe diarrhoeal cases or their guardians. Assent was taken from participants aged between 11 and 17 years. In the surveillance hospitals, consent was also obtained from patients with diarrhoea before collecting the stool specimen. The study protocol was reviewed and approved by the Ethics Committee of Sana'a University, Faculty of Medicine and Health Sciences.

RESULTS
The study includes 345 patients of sever diarrhoea in Sana'a city during a period of 12 days, starting in July 1 st 2017 and ending in July 12th 2017. The tested patients ages were ranged from 1 years to 65 years old, most of individuals were in age groups of <5 years (47.8%), followed by age group 5-10 years (43.1 %), while only 7.8% of the total were in age group 11-20 years and only 1.5% were in age group ≥ 21 years (  Table 2). There was slightly increasing in the rate of V. cholerae infection with increasing age, in which the highest rate occurred in age group 11-20 years old (15%), followed by 5-10 years old (9.4%), while the rates in age group < 5 years old was 6.2%, and in ≥ 21 years were zero% (  (Table 4). There was a highly significant increasing in the rate of V. cholerae infection with dispose sewages to the house surround environment (rate=21%, with OR= 3.4 times, CI=1.1 -10.9 times, PV=0.03). However, there was protective level of government sewage system against V. cholerae infections (Table 5).

DISCUSSION
This study provides data on prevalence and potential risk factors of cholera among severe diarrhoea in Sana'a city in Yemen which will be useful to inform decisions for effective control measures.
The study results show variability in rates at different age groups, in which there was slightly increasing in the rate of V. cholerae infection with increasing age (Table 4). Our study results is similar to that observed in Bangladesh that children cholera more frequently in older children compared to young children during diarrhoeal illness 4 . Higher rate of cholera in older patients might be related to those older children exposed to risk factors that related to out-door activities. 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 Jerry can bottles using (16.1%, with OR=3.1 times, CI=1.5-6.9, and PV=0.02) ( Table 4). Higher rate of cholera with Jerry can bottles using might be related to faecal contamination of drinking water sources or faecal contamination of the re-used jerry can bottles. There was a highly significant increasing in the rate of V. cholerae infection with dispose sewages to the house surround environment (rate=21%, with OR=3.4 times, CI=1.1 -10.9 times, PV=0.03) ( Table 5).This risk might be related to faecal contamination of drinking water. Bany Al-Harath distract 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. In this study, the prevalence of intestinal Entamoeba histolytica was 50.7%, in which 40.9% of them as single cause and 9.9% were suffering from co-infection with other micro-organisms, our study results were similar compared to previous studies done at Libya and others African countries in which intestinal Entamoeba histolytica was the most common cause of diarrhea among children 22,23 . High prevalence of intestinal Entamoeba histolytica is attributed by poor personal hygienic practices and poor environmental sanitation. Also E. histolytica and G. lamblia can directly transmit through food-handlers to consumers if ingested via contaminated food and water because cysts do not need environmental maturation 24,25 . Finally, for the confirmation of cholera cases, this study used a conventional culture method which remains the gold standard, but this procedure may yield false-negative results in case of inactivation of V. cholerae by in-vivo vibriolytic action of the phage and/or non-cultivability induced as a result of host response 26,27,28 . Rapid antigen-based diagnostic tests for cholera dipstick assays have identified 0-32% more cases than the conventional culture method in detecting V. cholerae antigens in stool samples 26,29,30,31 . By not accounting for culture-negative V. cholerae cases we are underestimating total cholera prevalence, but we did not adjust the prevalence calculations for culture negatives because we did not have molecular evidence from this population to estimate the magnitude of the correction.

CONCLUSION
Vibrio cholera infection rate, and intestinal protozoa infection rates were significantly high. The findings emphasize that there is cholera epidemic in Sana' city and diarrheal epidemic due to various diagnosed and non diagnosed pathogenic microorganisms which may predispose population of the study to significant health risks. Therefore, constant epidemiological surveillance and applying proper preventive measures through biannual routine parasitological tests and treatment of the infected cases along with the improvement of environmental sanitation are recommended.     Chi-square = > 3.9 (significant) PV Probability value = < 0.05 (significant) Chi-square = > 3.9 (significant) PV Probability value = < 0.05 (significant)