Study design and location
A rapid cross-sectional design was used to collect random biological samples of seawater, seafoods and stool from patient. The patient stool samples were collected 23 health facilities located at in the West Urban Region of Zanzibar and likewise seawater and seafood were collected in West Urban Region of Zanzibar at Kizingo handling site of fishermen.
Sample Collection Storage and Transportation
Hundred samples of marine water 250ml from sea show and from five kilometers from the seashorewas filtered using membrane filters with pore sizes of 0.5µm. One hundred samples of seafoods were collected at Kizingo handling site of fishermen in Zanzibar. Stool samples for this study were collected from 23 health facilities of West Urban region site in Zanzibar. The sample collection was performed from for a period of five months (October, 2019 – February, 2020). A total of 103 samples were randomly collected from diarrhea patients who volunteered to give 25g stool specimen, sterile plastic containers were used for collection of the samples, immediately after sample collection, each sample container was tightly closed, labelled and packed in a cool box at 4oC for transportation. All samples were transported to the to the microbiology department of the Pathology Laboratory at Mnazi Mmoja Hospital Zanzibar for further analysis using conventional microbiological and serology methods where they were stored in a refrigerator at 8o C prior to laboratory analysis.
Bacterial Culture, Isolation, and Identification
In the laboratory, the filters containing the seawater sample were then transferred on thiosulfate – citrate – bile – salt (TCBS) medium (Oxoid Humpshine UK) then were inoculated at 25oC for 24 hours. Presumptive Vibrio species triplicate count was expressed in colony forming units per milliliters (CFU/mL) of water yellow for V. cholerae colonies and green for V. parahaemolyticus colonies. V. vulnificus colonies appeared green on TCBS. The difference between V. vulnificus andV. parahaemolyticus was the formation of green colonies with a blue centre on TCBS by the later. TheV. alginolyticus that forms the yellow colonies was differentiated from V. cholerae by its ability to form large yellow mucoidal colonies on TCBS.
In addition, seafoods were cut in 5g small pieces aseptically and were shaken into Alkaline Peptone Water (APW) enrichment broth vigorously. The pieces are then removed aseptically with forceps then cultured onto TCBS medium and incubated under 37oC for 48 hours. Vibrio cultures were read after 24 and 48 hours then subjected to identification by colonial appearance. Gram staining, serology and biochemical tests were used to identify the bacteria causing diarrhoea. Ultimately, stool samples were cultured on TCBS agar inoculated in Alkaline Peptone Water at 37oC for 24 to 48 hours. The bacterial isolates were identified using oxidase test, Voges Proskauer test, and NaCl at 0% and 6%. For the oxidase test bacterial colonies were transferred with sterile glass rod to filter paper moistened with oxidase reagent. Rapid appearance of a dark purple color within few seconds was considered a positive reaction. The identified isolates were confirmed as described in the Centers for Infectious Disease Control Manual for cholera diagnosis (20).
Again, bacterial colonies were inoculated in 1% tryptone broth (Oxoid. Humpshine. UK) in the presence of 0 and 6% (wt/vol.) Sodium Chloride (NaCl) and incubated at 37oC for 18 – 24hours in a water bath shaker. This was the test to determine the requirement of Vibrios for Na+. Positive results were determined by examining turbidity. Voges Proskauer Testwas performed by inoculating vibrios in methyl-red and Voges Proskauer medium and incubated at 37oC for 48 hours.
The agglutination test was used for confirmation of isolates were tested with antisera - polyvalent, Inaba, Ogawa and 0139 (Mast Diagnosics, Merseyside, United Kingdom) according to manufacturer’s instructions. A loop - full colonies of 24 hours growth ofVibrio spp from heart infusion agar (HIA) was emulsified in small drop of normal saline on a clean glass slide and was mixed thoroughly. A drop of antiserum was then added and was mixed well to get a smooth homogenous mixture. The glass slide was tilted back and forth for observation of agglutination. For a positive reaction a strong clumping appeared within 1 minute.
Antimicrobial Susceptibility Testing
Kirby-Bauer disc diffusion method was used for antibiotic sensitivity tests. The 24hour growth culture, standardized to match the 0.5 McFarland standards were inoculated onto Mueller-Hinton agar plates using sterile cotton swabs. Using the same swab, the plates were rotated at 60 degrees and swabbing the surface of the plate. This procedure was repeated one or more times while avoiding hitting the sides of the petri-plate and creating aerosols. The inoculated plate was allowed to stand for 3 to 15 min before applying disks. The antimicrobial disks used were Ceftriaxone (CTX, 30 μg), Ciprofloxacin (CIP. 5 μg), Doxycycline (DOX, 30 μg), Chloramphenicol (C, 30 μg), Erythromycin (ERY, 20 μg), Ampicillin (AMP, 10 μg), Trimethoprim-sulfamethoxazole (SXT, 25 μg)/Co-trimoxazole (CO, 25 μg)), Tetracycline (TET, 20 μg ), Nalidixic acid (NA, 30 μg) and Azithromycin (AZM, 15 μg) which were placed on the agar plates. The plates were then incubated overnight at 35˚C for 18 hours. The drug with susceptible zone of inhibition appeared around the antibiotic disc. The size of the zone of inhibition is correlated to the level of antimicrobial activity. Zones of inhibitions of bacterial growth around each antibiotic disc were measured using a sharp-edged ruler based on the Standard Operating Procedure (SOP) adapted from Clinical and Laboratory Standards Institute (21) and results were reported as sensitive (S), intermediate (I) and resistance (R).
Data were analyzed using STATA version 14 (22). Descriptive statistics in frequencies and proportions were used to describe the magnitude of Vibrio cholerae, and other Vibrio species(spp) in seawater, seafood and stool from patient with diarrhoea. Correlations were used to determine the relationship between Vibrio species from different biological specimen. Patterns of proportions with similar frequencies from different specimen were used to determine the epidemiological linkages between Vibrio species from marine water, seafood and stool from patients with diarrhea.
Ethical approval was granted from the Zanzibar Medical Research Ethics Committee (Ref. No. ZAHREC/02/DEC/2018/6). Permission to conduct the study was sought from the respective health centre authorities. The information about the study was given in writings, and study representative explained the benefits, participation rights and freedom to withdraw from the study at any time. The consent was obtained from adult patients with diarrhoea aged above 18 years of age before collection of information. All patients involved in the study provided signed consents. The participants were assured of the confidentiality of the information and records of their samples. All participants were informed that information collected was not intended to be used for any other purpose except for research study.