A REVIEW ON DIAGNOSTIC METHODS FOR THE IDENTIFICATION OF VIBRIO CHOLERAE ABSTRACT

The severe diarrheal disease Cholera, has gained public health importance because of its life-threatening effect.

The detection of the causative agent of this disease (Vibrio cholera (V. Cholerae) O1 or O139) from a specimen

(stool, vomitus of food sample) remains a major concern in the world today. Phenotypic finger printing (the

conventional methods) of the toxigenic V. cholerae strain, remains the gold standard for the laboratory diagnosis

of cholera; especially during cholera epidemic outbreaks. Detection around the remote areas which are usually

rampaged by these outbreaks is usually difficult due to lack of required diagnostic facilities in small laboratories.

However, the use of phenotypic approaches have some major setbacks as they are usually labor-intensive and

time consuming. This delays treatment commencement especially in life threatening cases. To alleviate these

setbacks, rapid molecular typing techniques involving the Polymerase chain reaction (PCR) amplification,

hybridization methods, Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Multiple-

Locus Variable Number Tandem Repeat Analysis (MVLA), Fluorescent Amplified Fragment Length Polymorphism

(FAFLP), Whole Genome Sequencing (WGS) etc. represent promising tools for early detection of the pathogen

V. cholerae O1/O139 even in remote areas where laboratory resources are poor. Immunoassay-based

techniques like enzyme-linked immune-sorbent assay (ELISA), coagglutination, immunofluorescence, and quartz

crystal microbalance (QCM), are capital/labour intensive and expensive for low resource settings. Rapid

diagnostic tests based on immune-chromatography principle have also been developed for simultaneous

detection of V. cholerae serogroups O1 and O139. These test kits are easy to use, transport, and fast. All these

methods enable the subtyping of unrelated bacterial strains and they all operate with different accuracies,

discriminatory ability, and reproducibility. This review sought to address some of the methods used in diagnosing

the disease cholera, with the objective of identifying the best and easiest of the methods that can help to curb the

cholera problem (deaths) often encountered, especially in low resource settings in the developing countries

(Nigeria inclusive).

Keywords: A, Review, Diagnostic; Methods; identification; Vibrio cholerae INTRODUCTION

Cholera is an important life-threatening diarrheal disease, transmitted through contaminated water and food

(especially contaminated seafoods) [1]. The causative agent of this disease is the toxigenic strain of Vibrio

cholerae. Clinical manifestations range from voluminous stool, hypovolemic shock, to acidosis [2]. Cholera

outbreak is still a serious problem in some parts of the world today. In a review on cholera epidemiology, Tarh,

stated that the annual global estimate of cholera deaths ranged from 28,000 to 150,000 and the burden of cases

between the range of 3 to 5 million yearly. This, according to the author, has led to low socio-economic status in

the population living within the affected regions especially in Asia and Africa [3]. In those areas of the world, WHO

indicated that in 2015 alone, 172,454 cholera cases and 1304 deaths were recorded; 41% of these were reported

in Africa, while 37% and 21% respectively originated from Asia and the Americas [4]. The outbreak that occurred

in 2017, affected 34 countries that reported a total of 1,227,391 cases and 5654 deaths. About 179,835 of these

cases and 3220 deaths came from 14 African countries [3]

It has been reportedly estimated that approximately 200 serogroups of V. cholerae are known today, two (O1 and

0139) of which are toxigenic and are responsible for epidemic cholera outbreaks [5, 6]. Based on the A, B, and C

antigens in lipopolysaccharide, the O1 is classified as serotypes, Inaba, Ogawa, and Hikojima. In addition to the

above classification, the O1 is further classified into biotypes Classic and El Tor based on some biological and

biochemical properties [7, 1]. The O395 strain, which has been extensively used for molecular analysis of

virulence factors, has also been observed to be a classical serotype O1 strain of the Ogawa biotype [8].

Detection and identification of this toxigenic V. cholerae strain especially during cholera epidemic outbreaks is of

utmost importance to aid in the control and spread of the cholera epidemic. The lack of required diagnostic

facilities in small laboratories, located around the remote areas which are usually rampaged by these outbreaks,

is a cause for concern as this makes the detection of the bacterium difficult [9].

However, the identification of V. cholerae using conventional biochemical tests methods, after isolation from

selective agar plates remains one of the commonest diagnostic method still practiced even in these localities [10].

Other methods which require the use of phenotypic approaches have some major setbacks as they are usually

labor-intensive, time-consuming and longer waiting periods for the results which delays treatment commencement

especially in life threatening cases [11, 12).

However, to alleviate these setbacks usually encountered by the conventional microbiological culture methods,

molecular methods involving the Polymerase chain reaction (PCR) amplification, hybridization methods etc.

represent promising tools for early detection of the pathogen V. cholerae O1/O139 even in remote areas where

laboratory resources are poor [10]. These methods are less time consuming (13), but the PCR amplification and

other immunoassay-based techniques like enzyme-linked immune-sorbent assay (ELISA), coagglutination,

immunofluorescence, and quartz crystal microbalance (QCM), require skilled professionals and is also capital

intensive and expensive for low resource settings [6] Rapid diagnostic tests based on immune-chromatography

principle have also been reported for simultaneous detection of V. cholerae serogroups O1 and O139 [14]. These

test kits are easy to use, transport, and fast. This review addresses some of the methods used in diagnosing the

disease cholera with the objective of identifying the major causative agent (toxigenic Vibrio cholerae) especially in

low resource settings in the developing countries (Nigeria inclusive).

FIG 1: A SCHEMATIC REPRESENTATION OF THE DIFFERENT PHENOTYPIC FINGER PRINTING

METHODS USED IN THE DIAGNOSIS OF VIBRIO CHOLERAE. Source:[2] PHENOTYPIC FINGER PRINTING (THE CONVENTIONAL METHODS) The culture, isolation and identification of Vibrio cholerae serogroup O1 or O139 from a specimen (stool, vomitus

of food sample) remains the gold standard for the laboratory diagnosis of cholera. [15]. This conventional

identification of V. cholerae is often made possible when the right steps are followed appropriately and the right

materials used. This involves pre-enrichment of the sample in alkaline peptone water (APW), followed by culture

on the Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS) culture medium, or Tellurite medium, then isolation and

confirmation using a series of biochemical and serological tests to determine strain serotype [16]

ENRICHMENT Culture of the sample directly on the selective medium for the isolation of Vibrio species, has been successful in

some occasions, but the primary inoculation in an enrichment medium has proven to enhance greatly the

selectivity, with a lesser number of organisms after culture on the solid selective medium [17] Alkaline peptone

water (APW) has stood the test of time as the enrichment medium of choice for the isolation of vibrio species.

Many different modifications of this APW for the enhancement of species selection, have been reported by

several authors e.g. alkaline peptone water with 1 to 2% NaCl for 22 hours for the isolation of V. alginolyticus[ 18],

enrichment in Salt teepol broth (3% NaCl and 0.2% Teepol in V/5 M phosphate buffer) for the isolation of Vibrio

parahaemolyticus in marine specimens [17] etc. Alkaline bile peptone water has been recommended for V.

cholerae, but has not been evaluated for other species [17]. The variations commonly observed stem from the

type and concentration (0.1 to 2%), of peptone, salt concentration (0-3%), pH (8-9.2%) and supplementation with

electrolytes. The Brain Heart Infusion (BHI) broth, Tryptic Soy broth, and Luria broth have also been used with

some degree of success [17] Approximately 25 g of pulverized sample (seafood or vegetables) is weighed into a

500 ml conical flask containing 225 ml of APW and thoroughly mixed or blended for 2 minutes at high speed. The

homogenate is then incubated at 35 ±2°C for 6 to 8 hours or for up to 18 to 21 h at 42 ±0.2 °C in a water bath for

analysis of raw oysters [19].

CULTURE MEDIA

Culture and isolation of V. cholerae from specimens can be done using Thiosulphate Citrate Bile Salt Sucrose

Agar (TCBS), polymyxin mannose tellurite (PMT), or sodium dodecylsulphate polymyxin sucrose agar (SPS), as

selective media [20, 21]. On TCBS, after enrichment in APW at 35±2ºC overnight, [17] Vibrio Cholerae colonies

appear as shiny yellow color colonies due to sucrose fermentation [12]. However, V. parahaemolyticus colonies

on TCBS appear blue to green and green or yellow for V. vulnificus [22]. On Chrom Agar TM

Vibrio, (CHROMagar;

Paris, France) Vibrios appear mauve, dark blue and light blue respectively by use of chromogenic technology,

which is more accurate than TCBS [11]. Cellobiose-Polymyxin B Colistin Agar and its modified formulas, have

also been reported to be better than TCBS agar for isolation of V. vulnificus and V.cholerae [17] However, this

culture technique is very time-consuming, labour-intensive, and delay the result which can only be obtained

several days later [6].

ISOLATION AND CHARACTERIZATION OF ISOLATES

Dried Plates of TCBS agar, modified-CPC or CC agars may be prepared and loopful of the surface pellicle from

the APW homogenate culture transferred and spread on the surface of the dried plates to yield discrete colonies.

The plates are incubated for 18 to 24 hours at 35°±2°C. Modified CPC and CC may be incubated at 39-40°C for

18 to 24 hours. Typical colonies of V. cholerae on TCBS agar are large (2 to 3 mm), smooth, yellow and slightly

flattened with opaque centers and translucent peripheries but colonies of V. cholerae on mCPC or CC agar are

small, smooth, opaque, and green to purple in color, with a purple background on extended incubation [19].

Discrete colonies from the spread plates are picked and sub-culture into freshly prepared agar by streaking

technique to obtain pure cultures of the isolates. The purified isolates are cultured on agar slants and kept as

stock cultures in bijou bottles for further biochemical test.

FIG 2: VIBRIO CHOLERAE GROWING ON THIOSULPHATE CITRATE BILE SALT SUCROSE (TCBS) AGAR

PLATE. Source: [23]

GRAM STAINING

The representative colonies of the test bacteria are carefully examined microscopically to ascertain the cell

morphology. The chemical and physical nature of the bacterial cell wall determines whether a bacterium is Gram-

positive or Gram negative Vibrio cholerae [24] being a gram-negative curved bacillus, its cell wall contain thin

layer of peptidoglycan which are easily decolorize [25] A Gram-negative cell wall loses the purple color of crystal

violet primary stain, and retains the pink or red color of the counterstain (Safranin).

PROCEDURE FOR GRAM STAIN VISUAL AMRITA LABORATORIES UNIVERSALIZING EDUCATION.

VALUE @AMRITA [26]

A sterile wire loop is used to pick a loopfull of distilled water and placed on a grease-free microscope slide. The

loop is sterilized and then used to pick a bacteria colony and place on the water on the slide and emulsified to

form a smear, which is then allowed to air-dry. It is then heat fixed by passing the slide over a Bunsen flame

thrice, flooded with crystal violet (primary stain) and left to stand for one minute before being gently rinsed with

running water. It is again flooded with iodine (mordant) and allowed for one minute, rinsed with gentle stream of

running water. The smear is decolorized with acetone alcohol for few seconds and rinsed with water, and Safranin

(counterstain) applied for 30 seconds. It is then washed blotted and examined under oil immersion (x100) and

result recorded.

MOTILITY TEST

The natural endowment of some bacterial organisms with locomotors (flagella), gives them the capability of being

motile. This characteristic of theirs is the basis for the differentiation of bacteria into motile and non-motile bacteria

using the motility test.

In performing this test, approximately 6ml of the sterile nutrient agar is dispensed into a sterile test tube to prepare

a slant. A loop full of bacterial growth from 24hours stock culture is transferred with a sterile wire loop and

inoculated and stabbed down the center of the tube to half the depth of the medium. This is then followed by

inoculating the loosely sealed tubes at 37oC for 24 hours. The presence of a diffuse or hazy growth after

incubation, is indicative of a positive result.

BIOCHEMICAL TESTS

The difficulties faced during the biochemical identification and characterization of V. cholera, stems from their

diversified natural characteristics [17]. However, there are still some peculiar features that can be used to

differentiate them (V. cholera) from members of the Enterobacteriaceae family. One of these characteristic

features is the production of the enzyme cytochrome oxidase, which is the basis for their positive oxidase test.

Moreso, V. cholerae produces acid from the fermentation of glucose, maltose, mannitol, sucrose, and trehalose

but with without gas; a characteristic which differentiates them from other Vibrio species. A great majority of V.

cholerae trains produce a mucoid string when the colonies from a non-selective medium are emulsified in a drop

of 0.5% sodium deoxycholate. They are motile at 37 °C, metabolize lysine and ornithine but not arginine.

OXIDASE TEST [27]

The oxidase test is used to detect bacteria that produce cytochrome oxidase enzyme of the bacterial electron

transport chain. This enzyme oxidizes the reagent (tetramethyl-p-phenylenediaminedihydrochloride) to

iodophenol, a purple end product. Approximately 0.1g of the reagent is dissolved in 10ml of distilled water and

used immediately or stored in the fridge pending when it is to be used.

During the performance of the test, bacterial colonies from a 24hour culture on any non-carbohydrate-containing

medium, (not from thiosulfate citrate bile salts sucrose (TCBS) agar or TSI slants), is picked with a sterile wooden

applicator stick and smeared on a filter paper on which approximately 2 to 3 drops of 1% tetramethyl-p-

phenylenediamine have been impregnated and allowed to stand for about five minutes. The use of nichrome wire

loop may give a false positive reaction. The result is recorded within 10 seconds as positive reaction, is indicated

by the appearance of dark purple coloration. Any purple colour development after the stipulated time, is

disregarded. The test is usually positive for organisms of the genera Vibrio, Neisseria, Campylobacter,

Aeromonas, Plesiomonas, and Pseudomonas [27]

.

STRING TEST [28]

The string test is most frequently used to distinguish most Vibrio cholerae strains (which are positive), from

Aeromonas strains (which are usually negative). Using a glass microscope slide or plastic petri dish, an 18-

24hour growth from nutrient agar or any other non-inhibitory medium is mixed in a drop of 0.5% aqueous solution

of sodium deoxycholate or 3% potassium hydroxide. This solution lyses the bacterial cell suspension to release

the cell DNA that forms a viscous suspension if the result is positive. This causes the bacterial cells to lose

turbidity and be seen as a mucoid “string” when an inoculating loop is used to pick the suspension slowly away

from the slide.

KLIGLER’S IRON AGAR OR TRIPLE SUGAR IRON AGAR [29]

KIA, contains glucose and lactose while triple sugar iron agar (TSI) contains sucrose in addition to glucose and

lactose. These two are used as screening media and the reaction of V. cholerae on them is similar to those of

non-lactose-fermenting Enterobacteriaceae. The test is basically used for the differentiation of Enterobacteriaceae

from other Gram-negative bacteria by their ability to breakdown glucose, sucrose and lactose with the production

of sulphide from sodium thiosulphate. The preparation and inoculation of these media is the same. In the test

procedure, approximately 59.4g of the TSI agar is weighed and dissolved in 1000ml of distilled water and heated

over a Bunsen flame to dissolve completely. About 6ml volume of the heated agar is dispensed into terile test

tubes. The test tubes are then sealed immediately and sterilized at 121oC for 15 minutes in an autoclave. After

sterilization, the tubes are kept in a slant position to solidify. A sterile wire loop is used to pick the test organism

from the stock culture slant and smear on the surface of the agar. The wire loop is again sterilized and used to

stab the butt gently. The culture is then loosely covered (to avoid anaerobic conditions which may distort the

results if the caps are too tight) and then incubated at 35˚ to 37˚C for 18 to 24 hours. Positive results are seen as

alkaline slants and acid butts (K/A), no gas, no H2S) for KIA and acid or alkaline (Rare) slants and acid butt (A/A),

no gas, and no H2S for TSI.

FIG 3a: KLIGLER’S IRON AGAR (KIA) TUBES FIG 3b: TRIPLE SUGAR IRON AGAR TEST RESULTS With several reaction patterns Image source: Clark College A: Acid/Acid, Gas; B: Acid/Acid, No gas; [31] C: Alkaline/Acid; D: Alkaline /Acid, H2S+;

E: Alkaline/Alkaline [30]

TSI results

1. Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose and sucrose non-fermenter 2. Alkaline slant/Alkaline butt (K/K) i.e Red/Red = glucose, lactose and sucrose non-fermenter 3. Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+ or -) 4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2S (+

or -)

CARBOHYDRATEOXIDATIONFERMENTATIONTESTS [29] Carbohydrate fermentation is indicated by the ability of microorganisms to produce gas, acid and energy in the

form of ATP as the end product of carbohydrate metabolism. During the test period, Durham’s tubes are inserted

in an upside down position in an appropriate carbohydrate liquid medium inoculated with the test organism. These

tubes become filled with the liquid medium, but when there is gas produced, it is trapped inside the Durham’s

tubes, and this displaces a volume of medium from inside the tube which is proportional to the volume of the gas

produced. It can also be detected by the presence of air bubbles.

Test Procedure: A 1.0g weight of the appropriate sugar (D-glucose (monohydrate) sucrose, lactose, D-mannitol,

mannose, arabinose, or cellobiose) is weighed and poured into test tubes containing 6ml of peptone water,

(glucose and sucrose broths can be used) and sterilized. A sterile wire-loop is used to pick the test organism from

the slant of fresh growth and inoculate into the test tubes containing the broth and sugars. Durham’s tubes are

then inserted upside down, into the test tubes, which are then sealed and incubated at 35˚ to 37oC for 24 hours.

After incubation, the tubes are observed for acid production, or acid and gas production. If fermentation tests are

negative at 24 hours, they can be incubated for up to 7 days. Acid production is indicated by a pink color when

Andrade indicator is used in the medium. V. cholerae ferments both glucose and sucrose but does not produce

gas in either of these carbohydrate.

VOGUES PROSKAUER TEST

The Voges-Proskauer (VP) test is used to determine whether an organism produces acetylmethylcarbinol from

glucose fermentation. If present, acetylmethylcarbbinol is converted to diacetyl in the presence of α-naphthol,

strong alkaline (40% KOH), and atmospheric oxygen to form a pinkish red polymer. The V. cholerae O1 (classical

strains) are negative to Vogues–Proskauer test [32].

Approximately 17g of glucose phosphate broth is weighed and dissolved in 1000ml of distilled water and heated

over a Bunsen flame to dissolve completely. A 6ml volume of the broth is dispensed into test tubes, sealed

immediately and sterilized at 121oC for 15minutes in an autoclave. After sterilization the broth is allowed to cool

before it is inoculated with fresh growth from a 24hour culture of the test organism from the slant. The tubes are

again sealed and incubated at 37oC for 48 hours. After the incubation, about 1ml of 40% KOH followed by a 3ml

of 5% solution of α-naphthol, (Barits’s Reagent A and B) are added and the tubes observed for a red surface layer

within 2-5 minutes.

A modification of the reagents may involve the use of MR-VP broth that incorporates 1% NaCl, 5% alpha-naphthol

in absolute ethanol (reagent A), and 0.3% creatine in 40% KOH (potassium hydroxide) (reagent B). A cherry red

color indicates a positive reaction.

SALT BROTHS

Tubes of Muller Hinton or Tryptic Soy Broth are prepared to contain NaCl concentrations of 0% 3%, 6%, 8%, and

10% (nutrient broth base). The tubes are then inoculated very lightly (light enough to prevent visible turbidity) with

fresh growth and incubated at 35˚ to 37˚C for 18 to 24 hours. If no growth occurs after the period of incubation,

they may be incubated for up to 7 days. Only profuse growth in the tubes is considered as positive. Halophilic

Vibrio spp. do not grow in broth containing 0% NaCl, but all Vibrio spp. grow in broth containing 3% NaCl. Various

species have different salt tolerances that can be used for identification [33].

GROWTH AT 42°C.

Sterile Muller Hinton broth containing 2% NaCl is inoculated with a 24 hour broth culture qrowth and incubated in

water bath at 42°C for 24 hours. Only profuse growth is considered as positive. V. cholerae, V. parahaemolyticus,

V. alginolyticus, and V. vulnificus grow at 42°C [33].

DECARBOXYLASE/DIHYDROLASE REACTIONS.

The tests for amino acid utilizing enzymes may be carried out by inoculating broths that contain the required

amino acids (Arginine, lysine, ornithine) in the presence of 1% NaCl, (including a control of the broth without

amino acid) with a standardized overnight growth culture of the organism to be tested. The broth culture is then

over laid with 4 to 5 mm of sterile mineral oil and incubated at 35˚ to 37˚C for 24 to 48 hours, and above (for up to

7 days) in case the test results remain negative after 48hours. In positive results, the broth cultures in the tubes

appear purple (Alkaline reaction), and negative results appear yellow (acid reaction) (both in the tests and control

tubes) when the indicators cresol red or bromcresol purple are used [34]. The tests can also be performed on

solid media like Lysine Iron Agar and Aginine Glucose slants, by streaking the surface (slant) and stabbing the

culture about 2/3 to the bottom of the tube (butt). Positive reactions are seen as Purple (Alkaline reactions) in the

slant and butt while negative reactions are observed as purple (alkaline) slants and Yellow (Acid) Butts. V.

cholerae shows positive reactions for lysine and ornithine decarboxylase, and negative reactions for arginine

dihydrolase. Other Vibrio spp. are negative for lysine decarboxylase and positive for arginine dihydrolase.

Aeromonas spp. are variable for lysine decarboxylase and positive for arginine dihydrolase [33].

SUSCEPTIBILITY TO VIBRIOSTATIC COMPOUND O/129

The reagent 2, 4-diamino-6,7-diisopropyl-pteridine phosphate (O/129 or vibriostatic compound) differentiates

Vibrio (sensitive to O/129) from Aeromonas (resistant to O/129). Some clinical and environmental isolates of V.

cholerae O1 and non-O1 have been reported to be resistant to 10 and 150μ of O/129 [34] giving same results as

Aeromonas. Sensitivity to this compound is performed by spreading a standard overnight Mueller-Hinton broth

culture of the test organism on the surface of Mueller-Hinton agar plate which is left on the bench to adsorb

inoculum. Disc containing 10 and 150μ of O/129 are then placed on the surface of the plate containing the

organism and inverted plates are incubated for 18-24 hours at 35-37°C. Results are seen as Zones of growth

inhibition around the discs [33]

SEROGROPING OF V. CHOLERAE

Grouping of Vibrio Cholerae using antisera is necessary for the fast identification and differentiation of O1 from

O139. However, this is not actually needed for clinical purposes though, it may be very necessary in cases of

outbreaks of cholera. The presence of the somatic antigen on the cell wall, is the basis for the more than 130

serogroups of Vibrio cholerae already identified [35, 36]. Most epidemic out breaks and pandemics have been

associated with the O1 strains that agglutinate the O1 antisera, but acute watery diarrheal diseases have also

been traced to the other non-O1 sero-groups that are common environmental species. Since these non-O1

groups are not usually causes of epidemics, they are at times not usually reported when identified during

epidemiological investigations. Serotyping using agglutination with antisera to type-specific O antigens, has also

been used to differentiate the serotypes, Inaba, Ogawa, and Hikojima of the O1 serogroup of V. cholera. Vibrio

cholerae sero-groups that cannot agglutinate the polyvalent O1 antisera are not tested for these three serotypes

mentioned above. The agglutination reactions for the Ogawa and Inaba strains are usually observed at the same

time, but the the stronger and faster of the reactions are recorded against the serotype; since cross reactions of

the serotype is possible and often occurs slowly [35,36]

SLIDE AGGLUTINATION

In the slide agglutination test, a confluent overnight growth culture of the presumptive V. cholerae colonies (the

test organism) is tested on the slide or plate, for the presence of V. cholerae somatic O antigens using V.

cholerae specific polyvalent or monovalent O1 and O139 antisera. The colonies of the test organism on

nonselective agar medium are picked using an inoculating needle a wire or loop to make a smear in a small drop

of physiological saline on the slide. The smear is carefully observed after mixing by tilting to and fro for about 30

seconds to dismiss the presence of auto-agglutination (due to “rough” strains which and cannot be serotyped). At

the formation of a smooth homogenous suspension, approximately equal volumes of antiserum and growth

suspension are mixed (the volume of suspension may be as much as double the volume of the antiserum). To

conserve antiserum, volumes as small as 10μl can be used. The mixture is then tilted to and fro to observe for

agglutination. The appearance of a visible and very strong clump within 30 seconds to 1 minute is indicative of a

positive reaction [36]

BIOTYPING OF V. CHOLERAE O1 STRAINS

The V. cholerae O1 El Tor biotype isolated predominantly all over the globe is sometimes differentiated from its

other counterpart, the classical biotype (is scarcely isolated in many areas of the world, Bangladesh exclusive)

using bio-typing methods. This is very important during primary outbreaks of cholera for follow up in the

identification of the origin of the infectious strain, especially during epidemiological studies.

HEMOLYSIS TESTING

Haemolysin production is one of the tools used to differentiate the V. cholera O1 classical from the El Tor

biotypes. The production of hemolysin HlyA by some Vibrio cholera strains, endows this strains with the ability to

hemolyze sheep red blood cells. This phenotypic characteristic is the basis for the bio-typing of the strains. The

hemolysin is also considered as one of the virulence factors produced by Vibrio cholerae [37]. An example is a

thermo-labile direct hemolysin from some strains of the Vibrio cholera El Tor that have been noted (a simple

protein of molecular weight 20,000 ca), is cytotoxic, cardiotoxic, and rapidly lethal mimicking the thermostable

direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial

hemolysins [36].

Strong hemolytic activity is shown more by the Gulf Coast and the Australia clones of V. cholerae O1, El Tor

unlike the classical and El Tor strains from the rest of the world, including Latin America, which are non-

haemolytic ([36].

PLATE AND TUBE HEMOLYSIS ASSAY [36].

The inoculation of blood agar plates containing 5% to 10% sheep blood with bacterial colonies from a 24 hours

growth, and incubation at 35˚ to 37˚C for 18 to 24 hours, shows hemolysis (clear zones) around the colonies of V.

cholerae O1. This is an indication that the red blood cells around those colonies have been totally lysed.

Nonhemolytic V. cholerae on this medium appear with greenish clearing around areas of heavy growth but not

around well-isolated colonies (“hemo-digestion,”), due to metabolic by-product accumulation. These byproducts

which are inhibited by anaerobic incubation of the blood agar plate can be avoided by determining hemolysis only

around isolated colonies, when aerobic growth conditions are used.

In the tube assay, two well-characterized strains of V. cholerae (one strongly hemolytic and the other

nonhemolytic) are used as controls in the test.

Approximately 20 ml of sheep erythrocytes are washed in 25 ml of phosphate-buffered saline (PBS), 0.01 M, pH

6.8-7.2 repeatedly three times. The washed calls are then suspended in Phosphate buffered saline to give a 1%

(vol/vol) suspension of packed sheep erythrocytes in PBS. An overnight growth of the test and control organism

are inoculated into heart infusion broth (or Trypticase soy broths) with 1% glycerol (pH 7.4) and incubated at 35˚

to 37˚C for 24 hours. These are then centrifuged and the supernatants decanted using a Pasteur pipette. The

supernatants are divided into two equal portions, one part is heated to 56˚C for 30 minutes while the other part is

left unheated. Serial twofold dilutions of both the heated and unheated supernatants are made in PBS (1:2-

1:1,024). Then 0.5 ml of the 1% suspension of sheep erythrocytes in PBS is added to 0.5 ml of each dilution of

supernatant and incubated in a water bath at 37˚C for 2 hours. The suspensions are then kept at 4˚C for 24hours

and examined. The highest dilutions with complete hemolysis are then recorded as the hemolysin titers Non-

hemolyzed red blood cells will settle to the bottom of the test tube and form a “button.”

The heated tubes will show no hemolysis, since the hemolysin of V. cholerae is heat-labile, and if present, is

inactivated by the 56˚C incubation. Titers of 2 to 8 are considered intermediate, and titers of 16 or above are

strongly positive.

FIG 4: HEMOLYTIC AND NON-HEMOLYTIC ACTIVITY OF VIBRIO CHOLARAE

Source: [37].

VOGES-PROSKAUER TEST

The Voges-Proskauer test as previously described differentiates between the El Tor which generally show

positive results and classical biotype of V. cholerae O1 which usually are negative [32]

POLYMYXIN B SENSITIVITY [39]

Sensitivity to the polymyxin B is used to differentiate the El Tor biotype of V. cholerae O1 which is usually

resistant to 50-unit polymyxin B disk concentration while the Classical strains are usually sensitive to 50-unit

polymyxin B and will give a zone of inhibition. The test is usually performed by picking four to five colonies of the

test bacteria from overnight growth on agar and inoculating into broth (Mueller-Hinton broth, heart infusion broth,

or tryptone soy broth). The inoculum is incubated at 35˚C for about 18-24hours, and then the turbidity adjusted

properly to about 0.5 McFarland standard density. A sterile cotton swab is used to adsorb the broth culture by

dipping it into the culture tube. The excess inoculum is removed by pressing the swab against the walls of the

tube. The bacterial adsorbed swab is then streaked all over the entire surface of the medium in a circular manner

turning the plate about 60 degrees after each streak as well as around the edge of the agar surface, for uniform

spread of the inoculum. The placement of a 50 unit disc of polymyxin-B is at the center on the surface of the plate.

Thereafter, the plate is inverted, incubated overnight at 35° ±2°C, and the results recorded. The Classical strains

usually show a sensitivity with a Zone diameter of >12 mm.

HEMAGGLUTINATION (DIRECT TEST) [35].

In the Hemagglutination test, the fresh chicken or sheep red blood cells are washed with Phosphate buffered

saline as in hemolysin determination described above. Then a 2.5% (vol/vol) suspension of washed centrifuged

red cells is made in normal saline after the final wash. Thereafter, loopful of the red cell suspension is placed on a

microscope glass slide and portion of the growth of the test bacteria from a nonselective agar slant is added to the

red cells using a wire loop, and mixed. If agglutination of the red cells occurs within 30 to 60 seconds, it indicates

that the test is positive (El Tor strain present). If there is a non-hemagglutinating reaction it is indicative of the

presence of the classical strain. Control strains are always included with every new suspension of red cells in

every batch of test performed. Strains of classical V. cholerae O1 that have aged in the laboratory or have

undergone repeated passage in broth may cause hemagglutination and should not be used as controls.

BACTERIOPHAGE SUSCEPTIBILITY [35].

The susceptibility of V. cholerae serogroup O1 to a specific bacteriophage can be used to differentiate the

Classical strains of V. cholerae O1 (sensitive to cholera bacteriophage “Classical IV”); from the El Tor isolates

which are susceptible to bacteriophage “El Tor 5.”

The use of bacteriophage in biotyping of V. cholerae O1 is briefly described as follows. The isolate to be tested is

grown overnight in pure culture on a non-inhibitory medium. From the overnight growth, brain heart infusion broth

(or Trypticase soy broth) is inoculated and incubated for 6 hours at 35˚ to 37˚C. A lawn of bacteria in log-phase

growth (OD=0.1) is then seeded onto the surface of a brain heart infusion agar plate by dipping a cotton swab into

the 6-hour broth and lightly inoculating (swabbing) the entire surface of the plate. Positive and negative control

strains should also be included. A drop of the bacteriophage diluted to routine test dilution (a measure of

concentration of active bacteriophage particles) is applied to the bacterial lawn. The plate is incubated overnight

and read the next day. If the bacteria are susceptible to the bacteriophage they will be lysed, and there will be a

zone of lysis in the bacterial lawn.

TABLE 1: LABORATORY IDENTIFICATION OF VIBRIO CHOLERAE

Oxidase 100 String test 100 Kligler’s iron agar K/A, no gas, no H2S Triple sugar iron agar A/A, no gas, no H2S Glucosea (acid production) 100

Glucose (gas production) 0 Sucrose (acid production) 100 Lysinea 99 Argininea 0 Ornithinea 99 Growth in O% NaClb 100 Growth in 1% NaClb 100 Voges-Proskauera 75c

a Modified by the addition of 1% NaCl. b Nutrient broth base (Difco Laboratories) c Most isolates of V. cholerae serotype O1 biotype El Tor are positive in the VP test, whereas biotype classical

strains are negative.

Source: [35]

TABLE 2: DIFFERENTIATION OF CLASSICAL EL TOR BIOTYPES OF V. CHOLERA

SEROGROUP O1

Reaction

Property Classical El Tor

Voges-Proskauer (modified with 1% NaCl) - +

Zone around polymyxin B (50 U) + -

Agglutination of chicken erythrocytes - +

Lysis by bacteriophage:

Classical IV

El Tor V.

+

-

-

+

Source: [35]

Table 3: CHARACTERISTICS OF VIBRIO CHOLERA

Classification Method

Epidemic-associated Not epidemic-associated

Serogroups O1 Non-O1 (>130 exist)

Biotypes Classical, El Tor Biotypes not applicable

to non-O1 strains

Serotypes Ogawa, Inaba, Hikojima These 3 serotypes

not applicable to non-O1 strains

Antigens(Major O Factor Present) A,B A,C A,B,C

Toxin Produce cholera toxin a Usually do not produce cholera toxin;

sometimes produces other toxins

a Nontoxigenic O1 strains exist, but are not epidemic-associated

Source: [35]

RAPID METHODS FOR IDENTIFICATION OF VIBRIO CHOLERAE

Cholera Rapid diagnostic tests (RDTs) are onsite diagnostic tests, compressed into kits which are placed in the

group of reduced risk for regulatory oversight; with sensitivity, ranging from 58 to 100% and specificity, ranging

from 60 and 100% depending on the test and its organization. Some few examples of RDTs include; the Crystal

VC Dipstick (Arkray Health care Pvt., India; previously Span Diagnostics, Surat, India), SD Bioline Cholera Ag

O1/O139 RDT (Standard Diagnostics Inc., Korea) and Artron Vibrio cholerae O139 and O1ComboTes

t(ArtronLaboratoriesInc.,Canada) [14].

When Should Cholera Rapid Diagnostic Tests be used?

Since these tests can be done on the spot, usually when a clinical cases of cholera suspected and pending on

when the laboratory confirmation would be possible (especially in peripheral health centers and primary health

care during surveillance purposes), there is often increased specificity of clinical diagnosis of cholera and

improved positive predictive value. Therefore; Cholera RDTs are useful in early outbreak detection to give primary

signals, monitor outbreaks and seasonal peaks in highly endemic, initiate response measures (e.g. inform

authorities, mobilize resources and material, etc.).

In areas with ongoing outbreaks, RDTs Positive specimens are chosen from suspected cases for culture or PCR

during epidemic outbreaks for identification, characterization and genotyping. This is important for epidemiological

purposes [40]

RDT negative results give a clue that cholera is not present in that area. However, these simple and reliable V.

cholerae diagnostic techniques help epidemiologists, clinicians and health care workers in putting control

measures in place before an outbreak [41].

A DESCRIPTION OF THE DIRECT AND ENRICHED DIPSTICK TEST KIT Immuno-chromatographic rapid diagnostic tests for the simultaneous detection of V. cholerae O1 and O139 have

been repoted by Lekshmi et al. [42]. These authors described the cholera RDTs as either lateral flow units or

dipsticks which principally work by detecting either cholera toxin (CT) or antigenic lipopolysaccharide (LPS) of V.

cholerae.

Recently a dipstick rapid test, based on detection of lipopolysaccharide (LPS) antigen was introduced for

detecting V. cholerae directly from fecal samples [43]. The principle of this rapid dipstick test is based on the

detection of the V. cholerae O1 and O139 lipopolysaccharide on the cell surface by monoclonal antibodies. It

involves use of one step, vertical-flow immune-chromatography and antibodies conjugated with colloidal gold

particles for detection of bound antigens [44]. Previous reports have suggested that the APW enrichment step

facilitates high specificity and sensitivity of RDT reducing the incidences of false cholera outbreak alerts [45].

Subsequently, the steps involved in bacteria sub-culture and bacteriological identification are replaced by the

enriched RDT for detection reducing cost and diagnosis time [46]. However, dipstick does not provide for

antimicrobial sensitivity profiling and strains subtyping. According to WHO, positive results of rapid immunoassays

should be confirmed by the classic laboratory procedures [47].

The Crystal VC test kit (16IC101-10, Span Diagnostics, Surat, India), is a rapid cholera diagnostic dipstick test

method, which uses monoclonal antibodies specific for the lipopolysaccharides (LPS) of V. cholerae serogroups

O1 and O139 in a vertical flow immune-chromatography dipstick. The LPS detection level of this test method is

about 10 ng/ml for VC O1 and 50 ng/ml for VC O139. The minimum detectable limit of the kit reported is 106

Colony Forming Units (CFU) of VC O1/ml and 107 CFU of VC O139/ml (for both sero- groups to yield a positive

test). Crystal VC has separate lines for serotype O1, O139 and a control line. It has a sensitivity of about 90% and

a specificity of about 60–70% when used directly on patients’ fecal specimens [48].

Two drops of watery stool are added into the buffer provided with the kit, mixed together and four drops of the

stool-buffer mixture is placed into a test tube into which the dipstick is then placed vertically. The dipstick then

imbibes the liquid which spreads upwards and react with the antigen if V. cholerae is present to form two clear

visible lines within five minutes (apparent within 15 minutes). But when V. cholerae is not present, a single

(control) line will appear within a few minutes. The test can also be performed on stool that has been placed in a

broth of alkaline peptone water (APW) and incubated for five to 8 hours at 20-40°C for enrichment [45]. This aids

in the dilution of any material present in the stool sample that could possibly cross-react to give a false positive

line on the dipstick. The culture on APW also allows the V. cholera; if present, to grow luxuriantly, thereby

amplifying the Lipopolysaccharide (LPS) antigen and increasing the specificity of the test ([45].

Detailed Test Procedure

A 5 ml test tube with patient identifier is labeled and set aside. The sample processing vial is then unscrewed and

the sampling stick used to stab the sample (not scoop to avoid picking up particulate matter that may clog the

dipstick membrane) into the sample processing vial if the stool sample is solid, semisolid, or viscous stool. When

the stool is liquid, the transfer pipette is used to add 2 drops of sample to the sample processing vial, which is

then tightly recapped and shaken to mix contents. The outer end of the cap from the sample processing vial is

then cut and 4 drops of processed sample put into 5 ml test tube already labeled above. Carefully, the aluminum

pouch along perforated line is then opened and if there is no observable damage on the dipstick, it is labeled at

the top with the patient identifier and then place in the test tube, the arrows facing DOWN, with the end of the

dipstick (“the dipping area”) submerged in the processed sample. The arrows remains above the level of the

sample while the test is left to stand for about 15-30 minutes before removing the dipstick to read the results [49].

TABLE 4: RESULTS INTERPRETATION

Interpretation*

Pinkish red band observed

O1 O139 Control

A. Vibrio cholerae O139 detected - + +

B. Vibrio cholerae O1 detected + - +

C. Vibrio cholerae O1 and O139 detected + + +

D. Vibrio cholerae O1 and O139 not detected - - +

E. Invalid results + or - + or - -

Source: [50]

FIG 5: CRYSTAL® VC RAPID DIAGNOSTIC TEST (RDT) [50].

FIG 6: SCHEMATIC REPRESENTATION OF THE RESULTS

*Control band must appear for the result to be

considered valid source: [50]. MATRIX-ASSISTED LASER DESORPTION IONIZATION–TIME OF FLIGHT MASS SPECTROMETRY (MALDI-TOF-MS) MALDI-TOF-MS is one of the effective identification tool used in clinically microbiology for the identification of V.

cholerae and its serotypes. An important setback here is the requirement for a pure growth of the organism that

will require culture for at least 18–24 hours. However, this technique has the advantage that large numbers of

isolates, mostly in a hospital setting can be screened.

An 18–24 hours growth of bacterial cells is prepared in a suitable matrix and applied to a metal plate and

irradiated by a pulsed laser which triggers ablation and desorption of the sample and matrix. This causes the

protein molecules from bacterial cells to be ionized by protonation or deprotonation in the hot grid. The ions are

separated based on their mass-to-charge ratio, measured in the time-of-flight detector followed by the generation

of peptide mass fingerprint profile.

USE OF BIOSENSORS

Assay method by biosensors, makes use of single-stranded fluorescein-labelled ctxA, (rapid nucleic acid lateral

flow), produced by asymmetric PCR at ambient temperature for the detection of toxin producing Vibrio cholerae

[51]. Following a hybridization process, these sensors render easy, the capture of biosensor immobilized probes.

In the presence of conjugated gold nanoparticles, the captured amplicons bind and cause the development of a

red line. This assay has a sensitivity and specificity of 100% with the detection limit of 0.3 ng of genomic DNA or

10 CFU/ml with cultures of V. cholerae. Comparatively, the nanocoaxial-based electrochemical sensor which

works on the principle of electrochemical ELISA and differential pulse or square wave voltammetry in the

detection of CT, has a sensitivity range from 10 ng to 1 mg/ml, which is comparable to that of standard optical

ELISA in the detection of V. cholerae [52].

TABLE 5: DIAGNOSTIC TECHNIQUES FOR RAPID DETECTION OF VIBRIO CHOLERAE O1/O139

RDT Kit Sensitivity (%) Specificity (%)

Direct Enrichment [95% CI] [95% CI] DFA-COLTA 100 100 Cholera Smart O1 100 95–100 Bengal DFA O139 89 100 Bengal screen O139 78–100 95–100 Medicos 88 [81.0–94.0] 80 [73.0–95.0] SMART 83 [75.0–90.0] 88 [82.0–93.0] IP cholera O1/O139 95 [91.0–99.0] 89 [86.0–93.0] Cholera screen O1 60–100 85–100] SD Bioline 91–95 [81.3–96.6] 95–100 89.0–98.4] Cholkit* 98 [88.4–99.9] 96 [88.6–99.6]

Diagnostic techniques for rapid detection of Vibrio cholerae O1/O139 [52] THE SENSITIVE MEMBRANE ANTIGEN RAPID TEST (SMART) The Sensitive Membrane Antigen Rapid Test (SMARTTM, New Horizons Diagnostics, Columbia, MD, USA) is a

colorimetric immunoassay which unlike the conventional culture methods, (which take several days to complete)

is designed to detect the O1 antigen of V. cholerae in whole stool samples directly and reliably within minutes on

site. The assay method is very simple (can be performed by even untrained personnel) and less cumbersome

with 95% sensitivity and 100% specificity for the detection of V. cholerae O1 in a small field trial [53]

The assay works under the principle that a V. cholerae 01 suspected specimen if reacted to a colloidal-gold-

labeled monoclonal antibody, COLTA (University of Maryland Biotechnology Institute and the V. cholerae 01

antigen is present in a specimen, the antigen forms a complexes with anti-V. cholerae 01 monoclonal antibody

which diffuses to be subsequently captured and concentrated by a polyclonal-antibody-coated solid-phase matrix.

This is then visualized as a pink-to-red test spot developing from the deposition of colloidal gold. If the V. cholerae

01, is absent in the stool sample, no pink-to-red color is formed in the test spot because no complex is formed

[54].

The Cholera SMART kit. The Cholera SMART kit consists of a reaction vial (which contains a lyophilized

colloidal-gold-labeled monoclonal antibody, COLTA (Maryland Biotechnology Institute)), and a SMART device

which comprises of two compartments; the upper compartment (contains a membrane bounded by an area that

holds the swab used to transfer the test specimen from the reaction vial) and the lower compartment (that

contains a test spot coated with anti-V. cholerae 01 polyclonal antibody and a negative control spot coated with a

normal rabbit immunoglobulin). Other components of the test kit include; the reconstitution buffer (0.01 M

phosphate-buffered saline in 3% Tween 20, pH 7.3), extraction buffer (0.05 M Tris, 0.05 M EDTA and 0.1 M NaCl,

pH 8.2), and a specimen-filtering device consisting of a squeezable 2-ml plastic tube with a snap-on filter (1-,um

pore size) (that allows bacterial cells to pass through while retaining any interfering materials from the sample),

and swabs (100% Dacron) for reagent or sample transfer.

Procedure

During the process, 2 drops of reconstitution buffer are added first into the reaction vial, followed by 4 drops of a

watery stool specimen, released through the filtering device, into the reaction vial. For formed or semi formed

stool samples, the extraction buffer is used to liquefy it in the filtering device before 4 drops are released into the

reaction vial. The mixture in the reaction vial is imbibed with a sterile swab and placed in the upper compartment

of the SMART device which is then closed. The mixture from the swab then diffuses through the membrane onto

both the test and negative control spots in the lower compartment of the kit.

The antigen-antibody complex formed with colloidal gold is captured by the polyclonal anti-V. cholerae O1

antibody in the lower compartment in the presence of V. cholerae O1 antigen to form a red dot within 5 to 10

minutes. No color change is observed on the control spot. If the test is negative that is, no antigen present in the

sample, or antigen level is below the level of sensitivity of the test kit, no visible red colour is seen on the test spot

at the expiration of test time (<15 min) [53]

The MedicosTM Cholera Dip Stick (Advanced Diagnostics Inc., South Plainfield, NJ, USA) is also another rapid

test designed to detect V. cholerae O1 in whole stools. Although the assay method is known to have high

Sensitivity and Specificity, the specific mechanism employed for V. cholerae O1 detection in the laboratory has

not been found published Ramamurthy [52].

Table 6: THE PERFORMANCE AND LABELING OF THE VIBRIO CHOLERAE RAPID DIAGNOSTIC TEST PRODUCTS

Product name Antigens targeted

Comments

SD BIOLINE Cholera Ag O1/O139

O1 and O139 Among the best for analytical sensitivity (LPS). Lowest LOD for V. cholerae O139 strain (0.8 105 CFU/ml).Weak and some medium test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains. No integrated sampling/buffer system - subsequent steps to be carried out by end-user

Crystal VC (dipstick)*

O1 and O139 Among the best for analytical sensitivity (LPS). Weak test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains. Obvious background present in 46.1% of tests performed

VC O139 Cassette test

O139 Lowest LOD for V. cholerae O139 strain (0.8 105 CFU/ml) Obvious background present in 14.4% of tests performed.

VC O1-O Cassette test

O1 antigen Very low analytical sensitivity (LPS, V. cholerae reference strains) Obvious background present in 54.4% of tests performed.

VC O Combo Cassette test

O1, O1-Ogawa and O139

Weak and faint test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains. Visible groove in the test strip at test line 1 (V. cholerae O139), even before addition of sample. Blurred faint lines present at test lines 2 and 3 at delayed reading. Lower inter-observer agreement of reading (5.4% difference in reading positive versus negative)

VC O1 Cassette test O1 LPS Weak test line intensities for all but one V. cholerae O1 Inaba/Ogawa clinical strains

VC O1 Combo Cassette test

O1-Inaba and O1-Ogawa

Weak and faint test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains. Lower inter-observer agreement of reading (7.5% difference in reading positive versus negative)

VC O1 Strip test* O1 LPS antigen

Weak test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains No tubes for dipstick incubation included

Onsite Cholera Ag rapid test

O1 and O139 Lowest LOD for V. cholerae O139 strain (0.8 105 CFU/ml). Weak and faint test line intensities for V. cholerae O1 Inaba/Ogawa clinical strains. Cassette well labeled but too few space for writing patient identification

Source: [55]

FiG 7: BUFFERS PROVIDED FOR PRODUCT SD BIOLINE CHOLERA AG O1/O139 (LEFT) TO PRODUCT ONSITE CHOLERA AG RAPID TEST (RIGHT), PRODUCTS ARE IN THE ORDER AS LISTED IN TABLE 6.

FIG 8: THE RDT DEVICES FROM PRODUCT SD BIOLINE CHOLERA AG O1/O139 (LEFT) TO PRODUCT ONSITE CHOLERA AG RAPID TEST (RIGHT), AS LISTED IN TABLE 6. Source: [55] SD BIOLINE Cholera Ag O1/O139 Rapid Diagnostic Test Kit The SD BIOLINE Cholera Ag O1/O139 kit is a rapid, qualitative test for the detection of V.choleraeO1/O139 in

human fecal specimens. The SD BIOLINE Cholera Ag O1/O139 test contains a membrane strip, which is pre-

coated with mouse monoclonal anti-V.choleraO1 antibody on test line 1 (1) region and with mouse monoclonal

anti-V. cholera O139 antibody on test line 2 (2) region on the surface of the device. These lines in result window

are not visible before applying any samples. The Control Line is used for procedural control. Control Line should

always appear if the test procedure is performed properly and the test reagents of control line are working. A

purple Test Line will be visible in the result window if V. choleraeO1/ O139 antigen is present in the sample. When

a specimen is added to the test, V. choleraeO1/ O139 antigen in the specimen reacts with colloidal-gold-labeled

V. choleraeO1/ O139 specific antibodies and forms a complex of antigen-antibody colloidal gold conjugates. As

this complex migrates along the length of the result window by capillary action, the complex is captured by the V.

choleraeO1/ O139 specific monoclonal antibodies immobilized in the test line across the result window and

generates a colored line. In the absence of V. choleraeO1/ O139 in specimens, a complex is not formed and no

colored line appears in the result window of test device [56].

Test procedure

The buffer is transferred from the buffer bottle into the buffer vial with the pipette. Then the sample processing vial

is then unscrewed and the sampling stick used to stab the sample (not scoop to avoid picking up particulate

matter that may clog the dipstick membrane) into the sample processing vial if the stool sample is solid, semisolid,

or viscous. When the stool is liquid, the transfer pipette is used to add 2 drops of sample to the sample processing

vial, which is then tightly recapped and shaken to mix contents. After mixing, the buffer vial cap has to be

replaced by the nozzle cap and 3 drops of the sample can then be added to the cassette and results read within

15minutes. The test has a sensitivity and specificity of 100%. A colour change at the line indicated “1” and “C” on

the cassette, means that V. cholerae O1 is positive. A colour change at the line indicated “2” and “C” on the

cassette, means that V. cholerae O139 is positive. Colour changes at the line indicated “1”, “2”and “C” on the

cassette, means that V. cholerae O1 and O139 are positive. No colour change at the line indicated “1”, “2” but

colour in “C” on the cassette, means that no V. cholerae is detected. No colour change at the line indicated “1”,

“2” and “C”, colour change at the line indicated “1”, “2” but not at “C” on the cassette, means that the result is

nullified [52].

FIG 9: SD BIOLINE CHOLERA AG O1/O139 RAPID DIAGNOSTIC TEST KIT PROCEDURE Source: [56]. LOOP- MEDIATED ISOTHERMAL AMPLIFICATION DNA (LAMP) [57]

This assay is a new, easy method in which DNA is amplified specifically with high speed and performance in

isothermal conditions. Two internal and two external primers, that, together, identify six gene regions of target

DNA, the region which is amplified during a continuous process. LAMP can identify less than 6 copies of DNA in

the reaction mixture. It is simple and easy requiring only a primer, DNA polymerase and reaction mixture. The

test can be performed in a water bath or heat block, to yield reaction products such as mixture of DNA hairpin

structure with different sizes and cauliflower-like structure with multiple rings.

MOLECULAR METHODS FOR DETECTION AND IDENTIFICATION OF V. CHOLERAE There are a variety of rapid molecular typing techniques developed for the analysis of isolates from clinical

samples and these include the Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST),

Multiple-Locus Variable Number Tandem Repeat Analysis (MVLA), Fluorescent Amplified Fragment Length

Polymorphism (FAFLP) and Whole Genome Sequencing (WGS). They are all methods that use different

approaches that enable the subtyping of unrelated bacterial strains. They operate with different accuracies,

discriminatory ability, and reproducibilities [58]

FIG10: A SCHEMATIC REPRESENTATION OF SOME OF THE MOLECULAR METHODS USED IN THE DETECTION OF VIBRIO CHOLARAE. Source:[2]

PULSED-FIELD GEL ELECTROPHORESIS

Pulsed-Field Gel Electrophoresis, an epidemiological gold standard for typing V. cholera, among other molecular

fingerprinting techniques used for epidemiological detection of V. cholerae [59] has a high capacity to distinguish

between closely related Vibrio cholerae isolates. Results obtained by Pulsed-field gel electrophoresis can easily

be reproduced in other laboratories with cheaper laboratory setups [59].The procedure works under the principle

that, the cleavage of highly purified genomic DNA by the restriction endonuclease enzyme yields restriction

fragments which are then better resolved in to separate fragments by passing them through a pulsed-field

(alternating electric fields). In this way bigger DNA fragments (ranging from 30 kb to 1 M), are separated easily

[60]. This technique though good, requires skilled personnel, is time consuming, laborious and very slow.

Moreover, the results can easily be changed by small mutations at the restriction sites. Also, PFGE cannot

differentiate the seemingly identical band sizes with only small differences of less than 5% [59]. Teh et al. [61],

demonstrated that PFGE in combination with multi-locus VNTR (MLVA) can provide more information on

epidemiological relatedness and differences among isolates from different sources or geographical regions [2].

RIBOTYPING

This technique can be used alone or together with others when studies involving the molecular descent or origin

of V. cholerae necessary. In this method, the bacterial chromosome is first digested into component fragments by

restriction enzymes. These resulting component fragments are then electrophoresed using gel electrophoresis

and transferred to a membrane for hybridization with labeled probes complementary to the 16S and 23S rRNAs

[2].

POLYMERASE CHAIN REACTION (PCR)

PCR methods for the diagnosis of each Vibrio species requires specific expected virulence factor genes as

genetic markers. The ctxA gene encoding the A subunit of cholera toxin for the detection of cholera toxin [15],

the thermo-stable direct hemolysin (tdh) gene and the thermo-stable direct hemolysin-related hemolysin (trh)

gene for V. parahaemolyticus and the cytotoxin-haemolysin (vvhA) gene for V. vulnificus [17]

PCR requires that the crude template be prepared by boiling to lyse the cells. This is then followed by the

amplification of the genomic regions within this template using PCR pimers specific to V. cholerae. The target is

the internally transcribed spacer (ITS) region between 16S and 23S rDNA or the outer membrane protein subunit

W (ompW). Confirmed V. cholerae virulence genes are analyzed by gel electrophoresis and visualized under UV

light with ethidium bromide. Positive and negative controls are included and run in parallel.

PROCEDURE

PREPARATION OF CRUDE DNA TEMPLATE BY BOILING

From a broth culture, a 1-ml amount of overnight growth at 35°C is centrifuged and suspended in 1-ml of sterile

water. From agar plates, a loopful of pure culture (50–100 colonies) of a suspected V. cholerae I suspended into

300-µl of sterile water and vortex vigorously. The suspension is then diluted 1:10 and 1:1000 using sterile distilled

water in a sterile 2-ml micro centrifuge tube, which is place into a boiling water bath for 10 minutes. The tube is

then allowed on the bench to cool to room temperature (for about 30 minutes). The crude DNA template is treated

with 10-mg/mL bovine serum albumin (BSA) (4-µl per 100-µl supernatant) to limit PCR inhibitors and yield more

accurate results.

VIBRIO CHOLERAE-SPECIFIC PCR - INTERNAL TRANSCRIBED SPACER (ITS) [62]

This PCR method is used to confirm isolates of V. cholerae by targeting the Internal Transcribed Spacer (ITS)

region between 16S and 23S rDNA or the outer membrane protein subunit W (ompW) specific to V. cholerae.

V. cholerae- specific ITS (Internal Transcribed Spacer region) PCR is set up to contain a total reaction volume of

25-µl, containing 5 µl template prepared above, 1X reaction buffer, 200 µM dNTPs, 800-nM primers (pVC-F2,

pVCM-R1), 0.625 U Taq DNA polymerase.

The above V. cholerae-specific ITS target is amplified by the following cycle conditions:

Initial denaturation : 1 min at 94°C (initial denaturation)

30 cycles: 1 min at 94°C (denaturation)

1 min at 60°C (annealing)

1 min at 72°C (extension)

Final extension: 10 min at 72°C.

The PCR product is run out on a 1.5% agarose gel in 1X TAE for 1–2 hrs at 5-V/cm (CPMB 2.5A) and the gel

stained in 1 µg/ml ethidium bromide staining solution for 15 min. After this, it is distained in distilled water for 15

min and the gel viewed using a handheld UV lamp, trans illuminator, or gel documentation system to visualize the

products (V. cholerae 16S-23S rDNA intergenic spacer region amplicon is 300-bp in size). Screen ITS-PCR

confirms V. cholerae isolates for the virulence-associated factors.

MULTIPLEX PCR ASSAY FOR DETECTION OF ompW (V. CHOLERAE-SPECIFIC) AND ctxA

(TOXIGENICITY) [63]

This protocol detects V. cholerae-specific ompW sequence as well as the ctxA gene of the cholera toxin. The

ompW-ctxA multiplex PCR is set up in a total reaction volume of 25-µl, containing the following: 10 to 20 ng of

crude DNA template or extracted genomic DNA (DNA extraction using phenol, chloroform, and isoamyl alcohol),

1X reaction buffer, 250-µM dNTPs, 1.2-pmol/µl of ompW (ompW-F, R) primers, 0.25-pmol/µl ctxA (ctxA-F, -R)

primers and 0.625-U Taq polymerase. The targets are amplified with the following cycle conditions:

Initial denaturation: 5 min at 94°C

30 cycles: 30 sec at 94°C (denaturation)

30 sec at 64°C (annealing)

30 sec at 72°C (extension)

Final extension 7 min at 72 °C.

The PCR product is run out on a 1.5% agarose gel in 1X TAE for 1–2 hrs at 5-V/cm (CPMB 2.5A). The gel is

stained for 15 minutes in 1 µg/ml ethidium bromide staining solution, and destained for15 minutes in distilled

water. Visualization of the products is by viewing the gel under UV light. The ompW and ctxA amplicons are 588

and 302-bp in length, respectively.

REAL-TIME PCR (TAQMAN ASSAY) FOR DETECTION OF V. CHOLERAE [64] The TaqMan assay is a real-time PCR method that can be used to detect V. cholerae in pure cultures, seawater,

raw oysters and other samples. It works under the principle that the DNA polymerase of Thermus aquaticus, by its

5' exonuclease activity hydrolyzes an internal probe labeled with a fluorescent reporter dye (FAM, Cy3, Cy5, TET)

and a quencher dye. The reporter dye, is separated from the quencher dye during PCR amplification and

hydrolyzation of the probe to produce a level of fluorescence which is proportional to the amount of template DNA

present in the reaction. Because it is quantitative, sensitive, and rapid, it is used to detect V. cholerae with primers

and probes directed toward the non-Classical specific hemolysin (hylA) gene of V. cholerae O1, O139 and non-

O1/O139 as well as for detection of the ctxA gene in seawater and oysters with the sensitivity of <10 CFU per

reaction. Discrimination of culturable, Viable But Non-Culturable (VBNC), dead cells, as well as naked DNA is not

possible with Real-time PCR. This as well as some inhibitory substances present in some samples such as

seawater, can interfere with the results and give false positive outcome during the assay [36]. Their requirements

for an expensive thermal cycler with fluorescence detectors make the use of this assay scarce [65].

In the Real Time PCR, the total amplification reaction mixture is 50-µl per sample. This is constituted by adding

the following components; 2.5µl DNA sample (100 ng/µl), 1X TaqMan buffer A, 5 mM MgCl2, 200 µM (each)

dATP, dCTP, and dGTP; 400 µM dUTP, 0.02 µM hylA-probe, 0.3 µM hylA-F primer, 0.3 µM hylA-R primer, 1 U of

AmpErase uracil N-glycosylase, 2.5 U of AmpliTaqGold DNA polymerase.

The Amplification of the targets is by using the following cycle conditions:

Initial hold: 5 min at 50°C

Initial denaturation: 5 min at 94°C

40 cycles: 20 sec at 95°C

1 min at 60°C

The increase in fluorescence throughout the amplification cycles is monitored using a computer software available

with Real-Time PCR machines. The results are reported as Ct value (which is the number of amplification cycles

required to detect a fluorescent signal above a given threshold.). They are inversely proportional to the amount of

target nucleic acid in the sample. Values which are below 29 indicate that the target nucleic acid in the sample is

plenty and this is a strong positive reaction. The Ct values which range from 30 to 35 are considered as

moderately reactions positive reactions, which indicate that the target nucleic acid is in small amounts. are

considered weak reactions When the target nucleic acid in the sample is in negligible quantities or is absent, the

reaction is usually weak, with Ct values ranging from 38 to 40 [36].

SYBR GREEN ASSAY FOR DETECTION OF V. CHOLERAE [66]

This is assay is an alternative Real-Time PCR which uses a dsDNA binding dye (SYBR Green) to develop a

multiplex real-time PCR of four and six target genes for V. cholerae and other vibrios in seafoods, sea, estuarine

and river water samples. In this method, the amplification products are differentiated by the analysis of their

melting temperatures.

The target amplification reaction mixture is 25 µl, consisting of 1 µl template DNA, 0.2 µM of each primers, 2.5 µl

of Light Cycler Fast Start DNA Master SYBR Green I (Roche Diagnostics, Laval, Quebec, Canad a),3.75 mM

MgCl2 (final concentration).

The Multiplex PCR is prepared as follows; Amplification reaction mixtures: 25 µl containing: 1 µl template DNA,

0.08 µM each rtxA primers; 0.15 µM each epsM primers, 0.40 µM each mshA primers, 0.20 µM each tcpA

primers; 3 µl of Light Cycler Fast Start DNA Master SYBR Green I, 4.0 mM MgCl2 (final concentration).

The targets are amplified with the following cycle conditions:

initial denaturation: 150 sec at 95°C

45 cycles (single assays) 15 sec at 95°C

35 cycles (multiplex assay): 30 sec at 60°C

30 sec at 72 °C

Results are interpreted using designated computer software. Just like it is done with the TaqMan assay, results

are expressed in Ct values, but since in multiplex SYBR Green real-time PCR, the amplification products are

distinguished based on their melting temperature (Tm), the Tm values of each of the products are calculated at the

completion of PCR amplification monitoring the fluorescence in increasing temperature between 60°C and 95°C,

at a given rate. The Tm peaks are calculated based on the initial fluorescence curve [36].

COLONY BLOT HYBRIDIZATION WITH LABELED RNA OR DNA PROBES

The colony blot hybridization assay uses the colony lift procedure to immobilize DNA from bacterial colonies onto

nitrocellulose or nylon filters for fast analysis of a greater amount of colonies, for genetic elements of interest by

hybridization. In this assay, the presence of 50 to 150 well-defined viable, culturable cells of V. cholerae colonies

(which are about 2.0–3.0-mm in size) from an overnight growth from the sample, cultured on a non-selective

medium is of utmost importance. Its advantage over PCR is that isolation is performed simultaneously with blot

preparation and enumeration can be performed more easily. Nitrocellulose (or nylon) membranes (autoclaved by

sandwiching them between two pieces of filter paper for 15 minutes) are overlaid, lifted, and treated to bind RNA

or DNA (being cautious of RNase or Dnase contamination). The blots are then hybridized with labeled probe

specific for V. cholerae (and V. mimicus), 5’ ACTTTGTGAGATTCGCTCCACCTCG-3’ or toxigenic V. cholerae

(ctxA). Probes that are labelled with fluorochromes are better used in this assay when fluorochrome detecting

devices such as variable mode imager Typhoon, GE Healthcare, or Dark Reader (Clare Chemical Research,

Colorado, USA) are available. Alternatively in the absence of fluorochrome detecting devices however, the DIG

system (Roche) is used. The V. cholerae-specific RNA colony blot/hybridization protocol is presented first and

then a ctxA-DNA colony blot/DIG (Roche) hybridization protocol is presented.

Spread-plate preparation

For colony preparation, three serial fold dilutions of the sample cultured in an enrichment medium (APW) are

spread on the surface of a nonselective medium. If the colonies are to be counted, the sample is serially diluted

and cultured directly without enrichment or 100–500-ml of water is filtered through the 0.22-µm nylon membranes

and overlaid onto an agar plates and incubated (plate and membrane overnight at 30°C)

The membranes are then marked using a lead pencil with Blot ID (e.g., medium, sample, and dilution) that

matches plate to be lifted and orientation marks (asymmetrical). They are overlaid starting from the center to

avoid air bubbles and allowed to stand for at least 15 minutes for transfer to occur. A replica-plate is prepared on

a fresh modified nutrient agar plate, transferring orientation markings using the same membrane.

SDS, SSC, and Pyrex dishes (for holding filter paper) are reheated to 65°C. The membrane, placed with colony

side up on the just slightly larger filter paper is pre-wetted with 10% SDS (but not saturated to prevent colonies

from over-swelling and losing their circular shape), enclosed in a Pyrex dish, for 5 minutes at 65°C. This Pyrex

dish containing wetted filter paper and membrane is covered with Saran wrap to avoid drying. The membrane, on

a fresh filter paper in a Pyrex dish is wetted with 3X SSC, and incubated for 15 min at 65°C. The Pyrex dish is

also cover with Saran wrap. The membranes are then air-dried for 10 minutes on filter paper and bake for 15 min

at 70°C.

For the RNA-Colony Blot Hybridization, the membranes are washed three times in adequate pre-washing solution

for 15 minutes at room temperature. Again they are washed for 1.5 hours at 60°C in prewashing solution, and

rinsed in DEPC-treated water.

If hybridization solution base forms a precipitation upon maintaining at room temperature, it is heated to 40–50°C

to re-suspend it.

The membranes is pre-hybridized for 30 min at 60°C in hybridization solution at a ratio of 10-ml pre-hybridization

solution per 100-cm2 of blot membrane (85 mm-membranes have a surface area of ~60-cm

2). The pre-

hybridization solution is then removed and the hybridization solution plus probe (32-µl/10-ml solution) at a ratio of

10-ml hybridization solution per 100-cm2 blot added. The Hybridization is done at 16–20 hours at 60°C and the

membranes washed for 30 minutes at 60°C in washing solution. Viewing and imaging of the membrane is done

using Typhoon Scanner or Dark Reader.

For Colony Blot Hybridization with using DIG-labeled ctx DNA probe this protocol targets only toxigenic strains of

V. cholerae and the presence of ctxA is confirmed by hybridization using a ctxA-specific DNA-probe. The ctxA

probe can be produced from PCR, using the pCTA primer set or from EcoRI digestion of plasmid, pKTN901,

which contains a 540-bp XbaI-ClaI fragment of ctxA [67].

The blot is prepared as in For the RNA-Colony Blot Hybridization and the plates incubated for 18-24hours at 37°C

The membranes are then marked using a lead pencil with Blot ID (e.g., medium, sample, and dilution) that

matches plate to be lifted and orientation marks (asymmetrical). They are overlaid starting from the center to

avoid air bubbles and allowed to stand for at least 15 minutes for transfer to occur.

It is transferred onto a new nonselective medium plate keeping colony side up and incubated for 3 hour at 37°C,

rapped with parafilm and kept at 10–15°C. The master plates can remain in this state for about 14 days. The

membranes, placed with the colony side up, on Whatman filter paper No: 3 pre-soaked with lysis buffer is

incubated for 10 minutes at room temperature. It is removed from the lysis buffer and placed on Whatman filter

paper No: 3 presoaked with neutralization solution. It is removed from the neutralization solution, placed on

Whatman filter paper No:3 and air dried for about 30 minutes. The bacterial cells are immobilized onto the

membrane using a UV cross-linker or trans-illuminator (If the membranes are not very dry, they are cross-linked

at an output intensity of 120-mJ/cm2, the optimal or auto-crosslink setting on commercially calibrated machines).

If calibrated trans-illuminators or hand-held UV lamps are used, 1 min for 254-nm lamps or 3 min for 302-nm

lamps will be adequate.

The blot is the washed in 1X SSC buffer twice and air dried for about 30 minutes, while 100-ml Proteinase-K

solution is used to treat the membranes by gentle shaking for 30 minutes at 42°C.

The filters are washed thrice in 1X SSC in a shaking water bath at room temperature for 10 min each and then left

to air dry for about 30 minutes.

During the pre-hybridization and hybridization phase, the DIG-Easy Hybridization buffer is first preheated to 42°C.

Then the blots are pre-hybridized in preheated DIG-Easy hybridization buffer for 30 minutes at 42°C with gentle

shaking. Boiling for 5 minutes followed by fast cooling on ice, denatures the DIG-labeled probes. After the

denaturation, the pre-hybridization solution is removed and fresh DIG-Easy Hybridization solution and the

denatured probe (25 ng/ml solution) are added and incubated for 18-24hours at 42°C. .

The hybridization solution containing the probe is removed and can be reused as many times as possible stored

at about 2 months at −20°C.

The membrane is then washed twice in Stringency Wash Solution I for 5 minutes at room temperature with

continuous shaking, followed by another two times washing in Stringency Wash Solution II for 15 min at 65°C,

again with continuous shaking.

It is then rinsed for 5 minutes in Washing Buffer, incubated for 30 minutes at 25°C in Blocking Solution, again

incubated for 30 minutes at 25°C in Antibody Solution, washed twice in Washing Buffer for 15 minutes each,

equilibrated in Detection Buffer for 5 min at 25°C, place in hybridization pouch, CSPD added, covered and

incubate at room temperature for 5 minutes. After this period of incubation, the excess liquid from pouch is

drained out. It is sealed, incubated at 37°C for 10 minutes and exposed to X-ray film for 20 minutes at room

temperature before developing it. If the film is under- or over-exposed, the exposure process is repeated at

varying exposure time.

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) In this technique (Amplified fragment length polymorphism (AFLP), double stranded adaptors are used to join the

sticky ends fragments of genomic DNA that have been cut by two different restriction endonucleases. These

ligated restriction fragments are then selectively PCR amplified, with the side adaptor sequences used to bind the

primer. Resolution is possible with an automated DNA sequencer, while the resulting band patterns can be used

to compare and detect the genetic relatedness amongst the bacterial strains [2].

SEQUENCING BASED FINGERPRINTING

VARIABLE NUMBER OF TANDEM REPEATS ANALYSIS

Each individual V. cholerae isolate possesses some particular genetic characteristics which appear as a variable

number of short DNA sequence motifs, repeated in tandem at a specific locus. These tandem repeats (VNTRs)

or simple sequence repeats (SSRs), serve as areas where the differentiation among V. cholerae isolates is easily

obtained due to the high level of genomic polymorphism occurring there [2]. Sequencing based on genotypic

analysis of these repeats helps to solve some of the short comings observed with the PFGE, CTX-genotyping and

ribotyping (which show that most pathogenic V. cholerae O1 and O139 isolates possess similar genetic profiles).

MULTI-LOCUS VARIABLE TANDEM REPEAT ANALYSIS

Multi-locus variable tandem repeat analysis is an example of sequencing based typing. It targets five or six loci in

the V. cholerae genome, each of which has a tandem repeat, that contain six or seven nucleotides with the

capability of being repeated 4–31 times. Each tandem repeats length denotes a five-digit genotype which

represents an allele number in the corresponding locus for the five different loci. V. cholerae strains which are

presumptively identified to be distinct at a unit locus in a five-digit genotype, are believed to have stemmed from

the same clone; meaning they are derivatives of a single ancestral origin. Furthermore, strains identified with

genotypes that differ at two or more loci are believed to have stemmed from different clones, showing that they

evolved from different ancestors [2]. A Variation of the Multi-locus sequence typing is the ribosomal multi-locus

sequence typing which is based on variations of the 53 genes encoding the bacterial ribosome protein subunits

(rps genes). This sequencing method gives a better discrimination of the target genes from amongst the different

bacterial strains investigated [68].

The polymerase chain reaction is a better method to the laborious and time consuming (biochemical tests)

conventional diagnosis, whose results, at times, become so complicated and difficult to interpret [36]. PCR

although rapid, sensitive and distinguish virulent from avirulent strains [69] are faced with limitations, such as high

thermal cycles, use of expensive thermocycler machine, requirements for skilled professionals. All of these

disadvantages make diagnosis in low resource areas of the globe (where most of the times cholera out breaks

are reported) difficult [70,13).

IMMUNOLOGICAL METHODS FOR DIRECT DETECTION OF V. CHOLERAE IN ENVIRONMENTAL SAMPLES The direct detection of V. cholerae in samples most especially from environmental samples (where the bacterium

most frequently enters into the viable but non-culturable (VBNC) state) is the best bet. This is because other

methods like the conventional culture methods cannot detect the bacteria in VBNC state.

Immunoassay-based techniques used for the detection of V. cholerae are such as enzyme-linked immuno-

sorbent assay (ELISA), reverse passive latex agglutination test (RPLA), and immuno-chromatographic test (IC)

require sophisticated instruments, long assay time, and skilled personnel (6).

Fluorescent In-Situ Hybridization (FISH) Detection of V. cholerae

Fluorescent In-Situ Hybridization (FISH) is a direct quantification methods that allows quick estimates and highly

accurate detection of taxa-specific nucleic acid followed by visualization using microscopy without the need to

enrich and culture. This is accomplished this with the aid of a fluorescently-labeled oligonucleotide probe that is

visualized under epifluorescence or confocal laser scanning microscopy. Both culturable and non-culturable

(VBNC) V. cholerae cells can be detected.

Procedure

Sample preparation

About 500 to 1000 ml of water is filtered through a 0.2 µm polycarbonate membrane, and the attached cells re-

suspended in 5 ml 1X PBS. About 1 ml of 1X PBS re-suspended cells are put in micro-centrifuge tube and

centrifuged at 13,000 g for 10 minutes. The supernatant is discarded, the cells re-suspended in 250 µl of 1X PBS

and fixed with 750 µl of fresh 4% paraformaldehyde solution. The fixed cells are then incubated at 22–25°C) for

1hour.The cell solution is again centrifuged at 13,000 g for 5 minutes to pellet the cells. After this, the supernatant

is discarded and then the cells washed two times with 1X PBS. Further centrifugation concentrates the cells which

are re-suspended in 100 µl PBS-ethanol solution and stored at −20°.

Hybridization

Multi-well slides, dipped in 0.01% PLL solution for 10 minutes are air-dried, spotted with 5 µl aliquots of fixed cells

and again air-dried. They are washed for 3 minutes each in successive ethanol solutions (50, 80 and 96%) and

then air-dried. A Humid chamber, slides and filter paper soaked with hybridization solution are warmed at 45°C for

10 minutes. Then 5 µl aliquot of Hybridization solution containing 5 ng/µl of labeled probe are added to the wells.

The hybridization is done at 45°C for 24 hours in the humid chamber using hybridization soaked paper. The

washing buffer solution is heated at 45°C for 10 minutes, the slides washed and incubated at 45°C for 10 minutes.

Washing of the slides is again done in sterile deionized water and air-dried at room temperature. An anti-fading

agent is added to each well and covered with cover slips. The visualization of fluorescence is done using the

epifluorescence microscope

DIRECT FLUORESCENT ANTIBODY – DIRECT VIABLE COUNT (DFA-DVC) METHOD The direct fluorescent antibody staining method is rapid method which allows the detection of V. cholerae

serogroup O1 and O139 within 8 hours. When this technique is coupled with the direct viable count method, the

differentiation of culturable, viable cells from viable but non-culturable cells (VBNC) of V. cholerae is easy. This

method works under the principle that; the combined incubation of the samples with yeast extract in the presence

of nalidixic acid, allows the actively viable, substrate-responsive cells to become enlarged and elongated. A small

amount of the cell suspension, when air dried on a glass microscope slide and stained with fluorescent labeled

monoclonal antibody, raised against ‘A’ factor of V. cholerae O1 lipopolysaccharide (reacts with both serotypes,

Ogawa and Inaba.) and viewed under the epi-fluorescent microscope, the elongated cells of V. cholerae O1 or

O139, (based on the type of antibody used) appear bright green, showing a fluorescing periphery and a dark

interior.

Procedure

To 1 ml concentrated water or homogenized plankton sample is added 10-µl of yeast extract and 10-µl nalidixic

acid solutions. The mixture is frozen at -20°C and 1-ml a duplicate sample also prepared for confirmation using

PCR.

The mixture is incubated at 25°C for 6 -24hours, fixed with formaldehyde to a final concentration of 3% (v/v) and

incubated for 30 minutes at 22-25oC in the dark. Fixed cell at 4°C in the dark have a shelf life of up to 6 months.

About 5 to 10 µl of the fixed sample is spread onto a clean glass microscope slide and air dried for about 15–20

minutes. It is then fix by adding 5 µl methanol and air dried for 1–5 minutes. A 10-µl of reconstituted fluorescein-

isothiocyanate (FITC)-conjugated specific DFA as supplied by Cholera DFA or Bengal DFA kit is added and

incubated for 30 minutes at 37°C in a humid chamber while shielding slide from direct light. The slide is washed

with about 50 ml PBS (each), air dried in the dark for about 15–20 minutes, and mounted with one drop of kit-

provided Fluorescent Mounting Medium. The mounted slide is then covered with a cover slip and viewed under an

epi-fluorescent microscope.

INDIRECT FLUORESCENT ANTIBODY (IFA) METHOD

Procedure

Prepare and fix samples

The test sample is added to a Teflon-coated multi-well slide, air dried for 15–20 minutes at room temperature,

fixed with 95% ethanol and again air dried for 5–10 minutes at room temperature. It is then heated for 10 minutes

in a 55°C incubator (could be kept for up to 1 month at −70°C), rinsed with approximately 50-ml of PBS and air

dried for 15–20 minutes. The humid chamber is then heated in the incubator at 35°C for 15 minutes. About 1 to 2

drops of a 1:20 dilution of Fluorescent Antibody (FA) Rhodamine Counterstain is added to each dry sample well

and incubated in the humid chamber for 30 minutes at 35°C. While controlling the amount of light entering the

chamber the slide is slowly flooded with about 50 ml of PBS and washed for 10 minutes at room temperature. It is

then removed, rinsed again briefly in PB, and allowed to air dry for 15–20 minutes. This id followed by the addition

of 5–10-µl of V. cholerae O1-specific antiserum and incubation in a humid chamber for 30 minutes at 35°C. Again

slowly, with about 50 ml of PBS, the slide is flooded, and washed with PBS for 10 minutes at room temperature. It

is then removed, rinsed again briefly in PB, and allowed to air dry for 15–20 minutes. Undiluted FITC-conjugated

anti-rabbit globulin goat serum (1–2 drops) is added and incubated in a humid chamber for 30 minutes at 35°C.

Slowly, with about 50 ml of PBS, the slide is flooded, and washed with PBS for 10 minutes at room temperature. It

is then removed, rinsed again briefly in PB, and allowed to air dry for 15–20 minutes. A low fluorescence, anti-

quenching mounting medium, such as Citifluor AF1 is then used to mount each of the slides which is covered with

a cover slip and immediately viewed using epifluorescent microscope with a FITC band-pass filter.

COAGGLUTINATION TEST (COAT) Co-agglutination is a serological test which detects of microgram quantities of soluble antigen within minute cell

walls of clinical specimens [65]. Protein-A positive Staphylococcus aureus Cowan 1 strain sensitized with high

titer V. cholerae O1 antiserum detects pathogens directly from stools with 94% positivity compared to the

conventional culture method. When the sample is enriched, V. cholerae O1 and O139 are detected with high

sensitivity (97% and 92%, respectively) and specificity (99% and 100%, respectively by this co-agglutination

method [12].

3.5 LATEX AGGLUTINATION TEST Swapna [17], reported that latex agglutination assay was 98% accurate for V. cholerae O1 and 100% accurate for

non-O1 strains, with 100% specificity for both. This latex test according to the above researcher, is less

complicated and less time consuming than ELISA. Latex particles sensitized with the affinity purified antibodies

are used as a reagent for the rapid identification of the bacterium. Under this condition, V. cholerae can be

identified immediately after suspected colonies are observed on TCBS agar [17]

Magnetic sandwich immunoassay [5]

FIG 11: SCHEMATIC REPRESENTATION OF THE MAGNETIC SANDWICH IMMUNOFILTRATION ASSAYSource: [5]

The sandwich immunoassay previously described by Zhu et al. [71], directly uses super paramagnetic beads as a

marker to qualitatively and quantitatively measure CTB inside a sample. These beads act as markers for the

magnetic frequency mixing detection. There are various kinds of beads that can be used; the hydrodynamic

diameter 75 nm and B hydrodynamic diameter 1010 nm, all from micromod Partikeltechnologie GmbH (Rostock,

Germany)

The first coating antibody is trapped on a porous support made of polyethylene matrix. When the solution

containing the cholera toxin is passed in the over the matrix in gravity flow, the B subunit of the Cholera toxin

(CTB) is then trapped by the first antibody. This same bound CTB is also again bound by the second antibody

(the bio-tinylated secondary antibody) when it is flushed over the matrix. This second antibody is then to the

streptavidin-coated super-paramagnetic beads (MB, orange) through the biotin moiety of the secondary antibody.

Usually, Monoclonal antibodies (mAb) generated by hybridoma clones are used for the test.

CONCLUSION Since the dangerously severe, watery diarrheal disease cholera, has become a health challenge in many parts of

the globe, its diagnosis has also become a pertinent issue. Although the conventional diagnostic methods still

remain the gold standard for the laboratory diagnosis of cholera; especially during cholera epidemic outbreaks,

the rapid molecular methods aid detection to the species level. However, the use of phenotypic approaches as

well as the rapid molecular methods have some major setbacks as they are usually labor-intensive, capital

expensive, time-consuming, requiring skilled personnel and heavy equipment. Therefore; Cholera RDTs (onsite

diagnostic test in kits) which are usually very useful in early outbreak detection to give primary signals, monitor

outbreaks and seasonal peaks in highly endemic areas. This initiate response measures (e.g. help

epidemiologists, clinicians and health care workers to mobilize resources and material in putting control measures

in place before an outbreak) These test kits are easy to use, transport, and fast ; an important tool for fast

epidemiological response to a cholera outbreak.

REFERENCE LIST [1] Tarh JE. An Overview of Cholera Epidemiology: A Focus on Africa; with a Keen Interest on Nigeria IJTDH

2019; 40(3): 1-17, DOI: 10.9734/IJTDH/2019/v40i330229 [2] Rahaman MH, Islam T, Colwell RR and Alam M Molecular tools in understanding the evolution of V. cholerae.

Front. Microbiol. 2015; 6:1040.doi: 10.3389/fmicb.2015.01040 [3] Tarh JE. Epidemiological Distribution of Different V. cholerae Strains Causing Cholera Disease in Endemic Countries: A Review JAMMR 2019; 31(5): 1-15, DOI: 10.9734/JAMMR/2019/v31i530298 [4] Diaconu K, Falconer J, May FO, Jimenez M, Matragrano J, Njanpop-Lafourcade B, Ager A. Cholera diagnosis

in human stool and detection in water: Protocol for a systematic review of available technologies. Systematic Reviews. 2018; 7: 29. DOI 10.1186/s13643-018-0679-8.

[5] Achtsnicht S, Neuendorf C, Faßbender T, Nölke G, Offenhäusser A, Krause HJ, Schröper F. Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection. PLoS One. 2019; 14(7):e0219356. doi: 10.1371/journal.pone.0219356. PMID: 31276546; PMCID: PMC6611628.

[6] Rahman M, Heng LY, Futra D, Ling TL. Ultrasensitive biosensor for the detection of V. cholerae DNA with polystyrene-coacrylic acid composite nanospheres. Nanoscale Res Lett. 2017; 1-12. DOI 10.1186/s11671-017-2236-0.

[7] Bayat M,

Khabiri A,

and Hemati B. Development of IgY-Based Sandwich ELISA as a Robust Tool for Rapid

Detection and Discrimination of Toxigenic V. cholerae Can J Infect Dis Med Microbiol. 2018; 2018: 4032531. doi: 10.1155/2018/4032531

[8] Proteomes - Vibrio cholerae serotype O1 (strain ATCC 39541 / Classical Ogawa 395 / O395) Overview January 15, 2020 https://www.uniprot.org/proteomes/UP000000249

[9] Islam MT, Khan AI, Sayeed MA, Amin J, Islam K, Alam N, et al. Field evaluation of a locally produced rapid diagnostic test for early detection of cholera in Bangladesh. PLoS Negl Trop Dis. 2019; 13(1): e0007124. https://doi.org/10.1371/journal.pntd.0007124

[10] Li R, Chiou J, Chan EW-C and Chen S A Novel PCR-Based Approach for Accurate Identification of Vibrio

parahaemolyticus. Front. Microbiol. 2016; 7:44. doi: 10.3389/fmicb.2016.00044

[11] Sun. Conventional and molecular detection of V. cholerae isolated from environmental water with the prevalence of antibiotic resistance mechanisms. Inter J Res Pharm Sc. 2019; July 28 [12] Ramamurthy T, Das B, Chakraborty S, Mukhopadhyay AK, Sack DA. Diagnostic techniques for rapid

detection of V. cholerae O1/O139. Vaccine. 2019. https://doi.org/10.1016/j.vaccine.2019.07.099. [13] Yu CY, Ang GY, Chua AL, Tan EH, Lee SY, Falero-Diaz G. Dry-reagent gold nanoparticle-based lateral flow

biosensor for the simultaneous detection of V. cholerae serogroups O1 and O139. J Microbiol Methods. 2011; 86(3): 277–282

[14] Matias WR, Julceus FE, Abelard C, Mayo-Smith L M, Franke MF, Harris JB, et al. (2017)Laboratory

evaluation of immuno-chromatographic rapid diagnostic tests for cholera in Haiti. PLoS ONE. 2017; 12(11):e0186710. https://doi.org/10.1371/journal.pone.0186710

[15] Keddy KH, Sooka A, Parsons MB, Njanpop-Lafourcade BM, Fitchet K., Smith AS. Diagnosis of V. cholerae O1 Infection in Africa. The J Infect Dis. 2016; (S1): S23–31. DOI: 10.1093/infdis/jit196

[16] Al-Shukri MSM, Abdul-Lateef LA. Molecular detection of cholera toxin genes in V. cholerae infection in

human JCPS. 2017; 10 (2): 972-979

[17] Swapna M. Isolation, identification and differentiation of common Vibrio species in sea food samples. (Master’s Thesis). 2017; P.V. Narsimha Rao Telangana Veterinary University, Hyderabad. India.

[18] Centers for Disease Control and Prevention Laboratory Methods for the Diagnosis of V. cholerae Iv. Isolation of Vibrio Cholerae from Fecal Specimens. 16-26

[19] Kaysner CA. and DePaola, Jr A. Bacteriological Analytical Manual Chapter 9. Vibrio. Authors: Revision History: Chapter 9 substantially rewritten and revised May 2004. Content current as of 10/31/2017

[20] May D, Madigan T, Landinez L, Tan J, and Mcleod C. Assessment of Methodologies for the Enumeration of Pathogenic Vibrio species in Australian Prawns seafood CRC Project: 2009/787.Prawn Market Access Defenders South Aust Res Dev Inst. January 2014; 1-78

[21] Tarh JE, Mboto CI, Asikong BEE and Iroegbu CU. V. cholerae Incursion in Africa, the Journey So Far J Sci Res Rep. 2019; 25(3-4): 1-12, DOI: 10.9734/JSRR/2019/v25i3-430181

[22] Sagar Aryal. Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar- Composition, Principle, Uses, Preparation and Colony Morphology. Last updated: August 15, 2019 Microbiology info.com. [23] Cholera prevention and control/Cholera/Diagnosis and Detection%20%20 Cholera%20%20 CDC.htm

[24] Vibrio cholerae gram stain CDC.jpg. This page was last edited on 14 December 2017, at 06:48. https://commons.wikimedia.org/wiki/File:V. cholerae_gram_stain_CDC.jpg

[25] Sajeev H. What is the role of Gram staining stool exam in the diagnosis of cholera? Updated: Nov 02, 2018 Medscape Wednesday, April 22, 2020 [26] Visual Amrita Laboratories Universalizing Education Value @Amrita) https://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2

[27] Sagar Aryal. Oxidase Test- Principle, Uses, Procedure, Types, Result Interpretation, Examples and Limitations. Last updated: September 27, 2018. Microbiology info.com

[28] Acharya Tankeshwar. String test for Laboratory diagnosis of V. Cholerae. Bacteriology, Biochemical tests in Microbiology, Laboratory Diagnosis of Bacterial Disease. January 23, 2015

[29] Acharya Tankeshwar. Oxidation Fermentation test for Laboratory diagnosis of V. Cholerae 2016. January 23, 2015 Bacteriology, Biochemical tests in Microbiology, Laboratory Diagnosis of Bacterial Disease

[30] Acharya Tankeshwar. KIA test for Laboratory diagnosis of V. Cholerae. 2019. January 23, 2015 Bacteriology, Biochemical tests in Microbiology, Laboratory Diagnosis of Bacterial Disease

[31] Acharya Tankeshwar. TSI test for Laboratory diagnosis of V. Cholerae 2013. 3 January 23, 2015. Bacteriology, Biochemical tests in Microbiology, Laboratory Diagnosis of Bacterial Disease

[32] Weil AA and. Harris JB in Molecular Medical Microbiology (Second Edition), 2015. Biotype Designation of V. cholerae O1 Pages 2097-2145 Imprint Academic Press DOI https://doi.org/10.1016/C2010-1-67744-9 [33] Elliot EL., Kaysner CA., and Tamplin ML. Chapter 9. V. cholerae, V. parahaemolyticus, V. vulnificus, and

Other Vibrio spp. FDA Bacteriological Analytical Manual 7th Edition/1992

[34] Aqila MRA, Izza MA, Syarifuddin M. V. cholerae Test on Fishery Products at Cirebon Test and Application of

Fishery Products Technical Unit Agency, West Java, Indonesia World Scientific News. An Inter Sc 2019; J 119 192-203

[35] Centers for Disease Control and Prevention (CDC). (nd). Laboratory Methods for the Diagnosis of V. cholera. Vi. Laboratory Identification of V. Cholerae.38-66 [36] Huq A, Haley BJ, Taviani E, Chen A, Hasan NA, Colwell RR. Detection, isolation, and identification of V

cholerae from the environment. Curr Protoc Microbiol. (2012: 1-58. doi:10.1002/9780471729259.mc06a05s26.

[37] Fan Y, Li Z, Li Z. et al. Non-hemolysis of epidemic El Tor biotype strains of V. cholerae is related to multiple functional deficiencies of hemolysin A. Gut Pathog. 2019; 11, 38 https://doi.org/10.1186/s13099-019-0316-7

[38] Honda T and Finkelstein RA. Purification and characterization of a hemolysin produced by V cholerae biotype El Tor: Another toxic substance produced by cholera vibrios. Infect Imm.1980; 26(3):1020-7 DOI: 10.1128/IAI.26.3.1020-1027.1979PubMed

[39] Samanta P, Rudra NS, Chowdhury G, Arindam N, Sounak S, Shanta D, et al, Dissemination of newly emerged polymyxin B sensitive V. cholerae O1 containing Haitian-like genetic traits in different parts of India. J Med Microbiol. 2018; 67: 1326–1333

[40] GTFCC Surveillance Laboratory Working Group Interim Technical Note. The Use of Cholera Rapid Diagnostic Tests November 2016 GTFCCsecretariat@who.int [41] Yamasaki E, Sakamoto R, Matsumoto T, Morimatsu F, Kurazono T, Hiroi T, et al. Development of an

immune-chromatographic test strip for detection of cholera toxin. BioMed Res Inter. 2013; 1-7. http://dx.doi.org/10.1155/2013/679038.

[42] Lekshmi N, Joseph I, Ramamurthy T, Thomas S. Changing facades of V. cholerae: An enigma in the

epidemiology of cholera. Ind J Med Res. 2017; 133-141. DOI: 10.4103/ijmr.IJMR_280_17.

[43] Chakraborty S, Alam M, Scobie HM, Sack DA. Adaptation of a simple dipstick test for detection of V. cholerae O1 and O139 in environmental water. Front Microbiol. 2013; 1-3. doi: 10.3389/fmicb.2013.00320.

[44] Nyangena OL. Evaluation of enriched rapid diagnostic method for detection of V. cholerae and antibiotic susceptibility patterns of serotypes in Juba, South Sudan. (Master’s thesis). 2018; School of Medicine, Kenyatta University. South Sudan.

[45] Debes A, Subhra C, Ali M, Sack DA. Manual for Detecting V. cholerae O1 and O139 from Fecal Samples and from Environmental Water Using a Dipstick Assay. (Lecture mannual) March 28, 2014. Johns Hopkins Bloomberg School of Public Health 615 N. Wolfe Street / E5537, Baltimore, MD 21205, USA From DOVE Project www.stopcholera.org [46] Debes AK, Ateudjieu J, Guenou E, Ebile W, Sonkoua IT, Njimbia AC, Sack DA. Clinical and environmental

surveillance for V. cholerae in resource constrained areas: application during a 1-year surveillance in the far north region of Cameroon. The Amer J Trop Med Hyg. 2016; 94 (3):537-543.

[47] Dick MH, Guillerm M, Moussy F, Chaignat CL. Review of two decades of cholera diagnostics–how far have we really come? PLoS Negl Trop Dis. 2012; 6 (10): 1-8.

[48] Ley B, Khatib AM, Thriemer K, von Seidlein L, Deen J, Mukhopadyay A, et al, Evaluation of a rapid dipstick (Crystal VC) for the diagnosis of cholera in Zanzibar and a comparison with previous studies. PLoS ONE. 2012; 7(5):e3693. [49] CDC. Cholera-V. cholerae infection. Crystal® VC Rapid Diagnostic Test (RDT) Procedure Page last reviewed: July 20, 2018 https://www.cdc.gov/cholera/index.html [50] Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID) Page last reviewed: July 20, 2018. [51] Ang GY, Yu CY, Yean CY. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor. Biosens Bioelectr. 2012;8: 151–6. [52] Ramamurthy T, Bhabatosh D, Subhra C, Mukhopadhyay AK, Sack TD, Ramamurthy A. et al. / Vaccine. 2020; 38: A73–A82 [53] Hasan JAK, Huq A, Tamplin, ML, Siebeling RJ and Colwell' RR. Novel Kit for Rapid Detection of V. cholerae 01. J Clin Microbiol. 1994; 32 (1): 249-252A [54] Hasan, JAK, Loomis L, Huq A, Bernstein D, Tamplin, ML, Siebeling RJ and Colwell' RR. Development of a fast, simple, and sensitive immunoassay to detect V. cholerae 01 from clinical samples using SMARTT kit, abstr. C-1992; 118: 440. Abstr. 92nd Gen. Meet. Am. Soc. Microbiol. 1992. American Society for Microbiology, Washington, D.C. [55] Barbé B and Jan J, Institute of Tropical Medicine. Report June 20, 2015 150620_BB_Report V. cholerae RDTs_WHO. Laboratory evaluation of a selection of rapid diagnostic tests for Vibrio cholera

[56]Cholera Ag 01-0139 En Sp편집 – Biodiagnosticos www.biodiagnosticos.com › sd › cho.

[57] Barzamini B, Majid M and Nazila AS. Rapid Identification of V. cholerae Bacteria. LAMP and PCR methods in

Water and Waste water Biosc Biotech Res Asia, 2015; 12(1):569-579. DOI:http://dx.doi.org/10.13005/bbra/1699

[58] UK Standards for Microbiology Investigations. Identification of Vibrio and Aeromonas species. Protecting and

Improving the Nation’s Health. Public Health England. NHS. Bacteriol – Ident. 19; 3: 1 14.04.15

[59] Sabat AJ, Budimir A, Nashev D, Sá-Leão R, vanDijl JM, Laurent, F, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. EuroSurveill. 2013; 18: 20380.

[50] Goering RV. Pulsed field gel electrophoresis: are view of application and interpretation in the molecular epidemiology of infectious disease. Infect. Genet. Evol. 2010; 10, 866–875.doi:10.1016/j.meegid.2010.07.023

[61] Teh CSJ, Chua KH and Thong KL. Multiple-locusvariable- number tandem repeat analysis of V. cholerae in comparison with pulsed field gel electrophoresis and virulotyping. J. Biomed. Biotechnol. 2010; 817190. doi: 10.1155/2010/817190

[62] Chun J, Huq A, et al. Analysis of 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Appl Environ Microbiol. 1999; 65: 2202–2208. [PMC free article] [PubMed] [Google Scholar]

[63] Nandi B, Nandy R, et al. Rapid method for species-specific identification of V. cholerae using primers targeted to the gene of outer membrane protein OmpW. J. Clin. Micro. 2000;38(11):4145–4151. [PMC free article] [PubMed] [Google Scholar]

[64] Lyon WJ. TaqMan PCR for detection of V. cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater. Appl Envir Microbiol. 2001; 67(10):4685–4693. [PMC free article] [PubMed] [Google Scholar]

[65] Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing V. cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008; 8, 94. doi:10.1186/1471-2180-8-94.

[66] Gubala AJ. Multiplex real-time PCR detection of Vibrio cholerae. J Microbiol Meth. 2006; 65(2):278–293. [PubMed] [Google Scholar]

[67] Kaper J, Morris J, Tenover F. et al. DNA probes for infectious diseases. Boca Raton, FL: CRC Press; 1988. DNA probes for pathogenic Vibrio species. 65–77. [Google Scholar]

[68] Hounmanou YMG, Leekitcharoenphon P, Hendriksen RS, Dougnon TV, Mdegela RH, Olsen JE and Dalsgaard A Surveillance and Genomics of Toxigenic Vibrio cholerae O1 From Fish, Phytoplankton and Water in Lake Victoria, Tanzania. Front. Microbiol. 2019; 10:901. doi: 10.3389/fmicb.2019.00901

[69] Ranjbar R, Naghoni A, Afshar D, Nikkhahi F, Mohammadi M. Rapid molecular approach for simultaneous

detection of Salmonella spp., Shigella spp., and V. cholera. Osong Public Health Research Persp. 2016; 7(6): 373-377 http://dx.doi.org/10.1016/j.phrp.2016.10.002.

[70] Herfehdoost GR, Kamali M, Javadi HR, Zolfagary D, Emamgoli A, Choopani A, Ghasemi B, Hossaini S.

Rapid detection of V. cholerae by Polymerase Chain Reaction based on nanotechnology method. J App Biotechnol Rep. 2014; 1(2): 59-62

[71] Zhu K, Dietrich R, Didier A, Doyscher D, Märtlbauer E, Recent Developments in Antibody-Based Assays for

the Detection of Bacterial Toxins. Toxins 2014;6(4):1325–1348. 10.3390/toxins6041325 [PMC free article] [PubMed] [CrossRef] [Google Scholar]