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DI 02-60

Rapid diagnosis of cholera
based on immunochromatography

Inventors: Farida Nato et al.

Description of invention:

Cholera is a re-emerging disease. The exact magnitude of their incidence in the world is unknown because of under reporting of cases and other limitations of the surveillance systems. There is no simple and rapid assay for the diagnosis of this disease, and quite often the samples take several days or weeks after their collect before their arrival to the laboratory. Patients’ treatment is efficient if started before 24 to 48 hours after the onset of the disease. Without treatment, the lethality is 20 to 50% for cholera.
Cholera is a food-born diarrheal disease caused by Vibrio cholerae. Of the 155 known serogroups, only the O1 and the new O139 serogroups are responsible of cholera. The current 7th pandemic is due to the serogroup O1 that disseminated in Asia from 1961, in Africa from 1970 and in South America from 1991. In 1998, about 300 000 cases (10 000 deaths) were declared to WHO (a doubling of cases compared to 1997). In 1999, for the first time cholera reached the island of Madagascar, where 23, 000 cases were notified in one year.
It is likely that the 8th pandemic of cholera has already started. Indeed in 1992, outbreaks in India and Bangladesh were caused by the new O139 serogroup, and 11 countries in Asia have been invaded in 1994. After a period of silence, this strain re-emerged in Calcutta in 1996 and in Bangladesh in 1997. There is no cross-protection between the two serogroups O1 and O139.
When cholera is suspected in a given region, it is necessary to confirm the first cases by the bacteriology. When the epidemic is installed, all the suspected cases of cholera are treated and declared without biological confirmation, because of the overloading of the laboratory.
A rapid diagnosis of cholera is essential :
- To treat the patients as soon as possible,
- To provide a rapid chemoprophylaxis to the contact subjects,
- To convince the public health authorities to implement control measures for stopping transmission (chlorination of drinking water, public hygiene measures).
In practice today, these measures are initiated after the disease has been laboratory confirmed:
- To train the health staff and inform the population in the symptoms of the diseases and in the basic preventive methods,
Because the majority of the world population is naïve to the serogroup O139, the risk of explosion is very high.
We have developped a rapid assay for the diagnosis of both the O1 and O139 serogroups.

Principle of the assay
The principle is based on Immunochromatography with the colloïdal gold particles. The Mab is conjugated on immunogold particles, used as mobile phase immunosensor. The another antibody is immobilised in line onto nitrocellulose membrane. The antigen-antibody complex (sandwich assay) is visualised as a red capture line. A second antigen?antibody system with (mouse IgG and anti-mouse IgG) will be used as internal control. A negative sample will appear as one red line, and a positive sample will appear as two red lines.

Two new rapid assays "ready to use" for the diagnosis of cholera using monoclonal antibodies against LPS of Vibrio cholerae O1 and O139 have been developed and evaluated.
The specificity determined on 130 cultures of bacteria species (other serogroups of V. cholerae; other species of Vibrio, Aeromonas, Plesiomonas, Campylobacter, Yersinia and Enterobacteriacae was 100% for the two assays. On 74 stool samples (normal, Salmonella and Shigella positive), the specificity was respectively of 97% and 96% for the O1 and O139 dipsticks.
The sensitivity of O1 dipstick was 98% in Madagascar (66 V. cholerae O1 stool samples) and 94% in Bangladesh (52 V. cholerae O1 stool samples). The sensitivity of O139 dipstick determined on 91 V. cholerae O139 stool samples in Bangladesh was 100%.

These tests were stable at 60°C for 21 days and therefore can be sent in humidity proof bags and stored at room temperature in cholera endemic countries.

These dipstiks are rapid (15mn), simple and easy to perform at patient’s bedside by health workers. Both assays were used directly on stool samples (crude or diluted in PBS) without a previous extraction.
- In developing countries where a cold chain is not readily available, dipsticks can be sent and stored at room temperature,
- Homogeneity of the reagents due to the use of monoclonal antibodies.

Possible applications
Applications for individual diagnosis of patients (rapid treatment and emergency measures to prevent the extension of the disease), epidemiological surveillance of outbreak and environment (water).
The diagnostic value of both assays was excellent. These new diagnostic tools will be very useful for the epidemiological surveillance of cholera, especially for the surveillance of the extension of the O139 serogroup.

Nato F., Boutonnier A., Rajerison M., Grosjean P., Dartevelle S., Guénolé A., Bhuiyan N.A., Sack D.A., Nair G.B., Fournier J.M., & Chanteau S.
One-step immunochromatographic dipstick tests for rapid detection of Vibrio cholerae O1 and O139 in stools samples.
Clinical and Diagnostic Laboratory Immunology, in press

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