In this study, the sensitivity of the established IFAG method for detection of V. cholerae
O1 and O139 in laboratory culture was 103
CFU/ml; the method was also highly specific. In the mixed bacterial solution containing 10 species of interfering bacteria and the target V. cholerae
O1 and O139 strains, IFAG was able to specifically detect fluorescent aggregates of V. cholerae
O1 and O139 without cross-reactivity. Furthermore, the aggregates were verified to be V. cholerae
O1 and O139 in the subsequent culture and serum agglutination tests, showing high specificity. Hasan et al (18
). used DFA to detect V. cholerae
O139; because the water samples were filtered and concentrated in that study, the sensitivity of V. cholerae
O139 detection reached 1.5 × 102
CFU/ml and the specificity was 100%. In this study, because neither the pure cultures nor on-site water samples were filtered or concentrated, the sensitivity of detection was one log lower than that of Hasan et al., but our assay had the same specificity.
A number of methods have been evaluated for detection of V. cholerae
O1 and O139 in samples from environmental waters. The colloidal gold technique was used to easily screen V. cholerae
, but the sensitivity is low (requiring the bacteria concentration in samples to be more than 105
CFU/ml). Conventional culture requires a larger sample size, for which preprocessing steps for isolation and detection are laborious and cumbersome. The real-time PCR technique has high sensitivity in detecting V. cholerae
, but it still requires culture and isolation of sample after PCR detection when the strain is needed. The IFAG assay has high specificity and a higher isolation rate than the conventional culture and should be a good choice for environmental surveillance of V. cholerae
, especially surveillance and study based on obtaining isolates from environmental samples. We previously performed studies on the use of IFAG for detection of V. cholerae
by using antisera to V. cholerae
O1 and O139 (28
). Here we analyzed the enriched broth of estuarine water samples and used monoclonal antibodies to serogroups O1 and O139 in IFAG. We determined that the IFAG percentage positive was higher than that of conventional culture while the assay time was less than that of conventional culture methods. Additionally, with its high specificity, IFAG can be used to improve the isolation rate for V. cholerae
and efficiency when dealing with a large number of samples, since only IFAG-positive samples are forwarded for further isolation. Also, the isolation rate could be increased by drawing the fluorescent aggregates of bacteria in the IFAG-positive samples, compared to that for conventional culture.
In this study, 22 out of 29 IFAG-positive water samples were positive by subsequent culture of fluorescent aggregates, but for 7 samples, there was a failure to culture V. cholerae
from the fluorescent aggregates (Table ). Among possible reasons for the inability to obtain culture from some aggregates, one may be the presence of viable but nonculturable (VBNC) V. cholerae
in the waters. Studies by Binsztein et al (6
) suggested that in the interim period between cholera epidemics, V. cholerae
is still present in the environment in a VBNC state, indicating that even if fluorescent aggregates of bacteria are detected, no viable bacteria can be obtained by culture. The second reason is that even if fluorescent aggregates were obtained, they may have been suppressed in the gentamicin plate of subsequent culture or no colony could be formed in the gentamicin agar by a single colony due to interference from other bacteria. The third reason may be that the colonies were false positive due to incomplete washings and the presence of nonspecific fluorescence.
The IFAG percentage positive (19.9%) was lower than the real-time PCR positive rate (29.5%). The discordance between the two methods (where the real-time PCR gave a positive result and the IFAG result was negative) was found mainly in samples with higher CT values by real-time PCR detection. At low V. cholerae concentrations, the sensitivity of the assay or the formation of fluorescent aggregates of bacteria may have been affected by the composition of estuarine water after enrichment. Moreover, this study used a small amount (10 μl) of enrichment broth for IFAG detection, and this small sample could possibility result in weak detection.
In environmental monitoring of cholera, a large number of samples is often required. Enrichment, isolation and identification, and conventional culture methods are labor intensive, with many time-consuming steps. This study demonstrated that during handling of samples for real-time fluorescent PCR screening, the real-time PCR-positive samples can be used for isolation with the use of IFAG. This process not only reduces the workload of detection but also improves the isolation rate.
Nontoxigenic V. cholerae
was isolated from the estuary water of the Pearl River in this study. Currently China has a smaller number of reported cholera cases each year than before, less than 200 cases during the most recent 3 years. In the year we conducted this study, fewer than 10 cases of cholera were found in the Guangdong province. In the interim period between cholera outbreaks, V. cholerae
bacteria present mainly as nontoxigenic strains (20
). However, if nontoxigenic strains abnormally increase during environmental monitoring, it would suggest that toxigenic strains may also be increasing under the same environmental conditions, which are suitable for propagation of the pathogen. Therefore, the method described in our study would help to more efficiently accumulate monitoring data for V. cholerae
in environmental waters, which can provide a basis for early warnings of cholera outbreaks.