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Keywords:

  • fecal microbiota;
  • healthy children;
  • Vibrio cholerae/mimicus

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. DISCLOSURE
  5. REFERENCES

During a double-blind, randomized, placebo-controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.

List of Abbreviations: 
CFU

colony forming unit

E. coli

Escherichia coli

RT-qPCR

reverse transcription rRNA-targeted quantitative PCR

V

cholerae/mimicus

Vibrio

cholerae/mimicus

Toxigenic Vibrio cholerae O1 and O139 cause the acute dehydrating diarrheal disease, cholera. Contaminated water and food, poor hygiene and sanitation, a lack of safe drinking water and the presence of well-documented environmental reservoirs play important roles in the appearance and subsequent spread of cholera. Most often, a convergence of optimal environmental and conducive epidemiological conditions results in explosive outbreaks of this disease. The roles of asymptomatic infections or short-term carriers in the spread of cholera has been speculated about, but never proven. One reason may be that it is difficult to document the presence of V. cholerae in asymptomatic infections because there are so few organisms that they are well below the threshold of detection of conventional culture methods. This study reports the chance finding of V.cholerae/mimicus during examination of fecal microbiota of healthy children in an urban slum during a randomized, controlled, probiotic trial. We detected V. cholerae/mimicus by using a sensitive culture independent method. We also discuss the importance of this finding in the local, regional and global transmission of cholera.

We conducted a double-blind, randomized, placebo-controlled, probiotic trial among 3758 children between 1 and 5 years of age in an urban slum in Kolkata, India over 24 weeks between 12 July 2007 and 5 January 2008 (1). The study involved 12 weeks of intervention with a probiotic drink versus an identical nutrient drink without the probiotic strain and a further 12 weeks of follow-up observation. During the 24 week study period, we examined feces of 133 randomly selected healthy children from both groups periodically (five times at 6 weekly intervals) to analyze the fecal microbiota, including investigating for the presence of enteric pathogens. Community health workers helped to collect the feces and transport them at 4°C to the laboratory. We extracted RNA from each fecal sample and performed a sensitive RT-qPCR to detect V. cholerae/mimicus and other pathogens as described previously (2). The sensitivity of the RT-qPCR was about 103 CFU/g of fecal sample, which is below the threshold of culture based techniques (2). One of the limitations of this technique was its inability to discriminate V.cholerae from V.mimicus.

We detected V. cholerae/mimicus in the feces of 70 (52.6%) of the 133 healthy children sampled five times each during the 24 weeks and extrapolated bacterial counts as shown in Table 1. We detected V. cholerae/mimicus in all five fecal specimens collected from one child during the study. Among the 70 children positive for V.cholerae/mimicus, we also detected Campylobacter coli/jejuni in 38 (54.3%) of the stool samples. We detected other enteric pathogens (different pathotypes of diarrheagenic Escherichia coli and rotavirus) sporadically during the study (Table 2).

Table 1.  Detection of Vibrio cholerae/mimicus in feces collected from 133 healthy children
Detection frequencyDetection rate(%)Bacterial count
  1. aNumber of positive subjects/Number of subjects tested

  2. bMean ± SD, log10 cells/g feces

142/133a31.64.2 ± 1.5b
221/13315.83.8 ± 1.0
36/1334.54.5 ± 1.3
40/1330.0
51/1330.84.3 ± 0.9
Total70/13352.64.1 ± 1.3
Twice or more28/13321.14.0 ± 1.1
Twice or more in a row17/13312.84.2 ± 1.1
Table 2.  Detection of other pathogens in feces collected from 70 carriers of V. cholerae/mimicus
PathogensDetection ratea(%)Bacterial (viral) countsb
  1. a Number of the positive carriers/number of total carriers

  2. b Mean ± SD, log10 cells (particles)/g feces

Enterotoxigenic E. coli1/701.4
Enterohemorrhagic E. coli1/701.4
Enterohemorrhagic E. coli2/702.9
 and/or enteropathogenic   
 E. coli   
Campylobacter jejuni/coli 38/7054.37.5 ± 1.6
Rotavirus2/702.95.4
Total41/7058.6

In the 1960s, short term human carriers of V. cholerae O1 who intermittently excreted the pathogen in the absence of overt disease were documented in several studies conducted in the Philippines (1964–66), and in Kolkata (then Calcutta), India (1966–67) (3, 4). These studies were based on isolation of the pathogen by culture and led the World Health Organization Expert Committee on Cholera to conclude that “the carrier often serves as the source of infection and can be of great importance in the persistence of the disease and in its transmission, within a given population or even between neighboring countries” (5). Therefore, at that time human-to-human transmission was thought to be the mode of spread of cholera. In the 1970s, however, Colwell and coworkers (6) demonstrated that V. cholerae is autochthonous to the aquatic environment and highlighted the role of the environment in the origin, maintenance and transmission of cholera.

The rRNA–targeted quantitative PCR (RT-qPCR) used in this study is limited by its inability to distinguish between V. cholerae and V. mimicus, which are closely related as shown by comparative genomic analysis (7) but whose impact on human disease may be different. Both at the community level and at the infectious diseases hospital located close to the urban slums in Kolkata at which the fecal samples from healthy children were examined for this study, V. cholerae O1 is the main pathogen identified, whereas V. mimicus is rarely isolated as a cause of diarrhea (8). The annualized cholera incidence in slum settings in Kolkata, which have a high population density and where residents do not have sufficient water supply or sanitary facilities, is around 1.6 per 1000 population (9). It is therefore rational to assume that most of the rRNA-positive feces contained V. cholerae rather than V. mimicus. However, the V. cholerae detected by the RT-qPCR technique used in this study should be more extensively assessed to determine their serotypes, virulence potential and cholera toxin production. Further to confirm the conclusions reached based on the culture independent methods used in this study, efforts need to be made to isolate V. cholerae O1 using recently developed specific techniques for shifting viable but non-culturable V. cholerae into a culturable state by co-culturing them with eukaryotic cells (10). We are planning such studies to further explore asymptomatic carriage of V. cholerae.

The data emerging from this study shows substantial asymptomatic infection (carriage) of V. cholerae/mimicus in children in urban slum settings. Recent studies in Bangladesh have shown that asymptomatic cases represent roughly half of all cases and that these might contribute to the spread of the organism (11). V. cholerae has been isolated from rectal swabs of healthy family members who have had contact with cholera patients (12). Given the current debate on transmission of cholera following the cholera outbreak that started in October 2010 in Haiti, a previously cholera-free Caribbean island (13), this study emphasizes the importance of cholera carriers in the transmission of cholera. Morris has proposed a model of transmission in which environmental triggers lead to increases in V. cholerae in environmental reservoirs with “spill over” into human populations (14), after which transmission occurs primarily by fast transmission from person to person taking advantage of the hyperinfectious state without a return to the environment (15). Therefore, asymptomatic infections in cholera endemic areas play a role in the spread of cholera, especially when there is mass movement of populations without overt symptoms of cholera from cholera endemic to cholera-free areas. Such an event could pose a real risk of the spread of cholera.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. DISCLOSURE
  5. REFERENCES

This study was supported by the Japan Initiative for Global Research Network on Infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology, Japan.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. DISCLOSURE
  5. REFERENCES

No authors have any conflicts of interest to declare.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. DISCLOSURE
  5. REFERENCES
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