JCM Accepts, published online ahead of print on 25 August 2010 J. Clin. Microbiol. doi:10.1128/JCM.00616-10 Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Running title: Diagnostic hindrances in cholera
Diagnostic Limitations to Accurate Diagnosis of Cholera
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Munirul Alam1*, Nur A Hasan1, Marzia Sultana1, G. Balakrish Nair1,2, A. Sadique1, A. S. G. Faruque1, Hubert Ph. Endtz1, R. B. Sack3, A. Huq4, R. R. Colwell3,4,5, Hidemasa Izumiya6, Masatomo Morita6, Haruo Watanabe6, and Alejandro Cravioto1.
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International Center for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh; National Institute of cholera and Enteric Diseases, Kolkata, India; 3Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 4Maryland Pathogen Research Institute, University of Maryland, MD; 5Center for Bioinformatics and Computational Biology, University of Maryland, MD; 6Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan. 2
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*Corresponding Author: Munirul Alam
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Enteric and Food Microbiology Laboratory,
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Lab Sciences Division,
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International Center for Diarrheal Disease Research, Bangladesh,
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Dhaka,
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Bangladesh
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Tel.: +88-02-8860523 – 32 Fax: +88-02-8812529, 8821116
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E-Mail. :
[email protected]
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1
Running title: Diagnostic hindrances in cholera 1
Abstract
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The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent
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diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although
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more than 40% of the suspected cases failed to show cholera etiology by conventional culture
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methods (CM). In the present study, suspected cholera stools collected from every 50th patient
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during an acute diarrheal outbreak were analyzed extensively using different microbiological and
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molecular tools to determine etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1
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by CM, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay;
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all but three samples positive for V. cholerae O1 by CM were also positive for V. cholerae O1 by
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DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V.
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cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct
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fluorescent antibody (DFA), 36 (73%) amplified primers for the genes wbeO1 and ctxA by
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multiplex-polymerase chain reaction (M-PCR), and 31 (63%) showed El Tor-specific lytic phage
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on plaque assay (PA). Each of these methods allowed cholera etiology to be confirmed for 97%
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of the stool samples. The results suggest that the suspected cholera stools that fail to show
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etiology by CM during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by
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in vivo vibriolytic action of the phage and/or non-culturability induced as a host response.
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Key Words: Diarrhea, Cholera, Diagnosis, V. cholerae
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Introduction 2
Running title: Diagnostic hindrances in cholera 1 2
Cholera is a harsh disease, the fundamental clinical feature of which is a severe dehydrating
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diarrhea that can lead to rapidly progressing dehydration and death. The recent cholera epidemics
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that occurred in South America (6), Asia (7), and sub-Saharan Africa (17) affected millions of
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people with a high mortality rate. The World Health Organization (WHO) annual figures on
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global cholera incidence (26), which are based on official cases reported by affected countries,
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are believed to be underestimated due to limitations related to a lack of adequate surveillance
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systems. In addition, the actual number of cholera cases globally is estimated to be much higher
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than officially reported (22) because outbreaks are often not reported to avoid the risk of travel
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and trade embargoes on the affected country. Prompt and accurate diagnosis of V. cholerae is a key step in cholera outbreak
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surveillance that can greatly influence rapid intervention and prevention to minimize disease
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spread and mortality. Conventional culture methods (CMs) currently used for diagnosis of V.
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cholerae remain the gold standard but this procedure is not precise and requires highly skilled
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technicians and laboratory infrastructure. In remote cholera-endemic settings where modern
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laboratory facilities are often non-existent, simple dark-field microscopy to detect cells showing
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characteristic darting-motility is used to identify V. cholerae in stool specimens. Diagnostic tests
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known as cholera dipstick (DS) assays, which involve either cholera toxin (3) or
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lipopolysaccharide (LPS) antigens (14,20), have been introduced for rapid bedside detection of
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V. cholerae in cholera stools. DS tests produce results comparable with those from the CMs;
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perhaps because both require the physical presence of V. cholerae cells, although only readily
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culturable cells respond when CMs are used. Fluorescent monoclonal antibody (5) and PCR-
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Running title: Diagnostic hindrances in cholera 1
based methods (12) have been proposed for detecting the cholera bacterium, including non-
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culturable cells present in stools.
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In recent diarrheal outbreaks in Bangladesh, analysis of acute diarrhea cases showed V.
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cholerae to be the most commonly identified causative agent, even when approximately half of
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the stool samples from suspected cholera patients did not have any defined etiology (21). In the
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present study, stool samples collected from suspected cholera patients who had fallen ill during
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recent seasonal outbreaks in Bangladesh were subjected to extensive analyses by CMs,
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multiplex-PCR, and direct fluorescent antibody (DFA) to determine etiology. The stool samples
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were also analyzed by plaque assay for the presence of vibriolytic phages in the stools.
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Materials and Methods
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Collection of stool specimens. Suspected cholera stool specimens were collected from
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patients enrolled in the 2% systematic routine surveillance system at the Clinical Research and
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Service Centre of the International Centre for Diarrheal Disease Research, Bangladesh
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(ICDDR,B). In this surveillance system, every 50th patient attending the hospital is screened for
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major enteric pathogens. The Dhaka hospital of the ICDDR,B identifies cases of cholera
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throughout the year and treats patients during large outbreaks of the disease. According to the
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hospital surveillance records, about 88% of the patients report to the ICDDR,B Dhaka hospital
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within
