Ghana is affected by regular cholera epidemics and an annual average of 3,066 cases since 2000. In 2014, Ghana experienced one of its largest cholera outbreaks within a decade with more than 20,000 notified infections. In order to attribute this rise in cases to a newly emerging strain or to multiple simultaneous outbreaks involving multi-clonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012. Serotypes, biotypes, antibiotic susceptibilities were determined for 92 Vibrio cholerae isolates collected in 2011,2012 and 2014 from Southern Ghana. For a subgroup of 45 isolates pulsed-field gel electrophoresis, multilocus sequence typing and multilocus-variable tandem repeat analysis (MLVA) were performed. Eighty-nine isolates (97%) were identified as ctxB (classical type) positive V. cholerae O1 biotype El Tor and three (3%) isolates were cholera toxin negative non-O1/non-O139 V. cholerae. Among the selected isolates only sulfamethoxazole/trimethoprim resistance was detectable in 2011, while 95% of all 2014 isolates showed resistance towards sulfamethoxazole/trimethoprim, ampicillin and reduced susceptibility to ciprofloxacin. MLVA achieved the highest subtype discrimination, revealing 22 genotypes with one major outbreak cluster in each of the three outbreak years. Apart from those clusters genetically distant genotypes circulate during each annual epidemic. This analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs. Rapidly emerging multidrug resistance must be monitored closely.
The World Health Organization (WHO) estimates that 3–5 million annual cases of cholera occur worldwide, resulting in 100,000–120,000 deaths [1]. Since its introduction to Africa during the current seventh cholera pandemic in 1970, Vibrio cholerae caused regular vast epidemics across the continent with cumulative 3,762,902 case-notifications to WHO by 2013 [2]. In 2013 alone, 22 African countries reported 56,329 cholera cases. However, these numbers are considered to be substantially underestimated due to poorly functioning national epidemiological and laboratory surveillance systems, which are not able to detect the majority of mild disease presentations. Furthermore, systematic underreporting is common to avoid economic and political damage [3,4]. The first cholera case has been reported from Ghana in 1970 [5]. With 90% of the population at risk for cholera transmission, Ghana always ranks among the most affected countries on the African continent [4]. Ghana notified an annual average of 3,066 (range: 50–10,628) cholera cases between 2000 and 2013 with an overall case fatality rate of 1. 7% [2]. In the year 2014, Ghana experienced an exceptionally large cholera outbreak with 28,975 infections notified to the World Health Organization between June and November [6]. Causes of this sudden increase in case numbers might be very diverse and calls for investigation in a timely manner so as to implement preventive measures accordingly. This recent epidemic might be explained by the extrinsic introduction of a new V. cholerae strain into a susceptible population, as seen for cholera outbreaks in Zimbabwe and Haiti in the years 2009 and 2011 respectively [7,8]. Alternatively, multiple, endemic, genetically non-related strains might be responsible for concomitant epidemics, as reported from Kenya [9]. In the latter scenario V. cholerae might persist in aquatic reservoirs and regular epidemics are triggered by low hygienic conditions and climatic factors such as increased rainfall or flooding [10–12]. In recent years, a number of molecular typing tools have been developed to elucidate the source and evolution of V. cholerae outbreaks [13]. This study aims to describe the 2014 cholera epidemic in Ghana and uses molecular subtyping techniques to detect responsible newly emerging or multi-clonal strains, which are then compared to strains that circulated during the 2011 and 2012 epidemics. Results will advise public health authorities whether to focus on monitoring of endemic environmental reservoirs or rather on surveillance of mobile populations, with cross-border epidemiological collaborations to prevent importation of V. cholerae.
Within the Ghana Health Service, the Disease Surveillance Service supported by the National Public Health & Reference Laboratory (NPHRL) conducts cholera surveillance in Ghana. A case of cholera is defined according to the WHO standard case definition: If cholera is not known to be present in the area, a case of cholera is considered in a patient ≥5 years with severe dehydration or death from acute watery diarrhea, while during a cholera epidemic every patient aged ≥5 years with acute watery diarrhea and/or vomiting is considered as a case. Standardized line lists with suspected cholera cases are provided on a weekly basis by the District Health Management Teams to the Central Disease Surveillance Service in Accra, which collates information on name, place of residence, sex, age, disease onset, disease outcome and hospitalization of cases. A small subset of specimens is usually tested in peripheral laboratories by various methods and results are notified in the line lists. For laboratory confirmation, district and regional laboratories are encouraged to send suspected cholera stool samples to the NPHRL, where culture and serotype identification is performed. For the purposes of this study, all suspected cholera cases in the surveillance database from six Southern Ghanaian regions (Greater Accra, Central, Western, Eastern, Ashanti, Volta) from the year 2014 were extracted and described by sex, age and place of residence. Continuous variables were summarized as means with standard deviation (median with interquartile range for non-normally distributed variables) and dichotomous or categorical variables were summarized as proportions/percentages. Missing values were excluded from the analysis, thus the denominators for some comparisons differ. Surveillance data were computerized using Excel (Microsoft, USA) and statistical analysis was performed with Stata v. 12. 1 (Statacorp, Texas, USA). For spatio-temporal visualisation case data were imported into Arc GIS 10. 0 (ESRI: ArcGis Desktop: Release 10. 2011). Suspected cases were linked to the respective notifying districts. For the spatio-temporal visualisation, case data/district were then aggregated in six temporal groups, each covering five outbreak weeks.
A total of 20,185 cases of cholera were reported to the Ghanaian Disease Surveillance Center from the Ashanti-, Central-, Eastern-, Greater Accra-, Western- and Volta regions in 2014. The date of disease onset was reported for 20,120 cases, the remaining 65 cases were removed from the dataset for all other analyses. The earliest reported onset date was the 20th of May 2014 and the latest was the 11th of December 2014, with a peak number of 2,853 cases in the 35th calendar week (25–31 August; Fig 1). Age was reported for 19,863 cases and distributed with a median age of 26 years and an interquartile range (IQR) of 20–35 years. The median age of cases did not change during the course of the outbreak. The majority of cases was male (58. 4%; n = 11,796), and median age was not markedly different between males (26 years; IQR 20–35) and females (25 years; IQR 19–35). The case fatality rate (CFR) was 0. 8% (165 deaths) with a higher median age among deceased of 34 years (IQR 24–47). Laboratory testing was performed in regional laboratories with rapid diagnostic tests from different suppliers for 496 out of 20,120 (2. 5%) suspected cases with a positivity rate of 53% (264/496). Spatio-temporal analysis traced back the first cases during the first five weeks of the outbreak to four districts in the Eastern and Greater Accra regions (Fig 2). During the peak of the outbreak (outbreak week 6–20) the majority of cases were centred around the city of Accra, spreading in northward-direction to the Ashanti region and to the East along the coast. During the last 10 weeks of the outbreak, only three districts within the Ashanti and the Volta region reported cases. Antimicrobial susceptibility testing identified mono-resistance towards sulfamethoxazole/trimethoprim in 100% (18/18) of isolates from 2011 (Table 1). The resistance pattern changed in 2012 with 75% (9/12) of isolates expressing resistance against sulfamethoxazole/trimethoprim, nalidixic acid in combination to reduced susceptibility against ampicillin and ciprofloxacin. The same resistance profile was detected in 95% (59/62) of isolates in 2014. No resistance against chloramphenicol, gentamycin or tetracycline was observed for any of the isolates. The selected 92 V. cholerae isolates consisted of 88 (95. 7%) V. cholerae O1 biotype El Tor serotype Ogawa, 1 (1. 1%) V. cholerae O1 biotype EL Tor serotype Inaba and 3 (3. 3%) non-01/non-O139 V. cholerae. Apart from the non-O1/non-O139 V. cholerae strains with no cholera toxin production, all isolates produced the classical type cholera toxin. Out of these 92 isolates, 11 isolates from 2011, eight isolates from 2012 and 26 isolates from 2014 were genotyped by MLST, PFGE and MLVA (S1 Fig). All V. cholerae O1 belonged to the sequence type (ST) 69, while the three non-O1/non-O139 isolates presented with new alleles (isolate 059: metE allele 108; isolate 160: metE allele 109; and isolate 169: metE allele 110 and pyrC allele 85) and could not be allocated to any known ST. Theses strains were assigned new ST numbers (isolate 059: ST211, isolate 160: ST212 and isolate 169: ST213; corresponding to IDs 196,197 and 198 in the V. cholerae pubmlst database: http: //pubmlst. org/vcholerae/), which are closest related to the already known ST40 and ST39. As shown in Fig 3, using a cutoff of 95%, PFGE divided all strains into three main clusters and 11 pulsotypes (Fig 3). All 2011 isolates clustered together (cluster A) as 4 different pulsotypes. Isolates from 2012 belonged to 4 different pulsotypes dispersed in clusters A, B and C. All V. cholerae O1 from 2014 showed the same pulsotype and were grouped in cluster B. Finally, the non-O1/non-O139 isolates collected in 2014 showed different pulsotypes and were grouped outside clusters A, B or C. Neither regional nor district specific pulsotypes had been detected. MLVA differentiated the isolates into three clonal complexes (CC1-CC3) and 22 genotypes (Fig 4). The majority of 22 out of 26 isolates from 2014 clustered within CC1 together with one isolate from 2012. Distinct from the CC1 were the three non-O1/non-O139 and one O1 V. cholerae isolate (Isolate 146) from Ledzekuku-Krowor District (Greater Accra Region). For 2012, five out of eight isolates grouped together in CC2, while the other three isolates were genetically distinct, within or close to CC1 and CC3. Similarly, 10 of the 11 isolates from 2011 were classified into CC3, and one strain clustered outside with three alleles difference to the other CC3 strains. Analogous to the PFGE distribution, subtypes did not cluster by region or by district.
The Ghanaian Cholera outbreak in 2014 was caused by Vibrio cholerae O1 biotype El Tor carrying the classical cholera toxin. Molecular subtyping data of three outbreak years illustrate major annual outbreak clusters with co-circulating genetically distant genotypes, which might hint to an endemic reservoir of V. cholerae in Ghana. Public health authorities must be vigilant and take steps to prevent cholera transmission through aquatic reservoirs, particularly within urban agglomerations during the start of the rainy season. Considering the rapidly emerging multidrug resistance among V. cholerae isolates, laboratories are encouraged to monitor antimicrobial susceptibility closely.
The bacterium Vibrio cholerae is mainly transmitted faecal-orally via human-to-human contact or via environmental water sources in which V. cholerae is able to persist. West Africa, including Ghana, is regularly affected by Cholera epidemics, in particular during rainy seasons. In 2014, Ghana experienced an exceptionally large outbreak with over 20,000 cases, which raised questions about newly emerging V. cholerae strains in this region. In this study, we described the duration, the geographical spread and demographics of the outbreak using data from the National Ghanaian Surveillance system. Further, we characterized outbreak isolates from the outbreak years 2011,2012 and 2014 by three different subtyping methods. These analyses revealed strains with different genetic background and increasing antibiotic resistance circulating during each outbreak year. These data suggest that V. cholerae has an endemic reservoir in the environment and selection pressure results in a highly heterogeneous population of V. cholerae with a few strains evolving into pathogenic clones during each outbreak period. Public health authorities must be vigilant to prevent cholera transmission through aquatic reservoirs, particularly within urban agglomerations during the start of the rainy season. The rapidly emerging antibiotic resistance has to be monitored closely.
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Molecular Epidemiology and Antibiotic Susceptibility of Vibrio cholerae Associated with a Large Cholera Outbreak in Ghana in 2014