For almost 50 years sub-Saharan Africa, including Uganda, has experienced several outbreaks due to Vibrio cholerae. Our aim was to determine the genetic relatedness and spread of strains responsible for cholera outbreaks in Uganda. Sixty-three V. cholerae isolates collected from outbreaks in Uganda between 2014 and 2016 were tested using multiplex polymerase chain reaction (PCR), multi-locus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS). Three closely related MLVA clonal complexes (CC) were identified: CC1,32% (20/63); CC2,40% (25/63) and CC3,28% (18/63). Each CC contained isolates from a different WGS clade. These clades were contained in the third wave of the 7th cholera pandemic strain, two clades were contained in the transmission event (T) 10 lineage and other in T13. Analysing the dates and genetic relatedness revealed that V. cholerae genetic lineages spread between districts within Uganda and across national borders. The V. cholerae strains showed local and regional transmission within Uganda and the East African region. To prevent, control and eliminate cholera, these countries should implement strong cross-border collaboration and regional coordination of preventive activities.
Vibrio cholerae remains a major cause of morbidity and mortality globally [1]. There have been seven cholera pandemics since the disease was recognized as a global threat [2]. The English record of pandemics of cholera started in 1816, but cholera as a disease goes back centuries in Indian literature [3]. The organism responsible for cholera outbreaks, V. cholerae, was cultured over 130 years ago by Robert Koch (1884) in India [4] and its epidemiology in England was described by John Snow in 1886 [5]. Over time, considerable knowledge and skills in the management of this deadly infectious disease have accumulated leading to better prevention and control of epidemics [6–8]. Industrialized countries essentially have eliminated cholera as a public health problem through improved water and sanitation [9]. Nonetheless, this enteric bacterium continues to cause deaths and suffering in many countries [10–12]. Sub-Saharan Africa bears the highest reported cholera disease burden [13]. The ongoing outbreaks in Africa and elsewhere in the world are part of the seventh pandemic caused by the V. cholerae O1, El Tor lineage [14,15]. Genetic differences among isolates allow for a greater understanding of the transmission of the bacteria within and between geographic regions and time periods [16]. Two methods, multilocus variable-number tandem-repeat analysis (MLVA) [17,18] and whole genome sequencing (WGS) [19], provide sufficient genetic differentiation to distinguish between the isolates across different places and times. Less complex methods such as culture, biochemical and serological tests to detect, confirm and describe V. cholerae [20], do not permit accurate tracking of the spread of specific genetic lineages. Yet these are the only methods available in most African countries including Uganda [21]. The goal of this study was to analyze V. cholerae isolates responsible for cholera outbreaks that occurred between 2014–2016 in Uganda using multiplex PCR, MLVA and WGS to determine the genetic relatedness and spread of V. cholerae isolates from different outbreaks in Uganda.
A cross-sectional study was conducted using all available viable V. cholerae isolates collected during cholera outbreaks in Uganda between 2014 and 2016 and kept frozen (-80°C) at the Central Public Health Laboratory (CPHL) in Kampala. In addition, aggregated epidemiological cholera surveillance data for the years 2014–2016 were reviewed and used to generate Epi-maps that contextualized the epidemic spread and transmission of cholera.
A total of 63 V. cholerae isolates for the years 2014–2016 were tested. The isolates were from 9 locations: 8 districts in Uganda and a ninth from patients who acquired their illness in Juba, South Sudan, and were treated in Uganda. All 63 isolates tested positive for ompW, toxR and ctxA indicating the presence of V. cholerae virulence genes. The isolates included both V. cholerae Inaba (63%) and Ogawa (34%) serotypes as shown in Table 1. All 63 V. cholerae isolates were genotyped using MLVA. Three clonal complexes (CC) were identified circulating in Uganda. MLVA CC1 contained 32% (20/63); MLVA CC2,40% (25/63); and MLVA CC3,28% (18/63) of the isolates. The three MLVA CCs are shown in Fig 1. The spatial distribution of MLVA CCs in Uganda reveals the presence of multiple genetic lineages within outbreaks and genetically defined connections between outbreaks (Fig 2). Two lineages were observed in 2014, when CCs 1 & 3 were isolated in Arua and Moyo districts in northwest Uganda. In 2015, CCs 1 & 3 were observed in Hoima and CCs 1 & 2 were isolated in Kasese district in southwest Uganda. Each separate CC identified one of three genetically related series of outbreaks. First, isolates from CC3 were observed in June 2015 in individuals from Juba, South Sudan, and later in July 2015 in nearby Arua district, Uganda. Additional isolates were seen further south in September 2015 in Hoima on Lake Albert in Uganda. A second outbreak, defined by CC2, was initially identified in April 2015 in Kasese district in western Uganda, and subsequently in November 2015 in Wakiso district in central Uganda, in December 2015 in Kampala district in central Uganda and in December 2015 in Moroto district in northeastern Uganda. This outbreak persisted into January 2016 when it was found in Kampala and Mityana in central Uganda and in Mbale district in eastern Uganda. A third outbreak, defined by CC1, contained isolates collected in May and July 2015 in Kasese district, Uganda, and in June 2015 in individuals from Juba, South Sudan. WGS genotyping of ten isolates indicated that the DNA was typical of the third wave of the seventh pandemic containing the classical allele of ctxA (S2 Table). The Ugandan DNA sequences belonged to three distinct clades. Within these distinct clades, the Ugandan sequences differed by five or fewer nucleotides (Fig 3). Two clades were contained in the transmission event (T) 10 lineage and the other was contained in T13; no Ugandan isolate sequences were contained in a third African lineage T12. The Ugandan clades were closely related to each other and to sequences from Democratic Republic of Congo and Tanzania (Fig 3). Clade 2 sequences from Kasese district in April 2015 were related most closely to sequences from Mbale district in January 2016 and secondarily to sequences from i) the Democratic Republic of Congo and ii) epidemic isolates from Dar es Salaam, Tanzania in August 2015 which spread across Tanzania during 2015. Clade 3 sequences from Arua and Moyo districts, Uganda in April and May 2014 and Clade 1 sequences from Kasese district, Uganda in April and May 2015 were related closely to sequences from an outbreak in January 2015 in Kigoma, Tanzania. The distance between the Ugandan and Tanzanian clades was nine or fewer nucleotides.
No V. cholerae isolates were collected and tested from a cholera outbreak in 2016 in northwestern Uganda that started with the influx of South Sudan refugees and was restricted to districts where the refugees settled and their immediate neighborhoods. However, since this outbreak was restricted to a few districts in northwestern Uganda with refugees, it is unlikely that this had an effect on the findings of this study. The cholera outbreaks in Uganda were due to genetically diverse V. cholerae O1 isolates from two introductions from wave 3 of the seventh pandemic carrying the classical El Tor toxin gene. The V. cholerae strains showed local and regional transmission within Uganda and East Africa. Interventions to prevent, control, and eliminate cholera in Uganda and throughout East Africa should be strengthened with a focus on regional collaboration.
Cholera, an acute diarrheal disease, essentially was eliminated in the western world many decades ago, but has continued to cause many deaths in sub-Saharan Africa, South America and Asia. Cholera diagnosis in most countries in sub-Saharan Africa, including Uganda, is by stool culture, serology and biochemical methods. These testing methods are unable to establish the relatedness, virulence and spread of Vibrio cholerae in region. To determine the spread, relatedness and virulence of V. cholerae responsible for the various cholera outbreaks in Uganda, we used DNA-based testing methods. We tested 63 V. cholerae isolates from samples collected in Uganda from 2014–2016. Our results showed three distinct lineages of genetically related cholera-causing bacteria. These organisms showed internal spread in Uganda and cross-border spread to neighboring countries in East Africa. These findings provide a valuable baseline and help define the context for directing control measures and technologies for cholera prevention in East Africa.
medicine and health sciences
ecology and environmental sciences
pathology and laboratory medicine
pathogens
vibrio
tropical diseases
microbiology
geographical locations
uganda
bacterial diseases
vibrio cholerae
tanzania
aquatic environments
bodies of water
neglected tropical diseases
molecular biology techniques
infectious disease control
bacteria
bacterial pathogens
africa
research and analysis methods
infectious diseases
cholera
artificial gene amplification and extension
medical microbiology
lakes
marine and aquatic sciences
microbial pathogens
molecular biology
people and places
freshwater environments
polymerase chain reaction
earth sciences
biology and life sciences
organisms
Molecular characterization of Vibrio cholerae responsible for cholera epidemics in Uganda by PCR, MLVA and WGS