A Molecular and Epidemiological Study of Cholera Outbreak in Sulaymaniyah Province, Iraq, in 2022

Cholera is an enteric bacterial infectious disease caused by a Gram-negative, motile, flagellated bacterium Vibrio cholerae (Harris et al. 2012). The infection occurs from consuming vegetables and/or drinking water contaminated with the pathogen (Harris et al. 2012). The disease is particularly a problem in developing countries where poor sanitation, and lack of awareness and/or access to clean drinking water exist (Ali et al. 2015; Ramazanzadeh et al. 2015).

More than 200 serogroups of the bacteria have been identified so far, and cholera outbreaks mainly occur during the hot months of summer elicited by the O1 or O139 biotype (Ali et al. 2015; Ramazanzadeh et al. 2015). Severe watery diarrhea, known as “rice-watery” stool, odorless or of a mild fishy smell, with or without vomiting, are the major symptoms of the disease (Harris et al. 2012; Endris et al. 2022). Among different age groups, children below five years old are the most vulnerable to cholera compared to adults or the elderly (Deen et al. 2008). Additionally, people suffering from other diseases such as gastric disease and/or those individuals with type-O blood groups are at higher risk of getting cholera than other blood group types (Braunwald et al. 2001).

Cholera is considered a global health issue, and it has affected most parts of the world since the early of the nineteenth century (Harris et al. 2012). A majority (~ 99%) of cholera cases have been recorded in Africa and Southern Asia (Deen et al. 2008; Endris et al. 2022). In 2006, more than two million cholera cases with 6,311 deaths were recorded in 52 countries (WHO 2007a). Since 2020, 323,320 cholera cases have been reported in 27 countries, with 857 deaths (WHO 2021). In Iraq, the first recorded cholera epidemic dates back to 1820 in Basra, which resulted in many deaths (Al-Abbassi et al. 2005). Since 1966, cholera has been considered an endemic disease in different parts of Iraq (Hussain and Lafta 2019). In 2007, a study was conducted among 6,399 children suspected of cholera in the city of Kirkuk-Iraq and showed that 326 (5.1%) of them were infected with V. cholerae, of which five had died from the infection (Noaman et al. 2011). In another study, 4,667 cholera cases were reported in Iraq in 2007; 136 (2.9%) of them were recorded in the capital city of Baghdad, and about 33% of the confirmed cases were individuals between the age of (0–4) and (15–45) years old (Khwaif et al. 2010).

In 2022, a new cholera outbreak hit different cities of Iraq. According to the official data of the World Health Organization (WHO), 449 confirmed cholera cases were recorded in Iraq (WHO 2022). Here, we report the cholera outbreak data analysis and molecular identification of the pathogen in Sulaymaniyah from the beginning of June to the end of July 2022.

Isolation and identification of the pathogen using biochemical and serological tests. All the 414 positive cases recorded within two months study were confirmed using bacteriological and serological tests. Initially, isolation and preliminary identification of V. cholerae were carried out by culturing aliquot from APW on the TCBS media and checking for the characteristic colonies of V. cholerae. The preliminary indications of the V. cholerae were yellow, shiny, round, smooth, and slightly flattened colonies on the TCBS media (Fig. 1).

Fig. 1.

To identify the bacteria the oxidase test, KIA test, and serological tests were used. The V. cholerae isolates were oxidase positive (Fig. 2A); acid/alkaline, did not produce gas nor H₂S in the KIA test (Fig. 2B). All the positive tested samples were agglutination positive with monovalent O1 antiserum. The biotype O139 was not detected among the tested samples, and the majority of the samples reacted to monovalent Ogawa antiserum (Fig. 2C).

Fig. 2.

Identification of V. cholerae using molecular and phylogenetic analysis. The partial sequences of the 16S rRNA genes from three random bacterial isolates were used for molecular identification of the etiological agent of the outbreak. DNA sequencing and phylogenetic analysis revealed that the isolates belonged to V. cholerae species. The partial sequences of the three isolates were submitted to the GenBank with the following strain names: V. cholerae strain Suli1 (Accession number: OP470022), V. cholerae strain Suli2 (Accession number: OP470023), and V. cholerae strain Suli3 (Accession number: OP470024). Additionally, the phylogenetic tree showed that the 16S rRNA sequences of the isolates formed a clade with other V. cholerae strain ATCC^® 14035^™ (NR 119302), V. cholerae strain CECT 514 (NR 044853), and V. cholerae strain RC782 (NR 044050) (Fig. 3).

Fig. 3.

Epidemiology of the disease. During two months of the study, 4,754 suspected cases were admitted to the Shar hospital/Sulaymaniyah city, Iraq (3,474 cases in June and 1,280 in July). The percentage of males to females in June and July were 51%:49% and 47%:53%, respectively. It was noticed that the majority of the patients were 20–44 years old (61.5% in June and 61.8% in July) (Table I). The number and the percentage of the suspected age groups are shown in Table I. Out of 3,474 suspected cholera cases in June, 391 (11.3%) were diagnosed as cholera-positive, of which 222 were males, and 169 were females. Whereas the number of positive cases decreased to only 23 (1.8%) cases out of 1,280 suspected cholera cases in July of which 14 of them were male, and nine were female (Fig. 4).

Fig. 4.

The mean and the standard deviation of the male and female cholera cases were 56.61 ± 52.8 and 55.4 ± 46.37 in June, respectively. In July, the mean and the standard deviation data were 19.22 ± 7.54 and 22.06 ± 10.3 for males and females, respectively.

Statistically, the result (Mann-Whiney U-test) of the comparison between both genders for each month showed that there is no significant difference (p-value > 0.05) among the genders (Fig. 5A).

Fig. 5.

The data of male and female genders that were admitted into the hospital in both months were further analyzed for both June and July. The results have demonstrated that there was a significant difference (p-value < 0.05) in the number of males in June and July as well as the number of females in June and July that were admitted to the hospital (Fig. 5B). Furthermore, the mean and standard deviation for the number of males in June and July were 58.50 ± 52.64 and 18.62 ± 8.16 respectively; and for females in June and July, were 57.30 ± 45.99 and 21.37 ± 10.86, respectively.

Cholera is among the serious global health issues, particularly in Africa and southern Asia; according to WHO, it infects around 1.3 to 4 million people and causes between 21,000 to 143,000 deaths worldwide each year (Chowdhury et al. 2022). Several cholera outbreaks have been recorded in Iraq; the latest one was started at the end of May 2022, and according to a WHO report, 449 confirmed cases in Iraq by 24 July 2022 (WHO 2022). Here, we carried out a molecular and epidemiological study of the latest cholera outbreak in the Sulaymaniyah province of Iraq for the first time. The data presented here were from the public Shar hospital during the outbreak peak from the beginning of June to the end of July 2022.

Bacteriological, biochemical, and molecular identification of the isolates confirmed that all the isolates from this study belonged to the same biotype. Therefore, the etiological agent of the cholera outbreak in the Sulaymaniyah province in 2022 was V. cholerae biotype O1, and Ogawa was a dominant serotype. Preliminary identification showed that these bacteria were oxidase positive and changed the TCBS media’s color from green to yellow; they produced acid/base, but did not produce gas nor H₂S in the KIA test (Swanson and Gillmore 1964; Lányi 1988). Moreover, serological tests revealed that isolates belonged to biotype O1, and Ogawa was a dominant serotype. Previously, it has been reported that the cholera outbreak of 2015 in Baghdad was caused by V. cholerae O1, biotype El Tor, and serotype Inaba (Jameel et al. 2016). Another study of 80 clinical and five environmental V. cholerae isolates from the outbreak in Iraq in 2007–2009 revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotype, and 12.5% to the Non-O1 serotype, while all the environmental isolates belonged to the Non-O1 serotype (Saleh et al. 2011). In 1995, WHO reported a cholera outbreak in Sulaymaniyah governorate that resulted in 264 confirmed cases and three deaths. The etiological agent was identified as V. cholerae O1 serotype Ogawa (WHO 1995). A comprehensive review of the cholera outbreaks in the Kurdistan region covering seven cholera outbreaks between 1995 and 2022 indicated that both Inaba and Ogawa serotypes of V. cholerae, O1, El Tor individually caused these outbreaks (Sidiq 2022). In the outbreaks in 1995, 1998, 1999, and 2012 in Iraq and the Kurdistan Region the serotype Ogawa was dominant, while only the outbreaks of 2007 and 2015 were caused by the Inaba serotype (WHO 1995; Yassin 2002; Al-Abbassi et al. 2005; Kami 2007; WHO 2007b; Agha et al. 2008; Health Cluster 2015; Zgheir et al. 2019).

Molecular identification of the three randomly selected isolates revealed that these isolates were V. cholerae, and all three isolates were closely related to each other and also to V. cholerae strains ATCC^® 14035^™ (NR_119302), CECT 514 (NR_044853), and RC782 (NR_044050). The high sequence identity (> 99.9%) of the randomly selected V. cholerae isolates could suggest a common source origin of the pathogen before spreading in the city. Since no environmental sampling was tested in this study, the exact source of the infections remains to be determined. However, based on experimental data reported by WHO on 24 July, 11% of the water samples collected from different sources in Iraq were contaminated with V. cholerae (WHO 2022).

Additionally, the polluted water of the Tanjero River, which has been used to irrigate leafy vegetables in the region, can be a possible source of infection. Various waste materials and pollutants are constantly dumped into the Tanjero River in Sulaymaniyah, including sewage, industrial, and agricultural wastes (Aziz et al. 2012; Rahman et al. 2021). Previous studies have demonstrated that polluted lakes, rivers, and food were sources of cholera outbreaks in different regions of the world (Albert et al. 1997; Griffith et al. 2006; Bompangue et al. 2008; Dinede et al. 2020). It supports the hypothesis further that using polluted water of the Tanjero River for irrigation could have been the cause of the cholera outbreak in Sulaymaniyah.

The epidemiological data demonstrated that the number of people admitted to the hospital with cholera symptoms was significantly higher in June than in July: 3,474 and 1,280, respectively. The number of confirmed cholera-positive cases was also higher in June than in July: 391 and 23, respectively. Generally, acute diarrhea and vomiting conditions among people are more prevalent during summer due to contaminated food and drinking water usage. People drink more water, possibly contaminated, during summer to replace lost fluid because of the hot weather, which can also favor the growth of microorganisms on foods and cause food poisoning (Ramos-Alvarez and Sabin 1958; Anderson et al. 2020). Additionally, cholera outbreaks in summer could be due to the hot atmospheric air and low water level, which provide perfect conditions for bacterial growth (Asadgol et al. 2019). However, decreasing the number of cholera case in July could be due to the governmental action plan against the outbreak, such as prohibiting using contaminated products, treating contaminated water sources, and raising public awareness (Jensen et al. 2006; Panja et al. 2016).

Moreover, lytic bacteriophages can destroy the bacteria soon after being discharged into the water following their spread (Jensen et al. 2006; Panja et al. 2016). Some studies have found that vibrio phage can limit bacterial density and growth, and the severity of outbreaks can largely depend on the density of the phage (Jensen et al. 2006; Ujah et al. 2015; Panja et al. 2016; Asadgol et al. 2019). The transmission of cholera from person to person is more severe than those acquired from the environment (Harris et al. 2012).

In our study, only a small portion 391 (11.3%), and 23 (1.8%)) of the suspected individuals were confirmed as cholera cases in both June and July, respectively. No death was reported in the 2022 cholera outbreak in the Sulaymaniyah province; however, three deaths were recorded in the whole of Iraq; two of them were in the city of Kirkuk and one in Baghdad (WHO 2022). Besides V. cholerae, other pathogens such as Entoameba histolytica can be another causative agent of acute diarrhea and amebiasis is an important public health problem worldwide (Hameed et al. 2021). Previously, E. histolytica was identified among children suffering from diarrhea in the Kirkuk and Duhok provinces of Iraq (Hussein and Meerkhan 2019; Hameed et al. 2021). In addition to E. histolytica, Giardia lamblia could be a reason for acute diarrhea, and the parasite has been previously isolated from the Euphrates River in AL-Nasiriyah City in Southern Iraq (Al Jassas et al. 2022). The enteric parasites heavily contaminate the leafy vegetables in Baghdad city (Khalil 2019). Significant reasons for acute diarrhea and cholera outbreak are contaminated water sources, especially from artesian wells, house tanks, shallow wells in mosques, or water from tankers sold to people (WHO 2022). Several factors are involved in cholera’s prevalence, including poor sanitation and hygiene, lack of qualified healthcare, poor infrastructure, and contamination of drinking water sources with sewage system (Ujah et al. 2015; Oguttu et al. 2017).

Moreover, our results showed that the numbers of cholera cases were slightly, but not significantly, higher among males than females; people aged 20–44 were more affected by the disease. It could be because people at this age, especially males, are more frequently eating outside, which might have increased their chances of getting exposed to different pathogens, including V. cholerae. A study of a cholera outbreak in Uganda in 2011 indicated that cholera cases were higher in males than females by 1.6 times (Bwire et al. 2017). A prospective study in fishing villages in Uganda between 2011–2015 covering ten cholera outbreaks showed that case-fatality ratio was significantly higher among males than females (Bwire et al. 2017).

The recurrent cholera outbreak in Iraq indicates that efforts are needed to establish safe drinking water, proper sanitation, and treatment of sewage water, strengthen primary health care, establish disease surveillance mechanism, and improve the nation’s awareness and preparedness to detect and respond to cholera outbreaks in the future rapidly.

A cholera outbreak is a bacterial disease caused by the Gram-negative bacterium V. cholerae after consuming contaminated food and water. Here for the first time, we reported molecular and epidemiological data on the 2022 cholera outbreak in the Sulaymaniyah province that revealed the incidence rate and identified the strain as V. cholerae Ogawa serotype. We found that the number of diagnosed people was higher in June compared to July. Also the majority (> 60%) of the cholera cases were recorded among people aged 20–44 years in both months. This study provides an insight into the incidence rate of the cholera outbreak in Sulaymaniyah province during June and July 2022. It reveals the causative bacteria strain than can help authorities develop preventive strategies to stop such outbreaks in the future.