# Outbreak of Imported Seventh Pandemic Vibrio cholerae O1 El Tor, Algeria, 2018

Nabila Benamrouche, Chafika Belkader, Elisabeth Njamkepo, Sarah Sihem Zemam, Soraya Sadat, Karima Saighi, Dalila Torkia Boutabba, Faiza Mechouet, Rym Benhadj-Slimani, Fatma-Zohra Zmit, et al.

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Nabila Benamrouche, Chafika Belkader, Elisabeth Njamkepo, Sarah Sihem Zemam, Soraya Sadat, et al. Outbreak of Imported Seventh Pandemic Vibrio cholerae O1 El Tor, Algeria, 2018. Emerging Infectious Diseases, 2022, 28 (6), pp.1241-1245. 10.3201/eid2806.212451. pasteur-03705990

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# Outbreak of Imported Seventh Pandemic *Vibrio cholerae* O1 El Tor, Algeria, 2018

Nabila Benamrouche, Chafika Belkader, Elisabeth Njamkepo, Sarah Sihem Zemam, Soraya Sadat, Karima Saighi, Dalila Torkia Boutabba, Faiza Mechouet, Rym Benhadj-Slimani, Fatma-Zohra Zmit, Jean Rauzier, Farid Kias, Souad Zouagui, Corinne Ruckly, Mohamed Yousfi, Amel Zertal, Ramdane Chouikrat, Marie-Laure Quilici, François-Xavier Weill

After a lull of >20 years, Algeria experienced a cholera outbreak in 2018 that included 291 suspected cases. We found that outbreak isolates were *Vibrio cholerae* O1 serotype Ogawa from seventh pandemic El Tor sublineage AFR14, which corresponds to a new introduction of cholera into Africa from South Asia.

Cholera, a life-threatening diarrheal disease, is caused by *Vibrio cholerae* O1, or more rarely O139, serogroup bacteria that produce cholera toxin (CTX) and induce rapid massive loss of body fluids (1). Cholera has been a serious public health problem since its introduction into Africa in 1970, during the seventh cholera pandemic (2). This pandemic, caused by the novel *V. cholerae* O1 lineage El Tor (seventh pandemic El Tor), began in Indonesia in 1961 (2,3). After 60 years, ≈2.9 million cholera cases and ≈95,000 deaths still occur annually (4,5). During 2009–2012, nearly 60% of global cholera cases and deaths occurred in sub-Saharan Africa, but North Africa was considered cholera-free (5).

Algeria is a large country (2,381,741 km²) in North Africa (6). The World Bank (https://www.world-bank.org) ranks Algeria as the third largest economy

in the Middle East and North Africa region. In 2018, Algeria had ≈42.2 million inhabitants, ≈30.6 million of whom lived in urban areas (Macrotrends LLC, https://www.macrotrends.net).

Algeria reported cholera cases to the World Health Organization from 1971 (1,332 cases, 110 deaths) through 1994 (118 cases, 4 deaths), with a peak in 1979 (2,513 cases, 94 deaths) (Global Health Observatory, https://www.who.int/data/gho) (Figure 1). After a lull of >20 years, on August 23, 2018, the country's ministry of health announced a cholera outbreak in north Algeria (7). During August 7–September 27, 2018, Algeria reported 291 suspected cholera cases, including 270 persons who were hospitalized, in 7 wilayas (provinces): 6 in north-central Algeria (Bouira, Blida, Algiers, Tipaza, Aïn Defla, and Médéa) and 1 in northwest Algeria (Oran).

We used conventional microbiology and whole-genome sequencing to characterize virulence and antimicrobial resistance of clinical and environmental isolates collected during this outbreak. We also performed a phylogenomic analysis of >1,200 seventh pandemic El Tor genomes to determine whether the 2018 outbreak in Algeria was caused by a sublineage previously circulating in the country, a sublineage circulating in sub-Saharan Africa, or a new sublineage imported from elsewhere.

## The Study

The Enterobacteria Laboratory of the Institut Pasteur d'Algérie performed microbial analyses for case confirmation (Appendix 1 Table 1, https://wwwnc.cdc.gov/EID/article/28/6/21-2451-App1.xlsx; Appendix 2, https://wwwnc.cdc.gov/EID/article/28/6/21-2451-App2.pdf). During August 14–September 27, 2018, this laboratory received 695 stool

Author affiliations: Institut Pasteur d'Algérie, Algiers, Algeria (N. Benamrouche, C. Belkader, S.S. Zemam, S. Sadat, D.T. Boutabba, R. Benhadj-Slimani, F. Kias); University of Algiers (I, Algiers (N. Benamrouche, F. Mechouet, F.-Z. Zmit, A. Zertal); Institut Pasteur, Université Paris Cité, Paris, France (E. Njamkepo, J. Rauzier, C. Ruckly, M.-L. Quilici, F.-X. Weill); Public Hospital Establishment of Boufarik, Blida, Algeria (K. Saighi, M. Yousfi, R. Chouikrat); Specialized Hospital Establishment El Hadi Flici, Algiers (F. Mechouet, F.-Z. Zmit, A. Zertal); University Hospital of Oran and University of Oran I, Oran, Algeria (S. Zouagui)

DOI: https://doi.org/10.3201/eid2806.212451
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Cholera cases and seventh pandemic Vibrio cholerae O1 biotype El Tor sublineages, Algeria, 1971–2018. A) Number of cholera cases reported to the World Health Organization (WHO) by Algeria per year. For 2018, no cases were reported to WHO, but 291 suspected cases are indicated. B) Number of sequenced genomes detected from various sublineages per year of isolation. Orange circles indicate AFR1, green indicate AFR7, blue indicates AFR 8, purple AFR9, gold AFR14. Numbers in circles indicate the number of isolates.</figcaption></figure>

samples from hospitals or hygiene laboratories in 7 wilayas, 277 from suspected case-patients and 418 from case-contacts, as well as 24 clinical isolates (14 from patients and 10 from case-contacts) and 5 environmental isolates (2 from wastewater, 2 from public drinking water sources, and 1 from stored water) for confirmation. In all, we confirmed 97/291 (33.3%; 95% CI 28.2%–38.9%) suspected cases as V. cholerae O1 El Tor serotype Ogawa carrying the *ctxA* gene and 29/428 (6.8%; 95% CI 4.8%–9.6%) case-contacts as asymptomatic carriers. Of the 5

environmental isolates, we also confirmed 2 from wastewater and 1 from stored water as serotype Ogawa.

All V. cholerae O1 isolates had the same antimicrobial resistance profile: resistance to streptomycin, sulfamethoxazole, trimethoprim, sulfamethoxazole/trimethoprim, nalidixic acid; decreased susceptibility to ciprofloxacin; and intermediate resistance to chloramphenicol and nitrofurantoin (Table). However, isolates were susceptible to doxycycline, azithromycin, β-lactams, and colistin.

<table><caption>**Table.** Characteristics of Vibrio cholerae O1 epidemic strain, Algeria, 2018*</caption><thead><tr><th>Category</th><th>Strain characteristic</th></tr></thead><tbody><tr><td>Serogroup, serotype, biotype</td><td>O1, Ogawa, El Tor</td></tr><tr><td>Genomic wave</td><td>3</td></tr><tr><td>Sublineage</td><td>Seventh pandemic V. cholerae O1 biotype El Tor</td></tr><tr><td>Genetic markers</td><td>ctxB7, tcpA<sup>CIRS101</sup>, VSP-IIΔ‡</td></tr><tr><td>AMR profile, antimicrobial drug (MIC)†</td><td></td></tr><tr><td>Streptomycin (64–128 mg/L)</td><td>Resistant</td></tr><tr><td>Sulfamethoxazole (1,024 mg/L)</td><td>Resistant</td></tr><tr><td>Trimethoprim/sulfamethoxazole (32 mg/L)</td><td>Resistant</td></tr><tr><td>Trimethoprim (32 mg/L)</td><td>Resistant</td></tr><tr><td>Chloramphenicol (16 mg/L)</td><td>Intermediate</td></tr><tr><td>Nalidixic acid (256 mg/L)</td><td>Resistant</td></tr><tr><td>Ciprofloxacin (0.25 mg/L)</td><td>Decreased susceptibility</td></tr><tr><td>Nitrofurantoin (64 mg/L)</td><td>Intermediate</td></tr><tr><td>Colistin (2 mg/L)</td><td>Susceptible</td></tr><tr><td>Horizontally acquired AMR elements</td><td>ICEVchInd5</td></tr><tr><td>Horizontally acquired AMR genes</td><td>strAB, floR, sul2, dfrA1</td></tr><tr><td>Chromosomal gene mutations, AMR phenotype</td><td></td></tr><tr><td>gyrA_S83I and parC_S85L</td><td>Resistance to nalidixic acid; decreased susceptibility to ciprofloxacin</td></tr><tr><td>nfSA_R169C and nfsB_Q5Stop</td><td>Intermediate susceptibility to nitrofurantoin</td></tr><tr><td>vpraD_D89N</td><td>Susceptibility to colistin</td></tr></tbody></table>

<footnote>*Data were collected from 20 sequenced outbreak isolates. AMR, antimicrobial resistance; ICEVchInd5, integrative conjugative element of the SXT/R391 family; VSP-IIΔ, deletion in Vibrio seventh pandemic island II.</footnote>

<footnote>†MICs according to Clinical and Laboratory Standards Institute (https://clsi.org/media/1450/m45ed3_sample.pdf).</footnote>

<footnote>‡Deletion encompassing VC_0495-0512 according to GenBank accession no. AE003852.</footnote>
We used whole-genome sequencing, comparative genomics, and phylogenetic analysis to characterize a selection of 20 *V. cholerae* O1 isolates, 17 clinical and 3 environmental (Appendix 1 Tables 2,

3; Appendix 2). We placed these isolates in context with a global collection of 1,265 seventh pandemic El Tor genomic sequences (Appendix 1 Table 4), including 23 isolates collected in Algeria during

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Phylogenomic analyses of Vibrio cholerae O1 El Tor isolates from Algeria, 2018. A) Maximum-likelihood phylogeny for 1,285 seventh pandemic V. cholerae biotype El Tor genomic sequences. A6 was used as the outgroup (Appendix 1, Table 4, https://wwwnc.cdc.gov/EID/article/28/6/21-2451-App1.xlsx). Genomic waves and acquisition of ctxB7 allele are indicated. Sublineages previously introduced into Africa (AFR1, AFR3–AFR13) are shown at the right of the tree. Column 1 indicates the geographic origins of the isolates; column 2 indicates isolates from the 2018 cholera outbreak in Algeria, all of which belong to a new seventh pandemic wave 3 sublineage AFR14. B) Maximum-likelihood phylogeny for 115 wave 3 ctxB7 isolates belonging to the distal part of the tree in panel A. N16961 was used as the outgroup (Appendix 1, Table 4). The isolates belonging to AFR14 from the 2018 cholera outbreak in Algeria are shown in red. Acquisition of the polymyxin susceptibility-associated single nucleotide variant in vprA (D89N) is indicated. Blue dots indicate bootstrap values >90%. Scale bars indicate the number of nucleotide substitutions per variable site.</figcaption></figure>
1971–1997. We constructed a maximum-likelihood phylogeny of 1,285 genomes with 10,339 single-nucleotide variants (SNVs) evenly distributed over the nonrepetitive, nonrecombinant core genome. All the isolates recovered in Algeria during 2018 belonged to the seventh pandemic El Tor lineage and clustered in the wave 3 clade containing isolates carrying the *ctxB7* allele (Figure 2, panel A) (3). The 2018 isolates did not belong to sublineages previously found in Algeria, including AFR1, which circulated during the 1970s and early 1980s; AFR7, which circulated during the mid- to late-1980s and early 1990s; or AFR8 and AFR9, which circulated during the mid-1990s (Figures 1, 2) (8). The 2018 isolates also did not belong to other sublineages found in Africa, including the most recently introduced AFR13 sublineage, previously known as T13 (8–11). AFR13 has been circulating in eastern Africa since 2015 and in Yemen since 2016 (Figure 1). A second phylogeny, restricted to 115 wave 3 *ctxB7* isolates from the distal part of the global tree, showed the 2018 isolates from Algeria are closely related to isolates from South Asia collected during 2017–2018 in India and Bangladesh (Figure 2, panel B). This finding suggests the 2018 cholera outbreak in Algeria was cause by a newly imported strain (sublineage AFR14) from South Asia, rather than resurgence of any sublineage previously in Algeria or introduction of a sublineage circulating elsewhere in Africa.

The median pairwise distance between the 20 isolates recovered during the 2018 outbreak was 2.5 (range 0–8) core-genome SNVs. All 20 isolates had similar genomic features (Table), including the toxin-coregulated pilus subunit A gene variant, *tcpA*<sup>CIRS101</sup>, a deletion (ΔVC_0495–0512) in the *Vibrio* seventh pandemic island II (VSP-II), and an SXT/R391 integrating conjugating element (ICE), called ICEVchInd5, encoding resistance to streptomycin (*strAB*), sulfonamides (*sul2*), trimethoprim (*dfrA1*), sulfamethoxazole/trimethoprim (*dfrA1* and *sul2*), and intermediate resistance to chloramphenicol (*floR*) (8). The Algeria isolates had mutations of *VC_0715*, resulting in the R169C substitution, and *VC_A0637*, resulting in the premature stop codon (Q5Stop) conferring intermediate nitrofuran resistance. Isolates also had mutations of the DNA gyrase, *gyrA* (S83I), and topoisomerase IV, *parC* (S85L), genes conferring resistance to nalidixic acid and decreased susceptibility to ciprofloxacin (8,9). In addition, isolates had a specific nonsynonymous SNV in the *vprA* gene (*VC_1320*), which resulted in the D89N substitution, conferring susceptibility to polymyxins (9), as reported for the AFR13 sublineage, although resistance to polymyxin B has been

used as a marker of *V. cholerae* O1 biotype El Tor since the seventh pandemic began (12).

## Conclusions

The seventh pandemic El Tor wave 3 clade, containing isolates carrying the *ctxB7* allele, emerged in South Asia earlier this century (9,13) and has been exported from Asia ≥4 times: to West Africa in 2007 (AFR12 sublineage) (8), Haiti in 2010 (14), East Africa in 2013–2015 (AFR13) (9,10), and now North Africa (AFR14). Polymyxin-susceptible seventh pandemic El Tor isolates with a *vprA* mutation encoding the D89N substitution were identified in South Asia in 2012 (15), spread to Eastern Africa and Yemen (AFR13) (9,10), and then spread to Algeria (AFR14).

Algeria controlled disease spread more swiftly in 2018 than during previous seventh pandemic El Tor introductions. The ministry of health led the epidemic response, initiated an emergency action plan at national and local levels, and enhanced epidemiologic surveillance and reporting. A health surveillance unit coordinated response actions and implemented recommendations. Designated hospitals managed suspected case-patients in isolation wards. Persons with suspected *V. cholerae* were hospitalized, isolated, rehydrated, and treated with doxycycline, erythromycin, azithromycin, ceftriaxone, or ciprofloxacin; patients were released only after a negative *V. cholerae* culture. Case-contacts were systematically screened, and asymptomatic carriers received chemoprophylaxis. In affected areas, the ministry of health reinforced bacteriologic monitoring of water sources, including drinking water, bore holes, wells, springs, and wadi (ravines that are dry except during rainy seasons), and took corrective action for sources with poor bacteriologic quality.

In summary, *V. cholerae* O1 isolates collected during a 2018 cholera outbreak in Algeria were a seventh pandemic El Tor sublineage, AFR14, newly introduced into Africa from South Asia. Our findings suggest that, in addition to appropriate control and prevention measures during outbreaks, such as those used in Algeria, reducing the burden of cholera in South Asia might aid in long-term control of cholera in Africa.

## Acknowledgments

We thank all the participants and particularly Hamza Letlout of Public Health Facility of Tipaza and Ahlem Toua of Institut Pasteur of d'Algérie, Annex of Oran, for sending stool samples or *Vibrio cholerae* strains and Abdelali Meftah of Public Hospital Establishment of Boufarik for his assistance during this epidemic.
This work was supported by the Pasteur Institute of
Algeria, Institut Pasteur, and by the French Government's
Investissement d'Avenir program, Laboratoire
d'Excellence, Integrative Biology of Emerging Infectious
Diseases (grant no. ANR-10-LABX-62-IBEID).

About the Author

Dr. Benamrouche is a medical microbiologist, head of the Enterobacteria Laboratory at the Pasteur Institute of Algeria, Algiers, Algeria, Pasteur International Network, and a senior lecturer on the Faculty of Medicine, University of Algiers I, Algiers, Algeria. Her research interests include the surveillance and epidemiology of enteric bacterial pathogens and antimicrobial resistance.

References

1. Clemens JD, Nair GB, Ahmed T, Qadri F, Holmgren J. Cholera. Lancet. 2017;390:1539-49. https://doi.org/10.1016/S0140-6736(17)30559-7

2. Barua D, Greenough WB, editors. Cholera. New York: Plenum: 1992.

3. Mutreja A, Kim DW, Thomson NR, Connor TR, Lee JH, Kariuki S, et al. Evidence for several waves of global transmission in the seventh cholera pandemic. Nature. 2011;477:462-5. https://doi.org/10.1038/nature10392

4. World Health Organization. Cholera key facts [cited 2022 Mar 25]. https://www.who.int/news-room/fact-sheets/detail/cholera

5. Ali M, Nelson AR, Lopez AL, Sack DA. Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis. 2015;9:e0003832. https://doi.org/10.1371/journal.pntd.0003832

6. United Nations. Algeria country profile [cited 2022 Mar 25]. http://data.un.org/en/iso/dz.html

7. Reliefweb. WHO: cholera – Algeria (14 September 2018) [cited 2022 Mar 25]. https://reliefweb.int/report/algeria/who-cholera-algeria-14-september-2018

8. Weill FX, Domman D, Njamkepo E, Tarr C, Rauzier J, Fawal N, et al. Genomic history of the seventh pandemic of cholera in Africa. Science. 2017;358:785-9. https://doi.org/10.1126/science.aad5901

9. Weill FX, Domman D, Njamkepo E, Almesbahi AA, Naji M, Nasher SS, et al. Genomic insights into the 2016–2017 cholera epidemic in Yemen. Nature. 2019;565:230–3. https://doi.org/10.1038/s41586-018-0818-3

10. Mashe T, Domman D, Tarupiwa A, Manangazira P, Phiri I, Masunda K, et al. Highly resistant cholera outbreak strain in Zimbabwe. N Engl J Med. 2020;383:687-9. https://doi.org/10.1056/NEJMc2004773

11. Oprea M, Njamkepo E, Cristea D, Zhukova A, Clark CG, Kravetz AN, et al. The seventh pandemic of cholera in Europe revisited by microbial genomics. Nat Commun. 2020;11:5347. https://doi.org/10.1038/s41467-020-19185-y

12. Herrera CM, Crofts AA, Henderson JC, Pingali SC, Davies BW, Trent MS. The Vibrio cholerae VprA-VprB two-component system controls virulence through endotoxin modification. MBio. 2014;5:e02283-14. https://doi.org/10.1128/mBio.02283-14

13. Naha A, Pazhani GP, Ganguly M, Ghosh S, Ramamurthy T, Nandy RK, et al. Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB in Vibrio cholerae O1 strains isolated from Kolkata, India. J Clin Microbiol. 2012;50:1733-6. https://doi.org/10.1128/JCM.00387-12

14. Domman D, Quilici ML, Dorman MJ, Njamkepo E, Mutreja A, Mather AE, et al. Integrated view of Vibrio cholerae in the Americas. Science. 2017;358:789-93. https://doi.org/10.1126/science.aao2136

15. Samanta P, Ghosh P, Chowdhury G, Ramamurthy T, Mukhopadhyay AK. Sensitivity to polymyxin B in El Tor Vibrio cholerae O1 strain, Kolkata, India. Emerg Infect Dis. 2015;21:2100-2. https://doi.org/10.3201/eid2111.150762

Address for correspondence: Nabila Benamrouche, Institut Pasteur d'Algérie, Route du petit Staoueli Dely Ibrahim, 16000 Alger, Algeria; email: n_benamrouche@yahoo.fr; François-Xavier Weill, Institut Pasteur, 28 rue du Dr Roux, F-75015 Paris, France; email: francois-xavier.weill@pasteur.fr