# Imported Cholera Cases, South Africa, 2023

Anthony Smith, Phuti Sekwadi, Linda Erasmus, Christine Lee, Steven Stroika, Sinenhlanhla Ndzabandzaba, Vinitha Alex, Jeremy Nel, Elisabeth Njamkepo, Juno Thomas, et al.

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Anthony Smith, Phuti Sekwadi, Linda Erasmus, Christine Lee, Steven Stroika, et al. Imported Cholera Cases, South Africa, 2023. Emerging Infectious Diseases, 2023, 29 (8), pp.1687-1690. 10.3201/eid2908.230750. hal-04613480

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<figure><img src="image_1.png" /></figure>
## References

1. Abrahamian FM, Goldstein EJ. Microbiology of animal bite wound infections. Clin Microbiol Rev. 2011;24:231-46. https://doi.org/10.1128/CMR.00041-10

2. Miller AO, Buckwalter SP, Henry MW, Wu F, Maloney KF, Abraham BK, et al. *Globicatella sanguinis* osteomyelitis and bacteremia: review of an emerging human pathogen with an expanding spectrum of disease. Open Forum Infect Dis. 2017;4:ofw277. https://doi.org/10.1093/ofid/ofw277

3. Vandamme P, Hommez J, Snauwaert C, Hoste B, Cleenwerck I, Lefebvre K, et al. *Globicatella sulfidifaciens* sp. nov., isolated from purulent infections in domestic animals. Int J Syst Evol Microbiol. 2001;51:1745-9. https://doi.org/10.1099/00207713-51-5-1745

4. Edwards KJ, Logan JMJ, Langham S, Swift C, Gharbia SE. Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples. J Med Microbiol. 2012;61:645-52. https://doi.org/10.1099/jmm.0.041764-0

5. Dewhirst FE, Klein EA, Bennett ML, Croft JM, Harris SJ, Marshall-Jones ZV. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences. Vet Microbiol. 2015;175:294-303. https://doi.org/10.1016/j.vetmic.2014.11.019

6. European Committee on Antimicrobial Susceptibility Testing (EUCAST). Antimicrobial susceptibility tests on groups of organisms or agents for which there are no EUCAST breakpoints. December 2021 [cited 2023 May 16]. https://www.eucast.org/clinical_breakpoints_and_dosing/when_there_are_no_breakpoints

7. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114-20. https://doi.org/10.1093/bioinformatics/btu170

8. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455-77. https://doi.org/10.1089/cmb.2012.0021

9. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9:5114. https://doi.org/10.1038/s41467-018-07641-9

Address for correspondence: Nick K. Jones, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 0QQ, UK; email: nicholas.jones20@nhs.net

# Imported Cholera Cases, South Africa, 2023

Anthony M. Smith, Phuti Sekwadi, Linda K. Erasmus, Christine C. Lee, Steven G. Stroika, Sinenhlanhla Ndzabandzaba, Vinitha Alex, Jeremy Nel, Elisabeth Njamkepo, Juno Thomas, François-Xavier Weill

Author affiliations: University of Pretoria, Pretoria, South Africa (A.M. Smith); National Institute for Communicable Diseases, Johannesburg, South Africa (A.M. Smith, P. Sekwadi, L.K. Erasmus, J. Thomas); US Centers for Disease Control and Prevention, Atlanta, Georgia, USA (C.C. Lee, S.G. Stroika); National Heath Laboratory Service, Johannesburg (S. Ndzabandzaba, V. Alex); University of the Witwatersrand, Johannesburg (S. Ndzabandzaba, V. Alex, J. Nel); Institut Pasteur, Université Paris Cité, Paris, France (E. Njamkepo, F.-X. Weill)

DOI: https://doi.org/10.3201/eid2908.230750

Since February 2022, Malawi has experienced a cholera outbreak of >54,000 cases. We investigated 6 cases in South Africa and found that isolates linked to the outbreak were *Vibrio cholerae* O1 serotype Ogawa from seventh pandemic El Tor sublineage AFR15, indicating a new introduction of cholera into Africa from south Asia.

The seventh cholera pandemic arrived in Africa during 1970, and the related cholera strain, *Vibrio cholerae* O1 biotype El Tor (7PET), has since become endemic in many countries in Africa (1-3). As of March 20, 2023, at least 24 countries globally reported ongoing cholera cases. Several countries in southeastern Africa, in particular Malawi and Mozambique, were experiencing outbreaks. In addition, outbreaks were spreading regionally, including to Tanzania, Zambia, Zimbabwe, and South Africa. The largest active cholera outbreak on the continent was in Malawi: 54,841 cases and 1,684 deaths reported during February 28, 2022-March 20, 2023 (4).

South Africa is not considered endemic for cholera; previous outbreaks have typically been associated with importation events. However, cholera remains under active surveillance in South Africa. The National Institute for Communicable Diseases is notified of all suspected cases. All *V. cholerae* isolates are submitted to the Centre for Enteric Diseases, which provides further laboratory investigation, including phenotypic and genotypic characterization (Appendix 1, https://wwwnc.cdc.gov/EID/article/29/8/23-0750-App1.pdf) (5). Ethics approval was obtained from the Human Research Ethics Committee, University of the Witwatersrand, Johannesburg, South Africa (protocol reference no. M210752).
## References

1. Abrahamian FM, Goldstein EJ. Microbiology of animal bite wound infections. Clin Microbiol Rev. 2011;24:231-46. https://doi.org/10.1128/CMR.00041-10

2. Miller AO, Buckwalter SP, Henry MW, Wu F, Maloney KF, Abraham BK, et al. *Globicatella sanguinis* osteomyelitis and bacteremia: review of an emerging human pathogen with an expanding spectrum of disease. Open Forum Infect Dis. 2017;4:ofw277. https://doi.org/10.1093/ofid/ofw277

3. Vandamme P, Hommez J, Snauwaert C, Hoste B, Cleenwerck I, Lefebvre K, et al. *Globicatella sulfidifaciens* sp. nov., isolated from purulent infections in domestic animals. Int J Syst Evol Microbiol. 2001;51:1745-9. https://doi.org/10.1099/00207713-51-5-1745

4. Edwards KJ, Logan JMJ, Langham S, Swift C, Gharbia SE. Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples. J Med Microbiol. 2012;61:645-52. https://doi.org/10.1099/jmm.0.041764-0

5. Dewhirst FE, Klein EA, Bennett ML, Croft JM, Harris SJ, Marshall-Jones ZV. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences. Vet Microbiol. 2015;175:294-303. https://doi.org/10.1016/j.vetmic.2014.11.019

6. European Committee on Antimicrobial Susceptibility Testing (EUCAST). Antimicrobial susceptibility tests on groups of organisms or agents for which there are no EUCAST breakpoints. December 2021 [cited 2023 May 16]. https://www.eucast.org/clinical_breakpoints_and_dosing/when_there_are_no_breakpoints

7. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114-20. https://doi.org/10.1093/bioinformatics/btu170

8. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455-77. https://doi.org/10.1089/cmb.2012.0021

9. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9:5114. https://doi.org/10.1038/s41467-018-07641-9

Address for correspondence: Nick K. Jones, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 0QQ, UK; email: nicholas.jones20@nhs.net

# Imported Cholera Cases, South Africa, 2023

Anthony M. Smith, Phuti Sekwadi, Linda K. Erasmus, Christine C. Lee, Steven G. Stroika, Sinenhlanhla Ndzabandzaba, Vinitha Alex, Jeremy Nel, Elisabeth Njamkepo, Juno Thomas, François-Xavier Weill

Author affiliations: University of Pretoria, Pretoria, South Africa (A.M. Smith); National Institute for Communicable Diseases, Johannesburg, South Africa (A.M. Smith, P. Sekwadi, L.K. Erasmus, J. Thomas); US Centers for Disease Control and Prevention, Atlanta, Georgia, USA (C.C. Lee, S.G. Stroika); National Heath Laboratory Service, Johannesburg (S. Ndzabandzaba, V. Alex); University of the Witwatersrand, Johannesburg (S. Ndzabandzaba, V. Alex, J. Nel); Institut Pasteur, Université Paris Cité, Paris, France (E. Njamkepo, F.-X. Weill)

DOI: https://doi.org/10.3201/eid2908.230750

Since February 2022, Malawi has experienced a cholera outbreak of >54,000 cases. We investigated 6 cases in South Africa and found that isolates linked to the outbreak were *Vibrio cholerae* O1 serotype Ogawa from seventh pandemic El Tor sublineage AFR15, indicating a new introduction of cholera into Africa from south Asia.

The seventh cholera pandemic arrived in Africa during 1970, and the related cholera strain, *Vibrio cholerae* O1 biotype El Tor (7PET), has since become endemic in many countries in Africa (1-3). As of March 20, 2023, at least 24 countries globally reported ongoing cholera cases. Several countries in southeastern Africa, in particular Malawi and Mozambique, were experiencing outbreaks. In addition, outbreaks were spreading regionally, including to Tanzania, Zambia, Zimbabwe, and South Africa. The largest active cholera outbreak on the continent was in Malawi: 54,841 cases and 1,684 deaths reported during February 28, 2022-March 20, 2023 (4).

South Africa is not considered endemic for cholera; previous outbreaks have typically been associated with importation events. However, cholera remains under active surveillance in South Africa. The National Institute for Communicable Diseases is notified of all suspected cases. All *V. cholerae* isolates are submitted to the Centre for Enteric Diseases, which provides further laboratory investigation, including phenotypic and genotypic characterization (Appendix 1, https://wwwnc.cdc.gov/EID/article/29/8/23-0750-App1.pdf) (5). Ethics approval was obtained from the Human Research Ethics Committee, University of the Witwatersrand, Johannesburg, South Africa (protocol reference no. M210752).
<table><caption>**Table.** Characteristics of cholera cases and classification of Vibrio cholerae O1 serotype Ogawa sequence type 69 isolates from patient fecal samples, Gauteng Province, South Africa, 2023</caption><thead><tr><th>Case</th><th>Date sample collected</th><th>Cholera case classification</th><th>Comment on case classification</th><th>Patient age, y/sex</th><th>Clinical manifestations</th></tr></thead><tbody><tr><td>1</td><td>2023 Feb 1</td><td>Imported case</td><td>Infected in Malawi</td><td>37/F</td><td>Acute diarrhea and dehydration</td></tr><tr><td>2</td><td>2023 Feb 2</td><td>Imported case</td><td>Infected in Malawi</td><td>44/F</td><td>Mild diarrhea</td></tr><tr><td>3</td><td>2023 Feb 5</td><td>Related to imported case</td><td>Close household contact of case-patient 1 (direct link to imported case)</td><td>41/M</td><td>Acute diarrhea and dehydration</td></tr><tr><td>4</td><td>2023 Feb 16</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>27/M</td><td>Acute diarrhea and dehydration</td></tr><tr><td>5</td><td>2023 Feb 12</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>23/M</td><td>Mild diarrhea</td></tr><tr><td>6</td><td>2023 Feb 23</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>19/F</td><td>Mild diarrhea</td></tr></tbody></table>

As of February 28, 2023, a total of 6 cholera cases in South Africa had been laboratory confirmed by the Centre for Enteric Diseases; fecal samples were collected from patients February 1–23, 2023. All cases

occurred in Gauteng Province (Table); 3 case-patients were female (19–44 years of age) and 3 male (23–41 years of age). Cases 1–3 were imported or import-related cases. Case-patients 1 and 2 (sisters) left Johan-

<figure><img src="image_1.png" /><figcaption>**Figure.** Maximum-likelihood phylogeny of Vibrio cholerae O1 El Tor isolates collected in South Africa, 2023, compared with 1,443 reference seventh pandemic V. cholerae El Tor (7PET) genomic sequences. A6 was used as the outgroup. The genomic waves and acquisition of the *ctxB7* allele are indicated. Color coding indicates the geographic origins of the isolates; sublineages previously introduced into Africa (AFR1, AFR3–AFR14) are shown at right. A magnification of the clade containing the 6 isolates from South Africa (red text) is shown at right. For each genome, name (or accession number), country where contamination occurred, and year of sample collection are shown at the tips of the tree. The 6 isolates collected in South Africa belong to a new 7PET wave 3 sublineage called AFR15. Blue dots indicate bootstrap values ≥90%. Scale bars indicate number of nucleotide substitutions per variable site.</figcaption></figure>
<table><caption>**Table.** Characteristics of cholera cases and classification of *Vibrio cholerae* O1 serotype Ogawa sequence type 69 isolates from patient fecal samples, Gauteng Province, South Africa, 2023</caption><thead><tr><th>Case</th><th>Date sample collected</th><th>Cholera case classification</th><th>Comment on case classification</th><th>Patient age, y/sex</th><th>Clinical manifestations</th></tr></thead><tbody><tr><td>1</td><td>2023 Feb 1</td><td>Imported case</td><td>Infected in Malawi</td><td>37/F</td><td>Acute diarrhea and dehydration</td></tr><tr><td>2</td><td>2023 Feb 2</td><td>Imported case</td><td>Infected in Malawi</td><td>44/F</td><td>Mild diarrhea</td></tr><tr><td>3</td><td>2023 Feb 5</td><td>Related to imported case</td><td>Close household contact of case-patient 1 (direct link to imported case)</td><td>41/M</td><td>Acute diarrhea and dehydration</td></tr><tr><td>4</td><td>2023 Feb 16</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>27/M</td><td>Acute diarrhea and dehydration</td></tr><tr><td>5</td><td>2023 Feb 12</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>23/M</td><td>Mild diarrhea</td></tr><tr><td>6</td><td>2023 Feb 23</td><td>Locally acquired indigenous case</td><td>No travel history; no evidence of direct link to an imported case</td><td>19/F</td><td>Mild diarrhea</td></tr></tbody></table>

As of February 28, 2023, a total of 6 cholera cases in South Africa had been laboratory confirmed by the Centre for Enteric Diseases; fecal samples were collected from patients February 1–23, 2023. All cases

occurred in Gauteng Province (Table); 3 case-patients were female (19–44 years of age) and 3 male (23–41 years of age). Cases 1–3 were imported or import-related cases. Case-patients 1 and 2 (sisters) left Johan-

<figure><img src="image_2.png" /><figcaption>**Figure.** Maximum-likelihood phylogeny of *Vibrio cholerae* O1 El Tor isolates collected in South Africa, 2023, compared with 1,443 reference seventh pandemic *V. cholerae* El Tor (7PET) genomic sequences. A6 was used as the outgroup. The genomic waves and acquisition of the *ctxB7* allele are indicated. Color coding indicates the geographic origins of the isolates; sublineages previously introduced into Africa (AFR1, AFR3–AFR14) are shown at right. A magnification of the clade containing the 6 isolates from South Africa (red text) is shown at right. For each genome, name (or accession number), country where contamination occurred, and year of sample collection are shown at the tips of the tree. The 6 isolates collected in South Africa belong to a new 7PET wave 3 sublineage called AFR15. Blue dots indicate bootstrap values ≥90%. Scale bars indicate number of nucleotide substitutions per variable site.</figcaption></figure>
nesburg on January 15, 2023, and traveled together to Chinsapo, Lilongwe, Malawi, in one of the districts reporting active outbreaks, where they stayed until their departure on January 29, 2023. Both women reported onset of symptoms within 12 hours of departure during the bus trip back to Johannesburg. Case-patient 3 was a close household contact of case-patient 1. Case-patients 4–6 acquired infection locally and were classified as indigenous cases; none had travelled or had any link to the imported or import-related cases or to one another. We identified isolates associated with all 6 cases as *V. cholerae* O1 serotype Ogawa and all were PCR-positive for the cholera toxin-producing gene.

We used whole-genome sequencing, comparative genomics, and phylogenetic analysis to further characterize the isolates (Appendix 2 Tables 1, 2, https://wwwnc.cdc.gov/EID/article/29/8/23-0750-App1.xlsx). The 6 *V. cholerae* O1 isolates had similar genomic features, including the toxin-coregulated pilus gene subunit A gene variant, *tcpA*<sup>CIRS101</sup>, a deletion (ΔVC_0495-VC_0512) within the vibrio seventh pandemic island II (VSP-II), and an SXT/R391 integrating conjugating element called ICEVchInd5, encoding resistance to streptomycin (*strAB*), sulfonamides (*sul2*), trimethoprim (*dfrA1*), and trimethoprim/sulfamethoxazole (*dfrA1* and *sul2*) and resistance or intermediate resistance to chloramphenicol (*floR*). The isolates also had mutations of *VC_0715* (resulting in the R169C substitution) and *VC_A0637* (resulting in the premature stop codon Q5Stop) conferring nitrofuran resistance, and of the DNA gyrase, *gyrA* (S83I), and topoisomerase IV, *parC* (S85L) genes, conferring resistance to nalidixic acid and decreased susceptibility to ciprofloxacin (3,6). The isolates also had a specific nonsynonymous single-nucleotide variant (SNV) in the *vprA* gene (*VC_1320*) (resulting in the D89N substitution), conferring susceptibility to polymyxins (6).

To place these 6 isolates into a global phylogenetic context, we constructed a maximum-likelihood phylogeny of 1,443 genomes (Appendix 2 Table 3) with 10,679 SNVs evenly distributed over the nonrepetitive, nonrecombinant core genome. All isolates from South Africa clustered together (median pairwise distance of 4 [range 0–8] core-genome SNVs) in the 7PET lineage wave 3 clade, containing isolates carrying the *ctxB7* allele (Figure) (6). However, those isolates did not belong to any of the sublineages previously found in Africa (AFR1 and AFR3–AFR 14) (Figure) (3,6,7); instead, they tightly grouped with genomes of south Asia variants, suggesting that the 2022–2023 cholera outbreak in Malawi and cases in South Africa in our study were associated with a newly imported 7PET strain, sublineage AFR15, from south Asia. All but 1

of the closest genomes were either collected locally and identified in Pakistan during June–December 2022 or detected within the framework of cholera surveillance in the United States or Australia (8).

In conclusion, we show that isolates from cases in South Africa, which have been linked to the 2022–2023 cholera outbreak in Malawi, belong to the seventh pandemic El Tor sublineage AFR15. Those cases did not result from resurgence of a strain previously circulating in any region of Africa but were caused by a cholera agent newly introduced into Africa from south Asia. This finding offers valuable information to all public health authorities in Africa. Genomic microbial surveillance and cross-border collaborations have a key role to play in identifying new cholera introductions, areas prone to cholera importation, and the main routes of cholera circulation. All of these elements are key to better understanding cholera epidemiology in Africa.

## Acknowledgement

We thank the Gauteng Department of Health for their contributions.

This study was made possible by support from the SEQAFRICA project, which is funded by the UK Department of Health and Social Care's Fleming Fund using UK aid.

## About the Author

Dr. Smith is employed as a principal medical scientist at the Centre for Enteric Diseases, National Institute for Communicable Diseases, South Africa. He also holds the appointment of extraordinary professor with the University of Pretoria, South Africa. His interests include surveillance and epidemiology of enteric bacterial pathogens in South Africa.

## References

1. Mintz ED, Tauxe RV. Cholera in Africa: a closer look and a time for action. J Infect Dis. 2013;208(Suppl 1):S4–7. https://doi.org/10.1093/infdis/jit205

2. 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

3. 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

4. World Health Organization (WHO). 2023. Multi-country outbreak of cholera, external situation report #1–28 March 2023 [cited 2023 Apr 17]. https://www.who.int/publications/m/item/multi-country-outbreak-of-cholera-external-situation-report-1--28-march-2023

5. Smith AM, Weill FX, Njamkepo E, Ngomane HM, Ramalwa N, Sekwadi P, et al. Emergence of Vibrio cholerae
nesburg on January 15, 2023, and traveled together to Chinsapo, Lilongwe, Malawi, in one of the districts reporting active outbreaks, where they stayed until their departure on January 29, 2023. Both women reported onset of symptoms within 12 hours of departure during the bus trip back to Johannesburg. Case-patient 3 was a close household contact of case-patient 1. Case-patients 4–6 acquired infection locally and were classified as indigenous cases; none had travelled or had any link to the imported or import-related cases or to one another. We identified isolates associated with all 6 cases as *V. cholerae* O1 serotype Ogawa and all were PCR-positive for the cholera toxin-producing gene.

We used whole-genome sequencing, comparative genomics, and phylogenetic analysis to further characterize the isolates (Appendix 2 Tables 1, 2, https://wwwnc.cdc.gov/EID/article/29/8/23-0750-App1.xlsx). The 6 *V. cholerae* O1 isolates had similar genomic features, including the toxin-coregulated pilus gene subunit A gene variant, *tcpA*<sup>CIRS101</sup>, a deletion (ΔVC_0495-VC_0512) within the vibrio seventh pandemic island II (VSP-II), and an SXT/R391 integrating conjugating element called ICEVchInd5, encoding resistance to streptomycin (*strAB*), sulfonamides (*sul2*), trimethoprim (*dfrA1*), and trimethoprim/sulfamethoxazole (*dfrA1* and *sul2*) and resistance or intermediate resistance to chloramphenicol (*floR*). The isolates also had mutations of *VC_0715* (resulting in the R169C substitution) and *VC_A0637* (resulting in the premature stop codon Q5Stop) conferring nitrofuran resistance, and of the DNA gyrase, *gyrA* (S83I), and topoisomerase IV, *parC* (S85L) genes, conferring resistance to nalidixic acid and decreased susceptibility to ciprofloxacin (3,6). The isolates also had a specific nonsynonymous single-nucleotide variant (SNV) in the *vprA* gene (*VC_1320*) (resulting in the D89N substitution), conferring susceptibility to polymyxins (6).

To place these 6 isolates into a global phylogenetic context, we constructed a maximum-likelihood phylogeny of 1,443 genomes (Appendix 2 Table 3) with 10,679 SNVs evenly distributed over the nonrepetitive, nonrecombinant core genome. All isolates from South Africa clustered together (median pairwise distance of 4 [range 0–8] core-genome SNVs) in the 7PET lineage wave 3 clade, containing isolates carrying the *ctxB7* allele (Figure) (6). However, those isolates did not belong to any of the sublineages previously found in Africa (AFR1 and AFR3–AFR 14) (Figure) (3,6,7); instead, they tightly grouped with genomes of south Asia variants, suggesting that the 2022–2023 cholera outbreak in Malawi and cases in South Africa in our study were associated with a newly imported 7PET strain, sublineage AFR15, from south Asia. All but 1

of the closest genomes were either collected locally and identified in Pakistan during June–December 2022 or detected within the framework of cholera surveillance in the United States or Australia (8).

In conclusion, we show that isolates from cases in South Africa, which have been linked to the 2022–2023 cholera outbreak in Malawi, belong to the seventh pandemic El Tor sublineage AFR15. Those cases did not result from resurgence of a strain previously circulating in any region of Africa but were caused by a cholera agent newly introduced into Africa from south Asia. This finding offers valuable information to all public health authorities in Africa. Genomic microbial surveillance and cross-border collaborations have a key role to play in identifying new cholera introductions, areas prone to cholera importation, and the main routes of cholera circulation. All of these elements are key to better understanding cholera epidemiology in Africa.

## Acknowledgement

We thank the Gauteng Department of Health for their contributions.

This study was made possible by support from the SEQAFRICA project, which is funded by the UK Department of Health and Social Care's Fleming Fund using UK aid.

## About the Author

Dr. Smith is employed as a principal medical scientist at the Centre for Enteric Diseases, National Institute for Communicable Diseases, South Africa. He also holds the appointment of extraordinary professor with the University of Pretoria, South Africa. His interests include surveillance and epidemiology of enteric bacterial pathogens in South Africa.

## References

1. Mintz ED, Tauxe RV. Cholera in Africa: a closer look and a time for action. J Infect Dis. 2013;208(Suppl 1):S4–7. https://doi.org/10.1093/infdis/jit205

2. 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

3. 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

4. World Health Organization (WHO). 2023. Multi-country outbreak of cholera, external situation report #1–28 March 2023 [cited 2023 Apr 17]. https://www.who.int/publications/m/item/multi-country-outbreak-of-cholera-external-situation-report-1---28-march-2023

5. Smith AM, Weill FX, Njamkepo E, Ngomane HM, Ramalwa N, Sekwadi P, et al. Emergence of Vibrio cholerae
O1 sequence type 75, South Africa, 2018–2020. Emerg Infect Dis. 2021;27:2927–31. https://doi.org/10.3201/eid2711.211144

6. 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

7. Benamrouche N, Belkader C, Njamkepo E, Zemam SS, Sadat S, Saighi K, et al. Outbreak of imported seventh pandemic Vibrio cholerae O1 El Tor, Algeria, 2018. Emerg Infect Dis. 2022;28:1241–5. https://doi.org/10.3201/eid2806.212451

8. Sim EM, Martinez E, Blackwell GA, Pham D, Millan G, Graham RMA, et al. Genomes of Vibrio cholerae O1 serotype Ogawa associated with current cholera activity in Pakistan. Microbiol Resour Announc. 2023;12:e0088722. https://doi.org/10.1128/mra.00887-22

Address for correspondence: Anthony Smith, Centre for Enteric Diseases, National Institute for Communicable Diseases, Private Bag X4, Sandringham, 2131, Johannesburg, South Africa; email: anthonys@nicd.ac.za

<table><caption>**Table.** Characteristics and key indicators for a surveillance study among asymptomatic healthcare workers during SARS-CoV-2 Omicron surge, Germany, 2022*</caption><thead><tr><th>Characteristic</th><th>Value</th></tr></thead><tbody><tr><td>Total no. PCR tests</td><td>398,784</td></tr><tr><td>Median no. tests/wk (IQR)</td><td>7,559 (6,834–8,139)</td></tr><tr><td>Total no. PCR-positive tests</td><td>3,555</td></tr><tr><td>Median positivity rate, % (IQR)</td><td>0.9 (0.45–1.17)</td></tr><tr><td>Minimum, January 3–9</td><td>0.25</td></tr><tr><td>Maximum, March 14–20</td><td>1.89</td></tr><tr><td>Total no. HCWs tested</td><td>9,515</td></tr><tr><td>No. (%) infected</td><td>2,782 (29.2)</td></tr><tr><td>No. (%) HCWs with ≥2 infections</td><td>463 (4.87)</td></tr><tr><td>Sex, no. (%)</td><td></td></tr><tr><td>M</td><td>705 (25.3)</td></tr><tr><td>F</td><td>2,077 (74.7)</td></tr><tr><td>Median age, y (IQR)</td><td>42 (30–53)</td></tr><tr><td>Median Ct value (IQR)</td><td>30 (27–32)</td></tr><tr><td>No. (%) completing immunization regimen†</td><td>8,926 (93.8)</td></tr></tbody></table>

*Ct, cycle threshold; HCW, healthcare worker.

†As of March 2022 (Appendix Table 2, https://wwwnc.cdc.gov/EID/article/29/8/23-0156-App1.pdf).

## Asymptomatic Healthcare Worker PCR Screening during SARS-CoV-2 Omicron Surge, Germany, 2022

Ralph Bertram, Wolfgang Hitzl, Eike Steinmann, Joerg Steinmann

Author affiliations: Paracelsus Medical University, Nuremberg, Germany (R. Bertram, J. Steinmann); Paracelsus Medical University, Salzburg, Austria (W. Hitzl); Ruhr University Bochum, Bochum, Germany (E. Steinmann)

DOI: https://doi.org/10.3201/eid2908.230156

During 2022, a total of 9,515 asymptomatic healthcare workers of a large hospital in Germany underwent SARS-CoV-2 PCR screening twice weekly. Of 398,784 saliva samples, 3,555 (0.89%) were PCR positive (median cycle threshold value 30). Early identification of infected healthcare workers can help reduce SARS-CoV-2 transmission in the hospital environment.

COVID-19, caused by the SARS-CoV-2 virus, results in acute pulmonary and extrapulmonary manifestations and frequently causes long-term sequelae (1). In Germany, ≈38.5 million SARS-CoV-2

infections and ≈174,000 COVID-19 deaths had been reported through May 2023 (2). Among those, ≈30.2 million infections and ≈47,000 deaths occurred during 2022, when SARS-CoV-2 Omicron variant dominance was accompanied by a mean hospitalization incidence of 5.87 (2). SARS-CoV-2 infection rates among hospitalized patients were reported to be ≈10%–15% (3). Healthcare workers (HCWs) also were exposed to an elevated risk of acquiring and shedding SARS-CoV-2 infections (4). Regular SARS-CoV-2 testing of asymptomatic HCWs has been found to reduce viral transmission to patients and coworkers (5). We report data from a systematic SARS-CoV-2 PCR screening program comprising >9,500 HCWs in a large hospital in Germany during 2022.

Klinikum Nürnberg is a tertiary care hospital with 2,233 beds at 2 sites in Nuremberg, Germany, and cares for ≈100,000 inpatients and ≈170,000 outpatients per year. During January–November 2022, all 9,515 hospital staff were instructed to participate in a government-mandated regular SARS-CoV-2 PCR screening program. According to federal law in Germany, participation was mandatory irrespective of the level of working exposure risk or vaccination status (Table; Appendix Table 1, https://wwwnc.cdc.gov/EID/article/29/8/23-0156-App1.pdf).

Asymptomatic HCWs collected saliva samples twice weekly via self-sampling using a reliable gaging method (6); part-time workers collected samples less frequently. Samples were subjected to PCR testing by an external provider, and turnaround time between sampling and electronic reporting was ≈24–38 h. However, staff with acute COVID-19 symptoms were immediately PCR tested in house. Persons with PCR-verified
O1 sequence type 75, South Africa, 2018–2020. Emerg Infect Dis. 2021;27:2927–31. https://doi.org/10.3201/eid2711.211144

6. 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

7. Benamrouche N, Belkader C, Njamkepo E, Zemam SS, Sadat S, Saighi K, et al. Outbreak of imported seventh pandemic Vibrio cholerae O1 El Tor, Algeria, 2018. Emerg Infect Dis. 2022;28:1241–5. https://doi.org/10.3201/eid2806.212451

8. Sim EM, Martinez E, Blackwell GA, Pham D, Millan G, Graham RMA, et al. Genomes of Vibrio cholerae O1 serotype Ogawa associated with current cholera activity in Pakistan. Microbiol Resour Announc. 2023;12:e0088722. https://doi.org/10.1128/mra.00887-22

Address for correspondence: Anthony Smith, Centre for Enteric Diseases, National Institute for Communicable Diseases, Private Bag X4, Sandringham, 2131, Johannesburg, South Africa; email: anthonys@nicd.ac.za

<table><caption>**Table.** Characteristics and key indicators for a surveillance study among asymptomatic healthcare workers during SARS-CoV-2 Omicron surge, Germany, 2022*</caption><thead><tr><th>Characteristic</th><th>Value</th></tr></thead><tbody><tr><td>Total no. PCR tests</td><td>398,784</td></tr><tr><td>Median no. tests/wk (IQR)</td><td>7,559 (6,834–8,139)</td></tr><tr><td>Total no. PCR-positive tests</td><td>3,555</td></tr><tr><td>Median positivity rate, % (IQR)</td><td>0.9 (0.45–1.17)</td></tr><tr><td>Minimum, January 3–9</td><td>0.25</td></tr><tr><td>Maximum, March 14–20</td><td>1.89</td></tr><tr><td>Total no. HCWs tested</td><td>9,515</td></tr><tr><td>No. (%) infected</td><td>2,782 (29.2)</td></tr><tr><td>No. (%) HCWs with ≥2 infections</td><td>463 (4.87)</td></tr><tr><td>Sex, no. (%)</td><td></td></tr><tr><td>M</td><td>705 (25.3)</td></tr><tr><td>F</td><td>2,077 (74.7)</td></tr><tr><td>Median age, y (IQR)</td><td>42 (30–53)</td></tr><tr><td>Median Ct value (IQR)</td><td>30 (27–32)</td></tr><tr><td>No. (%) completing immunization regimen†</td><td>8,926 (93.8)</td></tr></tbody></table>

*Ct, cycle threshold; HCW, healthcare worker.

†As of March 2022 (Appendix Table 2, https://wwwnc.cdc.gov/EID/article/29/8/23-0156-App1.pdf).

## Asymptomatic Healthcare Worker PCR Screening during SARS-CoV-2 Omicron Surge, Germany, 2022

Ralph Bertram, Wolfgang Hitzl, Eike Steinmann, Joerg Steinmann

Author affiliations: Paracelsus Medical University, Nuremberg, Germany (R. Bertram, J. Steinmann); Paracelsus Medical University, Salzburg, Austria (W. Hitzl); Ruhr University Bochum, Bochum, Germany (E. Steinmann)

DOI: https://doi.org/10.3201/eid2908.230156

During 2022, a total of 9,515 asymptomatic healthcare workers of a large hospital in Germany underwent SARS-CoV-2 PCR screening twice weekly. Of 398,784 saliva samples, 3,555 (0.89%) were PCR positive (median cycle threshold value 30). Early identification of infected healthcare workers can help reduce SARS-CoV-2 transmission in the hospital environment.

COVID-19, caused by the SARS-CoV-2 virus, results in acute pulmonary and extrapulmonary manifestations and frequently causes long-term sequelae (1). In Germany, ≈38.5 million SARS-CoV-2

infections and ≈174,000 COVID-19 deaths had been reported through May 2023 (2). Among those, ≈30.2 million infections and ≈47,000 deaths occurred during 2022, when SARS-CoV-2 Omicron variant dominance was accompanied by a mean hospitalization incidence of 5.87 (2). SARS-CoV-2 infection rates among hospitalized patients were reported to be ≈10%–15% (3). Healthcare workers (HCWs) also were exposed to an elevated risk of acquiring and shedding SARS-CoV-2 infections (4). Regular SARS-CoV-2 testing of asymptomatic HCWs has been found to reduce viral transmission to patients and coworkers (5). We report data from a systematic SARS-CoV-2 PCR screening program comprising >9,500 HCWs in a large hospital in Germany during 2022.

Klinikum Nürnberg is a tertiary care hospital with 2,233 beds at 2 sites in Nuremberg, Germany, and cares for ≈100,000 inpatients and ≈170,000 outpatients per year. During January–November 2022, all 9,515 hospital staff were instructed to participate in a government-mandated regular SARS-CoV-2 PCR screening program. According to federal law in Germany, participation was mandatory irrespective of the level of working exposure risk or vaccination status (Table; Appendix Table 1, https://wwwnc.cdc.gov/EID/article/29/8/23-0156-App1.pdf).

Asymptomatic HCWs collected saliva samples twice weekly via self-sampling using a reliable gaging method (6); part-time workers collected samples less frequently. Samples were subjected to PCR testing by an external provider, and turnaround time between sampling and electronic reporting was ≈24–38 h. However, staff with acute COVID-19 symptoms were immediately PCR tested in house. Persons with PCR-verified