Application of genomic sequencing in a disease outbreak investigation; A case of cholera in Kayunga and Namayingo, 2023

Authors: Ritah Namusoosa1,2*, Samuel Gidudu1, Leah Naluwagga Baliruno1,2, Martha Pedun3, Alisen Ayitewala2, Grace Najjuka2, Alex Riolexus Ario1; Institutional affiliations:1Uganda Public Health Fellowship Program-Laboratory Leadership Program; Uganda National Institute of Public Health; Ministry of Health Kampala, Uganda, 2National Health Laboratory and Diagnostics Services; Ministry of Health, Kampala, Uganda, 3African Society for Laboratory Medicine; Addis Ababa, Ethiopia; Correspondence*: Tel: +256785842878, Email: rnamusoosa@uniph.go.ug

Summary

Background: On 19 July 2023, Uganda declared a Cholera outbreak in districts of Kayunga and Namayingo. We describe how we used genomic sequencing to ascertain the causative agents and drug resistance profile in this outbreak investigation.

Methods:  The national rapid response team worked with the district laboratory focal persons and hub coordinators to collect and refer samples through the hub system for testing. Staff from the National Microbiology Reference Laboratory (NMRL) and National Genomic Sequencing Laboratory tested samples for Vibrio cholerae and antibiotic resistance.

Results: Vibrio cholerae serotype 01 Ogawa isolate belonging to ST69 Multiple Locus Sequence Type (MLST) was identified. Isolates carried the IncC plasmid, known for harboring antibiotic-resistance genes. Identical drug resistance gene markers indicating resistance to multiple drugs included streptomycin, ampicillin, ceftriaxone, chloramphenicol, trimethoprim, erythromycin, azithromycin, sulfisoxazole, and co-trimoxazole.

Conclusion: Genomic sequencing did not establish a definitive transmission link between cases in the two districts, indicating potential diverse sources.


Background

Genomic sequencing provides an innovative approach in identifying and characterizing strains in disease outbreak investigations (1),(2). This approach has successfully been applied in ebola virus  disease, COVID-19 pandemic, seasonal influenza, and zika virus outbreak investigations (3). Although it’s been recommended for Cholera surveillance and investigation (4), genomic sequencing has hardly been used for this purpose in Uganda. We describe how we used genomic sequencing to ascertain the causative agents and drug resistance profile in this outbreak investigation.

 Methods

We analyzed culture and antibiotic and genomic testing data for samples collected from suspected cases during the cholera outbreak in Kayunga and Namayingo in July 2023.  A total of 144 stool samples were collected, 118 samples from Kayunga and 26 samples from Namayingo. The samples were collected in sterile stool containers and transported to the reference laboratories using the national sample transport system (hub system) in Cary- Blair transport media under cold chain (2-8OC) following triple packaging principles. The samples were accompanied with cholera case investigation forms that captured data variables which included age, sex, district, health facility, sample type and date of sample collection. The laboratory generated data of whole genome sequencing (WGS) WGS, culture results as well as antimicrobial susceptibility test results of which was then merged with the metadata for the downstream analysis

Culture and sensitivity were performed by the national microbiology reference laboratory (NMRL) using conventional biochemical methods for identification and disk diffusion for antimicrobial susceptibility testing. The resultant isolates were then subjected to whole genome sequencing at national genomic sequencing laboratory. For whole genome sequencing, DNA extraction was done using the Qiagen extraction kit followed by DNA quantification using quibit. Library preparation was done using Illumina DNA preparation kit and sequenced using the Miseq platform.  WGS data was analyzed using the back-page pipeline.

Ethical considerations

This was in response to the cholera outbreak in Kayunga and Namayingo. The office of the Center for Global Health, US Center for Disease Control and Prevention determined that this activity was not human subject research and its primary intent was for disease control. We sought permission and administrative clearance from the National Health Laboratory and Diagnostic Services (NHLDS), Kayunga Regional Referral Hospital and the district health authorities to conduct the investigation.

Results

Of the 144 samples referred, 59 samples tested positive by culture and sensitivity. Genomic sequencing revealed Vibrio cholerae serotype 01 Ogawa of ST69 Multiple Locus Sequence Type (MLST), a predominantly local endemic sequence, among isolates from both districts. All the isolates carried the IncC plasmid, a harboring antibiotic-resistance gene and forty-nine virulence factor- genes, contributing to the pathogenicity of vibrio cholerae. Identical drug resistance gene markers indicating resistance to multiple drugs included; streptomycin, ampicillin, ceftriaxone, chloramphenicol, trimethoprim, erythromycin, azithromycin, sulfisoxazole and co-trimoxazole. From the phenotypic results, doxycycline, the recommended first-line treatment, showed no signs of resistance. Ciprofloxacin, an alternative treatment, exhibited a 2% resistance rate. However, erythromycin, azithromycin, and trimethoprim/sulfamethoxazole demonstrated a resistance rate exceeding 96.5%, emphasizing the significance of associated gene markers.

Discussion

Whole genome sequencing revealed the persistence of a local endemic strain in the country. The presence of common antibiotic resistance markers among all isolates raises concerns about the potential for widespread resistance as these genes harbor integrating conjugative elements, plasmids, superintegron, transposable elements, and insertion sequences which are the key carriers of genetic traits encoding AMR function(5). The resistance to erythromycin, azithromycin, and trimethoprim/sulfamethoxazole highlights the importance of understanding the genetic basis for resistance, factors underlying the expansion of these resistant phenotypes as observed in other countries (6), (7) and the need for alternative treatment options. The absence of resistance to doxycycline aligns with current treatment guidelines, while the 2% resistance to ciprofloxacin suggests cautious consideration when selecting alternative treatments. The lack of resistance to chloramphenicol raises questions about the potential impact of the dfrA1 gene marker. Further investigation is required to determine the relationship between this marker and chloramphenicol resistance (5).

Study limitations

We did not sample all cases due to logistical challenges and there were no environmental samples sequenced to enable us have detailed transmission linkages for the two districts.

Conclusion

Genomic sequencing played a pivotal role in understanding the Cholera outbreak in Kayunga and Namayingo. Genomic sequencing did not establish a definitive transmission link between cases in the two districts, indicating potential diverse sources. However, shared characteristics among the Vibrio cholerae isolates, such as MLST results, plasmid presence, resistance markers, and virulence factors, strongly indicated a common origin. We recommend utilization of this technology in subsequent disease outbreak investigation including outbreaks of bacterial origin in nature.

Conflict of interest

The authors declare no conflict of interest.

Authors contribution

RN: participated in the conception, design, analysis, interpretation of the study and wrote the draft bulletin; SG, LNB,MP, AA, and GN reviewed the report, reviewed the drafts of the bulletin for intellectual content and made multiple edits to the draft bulletin; SG, RN, and ARA reviewed the final bulletin to ensure intellectual content and scientific integrity. All authors read and approved the final bulletin.

Acknowledgements

We thank the ministry of health for permitting us to respond to this outbreak. We thank Kayunga Regional Referral Hospital, Kayunga and Namayingo District Local Governments for granting us permission and overall guidance to the team.

Copyright and licensing

All material in the Uganda Public Health Bulletin is in the public domain and may be used and reprinted without permission. However, citation as to source is appreciated. Any article can be reprinted or published. If cited a reprint, it should be in the original form.

Reference

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