A novel post-mortem pathogen discovery program detects an outbreak of Echovirus E7: Uganda, 2022-2023

Abstract

Objectives: Utilizing post-mortem examination for routine monitoring of infectious diseases and pandemic preparedness is a common-sense, yet uncommon, public health measure. Here, we established a novel mortuary surveillance program in Uganda that leverages the unbiased nature of metagenomic next-generation sequencing (mNGS) to detect pathogens in recently deceased individuals.

Methods: Between October 2022 and December 2023, specimens and patient metadata were collected from 2,607 individuals across five mortuary sites around Kampala. Specimens were pre-screened for hemorrhagic fever viruses by RT-qPCR and a subset (n = 134) of RT-qPCR negatives were sequenced by mNGS.

Results: A variety of DNA (herpes, parvovirus, bufavirus) and RNA (Saffold, Salivirus, HAV) viruses, vectored (Bartonella, Rickettsia) and nosocomial (Enterobacter, Klebsiella) bacterial infections, and potentially lethal respiratory pathogens (e.g., Cryptococcus neoformans, Corynebacterium diphtheria) were detected. A localized outbreak of Enterovirus B (EV-B), specifically a recombinant Echovirus E7, was observed in Kampala. An epidemiologic assessment indicated that most identified pathogens were acquired via direct and/or indirect contact (e.g., fecal-oral, fomites) and that other modes of transmission (e.g., food-borne, insect-vectored) played a less significant role.

Conclusion: Integration of mortuary surveillance, coupled with mNGS, into public health systems represents a powerful strategy for identifying unrecognized outbreaks and monitoring the (re-) emergence of infectious diseases.

Keywords: Uganda; echovirus; enterovirus; metagenomics; mortuary surveillance.

Figure 1

Mortuary surveillance study design. 2,607 specimens were collected from deceased individuals arriving at 5 mortuaries in Kampala from 31 October 2022 to 12 December 2023. Specimen types included blood and/or nasopharyngeal/oral swabs–specimen aliquots first sent to the National VHF Reference laboratory to screen for Ebola, Marburg, CCHF, and RVF viruses. Positive PCR results for any of the VHF viruses is reported in the Uganda MoH Results Dispatch System (RDS) to inform response as well as to the UVRI Director, Public Health Emergency Operations Center (PHEOC) and the APDC. Specimens identified as PCR negative proceed to the metagenomics sequencing pipeline to detect known or novel pathogens of pandemic potential. A total of 134 VHF-negative samples were selected for mNGS, from October 2022 to February 2023. Fastq deep sequence files generated from the mNGS were analyzed using the DiVir3 bioinformatics analysis pipeline.

Figure 2

Specimen collection characteristics. (A) Accounting of the individuals enrolled in mortuary surveillance during the study period, summarized weekly with collection facility and detected VHF cases denoted. A timeline of the surveillance program’s major events is shown at the top. (B) Healthcare status of deceased individuals prior to death.

Figure 3

Detection of various pathogens by NGS, including an outbreak of Echovirus E7. (A) Genome coverage of selected pathogens processed by both mNGS and teNGS. (B) Geographical distribution of the detected recombinant Enterovirus B cases (i.e., place where the patient lived or became ill) with relevant case metadata (e.g., collection date). (C) Maximum likelihood phylogeny was performed on VP1 (top) and 3D (bottom) regions extracted from alignments of 1,766 complete Enterovirus B genomes. Branches containing the Kampala strain are shown. Ultrafast Boostrapping support values are shown only at those nodes where support is below 90%.

Figure 4

Taxonomic and transmission profile of the pathogens detected during this study. (A) Pie charts depicting the (co-) infection profile of the 134 sequenced specimens (left) and the genera of those pathogens (right). (B) Venn diagram depicting the distinct or shared transmission routes of the detected pathogens. Each pathogen also has a color key describing its various possible clinical presentations.