Case report
Whole-genome sequencing of Neisseria gonorrhoeae in a forensic transmission case

https://doi.org/10.1016/j.fsigen.2019.07.003Get rights and content

Highlights

  • Potential case of sexual abuse of a child with transmission of gonorrhoeae.

  • Isolates cultured from suspect, victim and two local controls.

  • PFGE and MLST analyses were not discriminatory enough.

  • Whole genome sequencing (WGS) revealed complete identity between suspect and victim.

  • First published case of bacterial WGS analysis used in court as evidence for child abuse.

Abstract

Molecular epidemiology and phylogenetic analyses are frequently used in the investigation of viral transmission cases in forensic contexts. Here, we present the methods and results of the analysis of a bacterial transmission episode in an alleged child abuse case using complete genome sequences obtained by high-throughput sequencing (HTS) methods.

We obtained genomes of Neisseria gonorrhoeae from the victim, the suspect, and 29 unrelated controls. The analysis of the genomes revealed that the victim and suspect isolates had identical sequences in both the bacterial chromosome and the single plasmid present in them. One of the local controls was very similar (differing in only 2 SNPs) to the case sequences, but the remaining controls were very divergent. Additional cases of identity and very high similarity among controls were observed occasionally, pointing at recent transmission cases.

These results were more discriminative than the previous molecular epidemiology analyses performed at the hospital’s Microbiology Service, as Multi-Locus Sequence Typing (MLST) could not distinguish between the suspect/victim and the controls isolates, and Pulse Field Gel Electrophoresis (PFGE) was not able to distinguish between the suspect/victim and one of the local controls.

These results lead us to conclude that complete bacterial genome sequences obtained with HTS technologies may be a valuable tool for establishing recent transmission cases and, although more studies are needed, they have a great potential for being used in forensic analyses.

Section snippets

Background

In the last decades, molecular epidemiology analyses have been applied to the study of virus transmission cases and outbreaks in forensic settings [[1], [2], [3], [4], [5], [6]]. This methodology was introduced in the forensic field by Ou and colleagues [1], who demonstrated the transmission of human immunodeficiency virus (HIV) by a dentist to some of his patients. The first case in a criminal court was presented by Metzker and colleagues [4], whose evidence contributed to the conviction of a

N. gonorrhoeae culture and detection

The first case detected was a young girl who attended her ambulatory general pediatrician because of purulent vaginal secretions. In the Gram stain of the vaginal exudate, gram-negative cocci compatible with gonococcus were observed and cultivated after 48 h in blood and chocolate agar plates (bioMérieux, Marcy-l'Étoile, France) in a 5% CO2 enriched atmosphere. Species identification was performed by MALDI-TOF (Bruker Diagnostics, Germany). Candida albicans was also isolated in the vaginal

Results

The SpeI and NheI PFGE profiles of the suspect and the victim were indistinguishable (Table 1, Supplementary Fig. 1). Surprisingly, of the 12 control isolates studied, one apparently epidemiologically unrelated isolate showed a PFGE pattern with 100% similarity after restriction with both enzymes (isolate identified as local control 3, LC3). So, these three isolates along with two additional local control isolates showing high similarity with the PFGE patterns of the isolates under

Discussion

The use of molecular epidemiology to investigate cases of transmission in a forensic context has become increasingly frequent. Since the pioneering study by Ou and colleagues [1], the analyses have become more complex, both in the molecular technologies used to obtain the sequence information and in the ensuing bioinformatics, evolutionary and statistical methods applied to that information. Although this type of studies usually deals with deliberate or unintentional transmission of viruses,

Conclusions

Sexual abuses in children, particularly those occurring in the family setting, are a matter of concern for judicial authorities. The presence of a sexually transmitted infection can be used to support allegations of sexual abuse, but the particular significance of the identification of a sexually transmitted agent as an evidence of possible child sexual abuse varies by the type of pathogen. Postnatal acquired gonorrhea usually suggests some kind of sexual contact [47,48]. Although traditional

Acknowledgements

This work was funded by projects BFU2017-89594-R from MICINN (Spanish Government) and Prometeo2016-122 (Generalitat Valenciana). CFC benefits from an FPI predoctoral contract (BES-2015-074204) from MICINN (Spanish Government). The funding agencies had no role in design, development nor submission of this research for publication. HTS at FISABIO-Plataforma de Secuenciación used equipment co-funded by the EU, ERDF program for the Comunidad Valenciana 2014-2020.

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