Detection of Saliva by Immunoaffinity and Mass Spectrometry (SIMS)

December 13, 2023 updated by: University Hospital, Montpellier

Detection of Specific Salivary Proteins by Immunoaffinity and Mass Spectrometry

The identification of saliva in genital area during a criminal investigation can be a critical component in the prosecution of a sexual assault in France, as non-consensual oral-genital intercourses have been considered as crimes since 2021.The development of highly specific methods for saliva detection is therefore crucial as the commonly employed screening methods lack specificity. Protein mass spectrometry has proven to be a sensitive and specific method but is particularly time consuming. A faster and more sensitive hybrid approach using automated immunoaffinity mass spectrometry (IP-LC-MS/MS) has been recently developed and has been found to be particularly performant for the detection of a seminal fluid protein (semenogelin), allowing a high-throughput seminal fluid identification in semen samples. Like semenogelin, specific salivary proteins such as histatin type 1, cystatin D or proline-rich proteins (PRPs) could be detected using this promising approach, which has never been tested on saliva samples. In collaboration with the Clinical Proteomics Platform and the Department of Reproductive Medicine of the University Hospital of Montpellier, we aim to develop a protocol for the detection of specific saliva proteins by IP-LC-MS/MS in sexual assault-type samples.

Study Overview

Status

Not yet recruiting

Conditions

Intervention / Treatment

Detailed Description

Each participant will be contacted by a phone call the day before the visit, to present for a presentation of the study.The day of the visit, two types of samples will be collected :

  • Saliva samples: on healthy volunteers in the Department of Legal Medicine (2 samples of 1.5 - 2 mL are collected for each volunteer).
  • Vaginal samples : on women consulting in the Department of Reproductive Medicine (2 dry swabs on each patient). 2 groups, One group with vaginal secretion fluid samples only, and one group with vaginal secretion fluid + sperm samples.

Study Type

Interventional

Enrollment (Estimated)

35

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact Backup

Study Locations

  • France
      • Montpellier, France, 34295
        • PEYRON

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

Yes

Description

Inclusion Criteria:

Saliva Samples

  • Men and women aged 18 or more

Vaginal samples :

  • Women aged 18 or more
  • No unprotected vaginal sexual intercourse during the 10 days prior to the visit (group 1)
  • Unprotected vaginal sexual intercourse with ejaculation in the 24 hours prior to the visit (group 2)

Exclusion Criteria:

Saliva Samples :

  • Active pathology of the saliva glands (infection, tumor)
  • Unprotected oral-genital sexual intercourse in the 24 hours prior to the visit
  • Failure to obtain written informed consent after a reflection period
  • Pregnant or breast feeding women
  • Adult protected by law or under guardianship or curatorship
  • No affiliation to the French Social Security System or no benefit from such a system

Vaginal Samples :

  • Oral-genital sexual intercourse (cunnilingus) in the 24 hours prior to the visit
  • Failure to obtain written informed consent after a reflection period
  • Pregnant or Breastfeeding women
  • Adult protected by law or under guardianship or curatorship
  • No affiliation to the French Social Security System or no benefit from such a system

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Other
  • Allocation: Non-Randomized
  • Interventional Model: Parallel Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Other: Saliva

Collection of two saliva samples (approx. 1.5 to 2 mL each) by passive drooling in healthy volunteers

These samples will be centrifuged and the supernatant collected.
Other: Saliva collection
Collection of 2 samples of 1.5 to 2mL of saliva by passive salivation in healthy volunteers
Other: IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry

sample preparation and analysis :

  • Impregnation of the tips (absorbent cotton) of sterile dry swabs and vaginal swabs with controlled quantities of saliva.
  • Immunocapture of salivary proteins of interest (histatin type 1, cystatin D, PRPs) by protein A affinity purification
  • LC-MS/MS analysis of eluted purified peptides (Multiple Reaction Monitoring mode)

Dilutions will be made from 10 to 10 (1/10, 1/100, 1/1000...), then after reaching a detectability threshold, specified by a second more precise analysis (e.g. if no signal at 1/1000, analysis at 1/500 then 1/250 etc.). Analytical sensitivity will be tested on three samples of each type (salivary, vaginal, vaginal + semen) to ensure reproducibility of results.

Operators, unaware of the presence of saliva in the samples, will then carry out saliva-specific protein detection analysis using the IP-LC-MS/MS method.
Other: Vaginal secretions
In order to be as close as possible to real life, two vaginal swabs (introduced through the vaginal orifice over a length of 2 to 3 cm) will be taken with a sterile dry swab during the gynecological examination (prior to endovaginal ultrasound or any other endovaginal procedure) of patients included in the Reproductive Medicine Service.
Other: IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry

sample preparation and analysis :

  • Impregnation of the tips (absorbent cotton) of sterile dry swabs and vaginal swabs with controlled quantities of saliva.
  • Immunocapture of salivary proteins of interest (histatin type 1, cystatin D, PRPs) by protein A affinity purification
  • LC-MS/MS analysis of eluted purified peptides (Multiple Reaction Monitoring mode)

Dilutions will be made from 10 to 10 (1/10, 1/100, 1/1000...), then after reaching a detectability threshold, specified by a second more precise analysis (e.g. if no signal at 1/1000, analysis at 1/500 then 1/250 etc.). Analytical sensitivity will be tested on three samples of each type (salivary, vaginal, vaginal + semen) to ensure reproducibility of results.

Operators, unaware of the presence of saliva in the samples, will then carry out saliva-specific protein detection analysis using the IP-LC-MS/MS method.

Other: Vaginal secretion collection
Collection of vaginal secretions with 2 dry swabs in women

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Reproducibility between saliva samples
Time Frame: 3 months

Intra-assessor agreement on saliva samples, which involves testing the first sample and retesting the second sample

The study's primary endpoint will be to measure the reliability of the protocol, consisting of reproducibility or intra-assessor agreement,for the detection of specific salivary proteins (histatin type 1, PRPs, cystatin D) using the IP-LC-MS/MS method on adult saliva samples.

Intra-assessor agreement (test/retest) will be measured by their means +/- standard deviation.

Several analyses (between 3 or 4) on the same sample will be carried out to determine whether the results are identical
3 months
Intermediate fidelity between technicians on saliva samples
Time Frame: 3 months

Inter-assessor agreement(Cohen's kappa coefficient) on saliva samples, which involves testing several samples by at least 2 different technicians

The study's primary endpoint will be to measure the reliability of the protocol, consisting of intermediate fidelity or inter-assessor agreement (Cohen's kappa coefficient) for the detection of specific salivary proteins (histatin type 1, PRPs, cystatin D) using the IP-LC-MS/MS method on adult saliva samples.

The kappa coefficient gives a score ranging from 0 to 1. If the coders totally agree, κ = 1. If they totally disagree (or agree due solely to chance), κ ≤ 0.
3 months

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Reproducibility between vaginal samples soaked with saliva
Time Frame: 3 months

Intra-assessor agreement on vaginal samples soaked with saliva

The secondary endpoints, will consist of repeating the primary endpoints (i.e. reproducibility and intermediate fidelity) on the vaginal samples impregnated with sperm or not.

Again, the same sample will be tested several times (between 3 or 4) to see if the results come back similar.
3 months
Intermediate fidelity between technicians on vaginal samples soaked with saliva
Time Frame: 3 months

: Inter-assessor agreement (Cohen's kappa coefficient) on vaginal samples soaked with saliva

The secondary endpoints, will consist of repeating the primary endpoints (i.e. reproducibility and intermediate fidelity) on the vaginal samples impregnated with sperm or not.

The kappa coefficient gives a score ranging from 0 to 1. If the coders totally agree, κ = 1. If they totally disagree (or agree due solely to chance), κ ≤ 0.
3 months
Analytical Sensibility
Time Frame: 3 months
Lowest saliva concentration (µL) detected by the method on saliva samples and on vaginal samples soaked with saliva in controlled condition
3 months
Diagnostic sensibility
Time Frame: 3 months
Analysis of saliva samples and vaginal samples soaked with saliva in controlled condition, to determine the rate of detection of true positives and false negatives
3 months
Diagnostic specificity
Time Frame: 3 months
Analysis of vaginal samples and dry samples in controlled condition, to determine the rate of false positives and true negatives.
3 months

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

University Hospital, Montpellier

Investigators

  • Study Director: Pierre-Antoine PEYRON, PI, University Hospital, Montpellier

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Helpful Links

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Estimated)

January 15, 2024

Primary Completion (Estimated)

July 15, 2024

Study Completion (Estimated)

December 31, 2024

Study Registration Dates

First Submitted

August 18, 2023

First Submitted That Met QC Criteria

December 13, 2023

First Posted (Estimated)

December 27, 2023

Study Record Updates

Last Update Posted (Estimated)

December 27, 2023

Last Update Submitted That Met QC Criteria

December 13, 2023

Last Verified

December 1, 2023

More Information

Terms related to this study

Keywords

Other Study ID Numbers

  • RECHMPL23_0098

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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