LITERATURE REVIEW
1.9 The Current Status of Forensic Fluid and Tissue Identification
1.9.2 The Use of Biological Markers
1.9.2.3 Microbial Biomarkers
Human bodies do not escape the ubiquitous presence of microorganisms. Microbes including viruses, fungi, bacteria, protozoa and parasites aid in food digestion and human health. They are located outside of, and within the body including hair, skin, the vagina, intestines and mouth. Variations of the human microbiome are attributed to illnesses such as obesity, periodontitis, vaginosis and inflammatory bowel disease (Jorth et al., 2014). Studies have shown that specific microbes may be found in particular human body sites and fluids.
This is now being exploited profusely for indications of body sites, fluids and tissues such as saliva, skin, nasal area, stool and vaginal fluids (Benschop et al., 2012; Huse et al., 2012).
The specificity of microbial biomarkers is found to be best for vaginal fluids and menstrual secretions (Benschop et al., 2012; Choi et al., 2003; Fleming and Harbison, 2010; Giampaoli et al., 2012). Akutsu and colleagues (2012) reported PCR detection of 16S-rRNA genes of Lactobacillus jensenii and L. crispatus in not only vaginal fluid but also female urine samples. The presence of L. gasseri and Gardnerella vaginalis was also found in semen. L.
gasseri has proven to be unreliable marker, being detected in vaginal fluid (Fleming and Harbison, 2010) as well as semen (Akutsu et al., 2012) and furthermore, the use of bacterial RNA is a drawback. Primers may target not only the sequence of interest but also any species-specific region. In RNA-based assays, sequences must first be transcribed (Sijen, 2014). Giampaoli and colleagues (2012) described multiplex RT-PCR detection of microflora DNA obtained from vaginal and faecal samples. Enterococcus spp. was most prevalent in faecal samples. Although strong signals for L. crispatus and L. gasseri were detected in vaginal fluid, signals for L. gasseri were also detected for faecal samples.
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Lactobacilli demonstrate potential as a vaginal fluid marker however, Pavlova and Tao (2000) demonstrated that benzo-(a)-pyrene-diol epoxide from smoking disrupts naturally occurring Lactobacillus spp. Antibiotics such as erythromycin causes changes in the vaginal microbial communities (Choi et al., 2003; Witkin et al., 2007). Deliberation exists with the strategy due to intra-individual variability; bacterial fingerprints may differ from hands of a single individual, leading to confusion (Kuczynski et al., 2010). Microbes are easily transferred, by touch or contact of another human or surfaces. Vaginal microflora can transfer to the male penis or groin (Benschop et al., 2012; Flores et al., 2011). Complexity arises in use of microbial markers since the microbiota at a body site does not necessarily relate to microbiota from a crime stain. Compositions may vary due to degradation, contamination and growth of many airborne species upon release from the human body (Sijen, 2014). Also, Dewhirst et al. (2011) showed that approximately 16% of oral microbiota is shared between canines and humans; including Mogibacterium timidum, M. diversum, Treponoma amylovorum, T. vincenti, three streptococcal species and numerous others. Such findings greatly compromise human specificity.
58 1.10 Scope of the Present Study
The most prevalent methods to analyse body fluids found at crime scenes are limited by lack of specificity and sensitivity, consumption of valuable biological material and are largely presumptive. However the precise identification of body fluids found at crime scenes provides vital information that can support a link between sample donors and actual criminal acts. Reports of whole genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show varying DNA methylation profiles according to the type of cell or tissue. Thus, body fluid-specific differential DNA methylation is a promising indicator for precise body fluid identification. Even though forensic tDMR-based body fluid identification shows much potential, not many tDMRs have been confirmed for this purpose. Studies have confirmed tDMRs for semen identification however specific tDMRs for blood, saliva and vaginal fluid have yet to be validated. The development of new tDMRs is the best way forward for accurate identification of biofluids for forensic applications. Accordingly, novel tDMRs were searched for based on the analysis of differential gene expression in human tissues and its correlation with DNA methylation.
The tDMRs were targeted to develop primers for use in Methylation-Specific Restriction Enzyme (MSRE-PCR) for efficient identification of saliva, blood, semen and vaginal fluid.
The identification of additional body fluid tDMRs is expected to spur the improvement of promising body fluid identification methods for forensic applications.
Additionally, researchers have provided resounding evidence of differential DNA methylation between population groups. Therefore, it is worthwhile to validate previously documented tDMR-based markers on the South African population. For this, methylation profiles of four previously reported tDMRs in saliva obtained from Blacks, Indians, Whites and Coloureds in South Africa were evaluated, to determine if there is differential methylation of targeted tDMRs among ethnic groups. Significant differences could assist forensic analysts in future, to narrow down their search for sample donors.
59 1.10.1 Hypotheses tested
It is hypothesized that the identification of new tissue-specific differentially methylated regions (tDMRs) in human body fluids would allow the development of markers to differentiate between body fluids.
It is further hypothesized that DNA methylation profiles of saliva, using selected tissue-specific differentially methylated regions (tDMRs) for USP49, DACT1, L81528 and PFN3 genes differ among the diverse South African population and thus may be of significance in forensic casework.