One of the central applications of forensic entomology is the determination of the post mortem interval (PMI) from arthropod evidence associated with a corpse. Estimations of the PMI are based on succession and developmental patterns of specific species that visit the body. As first colonisers, Calliphoridae (blow flies) are often used by forensic entomologists to determine the PMI however, developmental rates of visiting fauna differ substantially which makes correct species identification vital. Traditional methods of identification which assign species based on keys that capitalise on morphological differences are insufficient for closely related species, especially during immature stages of the lifecycle or when the specimen is damaged. Molecular identification such as DNA barcoding has therefore become a popular method of identifying species. DNA barcoding characterises species by sequencing and analysing specific regions in the genome. This technique has been used to characterise species in various countries including parts of South Africa. Its application has also been demonstrated in a forensic setting but data for the Western Cape is minimal. This study therefore aimed to assess the utility of DNA barcoding for species level determination of four blow fly species common to the Western Cape of South Africa (Chrysomya chloropyga, Chrysomya albiceps, Chrysomya marginalis, and Lucilia sericata) as well as its ability to identify immature specimens. Ten adult specimens from each species were morphologically and molecularly identified using microscopy and DNA barcoding respectively. The standard DNA barcode, cytochrome c oxidase subunit I (COI) and a secondary marker, the second internal transcribed spacer (ITS2) were analysed. Phylogenetic analyses for both barcodes showed high interspecific divergence values which are desirable for species level differentiation by DNA barcoding. COI sequences from adult flies were also submitted and searched against BOLD for identification and only genus level identity could be achieved, indicating that, COI alone may be insufficient to discriminate between closely related species. DNA sequences from the adult specimens were then used as reference sequences for identification of seven unknown immature specimen using DNA barcoding of both COI and ITS2. Sequence similarity was assessed and identity was assigned based on >98% similarity scores, and all immatures were successfully identified. The use of more than one DNA marker to complement morphological data ensures higher confidence of species level identification. This method provides a reliable and consistent tool for entomologists to use for species identification which results in higher levels of accuracy in PMI estimations.

Road transportation systems are a global developmental achievement. However, with them comes increased morbidity and mortality rates in the form of road traffic accidents. In South Africa, there is a need to characterize road traffic accidents and the injuries associated with them, to determine the preventative mechanisms required to reduce their morbidity and mortality rates. A brief review of fatal road traffic accidents from a global perspective is presented, highlighting the current literature surrounding the prevalence, demographics and blunt force trauma injuries associated with road traffic accidents in South Africa. There is limited research regarding the prevalence and characteristics of road traffic accidents. The objective of this study was to determine the prevalence of fatal road traffic accidents, necessitating the need for research, particularly at the regional level. A retrospective analysis was therefore conducted of all fatal road traffic accident related deaths autopsied at Salt River Mortuary (which services the West Metropole region of Cape Town, South Africa) from January 1st , 2013 to December 31st , 2014. The mean prevalence of road traffic accidents for the reviewed period was 15.9 / 100 000 population. The majority of road traffic accident victims were males who fell in the age group of 30 – 49 years. Over the two-year period, the majority of road traffic accident victims were pedestrians with elevated blood alcohol concentration levels. The head and facial regions of victims commonly exhibited external injuries, while the majority of fractures and organ injury were seen in the head and chest regions. There are limited studies which have investigated the blunt force trauma injuries associated with road traffic accidents in South Africa, and there is a need for further research. Interventions are of paramount importance to decrease fatal road traffic accidents, particularly amongst pedestrians as a road user. This study presents recent data on road traffic accidents for the West Metropole region of Cape Town (South Africa).

Background: The Republic of South Africa (RSA) is facing a rape crisis, with women and children being the most vulnerable. DNA evidence is used to aid in suspect identification and to confirm sexual contact. The collection of biological forensic evidence after a rape incident is routine in RSA, however, its contribution to the outcome of rape cases is still unclear. This study aimed to investigate the use of Sexual Assault Evidence Collection Kits (SAECKs) and their impact on the outcome of rape cases. Methods: Data for this study was collected by conducting a retrospective review of rape cases (n=98) prosecuted and finalised at the Western Cape High Court between 1st January 2012 and 31st December 2016. Court dockets were examined, and data was collected using a set of predefined variables. Backward logistic regression was used to analyse whether there is an association between the accused being found guilty and the use of SAECKs and the presentation of DNA evidence. Main findings: In 61% (n=60/98) of the cases a SAECK was used during the medical examination and a DNA report was generated and available for use in 77% (n=46/60) of these cases. There was a higher conviction rate when a SAECK was used (88%, n=53/60) in comparison to when it was not used (53%, n=8/15). Contrary to our expectation, the use of SAECKs was not found to be positively associated with the conviction when using backward logistic regression. This means the use of a SAECK decreased the odds of finding the defendant guilty of rape, albeit by a small degree (OR=0.196, p-value=0.040). DNA was not associated with the case outcome. The majority of cases were stranger rapes (64%,n=63/98), that occurred outdoors (64%,n=61/95)and mostly in the township locations such as Khayelitsha, Philippi, Nyanga, areas located on the Cape Flats, with only a single perpetrator. Injuries were documented in 50% of the cases (n=49)and almost 48% (n=46/95) of the victims were coerced and threatened with a weapon. Our binary model found only the location of incidence (p-value= 0.006) to be significantly associated with case outcome with an odds ratio of 19.827. Conclusions: The findings from this study are significant as they point out that health care practitioners are adequately trained and equipped to provide quality health care responses and to work effectively with police officers. SAECKs are utilised routinely during rape investigations. However, there are various factors relating to the victim, perpetrator and assault characteristics that influence the judge's verdict. This study raised questions relating to how those factors are measured or weighed and are predictive of case outcome. Although we found no positive associations between the variables investigated (except the location of the incident) and court outcome, future research, including larger sample size, should investigate these variables in order to obtain true significance, especially concerning the use of SAECKs and its impact on the court outcome.

Forensic entomologists rely on insect development and successional data to estimate the post-mortem interval (PMI). Ante-mortem drug use prior to an individual's death may result in drug transmission to feeding insects and subsequent alteration of their development, thereby altering PMI estimates. This study investigated the influence of Ritalin (methylphenidate hydrochloride) (MPH) on the development of Chrysomya chloropyga. C. chloropyga larvae were reared on pig liver treated with MPH and exhibited a trend of expedited larval development and prolonged pupal development. Conservatively, the results suggest that MPH may expedite the larval stage by up to 17 hours and prolong the pupal stage by up to 16 hours. These preliminary findings suggest that, at the concentration investigated, MPH may alter the duration of C. chloropyga developmental stages, and consequently PMI estimates if MPH is not detected or its effects not considered. Furthermore, MPH was detected in both frozen and ethanol preserved specimens. MPH could still be detected from treated larvae, after 3.5 days incubation at ~30°C. This may suggest an improved stability of MPH in insects. Moreover, detection of MPH from ethanol preserved specimens suggests the qualitative toxicological utility of specimens maintained in this preservation liquid, despite stability and self-extraction concerns. These are the first entomotoxicological data on MPH generated for blow flies local to the Western Cape, South Africa.

Hair has, over recent years, become widely recognised as an alternate or complementary matrix to blood and urine for drug analysis. Hair analysis offers a wider detection window after drug exposure than blood or urine testing and can provide a long-term history of an individual’s drug use. There are several practical applications of hair analysis for drugs including workplace drug testing, doping control, driving licence re-granting, drug-related deaths and drug-facilitated crimes. As a result hair analysis is currently being performed within various toxicological fields in laboratories around the world. However, before the start of this study no hair analysis for drugs was being performed in South Africa. Therefore, the main aim of this study, as stated in Chapter 1, was to develop a method for the detection and quantification of drugs of abuse, specifically methamphetamine and amphetamine, in hair using High Performance Liquid Chromatography coupled to Mass Spectrometry.

Violence and resulting injuries are critical health burdens worldwide, accounting for the death of millions of individuals annually. The literature reports an association between drug use and violence, providing data indicating that the use of psychoactive substances increases the risk of morbidity and mortality due to violent acts. South Africa has a long history of violence, with one of the highest rates of recorded violence- and injury-related deaths in the world. This is complicated by an increase in illicit substance use and abuse, particularly in the Cape Town Metropole, located within the Western Cape Province. The use of toxicological findings from victims of violent death (homicides, suicides, and accidents) to examine community-specific drug-related violence is slowly increasing in different parts of the world. In South Africa, however, monitoring drug trends in violent fatalities using toxicological analysis is uncommon, and hence drug toxicology of violent-related fatalities is limited. Divided into three contextual sections, this research study focuses on the post-mortem toxicology of violent deaths in a South African setting. The first section provides a general idea of the research problem and an initial development of the investigation process. The second section provides a theoretical basis for performing routine toxicological analyses in deaths due to violence, reports important research work conducted in the field worldwide, and emphasizes the need to monitor toxicological data derived from violent fatalities in Cape Town, South Africa. The last section, in the form of a manuscript, presents the overall research study including the methodology, outcomes, and concluding findings in a concise and illustrative manner. The primary aim of this pilot study was to investigate the prevalence and characteristics of illicit substances in violent fatalities (homicides, suicides, and accidents) of the Salt River mortuary in Cape Town, South Africa. The objectives were to conduct a comprehensive drug toxicology analysis to generate qualitative and comparative data from the aforementioned cases. In addition, this study investigated the dynamics between psychoactive substance use and violent deaths in terms of toxicological trends, and the demographics and circumstances of death of the victim. Lastly, the author discusses potential qualitative associations between illicit substances and violence-related deaths in a South African setting, and provide suggestions for future toxicological analyses in these fatalities.

Drug usage, both of a recreational or pharmaceutical nature, is common, however the abuse of such substances is an international problem. In the Western cape, South Africa, the burden of drug-related fatalities is high compared to the rest of the country. The provincial Forensic Pathology Service may encounter cases where drug-related fatalities are unclear whether death was accidental or suicidal, or drug toxicity is inconsistent with the medical/social history. This may be due to genetic alterations with drug metabolism and it has been suggested that genetic analyses may be the next step in these cases. However, toxicology results from the National Forensic Chemistry Laboratory in the Western Cape may be delayed by months to years, meaning that upon interpretation of toxicology results, there is no chance to obtain another blood sample from the deceased individual for genetic analysis. It was therefore important to determine the suitability of blood samples collected and handled in toxicology environments for subsequent genetic tests. Previously, blood samples from 30 post-mortem cases were collected into two red-top (no additives), two grey-top (sodium fluoride/potassium oxalate) and one purple-top (EDTA) tubes. Samples from one red-top and one grey-top tube underwent toxicological analysis, followed by DNA analysis, while the remaining tubes (controls) underwent DNA analysis immediately. All samples were then stored for approximately one year, prior to this study. The DNA analysis was repeated on all blood samples (n = 150) and results were assessed in terms of storage time and tube type. DNA was not significantly degraded in any of the samples; however, DNA from red-top tubes had significantly lower concentrations compared to that from grey-top tubes (p < 0.001), regardless of whether the sample had undergone toxicological analysis. The very low yields of DNA from red-top tubes posed substantial challenges for PCR-based analysis, resulting in poor quality Sanger sequencing results. Some DNA from grey-top tubes, passed the quality assessments and hence further work is required to provide an informed decision on which tube type is better suited for genetic analyses.

Introduction: Road traffic accidents (RTAs) and associated morbidity and mortality are a global public health burden. Literature reports on an association between drugs and/or alcohol intoxication and traffic collisions. In South Africa (SA), where drug use and abuse are prevalent, annual RTAs rates are higher than the average global burden. Toxicological analyses in cases of RTA fatalities are not performed routinely in SA (apart from alcohol analysis), thus understanding the burden of other drug impairment on road traffic deaths is limited. Aim: A prospective toxicological analysis was performed in a cohort of road traffic fatality cases (drivers, passengers, pedestrians, motorcyclists and cyclists) from Salt River Mortuary in Cape Town, SA. The objectives were to perform drug screening in these cases to preliminary investigate detected substances as well as to evaluate the demographics and circumstances of death of the aforementioned cases. Methods: A systematic review was first performed to investigate the prevalence of drugs in internationally reported RTA fatalities. For the prospective study, post-mortem specimens including blood, vitreous humor, urine and bile were collected from cases in which next-of-kin consent was obtained. All samples were analysed using liquid chromatography-quadrupole time-of -flight mass spectrometry (LC/QTOF-MS). Results: Thirty cases were analysed over 3 months, of which most were male, pedestrians and between the age group of 31-40 years. The most prevalent cause of death was multiple blunt force injuries to the body. Single vehicle crashes were predominant particularly among the pedestrians and motorcyclists whereas drivers were mostly involved in multiple vehicle crashes. Substances (other than ethanol) were detected in 90% (n=27) of the cases. A broad range of drug groups were detected, and the most prevalent specific legal substances were caffeine (66.7%) and nicotinamide (53.3%) and illegal substances were methaqualone (10.0%) and methamphetamine (6.67%). Multiple cases indicated the detection of impairing substances even if consumed therapeutically, such as codeine, chlorpheniramine, diphenhydramine and zopiclone. Discussion: This study was the first to the author’s knowledge to report on prospective toxicological findings in road traffic accident cases in Cape Town. Although this was a pilot study, the results were in line with findings from other international studies, together with findings of prominent abused drugs within Western Cape (e.g. methaqualone and methamphetamine). While this study made no inferences of drug intoxication to cause of death, it has set a basis for future research in this topic and the development of a standardised protocol for the routine analyses of such cases in SA.

Includes abstract. Includes bibliographical references.

Extracting forensically useable DNA from human remains recovered from an open marine environment is problematic, and in some cases, impossible. The reason is unclear given the lack of research on marine decomposition, DNA survival in seawater, and possible methods to optimise the DNA extraction workflow. Compounding this problem is the fact that South Africa experiences a high number of unidentified human bodies entering its mortuaries each year, and these individuals often remain unidentified. The aim of the study was to extract forensically useable DNA from pig (Sus scrofa) teeth submerged in-situ in an open marine environment, by a process of optimisation and implementation. Detailed environmental information was available for this study. A DNA extraction technique was developed and optimised on “fresh” control pig teeth (n = 13). The developed methods for decontamination, tooth sampling, and the optimised DNA extraction protocol were successfully performed on these, with forensically useable DNA obtained. However, this was not the case for the subsample of experimental pig teeth (n = 6) tested. Implementation of the developed method on a larger sample of experimental teeth (n = 28) was warranted to assess the recovery of nDNA and mtDNA. Amplification of nDNA by qPCR was successful in 60% (17/28) of samples for a 96 bp fragment, and in 46% (13/28) for 200 bp. By comparison, mtDNA showed a detection rate of 57% (16/28) for a 486 bp fragment via PCR amplification. In seven samples mtDNA was detected where nDNA was not, demonstrating improved survivability in seawater. Colder and more stable seawater temperatures is hypothesised to have preserved molecular elements. DNA hydrolysis and the possibility of DNAase activity from marine bacteria, may have contributed to poor DNA preservation in the other samples. Recovery of DNA from teeth submerged in an open marine environment is complex and requires further investigation in human samples to improve the identification process for individuals who have died at sea.