Mortality due to respiratory infections: an alert study before COVID-19 pandemic.

Objective: Respiratory tract infections remain a common problem in clinical practice with high morbidity and mortality worldwide. In Portugal, pneumonia was the third leading death cause in 2018. Due to COVID-19 pandemic, there is a growing concern about the burden of respiratory diseases and preventable risk factors. The present study started before the pandemic and its aim was to determine the occurrence of pneumonia/bronchopneumonia in a postmortem series and to characterize its circumstantial context. Methods: A retrospective anatomopathological study was performed on cases with acute pneumonia/bronchopneumonia at the Medicolegal Portuguese Institute (2011-2017). Results: In an autopsy series of 737 patients, 521 were male and 675 presented comorbidities. The mean age was 63.87 ± 19.8 years. The most common acquisition site was community (65.1%), as natural death (65.5%). Concerning the manner of death, most cases (48.0%) were sudden deaths, followed by accidents (29.2%). A statistically significant association was observed between the medicolegal etiology and the place of infection acquisition, with higher prevalence of natural obitus (91.0%) in community-acquired pneumonia/bronchopneumonia versus higher prevalence of violent obitus in hospital-acquired pneumonia/bronchopneumonia (82.1%) (p < 0.001). Conclusions: Forensic anatomopathological postmortem data may contribute to better understand community and hospital pulmonary infections.

Deoxyribonucleic Acid Extraction and Quantification from Human Saliva Deposited on Foods with Bitemarks.

AIM: The aim of this study is to evaluate the potential of deoxyribonucleic acid (DNA) recovery from bite marks in foods, in different collection types, from DNA quantification. MATERIALS AND METHODS: The sample consisted of 80 swabs, obtained from 20 cheese pieces, bitten by the same person, using the double-swab technique in the center and the periphery of the bite. A statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS) statistical software version 20.0, with values of p < 0.05 being considered statistically significant. RESULTS: The DNA was recovered in all cheese pieces, regardless of the collection types and the bite region. However, the comparative analysis of DNA recovery potential in the four swabs allowed us to infer that the collections in the central region of the bite (DC and WC) were the ones that presented better precision, in addition to extracting a higher DNA concentration, the dry swab being in the center of the bite which presented better results. CONCLUSION: The results proved the effectiveness of the double-swab technique for collecting genetic materials in bite marks; however, in the food used, a single collection at the center of the bite would be enough, optimizing the resources and time needed for the analysis. CLINICAL SIGNIFICANCE: Due to the difficulties of physically comparing a site of a skin lesion and the dental arches of the suspect, the evidence of DNA in saliva has been used to indicate the perpetrator of the bite. In addition, the collection, preservation, and isolation of saliva DNA can be done at low cost and provide flexibility for clinical and laboratory workflow.

Human settlement history between Sunda and Sahul: a focus on East Timor (Timor-Leste) and the Pleistocenic mtDNA diversity.

BACKGROUND: Distinct, partly competing, "waves" have been proposed to explain human migration in(to) today's Island Southeast Asia and Australia based on genetic (and other) evidence. The paucity of high quality and high resolution data has impeded insights so far. In this study, one of the first in a forensic environment, we used the Ion Torrent Personal Genome Machine (PGM) for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice. RESULTS: In this first representative investigation on the mitochondrial DNA (mtDNA) variation of East Timor (Timor-Leste) population including >300 individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup P1, an indigenous lineage of the Southwest Pacific region. Our results suggest a colonization of southern Sahul (Australia) >37 kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern Sahul (New Guinea) followed by westward migrations <28 kya. CONCLUSIONS: The temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated "later" events of (South) East Asian origin pinpoints a highly dynamic migratory phase.

Assessment of IrisPlex-based multiplex for eye and skin color prediction with application to a Portuguese population.

DNA phenotyping research is one of the most emergent areas of forensic genetics. Predictions of externally visible characteristics are possible through analysis of single nucleotide polymorphisms. These tools can provide police with "intelligence" in cases where there are no obvious suspects and unknown biological samples found at the crime scene do not result in any criminal DNA database hits. IrisPlex, an eye color prediction assay, revealed high prediction rates for blue and brown eye color in European populations. However, this is less predictive in some non-European populations, probably due to admixing. When compared to other European countries, Portugal has a relatively admixed population, resulting from a genetic influx derived from its proximity to and historical relations with numerous African territories. The aim of this work was to evaluate the utility of IrisPlex in the Portuguese population. Furthermore, the possibility of supplementing this multiplex with additional markers to also achieve skin color prediction within this population was evaluated. For that, IrisPlex was augmented with additional SNP loci. Eye and skin color prediction was estimated using the multinomial logistic regression and binomial logistic regression models, respectively. The results demonstrated eye color prediction accuracies of the IrisPlex system of 90 and 60% for brown and blue eye color, respectively, and 77% for intermediate eye color, after allele frequency adjustment. With regard to skin color, it was possible to achieve a prediction accuracy of 93%. In the future, phenotypic determination multiplexes must include additional loci to permit skin color prediction as presented in this study as this can be an advantageous tool for forensic investigation.

Biological Evidence Management for DNA Analysis in Cases of Sexual Assault.

Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody). Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis.

LC-MS-MS-MS3 for the determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samples.

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography-tandem mass spectrometry methodology (LC-MS-MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d., 1.8 µm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS-MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

Sudden death in young South European population: a cross-sectional study of postmortem cases.

To describe the annual incidence and the leading causes of sudden non-cardiac and cardiac death (SCD) in children and young adult Portuguese population. We retrospectively reviewed autopsy of sudden unexpected deaths reports from the Portuguese National Institute of Legal Medicine and Forensic Sciences' database, between 2012 and 2016, for the central region of Portugal, Azores and Madeira (ages 1-40: 26% of the total population). During a 5-year period, 159 SD were identified, corresponding to an annual incidence of 2,4 (95%confidence interval, 1,5-3,6) per 100.000 people-years. Victims had a mean age of 32 ± 7 years-old, and 72,3% were male. There were 70,4% cardiac, 16,4% respiratory and 7,5% neurologic causes of SD. The most frequent cardiac anatomopathological diagnosis was atherosclerotic coronary artery disease (CAD) (33,0%). There were 15,2% victims with left ventricular hypertrophy, with a diagnosis of hypertrophic cardiomyopathy only possible in 2,7%. The prevalence of cardiac pathological findings of uncertain significance was 30,4%. In conclusion, the annual incidence of SD was low. Atherosclerotic CAD was diagnosed in 33,0% victims, suggesting the need to intensify primary prevention measures in the young. The high prevalence of pathological findings of uncertain significance emphasizes the importance of molecular autopsy and screening of first-degree relatives.

Medico-Legal Examination of Sexual Assaulted Victims Unable to Consent in Portugal: Ethical Decision-Making.

Medical decision-making is a complex task in any field. In the medico-legal examination of victims that have (allegedly) been sexually assaulted there are many specific variables and features influencing the decision. It is essential to complement the clinical intervention with a forensic approach. Clinical parameters such as the victim's physical and cognitive state along with circumstantial information such as the elapsed time from the event and the type of abuse (described or suspected) grant different levels of priority to the forensic medical assessment. In such cases, forensic medical doctors or other medical doctors responsible for attending to the victim may have to decide whether to perform the examination prior to a judicial analysis of the case if consent cannot be obtained. This implies the need to deliberate about performing the examination and/or reporting the case to legal authorities. This article discusses the forensic medical decision-making process in cases of alleged recent sexual assault of victims who are legally unable to consent or unable to consent for other reasons. We aimed to identify possible ethical problems that can arise in this context and discuss which elements should be considered by medical doctors when making decisions about such cases. The Portuguese legal framework of medico-legal examinations is analyzed. The authors also make considerations about reporting these cases from a legal point of view. The discussion turns to an ethical perspective where possible ethical problems arising from medical deliberation are identified. Issues about legally incompetent victims and incompetent victims due to other reasons are addressed. A decision-making tree, based on the problems identified, is proposed.

A Blood-Bone-Tooth Model for Age Prediction in Forensic Contexts.

The development of age prediction models (APMs) focusing on DNA methylation (DNAm) levels has revolutionized the forensic age estimation field. Meanwhile, the predictive ability of multi-tissue models with similar high accuracy needs to be explored. This study aimed to build multi-tissue APMs combining blood, bones and tooth samples, herein named blood-bone-tooth-APM (BBT-APM), using two different methodologies. A total of 185 and 168 bisulfite-converted DNA samples previously addressed by Sanger sequencing and SNaPshot methodologies, respectively, were considered for this study. The relationship between DNAm and age was assessed using simple and multiple linear regression models. Through the Sanger sequencing methodology, we built a BBT-APM with seven CpGs in genes ELOVL2, EDARADD, PDE4C, FHL2 and C1orf132, allowing us to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 6.06 years, explaining 87.8% of the variation in age. Using the SNaPshot assay, we developed a BBT-APM with three CpGs at ELOVL2, KLF14 and C1orf132 genes with a MAD of 6.49 years, explaining 84.7% of the variation in age. Our results showed the usefulness of DNAm age in forensic contexts and brought new insights into the development of multi-tissue APMs applied to blood, bone and teeth.

Age prediction in living: Forensic epigenetic age estimation based on blood samples.

DNA methylation analysis in a variety of genes has brought promising results in age estimation. The main aim of this study was to evaluate DNA methylation levels from four age-correlated genes, ELOVL2, FHL2, EDARADD and PDE4C, in blood samples of healthy Portuguese individuals. Fifty-three samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for CpG dinucleotide methylation status. Linear regression models were used to analyze relationships between methylation levels and chronological age. The highest age-associated CpG in each locus was chosen to build a multi-locus age prediction model (APM), allowing to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 5.35 years, explaining 94.1% of age variation. Validation approaches demonstrated the accuracy and reproducibility of the proposed multi-locus APM. Testing the APM in 51 blood samples from deceased individuals a MAD of 9.72 years was obtained. Potential differences in methylation status between samples from living and deceased individuals could exist since the highest age-correlated CpGs were different in some genes between both groups. In conclusion, our study using the bisulfite PCR sequencing method is in accordance with the high age prediction accuracy of DNA methylation levels in four previously reported age-associated genes. DNA methylation pattern differences between blood samples from living and deceased individuals should be taken into account in forensic contexts.

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals.

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.

Identification and Quantification of Antipsychotics in Blood Samples by LC-MS-MS: Case Reports and Data from Three Years of Routine Analysis.

Antipsychotic drugs (AP) are widely prescribed for the treatment of schizophrenia and psychosis. The pharmacological treatment of schizophrenia is often performed with the simultaneous use of two or more antipsychotic agents to achieve the desired control of psychotic symptoms Available AP include both conventional (typical) and new (atypical) antipsychotic medications. Atypical AP, such as quetiapine, now account for the vast majority of AP prescriptions. In forensic toxicology, AP are of considerable interest because of their potential abuse and their involvement in intoxications and suicides. The authors retrospectively examined AP positive cases detected in samples collected during autopsies performed in the Forensic Clinical and Pathology Service of National Institute of Legal Medicine and Forensic Sciences Centre Branch or in other autopsies carried out in the central region of Portugal, between January 2016 and December 2018. A quantitative liquid chromatography-tandem mass spectrometry assay was developed for the simultaneous determination of 16 AP (amisulpride, aripiprazole, chlorpromazine, clozapine, cyamemazine, fluphenazine, haloperidol, levomepromazine, melperone, olanzapine, paliperidone, promethazine, quetiapine, risperidone, sulpiride and ziprasidone) in blood samples of postmortem cases. The Laboratory of Forensic Chemistry and Toxicology received 3,588 requests for toxicological analysis: 1,413 cases were positive for drugs from which 351 (24.8%) cases were positive for AP, 60.1% from male individuals and 39.9% from female. Quetiapine was the most prevalent AP (36.5%) followed by olanzapine (20.8%). During this period, there were 25 postmortem cases with AP blood concentrations above therapeutic range, in which 36% of those are in agreement with the information received (psychological history or acute intoxication suspicion) and the manner of death was suicide. Our results point that antipsychotics are an increasingly prevalent class of drugs. AP must be measured not only in toxic concentrations but also in therapeutic levels in postmortem cases; therefore, it is important to come up with a sensitive method to cover the low therapeutic range in which AP are usually present.

A Novel Bioanalytical Method for the Determination of Opioids in Blood and Pericardial Fluid.

Opioids are the drugs most commonly detected in overdose deaths and the second most consumed worldwide. An analytical methodology has been optimized and fully validated for the determination of codeine, morphine, 6-acetylmorphine, 6-acetylcodeine, oxycodone, oxymorphone and fentanyl in whole blood and pericardial fluid. The internal standards used were codeine-d3, morphine-d3, 6-acetylmorphine-d3 and fentanyl-d5. Before solid-phase extraction, volumes of 250 µL of blood and pericardial fluid were subjected to a protein precipitation (with 750 µL of ice-cold acetonitrile) and a microwave-induced oximation was performed using a solution of 1% aqueous hydroxylamine hydrochloride in phosphate-buffered saline (1:2, v/v). Finally, the dried extracts were further derivatized with a solution of n-methyl-n-(trimethylsilyl) trifluoroacetamide + 5% trimethylchlorosilane under microwave irradiation. The chromatographic analysis was carried out using gas chromatography-mass spectrometry operating in electron impact and selected ion monitoring mode. For all analytes, the method was linear between 5 and 1,000 ng/mL with determination coefficients (r2) >0.99. Depending on the analyte and matrix, the limit of detection varies between 3 and 4 ng/mL. Intra- and intermediate precision (<20%) and bias (±20%) were acceptable for all analytes in both matrices. The stability of the substances in the studied matrices was guaranteed, at least, 24 h in the autosampler, 4 h at room temperature and 30 days after three freeze/thaw cycles. This methodology was applied to real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Dental evaluation specificity in orofacial damage assessment: A serial case study.

INTRODUCTION: The evaluation of medico-legal post-traumatic events has been increasing over the last decades. This study analysed the input of dental evaluation in orofacial damage assessment, highlighting the individual's biopsychosocial model, by a serial case study. It is aimed to analyse the physical as well as the psychological repercussions of traumatic events. It also aimed to relate the type of trauma impact with the individual's sequelae. MATERIAL AND METHOD: An observational and retrospective study was carried out of Portuguese medico-legal database. A serial case study was distinguished by the direction of the impact: frontal striking, lateral striking and clashing with a bidirectional (frontal-lateral). RESULTS AND DISCUSSION: 7 cases fulfilled the inclusion criteria, as a pilot study. They have in common the involvement of the 2 lower thirds of the face, including the temporomandibular joint. The consolidation of the maxillary bone fractures does not always correspond to restituto ad integrum. CONCLUSION: The impact direction may guide clinical examination in detecting permanent impairment, emphasizing temporomandibular joint disorders, as well as their association with psychosocial repercussions. The medical-dental examination is differentiating and relevant to the accomplishment of the general objective of damage assessment.

PowerPlex® fusion 6C system: internal validation study.

This work is aimed at describing the proceedings and parameters used to validate PowerPlex® Fusion 6C System, the polymerase chain reaction (PCR) amplification kit by Promega, for posterior implementation in the laboratorial routine of the Forensic Genetic Service. The PowerPlex® Fusion 6C System allows multiplex PCR, through simultaneous amplification and posterior detection by fluorescence of 27 loci. Characterization of the kit was made according to the laboratory's internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods. Some parameters were evaluated, such as specificity, analytical thresholds, sensitivity, precision, mixture studies, DNA control samples, a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number, changes in the reaction mixture for direct amplification. This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer. In some parameters, the results were better than expected.

Autosomal SNPs study of a population sample from Southern Portugal and from a sample of immigrants from Guinea-Bissau residing in Portugal.

In recent years, autosomal single nucleotide polymorphisms (SNPs) have been comprehensively investigated in forensic research due to their usefulness in certain circumstances in complementing short tandem repeats (STRs) analysis, or even for use on their own when analysis of STRs fails. However, as with STRs, in order to properly use SNP markers in forensic casuistic we need to understand the population and forensic parameters in question. As a result of Portugal's colonial history during the time of empire, and the subsequent process of decolonization, some African individuals migrated to Portugal, giving rise to large African and African-descendent communities. One of these groups is the community originating from Guinea-Bissau, that in 2014, was enumerated to consist of more than 17,700 individuals with official residency status, more than the third major city of Guinea-Bissau. In order to study the population and forensic parameters mentioned above for the two populations important to our casuistic, a total of 142 unrelated individuals from the South of Portugal and 90 immigrants from Guinea-Bissau (equally non related and all residing in Portugal) were typed with SNaPshot™ assay for all 52 loci included in the SNPforID 52plex.

Lack of gene-language correlation due to reciprocal female but directional male admixture in Austronesians and non-Austronesians of East Timor.

Nusa Tenggara, including East Timor, located at the crossroad between Island Southeast Asia, Near Oceania, and Australia, are characterized by a complex cultural structure harbouring speakers from two different major linguistic groups of different geographic origins (Austronesian (AN) and non-Austronesian (NAN)). This provides suitable possibilities to study gene-language relationship; however, previous studies from other parts of Nusa Tenggara reported conflicting evidence about gene-language correlation in this region. Aiming to investigate gene-language relationships including sex-mediated aspects in East Timor, we analysed the paternally inherited non-recombining part of the Y chromosome (NRY) and the maternally inherited mitochondrial (mt) DNA in a representative collection of AN- and NAN-speaking groups. Y-SNP (single-nucleotide polymorphism) data were newly generated for 273 samples and combined with previously established Y-STR (short tandem repeat) data of the same samples, and with previously established mtDNA data of 290 different samples with, however, very similar representation of geographic and linguistic coverage of the country. We found NRY and mtDNA haplogroups of previously described putative East/Southeast Asian (E/SEA) and Near Oceanian (NO) origins in both AN and NAN speakers of East Timor, albeit in different proportions, suggesting reciprocal genetic admixture between both linguistic groups for females, but directional admixture for males. Our data underline the dual genetic origin of East Timorese in E/SEA and NO, and highlight that substantial genetic admixture between the two major linguistic groups had occurred, more so via women than men. Our study therefore provides another example where languages and genes do not conform due to sex-biased genetic admixture across major linguistic groups.

Salvia divinorum: toxicological aspects and analysis in human biological specimens.

The identification and quantitation of the main psychoactive component of Salvia divinorum (salvinorin A) in biological specimens are crucial in forensic and clinical toxicology. Despite all the efforts made, its uncontrolled abuse has increased quickly, exposing its users' health to serious risks both in the short and long term. The use of alternative biological matrices in toxicological analyzes can be advantageous as complementary postmortem samples, or in situations when neither blood nor urine can be collected; they may be useful tools in those determinations, providing important information about prior exposure. The aim of this article is to present a brief summary of legal aspects of Salvia divinorum and salvinorin A, including the methods used for the determination of the latter in biological matrices.

Analysis of Delta9-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography-electrospray ionization mass spectrometry.

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for the confirmation of Delta(9)-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm(3), 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC+H(+)], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d(3)-THC+H(+)]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r(2)=0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette collection, 26 of the 40 cases had an undetectable THC.