Old meets new: Comparative examination of conventional and innovative RNA-based methods for body fluid identification of laundered seminal fluid stains after modular extraction of DNA and RNA.

The knowledge about the type of the body fluid/tissue that contributed to a trace can provide contextual insight into crime scene reconstruction and connect a suspect or a victim to a crime scene. Especially in sexual assault cases, it is important to verify the presence of spermatozoa. Victims often tend to clean their underwear/bedding after a sexual assault. If they later decide to report the crime to the police, in our experience, investigators usually do not send laundered items for DNA examination, since they believe that analysis after washing is no longer promising. As not only the individualization of traces on laundered items could be important in court, but also the type of biological material, we compared the potential of modular DNA and RNA extraction from the same specimen for simultaneous body fluid identification (BFI) and STR profiling of laundered items. BFI included the comparison of a broad range of conventional approaches, as wells as new molecular mRNA- and miRNA-based methods. The examination comprises the assessment of different fabrics and washing temperatures and multiple washing steps. Our results indicate that although conventional enzymatic and immunochromatographical approaches show limitations for BFI of laundered stains, the RSID test was sensitive enough to detect seminal fluid in 80% of all tested samples. Furthermore, the HY-Liter fluorescence analysis successfully detected spermatozoa, even in cloths that were washed twice. For the first time, it could be shown that a marker set of mRNAs can be applied for the identification of seminal fluid stained cotton and synthetic fiber fabrics that have been washed at 40 °C. Our experiments demonstrate that analysis of DNA and RNA of laundered items is feasible, giving the possibility to identify the perpetrator as well as the biological properties of the same laundered (seminal) stain under certain conditions.

Comprehensive examination of conventional and innovative body fluid identification approaches and DNA profiling of laundered blood- and saliva-stained pieces of cloths.

Body fluids like blood and saliva are commonly encountered during investigations of high volume crimes like homicides. The identification of the cellular origin and the composition of the trace can link suspects or victims to a certain crime scene and provide a probative value for criminal investigations. To erase all traces from the crime scene, perpetrators often wash away their traces. Characteristically, items that show exposed stains like blood are commonly cleaned or laundered to free them from potential visible leftovers. Mostly, investigators do not delegate the DNA analysis of laundered items. However, some studies have already revealed that items can still be used for DNA analysis even after they have been laundered. Nonetheless, a systematical evaluation of laundered blood and saliva traces that provides a comparison of different established and newly developed methods for body fluid identification (BFI) is still missing. Herein, we present the results of a comprehensive study of laundered blood- and saliva-stained pieces of cloths that were applied to a broad range of methods for BFI including conventional approaches as well as molecular mRNA profiling. The study included the evaluation of cellular origin as well as DNA profiling of blood- and saliva-stained (synthetic fiber and cotton) pieces of cloths, which have been washed at various washing temperatures for one or multiple times. Our experiments demonstrate that, while STR profiling seems to be sufficiently sensitive for the individualization of laundered items, there is a lack of approaches for BFI with the same sensitivity and specificity allowing to characterize the cellular origin of challenging, particularly laundered, blood and saliva samples.

Clinical and economic burden of Clostridium difficile infection in Europe: a systematic review of healthcare-facility-acquired infection.

PubMed, EMBASE and conference abstracts were reviewed systematically to determine the clinical and economic burden associated with Clostridium difficile infection (CDI) acquired and treated in European healthcare facilities. Inclusion criteria were: published in the English language between 2000 and 2010, and study population of at least 20 patients with documented CDI acquired/treated in European healthcare facilities. Data collection was completed by three unblinded reviewers using the Cochrane Handbook and PRISMA statement. The primary outcomes were mortality, recurrence, length of hospital stay (LOS) and cost related to CDI. In total, 1138 primary articles and conference abstracts were identified, and this was narrowed to 39 and 30 studies, respectively. Data were available from 14 countries, with 47% of studies from UK institutions. CDI mortality at 30 days ranged from 2% (France) to 42% (UK). Mortality rates more than doubled from 1999 to 2004, and continued to rise until 2007 when reductions were noted in the UK. Recurrent CDI varied from 1% (France) to 36% (Ireland); however, recurrence definitions varied between studies. Median LOS ranged from eight days (Belgium) to 27 days (UK). The incremental cost of CDI was £4577 in Ireland and £8843 in Germany, after standardization to 2010 prices. Country-specific estimates, weighted by sample size, ranged from 2.8% to 29.8% for 30-day mortality and from 16 to 37 days for LOS. CDI burden in Europe was most commonly described using 30-day mortality, recurrence, LOS and cost data. The continued spread of CDI and resultant healthcare burden underscores the need for judicious use of antibiotics.

Water resource use and management by the United States forest products industry.

The connections between forest products operations and water resources in the United States is considered and, where possible, quantified. Manufacture of wood, pulp, and paper products and the influences of forest management and forest products manufacture on water quality are discussed. Most fresh water in the US originates in forested areas. Responsible harvesting strategies, best management practices, and forest re-growth combine to minimize or eliminate changes in water availability and degradation of water quality due to harvesting. Relative to alternative land uses and large-scale disturbance events, forested areas produce the highest quality of fresh water. Water inputs for the manufacture of forest products total about 5.8 billion m(3) per year, an amount equal about 0.4% of the surface and groundwater yield from timberland. Approximately 88% of water used in manufacturing is treated and returned directly to surface waters, about 11% is converted to water vapor and released during the manufacturing process, and 1% is imparted to products or solid residuals. Extensive study and continued monitoring of treated effluents suggest few or no concerns regarding the compatibility of current effluents with healthy aquatic systems.

Short amplicon STR multiplex for stain typing.

We developed a short tandem repeat (STR) typing kit based on DNA database systems that are included in, for example, the Interpol Standard Set of Loci recommendations (i.e., TH01, VWA, D3S1358, FGA) and the gender typing system Amelogenin. Two different multiplex sets were tested using the fluorescent dyes FAM, JOE, and VIC. The PCR results were compared to the commercially available AmpFISTR Blue kit, which contains the STRs D3S1358, VWA, and FGA. The advantage of our multiplex compared with the Blue kit was the generation of shorter amplicons (<200 bp) and the higher combined power of discrimination.

A very long ACTBP2 (SE33) allele.

Analysing a buccal swab we found a long allele in the STR system ACTBP2. For confirmation we sequenced the isolated PCR product and found a sequence structure common in alleles of type III. Based on the repeat array the new allele is assigned as allele "49".

Population genetic comparisons of three X-chromosomal STRs.

The X-chromosomal short tandem repeats (STRs) DXS6800, DXS101 and DXS8377 were analysed in male and female population samples from Germany and Austria using a PCR multiplex approach. We investigated 135 family trios from Innsbruck (Austria) and surrounding areas and 50 families and further male and female samples from Ulm (Germany) and surrounding areas. The comparisons of the allele frequencies gave similar distributions for Innsbruck and Ulm although minor variations were found for some alleles. Additionally, some differences were found when comparing the allele frequencies of the male and female samples independently. The forensic efficiency values demonstrate that especially DXS101 and DXS8377 are highly informative markers for kinship analysis and deficiency cases. Based on the investigated meiotic events no new mutations were detected.

Unusual presentation of central nervous system relapse with oculomotor nerve palsy in a case of CD56-positive acute myeloid leukemia following allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) plays an important role in the treatment of infants and children with acute myelogenous leukemia (AML). Leukemic relapse after allo-SCT is responsible for a high rate of treatment failure. Extra-medullary relapse (EMR), without involvement of bone marrow, is rare compared to medullary relapse. CD56, the neural cell adhesion molecule, may contribute to the higher frequency of CNS relapse in CD56-positive AML. We observed an isolated EMR on the oculomotor nerve of a 17-month-old girl 12 weeks after cord blood transplantation (CBT), who was transplanted because of CD56-positive AML. Diagnosis of relapse was suspected clinically and confirmed by magnetic resonance imaging (MRI), and fluorescence-activated cell sorter (FACS) and chimerism analysis of cerebrospinal fluid (CSF). Therapy consisted of intra-thecal chemotherapy, CNS irradiation, and systemic immunomodulation by cyclosporin A (CsA) and basiliximab withdrawal. Twenty-one months after relapse, the patient shows full remission of symptoms and previously described oculomotor nerve infiltration.

Somatic mutations at STR loci--a reason for three-allele pattern and mosaicism.

Two families are analysed in which one of the parents exhibited a three-allele pattern at the ACTBP2 locus. Since the alleles were obviously segregated independently to the children, a generalised mosaicism must be assumed involving at least two tissues in one of them and at least four tissues in the other one. The intensity of the PCR amplified alleles in both three-allele individuals indicate an occurrence in a very early embryonic stage. Occurrence was most probably due to a single step mutation in both cases. Forensic implications would include paternity testing as well as stain analysis.

Assessment of shooting distance on the basis of bloodstain analysis and histological examinations.

A 28-year-old man was shot using a pump-gun. The main question to be resolved was whether the biological stain pattern on the suspect's trousers, and in particular the bloodstains, can provide evidence to assess the shooting distance between the suspect and the position of the victim's body. The biological stain pattern (i.e. bloodstains and brain tissue) showed backspatters from the shot entrance wound on the back of the head, while the victim was lying face down and the suspect was standing close behind his head.

Less is more--length reduction of STR amplicons using redesigned primers.

PCR primers closely flanking the repeat region were redesigned to reduce the amplicon length of the selected STRs down to approximately 100 bp for the shorter alleles (loci HumTH01, D10S2325, DYS19 and DYS391). Highly degraded DNA (e.g. formalin-fixed tissue) and very low amounts of DNA could be more successfully typed using the new redesigned primers compared to the established sequences generating longer amplicons.

Microsatellite structures in the context of human evolution.

Six microsatellite - or short tandem repeat (STR) - systems with uniform repetitive sequences (HumTH01, HumCD4, HumFES/FPS, HumF13B, HumTPO, HumLPL) and three compound repeat systems (HumVWA, HumFIBRA, D21S11) were used, including data from the literature, to determine genetic distances among eight populations worldwide. The TH01- and VWA homologous loci in nonhuman primates (chimpanzees, gorillas, orangutans, rhesus monkeys, ring-tailed lemurs) were compared and found to be shorter than in humans. Microsatellites of lower complexity were most efficient for the separation of major ethnic groups. The loci of higher complexity showed a leveling of the diversity differences among populations, which could be attributed to higher mutation rates.

D18S535, D1S1656 and D10S2325: three efficient short tandem repeats for forensic genetics.

Three short tandem repeat (STR) polymorphisms characterized by PCR product length < 175 bp were investigated. D18S535 and D1S1656 contained a 4 bp unit as basic repeat motif, D10S2325 a 5 bp unit. The heterozygosity rates were 0.76 (D18S535), 0.88 (D10S2325) and 0. 90 (D1S1656), leading to a combined discrimination power of 0.9999. In contrast to D10S2325 and D18S535, which showed a homogeneous repeat array without any variation in the repeat motifs, repeat length and sequence variation was found for D1S1656. Robust typing results could be observed for all three STRs using highly degraded DNA.

German shepherd dog is suspected of sexually abusing a child.

A rare case of provoked anal penetration of an 11-year-old boy by a male German shepherd dog was confirmed by the results of morphological, serological and molecular genetic investigations. These results were of great importance to refute the suspicion on two adults. Some serious doubts remained in the version of the course of the event as presented by the boy. Some weeks later when confronted by a psychologist, the boy admitted having deliberately stimulated the dog manually and caused the animal to penetrate him.

Tetranucleotide STR system D8S1132: sequencing data and population genetic comparisons.

In the present investigation of the D8S1132 locus 31 selected alleles were sequenced. In total there were 9 distinguishable alleles found to increase in size by regular 4 bp increments from 134 to 170 bp with a repeat array following the pattern (TCTA)n TCA (TCTA)n. One-third of the sequenced alleles exhibited an altered repeat sequence TCTG TCTA at the 3' flanking region of the repeat array. A nomenclature for the designation of D8S1132 alleles is proposed on the basis of this sequence data and in accordance with the ISFH recommendations. The allele distribution of the D8S1132 locus has been investigated in three German populations (Halle-, Münster-, and Wiesbaden area) with frequencies ranging from 0.004 to 0.24. No deviation from Hardy-Weinberg equilibrium could be observed. The heterozygosity was 0.83 and the discrimination power 0.96 for the Halle population.

Population genetic diversity in relation to microsatellite heterogeneity.

Nine populations (Germans, Turks, Moroccans, Ovambos, Ugandans, Chinese, Japanese, Papuans, and Australian Aborigines) were investigated using six microsatellite systems (HumCD4, Hum F13B, HumFES/FPS, HumTH01, HumVWA, and D21S11), so-called STRs (short tandem repeats). Allele frequency data and sequencing results were used to compare the population genetic diversity among these populations. The genetic differences varied depending on the STR applied. According to the systems investigated, we defined three categories of STR microvariation: LOMs (low microvariation systems), INMs (intermediate microvariation systems), and HIMs (high microvariation systems). LOMs (STRs: CD4, FES, F13B, TH01) are characterised by a number of repeats between 5-15 and a stable repeat sequence. INMs and HIMs each showed an increasing number of repeats and additional sequence variation in the repeat motifs. The rate of new mutations was associated with the extent of microvariation. The reconstruction of phylogenetic trees led to a clustering in an early split of the African populations followed by further branching of the Asian/Melanesian and the Caucasian groups.

DNA typing of epithelial cells after strangulation.

DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework.

Genetic variation at five STR loci in subpopulations living in Turkey.

Five short tandem repeat (STR) systems HumVWA, HumTH01, HumCD4, HumF13B and HumFES were investigated in 2 subpopulations living in Turkey (Laz Turks and Kurds). The population genetic data were compared to a Turkish population sample from the Adana area. A closer genetic relationship was found to the Laz Turks than to the Kurdish sample which was also confirmed by phylogenetic tree reconstruction with seven populations from three major ethnic groups (Caucasian, Asian and African). In contrast to the Laz and Adana populations the Kurdish sample showed relatively low heterozygosity values and deviations from Hardy-Weinberg equilibrium in four of the five systems.

[DNA degradation in formalin fixed tissues]. / DNA-Degradation in formalinfixierten Geweben.

The intensity of DNA degradation in fixed tissues is dependent on the fixation solution and the fixation time. The aim of this study was the investigation of DNA degradation over fixation times of up to 70 days in different tissues (muscle, brain, liver, bone) and with different formalin concentration (2%, 4%, 8%; unbuffered). An additional test was performed to see whether the fixed tissues could be individualized using PCR analysis. The smallest amounts of DNA were extracted from liver and brain and the largest from muscle and bone. The amount of DNA that could be extracted decreased with increasing formalin concentration, while at the same time DNA degradation increased. With the PCR-VNTR system HUMTH01, all fixed samples could be typed regardless of the fixation time and the formalin concentration.

Detection and significance of adenoviruses in cases of sudden infant death.

Respiratory tract infections have been thought to act as a trigger mechanism in sudden infant death. In 118 autopsy cases of infant death, paraffin-embedded or frozen lung tissues were investigated by means of a nested polymerase chain reaction (PCR) to detect adenovirus (AV) DNA. The primers used are general primers and allow the detection of most pathogenic adenoviruses with high specificity and sensitivity and independently of devitalization of viruses or degradation of viral DNA. For the investigation three groups were established: there were 13 cases of unnatural death, 78 cases of natural death without histological signs of interstitial pneumonia, and 27 cases with interstitial pneumonia. The first group was AV negative. In the group without interstitial pneumonia AV was detected in 10.2% of the cases. In the group with interstitial pneumonia the frequency of AV detection was almost 26%. The results obtained demonstrate an association between interstitial pneumonia and detection of AV DNA, indicating that AV may play an important part in pulmonary infection in infants. Histological evidence of interstitial pneumonia was not observed in all AV-positive cases, perhaps because nonspecific virus-related changes occurred only in early stages of viral infection. Comparison of the AV frequency in SIDS (25%) and non-SIDS cases (4%) indicates an association between pulmonary AV infections and sudden death. These results support the working hypothesis of respiratory infections acting as a trigger mechanism in sudden infant death.