Nonrandom Selection and Multiple Blood Feeding of Human Hosts by Anopheles Vectors: Implications for Malaria Transmission in Papua New Guinea.

Nonrandom selection and multiple blood feeding of human hosts by Anopheles mosquitoes may exacerbate malaria transmission. Both patterns of blood feeding and their relationship to malaria epidemiology were investigated in Anopheles vectors in Papua New Guinea (PNG). Blood samples from humans and mosquito blood meals were collected in villages and human genetic profiles ("fingerprints") were analyzed by genotyping 23 microsatellites and a sex-specific marker. Frequency of blood meals acquired from different humans, identified by unique genetic profiles, was fitted to Poisson and negative binomial distributions to test for nonrandom patterns of host selection. Blood meals with more than one genetic profiles were classified as mosquitoes that fed on multiple humans. The age of a person bitten by a mosquito was determined by matching the blood-meal genetic profile to the villagers' genetic profiles. Malaria infection in humans was determined by PCR test of blood samples. The results show nonrandom distribution of blood feeding among humans, with biased selection toward males and individuals aged 15-30 years. Prevalence of Plasmodium falciparum infection was higher in this age group, suggesting males in this age range could be super-spreaders of malaria parasites. The proportion of mosquitoes that fed on multiple humans ranged from 6% to 13% among villages. The patterns of host utilization observed here can amplify transmission and contribute to the persistence of malaria in PNG despite efforts to suppress it with insecticidal bed nets. Excessive feeding on males aged 15-30 years underscores the importance of targeted interventions focusing on this demographic group.

Bacterial Profiling of Soil For Forensic Investigations: Consideration of Ex Situ Changes in Questioned and Known Soil Samples.

Soil, being diverse and ubiquitous, can potentially link a suspect or victim to a crime scene. Recently scientists have examined the microbial makeup of soil for determining its origin, and differentiating soil samples is well-established. However, when soil is transferred to evidence its microbial makeup may change over time, leading to false exclusions. In this research, "known" soils from diverse habitats were stored under controlled conditions, while evidence soils were aged on mock evidence. Limited quantities of soil were also assayed. Bacterial profiles were produced using next-generation sequencing of the 16S rRNA gene. Overall, known soils stored open at room temperature were more similar to evidence soils over time than were known soils stored bagged and/or frozen. Evidence soils, even as little as 1 mg, associated with the correct habitat 99% of the time, accentuating the importance of considering ex situ microbial changes in soil for its successful use as forensic evidence.

Intra- and Inter-Element Variability in Mitochondrial and Nuclear DNA from Fresh and Environmentally Exposed Skeletal Remains.

Successful identification of skeletonized remains often relies upon DNA analyses, frequently focusing on the mid-diaphysis of weight-bearing long bones. This study explored intra-bone DNA variability using bovine and porcine femora, along with calcanei and tali. DNA from fresh and short-term environmentally exposed bone was extracted utilizing demineralization and standard lysis buffer protocols, and DNA quantity and quality were measured. Overall, femoral epiphyses, metaphyses, and the tarsals had more nuclear and mitochondrial DNA than did the femoral diaphyses. DNA loss was much more rapid in buried bones than in surface exposed bones, while DNA quality differed based on environment, but not bone region/element. The demineralization protocol generated more DNA in some bone regions, while the standard lysis was more effective in others, and neither significantly affected DNA quality. Taken together, these findings reinforce the importance of considering inter- and intra-bone heterogeneity when sampling skeletal material for forensic DNA-based identifications.

Time Radically Alters Ex Situ Evidentiary Soil 16S Bacterial Profiles Produced Via Next-Generation Sequencing,.

Previous research has revealed the potential of soil bacterial profiling for forensic purposes; however, investigators have not thoroughly examined fluctuations in microbial profiles from soil aged on evidence. In this research, soils collected from multiple habitats were placed on evidence items and sampled over time, and then bacterial profiles were generated via next-generation sequencing of the 16S rRNA locus. Bacterial abundance charts and nonmetric multidimensional scaling plots provided visual representation of bacterial profiles temporally, while supervised classification was used to statistically associate evidence to a source. The ex situ evidence soils displayed specific, consistent taxonomic changes as they aged, resulting in their drift in multidimensional space, but never toward a different habitat. Ninety-five percent of the 364 evidentiary profiles statistically classified to the correct habitat, with misclassification generally stemming from evidence type and increased age. Ultimately, understanding bacterial changes that occur temporally in ex situ soils should enhance their use in forensic investigations.

Plasticity of host selection by malaria vectors of Papua New Guinea.

BACKGROUND: Host selection is an important determinant of vectorial capacity because malaria transmission increases when mosquitoes feed more on humans than non-humans. Host selection also affects the outcome of long-lasting insecticidal nets (LLIN). Despite the recent nationwide implementation of LLIN-based malaria control program in Papua New Guinea (PNG), little is known about the host selection of the local Anopheles vectors. This study investigated the host selection of Anopheles vectors in PNG. METHODS: Blood-engorged mosquitoes were sampled using the barrier screen method and blood meals analyzed for vertebrate host source with PCR-amplification of the mitochondrial cytochrome b gene. Abundance of common hosts was estimated in surveys. The test of homogeneity of proportions and the Manly resource selection ratio were used to determine if hosts were selected in proportion to their abundance. RESULTS: Two thousand four hundred and forty blood fed Anopheles females of seven species were sampled from five villages in Madang, PNG. Of 2,142 samples tested, 2,061 (96.2%) yielded a definitive host source; all were human, pig, or dog. Hosts were not selected in proportion to their abundance, but rather were under-selected or over-selected by the mosquitoes. Four species, Anopheles farauti (sensu stricto) (s.s.), Anopheles punctulatus (s.s.), Anopheles farauti no. 4 and Anopheles longirostris, over-selected humans in villages with low LLIN usage, but over-selected pigs in villages with high LLIN usage. Anopheles koliensis consistently over-selected humans despite high LLIN usage, and Anopheles bancroftii over-selected pigs. CONCLUSIONS: The plasticity of host selection of an Anopheles species depends on its opportunistic, anthropophilic or zoophilic behavior, and on the extent of host availability and LLIN usage where the mosquitoes forage for hosts. The high anthropophily of An. koliensis increases the likelihood of contacting the LLIN inside houses. This allows its population size to be reduced to levels insufficient to support transmission. In contrast, by feeding on alternative hosts the likelihood of the opportunistic species to contact LLIN is lower, making them difficult to control. By maintaining high population size, the proportion that feed on humans outdoors can sustain residual transmission despite high LLIN usage in the village.

Mitochondrial DNA Profiling of Illegal Tortoiseshell Products Derived from Hawksbill Sea Turtles.

The hawksbill sea turtle (Eretmochelys imbricata) is a highly endangered species, commonly poached for its ornate shell. "Tortoiseshell" products made from the shell are widely, although illegally, available in many countries. Hawksbills have a circumglobal distribution; thus, determining their origin is difficult, although genetic differences exist geographically. In the research presented, a procedure was developed to extract and amplify mitochondrial DNA from tortoiseshell items, in an effort to better understand where the species is being poached. Confiscated tortoiseshell items were obtained from the U.S. Fish and Wildlife Service, and DNA from 56 of them was analyzed. Multiple mitochondrial haplotypes were identified, including five not previously reported. Only one tortoiseshell item proved to be of Atlantic origin, while all others corresponded to genetic stocks in the Indo-Pacific region. The developed methodology allows for unique, and previously unattainable, genetic information on the illegal poaching of sea turtles for the decorative tortoiseshell trade.

Next-Generation Sequencing of the Bacterial 16S rRNA Gene for Forensic Soil Comparison: A Feasibility Study.

Soil has the potential to be valuable forensic evidence linking a person or item to a crime scene; however, there is no established soil individualization technique. In this study, the utility of soil bacterial profiling via next-generation sequencing of the 16S rRNA gene was examined for associating soils with their place of origin. Soil samples were collected from ten diverse and nine similar habitats over time, and within three habitats at various horizontal and vertical distances. Bacterial profiles were analyzed using four methods: abundance charts and nonmetric multidimensional scaling provided simplification and visualization of the massive datasets, potentially aiding in expert testimony, while analysis of similarities and k-nearest neighbor offered objective statistical comparisons. The vast majority of soil bacterial profiles (95.4%) were classified to their location of origin, highlighting the potential of bacterial profiling via next-generation sequencing for the forensic analysis of soil samples.

Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.

Assessment and mitigation of DNA loss utilizing centrifugal filtration devices.

Maximizing DNA recovery during its isolation can be vital in forensic casework, particularly when DNA yields are expected to be low, such as from touch samples. Many forensic laboratories utilize centrifugal filtration devices to purify and concentrate the DNA; however, DNA loss has been reported when using them. In this study, all centrifugal filtration devices tested caused substantial DNA loss, affecting low molecular weight DNA (PCR product) somewhat more than high molecular weight DNA. Strategies for mitigating DNA loss were then examined, including pre-treatment with glucose, glycogen, silicone (RainX(®)), bovine serum albumin, yeast RNA, or high molecular weight DNA. The length of pre-treatment and UV irradiation of pre-treatment reagents were also investigated. Pre-treatments with glucose and glycogen resulted in little or no improvement in DNA recovery, and most or all DNA was lost after silicone pre-treatment. Devices pre-treated with BSA produced irregular and uninterpretable quantitative PCR amplification curves for the DNA and internal PCR control. On the other hand, nucleic acid pre-treatments greatly improved recovery of all DNAs. Pre-treatment time and its UV irradiation did not influence DNA recovery. Overall, the results show that centrifugal filtration devices trap DNA, yet their proper pre-treatment can circumvent that loss, which is critical in the case of low copy forensic DNA samples.

Collecting and analyzing DNA evidence from fingernails: a comparative study.

Forensic practitioners and crime laboratories regularly collect and analyze fingernail evidence; however, the best techniques for processing such evidence have not been established. In this study, numerous aspects of fingernail evidence processing-collection of exogenous cells, transportation, purification of DNA, and STR analysis-were analyzed using fingernails harboring applied blood or epithelial cells from scratchings. Autosomal STR mixtures resulted when fingernails were soaked or swabbed, while scrapings rarely generated mixtures but exhibited allelic dropout. Y-STRs yielded single source profiles, with scrapings again showing dropout. A silica-based kit extraction recovered significantly more exogenous DNA than did organic extraction, neither of which was affected by nail polish. Swabbing nails in succession resulted in some cross-contamination from exogenous material, while transporting nails together did not, although there was loss of exogenous cells. Optimized nail processing produced complete Y-STR profiles of male volunteers from female fingernails following scratchings.

The influence of swabbing solutions on DNA recovery from touch samples.

There has been minimal research into how to best obtain DNA from touch samples. Many forensic laboratories simply moisten a swab with water and use it for collecting cells/DNA from evidentiary samples. However, this and other methods have not been objectively studied in order to maximize DNA yields. In this study, fingerprints were collected using swabs moistened with water or laboratory or commercially available detergents, including sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, Formula 409(®) , and Simple Green(®) . Prints were swabbed, DNA isolated using an organic extraction, yields quantified, and relative yields compared. In all cases, the detergent-based swabbing solutions outperformed water, with SDS and Triton X-100 producing significant increases in yield. Short tandem repeat profiles were consistent with the individuals that placed them. Subsequent analysis of SDS concentrations for collecting touch DNA demonstrated an increase in DNA yield with increasing SDS concentration, with an optimal concentration of approximately 2%.

Osteological and molecular identification of Brucellosis in ancient Butrint, Albania.

Ancient skeletal remains can harbor unique information about past civilizations at both the morphological and molecular levels. For instance, a number of diseases manifest in bone, some of which have been confirmed through DNA analysis, verifying their presence in ancient populations. In this study, anthropological analysis of skeletal remains from the ancient Albanian city of Butrint identified individuals with severe circular lytic lesions on their thoracic and lumbar vertebrae. Differential diagnosis suggested that the lesions resulted from pathologies known to affect these skeletal regions, such as tuberculosis (TB) or brucellosis. Relevant bones of two adolescent males from the 10th to 13th century AD that displayed the lesions, along with unaffected individuals, were collected in the field. Genetic screening of the skeletal samples for TB was repeatedly negative, thus additional testing for Brucella spp.-bacteria of livestock and the causative agent of brucellosis in humans-was conducted. Two Brucella DNA markers, the IS6501 insertion element and Bcsp31 gene, amplified from the affected vertebrae and/or ribs, whereas all unaffected individuals and control samples were negative. Subsequent DNA sequencing confirmed the presence of the brucellar IS6501 insertion element. On the basis of the skeletal lesions, negative tests for TB, and positive Brucella findings, we report a confirmed occurrence of brucellosis in archaeologically recovered human bone. These findings suggest that brucellosis has been endemic to the area since at least the Middle Ages.

Investigative studies into the recovery of DNA from improvised explosive device containers.

Apprehending those who utilize improvised explosive devices (IEDs) is a national priority owing to their use both domestically and abroad. IEDs are often concealed in bags, boxes, or backpacks to prevent their detection. Given this, the goal of the research presented was to identify IED handlers through postblast DNA recovery from IED containers. Study participants were asked to use backpacks for 11 days, after which they served as containers for pipe bombs. Eleven postdeflagration backpack regions likely to be handled were swabbed and analyzed via mini-short tandem repeats (miniSTRs) and alleles were called blind. An experimental consensus method was examined in which profiles from all regions were considered, to help identify spurious drop-in/out. Results were correct for all loci, except one that remained ambiguous. The results show that recovering DNA from IED containers is a viable approach for aiding in the identification of those who may have been involved in an IED event.

Simplified field preservation of tissues for subsequent DNA analyses.

Successful DNA-based identification of mass disaster victims depends on acquiring tissues that are not highly degraded. In this study, multiple protocols for field preservation of tissues for later DNA analysis were tested. Skin and muscle samples were collected from decaying pig carcasses. Tissues were preserved using cold storage, desiccation, or room temperature storage in preservative solutions for up to 6 months. DNA quality was assessed through amplification of successively larger segments of nuclear DNA. Solution-based storage, including a DMSO/NaCl/EDTA mixture, alcohols, and RNAlater preserved DNA of the highest quality, refrigeration was intermediate, and desiccation was least effective. Tissue type and extent of decomposition significantly affected stored DNA quality. Overall, the results indicate that any tissue preservation attempt is far superior to delaying or forgoing preservation efforts, and that simple, inexpensive methods can be highly effective in preserving DNA, thus should be initiated as quickly as possible.

The conviction of Dr. Crippen: new forensic findings in a century-old murder.

Dr. Hawley Crippen was accused and convicted of murdering his wife in London in 1910. Key to the conviction was microscopic analysis of remains found in the Crippen's coal cellar, which were identified as Cora Crippen based on a scar she was said to have. Dr. Crippen was hanged, always proclaiming his innocence. In this study, genealogical research was used to locate maternal relatives of Cora Crippen, and their mitochondrial haplotypes were determined. Next, one of the pathology slides of the scar was obtained, DNA was isolated, and the haplotype was determined. That process was then repeated. Finally, both DNA isolates were assayed for repetitive elements on autosomes and repetitive elements specific to the Y chromosome. Based on the genealogical and mitochondrial DNA research, the tissue on the pathology slide used to convict Dr. Crippen was not that of Cora Crippen. Moreover, that tissue was male in origin.

Gene expression during blow fly development: improving the precision of age estimates in forensic entomology.

Forensic entomologists use size and developmental stage to estimate blow fly age, and from those, a postmortem interval. Since such estimates are generally accurate but often lack precision, particularly in the older developmental stages, alternative aging methods would be advantageous. Presented here is a means of incorporating developmentally regulated gene expression levels into traditional stage and size data, with a goal of more precisely estimating developmental age of immature Lucilia sericata. Generalized additive models of development showed improved statistical support compared to models that did not include gene expression data, resulting in an increase in estimate precision, especially for postfeeding third instars and pupae. The models were then used to make blind estimates of development for 86 immature L. sericata raised on rat carcasses. Overall, inclusion of gene expression data resulted in increased precision in aging blow flies.

Bacterial profiling of soil using genus-specific markers and multidimensional scaling.

Forensic identification of soil based on microbial DNA fingerprinting has met with mixed success, with research efforts rarely considering temporal variability or local heterogeneity in soil's microbial makeup. In the research presented, the nitrogen fixing bacteria rhizobia were specifically examined. Soils were collected monthly from five habitats for 1 year, and quarterly in each cardinal direction from the main collection site. When all habitats were compared simultaneously using Terminal Restriction Fragment Length Polymorphism analysis of the rhizobial recA gene and multidimensional scaling, only two were differentiated over a year's time, however pairwise comparisons allowed four of five soils to be effectively differentiated. Adding in 10-foot distant soils as "questioned" samples correctly grouped them in 40-70% of cases, depending on restriction enzyme used. The results indicate that the technique has potential for forensic soil identification, although extensive anthropogenic manipulation of a soil makes such identification much more tentative.

Polymerase resistance to polymerase chain reaction inhibitors in bone*.

Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone-derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone-derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains.

The correlation between skeletal weathering and DNA quality and quantity.

Mitochondrial DNA analysis of skeletal material is invaluable in forensic identification, although results can vary widely among remains. Previous studies have included bones of different ages, burial conditions, and even species. In the research presented, a collection of human remains that lacked major confounders such as burial age, interment style, and gross environmental conditions, while displaying a very broad range of skeletal degradation, were examined for both mitochondrial DNA (mtDNA) quality and quantity. Overall skeletal weathering, individual bone weathering, and bone variety were considered. Neither skeletal nor bone weathering influenced DNA quality or quantity, indicating that factors that degrade bone do not have the same effect on DNA. In contrast, bone variety, regardless of weathering level, was a significant element in DNA amplification success. Taken together, the results indicate that neither skeletal nor individual bone appearance are reliable indicators of subsequent mtDNA typing outcomes, while the type of bone assayed is.