DNA fingerprinting of glioma cell lines and considerations on similarity measurements.

Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

Use of DNA profiles for investigation using a simulated national DNA database: Part I. Partial SGM Plus profiles.

In traditional criminal investigation, uncertainties are often dealt with using a combination of common sense, practical considerations and experience, but rarely with tailored statistical models. For example, in some countries, in order to search for a given profile in the national DNA database, it must have allelic information for six or more of the ten SGM Plus loci for a simple trace. If the profile does not have this amount of information then it cannot be searched in the national DNA database (NDNAD). This requirement (of a result at six or more loci) is not based on a statistical approach, but rather on the feeling that six or more would be sufficient. A statistical approach, however, could be more rigorous and objective and would take into consideration factors such as the probability of adventitious matches relative to the actual database size and/or investigator's requirements in a sensible way. Therefore, this research was undertaken to establish scientific foundations pertaining to the use of partial SGM Plus loci profiles (or similar) for investigation.

The likelihood approach to compare populations : a study on DNA evidence and pitfalls of intuitions

The paper follows on from earlier work [Taroni F and Aitken CGG. Probabilistic reasoning in the law, Part 1: assessment of probabilities and explanation of the value of DNA evidence. Science & Justice 1998; 38: 165-177]. Different explanations of the value of DNA evidence were presented to students from two schools of forensic science and to members of fifteen laboratories all around the world. The responses were divided into two groups; those which came from a school or laboratory identified as Bayesian and those which came from a school or laboratory identified as non-Bayesian. The paper analyses these responses using a likelihood approach. This approach is more consistent with a Bayesian analysis than one based on a frequentist approach, as was reported by Taroni F and Aitken CGG. [Probabilistic reasoning in the law, Part 1: assessment of probabilities and explanation of the value of DNA evidence] in Science & Justice 1998.

One person with two DNA profiles: a(nother) case of mosaicism or chimerism.

Nuclear DNA markers, such as short tandem repeats (STR), are widely used for crime investigation and paternity testing. STR were used to determine whether a piece of tissue regurgitated by a dog was part of the penis of a dead, emasculated, man. Unexpectedly, when analyzing the recovered material and a blood sample from the deceased, five out of the 18 loci differed. According to the results, one could have concluded that these samples originated from two different persons. However, taking into account contextual information and data from complementary genetic analyses, the most likely hypothesis was that the deceased was a genetic mosaic or a chimera. Within a forensic genetic context, such genetic peculiarities may prevent associating the perpetrator of an offense with a stain left at a crime scene or lead to false paternity exclusions. Fast recognition of mosaics or chimeras, adapted sampling scheme, as well as careful interpretation of the data should allow avoiding such pitfalls.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Characterization of microsatellite loci and reliable genotyping in a polyploid plant, Mercurialis perennis (Euphorbiaceae).

For many applications in population genetics, codominant simple sequence repeats (SSRs) may have substantial advantages over dominant anonymous markers such as amplified fragment length polymorphisms (AFLPs). In high polyploids, however, allele dosage of SSRs cannot easily be determined and alleles are not easily attributable to potentially diploidized loci. Here, we argue that SSRs may nonetheless be better than AFLPs for polyploid taxa if they are analyzed as effectively dominant markers because they are more reliable and more precise. We describe the transfer of SSRs developed for diploid Mercurialis huetii to the clonal dioecious M. perennis. Primers were tested on a set of 54 male and female plants from natural decaploid populations. Eight of 65 tested loci produced polymorphic fragments. Binary profiles from 4 different scoring routines were used to define multilocus lineages (MLLs). Allowing for fragment differences within 1 MLL, all analyses revealed the same 14 MLLs without conflicting with merigenet, sex, or plot assignment. For semiautomatic scoring, a combination of as few as 2 of the 4 most polymorphic loci resulted in unambiguous discrimination of clones. Our study demonstrates that microsatellite fingerprinting of polyploid plants is a cost efficient and reliable alternative to AFLPs, not least because fewer loci are required than for diploids.

Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicilin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. To differentiate reliably among S. aureus isolates, we recently developed double locus sequence typing (DLST) based on the analysis of partial sequences of clfB and spa genes. In the present study, we evaluated the usefulness of DLST for epidemiological investigations of MRSA by routinely typing 1242 strains isolated in Western Switzerland. Additionally, particular local and international collections were typed by pulsed field gel electrophoresis (PFGE) and DLST to check the compatibility of DLST with the results obtained by PFGE, and for international comparisons. Using DLST, we identified the major MRSA clones of Western Switzerland, and demonstrated the close relationship between local and international clones. The congruence of 88% between the major PFGE and DLST clones indicated that our results obtained by DLST were compatible with earlier results obtained by PFGE. DLST could thus easily be incorporated in a routine surveillance procedure. In addition, the unambiguous definition of DLST types makes this method more suitable than PFGE for long-term epidemiological surveillance. Finally, the comparison of the results obtained by DLST, multilocus sequence typing, PFGE, Staphylococcal cassette chromosome mec typing and the detection of Panton-Valentine leukocidin genes indicated that no typing scheme should be used on its own. It is only the combination of data from different methods that gives the best chance of describing precisely the epidemiology and phylogeny of MRSA.

Use of forensic investigations in anti-doping.

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules. In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison. Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished. This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

Nestmate recognition and the genetic relatedness of nests in the ant Formica pratensis

Genetic relatedness of the mound-building ant Formica pratensis was determined by means of microsatellite DNA polymorphism, and its impact on nestmate recognition was tested in a population in Southern Sweden (Oeland). Recognition between nests was measured by testing aggression levels between single pairs of workers. The genetic distances of nests (Nei's genetic distance) and the spatial distance of nests were correlated and both showed a strong relation to the aggression behavior. Multiple regression analysis revealed a stronger impact of genetic relatedness rather than spatial distances on aggression behavior. Neighbouring nests were more closely related than distant nests, which may reflect budding as a possible spreading mechanism. The genetic distance data showed that nestmate recognition was strongly genetically influenced in F. pratensis.

Ecological temporal stability of Staphylococcus aureus nasal carriage.

We described the colonization dynamics of Staphylococcus aureus in a group of 266 healthy carriers over a period of approximately 1 year. We used precise genotyping methods, i.e., amplified fragment length polymorphism (AFLP), spa typing, and double-locus sequence typing (DLST), to detect changes in strain identity. Strain change took place rather rarely: out of 89 carriers who had initially been colonized, only 7 acquired a strain different from the original one. Approximately one-third of the carriers eliminated the colonization, and a similar number became newly colonized. Some of these events probably represent detection failure rather than genuine colonization loss or acquisition. Lower bacterial counts were associated with increased probability of eliminating the colonization. We have confirmed a high mutation rate in the spa locus: 6 out of 53 strains underwent mutation in the spa locus. There was no overall change in S. aureus genotype composition.

Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages.

To reliably differentiate among Staphylococcus aureus isolates we recently developed the Double Locus Sequence Typing (DLST) based on the analysis of partial sequences of clfB and spa genes. This method is highly discriminatory and gives unambiguous definition of types. The highly clonal population structure of S. aureus suggests that isolates with identical clfB or spa alleles belong to the same clonal complex (CC) defined by Multi-Locus Sequence Typing (MLST). To test this hypothesis as well as to investigate putative intra-CC genetic structure, we analyzed a total of 289 isolates (186 MSSA and 103 MRSA) with DLST-, spa- and MLST-typing. Among the 289 strains, 242 were clustered into 7 major MLST CCs, 40 into minor CCs and 7 were not grouped into CCs. A total of 205 DLST- and 129 spa-types were observed. With one exception, all DLST-clfB, DLST-spa and spa-type alleles were segregated into CCs. DLST-types sharing an identical allele (clfB or spa) were clustered using eBURST. Except for one strain, all isolates from each DLST cluster belonged to the same CC. However, using both DLST- and spa-typing we were not able to disclose a clear intra-CC structure. Nevertheless, the high diversity of these loci confirmed that they are good markers for local epidemiological investigations.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans.

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

The evolutionary host switches of Polychromophilus: a multi-gene phylogeny of the bat malaria genus suggests a second invasion of mammals by a haemosporidian parasite.

BACKGROUND: The majority of Haemosporida species infect birds or reptiles, but many important genera, including Plasmodium, infect mammals. Dipteran vectors shared by avian, reptilian and mammalian Haemosporida, suggest multiple invasions of Mammalia during haemosporidian evolution; yet, phylogenetic analyses have detected only a single invasion event. Until now, several important mammal-infecting genera have been absent in these analyses. This study focuses on the evolutionary origin of Polychromophilus, a unique malaria genus that only infects bats (Microchiroptera) and is transmitted by bat flies (Nycteribiidae). METHODS: Two species of Polychromophilus were obtained from wild bats caught in Switzerland. These were molecularly characterized using four genes (asl, clpc, coI, cytb) from the three different genomes (nucleus, apicoplast, mitochondrion). These data were then combined with data of 60 taxa of Haemosporida available in GenBank. Bayesian inference, maximum likelihood and a range of rooting methods were used to test specific hypotheses concerning the phylogenetic relationships between Polychromophilus and the other haemosporidian genera. RESULTS: The Polychromophilus melanipherus and Polychromophilus murinus samples show genetically distinct patterns and group according to species. The Bayesian tree topology suggests that the monophyletic clade of Polychromophilus falls within the avian/saurian clade of Plasmodium and directed hypothesis testing confirms the Plasmodium origin. CONCLUSION: Polychromophilus' ancestor was most likely a bird- or reptile-infecting Plasmodium before it switched to bats. The invasion of mammals as hosts has, therefore, not been a unique event in the evolutionary history of Haemosporida, despite the suspected costs of adapting to a new host. This was, moreover, accompanied by a switch in dipteran host.

Tunisian population data on 10 Y-chromosomal loci.

Alleles and haplotypes frequencies for 10 Y-chromosome STR loci (DYS19, DYS385 I/II, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438 and DYS439), included in the Y-Plex6 and Y-Plex5 kits were determined for a Tunisian population sample of 100 male individuals.