Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Considerações sobre os Thallosporados: o gênero Trichosporon: "Trichosporon minor" n. sp. produtor da piedra axilar

O autor estuda o gênero trichosporon, sob o ponto de vista da sistemática e como produtor da piedra branca e de micoses internas generalisadas do tipo gomoso. Estabelece a prioridade do termo Trichosporon, como foi descrito primeiramente, por BEHREND, devendo-se abandonar a designação posterior de Trichosporum, dada por VUILLEMIN. Estuda a distinção das duas piedras – a piedra branca ou trichosporica e a piedra negra ou asterinica. As lesões produzidas pelos dois cogumelos no pelo parasitado são consideradas como fator indispensável para a fixação do parasito no pêlo e para a formação ulterior no nódulo. Por iim descreve uma nova espécie de piedra, a piedra axilar, produzida pelo Trichosporon minor n. sp. Trichosporon minor n. sp. – cogumelo produtor da piedra axilar, formando nos cabelos desta região pequenos nódulos de coloração acinzentada, de consistência dura e tamanho variável e constituídos de células arredondadas, elíticas ou alongadas, medindo 0,5 a 4 μde diâmetro e algumas germulantes formando cadeias de dois a quatro elementos. Descola a epidermícula do cabelo, onde se fixa, sem entretanto penetrar nas camadas mais profundas. Nos meios artificiais de cultura dá colônias a princípio chatas de superfície húmida e aveludada com franjas espessas e salientes ou colônias vermiculares, ou culturas brancas, chatas, semelhantes a culturas de actinomyces. Não fermenta os açúcares; gelifica tardiamente a gelatina.

The metazoan parasites of Stellifer minor (Tschudi, 1844): an ecological approach

A quantitative and qualitative analysis of the parasite fauna of the sciaenid Stellifer minor (Tschudi) from Chorrillos, Peru, was made. Some characteristics of the infectious processes, in terms of intensity and prevalence of infection, as a function of host sex and size, are given. Moreover, comments on the characteristics of the parasite fauna, related with host role in the marine food webs are included. The parasite fauna of Stellifer minor taken of Chorrillos, Peru, include the monogeneans Pedocotyle annakohni, Pedocotyle bravoi, Rhamnocercus sp. and Cynoscionicola sp., the digenean Helicometra fasciata, the adult acantocephalan Rhadinorhynchus sp. and the larval Corynosoma sp., the nematode Procamallanus sp., the copepods Caligus quadratus, Clavellotis dilatata and Bomolochus peruensis and one unidentified isopod of the family Cymothoidae. A distinctive characteristic of the parasite fauna (Metazoa) of S. minor is the almost absence of larval forms.

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae)

Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

Development of nuclear DNA probes for the typing of Trypanososma cruzi

We have developed and tested a new way of typing Trypanosoma cruzi, mamely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Soutern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis a higher degree of heterogeneity was observed at this level.

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations

Four Trypanosoma cruzi strains from zymodermes A, B, C and D were successively clonedon BHI-LIT-agar-blood BLAB). Twenty clones from the first generation (F1), 10 from The second (F2) and 4 from the third (F3) from the strains A138, B147 and C23 were isolated. The D150 strain provied 29 F1 and F2 clones. The strains and clones had their isoenzyme and K-DNA patterns determined. The clones from A138, Bl47 and C231 strains presented isoemzyme and K-DNA patterns identical between thewmselves and their respective parental strains. Therefore showing the homogenety and stability of isoenzyme and K-DNA patterns after successive cloning. The Dl50 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a K-DNA pattern identical to its parental strain. The remining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homonogeous populations. Their experimental use will make future results more reliable and reproducible.

A new repetitive DNA sequence from Trypanosoma cruzi

Tandemly repeated DNA sequences are found in the genome of higher eukaryotes, and have also been demonstrated in Trypanosoma cruzi. Repeated DNA sequences are potentially useful for the diagnostic detection of T. cruzi (A. Gonzales et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 3356-3360). We have isoleted two clones from a genomic library of T. cruzi (Y strain) that contain, in one clone a family of at least seven copies of a repetitive sequence of approximately 600 base pairs, and in the other an independent copy of the same sequence. One copy of the repetition (HSP) and the independent clone (HCR) were sequenced by the Sanger procedure (Fig.). This sequence hybridized to four strains of T. cruzi tested and did not hybridize to eleven species of trypanosotids from five different Genera, being a good candidate for diagnostic assays. GenBank accession numbers: HSP#m31919, HCR#31920.

Kudoa sciaenae (Myxozoa: Multivalvulidae) cysts distribution in the somatic muscles of Stellifer minor (Tschudi, 1844) (Pisces: Sciaenidae)

The distribution of Kudoa sciaenae cysts (Myxozoa), in terms of intensity and prevalence, in the somatic muscles of the sciaenid Stellifer minor, shows an apparent preference for the anterior body region, including the head. The observed preference seems to be a consequence of the differential distribution of muscle mass, in the defined area, because when density (cyst/g dry muscle), is considered, all the somatic areas, but not cephalic area, do no show significant differences in terms of mean intensity and prevalence.

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CVM) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CVM from urine and the technical advantage of each is discussed.

Experimental heteroxenous cycle of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae) in white mice and in cats

Reports of natural infections of sylvatic carnivores by adult worms of species similar to Lagochilascaris minor in the Neotropical region led to attempts to estabilish experimental cycles in laboratory mice and in cats. Also, larval development was seen in the skeletal muscle of an agouti (Dasyprocta leporina) infected per os with incubated eggs of the parasite obtained from a human case. In cats, adult worms develop and fertile eggs are expelled in the feces: in mice, larval stages of the parasite develop, and are encapsulate in the skeletal muscle, and in the adipose and subcutaneous connective tissue. From our observations, we conclude that the larva infective for the mouse is the early 3rd stage, while for the final host the infective form is the later 3rd stage. A single moult was seen in the mouse, giving rise to a small population of 4th stage larvae, long after the initial infection.