Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori

An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori. For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H. pylori clinical isolate. The animals were divided into two groups. Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2). Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group. Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi). Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination. Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach. The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen. However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus. In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi. Our results showed that coccoid H. pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10³ trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Genetic polymorphism of the serine rich antigen N-terminal region in Plasmodium falciparum field isolates from Brazil

In this work we investigated the frequency of polymorphism in exon II of the gene encoding most of the amino-terminal region of the serine rich antigen (SERA) in Plasmodium falciparum field samples. The blood samples were colleted from P. falciparum infected individuals in three areas of the Brazilian Amazon. Two fragments have been characterized by polymerase chain reaction: one of 175 bp corresponding to the repeat region with 5 octamer units and one other of 199 bp related to the 6 repeat octamer units of SERA protein. The 199 bp fragment was the predominant one in all the studied areas. The higher frequency of this fragment has not been described before and could be explained by an immunological selection of the plasmodial population in the infected individuals under study. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitor antibodies, data reported here suggest that the analysis of the polymorphism of P. falciparum isolates in different geographical areas is a preliminary stage before the final drawing of an universal vaccine against malaria can be reached.

The human immunodeficiency virus preventive vaccine research at the French National Agency for acquired immunodeficiency syndrome research

The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the French National Agency for AIDS research (ANRS) has been committed to an original program combining basic science and clinical research. The HIV preventive vaccine research program run by the ANRS covers upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. Most researchers in 2004 believe that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, the ANRS has set up 15 phases I and II clinical trials in order to evaluate the safety and the capacity of the candidate vaccines for inducing cellular immune responses. The tested candidate vaccines were increasingly complex recombinant canarypox viruses (Alvac) containing sequences coding for certain viral proteins, utilized alone or combined with other immunogens (whole or truncated envelope proteins). ANRS has also been developing an original strategy based on the utilization of lipopeptides. These comprise synthetic fragments of viral proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches promptly allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

The neuraminidase gene is present in the non-toxigenic Vibrio cholerae Amazonia strain: a different allele in comparison to the pandemic strains

The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.

Detection of Rickettsia rickettsii in the tick Amblyomma cajennense in a new Brazilian spotted fever-endemic area in the state of Minas Gerais

The present study evaluated rickettsial infection in Amblyomma spp. ticks collected in a farm in Coronel Pacheco, a Brazilian spotted fever (BSF) endemic area. A total of 78 A. cajennense and 78 A. dubitatum free-living adult ticks were collected and tested by polymerase chain reaction (PCR) targeting a fragment of the rickettsial gene gltA. Only one pool of three A. cajennense ticks showed the expected product by PCR. This pool was further tested by PCR using sets of primers targeting the rickettsial genes gltA, ompA, and ompB. All reactions yielded the expected bands that by sequencing, showed 100% identity to the corresponding sequences of the Rickettsia rickettsii gene fragments gltA (1063-bp), ompA (457-bp), and ompB (720-bp). The minimal infection rate of R. rickettii in the A. cajennense population was 1.28% (at least one infected tick within 78 ticks).The present study showed molecular evidence for the presence of R. rickettsii in A. cajennense from a BSF-endemic area in Coronel Pacheco, state of Minas Gerais. Although R. rickettsii has been previously reported infecting A. cajennense ticks in Brazil and other Latin American countries, the present study performed the first molecular characterization of R. rickettsii from the tick A. cajennense.

Taeniasis-cysticercosis in Southern Ecuador: assessment of infection status using multiple laboratory diagnostic tools

Taenia solium-taeniasis and cysticercosis were studied in the human and porcine populations of a rural community in the Southern Ecuadorian Andes. From the 1059 inhabitants, 800 serum samples and 958 stool samples could be collected. In addition, 646 from the estimated 1148 pigs were tongue inspected. Circulating antigen was detected by enzyme linked immunosorbent assay (Ag-ELISA) in 2.25% of the human population, whereas intestinal taeniasis was detected in 1.46% by the formalin-ether technique. Following treatment and recovery of tapeworm fragments these were all identified as T. solium. Porcine cysticercosis was diagnosed in 3.56% of the pigs by tongue inspection. In addition, enzyme linked immunoelectrotransfer blot (EITB) was performed on a subset group of 100 humans to confirm the results of the Ag-ELISA. One hundred serum samples from pigs were also analysed by EITB. It appeared that 43 and 74% of humans and pigs had antibodies against T. solium cysticerci, respectively. It is concluded that contrary to the high exposure of the human population to T. solium that is suggested by EITB, the number of active cysticercosis cases, diagnosed by Ag-ELISA, was low, which may indicate endemic stability. The further use of complementary diagnostic methods for a better understanding of the epidemiology of T. solium is suggested.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Paleogenetic and taphonomic analysis of human bones from Moa, Beirada, and Zé Espinho Sambaquis, Rio de Janeiro, Brazil

The present paper discusses mtDNA and taphonomy of human remains from Moa, Beirada, and Zé Espinho sambaquis of Saquarema, state of Rio de Janeiro, Brazil. New human bone dating by 14C-AMS for Moa archeological site (3810+50 BP - GX-31826-AMS) is included. Preservation of microscopic lamellae and DNA is not related to the macroscopic integrity of the bones. Results here suggest that the preservation of amplifiable DNA fragments may have relation to the preservation of the lamellar arrangement as indicated by optical microscopic examination (polarized light). In 13 human bone fragments from Moa, Beirada, and Zé Espinho it was possible to sequence mtDNA from the 3 individuals of Moa, and from 1 of 4 individuals of Beirada, whose bones also show extensive areas with preserved lamellar structures. The 6 human bone fragments of Zé Espinho and 3 of the 4 fragments of Beirada showed extensive destruction of cortical microstructure represented by cavities, intrusive minerals, and agglomerated microscopic bodies of fungi and bacteria; it was not possible to extract mtDNA from these samples. The results support the hypothesis that the preservation of the microscopic osteon organization is a good predictor for DNA preservation. It was also confirmed the C haplogroup antiquity in Brazil.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.