Low-stringency Single Specific Primer PCR Research Articles

Molecular Detection and Characterization of Novel Lipase Genes of the Lipolytic Yeast Candida palmioleophila

In this study we analyzed the genetic variability of lipase gene sequences from eight oil and grease-degrading strains of Candida palmioleophila and to relate it to their degrading ability. The genetic variability of lipase genes was analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and low-stringency single specific primer- PCR (LSSP-PCR), in order to obtain specific DNA fingerprints from each strain, which were subsequently compared by bioinformatic programs. DNA fingerprints were contrasted to the ability of each strain to remove palm oil in liquid culture. The results showed that at least three genes encoding lipases are present in C. palmioleophila, two of them resembling the LIP2 and LIP6 genes of C. albicans. DNA fingerprints obtained by LSSP-PCR revealed differences in the sequences of C. palmioleophila lipase genes, which allowed to group the strains according to their degrading activity. C. palmioleophila strains SACL05, SACL08 and SACL11, which showed the highest removal of palm oil after 72 h (77 to 79 % removal), were grouped in a single clade in dendrograms. Similarly, strains SACL01, SACL03, SACL06 and SACL09, which showed intermediate removal activity of palm oil (54 to 76%) grouped in a different clade. This suggests the genetic variability in lipase genes is directly related to the differences found in the efficiency of degradation of oils. On the other hand, DNA fingerprints obtained by PCR-RFLP did not allow to differentiate the strains and did not generate changes in the bands patterns between the analyzed strains. In conclusion, this study reported for the first time the detection and characterization of lipase genes from the lipolytic yeast Candida palmioleophila, and their association to the degradation of oils.

Genotypic profiles of Leishmania (Viannia) braziliensis strains from cutaneous leishmaniasis patients and their relationship with the response to meglumine antimoniate treatment: a pilot study.

Background: Forty-four strains isolated from a cohort of cutaneous leishmaniasis (CL) patients who did or did not respond to one course of treatment with meglumine antimoniate were investigated to explore genetic polymorphisms in parasite kinetoplast DNA minicircles. Leishmania (Viannia) braziliensis strains isolated from responder (R) and non-responder (NR) patients who acquired infection in Rio de Janeiro or in other Brazilian states were studied using low-stringency single-specific primer polymerase chain reaction (LSSP-PCR) to identify genetic polymorphisms. Results: Polymorphisms were observed in parasites recovered from patient lesions. No association was found between a specific genotype and R or NR patients. Phenetic analysis grouped the genotypes into three main clusters, with similarity indices varying from 0.72 to 1.00. Although no specific genotype association was detected, at least one group of L. (V.) braziliensis genotypes that circulates in Rio de Janeiro was discriminated in clusters I and III, showing phenotypes of good and poor responses to treatment, respectively. Cluster I comprised parasite profiles recovered from R patients from Rio de Janeiro and in cluster III, NR samples were prevalent. Cluster II comprised 24 isolates, with 21 from Rio de Janeiro and three from other states, equally distributed between R and NR patients. Additionally, we found that parasites sharing all common genetic characteristics acted differently in response to treatment. Conclusions: These results are of clinical-epidemiological importance since they demonstrate that populations of L. (V.) braziliensis that exhibit high levels of genetic similarity also display different phenotypes associated with meglumine antimoniate responses in cutaneous leishmaniasis patients.

High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil.

Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease. An epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture). T. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected. LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

First Evidence of Genetic Intraspecific Variability and Occurrence of Entamoeba gingivalis in HIV(+)/AIDS

Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4+ lymphocyte count (≤200 cells/mm3 (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(−) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4+ lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated from HIV patients.

American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis: assessment of parasite genetic variability at intra- and inter-patient levels

BackgroundThe genetic variability of Leishmania (Viannia) braziliensis was assessed at intra and interpatient levels of individuals with different clinical manifestations of American tegumentary leishmaniasis (ATL).MethodsFifty-two samples, of which 13 originated from cutaneous lesions and 39 from mucosal lesions, provided by 35 patients, were examined by low-stringency single-specific-primer PCR (LSSP-PCR) and phenetic analysis. Genetic variability of L. (V.) braziliensis, in kinetoplast DNA (kDNA) signatures, was compared both from different patients and from different lesions of the same patient. Phenetic analysis was performed to evaluate the degree of heterogeneity of the kDNA minicircles. In order to evaluate inter and intrapatient L. (V.) braziliensis genetic variability, the percentage of shared bands and analysis of the coefficients of similarity were analyzed.ResultsDifferent genetic profiles, representing kDNA signatures of the parasite, were obtained by LSSP-PCR analysis of each sample. Phenetic analysis grouped genetic profiles of different levels of differentiation from more similar to most divergent. The percentage of shared bands at the inter and intrapatient levels was 77% and 89%, respectively. Comparison of the average inter and intrapatient coefficients of similarity and their standard deviations were statistically significant (p < 0.001).ConclusionGenetic variability at the intrapatient level was less pronounced than that between different patients. A conceptual model was proposed to better understand the complexity at both levels.

Canine Cutaneous Leishmaniasis: Dissemination and Tissue Tropism of Genetically DistinctLeishmania (Viannia) braziliensisPopulations

Little is known regarding the internal dissemination of initial cutaneous lesions and tissue tropism of Leishmania (Viannia) braziliensis populations in naturally infected dogs. The aim of this study was to investigate genetic polymorphisms of L. (V.) braziliensis populations in different anatomic sites of naturally infected dogs by using polymerase chain reaction (PCR) and low-stringency single specific primer-PCR (LSSP-PCR) techniques. The amplified products were analyzed by LSSP-PCR to investigate the genetic variability of the parasite populations present in different anatomical sites. Twenty-three out of the 52 samples gave PCR-positive results. The existence of L. (V.) braziliensis strains that remained restricted to cutaneous lesions and others showing characteristics of dissemination to internal organs and healthy skin was observed. LSSP-PCR and numerical analyses revealed that parasite populations that do not disseminate were genetically similar and belonged to a separate phenetic cluster. In contrast, populations that showed spreading to internal organs displayed a more polymorphic genetic profile. Despite the heterogeneity, L. (V.) braziliensis populations with identical genetic profiles were observed in popliteal and cervical lymph nodes of the same animal. Our results indicate that infection in dogs can be manifested by dissemination and tissue tropism of genetically distinct populations of L. (V.) braziliensis.

Genetic polymorphism in Leishmania (Viannia) braziliensis detected in mucosal leishmaniasis of HIV-infected and non-HIV-infected patients

The genetic polymorphism of Leishmania (Viannia) braziliensis detected in cases of mucosal leishmaniasis (ML) from HIV-infected and non HIV-infected patients was evaluated. Nine samples from three HIV-infected patients and five samples from five non HIV-infected patients were analysed by polymerase chain reaction (PCR), low-stringency single-specific primer PCR (LSSP-PCR) and phenetic analysis. The presence of L. (V.) braziliensis DNA was detected in all samples by specific PCR assay. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was investigated by LSSP-PCR. Phenetic analysis grouped the genetic profiles into two distinct clusters, which discriminated between samples obtained from HIV-infected and non HIV-infected patients. In two HIV-infected patients, identical genetic profiles were detected in lesions biopsied at different times after the treatment of the initial lesion. Interestingly, genetically divergent profiles were detected in the cutaneous and mucosal lesions of the same HIV-infected patient collected at the same time. This is the first work comparing genetic polymorphism of L. (V.) braziliensis in cases of mucosal leishmaniasis from HIV-infected and non HIV-infected patients.

KDNA genetic signatures obtained by LSSP-PCR analysis of Leishmania (Leishmania) infantum isolated from the new and the old world.

BackgroundVisceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region.Principal FindingsKDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World.ConclusionsLSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.

Molecular Characterization by LSSP‐PCR and DNA Sequencing of a Pathogenic Isolate of Leptospira interrogans from Brazil

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31-21%), Hardjo Norma (46-40%), Hardjo hardjobovis (18-10%), Mini (8-4%) and Wolffi (7-4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three-dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single-specific primer polymerase chain reaction (LSSP-PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.

Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

BackgroundChagas disease is a neglected illness, with limited treatments, caused by the parasite Trypanosoma cruzi. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by T. cruzi. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.ResultsThe differential genes expression of a sensitive Trypanosoma cruzi strain and its induced in vitro benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC50 ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.ConclusionHere, we propose NTR as the major marker of the appearance of resistance to BZ.

KDNA minicircle signatures of Leishmania (Viannia) braziliensis in oral and nasal mucosa from mucosal leishmaniasis patients

Polymerase chain reaction (PCR) and low-stringency single-specific primer PCR (LSSP-PCR) analyses were used to detect Leishmania (Viannia) braziliensis DNA and investigate kDNA signatures of parasite populations present in oral and nasal mucosa lesions from mucosal leishmaniasis patients. A total of 25 samples from 22 patients were processed by specific PCR/hybridization assays. Parasite DNA was detected in all samples analyzed. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was also investigated by LSSP-PCR. Similar kDNA signatures were observed in parasites recovered from nasal and oral mucosa lesions of the same patient. In contrast, genetically divergent profiles were detected in lesions from patients biopsied at different times within a period of 1 year. This is the first work to report genetic typing of L. (V.) braziliensis directly from human oral and nasal mucosal lesions.

Differentiation of Plum pox virus isolates by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis of HC-Pro genome region

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR were assessed for suitability and reliability in genotyping of Plum pox virus (PPV) isolates. Examined PPV isolates included 16 PPV-D, 12 PPV-M, and 14 PPV-Rec isolates collected in Czech Republic. The analysis was performed on the helper component protease (HC-Pro) region of the PPV genome. SSCP and LSSP-PCR allowed the differentiation of PPV strain, but SSCP was not able to distinguish isolates within the same strain. The individual genotyping of each PPV isolate was obtained by LSSP-PCR. Nevertheless, both SSCP and LSSP-PCR techniques are suitable for preliminary screening of genetic variability of plant RNA viruses.

Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes

A low-stringency single-primer polymerase chain reaction (LSSP-PCR) typing procedure targeted to the intergenic regions of spliced-leader genes (SL) was designed to profile Trypanosoma cruzi I stocks from endemic regions of Colombia. Comparison between SL-LSSP-PCR profiles of parasite DNA from vector faeces and cultures isolated from those faeces showed more conservative signatures than profiles using LSSP-PCR targeted to the minicircle variable regions (kDNA). This was also observed by analysing 15 parasite clones from one stock as well as serial samples of a same stock after in vitro culturing or inoculation into mice. Thus, SL-LSSP-PCR appears more appropriate than kDNA-LSSP-PCR for reliable typing of major T. cruzi I groups from in vitro cultured stocks and triatomine faeces. SL-LSSP-PCR grouped 46 of 47 T. cruzi I Colombian stocks according to their geographical procedences in four clusters: Cluster Cas from Casanare Department, Cluster Mg from Northern Magdalena department, Cluster Mom from Momposina Depression in Southern Magdalena and finally Cluster NW from northwestern Colombia, including Sucre, Chocó, Córdoba and Antioquia departments. Sequence analysis identified punctual mutations among amplicons from each cluster. Within Cluster Mg, sequence polymorphism allowed association with different sylvatic vector species. Novel SL sequences and LSSP-PCR profiles are reported from T. cruzi I infecting Eratyrus cuspidatus, Panstrongylus geniculatus and Rhodnius pallescens vectors.

Genetic polymorphism in Trypanosoma cruzi I isolated from Brazilian Northeast triatomines revealed by low-stringency single specific primer–polymerase chain reaction

Different molecular markers have been employed for typing Trypanosoma cruzi strains from endemic areas of Chagas disease. The low-stringency single specific primer-polymerase chain reaction (LSSP-PCR) has been a sensitive and informative technique that uses the variable region of kinetoplast DNA minicircles as a genetic marker, allowing detection of DNA sequence variation. In the present study, we analyzed the intra-lineage genetic variability of the T. cruzi strains obtained from triatomine feces collected on filter paper FTA card by LSSP-PCR. The hybridization of the PCR products with a probe for the subgenus Schizotrypanum and a clone-specific probe from Dm28c confirmed the subgenus as T. (S.) cruzi and respective lineages as T. cruzi I. Phenetic analysis showed the presence of three clusters that diverged by different coefficients of similarity. Thirteen T. cruzi I genotypes were observed circulating among Triatoma pseudomaculata and Rhodnius nasutus from peridomiciliary and natural environments in five peri-urban and urban localities of Jaguaruana, Ceará, Brazil. These data indicate the importance of the circulation of T. cruzi I genotypes among T. pseudomaculata and R. nasutus in different environments and the possible risk of Chagas disease domestic transmission.

Evaluation of LSSP-PCR for identification of Leptospira spp. in urine samples of cattle with clinical suspicion of leptospirosis

We evaluated the use of low-stringency single specific primer PCR (LSSP-PCR) for genetically typing Leptospira directly from urine samples of cattle with clinical suspicion of leptospirosis. Urine samples obtained from 40 cattle with clinical suspicion of leptospirosis were amplified by specific PCR using the following primers: Internal 1/Internal 2 and G1/G2. The internal primers were designed from the gene sequence of the outer membrane lipoprotein Lip32 from Leptospira kirschneri, strain RM52. The PCR products were amplified with these two pairs of primers, which had approximately 497 and 285 bp, respectively, and were subsequently used as a template for LSSP-PCR analysis. The genetic signatures from the leptospires which were present in the urine samples allowed us to make a preliminary identification of the leptospires by comparing the LSSP-PCR profiles obtained directly from urine samples with those from reference leptospires. The LSSP-PCR profiles obtained with the Internal 1 primer or with the G1 primer allowed the grouping of the leptospires into serogroups. LSSP-PCR was found to be a useful and sensitive approach capable of identifying leptospires directly from biological samples without the need for prior bacterial isolation. In conclusion, the LSSP-PCR technique may still be helpful in discriminating serogroups of Leptospira from different animal reservoirs, since the early identification of carrier animals and information on the shedding state are crucial to prevent the spread of leptospiral infection to other animals and humans.

Variability of Kinetoplast DNA Gene Signatures of Trypanosoma cruzi II Strains from Patients with Different Clinical Forms of Chagas' Disease in Brazil

The clinical course of Chagas' disease varies widely among different patients and geographic regions. For reasons that are not completely understood but involve host and parasite factors, some patients never develop the disease while others present cardiac and/or gastrointestinal symptoms. Many studies have been conducted in order to correlate the genetic variability of the parasites with the clinical forms of the disease, but no conclusive data have been obtained. Our research aims at characterizing the genetic profiles of Trypanosoma cruzi isolates recently obtained from 70 chagasic patients who either showed pathological lesions with symptoms of various intensities or were asymptomatic. All patients came from an area where Chagas' disease is endemic in southeast Brazil where vectorial transmission has been controlled and different clinical forms of the disease can be found. The molecular characterization of parasites evaluated the polymorphisms of the 3' region of the 24Salpha rRNA gene and the variability of kinetoplast DNA (kDNA) minicircles of T. cruzi populations by low-stringency single specific primer PCR. Data presented here provide a strong correlation between T. cruzi II and human infection in this region. However, a high degree of variability was observed within T. cruzi II, as demonstrated by intense kDNA polymorphism among all clinical forms and also within each of them, irrespective of the intensity of pathological processes.

The Trypanosoma rangeli histone H2A gene sequence serves as a differential marker for KP1 strains

Trypanosoma rangeli has recently been divided in two primary lineages denoted as KP1(+) and KP1(−) strains because of epidemiological and evolutionary interest in the molecular differentiation of these two groups. We report the molecular characterization of the genes encoding histone H2A protein from a T. rangeli KP1(+) strain (H14), its comparison to T. rangeli KP1(−) strain (C23) histone H2A coding genes [Puerta, C., Cuervo, P., Thomas, M.C., López, M.C., 2000. Molecular characterization of the histone H2A gene from the parasite Trypanosoma rangeli. Parasitol. Res. 86, 916–922], and its application in a low-stringency single specific primer polymerase chain reaction (LSSP-PCR) assay to differentiate these parasite groups. The results show that the locus encoding the H2A protein in the H14 strain is formed by at least 11 gene units measuring 799 nucleotides in length, organized in tandem, and located in two chromosomes of approximately 1.9 and 1.1 Mb in size. Remarkably, in KP1(−) strains these genes are on pairs of chromosomes of about 1.7 and 1.9 Mb. In addition, there is a hybridization signal in the compression region above 2.1 Mb in all T. rangeli strains. Therefore, the chromosomal location of these genes is a useful marker to distinguish between KP1(+) and KP1(−) T. rangeli strains. The alignment of the H2A nucleotide sequences from H14 and C23 strains showed an identity of 99.5% between the coding regions and an identity of 95% between the non-coding regions. The deduced amino acid sequences proved to be identical. Based on 5% of the difference between the intergenic regions, we developed a LSSP-PCR assay which can differentiate between KP1(+) and KP1(−) strains.

Análisis por LSSP-PCR de la variabilidad genética de Trypanosoma cruzi en sangre y órganos de ratones.

Chagas' disease, caused by Trypanosoma cruzi, has a variable clinical course, ranging from asymptomatic infection to chronic disease. Trypanosoma cruzi has a clonal population structure, although infecting strains are often multiclonal. Genetic variability of T. cruzi may be one of the determinant factors in differential tissue tropism and consequently of the clinical forms of the disease. To examine this possibility, mice were infected with two Colombian T. cruzi strains to determine the distribution of genetic variants in blood and organs. The sensitivity of three molecular markers was evaluated, and the genetic variability of the clones was determined. The latter was attained by means of low-stringency single specific primer polymerase chain reaction (LSSP-PCR) using a kinetoplast DNA (kDNA) marker. The kDNA signatures obtained with the LSSP-PCR were analyzed by neighbor-joining. The presence of the two lineages of T. cruzi was confirmed, as well as the multiclonal character of the two strains. The most sensitive marker was the kDNA. The most affected organ was the heart, which showed the greatest number of positive results with the three markers. Genetic differences were noted between the clones from the blood and organs of infected mice. These results support the value of the LSSP-PCR technique for the study of the molecular epidemiology of Chagas' disease.

Potential application of low-stringency single specific primer-PCR in the identification ofLeptospirain the serum of patients with suspected leptospirosis

In this study we tested the potential use of low-stringency single specific primer-PCR (LSSP-PCR) for genetically typing Leptospira directly from biological samples. Serum samples obtained from 29 patients with clinically suspected leptospirosis were amplified by specific PCR, using the previously selected G1 and G2 primers. The PCR products of approximately 300 bp were subsequently used as a template for LSSP-PCR analysis. We were able to produce genetic signatures from the leptospires present in the human samples, which permitted us to make a preliminary identification of the infective serovar by comparing the LSSP-PCR profiles obtained directly from serum samples with those from reference leptospires. Thus, LSSP-PCR has the potential to become a useful diagnostic tool for identifying leptospires in biological samples without the need for bacteria isolation and culture.

Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae)

To understand the interaction of Trypanosoma cruzi with caviomorph rodents, which supposedly have an ancient co-evolutionary history with this parasite, experimental infection of laboratory reared Trichomys apereoides with several isolates of both genotypes of the parasite was studied. Parasitemia, pattern of hematic cells, specific humoral immune response, histopathological features and parasite clearance were appraised. T. apereoides maintained stable infections independent of the T. cruzi genotype as demonstrated by positive PCR results in analyses of several tissues after a 5 months follow-up. The acute phase was characterized by abundant and disseminated presence of amastigotes, vacuolization and/or myocytolysis. Lymphocytosis was a common feature. The chronic phase was characterized mainly by lymphomacroeosinophilic infiltrates independent of the inoculated T. cruzi isolate. T. cruzi of different genotypes did not show any tissular preference in T. apereoides. Index Descriptors and Abbreviations: Trypanosoma cruzi; Changes’ disease; Echimyidae; Trichomys apereoides; Histopathology; bp, base pairs; DIG-labeled DNA, DNA probes with digoxygenin-labeled deoxynucleotides; DNA, deoxyribonucleic acid; Ethylene diamine tetra acetic acid; FITC, fluorescein isothiocyanate; IFA, immunofluorescence assay; HE, hematoxylin–eosin; IgG, immunoglobulin G; kDNA, kinetoplast deoxyribonucleic acid; LCSSP-PCR, low-stringency single specific primer PCR; LIT, liver infusion tryptose medium; NNN, Novy–Mc Neal–Nicole medium; PCR, polymerase chain reaction; PI, post-infection; SSC, saline sodium citrate solution; TAE, trisma acetate buffer; Taq, thermostable DNA polymerase