A rapid and simple method to detect ESBL in Enterobacter cloacae based on MIC of cefepime

INTRODUCTION: The aim of this study was to identify a rapid and simple phenotypic method for extended-spectrum β-lactamase (ESBL) detection in Enterobacter cloacae. METHODS: A total of 79 consecutive, non-repeated samples of E. cloacae were evaluated. Four phenotypic methods were applied for ESBL detection, results were compared to multiplex polymerase chain reaction (PCR) as the gold standard reference method: 1) ceftazidime and cefotaxime disks with and without clavulanate, both with boronic acid added; 2) disk approximation using cefepime and amoxicillin/clavulanate; 3) ESBL screening by minimum inhibitory concentration (MIC) ≥ 16µg/mL and 4) by MIC ≥ 2µg/mL for cefepime. RESULTS: Method 4 showed the best combination of sensitivity (100%) and specificity (94%). CONCLUSIONS: MIC ≥ 2µg/mL for cefepime would be very useful for the phenotypic detection of ESBL in samples of E. cloacae.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Gender-Based Differences in Cardiac Remodeling and ILK Expression after Myocardial Infarction

Background: Gender can influence post-infarction cardiac remodeling. Objective: To evaluate whether gender influences left ventricular (LV) remodeling and integrin-linked kinase (ILK) after myocardial infarction (MI). Methods: Female and male Wistar rats were assigned to one of three groups: sham, moderate MI (size: 20-39% of LV area), and large MI (size: ≥40% of LV area). MI was induced by coronary occlusion, and echocardiographic analysis was performed after six weeks to evaluate MI size as well as LV morphology and function. Real-time RT-PCR and Western blot were used to quantify ILK in the myocardium. Results: MI size was similar between genders. MI resulted in systolic dysfunction and enlargement of end-diastolic as well as end-systolic dimension of LV as a function of necrotic area size in both genders. Female rats with large MI showed a lower diastolic and systolic dilatation than the respective male rats; however, LV dysfunction was similar between genders. Gene and protein levels of ILK were increased in female rats with moderate and large infarctions, but only male rats with large infarctions showed an altered ILK mRNA level. A negative linear correlation was evident between LV dimensions and ILK expression in female rats with large MI. Conclusions: Post-MI ILK expression is altered in a gender-specific manner, and higher ILK levels found in females may be sufficient to improve LV geometry but not LV function.

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.

Molecular approaches to malaria and Babesisosis diagnosis

The development of additional methods for detecting and identifuing Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. To preliminarily evaluate sunthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK)was selected from published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzymelinked synthetic DNA assay for P. falciparum detected 30 parasites/mm(cúbicos) from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min. exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.

New methods for the diagnosis of Babesia bigemina infection

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currentlyused methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These testes have been used the fild but because they lack sensitivity and specificity, never and improved methods of diagnosis are being developed. The quantitative buffy coat (OBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with grater sensitivity and specificity than previously described serological tests. Similary, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of diferentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Molecular Epidemiologic Typing Systems of Bacterial Pathogens: Current Issues and Perpectives

The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s )? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection). Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

Molecular characterisation of intermediate snail hosts and the search for resistance genes

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 µg/ml for macrophages and a few of them maintained this cytotoxicity even at 10 µg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1µg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

The clinical value of an in-house cytomegalovirus nested polymerase chain reaction (CMV-PCR) and a commercial molecular assay hybrid capture CMV DNA assay (HCA) was evaluated in monitoring a group of renal transplant patients for six months follow up. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of nested CMV DNA PCR assay and HCA at the beginning of the study were 70, 42.9, 46.7, 66.7, and 60, 78.6, 66.7, and 73.3% respectively. After six months, they were 80, 66.7, 80, 66.7 for CMV PCR and 73.3, 88.9, 91.7, 66.7% for HCA respectively. These results indicate that in monitoring and predicting CMV infections in renal transplant recipients, not only qualitative but also quantitative assays must be used together in order to decide the preemptive strategies.

Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.

Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates

A multiplex polymerase chain reaction (PCR) assay was performed on 167 thermophilic campylobacters isolated from non-human primates. Samples were first identified by phenotypic methods resulting in 64 Campylobacter jejuni and 103 C. coli strains. Four strains identified biochemically as C. coli, were then determined to be C. jejuni by PCR. Comparison of methodologies showed that the main discrepancies were attributed to the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Analysis of data showed that the application of phenotypic methods should be supplemented by a molecular method to offer a more reliable Campylobacter identification.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.