Candida dubliniensis identification in Brazilian yeast stock collection

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42°C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42°C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Standardization of in-house polymerase chain reaction for the identification of Mycobacterium tuberculosis at the reference tropical disease hospital in the State of Goiás, Brazil

This study compares smear, growth in Lowenstein-Jensen medium, and in-house polymerase chain reaction (PCR) techniques for the detection of Mycobacterium tuberculosis. A total of 72 specimens from 72 patients with clinical symptoms of tuberculosis, including 70 sputum and two bronchial aspirate samples, were tested in parallel by smear, culture, and in-house PCR techniques. From these, 48 (66.6%) were negative by the 3 methods, 2 (2.8%) were smear positive and negative by culture and in-house PCR, 11 (15.3%) were both smear and culture negative, and in-house PCR positive, 7 (9.7%) were positive by the 3 methods, 2 (2.8%) were positive by smear and culture, and negative by PCR, 2 (2.8%) were positive by culture and PCR, but smear negative. After the resolution of discrepancies in PCR results, the sensitivity and specificity for in-house PCR technique to M. tuberculosis relative to the culture, were 81.8% and 81.9%, respectively. These results confirm that this method, in-house PCR, may be a sensitive and specific technique for M. tuberculosis detection, occurring in both positive and negative smear and negative cultures.

Blackflies (Diptera: Simuliidae) of Southern Guyana with keys for the identification of adults and pupae: a review

A revision is made of the previously poorly studied blackfly fauna from the south-western border of Guyana with Brazil. Notes on the biosystematics of the species found are provided, together with keys and illustrations based on their morphology. Of the 14 species recorded, eight are anthropophilic and two of these (Simulium oyapockense s.l. and S. guianense s.l.) are proven vectors of human onchocerciasis in the nearby Amazonia focus of the disease in neighbouring Brazil.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Identification of the best ovitrap installation sites for gravid Aedes (Stegomyia) aegypti in residences in Mirassol, state of São Paulo, Brazil

This study aimed at identifying the best ovitrap installation sites for gravid Aedes aegypti in Mirassol, state of São Paulo, Brazil. Ovitraps were installed in ten houses per block over ten blocks. Four ovitraps were placed per residence, one in the bedroom, one in the living room, and two outdoors with one in a sheltered area and one in an outside site. Each week for eleven weeks, visits were made to examine the ovitraps and to change the paddles used for egg-laying. Eggs were analyzed according to the trap location. The results showed that the outdoor sites received significantly more oviposition than indoor sites. Additionally, in respect to the outdoor sites, the outside site received significantly more oviposition than the sheltered site. A b correlation was observed between positive traps and egg numbers. The results are discussed with respect to the best installation site of the traps and their implications in surveillance and control of dengue vectors.

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.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5´LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5´LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.

Identification of sex pheromones of Lutzomyia longipalpis (Lutz & Neiva, 1912) populations from the state of São Paulo, Brazil

In Brazil, four populations of Lutzomyia longipalpis each producing different sex pheromones are recognised. It has been suggested that these chemotype populations represent true sibling species. In this study we present the results of an analysis, by coupled gas cromotography - mass spectrometry, of the pheromones of males L. longipalpis from two different municipalities of the state of São Paulo. Our study showed that L. longipalpis from these two municipalities produced different sex pheromones from each other. This coupled with the remarkable difference between the epidemiological situation in Araçatuba and Espírito Santo do Pinhal, suggests that the (S)-9-methylgermacrene-B and cembrene-1 populations may have different vectorial capacities.

Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci

Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bacteremia, specially in patients with indwelling devices or those submitted to invasive medical procedures. The identification of species and the accurate and rapid detection of methicillin resistance are directly dependent on the quality of the identification and susceptibility tests used, either manual or automated. The objective of this study was to evaluate the accuracy of two automated systems MicroScan and Vitek - in the identification of CoNS species and determination of susceptibility to methicillin, considering as gold standard the biochemical tests and the characterization of the mecA gene by polymerase chain reaction, respectively. MicroScan presented better results in the identification of CoNS species (accuracy of 96.8 vs 78.8%, respectively); isolates from the following species had no precise identification: Staphylococcus haemolyticus, S. simulans, and S. capitis. Both systems were similar in the characterization of methicillin resistance. The higher discrepancies for gene mec detection were observed among species other than S. epidermidis (S. hominis, S. saprophyticus, S. sciuri, S. haemolyticus, S. warneri, S. cohnii), and those with borderline MICs.

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.

Identification of the Boudicca and Sinbad retrotransposons in the genome of the human blood fluke Schistosoma haematobium

Schistosomes have a comparatively large genome, estimated for Schistosoma mansoni to be about 270 megabase pairs (haploid genome). Recent findings have shown that mobile genetic elements constitute significant proportions of the genomes of S. mansoni and S. japonicum. Much less information is available on the genome of the third major human schistosome, S. haematobium. In order to investigate the possible evolutionary origins of the S. mansoni long terminal repeat retrotransposons Boudicca and Sinbad, several genomes were searched by Southern blot for the presence of these retrotransposons. These included three species of schistosomes, S. mansoni, S. japonicum, and S. haematobium, and three related platyhelminth genomes, the liver flukes Fasciola hepatica and Fascioloides magna and the planarian, Dugesia dorotocephala. In addition, Homo sapiens and three snail host genomes, Biomphalaria glabrata, Oncomelania hupensis, and Bulinus truncatus, were examined for possible indications of a horizontal origin for these retrotransposons. Southern hybridization analysis indicated that both Boudicca and Sinbad were present in the genome of S. haematobium. Furthermore, low stringency Southern hybridization analyses suggested that a Boudicca-like retrotransposon was present in the genome of B. truncatus, the snail host of S. haematobium.