Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/µL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Cost-effective analysis of different algorithms for the diagnosis of hepatitis C virus infection

We compared the cost-benefit of two algorithms, recently proposed by the Centers for Disease Control and Prevention, USA, with the conventional one, the most appropriate for the diagnosis of hepatitis C virus (HCV) infection in the Brazilian population. Serum samples were obtained from 517 ELISA-positive or -inconclusive blood donors who had returned to Fundação Pró-Sangue/Hemocentro de São Paulo to confirm previous results. Algorithm A was based on signal-to-cut-off (s/co) ratio of ELISA anti-HCV samples that show s/co ratio ³95% concordance with immunoblot (IB) positivity. For algorithm B, reflex nucleic acid amplification testing by PCR was required for ELISA-positive or -inconclusive samples and IB for PCR-negative samples. For algorithm C, all positive or inconclusive ELISA samples were submitted to IB. We observed a similar rate of positive results with the three algorithms: 287, 287, and 285 for A, B, and C, respectively, and 283 were concordant with one another. Indeterminate results from algorithms A and C were elucidated by PCR (expanded algorithm) which detected two more positive samples. The estimated cost of algorithms A and B was US$21,299.39 and US$32,397.40, respectively, which were 43.5 and 14.0% more economic than C (US$37,673.79). The cost can vary according to the technique used. We conclude that both algorithms A and B are suitable for diagnosing HCV infection in the Brazilian population. Furthermore, algorithm A is the more practical and economical one since it requires supplemental tests for only 54% of the samples. Algorithm B provides early information about the presence of viremia.

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Distribution of the human leukocyte antigen class II alleles in Brazilian patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a global medical problem. The current standard of treatment consists of the combination of peginterferon plus ribavirin. This regimen eradicates HCV in 55% of cases. The immune response to HCV is an important determinant of disease evolution and can be influenced by various host factors. HLA class II may play an important role in immune response against HCV. The objective of the present study was to determine the distribution of HLA class II (DRB1 and DQB1) alleles, their association with chronic HCV infection and their response to interferon therapy. One hundred and two unrelated white Brazilian patients with chronic HCV infection, 52 responders (45 males and 7 females) and 50 non-responders (43 males and 7 females) to antiviral treatment, were included in the study. Healthy Brazilian bone marrow donors of Caucasian origin from the same geographic area constituted the control group (HLA-DRB1, N = 99 and HLA-DQB1, N = 222 individuals). HLA class II genotyping was performed using a low-resolution DRB1, DQB1 sequence-specific primer amplification. There were higher frequencies of HLA-DRB1*13 (26.5 vs 14.1%) and HLA-DQB1*02 (52.9 vs 38.7%) in patients compared with controls; however, these were not significantly different after P correction (Pc = 0.39 and Pc = 0.082, respectively). There was no significant difference between the phenotypic frequencies of HLA-DRB1 (17.3 vs 14.0%) and HLA-DQB1 alleles in responder and non-responder HCV patients. The HLA-DRB1*07 allele was significantly more common in HCV patients (33.3 vs 12.1%) than in controls (Pc = 0.0039), suggesting that the HLA-DRB1*07 allele is associated with chronic HCV infection.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG-100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.

Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situhybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Discriminação de sorovares de Salmonella spp. isolados de carcaças de frango por REP e ERIC-PCR e fagotipagem do sorovar Enteriditis

Salmonelose é a infecção bacteriana de origem alimentar mais freqüente no Paraná, Brasil, e os surtos estão associados, principalmente, ao consumo de ovos, carne de aves e derivados. Os objetivos deste trabalho foram identificar os sorovares de Salmonella isolados de carcaças de frango e caracterizá-los molecularmente por REP e ERIC-PCR, assim como identificar os fagotipos de Salmonella Enteriditis. Dos 25 isolados de Salmonella spp. analisados, 18 foram identificados como Enteriditis, 4 como Braenderup, 2 como Worthington e 1 como infantis. Dos 18 isolados de Enteriditis, 14 foram PT4, 2 PT4a, 1 PT7 e 1 RDNC, por se tratar de colônia rugosa. REP-PCR forneceu padrão eletroforético distinto de 10 a 13 bandas distribuídas entre 120 e 2072 pb para cada sorovar diferente testado. A ERIC-PCR mostrou um padrão de 4 a 5 bandas entre 180 e 1000 pb e foi menos discriminativa quando comparada à REP-PCR. Os resultados encontrados confirmaram que a fagotipagem é uma ferramenta útil e discriminativa para o sorovar Enteriditis. Apesar do pequeno número de sorovares testados, os resultados sugerem que a REP-PCR parece ser um método atrativo a ser utilizado no futuro para a discriminação preliminar de sorovares de Salmonella.