Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 degrees C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Fluorescence images combined to statistic test for fingerprinting of citrus plants after bacterial infection

The citrus greening (or huanglongbing) disease has caused serious problems in citrus crops around the world. An early diagnostic method to detect this malady is needed due to the rapid dissemination of Candidatus Liberibacter asiaticus (CLas) in the field. This analytical study investigated the fluorescence responses of leaves from healthy citrus plants and those inoculated with CLas by images from a stereomicroscope and also evaluated their potential for the early diagnosis of the infection caused by this bacterium. The plants were measured monthly, and the evolution of the bacteria on inoculated plants was monitored by real-time quantitative polymerase chain reaction (RT-qPCR) amplification of CLas sequences. A statistical method was used to analyse the data. The selection of variables from histograms of colours (colourgrams) of the images was optimized using a paired Student's t-test. The intensity of counts for green colours from images of fluorescence had clearly minor variations for healthy plants than diseased ones. The darker green colours were the indicators of healthy plants and the light colours for the diseased. The method of fluorescence images is novel for fingerprinting healthy and diseased plants and provides an alternative to the current method represented by PCR and visual inspection. A new, non-subjective pattern of analysis and a non-destructive method has been introduced that can minimize the time and costs of analyses.

Prevalence, Incidence Density, and Genotype Distribution of GB Virus C Infection in a Cohort of Recently HIV-1-Infected Subjects in Sao Paulo, Brazil

Background: The results of previous studies elsewhere have indicated that GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus type 1 (HIV-1) due to similar transmission routes of both viruses. The aim of this study was to determine the prevalence, incidence density and genotypic characteristics of GBV-C in this population. Methodology/Principal Findings: The study population included 233 patients from a cohort primarily comprised of homosexual men recently infected with HIV-1 in Sao Paulo, Brazil. The presence of GBV-C RNA was determined in plasma samples by reverse transcriptase-nested polymerase chain reaction and quantified by real-time PCR. GBV-C genotypes were determined by direct sequencing. HIV viral load, CD4+ T lymphocyte and CD8+ T lymphocyte count were also tested in all patients. The overall prevalence of GBV-C infection was 0.23 (95% CI: 0.18 to 0.29) in the study group. There was no significant difference between patients with and without GBV-C infection and Glycoprotein E2 antibody presence regarding age, sex, HIV-1 viral load, CD4+ and CD8+ T cell counts and treatment with antiretroviral drugs. An inverse correlation was observed between GBV-C and HIV-1 loads at enrollment and after one year. Also, a positive but not significant correlation was observed between GBV-C load and CD4+ T lymphocyte. Phylogenetic analysis of the GBV-C isolates revealed the presence of genotype 1 and genotype 2, these sub classified into subtype 2a and 2b. Conclusion/Significance: GBV-C infection is common in recently HIV -1 infected patients in Sao Paulo, Brazil and the predominant genotype is 2b. This study provides the first report of the GBV-C prevalence at the time of diagnosis of HIV-1 and the incidence density of GBV-C infection in one year.

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

HEMOPLASMAS IN WILD CANIDS AND FEUDS IN BRAZIL

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of Sao Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.

Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches` broom pathogen Moniliophthora perniciosa

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches` broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabollite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

Gene expression profile of residual breast cancer after doxorubicin and cyclophosphamide neoadjuvant chemotherapy

In breast cancer patients, primary chemotherapy is associated with the same survival benefits as adjuvant chemotherapy. Residual tumors represent a clinical challenge, Lis they may be resistant to additional cycles of the same drugs. Our aim was to identify differential transcripts expressed in residual tumors, after neoadjuvant chemotherapy, that might be related with tumor resistance. Hence, 16 patients with paired tumor samples, collected before and after treatment (4 cycles doxorubicin/cyclophosphamide, AC) had their gene expression evaluated on cDNA microarray slides containing 4,608 genes. Three hundred and eighty-nine genes were differentially expressed (paired Student`s t-test, pFDR<0.01) between pre- and post-chemotherapy samples and among the regulated functions were the JNK cascade and cell death. Unsupervised hierarchical clustering identified one branch comprising exclusively, eight pre-chemotherapy samples and another branch, including the former correspondent eight post-chemotherapy samples and other 16 paired pre/post-chemotherapy samples. No differences in clinical and tumor parameters could explain this clustering. Another group of I I patients with paired samples had expression of selected genes determined by real-time RT-PCR and CTGF and DUSP1 were confirmed more expressed in post- as compared to pre-chemotherapy samples. After neoadjuvant chemotherapy some residual samples may retain their molecular signature while others present significant changes in their gene expression, probably induced by the treatment. CTGF and DUSP1 overexpression in residual samples may be a reflection of resistance to further administration of AC regimen.