Evaluation of Primary Resistance to HIV Entry Inhibitors Among Brazilian Patients Failing Reverse Transcriptase/Protease Inhibitors Treatment Reveal High Prevalence of Maraviroc Resistance-Related Mutations

Entry inhibitor is a new class of drugs that target the viral envelope protein. This region is variable; hence resistance to these drugs may be present before treatment. The aim of this study was to analyze the frequency of patients failing treatment with transcriptase reverse and protease inhibitors that would respond to the entry inhibitors Enfuvirtide, Maraviroc, and BMS-806. The study included 100 HIV-1 positive patients from one outpatient clinic in the city of Sao Paulo, for whom a genotype test was requested due to treatment failure. Proviral DNA was amplified and sequenced for regions of gp120 and gp41. A total of 80 could be sequenced and from those, 73 (91.3%), 5 (6.3%) and 2 (2.5%) were classified as subtype B, F, and recombinants (B/F and B/C), respectively. CXCR4 co-receptor use was predicted in 30% of the strains. Primary resistance to Enfuvirtide was found in 1.3%, following the AIDS Society consensus list, and 10% would be considered resistant if a broader criterion was used. Resistance to BMS-806 was higher; 6 (7.5%), and was associated to non-B strains. Strikingly, 27.5% of samples harbored one or more mutation among A316T, I323V, and S405A, which have been related to decreased susceptibility of Maraviroc; 15% of them among viruses predictive to be R5. A more common mutation was A316T, which was associated to the Brazilian B strain harboring the GWGR motif at the tip of V3 loop and their derivative sequences. These results may be impact guidelines for genotype testing and treatment in Brazil.

Primary resistance of human immunodeficiency virus type 1 in a reference center in Recife, Pernambuco, Brazil

To assess the prevalence of primary resistance of human immunodeficiency virus type 1 (HIV-1) to antiretrovirals, 84 patients chronically infected with HIV without prior antiretroviral treatment from Northeast Brazil were studied. Genotyping was performed using the ViroSeqTM Genotyping System. Thimidine analog mutations occurred in 3 (3.6%) patients. Accessory mutations related to NRTI occurred in 6 (7.1%) and related to PI in 67 (79.8%). Subtypes B (72.6%), F (22.6%), B/F 3 (3.6%), and C (1.2%) were detected. A low prevalence of major mutations related to NRTI in patients chronically infected by HIV was observed.

Fibroblast growth factors: An epigenetic mechanism of broad spectrum resistance to anticancer drugs

Based on the observation that removal of tumors from metastatic organs reversed their chemoresistance, we hypothesized that chemoresistance is induced by extracellular factors in tumor-bearing organs. By comparing chemosensitivity and proteins in different tumors (primary vs. metastases) and different culture systems (tumor fragment histocultures vs. monolayer cultures derived from the same tumor), we found elevated levels of acidic (aFGF) and basic (bFGF) fibroblast growth factors in the conditioned medium (CM) of solid and metastatic tumors. These CM induced broad spectrum resistance to drugs with diverse structures and action mechanisms (paclitaxel, doxorubicin, 5-fluorouracil). Inhibition of bFGF by mAb and its removal by immunoprecipitation resulted in complete reversal of the CM-induced chemoresistance, whereas inhibition/removal of aFGF resulted in partial reversal. Using CM that had been depleted of aFGF and/or bFGF and subsequently reconstituted with respective human recombinant proteins, we found that bFGF but not aFGF induced chemoresistance whereas aFGF amplified the bFGF effect. aFGF and bFGF fully accounted for the CM effect, indicating these proteins as the underlying mechanism of the chemoresistance. The FGF-induced resistance was not due to reduced intracellular drug accumulation or altered cell proliferation. We further showed that an inhibitor of aFGF/bFGF (suramin) enhanced the in vitro and in vivo activity of chemotherapy, resulting in shrinkage and eradication of well established human lung metastases in mice without enhancing toxicity. These results indicate elevated levels of extracellular aFGF/bFGF as an epigenetic mechanism by which cancer cells elude cytotoxic insult by chemotherapy, and provide a basis for designing new treatment strategies.

Evaluating budesonide efficacy in nasal polyposis and predicting the resistance to treatment

Cell resistance to glucocorticoids is a major problem in the treatment of nasal polyposis (NP). The objectives of this study were to observe the effect of budesonide on the expression of IL-1 beta, TNF-alpha, granulocyte macrophage-colony stimulating factor, intercellular adhesion molecule (ICAM)-1, basic fibroblast growth factor, eotaxin-2, glucocorticoid receptor (GR)-alpha, GR-beta, c-Fos and p65 in nasal polyps and to correlate their expression to clinical response. Biopsies from nasal polyps were obtained from 20 patients before and after treatment with topical budesonide. Clinical response to treatment was monitored by a questionnaire and nasal endoscopy. The mRNA levels of the studied genes were measured by real-time quantitative (RQ)-PCR. There was a significant decrease in the expression of TNF-alpha (P < 0.05), eotaxin-2 (P < 0.05) and p65 (P < 0.05) in NP after treatment. Poor responders to glucocorticoids showed higher expression of IL-1 beta (3.74 vs. 0.14; P < 0.005), ICAM-1 (1.91 vs. 0.29; P < 0.05) and p65 (0.70 vs. 0.16; P < 0.05) before treatment. Following treatment, IL-1 beta (4.18 vs. 0.42; P < 0.005) and GR-beta (0.95 vs. 0.28; P < 0.05) mRNA expression was higher in this group. Topical budesonide reduced the expression of TNF-alpha, eotaxin-2 and p65. Poor responders to topical budesonide exhibit higher levels of IL-1 beta, ICAM-1 and nuclear factor (NF)-kappa B at diagnosis and higher expression of both IL-1 beta and GR-beta after treatment. These results emphasize the anti-inflammatory action of topical budesonide at the molecular level and its importance in the treatment of NP. Nevertheless, IL-1 beta, ICAM-1 and NF-kappa B may be associated with primary resistance to glucocorticoids in NP, whereas higher expression of GR-beta in poor responders only after glucocorticoid treatment may represent a secondary drug resistance mechanism in this disease.

Analysis of Transmitted Resistance to Raltegravir and Selective Pressure among HIV-1-Infected Patients on a Failing HAART in Sao Paulo, Brazil

We studied the presence of primary resistance to raltegravir (RAL), natural polymorphisms, and selection pressure on HIV-1 integrase. We found a high frequency of integrase polymorphisms related to the resistance to RAL and sequence stability. Further studies are needed to determine the importance of these polymorphisms to RAL resistance.

High frequency of resistance to the drugs isoniazid and rifampicin among tuberculosis cases in the city of Cabo de Santo Agostinho, an urban area in Northeastern Brazil

The objective of the present study was to investigate the frequency and risk factors for developing multidrug-resistant tuberculosis in Cabo de Santo Agostinho, PE. This was a prospective study conducted from 2000 to 2003, in which suspected cases were investigated using bacilloscopy and culturing. Out of 232 confirmed cases of tuberculosis, culturing and antibiotic susceptibility tests were performed on 174. Thirty-five of the 174 cultures showed resistance to all drugs. The frequencies of primary and acquired resistance to any drug were 14% and 50% respectively, while the frequencies of primary and acquired multidrug resistance were 8.3% and 40%. Previous tuberculosis treatment and abandonment of treatment were risk factors for drug resistance. The high levels of primary and acquired resistance to the combination of isoniazid and rifampicin contributed towards the difficulties in controlling tuberculosis transmission in the city.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

A survey was performed to estimate the frequency of Escherichia coli and Shiga toxin-producing E. coli (STEC) in carcasses obtained from an abattoir in Brazil between February 2006 and June 2007. A total of 216 beef carcasses were sampled at three stages of the slaughter process-preevisceration, postevisceration, and postprocessing-during the rain and dry seasons, respectively. Of the carcasses sampled, 58%, were preevisceration E. coli positive, 38% were postevisceration positive, and 32% postprocessing positive. At the postprocessing stage, the isolation of E. coli was twice as high in the rain season. E. coli was isolated from 85 carcasses of which only 3 (1.4%) were positive for stx-encoding genes. No E. coli O157 serogroup isolates were detected. No antimicrobial resistance was found in nine of the isolates (10% of the total). The most frequent resistances were seen against cephalothin (78%), streptomycin (38%), nalidixic acid (36%), and tetracycline (30%). Multidrug resistance (MDR) to three or more antimicrobial agents was determined in 28 (33%) E. coli isolates. The presence of STEC and MDR strains among the isolates in the beef carcasses emphasizes the importance of proper handling to prevent carcass contamination.

Resistance to antiparasitic drugs: The role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Anti-schistosomal drugs: observations on the mechanism of drug resistance to hycanthone, and on the involvement of host antibodies in the mode of action of praziquantel

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.

Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.

Statistical methods for examining genetic influences of resistance to anti-epileptic drugs

It is generally accepted that genetics may be an important factor in explaining the variation between patients’ responses to certain drugs. However, identification and confirmation of the responsible genetic variants is proving to be a challenge in many cases. A number of difficulties that maybe encountered in pursuit of these variants, such as non-replication of a true effect, population structure and selection bias, can be mitigated or at least reduced by appropriate statistical methodology. Another major statistical challenge facing pharmacogenetics studies is trying to detect possibly small polygenic effects using large volumes of genetic data, while controlling the number of false positive signals. Here we review statistical design and analysis options available for investigations of genetic resistance to anti-epileptic drugs.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)