The emergence of YMDD mutants precedes biochemical flare by 19 weeks in lamivudine-treated chronic hepatitis B patients: an opportunity for therapy reevaluation

Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 ± 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 ± 14 and 60 ± 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 ± 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.

Genotyping of methicillin-resistant Staphylococcus aureus isolates obtained in the Northeast region of Brazil

Methicillin-resistant Staphylococcus aureus (MRSA) is a major agent of hospital infections worldwide. In Brazil, a multiresistant MRSA lineage (ST239-SCCmecIIIA), the so-called Brazilian epidemic clone (BEC), has predominated in all regions. However, an increase in nosocomial infections caused by non-multiresistant MRSA clones has recently been observed. In the present study, 45 clinical isolates of MRSA obtained from a university hospital located in Natal city, Brazil, were identified by standard laboratory methods and molecularly characterized using staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was carried out using CLSI methods. The MRSA isolates studied displayed a total of 8 different pulsed-field gel electrophoresis patterns (types A to H) with predominance (73%) of pattern A (BEC-related). However, MRSA harboring SCCmec type IV were also identified, 3 (7%) of which were genetically related to the pediatric clone - USA800 (ST5-SCCmecIV). In addition, we found a considerable genetic diversity within BEC isolates. MRSA displaying SCCmecIV are frequently susceptible to the majority of non-β-lactam antibiotics. However, emergence of multiresistant variants of USA800 was detected.

A rapid method for testing in vivo the susceptibility of different strains of Trypanosoma cruzi to active chemotherapeutic agents

A method is described which permits to determine in vivo an in a short period of time (4-6 hours) the sensitivity of T. cruzo strains to known active chemotherapeutic agents. By using resistant- and sensitive T. cruzi stains a fairly good correlation was observed between the results obtained with this rapid method (which detects activity against the circulating blood forms) and those obtained with long-term schedules which involve drug adminstration for at least 20 consecutive days and a prolonged period of assessment. This method may be used to characterize susceptibility to active drugs used clinically, provide infomation on the specific action against circulating trypomastigotes and screen active compounds. Differences in the natural susceptibility of Trypanosoma cruzi strains to active drugs have been already reported using different criteria, mostly demanding long-term study of the animal (Hauschka, 1949; Bock, Gonnert & Haberkorn, 1969; Brener, Costa & Chiari, 1976; Andrade & Figueira, 1977; Schlemper, 1982). In this paper we report a method which detects in 4-6 hours the effect of drugs on bloodstream forms in mice with established T. cruzi infections. The results obtained with this method show a fairly good correlation with those obtained by prolonged treatment schedules used to assess the action of drugs in experimental Chagas' disease and may be used to study the sensitivity of T. cruzi strains to active drugs.

The significance of variation in the susceptibility of Schistosoma mansoni to the antischistosomal drug oxamniquine

Clinical and laboratory evidence is reviewed which shows that there is a great deal of variation in the susceptibility of Schistosoma mansoni to oxamniquine. This variation occurs both among endemic regions and within endemic regions in Brazil and Kenya. It is genetically controlled. It is suggested that the parasite possesses a large capacity for developing resistance to the drug and that resistance will develop where sufficient drug pressure is maintained.

Antimicrobial susceptibility patterns of unusual nonfermentative gram-negative bacilli isolated from Latin America: report from the SENTRY Antimicrobial Surveillance Program (1997-2002)

The antimicrobial susceptibility of 176 unusual non-fermentative gram-negative bacilli (NF-GNB) collected from Latin America region through the SENTRY Program between 1997 and 2002 was evaluated by broth microdilution according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Nearly 74% of the NF-BGN belonged to the following genera/species: Burkholderia spp. (83), Achromobacter spp. (25), Ralstonia pickettii (16), Alcaligenes spp. (12), and Cryseobacterium spp. (12). Generally, trimethoprim/sulfamethoxazole (MIC50, < 0.5 µg/ml) was the most potent drug followed by levofloxacin (MIC50, 0.5 µg/ml), and gatifloxacin (MIC50, 1 µg/ml). The highest susceptibility rates were observed for levofloxacin (78.3%), gatifloxacin (75.6%), and meropenem (72.6%). Ceftazidime (MIC50, 4 µg/ml; 83.1% susceptible) was the most active beta-lactam against B. cepacia. Against Achromobacter spp. isolates, meropenem (MIC50, 0.25 µg/ml; 88% susceptible) was more active than imipenem (MIC50, 2 µg/ml). Cefepime (MIC50, 2 µg/ml; 81.3% susceptible), and imipenem (MIC50, 2 µg/ml; 81.3% susceptible) were more active than ceftazidime (MIC50, >16 µg/ml; 18.8% susceptible) and meropenem (MIC50, 8 µg/ml; 50% susceptible) against Ralstonia pickettii. Since selection of the most appropriate antimicrobial agents for testing and reporting has not been established by the NCCLS for many of NF-GNB species, results from large multicenter studies may help to guide the best empiric therapy.

Drug abuse among workers in Brazilian regions

OBJECTIVE: Many business organizations in Brazil have adopted drug testing programs in the workplace since 1992. Rehabilitation, rather than layoff and disciplinary measures, has been offered as part of the Brazilian employee assistance programs. The purpose study is to profile drug abuse among company workers of different Brazilian geographical regions. METHODS: Urine samples of 12,700 workers from five geographical regions were tested for the most common illicit drugs of abuse in the country: marijuana, cocaine, and amphetamine. Enzyme multiplied immunoassay technique (EMIT) and gas chromatography coupled with mass spectrometry (GC/MS) were the techniques utilized for urine testing. The distribution of collected urine samples according to geographical regions was: 72.0% southeast, 13.8% northeast, 7.9% south, 5.7% central west and 0.6% north. RESULTS: Of all samples analyzed, 1.8% was found to be positive for drugs: 0.5% from the south region, 1.1% from northeast, 1.2% from central west, 1.3% from north, and 2.2% from southeast. Of these, 59.9% was marijuana, 17.7% cocaine, 14.6% amphetamine, and 7.7% associated drugs. CONCLUSIONS: The distribution of drugs found in the samples shows a regional variation. Marijuana, however, was found in all regions. Cocaine was seen only in central west and southeast regions. Amphetamine was found in northeast, central west, and southeast regions.

Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol

Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum β-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

PREVALENCE OF DRUG RESISTANCE AND VIRULENCE FEATURES IN Salmonella spp. ISOLATED FROM FOODS ASSOCIATED OR NOT WITH SALMONELLOSIS IN BRAZIL

Salmonella is the most common etiological agent of cases and outbreaks of foodborne diarrheal illnesses. The emergence and spread of Salmonella spp., which has become multi-drug resistant and potentially more pathogenic, have increased the concern with this pathogen. In this study, 237 Salmonella spp., associated or not with foodborne salmonellosis in Brazil, belonging mainly to serotype Enteritidis, were tested for antimicrobial susceptibility and the presence of the virulence genes spvC, invA, sefA and pefA. Of the isolates, 46.8% were sensitive to all antimicrobials and 51.9% were resistant to at least one antimicrobial agent. Resistance to more than one antimicrobial agent was observed in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%) and nalidixic acid (16.9%). No strain was resistant to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxacin and imipenem. The invA gene was detected in all strains. Genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The gene sefA was detected in 31.6% of the strains and only among S. Enteritidis. Resistance and virulence determinants were detected in Salmonella strains belonging to several serotypes. The high rates of antibiotic-resistance in strains isolated from poultry products demonstrate the potential risk associated with the consumption of these products and the need to ensure good food hygiene practices from farm to table to reduce the spread of pathogens relevant to public health.

SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring.

THE SUSCEPTIBILITY OF RECENT ISOLATES OF Schistosoma mansoni TO PRAZIQUANTEL

Introduction: Schistosomiasis is a chronic disease caused by trematode flatworms of the genus Schistosoma and its control is dependent on a single drug, praziquantel (PZQ), but concerns over PZQ resistance have renewed interest in evaluating the in vitro susceptibility of recent isolates of Schistosoma mansoni to PZQ in comparison with well-established strains in the laboratory. Material and methods: The in vitro activity of PZQ (6.5-0.003 µg/mL) was evaluated in terms of mortality, reduced motor activity and ultrastructural alterations against S. mansoni. Results: After 3 h of incubation, PZQ, at 6.5 µg/mL, caused 100% mortality of all adult worms in the three types of recent isolates, while PZQ was inactive at concentrations of 0.08-0.003 µg/mL after 3 h of incubation. The results show that the SLM and Sotave isolates basically presented the same pattern of susceptibility, differing only in the concentration of 6.5 µg/mL, where deaths occurred from the range of 1.5 h in Sotave and just in the 3 h range of SLM. Additionally, this article presents ultrastructural evidence of rapid severe PZQ-induced surface membrane damage in S. mansoni after treatment with the drug, such as disintegration, sloughing, and erosion of the surface. Conclusion: According to these results, PZQ is very effective to induce tegument destruction of recent isolates of S. mansoni.

Comparison between tilt-table testing results performed during different periods of the day

OBJECTIVE: To evaluate the influences of circadian variations on tilt-table testing (TTT) results by comparing the positivity rate of the test performed during the morning with that of the test performed in the afternoon and to evaluate the reproducibility of the results in different periods of the day. METHODS: One hundred twenty-three patients with recurrent unexplained syncope or near-syncope referred for TTT were randomized into 2 groups. In group I, 68 patients, TTT was performed first in the afternoon and then in the morning. In group II, 55 patients, the test was performed first in the morning and then in the afternoon. RESULTS: The TTT protocol was the prolonged passive test, without drug sensitization. Twenty-nine (23.5%) patients had a positive result in at least one of the periods. The positivity rate for each period was similar: 20 (16.2%) patients in the afternoon and 19 (15.4%) in the morning (p=1.000). Total reproducibility (positive/positive and negative/negative) was observed in 49 (89%) patients in group I and in 55 (81%) in group II. Reproducibility of the results was obtained in 94 (90.4%) patients with first negative tests but in 10 (34%) patients with first positive tests. CONCLUSION: TTT could be performed during any period of the day, and even in the 2 periods to enhance positivity. Considering the low reproducibility rate of the positive tests, serial TTT to evaluate therapeutic efficacy should be performed during the same period of the day.

Comparison of the effect of two HMG CoA reductase inhibitors on LDL susceptibility to oxidation

OBJECTIVE: To study the differences between fluvastatin and pravastatin regarding LDL susceptibility to oxidation, plasma levels of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in hypercholesterolemic patients with established coronary heart disease (CHD). METHODS: A double-blind randomized parallel study was conducted that included 41 hypercholesterolemic outpatients with CHD treated at the Instituto de Cardiologia do Rio Grande do Sul. The inclusion criteria were LDL-C above 100 mg/dL and triglycerides below 400 mg/dL based on 2 measures. After 4 weeks on a low cholesterol diet, those patients that fullfilled the inclusion criteria were randomized into 2 groups: the fluvastatin group (fluvastatin 40 mg/day) and the pravastatin group (pravastatin 20 mg/day), for 24 weeks of treatment. LDL susceptibility to oxidation was analyzed with copper-induced production of conjugated dienes (Cu2+) and water-soluble free radical initiator azo-bis (2'-2'amidinopropanil) HCl (AAPH). Spectroscopy nuclear magnetic resonance was used for determination of lipids. RESULTS: After 24 weeks of drug therapy, fluvastatin and pravastatin significantly reduced LDL susceptibility to oxidation as demonstrated by the reduced rate of oxidation (azo and Cu) and by prolonged azo-induced lag time (azo lag). The TC, LDL-C, and TG reduced significantly and HDL-C increased significantly. No differences between the drugs were observed. CONCLUSION: In hypercholesterolemic patients with CHD, both fluvastatin and pravastatin reduced LDL susceptibility to oxidation.

Drug resistance in Schistosomiasis: a review

Drug resistance associated with the treatment of human schistosomiasis appears to be an emerging problem requiring more attention from the scientific community than the subject currently receives. Drug-resistant strains of Schistosoma mansoni have been isolated by various investigators as a result of laboratory experimentation or from a combination of field and laboratory studies. Review of this data appears to indicate that the lack of susceptibility observed for some of the isolated strains cannot be ascribed solely to previous administration of antischistosome drugs and thus further studies are required to elucidate this phenomena. Strains of S. mansoni have now been identified from Brazil which are resistant to oxamniquine, hycanthone and niridazole; from Puerto Rico which are resistant to hycanthone and oxamniquine; and from Kenya which are resistant to niridazole and probably oxamniquine. Strains derived by in vitro selection and resistant to oxamniquine and possibly to oltipraz are also available. All of these strains are currently maintained in the laboratory in snails and mice, thus providing for the first time an opportunity for indepth comparative studies. Preliminary data indicates that S. haematobium strains resistant to metrifonate may be occurring in Kenya. This problem could poise great difficulty in the eventual development of antischistosomal agents. Biomphalaria glabrata from Puerto Rico and Brazil were found to be susceptible to drug-resistant S. mansoni from each country.

Response of drug resistant isolates of Schistosoma mansoni to antischistosomal agents

The susceptibility of four isolates of Schistosoma mansoni (BH, MAP, MPR-1 and K) to four multiple doses of anti-schistosomal agents (hycanthone, niridazole, oxamniquire, and praziquantel) were evaluated in infected female Swiss albino mice. These schistosomal isolates had been maintained in the laboratory without further drug pressure for 20 to 30 generations. Multiple dosage regimens were used for each drug against each isolate of S. mansoni to generate ED50 (effective dose 50%) values. Results demonstrated that the K isolate is resistant to niridazole, the MPR-1 isolate to oxamniquine, and the MAP isolate to both hycanthone and oxamniquine. The BH isolate was susceptible to all drugs and was used as the reference isolate. All isolates were susceptible to praziquantel. The significance of the difference in response of the MPR-1 and MAP isolates is discussed. These results confirm the resistance of these isolates of S. mansoni of three schistosomicides and demonstrate that the resistance of these isolates are stable over long periods of time without exposure to drugs.