Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources

A comprehensive survey to document the presence of free-living amoebae of the genus Acanthamoeba was conducted in tap water and sea water sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus-specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potentially pathogenic strains in both sources. Thus, some of the amoebae in these aquatic habitats can act as opportunistic pathogens, could play a role in the diseases of aquatic organisms, and may present a risk to human health.     

Sex hormone-binding globulin in non-cirrhotic alcoholic patients during early withdrawal and after longer abstinence

In recently intoxicated non-cirrhotic male alcohol-misusing and -dependent patients, we studied, during early withdrawal and more prolonged abstinence, the rate of changes of sex hormones and their binding globulin (SHBG), the prevalence of hypo-androgenism and possible determinant factors of SHBG increase. Twenty-one alcoholics and 21 controls were studied. SHBG plasma levels, sex hormones (SH), cortisol, insulin and thyroid hormones were measured at admission and discharge. SHBG and SH were also determined on days 2, 4 and 7 after admission and on weeks 2, 6 and 12 after discharge. SHBG showed a 3-fold increase, decreasing slowly during the first 10 days, but remaining above control values. Luteinizing hormone was also increased. Free testosterone (Tf) was low at admission and correlated negatively with SHBG during the first 10 days. By day 10, Tf reached normal values, despite SHBG remaining elevated. The other sex hormones were normal. Neither insulin nor thyroid hormones correlated with SHBG. Cortisol was high at admission and then normalized. Clinical hypo-androgenism was found in 33-50% of patients, but did not correlate with SHBG or SH. During follow-up, nine patients relapsed. In those remaining abstinent, SHBG continued decreasing, reaching normal levels in the 12th week. In those who relapsed, SHBG remained high or even increased further. Gamma-glutamyltransferase showed similar but faster changes. We conclude that excessive alcohol ingestion is associated with marked increases of SHBG which slowly revert during abstinence. High SHBG does not fully explain the low Tf values or the presence of clinical hypo-androgenism in alcoholics. This SHBG response to ethanol makes it a potential marker of excessive alcohol intake.     

Irinotecan in combination with fluorouracil in a 48-hour continuous infusion as first-line chemotherapy for elderly patients with metastatic colorectal cancer: a Spanish Cooperative Group for the Treatment of Digestive Tumors study.

PURPOSE: Elderly patients constitute a subpopulation with special characteristics that differ from those of the nonelderly and have been underrepresented in clinical trials. This study was performed to determine the efficacy and safety of irinotecan (CPT-11) in combination with fluorouracil (FU) administered as a 48-hour continuous infusion twice a month in elderly patients. PATIENTS AND METHODS: Patients > or = 72 years old with metastatic colorectal cancer, Eastern Cooperative Oncology Group performance status of 0 to 1, no geriatric syndromes, and no prior treatment were treated every 2 weeks with CPT-11 180 mg/m2 plus FU 3,000 mg/m2 in a 48-hour continuous infusion. RESULTS: By intent-to-treat analysis, in 85 assessable patients, the objective response rate was 35% (95% CI, 25% to 46%), and stable disease was 33% (95% CI, 23% to 44%). Median time to progression was 8.0 months (95% CI, 6.0 to 10.0 months), and median overall survival time was 15.3 months (95% CI, 13.8 to 16.9 months). Toxicity was moderate. Grade 3 and 4 neutropenia, diarrhea, and asthenia were observed in 21%, 17%, and 13% of patients, respectively. Only one case of neutropenic fever occurred. There were two toxic deaths, one was a result of grade 4 diarrhea and acute kidney failure, and the other was a result of massive intestinal hemorrhage in the first cycle. The study of prognostic factors did not reveal any predictive factor of response. Response to treatment and baseline lactate dehydrogenase were the main factors conditioning progression-free and overall survival. CONCLUSION: Twice a month continuous-infusion CPT-11 combined with FU is a valid therapeutic alternative for elderly patients in good general condition.     

Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections

Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.     

Laparoscopic Cardial Calibration and Gastropexy for Treatment of Patients with Reflux Esophagitis: Pathophysiological basis and Result

Laparoscopic antireflux surgery is the gold standard procedure for treatment of patients with reflux esophagitis. The current results of the laparoscopic approach are absolutely comparables with the results obtained during the open surgery era. The Nissen, Nissen-Rossetti, or Toupet techniques are the more frequently used. We have performed cardial calibration and posterior gastropexy or Nissen fundoplication by the open approach with similar results. The purpose of this article is to present the anatomo-physiological basis for employing cardial calibration and posterior gastropexy in patients with reflux esophagitis. This study includes 108 symptomatic patients, 12 of them with associated extraesophageal manifestations ( posterior laryngitis). Endoscopic mild or moderate esophagitis was confirmed in 83 patients, Barrett’s esophagus in 12 patients, and type I or II hiatal hernia in 13 patients. All patients were also submitted to manometry, 24 hour intraesophageal pH monitoring, and barium swallow before and after surgery. Follow-up ranged from 12 to 36 months. There were no conversion, major intraoperative, or postoperative complications; nor were there any deaths. Postoperative dysphagia was present in 5% of cases. Symptomatic recurrence of reflux was observed in 10.3% and endoscopic presence of esophagitis in 12.3% of cases . Lower esophageal sphincter pressure increased significantly after surgery, even in patients with endoscopic recurrence. 24-hour intraesophageal monitoring improved after surgery, except in patients with objective recurrence of esophagitis. In conclusion, laparoscopic cardial calibration with posterior gastropexy presents comparable results to those reported after Nissen fundoplication and therefore could be another excellent therapeutic option in patients with reflux esophagitisThis paper was accepted for presentation as a free paper oral presentation for the last World Congress of ISS-SIC (cancelled).     

Open transthoracic or transhiatal esophagectomy versus minimally invasive esophagectomy in terms of morbidity, mortality and survival

Background Surgical treatment of esophageal cancer is associated with a high rate of morbidity and mortality even in specialized centers. Minimally invasive surgery has been proposed to decrease these complications. Methods The authors present their results regarding postoperative complications and the survival rate at 3 years, comparing the classic open procedures (transthoracic or transhiatal esophagectomy) with minimally invasive surgery. Surgical procedures were performed according to procedures published elsewhere. Results The study enrolled 166 patients who underwent surgery between 1990 and 2003. Open transthoracic surgery was performed for 60 patients. In this group of patients, postoperative mortality was observed in 11% of the cases. Major, minor, and late complications were observed in 61.6% of the patients, and the 3-year survival rate was 30% for this group. Open transhiatal surgery was performed for 59 patients. The morbidity, mortality, and 3-year rate were almost the same as for the transthoracic surgery group. For the 47 patients submitted to minimally invasive procedures (thoracoscopic and laparoscopic), the complications and mortality rates were significantly reduced (38.2% and 6.4%, respectively). For the patients submitted to minimally invasive surgery, the 3-year survival rate was 45.4%. It is important to clarify that the patients submitted to minimally invasive surgery manifested early stages of the diseases, and that this the reason why the morbimortality and survival rates were better. Conclusions The transthoracic and transhiatal open approaches have similar early and late results. Minimally invasive surgery is an option for patients with esophageal carcinoma, with reported results similar to those for open surgery. This approach is indicated mainly for selected patients with early stages of the disease. Presented as a “free paper” during the 9th World Congress of Endoscopic Surgery, Cancun, Mexico, 4-7 February, 2004     

Limited role for IVUS in the endovascular repair of aortoiliac aneurysms.

BACKGROUND: To determine the need for routine versus selective intraoperative IVUS during endovascular aortoiliac aneurysm (AIA) repair. METHODS: One-hundred and eighty-eight endovascular AIA repairs performed over a 5-year period were reviewed and included in the study. Surgeon-made aorto-uni-femoral grafts (n=78) and industry-made bifurcated or tube grafts (n=110) were used. In the initial 51 cases IVUS was routinely performed. In the latter 137 cases IVUS was used selectively. In this group graft deformities suspected on completion angiography or pullback pressure measurements were treated with balloon dilatation and stenting. IVUS was then performed only in the presence of a persistent pressure gradient or inconclusive angiographic findings. RESULTS: In the initial 51 cases IVUS revealed 20 lesions of which 8 were not initially detected angiographically and which required further treatment. In the latter 137 cases IVUS was necessary in only 1 case, and guided the treatment of an angiographically undetectable lesion. There have been no late episodes of graft compression, kinking, or thrombosis in the selective IVUS group. CONCLUSIONS: The use of pullback pressure measurements with a low threshold for angioplasty and stenting, especially in unsupported grafts, followed by the selective use of IVUS decreases the overall requirement for IVUS and its associated costs.     

Statins and Voriconazole Induce Programmed Cell Death in Acanthamoeba castellanii

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC₅₀s) and IC₉₀s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.     

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.