Treatment protocol for secondary esophageal reconstruction using 'supercharged' colon interposition flaps.

Locoregional esophageal cancer is currently treated with induction chemoradiotherapy, followed by esophagectomy with reconstruction, using a gastric conduit. In cases of conduit failure, patients are temporized with a cervical esophagostomy and enteral nutrition until gastrointestinal continuity can be established. At our institution, we favor reconstruction, using a colon interposition with a 'supercharged' accessory vascular pedicle. Consequently, we sought to examine our technique and outcomes for esophageal reconstruction, using this approach. We performed a retrospective review of all patients who underwent esophagectomy at our center between 2008 and 2018. We identified those patients who had a failed gastric conduit and underwent secondary reconstruction. Patient demographics, perioperative details, and clinical outcomes were analyzed after our clinical care pathway was used to manage and prepare patients for a second major reconstructive surgery. Three hundred and eighty eight patients underwent esophagectomy and reconstruction with a gastric conduit. Seven patients (1.8%) suffered gastric conduit loss and underwent a secondary reconstruction using a colon interposition with a 'supercharged' vascular pedicle. Mean age was 70.1 (±7.3) years, and six patients were male. The transverse colon was used in four cases (57.1%), left colon in two cases (28.6%), and right colon in one case (14.3%). There were no deaths or loss of the colon interposition at follow-up. Three patients (42.9%) developed an anastomotic leak, which resolved with conservative management. All patients had resumption of oral intake within 30 days. Utilizing a 'supercharging' technique for colon interposition may improve the perfusion to the organ and may decrease morbidity. Secondary reconstruction should occur when the patient's oncologic, physiologic, and psychosocial condition is optimized. Our outcomes and preoperative strategies may provide guidance for those centers treating this complicated patient population.

Chemical Modifications of Gold Surfaces with Basic Groups and a Fluorescent Nanoparticle Adhesion Assay To Determine Their Surface p Ka.

For pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces with pH-sensitive groups and to determine the p Ka of the modified gold surfaces using a fluorescent nanoparticle adhesion assay. To introduce pH-sensitive groups onto gold surfaces, we modified gold-coated silicon slides with four different bases: 4-mercaptopyridine (4-MP), 4-pyridylethylmercaptan (4-PEM), 4-aminothiophenol (4-ATP), and 2-mercaptoethylamine (2-MEA). To screen whether the modifications were successful, the binding of negatively charged fluorescently labeled nanoparticles to the positively charged surfaces was visualized by fluorescence microscopy and atomic force microscopy. Next, the p Ka of the modified surfaces was determined by quantifying the pH-dependent adhesion of the fluorescently labeled nanoparticles with fluorescence spectroscopy. Fluorescence microscopy showed that the gold surfaces were successfully modified with the four different basic molecules. Moreover, fluorescence spectroscopy revealed that fluorescently labeled negatively charged nanoparticles bound onto gold surfaces that were modified with one of the four bases in a pH-dependent manner. By quantifying the adsorption of negatively charged fluorescently labeled nanoparticles onto the functionalized gold surfaces and using the Henderson-Hasselbalch equation, the p Ka of these surfaces was determined to be 3.7 ± 0.1 (4-MP), 5.0 ± 0.1 (4-PEM), 5.4 ± 0.1 (4-ATP), and 7.4 ± 0.3 (2-MEA). We successfully functionalized gold surfaces with four different basic molecules, yielding modified surfaces with different p Ka values, as determined with a fluorescent nanoparticle adhesion assay.

Neoadjuvant chemoradiotherapy with concurrent cisplatin/5-fluorouracil is associated with increased pathologic complete response and improved survival compared to carboplatin/paclitaxel in patients with locally advanced esophageal cancer.

Trimodal therapy consisting of neoadjuvant chemoradiation followed by esophagectomy has become the standard of care in North America for locally advanced esophageal cancer. While cisplatin/5-fluorouracil has been a common concurrent chemotherapy regimen since the 1980s, its utilization has declined in recent years as the Chemoradiotherapy for Oesophageal Cancer Followed by Surgery Study (CROSS) trial regimen of carboplatin/paclitaxel has become widely adopted. The efficacy of the CROSS regimen compared to alternate chemotherapy choices, however, has rarely been evaluated when each is used as a component of a trimodal treatment approach. The aim of this study is to report our institutional experience with these two concurrent chemotherapy regimens at a specialized esophageal cancer center.We performed an Institutional Review Board-approved retrospective review of a prospectively maintained institutional foregut registry from a single National Cancer Institute-designated cancer center. Esophageal cancer patients who completed trimodal therapy with a chemotherapy regimen of either carboplatin/paclitaxel or cisplatin/5-fluorouracil were identified and divided into groups based on their chemotherapy regimens. Multivariable logistic regression was used to analyze pathologic complete response rates, while the Kaplan-Meier and Cox proportional hazards models were utilized to evaluate recurrence-free and overall survival. Analytical models were adjusted for age, clinical stage, radiation dose, histologic subtype (adenocarcinoma vs. squamous cell carcinoma), and time interval from completion of neoadjuvant therapy to surgery.One hundred and forty-two patients treated between January of 2000 and July of 2015 were identified as meeting inclusion criteria. Of this group, 87 had received the CROSS regimen of carboplatin/paclitaxel, while 55 had completed cisplatin/5-fluorouracil. Multivariable analysis demonstrated that the cisplatin/5-fluorouracil.group had an increased odds of pathologic complete response (odds ratio = 2.68, 95% confidence interval, P = 0.032), as well as significantly improved recurrence-free survival (hazard ratio = 0.39, 95% confidence interval 0.21-0.73, P = 0.003) and overall survival (hazard ratio = 0.46, 95% confidence interval 0.24-0.87, P = 0.016), compared to the carboplatin/paclitaxel group.Concurrent chemotherapy with cisplatin/5-fluorouracil in locally advanced esophageal cancer is associated with higher rates of pathologic complete response and improved recurrence-free and overall survival compared to the CROSS regimen of carboplatin/paclitaxel. This suggests that, for select patients, alternate neoadjuvant chemotherapy approaches, such as cisplatin/5-fluorouracil, merit reconsideration as potential primary treatment choices in the management of this highly morbid disease.

[Combining a classic monoamine oxidase inhibitor with a tricyclic antidepressant in therapy-resistant depression: a case report and literature review]. / Combineren van klassieke monoamine-oxidaseremmer en tricyclisch anti-depressivum bij therapieresistente depressie: gevalsbeschrijving en literatuuroverzicht.

BACKGROUND: Combining a monoamine oxidase inhibitor (MAOI) with a tricyclic antidepressant (TCA) is considered to be contra-indicated because there is a risk that the recipient develops a serotonin syndrome. An accidental clinical observation motivated us to search and study publications relating to the risk and effectivity of combining these two types of antidepressants.
AIM: To search and study articles on the risks and effectivity of combining the use of MAOIs and TCAs in the treatment of therapy-resistant depression.
METHOD: We searched in PubMed and also studied relevant articles that papers referred to in the database.
RESULTS: Because many case-reports have been misinterpreted, the patients' risk of developing a serotonin syndrome and other complications as a result of the combined use of MAOIs and TCAs is overestimated. The literature provides some evidence that the combination therapy may be effective for some patients who have not responded to TCA or MAO-I monotherapy. Combination therapy seems to be safe if monitored carefully and if TCAs with marked serotonergic affinity are avoided. To enhance safety, the MAOI should be added to a TCA or both the TCA and MAOI should be started simultaneously and titrated slowly.
CONCLUSION: The combination of a MAOI and a TCA can be a possible treatment for patients with treatment resistant depression when monotherapy with either a TCA or a MAOI has failed.

Worldwide Esophageal Cancer Collaboration: pathologic staging data.

We report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for patients with pathologically staged cancer of the esophagus and esophagogastric junction after resection or ablation with no preoperative therapy from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted de-identified data using standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 13,300 patients, 5,631 had squamous cell carcinoma, 7,558 adenocarcinoma, 85 adenosquamous carcinoma, and 26 undifferentiated carcinoma. Patients were older (62 years) men (80%) with normal body mass index (51%), little weight loss (1.8 kg), 0-2 ECOG performance status (83%), and a history of smoking (70%). Cancers were pT1 (24%), pT2 (15%), pT3 (50%), pN0 (52%), pM0 (93%), and pG2-G3 (78%); most involved distal esophagus (71%). Non-risk-adjusted survival for both squamous cell carcinoma and adenocarcinoma was monotonic and distinctive across pTNM. Survival was more distinctive for adenocarcinoma than squamous cell carcinoma when pT was ordered by pN. Survival for pTis-1 adenocarcinoma was better than for squamous cell carcinoma, although monotonic and distinctive for both. WECC pathologic staging data is improved over that of the 7th edition, with more patients studied and patient and cancer variables collected. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient, cancer, and treatment characteristics, and should direct 9th edition data collection. However, the role of pure pathologic staging as the principal point of reference for esophageal cancer staging is waning.

Worldwide Esophageal Cancer Collaboration: clinical staging data.

To address uncertainty of whether clinical stage groupings (cTNM) for esophageal cancer share prognostic implications with pathologic groupings after esophagectomy alone (pTNM), we report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for clinically staged patients from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted data using variables with standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 22,123 clinically staged patients, 8,156 had squamous cell carcinoma, 13,814 adenocarcinoma, 116 adenosquamous carcinoma, and 37 undifferentiated carcinoma. Patients were older (62 years) men (80%) with normal body mass index (18.5-25 mg/kg2 , 47%), little weight loss (2.4 ± 7.8 kg), 0-1 ECOG performance status (67%), and history of smoking (67%). Cancers were cT1 (12%), cT2 (22%), cT3 (56%), cN0 (44%), cM0 (95%), and cG2-G3 (89%); most involved the distal esophagus (73%). Non-risk-adjusted survival for squamous cell carcinoma was not distinctive for early cT or cN; for adenocarcinoma, it was distinctive for early versus advanced cT and for cN0 versus cN+. Patients with early cancers had worse survival and those with advanced cancers better survival than expected from equivalent pathologic categories based on prior WECC pathologic data. Thus, clinical and pathologic categories do not share prognostic implications. This makes clinically based treatment decisions difficult and pre-treatment prognostication inaccurate. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient characteristics, cancer categories, and treatment characteristics and should direct 9th edition data collection.

Worldwide Esophageal Cancer Collaboration: neoadjuvant pathologic staging data.

To address uncertainty of whether pathologic stage groupings after neoadjuvant therapy (ypTNM) for esophageal cancer share prognostic implications with pathologic groupings after esophagectomy alone (pTNM), we report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for pathologically staged cancers after neoadjuvant therapy from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted data using variables with standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 7,773 pathologically staged neoadjuvant patients, 2,045 had squamous cell carcinoma, 5,686 adenocarcinoma, 31 adenosquamous carcinoma, and 11 undifferentiated carcinoma. Patients were older (61 years) men (83%) with normal (40%) or overweight (35%) body mass index, 0-1 Eastern Cooperative Oncology Group performance status (96%), and a history of smoking (69%). Cancers were ypT0 (20%), ypT1 (13%), ypT2 (18%), ypT3 (44%), ypN0 (55%), ypM0 (94%), and G2-G3 (72%); most involved the distal esophagus (80%). Non-risk-adjusted survival for yp categories was unequally depressed, more for earlier categories than later, compared with equivalent categories from prior WECC data for esophagectomy-alone patients. Thus, survival of patients with ypT0-2N0M0 cancers was intermediate and similar regardless of ypT; survival for ypN+ cancers was poor. Because prognoses for ypTNM and pTNM categories are dissimilar, prognostication should be based on separate ypTNM categories and groupings. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient, cancer, and treatment characteristics and should direct 9th edition data collection.

Significant understaging is seen in clinically staged T2N0 esophageal cancer patients undergoing esophagectomy.

This study aimed to determine the impact of preoperative staging on the treatment of clinical T2N0 (cT2N0) esophageal cancer patients undergoing esophagectomy. We reviewed a retrospective cohort of 27 patients treated at a single institution between 1999 and 2011. Clinical staging was performed with computed tomography, positron emission tomography, and endoscopic ultrasound. Patients were separated into two groups: neoadjuvant therapy followed by surgery (NEOSURG) and surgery alone (SURG). There were 11 patients (41%) in the NEOSURG group and 16 patients (59%) in the SURG group. In the NEOSURG group, three of 11 patients (27%) had a pathological complete response and eight (73%) were partial or nonresponders after neoadjuvant therapy. In the SURG group, nine of 16 patients (56%) were understaged, 6 (38%) were overstaged, and 1 (6%) was correctly staged. In the entire cohort, despite being clinically node negative, 14 of 27 patients (52%) had node-positive disease (5/11 [45%] in the NEOSURG group, and 9/16 [56%] in the SURG group). Overall survival rate was not statistically significant between the two groups (P = 0.96). Many cT2N0 patients are clinically understaged and show no preoperative evidence of node-positive disease. Consequently, neoadjuvant therapy may have a beneficial role in treatment.

Preoperative carboplatin and paclitaxel-based chemoradiotherapy for esophageal carcinoma: results of a modified CROSS regimen utilizing radiation doses greater than 41.4 Gy.

Trimodality therapy for resectable esophageal and gastroesophageal junction cancers utilizing preoperative radiotherapy with concurrent carboplatin and paclitaxel-based chemotherapy is being increasingly utilized secondary to the results of the phase III CROSS trial. However, there is a paucity of reports of this regimen as a component of chemoradiotherapy in North America. We aim to report on our clinical experience using a modified CROSS regimen with higher radiotherapy doses. Patients with advanced (cT2-cT4 or node positive) esophageal or gastroesophageal junction carcinoma who received preoperative carboplatin/paclitaxel-based chemoradiotherapy with radiation doses of greater than 41.4 Gray (Gy) followed by esophagectomy were identified from an institutional database. Patient, imaging, treatment, and tumor response characteristics were analyzed. Twenty-four patients were analyzed. All but one tumor had adenocarcinoma histology. The median radiation dose was 50.4 Gy. Pathologic complete response was achieved in 29% of patients, with all receiving 50.4 Gy. Three early postoperative deaths were seen, due in part to acute respiratory distress syndrome and all three patients received 50-50.4 Gy. With a median follow-up of 9.4 months (23 days-2 years), median survival was 24 months. Trimodality therapy utilizing concurrent carboplatin/paclitaxel with North American radiotherapy doses appeared to have similar pathologic complete response rates compared with the CROSS trial, but may be associated with higher toxicity. Although the sample size is small and further follow-up is necessary, radiation doses greater than 41.4 Gy may not be warranted secondary to a potentially increased risk of severe radiation-induced acute lung injury.

Systemic or intrasynovial medication as singular or as combination treatment in horses with (peri-)synovial pain.

Records of lame horses (2004-2007) with (peri-)synovial pain of the metacarpophalangeal joint (MCPJ, n = 53), digital flexor tendon sheath (DFTS, n = 20), and distal interphalangeal joint (DIPJ, n = 31) were evaluated (n = 104). Cases were grouped according to the following treatments: (1) systemic administration of NSAIDs (n = 40); (2) intrasynovial administration of glucocorticosteroids (n = 30); (3) combination treatment (n = 34). Outcome was defined as successful when the owner reported that the horse had returned to its previous level of performance within a period of up to six months after treatment. Statistical evaluation was performed using Fisher's Exact tests and multivariable logistic regression analyses at P < 0.05. Successful outcome was significantly higher for horses in treatment group 3 (19/34, 55.9 per cent, P = 0.021) than in treatment groups 1 (11/40, 27.5 per cent, OR 0.21, 95 per cent CI 0.06 to 0.71) and 2 (8/30, 26.6 per cent, OR 0.18, 95 per cent CI 0.06 to 0.56). A significant association was only found with regard to the affected synovial structure (P = 0.025); relative to the DIPJ, involvement of the MCPJ and DFTS was four to five times more likely to provide a successful outcome (OR 4.18 and 5.59, 95 per cent CI 1.24 to 14.08 and 1.42-22.22, respectively).

Pesticides in groundwater and drinking water wells: overview of the situation in the Netherlands.

In the Netherlands, many of the fresh groundwater resources are vulnerable to pollution. Owing to high population densities and intensive farming practices, pesticide residues are found in groundwater at many places. Hence a number of drinking water abstraction wells contain pesticides residues, causing considerable costs for purification. The Water Framework Directive (WFD) requires countries to assess the chemical status of groundwater bodies and set up monitoring plans for groundwater quality, including pesticides. 771 groundwater samples were taken from monitoring wells in 2006 and analysed for a broad list of pesticides in order to fulfil these requirements. Pesticide were detected in 27% of samples, while in 11% the WFD limit of 0.1 microg/l was exceeded. In this paper, these and earlier measurements are evaluated further, considering also measurements in drinking water wells, information about the origin of measured pesticides and calculated trends in use and emissions. The measurements in the monitoring wells showed that where pesticides are used, 15-55% (minimal and maximal estimation) of the wells in shallow groundwater (1 to 20 m below soil surface) contain pesticides residues at concentrations above 0.1 microg/l. When the metabolites BAM and AMPA are excluded (as not relevant in human toxicological terms), the estimation range is 7-37%. These patterns observed in shallow groundwater are reflected by the occurrence of pesticides in vulnerable abstraction wells that are used for the production of drinking water. The WFD requires the determination of both status and trends. The design of current monitoring network is evaluated from this perspective. Several recommendations are made for more adequate and efficient monitoring.

Runoff and windblown vehicle spray from road surfaces, risks and measures for soil and water.

Soil and surface water along roads are exposed to pollution from motorways. The main pollutants are polycyclic aromatic hydrocarbons (PAH), mineral oil, heavy metals and salt. These pollutants originate from vehicles (fuel, wires, leakage), wear and degradation of road surfaces and road furniture (i.e. crash barriers) and the application of de-icing salts. Runoff, vehicle spray and dry deposition disperse these contaminants into the soft shoulder (verges) of the roads and surface water to a measurable distance of about 50 up to more then 150 m from the road. Despite many monitoring programs, little is known about the risks of this diffuse pollution for soil and water quality and the geochemical and physical factors which determine these risks. Also little is known about the effects of possible measures. Therefore, extensive research has been carried out at two local motorways. Specific measurements on runoff, vehicle spray and effects of measures have been carried out for one year (13 months). This resulted in several new insights. The pollutants appear to adsorb effectively to natural soils. In vulnerable areas groundwater can be protected by adjusting the policy to removing the contaminated upper topsoil of the verges. Discharges of runoff into local surface water are not recommended.

Increased polysialic acid neural cell adhesion molecule expression in human hippocampus of heroin addicts.

Chronic exposure to heroin is known to cause cognitive deficits. However, little is known about the underlying molecular mechanisms. It has been suggested that opiate-induced neurotoxicity as well as impaired plasticity and regeneration may be relevant. One of the target regions where regeneration still can be observed in the adult brain is the hippocampus. Since polysialic acid neural cell adhesion molecule is regarded as one of the key players involved in plasticity and regeneration of neural tissue, we analyzed polysialic acid neural cell adhesion molecule expression in the fascia dentate hilus of the human hippocampus of 29 lethally intoxicated heroin addicts and matched controls. Immunohistochemistry with an antibody directed against polysialic acid neural cell adhesion molecule revealed its expression in differently sized cells which could be identified as neurons and glial cells. We observed an increase in the percentage of polysialic acid neural cell adhesion molecule positive neurons in hippocampal hilus of heroin addicts compared with controls (P = 0.001).Interestingly, we also observed polysialic acid neural cell adhesion molecule expression in glial cells as evidenced by double immunofluorescence with glial fibrillary acidic protein and polysialic acid neural cell adhesion molecule using confocal laser scanning microscopy. The fraction of polysialic acid neural cell adhesion molecule positive glial cells was also higher in heroin addicts compared with controls (P = 0.009). In addition, within the group of addicts morphine blood concentrations showed a positive correlation with the percentage of polysialic acid neural cell adhesion molecule positive neurons (P = 0.04; r = 0.547). In conclusion, we observed an increase in polysialic acid neural cell adhesion molecule positive neurons and glial cells in hippocampi of heroin addicts. This might reflect an attempt to repair cell damage due to heroin exposure.

Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults.

Respiratory syncytial virus (RSV) accounts for the majority of respiratory virus infections, producing high mortality rates in immunocompromised patients with hematologic malignancies. The available methods for the rapid detection of RSV by antigen detection or PCR either lack sensitivity, require complex laboratory manipulation, or have not been evaluated in this patient population. To assess the applicability of a TaqMan-based real-time PCR technique for the detection of RSV A and B in immunocompromised adults, we developed a rapid, sensitive detection method that simultaneously detects RSV A and B and can be applied in routine diagnostics. The specificity of the assay was assessed using a panel of reference strains of other respiratory viruses and RSV. Electron microscopy-counted stocks of RSV A and B were used to develop a quantitative PCR format. Eleven copies of viral RNA could be detected for RSV A strain Long, and 14 copies could be detected for RSV B strain 9320, corresponding to 50% tissue culture infective doses of 0.86 and 0.34, respectively. The assay was evaluated on 411 combined nose and throat swabs derived from immunocompromised adults with or without signs of respiratory tract infection. The diagnostic efficacy of the TaqMan PCR determined on the clinical samples showed that this real-time PCR technique was substantially more sensitive than the combination of conventional viral culture and shell vial culture. None of the clinical specimens derived from patients without signs of respiratory illness were found to be positive for RSV by real-time TaqMan PCR.

Clinical diagnosis of influenza virus infection: evaluation of diagnostic tools in general practice.

BACKGROUND: With the development of new antiviral agents for influenza, the urge for rapid and reliable diagnosis of influenza becomes increasingly important. Respiratory virus infections are difficult to distinguish on clinical grounds. General practitioners (GPs) however, still depend on their clinical judgement. AIM: To evaluate the importance of clinical symptoms in the diagnosis of influenza virus infection. DESIGN OF STUDY: A multicentre questionnaire study. SETTING: Eighty-one patients from 14 general practices. METHOD: Patients with fever and at least one constitutional symptom and one respiratory symptom were included. A questionnaire with the medical history and clinical symptoms was completed and a combined nose-throat swab was taken. Virus culture, rapid culture, and polymerase chain reaction (PCR) amplification were performed on each specimen. Multivariate analysis was used to obtain the best predictive model. RESULTS: By using PCR, an increase was seen in the detection of the viral pathogens compared with the results of culture. In 42 out of 81 patients PCR was positive for influenza. A positive predictive value (PPV) of 75% was observed for the combination of headache at onset, feverishness at onset, cough, and vaccination status during the period of increase influenza activity. Criteria used by the ICHPPC-2 resulted in a PPV of 54%. The PPV for diagnosis made by the GP was 76%. CONCLUSION: Although influenza is difficult to diagnose on clinical grounds, the GPs in this study were able to diagnose influenza as such more accurately on their judgement than by the other criteria.

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR.

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE: It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS: Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS: The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION: This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy.

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription-PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500-15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Rabbit rectus femoris muscle for ischemia-reperfusion studies: an improved model.

The rabbit rectus femoris muscle was evaluated as a potential model for skeletal muscle reperfusion injury studies. Six white New Zealand rabbits were used. On one randomly selected hind limb, ischemia was induced by direct clamping of the rectus femoris muscle's vascular pedicle. On the other side, blood flow was interrupted by clamping the femoral artery above and below the origin of the vascular pedicle that supplies the rectus femoris muscle. The duration of normothermic ischemia was 4 hr and was followed by 24 hr of normothermic reperfusion. The interruption and restoration of blood flow was monitored using a laser flow meter. The rectus femoris muscles were weighed on a suspension spring balance prior to ischemia and at the end of reperfusion to estimate edema. The extent of muscle necrosis was determined using planimetry following staining with nitroblue tetrazolium. The muscle necrosis obtained by direct clamping of the vascular pedicle (66.9 +/- 14.3%) was significantly greater than that obtained by indirect clamping (18.6 +/- 11.4%) (P < 0.03 by t test). Unlike the indirect clamping technique, direct clamping achieved a good magnitude of muscle necrosis, thus allowing that specific model to be used in skeletal muscle reperfusion injury studies. The muscle weight gain observed in the direct clamping muscle group was 19.8 +/- 9.0% and was significantly greater than that observed in the opposite group being 6.3 +/- 6.5% (P < 0.05 by t test). The rabbit rectus femoris muscle is a suitable model for evaluating skeletal muscle reperfusion injury provided that direct clamping of the vascular pedicle is utilized.

Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay.

A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.