Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC.

An LTR-based quantitative PCR (qPCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and PBMC. TaqMan qPCR primers and probes were designed against the NCBI/LANL HIV-1 compendium database by analyzing sequences used in assays for sensitive cross-clade detection of HIV-1 as reported in the literature and elucidating regions of improved cross-subtype specificity. Inosine and mixed nucleotide bases were included at polymorphic sites. Real-time RT-qPCR and qPCR were performed on plasma viral RNA and cellular lysates. A step-up amplification approach to allow binding of primers across polymorphic regions showed improved sensitivity compared to universal cycling. Unlike a lead competing laboratory-developed assay, all major HIV-1 subtypes, and a wide range of recombinants from a 127-member diversity panel were detected and accurately quantified in spiked plasmas. Semi-nested PCR increased detection sensitivity even further. The assay was able to detect down to 88 copies/mL of HIV-1 in plasma with 95% efficiency or the equivalent of a single infected cell. The PCR assay will be valuable in studies that monitor very low viral levels including residual or break through HIV-1 in patients receiving antiretroviral therapy, in HIV-1 cure, and in other research studies.

Anaesthetic implications of the changing management of patients with mucopolysaccharidosis.

The mucopolysaccharidoses are a group of inherited metabolic disorders that are renowned for presenting clinical problems, particularly related to cardiac, airway, and skeletal abnormalities, in children during anaesthesia. The changing clinical management of the mucopolysaccharidoses can be described in three phases. An initial phase of accumulation and dissemination of knowledge about the management of this rare disease with a growing recognition that untreated Hurler syndrome and more severe forms of other phenotypes such as Hunter syndrome and Maroteaux-Lamy syndrome were associated with severe complications under anaesthesia. This was followed by a second phase reflecting the beneficial results of new treatments such as haemopoietic stem cell transplantation and enzyme replacement therapy. Early and successful transplantation has dramatically improved long-term outcome and reduced anaesthetic complications in children with Hurler syndrome. Enzyme replacement therapy is available for many forms of mucopolysaccharidosis. If commenced at an early age improvement in many organ systems may be observed with an improved quality of life. However, these current treatment regimens do not appear to improve neurocognitive dysfunction, or cardiac valvular or skeletal abnormalities. We are now entering a third phase where the partial benefits of these treatment regimens are resulting in an increasing number of older patients with partially corrected abnormalities, including difficult airways, presenting for ongoing treatment to a new and potentially unsuspecting group of clinicians. Major airway abnormalities may be encountered and current adult guidelines may need to be adapted. A multidisciplinary team approach involving paediatric and adult anaesthetists is recommended to optimise future management.

[Screening for colorectal cancer: a cost benefit analysis on a health prevention programme at the Boehringer Ingelheim Company]. / Darmkrebsscreening im betrieblichen Umfeld: Kosten-Nutzen-Analyse einer Maßnahme zur Gesundheitsförderung bei Boehringer Ingelheim.

BACKGROUND AND OBJECTIVE: In Germany, approximately 70.000 people are diagnosed with colorectal cancer every year. With early diagnosis the recovery rates are over 90 % and early intervention can significantly reduce the costs of medical treatment as well as the economic losses from worker productivity. We here present the organisational procedure for bowel cancer screening and have weighed the costs against benefits to employees, the company and the healthcare system. The screening costs are compared with economic benefits. METHODS: The target group for the study consisted of all 11.536 employees at the company's site in Germany. Volunteers were given a standardized questionnaire about the risk factors for colorectal cancers and an immunological fecal occult blood test (IFOBT). If risk factors for development of colorectal cancer were present or if the test result was positive, a colonoscopy was recommended in accordance with DGVS guidelines (German Society of Digestive and Metabolic diseases). RESULTS: A total of 4.287 employees (37.2 %) indicated an interest in undergoing screening; at the end of the period 3.958 complete datasets (2.296 men and 1.662 women, mean age 51.2 years) were available for evaluation. A colonoscopy was performed on 114 persons. Six cases of overt cancer were detected with three in the 36 - 50 age group and three in the 51 - 65 age group. Five of the six cases were stage T1 or T2. Adenomatous polyps were found and removed in 29 persons. The calculated cost benefit ratio was 1:2 for the company and 1:35 for the public health system. CONCLUSION: Using the example of colorectal screening, this study represents a cost benefit analysis of this preventative health measure in a company environment. The results show that even while taking into account the financial and personal commitment required, the cost benefit ratio is positive both for the company and for the healthcare system.

The anaesthetic management of children with anterior mediastinal masses.

Children with anterior mediastinal masses may experience serious complications during general anaesthesia. We retrospectively surveyed the records of children with an anterior mediastinal mass who had been admitted to our hospital over a 7 year period. The presence of pre-operative symptoms or signs, findings of any special investigations performed and the anaesthetic outcome were noted. All radiological investigations were studied and tracheal compression measured. The majority of patients presented with severe clinical signs. There was a poor relationship between clinical signs and size of tumour or tracheal compression on CT scan. Corticosteroids were used prior to diagnosis in 33% of patients, all of whom were considered high risk. A clear diagnosis was made in 95% of these patients. The overall complication rate was 20% and 5% of patients had a serious complication related to anaesthesia. Stridor was the only sign that predicted an anaesthetic complication. Peri-operative respiratory complications were confined to patients with an isolated tracheal cross-sectional area less than 30% normal or less than 70% and associated with bronchial compression.

Rotigotine transdermal system for perioperative administration.

We present a series of patients participating in clinical trials with the rotigotine transdermal system. All patients were scheduled for surgery with general anaesthesia unrelated to the trial procedure or to rotigotine. Perioperative administration of rotigotine appeared to be feasible and efficacious. No safety issues emerged from these observations.

Restriction in the repertoire of the immunoglobulin light chain subgroup in pathological cold agglutinins with anti-Pr specificity.

BACKGROUND AND OBJECTIVES: In cold agglutinin disease, monoclonal red blood cell autoantibodies, termed cold agglutinins, induce haemolysis in patients exposed to the cold. Commonly, these autoantibodies are directed against the developmentally regulated I/i blood groups. A second blood group system, the Pr system (located on glycophorins), is involved less frequently. Anti-Pr cold agglutinins recognize either alpha 2,3- or alpha 2,6-linked N-acetylneuraminic acid as the immunodominant group. Cold agglutinins of anti-I/i specificity show a remarkable restriction in their genomic repertoire of the immunoglobulin heavy and light-chain immunoglobulin-variable domain (i.e. exclusive use of VH4-34 in heavy chains). For anti-Pr cold agglutinins, preliminary data on the repertoire of the light-chain variable domain indicate a preference for the subgroup Vkappa IV. To elucidate restrictions in the light-chain variable-domain subgroup repertoire of anti-Pr cold agglutinins systematically, and to discuss these results in the context of their anti-Pr(1-3) subclassification and immunodominant sialic acid, light chains in 13 anti-Pr cold agglutinins were investigated. MATERIALS AND METHODS: The anti-Pr light chains were isolated using temperature-dependent absorption/elution techniques. Subsequently, they were subjected to N-terminal Edman degradation, and the light chain Vkappa subgroup was affiliated using the Kabat database. RESULTS: Five of 13 (38%) light chains belonged to Vkappa IV, five of 13 (38%) to Vkappa I and three of 13 (23%) to Vkappa III. Anti-Pr with Vkappa IV subgroup light chains exclusively recognized alpha 2,3-linked N-acetylneuraminic acid. CONCLUSIONS: Including data from the literature, the repertoire of the light-chain variable domain in pathological anti-Pr cold agglutinins exhibits a clear bias towards the use of the single germline gene-derived subgroup, Vkappa IV (eight of 17 or 47%). The association of Vkappa IV subgroup light chain-containing anti-Pr cold agglutinins with binding to alpha 2,3-, but not alpha 2,6-linked N-acetyneuraminic acid raises speculations about a possible role of subgroup-derived determinants in anti-Pr binding.

[Medical counseling of public health-insurances in questions of occupational diseases in regard to economical benefits--project of cooperation between AOK Hesse and the Medical Advisory and Expertising Service Hesse]. / Okonomischer Nutzen der Beratung gesetzlicher Krankenkassen in Berufskrankheitenfragen.

Since September 1998 exists a project of cooperation and consultation between the AOK Hesse and the Medical Advisory and Expertising Service Hesse with the aim to identify occupational diseases and to survey decisions of the Employer's Liability Insurance Association. The procedure is based on a computer-added recognition-system, a profound preparation of the single cases by the employees of the health-insurance and a very intensively carried out deliberation by Medical Doctors of occupational medicine. In a period of four and a half year 8391 cases have been reviewed of which 4859 have already been determined. An approval as occupational disease by the Employer's Liability Insurance Association has been determined in 1954 cases, in 2905 cases the acknowledgement has not been determined. Regarding the determined cases a recourse of 10,078,922.27 EUR has been realized. In regard to the invested small resources of personnel the procedure has proved itself as highly effective to discover and to assert recourses. Beside the economical aspects for the public health-insurance, other results of the project were the assurance of the entitlement to benefits of people coming down with occupational diseases or their relatives. New insights about the actual development of occupational diseases in Germany als well as their prevention can be proceeded.

Plasma concentrations of bupivacaine after combined spinal epidural anaesthesia in infants and neonates.

The unbound and bound plasma concentration of bupivacaine in 50 infants less than 55 weeks postconceptual age was determined following combined spinal and epidural anaesthesia (csea). Plasma concentrations were determined at 15-min intervals up to 60 min postspinal anaesthesia. Maximum plasma bupivacaine levels were recorded between 45 and 60 min post CseA. Total plasma concentrations above a toxic threshold level of 4 microg.ml(-1) were recorded in 4% of patients and above 2.5 microg.ml(-1) in 10% of patients. Unbound bupivacaine levels were greater than a presumed toxic level of 0.25 microg.ml(-1) in 16% of cases and above 0.3 microg. ml(-1) in 14% of cases. A wide range of protein binding was measured (varying from 53.8-98.2%) and could not be correlated with standard indicators of local anaesthetic binding. Two neonates had brief apnoeas in the immediate perioperative phase but no adverse cardiac or central nervous system events attributable to the performance of Csea were demonstrated.

The perioperative management of children with phaeochromocytoma.

The safe anaesthetic management of a child with a phaeochromocytoma requires an understanding of the pathophysiology of the disease, together with a thorough knowledge of its pharmacology, in order to avoid or minimize the potentially harmful cardiovascular changes that may occur during anaesthesia. Although there is a considerable amount of information on the management of the adult with phaeochromocytoma, much less has been written concerning children with the disease. Children differ significantly from adults in the incidence, location, presentation and management of this condition and these differences are discussed here together with some of the more controversial issues of management.

A comparison of rectal and intramuscular codeine phosphate in children following neurosurgery.

Codeine is frequently used for postoperative analgesia in children. Intramuscular injections are not ideal and the rectal route may be preferable. We compared rectal and intramuscular codeine administered following neurosurgery. 20 children (over 3 months) undergoing elective neurosurgical procedures, were randomized to receive either rectal or intramuscular codeine phospate (1 mg.kg-1) at the end of the procedure. Serum levels of codeine and morphine were assayed at intervals following administration (0, 30, 60, 120, 240 min). Fentanyl was the intraoperative analgesic and postoperative rescue analgesia was paracetamol, diclofenac and intramuscular codeine. The Children's Hospital of Eastern Ontario Pain Scale was used to assess analgesia. Peak codeine levels in both groups were observed at 30 min and morphine levels were consistently low. The plasma codeine levels were significantly greater at 30 and 60 min following intramuscular injection, and were associated with slightly better analgesia scores, but did not reach statistical significance. However, the peak plasma level occurred at similar times in both groups. Codeine is absorbed as rapidly via the rectal route compared with the intramuscular route but the peak levels are lower.

Serum affinity chromatography for the detection of IgG alloantibodies in a patient with high-titer IgM cold agglutinins.

BACKGROUND AND OBJECTIVES: High-titer cold autoagglutinins (CA) interfere with the detection of alloantibodies against red-blood-cell antigens. The diagnostic procedure is extremely difficult, especially under pressure of time. It was the purpose of the study to find out whether quantitative IgG purification from serum or plasma by affinity chromatography facilitates the detection of IgG alloantibodies in the presence of high-titer IgM CA. MATERIALS AND METHODS: Serum: IgM-chi CA, 0 degrees C titer = 65,000; five anti-IgG reactive alloantibodies. Affinity chromatography: HiTrap protein G 1-ml affinity column (Pharmacia Biotech, Uppsala, Sweden). RESULTS: All IgG alloantibodies were detectable in the eluate after affinity chromatography without interference of IgM CA. CONCLUSION: IgG alloantibodies in the presence of high-titer IgM CA can be easily and rapidly detected under routine conditions by quantitative IgG purification from serum or plasma by affinity chromatography.

Alpha 2,3-specific desialylation of human red cells: effect on the autoantigens of the Pr, Sa and Sia-l1, -b1, -lb1 series.

BACKGROUND AND OBJECTIVES: Pr1,2,3, PrM, Sa and Sia-l1, -b1, -lb1 are sialic acid (NeuNAc)-dependent antigens recognized by human cold agglutinins. Pr and Sa antigens are the O-glycans of glycophorins containing alpha 2,3NeuNAc (to galactose) and/or alpha 2,6NeuNAc (to galactosamine). The antigens of the Sia-l1, -b1, -lb1 complex are gangliosides that may carry alpha 2,3NeuNAc (to galactose) and/or alpha 2,8NeuNAc (to NeuNAc). We studied the NeuNAc groups involved in the antigens. MATERIALS AND METHODS: From 74 sera with cold agglutinins against NeuNAc-dependent antigens, anti-T-free preparations were made and tested against human red cells, treated with an alpha 2,3-specific recombinant sialidase. RESULTS: Most (51/62) Pr antigens use alpha 2,3NeuNAc, some (8/62) use alpha 2,6NeuNAc and a few (3/62) use both sialyl groups as immunodominant components on glycophorins. The immunodominant component of Sa and Sia-l1, -b1, -lb1 determinants was alpha 2,3NeuNAc in all cases. CONCLUSION: The red cell target structures for cold agglutinins against NeuNAc-dependent antigens have been identified. We propose a Pr nomenclature to reflect this. The binding of anti-Pr to gangliosides may be the basis for anti-Pr-induced peripheral neuropathy.

Identification of IgG alloantibodies in patients with high-titer IgM cold agglutinins by serum/plasma affinity chromatography.

The detection of IgG alloantibodies in the presence of high-titer cold autoagglutinins (CAs) can be extremely difficult, especially under pressure of time when transfusion of red blood cells is urgently needed. Here we demonstrate that IgG alloantibodies in the presence of high-titer IgM CAs can be easily detected by quantitative IgG purification from serum or plasma by affinity chromatography. In comparison with the routinely used methods for IgG alloantibody identification, affinity chromatography shows better or identical results and is the method leading to results most rapidly.