Development of a compounded propofol nanoemulsion using multiple non-invasive process analytical technologies.

Propofol is the preferred anaesthetic for induction and maintenance of sedation in critically ill mechanically ventilated COVID-19 patients. However, during the outbreak of the COVID-19 pandemic, regular supply chains could not keep up with the sudden increase in global demand, causing drug shortages. Propofol is formulated as an oil-in-water emulsion which is administered intravenously. This study explores the extemporaneous preparation of a propofol emulsion without specialized manufacturing equipment to temporally alleviate such shortages. A commercially available lipid emulsion (IVLE, SMOFlipid 20 %), intended for parenteral nutrition, was used to create a propofol loaded nanoemulsion via addition of liquid propofol drug substance and subsequent mixing. Critical quality attributes such as mean droplet size and the volume-weighted percentage of large-diameter (>5µm) droplets were studied. The evolution of droplet size and propofol distribution was monitored in situ and non-destructively, maintaining sterility, using Spatially Resolved Dynamic Light Scattering and Near Infrared Spectroscopy, respectively. Using response surface methodology, an optimum was found for a 4 % w/v propofol formulation with a ∼15 min mixing time in a flask shaker at a 40° shaking angle. This study shows that extemporaneous compounding is a viable option for emergency supply of propofol drug product during global drug shortages.

New unique PAT method and instrument for real-time inline size characterization of concentrated, flowing nanosuspensions.

With the rise of nanotherapeutics -and nano based products in general-, there has been an increasing need for better understanding and control of nano-particle (NP) synthesis and formulation processes. Size characteristics are often primary, if not critical, quality attributes of nanodispersions. Process Analytical Technology (PAT) tools for inline size characterization during dispersion processing are therefore highly desired. Traditional methods for NP sizing -based on Dynamic Light Scattering (DLS) - are typically ill-suited for direct inline application: (i) typical dispersion turbidities in process conditions often exceed by far the application limits for DLS (ii) agitation/flow typical for process conditions is incompatible with standard DLS and (iii) direct and convenient inline application requires a non-invasive PAT tool giving measurements on process relevant time scales. In this article we describe a new non-invasive PAT instrument - the NanoFlowSizer (patent pending)- which provides continuous, real-time, inline size and PSD characterization of concentrated and/or flowing nanodispersions in process environments. The instrument employs Fourier Domain low coherence interferometry, yielding path length resolved dynamic light scattering data of nanodispersions. Particle size characteristics can be analyzed from these data while effects of flow and/or multiple scattering are simultaneously characterized and accounted for. As first application examples we describe (i) real-time monitoring of NP size characteristics by remote measurement of mono and bi-disperse suspensions at different turbidities in a stirred beaker (ii) real-time monitoring of NP size characteristics using an online sampling loop with a micro-flow cell and (iii) real-time inline monitoring of size characteristics of a pharmaceutical nanoemulsion during industrial pilot scale nano-emulsification and for a pharmaceutical NP suspension during circulation, at flowrates ranging up to ~l/min.

Transfection by cationic gemini lipids and surfactants.

Diseases that are linked to defective genes or mutations can in principle be cured by gene therapy, in which damaged or absent genes are either repaired or replaced by new DNA in the nucleus of the cell. Related to this, disorders associated with elevated protein expression levels can be treated by RNA interference via the delivery of siRNA to the cytoplasm of cells. Polynucleotides can be brought into cells by viruses, but this is not without risk for the patient. Alternatively, DNA and RNA can be delivered by transfection, i.e. by non-viral vector systems such as cationic surfactants, which are also referred to as cationic lipids. In this review, recent progress on cationic lipids as transfection vectors will be discussed, with special emphasis on geminis, surfactants with 2 head groups and 2 tails connected by a spacer.

Graves' disease and celiac disease in a patient with myotonic dystrophy type 2.

We report a patient with progressive proximal muscle weakness in her legs, early-onset cataract and perceptive hearing loss, who was recently diagnosed with myotonic dystrophy type 2 (DM2). She also had two autoimmune disorders in her history, namely Graves' disease and celiac disease. Previous studies have shown a high frequency of autoimmune diseases (21%) in patients with DM2. This is the first report of a patient with DM2 and two autoimmune diseases which both have not yet been described in DM2. The cause of this association might be explained at DNA, mRNA and protein levels, including genetic mutation in flanking genes and the toxic effect of the DM2 mutation on proteins involved in inflammation. This case report widens the spectrum of autoimmune diseases in DM2 and has implications both for clinical practice and for research.

Symptomatic primary HIV infection in a 49-year-old man who has sex with men: beware of the window phase.

A 49-year-old man with a history of receptive unprotected anal intercourse with multiple anonymous men presented with a symptomatic primary HIV infection. Upon his initial visit the rapid HIV antibody screening test was negative but a p24 antigen test suggested a highly infectious phase in the HIV infection. An immunoblot assay confirmed the HIV diagnosis only 14 days later. Recent infections are characterised by a highly infectious phase and, if gone unnoticed, can have a large contribution to the ongoing transmission of HIV. Healthcare providers should be aware of primary HIV infection and the pitfalls in its diagnosis.

More learned irrelevance than perseveration errors in rule shifting in healthy subjects.

The present experiments examined the extent to which two possible sources of error affect healthy subjects' performance in a rule-shift task. All 115 participants first received a discrimination learning task, in which a pair of different visual stimuli was presented on each trial, one of which had to be identified as 'correct.' Each stimulus varied in two dimensions: a task-relevant and a task-irrelevant dimension. Feedback on correctness was given after each choice. After eight successive correct choices, the nature of the task-relevant dimension changed: the post-shift learning phase. Two types of error can occur in this phase: continued responding to the former relevant, but now irrelevant, dimension, a perseverative error, and non-responding to the former irrelevant, but now relevant, dimension, an error due to learned irrelevance. Different groups received a post-shift task in which none, one, or both of these two types of error could affect performance. The number of incorrect choices in the post-shift phase was significantly affected by learned-irrelevance errors but not by perseverative errors. An associative-learning model incorporating feedback-induced changes in both associative strength and saliency of the elements comprising the stimuli can explain these results.

Pigmented villonodular synovitis of the foot and ankle.

BACKGROUND: Pigmented villonodular synovitis (PVNS) is a rare disease of uncertain etiology usually affecting the synovium of weightbearing joints. METHODS: We retrospectively evaluated 11 patients who were diagnosed and treated for PVNS of the ankle and foot over a 13-year period with a minimum of 2-year followup. Four patients with ankle joint PVNS and one patient with PVNS of the fifth metatarsophalangeal joint were seen initially at our institution and were treated with surgery alone. Six patients with ankle joint PVNS were referred to our institution for recurrent PVNS lesions; two of these patients were treated with excision alone, and the other four patients had surgical excision followed by radiation therapy with dosages ranging from 3600-4000 cGy. RESULTS: No recurrence was noted at a mean followup of 9 years for primary lesions and 3.5 years for recurrent lesions. CONCLUSION: Based on these results, surgical excision of primary lesions and excision with postoperative radiation for recurrent lesions are recommended.

Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry.

The conformational stability of vancomycin group antibiotics (i.e., vancomycin and avoparcin) in aqueous solution has been studied. These complex glycopeptide antibiotics contain many chiral centers allowing the potential formation of stereoisomers. Using capillary electrophoresis these stereoisomers could be separated and detected by UV and/or mass spectrometry. Fresh aqueous samples of both vancomycin and avoparcin already contained a plethora of stereoisomers. Thermal degradation of the antibiotics was studied as well. For vancomycin thermal degradation led primarily to the formation of CDP-I and aglycons. In the case of avoparcin thermal degradation led mainly to the interconversion between stereoisomers. These antibiotic stereoisomers may exhibit different antibacterial efficacy. Solution-phase association constants of fresh and heated samples of these antibiotics and their bacterial cell wall mimicking receptors were determined by bioaffinity mass spectrometry and revealed that the heated samples exhibited, in general, a lower affinity. Minimum inhibitory concentrations (Micrococcus flavus) were determined and confirmed the decrease in antibacterial efficacy upon heating.

Sustained virological response in chronic hepatitis C patients after a 6- and a 36-month interferon-alpha2b treatment schedule: a multicenter, randomized, controlled study.

BACKGROUND: In patients with chronic hepatitis C (HCV) Interferon-alpha (IFN) treatment for 12-18 months is more effective than 6 months in inducing a sustained virological response. METHODS: In a multicenter, randomized, controlled trial, 88 patients with chronic HCV were enrolled (47 treated with IFN-alpha2b and 41 constituted an untreated control group). Treatment consisted of 5 million units (MU) IFN thrice a week (tiw) for 8 weeks and subsequently 2.5 MU IFN tiw for 16 weeks ('standard treatment'). After week 24 ('long-term treatment'), in virological non-responders treatment was continued using 5 MU IFN tiw for up to week 156, whereas in virological responders IFN was discontinued. In case of a virological relapse, treatment with 5 MU IFN tiw was restarted and continued up to week 156. RESULTS: Sustained virological response rate was 6/47 (13%) after standard treatment and increased to 19/47 (40%) after long-term treatment (McNemar paired test; P = 0.002). Of the 18 patients with a breakthrough or relapse during or after standard treatment, 14 (78%) became sustained virological responders upon long-term treatment. Of the 4 patients who did not have a sustained virological response after long-term treatment, 3 did not receive complete treatment due to side effects and/or non-compliance. In patients who failed to respond to standard treatment, no virological response was observed during long-term treatment. In the control group, no spontaneous clearance of HCV was observed. CONCLUSIONS: Long-term IFN (re)treatment enhanced the virological sustained response rate significantly and was particularly effective in patients with a breakthrough or relapse following standard treatment.

Characterization of the quantitative HCV NASBA assay.

A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.3) per 100 microl. The amplification efficiency was independent of the plasma matrix, but dependent on the HCV genotype. The HCV NASBA-QT assay was more than 10 times as sensitive as the bDNA assay while the quantitative results of both assays were highly concordant. The HCV NASBA-QT assay was comparable in sensitivity with the HCV MONITOR assay, but the HCV MONITOR assay yielded consistently lower values. It is concluded that the HCV NASBA-QT assay is a reliable assay for quantitative HCV-RNA detection in various settings.

Low levels of hepatitis C virus RNA in serum, plasma, and peripheral blood mononuclear cells of injecting drug users during long antibody-undetectable periods before seroconversion.

Screening of antibodies to hepatitis C virus (HCV) is widely used for monitoring the prevalence of HCV infections and to assess HCV infectivity. Among HCV-infected individuals in the general population, the interval between the detection of HCV RNA and the development of HCV antibodies is usually 5 to 6 weeks, but in rare cases, seroconversion may be prolonged up to 6 to 9 months. In this study, we tested for the presence of HCV RNA during the antibody-undetectable period of 19 drug-injecting HCV seroconverters to gain insight into the antibody-negative carrier status in this population. HCV seroconversion status was determined by testing the first and last serum samples obtained from each subject, using third-generation antibody screening and confirmation assays. Serial samples were tested for HCV-specific antibodies to establish the moment of seroconversion and HCV RNA by single reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA assay (bDNA) in serum. Plasma and peripheral blood mononuclear cells (PBMCs) were independently collected and tested for HCV RNA. HCV RNA-positivity was confirmed by Southern blot hybridization and sequencing of serial samples. The 19 HCV seroconverters had a mean follow-up of 5 years (range, 1 to 8 years). Of the 19, 4 were human immunodeficiency virus (HIV)-infected before HCV seroconversion. HCV RNA was detected in serum before seroconversion in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status. In 7 of these 12, the antibody-undetectable period was relatively short (2 to 10 months). The other 5, who were all HIV-negative before HCV seroconversion, had intermittent low levels of HCV RNA before seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent repeats of the experiments confirmed the presence of HCV RNA in serum, and in 3 of these individuals, HCV-positivity was confirmed in independently collected plasma and PBMC samples. Low levels of HCV RNA may be present during prolonged antibody-undetectable periods before seroconversion in a number of injecting drug users. Independent of HIV status, their immune system appears to be unable to respond to these low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV infection may not always elicit the rapid emergence of HCV antibodies and suggests that persistent low levels of HCV RNA (regardless of the genotype) may not elicit at all or delay antibody responses for prolonged periods of time.

Hepatitis G virus RNA and hepatitis G virus-E2 antibodies in Dutch hemophilia patients in relation to transfusion history.

The prevalence of hepatitis G virus (HGV)-RNA and HGV-E2 antibodies was studied in a cohort of Dutch hemophilia patients in relation to clotting products used, age, and coinfection with hepatitis C. Between 1991 and 1995, blood samples were taken from 294 patients with hemophilia A, B, or von Willebrand disease. From each patient one fresh frozen sample was tested for HGV cDNA polymerase chain reaction (PCR) and HCV cDNA PCR. Alanine aminotransferase (ALT) tests were performed on plasma samples of all patients. The presence of HGV-E2 antibodies was tested on plasma samples from a subset of 169 patients representing all age groups. Based on the origin and viral safety of the products used, three subgroups of patients were distinguished. Group A: patients who used viral noninactivated factors derived from small and large donor pools; group B: patients who used factors prepared with inadequate viral inactivation techniques derived from small and large donor pools; and group C: patients treated only with optimally viral inactivated large pool clotting factor or recombinant clotting factor concentrate. The prevalence of HGV-RNA was 18%. In group A patients the prevalence was 71%, in group B 50%, and in group C 6%. When related to age, the highest prevalence of HGV-RNA (35%) was seen in patients born between 1980 and 1989. The prevalence of HGV-E2 antibodies increased with age. Of HGV-RNA-negative patients born before 1950, 96% tested positive. HGV viremia did not affect ALT levels, neither in HCV-RNA positive nor in HCV-RNA negative patients. HGV infection is frequently seen in patients with hemophilia. In older age groups a lower rate of HGV-RNA positivity is seen coinciding with a higher rate of antienvelope antibodies.

Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification.

The influence of different anticoagulants and pre-amplification storage conditions on the stability of hepatitis C virus (HCV)-RNA, as detected by the quantitative HCV NASBA assay (NASBA-QT), was studied. The HCV-RNA load remained stable for at least 15 months when serum or plasma samples (EDTA and heparin) were directly frozen at -70 degrees C in lysis buffer. At 4 degrees C, the HCV-RNA load in serum or plasma stored with lysis buffer did not decline for at least 14 days. At 30 degrees C, however, the load declined significantly after 7 days. When clotted, whole blood was stored at 4 degrees C, the HCV-RNA load was stable for 72 h. However, when EDTA-anticoagulated whole blood was stored at 4 degrees C, the HCV-RNA load declined significantly after 48 h. In paired plasma and serum samples at baseline the HCV-RNA levels were similar. Heparin did not influence the efficiency of the HCV NASBA-QT assay. Clotted blood as well as EDTA or heparin anticoagulated blood can be used for quantifying HCV-RNA using the NASBA-QT assay. Blood samples should be stored at 4 degrees C after collection and serum or plasma separated within 24 h. Preferably, after separation, samples should be frozen in lysis buffer at -70 degrees C until NASBA-QT analysis.

Metastatic melanoma of gastrocolic ligament treated with minimally invasive approach.

A patient with metastatic melanoma of the gastrocolic ligament was rendered clinically tumor free using a minimally invasive procedure. The technique and the rationale for the procedure are reviewed.

Formation of antibodies to factor VIII in patients with hemophilia A who are treated with interferon for chronic hepatitis C.

OBJECTIVE: To evaluate the risk for development of antibodies to factor VIII (factor VIII inhibitors) during and after interferon therapy in patients with hemophilia A and chronic hepatitis C infection. DESIGN: Patients were divided into two treatment groups and an untreated control group. Test results from the two treatment groups were compared with those from the control group. SETTING: 3 clinical centers in the Netherlands. PATIENTS: 35 men with hemophilia A who had acquired hepatitis C through the use of plasma-derived clotting factor concentrates. MEASUREMENTS: Patients were tested for factor VIII inhibitors before the start of interferon therapy and every 6 months thereafter. RESULTS: 21 patients with hemophilia A received interferon therapy for chronic hepatitis C infection for a mean of 19.5 months (range, 0.5 to 36 months). In 2 patients, inhibitors were detected on one occasion (maximum titer, 1.2 Bethesda units/mL) during interferon therapy. In 3 patients who were known to have had inhibitors before interferon therapy, no anamnestic reaction was seen during treatment. In 3 of 14 untreated controls who were followed for a mean of 28 months (range, 18 to 40 months), inhibitors were detected on one occasion (maximum titer, 2.3 Bethesda units/mL). CONCLUSION: Long-term interferon therapy in patients with hemophilia did not increase the risk for development of factor VIII inhibitors.

International collaborative study on the second EUROHEP HCV-RNA reference panel.

Eighty-six laboratories participated in a collaborative study and tested the second EUROHEP HCV-RNA reference panel. The coded panel comprised 4 HCV-RNA positive plasma samples (one weak positive), 6 HCV-RNA negative plasma samples and two dilution series of HCV-RNA genotype 1 and 3 plasma standards. The 86 laboratories submitted 136 coded data forms for evaluation. Of these data sets 99 were tested using a PCR assay developed in-house, 28 using a commercially available HCV-PCR test (AMPLICOR, Roche Diagnostic Systems) and 9 using other amplification methods. Twenty-two data forms (16%) had faultless results, 39 (29%) missed the weak positive sample only and 75 data sets (55%) had false positive and/or false negative results. Participants using the commercial HCV-PCR test tended to reach a sufficient quality score more often than investigators using assays developed in-house (64% versus 45%, P = 0.11). The UNG system in the commercial HCV-PCR test did not prevent five laboratories generating false-positive results in the 6 HCV-RNA negative samples. Among the laboratories with satisfactory results, up to 10000-fold differences in sensitivity were observed in the dilution series. The 50% and 90% laboratories detection endpoints in the dilution series of the HCV genotype 1 plasma standard were approximately 600 genome equivalents per ml (geq/ml) and 7750 geq/ml according to a standard applied in a signal amplification assay (bDNA, Chiron). Our results suggest that the detection efficiency for genotype 3 by commercial HCV-RNA assays is lower than by the in-house assays. Internationally characterized HCV-RNA plasma standards should be made available for validation and standardization of HCV-RNA assays for HCV diagnosis and virological safety testing of blood products.

Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus.

BACKGROUND: In a confirmatory laboratory, the second-generation recombinant immunoblot assay (RIBA-2) was replaced by the third-generation RIBA (RIBA-3) in March 1993. The aim of this validation study was to compare the sensitivity and specificity of RIBA-2 and RIBA-3 in a routine setting, by using a validated hepatitis C virus (HCV) RNA polymerase chain reaction to establish plasma viremia. STUDY DESIGN AND METHODS: RIBA-2 testing was performed (March 1991-March 1993) in 593 HCV RNA-positive and 1498 HCV RNA-negative subjects. RIBA-3 testing was performed (March 1993-May 1994) in 220 HCV RNA-positive and 530 HCV RNA-negative subjects. All samples reacted for anti-HCV in enzyme-linked immunosorbent assay. RESULTS: In HCV RNA-positive individuals, the sensitivity of RIBA-3 was significantly higher than that of RIBA-2 (99.5% vs. 93.3%, p = 0.0005). This was not caused by inclusion of the NS5 antigen, but by a higher sensitivity of the antigens c33 and c100 (RIBA-2: 94.3% and 62.6%; RIBA-3: 99.5% and 88.6%). Replacement of the c22 and c100 recombinant proteins by synthetic peptides significantly reduced nonspecific reactivity against these antigens (p < 0.0001). Unfortunately, increased nonspecific reactivity against the modified c33 antigen and the new NS5 antigen canceled out this effect. Two-band reactivity occurred more often in nonviremic persons than in viremic persons (32.7% vs. 8.2%, p < 0.0001). Risk factors for HCV infection were less frequently observed in 11 blood donors with two-band reactivity than in 6 blood donors with other positive RIBA-3 patterns (18% vs. 83%, p = 0.03). CONCLUSION: The higher sensitivity of RIBA-3 significantly reduced the number of indeterminate test results in HCV RNA-positive persons. Confirmatory laboratories must be aware of the frequent occurrence of nonspecific, isolated reactivity and even nonspecific, two-band reactivity in anti-HCV enzyme-linked immunosorbent assay-reactive blood donors.