Detection of influenza A(H1N1)v virus by real-time RT-PCR.

Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic.

Investigation of the HotDog patient warming system: detection of thermal gradients.

OBJECTIVE: To assess the performance of an active patient-warming device. MATERIALS AND METHODS: Temperatures of an active patient-warming device (HotDog system) were measured at various time points using an infrared thermometer. The study was conducted in two phases: Phase 1 compared temperatures among four different areas of the warming blanket. Phase 2 compared conditions simulating different scenarios using a weighted patient simulator. RESULTS: Phase 1: Three out of four positions on the warming blanket had significantly different temperature measurements. Phase 2: Temperature output by the warming blanket was reduced: (1) in the absence of the patient simulator placed across the blanket (-1·9°C, P=0·013); (2) if the patient simulator was placed away from the blanket sensor (-2·0°C, P=0·009); and (3) if there was fluid between the patient simulator and warming blanket (-2·2°C, P=0·004). In a majority of measurements (95%), the set temperature of 43°C on the control unit was not reached (range, 29·8 to 42·9°C) and 2·3% of measurements were higher (range, 43·1 to 45·8°C) than the control unit set temperature of 43°C. CLINICAL SIGNIFICANCE: Measured temperatures on the active warming blanket did not reflect control unit settings. This could result in the potential for hyperthermic injury, ineffectual heating and uneven heat distribution.

Two cases of severe obstructive pneumonia associated with an HKU1-like coronavirus.

BACKGROUND: During the last few years a number of previously undescribed viruses, including human metapneumovirus, coronaviruses SARS, NL63 and HKU1, and bocavirus, were identified in nasopharyngeal samples from patients with signs of respiratory infections. These viruses may cause mild to life-threatening infections. OBJECTIVES: Nasopharyngeal samples from hospitalized pediatric patients with respiratory disease were analysed for the presence of coronaviruses and other well known and newly identified respiratory viruses. RESULTS: Two clinical cases of a severe obstructive pneumonia, which were associated with the presence of RNA of a novel variant (subtype) of HKU1 coronavirus in the nasopharyngeal aspirates, were identified. DISCUSSION: The detection of a HKU1-like coronavirus in pediatric patients in the current study complement the most recent independent finding of similar or closely related coronaviruses in patients with respiratory diseases in France (Vabret et al. 2006) and Norway (Jonassen et al., see accompanying manuscript). These observations indicate a wide dissemination of HKU1-like coronaviruses in Europe.

Antigen-specific cytokine response to hepatitis C virus core epitopes in HIV/hepatitis C virus-coinfected patients.

OBJECTIVE: Epidemiological data indicate that hepatitis C virus (HCV) infection runs a more rapid and severe course of disease in HIV-coinfected patients, probably because of an altered immune response. DESIGN: We investigated whether HCV-specific cytokine responses are affected by HIV coinfection. METHODS: Using triple colour flow cytometry on peripheral blood lymphocytes after stimulation with the four major immunodominant HCV core T cell epitopes, CT1-CT4, we determined intracytoplasmic production of IFN-gamma, IL-2, IL-4, IL-10 and CD30 expression, a putative surrogate marker of type 2 cells. Fifteen patients with asymptomatic HIV/HCV coinfection (group A), 15 patients with chronic HCV infection (group B) and 10 HIV-infected patients without hepatitis C (group C) were included in the study. RESULTS: In group A, HCV antigens induced significantly higher IL-2 and IFN-gamma production than groups B and C (P < 0.05). Groups A and B showed a similar induction of CD30, which was significantly higher than in group C (P < 0.001). Remarkably, in group A HCV antigens induced IL-4 production in addition to IL-10 and IFN-gamma in the CD30 subset, whereas in groups B and C no IL-4 induction was observed in this T cell subset (P < 0.002). CONCLUSION: Our data suggest that asymptomatic HIV coinfection importantly alters the HCV-specific cytokine response towards a greater production of proinflammatory type 1 cytokines. Moreover, the antiviral activity of type 1 cytokines may be modified by an increased production of type 2 cytokines in the CD30 subset. The altered cytokine pattern may contribute to the adverse natural course of hepatitis C in HIV coinfection.

Protection against parenteral HIV-1 infection by homozygous deletion in the C-C chemokine receptor 5 gene.

OBJECTIVES: To investigate the role of the CC chemokine receptor 5 (CCR5) for parenteral transmission of HIV-1. DESIGN: The prevalence of the delta32 deletion within the CCR5 gene was determined in a cohort of 207 patients, who had received documented amounts of non-antibody-tested and non-inactivated clotting factor concentrate. METHODS: Chromosomal DNA of haemophiliacs was isolated from whole blood. A portion of the CCR5 gene spanning the delta32 deletion was amplified by PCR. The resulting DNA fragments were analysed by agarose gel electrophoresis. RESULTS: The rate of HIV-1 infection was correlated strongly with increasing amounts of inoculated clotting factor concentrate. None of the HIV-positive patients (n = 129) had the delta32/delta32 genotype, whereas 12 out of 78 HIV-negative haemophiliacs had the homozygous delta32 deletion. CONCLUSIONS: The delta32/delta32 genotype was highly protective against HIV-1 infection, even in patients who had received millions of non-inactivated clotting factor units. As it is likely that in the early 1980s plasma pools were contaminated not only with monocyte-tropic HIV-1 strains, CCR5 appears to be the major mediator of HIV-1 infection. Furthermore, we conclude that there must be other protective mechanisms in multiply exposed non-infected haemophiliacs who have wild-type CCR5.

Parallel evolution in the V3 region of HIV type 1 after infection of hemophiliacs from a homogeneous source.

To determine the genetic diversification in the highly functional V3 loop, we followed up five hemophiliacs who were infected by a homogeneous HIV-1 population from a contaminated clotting factor lot. Initially, all patients displayed identical DNA sequences in this part of the proviral env gene. Therefore, this unique outbreak allows us to investigate the biological and genetic development of a common ancestor virus in different patients. A high degree of homology is maintained in the predominant sequences from 5 until 35 months after seroconversion. Only one patient showed a remarkable diversification 3 years postinfection. However, these genetic changes in the V3 region were not associated with disease progression. Discontinuous sequence changes were observed mainly in a region downstream of the V3 loop. Two positions in particular are involved in a sequence evolution within the V3 loop leading to the same amino acids in different patients. These directed changes occurred at sites that are reported to be critical for the specificity of antibodies (position 308) and viral cytopathicity (position 324). However, the parallel evolution was associated neither with differentiation of the viral phenotype nor with progression of the disease.

The genetic diversification of the HIV type 1 gag p17 gene in patients infected from a common source.

An evolutionary analysis was undertaken of HIV-1 gag p17 sequences taken from a small cohort of hemophilia B patients infected from a common batch of clotting factor concentrate. The sequence population found at seroconversion was highly homogeneous, suggesting that the infecting batch also contained little sequence variation. Genetic diversification was found in follow-up sequences taken approximately 3 years later and was generally found to be complex. Greater rates of synonymous to nonsynonymous substitution were observed, especially when comparing distantly related isolates, and the rate of synonymous substitution was used to estimate times of divergence for a number of isolates of HIV-1 including the origin of the subtypes A to H. The p17 region is therefore proposed as a useful marker for future epidemiological studies.

Transformation of rodent fibroblasts by ICP4, the major transactivating protein of herpes simplex virus type 1.

Rat fibroblasts were transfected with a plasmid containing the IE3 gene derived from the temperature sensitive HSV-1 mutant tsK. Three of four clones expressing biologically active, temperature-sensitive ICP4, formed a substantial number of colonies in soft agar at the permissive temperature. During the first passages of cells, the transformed state of the major proportion of transformed cells was dependent on the continuous activity of ICP4. In a smaller and distinct subpopulation of transformed cells, as well as after longer subcloning of cells, ICP4 was no longer required for the maintenance of the transformed state, pointing to the induction of stable genomic changes by ICP4. Our data show that ICP4 of HSV-1 is involved in transformation of fibroblasts. Transformed cells are, however, subject to intracellular and intercellular control mechanisms.

Detection of herpes simplex virus and adenovirus DNA by dot blot hybridization using in vitro synthesized RNA transcripts.

A new diagnostic assay was developed to detect herpes simplex virus (HSV) and adenovirus DNA in clinical specimens using in vitro synthesized radioactively labelled RNA transcripts from virus-specific DNA fragments cloned in transcription vector pSP 64/65. RNA probes derived from HSV-I-Eco-RI-G-DNA fragment show a sensitivity of less than 3 pg of whole plasmid DNA and hybridize only with DNA of HSV I and II, but not with other viral or cellular DNA. The analysis of 15 clinical specimens showed concordance with virus isolation, except for two culture-negative samples of cerebrospinal fluid of patients with suspected HSV encephalitis, which was confirmed by serology as well as by hybridization. Using RNA transcripts from adenovirus-2-Hind-III-D-DNA fragment, we attained a sensitivity of less than 3 pg of whole plasmid DNA. This probe detected different types of adenovirus, but failed to hybridize to other viral, bacterial or cellular DNA. Compared with the cell culture method this assay did not show any false-positive or false-negative results in 16 different clinical specimens. The technique is sensitive, specific and useful for screening clinical specimens and may be helpful in confirming the diagnosis of HSV encephalitis.

Recurrent herpetic keratitis during topical acyclovir application.

Dendritic herpetic keratitis developed in a 49-year-old patient during topical acyclovir treatment. A positive herpes simplex culture was obtained. After acyclovir was replaced by trifluorothymidine and interferon, the dendritic lesion disappeared and herpes simplex culture became negative. Six months later a carcinoma of the larynx was diagnosed. The acyclovir-resistant herpetic keratitis may be associated with the carcinoma because resistant herpes simplex virus strains are predominantly described in patients suffering from immune deficiency.

[Gitelman syndrome in children: true hypokalemia but false Bartter syndrome]. / Syndrome de Gitelman chez l'enfant: vraie hypokaliémie mais faux syndrome de Bartter.

BACKGROUND: Gitelman's syndrome or familial hypokalemia-hypomagnesemia and Bartter syndrome share some common features but their prognosis is quite different. CASE REPORT: Four unrelated children, aged 5 to 12 years, were studied because they suffered from muscle cramps and/or abdominal pain. Supportive findings included: hypokalemia (2.1 to 2.9 mmol/l), metabolic alkalosis (31 to 34 mmol/l), hyperkaliuresis (5.8 to 7.1 mmol/kg/day), hypomagnesemia (0.58 to 0.64 mmol/l), hypermagnesuria (0.19 to 0.23 mmol/kg/day), hypocalciuria (0.012 to 0.021 mmol/kg/day). Blood pressure contrasting with high renin activity (19.04 to 20.03 ng/ml/hr) was normal. Chloride fractional excretion after oral water supplementation was only slighty decreased and hypercalciuric response to furosemide administration was not observed. Supplementation with magnesium chloride failed to correct hypomagnesemia while potassium chloride improved hypokalemia. CONCLUSIONS: Age of onset, tetany manifestations, absence of growth retardation, hypermagnesuria despite, hypomagnesemia, hypocalciuria not improved by furosemide favor the diagnosis of Gitelman's syndrome rather than that of Bartter syndrome initially considered.

[Evaluation of electromyographic recordings in the detection of Duchenne's dystrophy carriers]. / Ocena wartosci automatycznego zapisu elektromiograficznego dla wykrywania nosicielstwa dystrofii Duchenne'a.

The purpose of this work was to compare the possibility of detecting subtle changes in muscular bioelectric activity which are observed in some carriers of Duchenne dystrophy gene using two methods: routine quantitative electromyography and automatic EMG recording (with ANOPS computer). Twenty-one confirmed dystrophy-gene carriers were examined Two muscles were studied in each case: m. biceps brachii and m. quadriceps femoris. There was no difference between the detectability of reduced potential durathion with both methods, but automatic recording made possible a much more accurate determination of the percent of polyphasic potentials. For example, using automatic recording 16.5% of polyphasic potentials were found in the quadriceps femoris muscle of carriers and 15.3% in the biceps brachii muscle, while in routine electromyography these values were respectively 9.9% and 8.9%. This parameter is particularly useful for recognition of very early and slight pathological processes--because of that automatic EMC recording seems to be superior to routine quantitative EMG in the detection of Duchenne dystrophy carriers in whom only very small changes may be expected. In the investigations carried out as yet the authors observed that the introduction of automatic EMG recording raised the detectability rate of gene carriers by 18.9% in relation to the rate of detection by means of CPK determination (previously the EMG raised this rate only by 5 to 9%).

Screening for amplification of specific DNA sequences in cultured mammalian cells.

Carcinogen- or herpes virus-induced amplification of integrated simian virus 40 DNA in transformed cell lines has been demonstrated previously by Southern blot analyses, dot hybridizations, and dispersed cell assays. Although these methods are quite reliable, large series of experiments require a considerable amount of time and effort. Therefore, an alternative method has been developed that allows one to carry out great numbers of separate gene-amplification experiments in the 96 wells of microtiter plates and to analyze the DNAs of the treated cells simultaneously after "replica blotting" onto membrane filters by hybridization with a cloned DNA probe.

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.

Herpes simplex virus causes amplification of recombinant plasmids containing simian virus 40 sequences.

Simian virus 40 (SV40) DNA, inserted into a plasmid vector, does not replicate when transfected into baby hamster kidney cells. However, when the recipient cells are superinfected with herpes simplex virus type 1 (HSV-1), extensive amplification of the introduced plasmid occurs. Deletion of the late SV40 region or part of the coding sequences of the small tumour (t) antigen has no effect on the efficiency of amplification, whereas manipulations affecting either the SV40 origin of replication or the integrity of large tumour (T) antigen substantially decrease HSV-induced amplification. Phosphonoacetic acid, an inhibitor of HSV DNA polymerase, strongly inhibits plasmid replication. Also, an HSV-1 mutant with a temperature-sensitive defect in the DNA polymerase gene (tsH) is unable to carry out amplification of test plasmids at the non-permissive temperature. On the other hand, a further mutant (tsS) causes SV40-plasmid amplification independent of the temperature, but this mutant fails to amplify a plasmid with an HSV origin at the non-permissive temperature. Thus, HSV-induced amplification of heterologous DNA is possible in the absence of HSV DNA replication. Since tsS putatively has a defect in the gene coding for an HSV origin-binding protein (UL9), this observation appears plausible. The implications for interaction between herpesviral replication functions and heterologous (possibly cellular) DNA sequences are discussed.

Thermosensitive UL9 gene function is required for early stages of herpes simplex virus type 1 DNA synthesis.

DNA replication of herpes simplex virus type 1 (HSV-1) is dependent on a virus-encoded sequence-specific origin-binding protein, the product of the UL9 reading frame. We have identified the mutations in the UL9 gene of three temperature-sensitive (ts) mutants of HSV-1 which are responsible for the ts phenotype (A90T in mutant tsS and V220M in tsR and tsX). The mutations are located in two different conserved helicase sequence motifs of UL9. Two further alterations (I204T and E280D) compared to the published sequence were found in the mutant, revertant and parental wild-type strain 17syn+ sequences and therefore seemed to be irrelevant for the ts phenotype. The ts function of the UL9 protein was required at early times during DNA synthesis whereas upward temperature shifts at later times did not considerably inhibit DNA synthesis.