The dynamics of human cytomegalovirus replication in vivo.

Cytomegalovirus (CMV) is generally described as a slowly replicating virus. During studies of immunocompromised patients, we observed rapid changes in the quantity of CMV DNA present in serial blood samples by quantitative-competitive polymerase chain reaction commensurate with a doubling time of <2 d. To further investigate the dynamics of replication in vivo, patients in three distinct situations were studied in detail: (a) those receiving intravenous ganciclovir; (b) those in whom ganciclovir-resistant strains appeared during long-term therapy; and (c) those in whom ganciclovir-resistant strains disappeared with alternative drug therapy. In all cases, it was possible to provide accurate estimates of the doubling time of CMV and its half-life of disappearance after antiviral chemotherapy. The results from all three approaches demonstrated that the doubling time/half-life of CMV in blood is approximately 1 d when frequent samples are collected. These results show that CMV DNA replication in vivo is a highly dynamic process. We conclude that the reputation of CMV as a slowly replicating virus based on the time taken to produce cytopathic effects in vitro is unwarranted. These findings have implications for the potency, dose, and duration of antiviral chemotherapy needed for the effective treatment of this important human pathogen.

Angle-corrected imaging transcranial doppler sonography versus imaging and nonimaging transcranial doppler sonography in children with sickle cell disease.

BACKGROUND AND PURPOSE: Nonimaging transcranial Doppler sonography (TCD) and imaging TCD (TCDI) are used for determination of the risk of stroke in children with sickle cell disease (SCD). The purpose was to compare angle-corrected, uncorrected TCDI, and TCD blood flow velocities in children with SCD. MATERIALS AND METHODS: A total of 37 children (mean age, 7.8 +/- 3.0 years) without intracranial arterial narrowing determined with MR angiography, were studied with use of TCD and TCDI at the same session. Depth of insonation and TCDI mean velocities with and without correction for the angle of insonation in the terminal internal carotid artery (ICA) and middle (MCA), anterior (ACA), and posterior (PCA) cerebral arteries were compared with TCD velocities with use of a paired t test. RESULTS: Two arteries were not found on TCDI compared with 15 not found on TCD. Average angle of insonation in the MCA, ACA, ICA, and PCA was 31 degrees , 44 degrees , 25 degrees , and 29 degrees , respectively. TCDI and TCD mean depth of insonation for all arteries did not differ significantly; however, individual differences varied substantially. TCDI velocities were significantly lower than TCD velocities, respectively, for the right and left sides (mean +/- SD): MCA, 106 +/- 22 cm/s and 111 +/- 33 cm/s versus 130 +/- 19 cm/s and 134 +/- 26 cm/s; ICA, 90 +/- 14 cm/s and 98 +/- 27 cm/s versus 117 +/- 18 cm/s and 119 +/- 23 cm/s; ACA, 74 +/- 24 cm/s and 88 +/- 25 cm/s versus 105 +/- 23 cm/s and 105 +/- 31 cm/s; and PCA, 84 +/- 27 cm/s and 82 +/- 21 cm/s versus 95 +/- 23 cm/s and 94 +/- 20 cm/s. TCD and angle-corrected TCDI velocities were not statistically different except for higher angle-corrected TCDI values in the left ACA and right PCA. CONCLUSION: TCD velocities are significantly higher than TCDI velocities but are not different from the angle-corrected TCDI velocities. TCDI identifies the major intracranial arteries more effectively than TCD.

Cloning and nucleotide sequence of the Brucella abortus groE operon.

The cloning and sequencing of the Brucella abortus groES and groEL genes are reported. The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon. Putative promoter and terminator sequences are also identified. The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.

Suppression of HIV p24 antigen and induction of HIV anti-p24 antibody by alpha interferon in patients with chronic hepatitis B.

Five HIV p24 antigen (p24Ag)-positive patients received alpha interferon during trials of therapy for hepatitis B. Four of these showed marked falls in p24Ag during treatment. One of the two patients who became p24Ag-negative [corrected] developed anti-p24 antibodies (anti-p24). Five out of nine p24Ag-negative HIV-antibody-positive patients showed a rise in anti-p24 titres during interferon therapy, whereas only two out of six untreated controls showed a similar rise. This study provides evidence that alpha interferon has anti-HIV activity in vivo.

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

BACKGROUND: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). OBJECTIVES: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. STUDY DESIGN: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. RESULTS: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log(10) viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. CONCLUSION: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

Longitudinal fluctuations in cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease.

Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log10 genomes/ml) vs asymptomatic patients (median 3.6 log10 genomes/ml, P=0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D-, median 5.0 log10), compared to those in the R-D- and R+D+ groups (P < 0.01 and <0.005). Odds ratios for disease per 0.25 log10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P=0.004), 6.60 (P=0.05) and 3.17 (P=0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.

Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase.

A 24 base pair oligonucleotide probe directly conjugated to alkaline phosphatase has been used to detect immobilised amplicons derived from a cytomegalovirus specific polymerase chain reaction (PCR). The sensitivity of detection using a highly amplified alkaline phosphatase detection system was four genome equivalents and was comparable to the limit of detection using agarose gel methods. The mean optical density at 492 nm of samples not known to contain cytomegalovirus DNA was 0.085 +/- 0.006 and was well separated from the optical density generated from four genome equivalents (absorption at 492 nm: 0.132). The assay was used to identify the presence of cytomegalovirus in blood DNA extracts from immunocompromised patients in whom conventional ethidium bromide stained agarose gel electrophoresis revealed the presence of multiple amplicons. Samples yielding an uninterpretable result at both neat and diluted 1 in 20 in the PCR gave rise to the highest proportion of positive results (68%) whilst samples that produced uninterpretable results neat but were negative at 1 in 20 and vice versa gave positive rates of 33.6 and 21.7%, respectively. The use of this assay for identifying cytomegalovirus specific PCR products in problematic samples is discussed.

Clinical experience with zidovudine for patients with acquired immune deficiency syndrome and acquired immune deficiency syndrome-related complex.

We have treated 113 patients with zidovudine since its licensure, 80 with acquired immunodeficiency syndrome and 33 with acquired immunodeficiency syndrome-related complex. This paper reports on the efficacy and toxicity observed in these patients. Improved well-being, reduced frequency and severity of opportunist infections were notable in the first year of follow-up. More rapid improvement in pulmonary physiological tests during recovery from Pneumocystis carinii pneumonia was also observed in treated patients. Patients with lower initial platelet counts showed early increases in platelet counts. There was a consistent fall in human immunodeficiency virus (HIV) p24 antigen during treatment, although not always to undetectable levels. CD4 cell counts showed a rise in the first months of treatment but these were not sustained, despite continuing clinical benefit. Neuropsychological and clinical evidence of benefit in HIV encephalopathy are described. We have analysed the factors influencing marrow toxicity and have found that low CD4 count and the intercurrent use of ganciclovir and dapsone increase myelotoxicity. We describe the clinical and biochemical features of the myopathy associated with long-term use of zidovudine and summarise our findings on dose-reduction associated meningo-encephalitis.

High blood pressure knowledge in an urban African-American community.

OBJECTIVE: To determine the level and determinants of knowledge of the risks for hypertension and the potential for its prevention in an urban African-American community. METHODS: In a survey of 397 African-American adults (18-73 years of age) at an urban community fair, we measured high blood pressure knowledge using a 12-item questionnaire designed at NIH for the assessment of high blood pressure knowledge among non-medical persons. RESULTS: The mean high blood pressure knowledge score for the overall sample was 83.1%. There were subgroup differences in the scores with significant associations between high blood pressure knowledge score and level of education (P = .002) and a personal history of hypertension (P = .009). CONCLUSION: We concluded that the participants exhibited a high, but variable, level of high blood pressure knowledge with a higher level of education and/or a personal history of hypertension having a significant association with greater blood pressure knowledge. The effects of the magnitude and mode of acquisition of high blood pressure knowledge on the control of high blood pressure and its related outcomes need to be examined in further studies.

Chemical inactivation of HIV on surfaces.

To assess whether alcohol and glutaraldehyde are effective disinfectants against dried HIV the virucidal effects of 70% alcohol (ethanol and industrial methylated spirit) and 1% and 2% alkaline glutaraldehyde were tested against cell associated and cell free HIV dried on to a surface. Virus stock (100 microliters) or 10,000 cultured C8166 T lymphocytes infected with HIV were dried onto sterile coverslips and immersed in 2% and 1% alkaline glutaraldehyde and 70% ethanol for 30 seconds and one, two, four, and 10 minutes, there being an additional time point of 20 minutes for cell free virus disinfected with 70% industrial methylated spirit. In addition, virus stock in neat serum was tested with 1% and 2% alkaline glutaraldehyde to see whether the fixative properties of glutaraldehyde impair its virucidal properties. Virus activity after disinfection was tested by incubating the coverslips (cell associated virus) or the coverslips and sonicated cell free virus with C8166 T lymphocytes. The lymphocytes were examined for the formation of syncytia and HIV antigens were assayed in the culture fluid. Both 2% and 1% alkaline glutaraldehyde inactivated cell free HIV within one minute; 2% alkaline glutaraldehyde also inactivated cell free virus in serum within two minutes, but a 1% solution was ineffective after 15 minutes' immersion. Cell associated HIV was inactivated by 2% alkaline glutaraldehyde within two minutes. Seventy per cent industrial methylated spirit failed to inactivate cell free and cell associated HIV within 20 and 15 minutes, respectively, and 70% ethanol did not inactivate cell free virus within 10 minutes. Seventy per cent industrial methylated spirit and ethanol are not suitable for surface disinfection of HIV. Fresh 2% solutions of alkaline glutaraldehyde are effective, but care should be taken that they are not too dilute or have not become stale when used for disinfecting HIV associated with organic matter.

Humoral immune responses of Africans to cysteine protease-related antigens of Plasmodium falciparum.

The Plasmodium falciparum serine repeat antigen (SERA) and serine repeat protein homologue (SERPH) contain highly conserved domains that appear to encode cysteine proteases or related proteins. Humoral immune responses against the protease domains of SERA and SERPH were evaluated. Malaria-immune Africans, but not nonimmune controls, demonstrated potent humoral responses against the protease domains. As the SERA and SERPH protease domains are likely accessible to circulating antibody, these results suggest that humoral responses to the domains may contribute to antimalarial immunity.

Protective immune responses against protease-like antigens of the murine malaria parasite Plasmodium vinckei.

The Plasmodium falciparum proteins serine repeat antigen (SERA) and serine repeat protein homologue (SERPH) have similarity in sequence with cysteine proteases in a well-conserved protease domain. We identified three SERA homologues from the murine malaria parasite Plasmodium vinckei and evaluated immune responses to the protease domains of these proteins. Mice that developed protective immunity to P. vinckei after serial infection and cure demonstrated humoral and cell-mediated responses against the SERA homologue protease domains. Mice immunized with Salmonella typhimurium expressing the protease domain of one of these antigens demonstrated cellular responses against the antigen and increased survival against lethal challenge with P. vinckei. Our results suggest that the protease domains of SERA and SERPH are worthy of additional study as potential components of a malaria vaccine.

Elimination of high titre HIV from fibreoptic endoscopes.

Concern about contamination of fibreoptic endoscopes with human immunodeficiency virus (HIV) has generated a variety of disruptive and possibly unnecessary infection control practices in endoscopy units. Current recommendations on the cleaning and disinfection of endoscopes have been formulated without applied experimental evidence of the effective removal of HIV from endoscopes. To study the kinetics of elimination of HIV from endoscope surfaces, we artificially contaminated the suction-biopsy channels of five Olympus GIF XQ20 endoscopes with high titre HIV in serum. The air and water channels of two instruments were similarly contaminated. Contamination was measured by irrigating channels with viral culture medium and collecting 3 ml at the distal end for antigen immunoassay. Endoscopes were then cleaned manually in neutral detergent according to the manufacturer's recommendations and disinfected in 2% alkaline glutaraldehyde (Cidex, Surgikos) for two, four, and ten minutes. Contamination with HIV antigens was measured before and after cleaning and after each period of disinfection. Initial contamination comprised 4.8 x 10(4) to 3.5 x 10(6) pg HIV antigen/ml. Cleaning in detergent achieved a reduction to 165 pg/ml (99.93%) on one endoscope and to undetectable levels (100%) on four. After two minutes in alkaline glutaraldehyde all samples were negative and remained negative after the longer disinfection times. Air and water channels, where contaminated, were tested after 10 minutes' disinfection and were negative. These findings underline the importance of cleaning in removing HIV from endoscope and indicate that the use of dedicated equipment and long disinfection times are unnecessary.

Radiologic differentiation of intraocular glass: evaluation of imaging techniques, glass types, size, and effect of intraocular hemorrhage.

OBJECTIVE: The accurate detection of intraocular foreign bodies is critically important in treating ocular trauma. The purpose of this study was to evaluate the efficacy of CT, MR imaging, and sonography in detecting seven types of glass varying in size and placed in three locations in the globe, and to examine the effect of intraocular hemorrhage. MATERIALS AND METHODS: Glass pieces were cut into 1.5-, 1.0-, and 0.5-mm pieces and implanted on the corneal surface and the anterior and posterior chambers of 42 fresh porcine eyes. Twenty-one eyes were scanned comparing axial CT, helical CT, and MR imaging. The remaining 21 eyes were scanned using helical CT and sonography after implantation in a simulated human skull before and after placement of blood in the anterior chamber (hyphema). RESULTS: Detection rates were 57.1% for helical CT, 41.3% for axial CT, and 11.1% for T1-weighted MR imaging (n = 63 fragments). Results were significant (p < 0.0001). Sonography detected 43% of glass fragments in the posterior chamber and 24% in the anterior chamber. Detectability was greatest for green beer bottle glass (90.3%) and least for spectacle glass (43.1%) (p < 0.0001). Detection rates for size ranged from 96.2% at 1.5 mm to 48.3% at 0.5 mm, which was also significant (p < 0.0001). On helical CT, anterior chamber glass was easiest to detect (91.7%) and corneal surface glass the most difficult (64.9%). Hyphema made no statistical difference (p < 0.0001). CONCLUSION: Helical CT was the most sensitive imaging modality for the detection of intraocular glass. The sensitivity of detection was unaffected by hyphema but was determined by the type of glass, size, and location.

Impact of a diagnostic workstation on workflow in the emergency department at a level I trauma center.

PURPOSE: When a computed tomography (CT) scan on a patient from the emergency department is completed at University of Medicine and Dentistry of New Jersey (UMDNJ)-University Hospital, a non-picture archiving and communication system (PACS) environment, formal diagnostic review cannot begin until the images are printed and transported to the on-call radiology resident. The time to reach a final diagnosis has been significantly reduced by the introduction of a single workstation in the on-call reading room. MATERIALS AND METHODS: Five radiology residents were studied. Each read 10 CT studies on film and 10 on a workstation. After a training period to familiarize the residents with the workstation, measurements were taken of the time required to read the examination and the time required for printing and transporting or networking the images. RESULTS: The average time required to transmit the images was reduced from approximately 40 minutes to 16 minutes. Interpretation times between the workstation and film were comparable. CONCLUSION: The addition of a single workstation significantly reduces the time required to reach a final diagnosis by obviating the need to print and transport the images to the on-call radiology resident. Such time savings can have a significant impact on the care of trauma patients.

Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation.

BACKGROUND: Cytomegalovirus (CMV) continues to be a major problem post-transplantation; early markers for predicting patients at risk of CMV disease are needed. Peak CMV load in the blood correlates with CMV disease but frequently occurs too late to provide prognostic information. METHODS: 359 transplant recipients (162 liver, 87 renal, and 110 bone marrow) were prospectively monitored for CMV DNA in the blood with qualitative and quantitative PCR. 3873 samples were analysed. The CMV load in the first PCR-positive sample and the rate of increase in CMV load in blood during the initial phase of replication were assessed as risk factors for CMV disease using logistic regression. FINDINGS: 127 of the 359 patients had CMV DNA in the blood and 49 developed CMV disease. Initial viral load correlated significantly with peak CMV load (R2=0.47, p=<0.001) and with CMV disease (odds ratio 1.82 [95% CI 1.11-2.98; p=0.02; 1.34 [1.07-1.68], p=0.01, and 1.52 [1.13-2.05], p=0.006, per 0.25 log10 increase in viral load for liver, renal, and bone-marrow patients, respectively). The rate of increase in CMV load between the last PCR-negative and first PCR-positive sample was significantly faster in patients with CMV disease (0.33 log10 versus 0.19 log10 genomes/mL daily, p<0.001). In multivariate-regression analyses, both initial CMV load and rate of viral load increase were independent risk factors for CMV disease (1.28 [1.06-1.52], p=0.01, per 0.25 log10 increase in CMV load and 1.52 [1.06-2.17], p=0.02, per 0.1 log10 increase in CMV load/mL daily, respectively). INTERPRETATION: CMV load in the initial phase of active infection and the rate of increase in viral load both correlate with CMV disease in transplant recipients; in combination, they have the potential to identify patients at imminent risk of CMV disease.

Quantification of CMV viraemia in a case of transfusion-related graft-versus-host disease associated with purine analogue treatment.

We report a patient who developed transfusion-associated graft-versus-host-disease (GvHD) and concurrent cytomegalovirus (CMV) infection, both complications thought to be related to severe T lymphocyte depletion induced by treatment with a purine analogue drug, fludarabine. CMV viraemia was detected by qualitative PCR and the viral load in positive samples was measured using a fully quantitative PCR assay. This quantitative assay enabled the evaluation of the efficacy of antiviral interventions based on the qualitative PCR result. The case illustrates the risks associated with the use of purine analogue drugs, as well as the value of quantitative CMV PCR assays for monitoring CMV infection in immunocompromised patients.

Recovery of the human immunodeficiency virus from fibreoptic bronchoscopes.

Ten bronchoscopes that had been used on patients with the acquired immunodeficiency syndrome were sampled to determine the nature and extent of microbial contamination. Samples were taken by irrigating the suction biopsy channel with modified viral transport medium and by swabbing the insertion tube. Sampling was repeated after they had been cleaned in detergent and after two minutes' disinfection in 2% alkaline glutaraldehyde. Before being cleaned the seven bronchoscopes tested by polymerase chain reaction were contaminated with the human immunodeficiency virus, though infectivity and antigen assays gave negative results. Other organisms identified were hepatitis B virus (1), commensal bacteria (9), and Pneumocystis carinii (4). Mean bacterial contamination was 2.27 log colony forming organisms per millilitre. Cleaning the bronchoscope before disinfection removed all detectable contaminants with a reduction in bacterial growth of up to 8 log colony forming units/ml.