Apolipoprotein-defined lipoprotein abnormalities in rheumatoid arthritis patients and their potential impact on cardiovascular disease.

OBJECTIVES: The aim of this study was to explore apolipoprotein-defined lipoproteins for abnormalities when comparing non-rheumatological controls to rheumatoid arthritis (RA) patients. METHODS: Apolipoprotein and lipoprotein profiles were measured on 94 RA patients and 79 controls by immunoturbidimetric procedures, electroimmunoassays, and immunoprecipitation. Differences between means were tested with a two-sided Student t test with Satterthwaite adjustment. p-values were adjusted for multiple comparisons using the Bonferroni procedure. RESULTS: RA patients had significantly higher levels of total cholesterol (TC), triglycerides (TG), and very low density lipoprotein cholesterol (VLDL-C) than controls, but no significant differences in the levels of high density lipoprotein cholesterol (HDL-C) and LDL-C. RA patients had significantly lower levels of apolipoprotein (apo)A-I and lipoprotein (Lp)A-I:A-II, but no difference in levels of LpA-I than normal controls. There was a significant difference in the levels of LpB:C but not in LpB:C:E between RA patients and controls. The main abnormality among apoB lipoproteins was the significantly increased concentration of the LpA-II:B:C:D:E subclass in RA patients in comparison with controls. The high levels of LpA-II:B:C:D:E are also reflected in significantly increased levels of apoC-III, and apoC-III bound to apoB lipoproteins. CONCLUSION: The LpA-II:B:C:D:E subclass has potential as a new marker for cardiovascular disease (CVD) in RA patients.

Apolipoprotein B-containing lipoprotein subclasses as risk factors for cardiovascular disease in patients with rheumatoid arthritis.

OBJECTIVE: The purpose of this study was to explore whether nontraditional risk factors, such as apolipoprotein C-III (Apo C-III) and its corresponding Apo B lipoprotein (Lp) subclasses, contribute to the risk of cardiovascular disease in rheumatoid arthritis (RA) patients. METHODS: Apolipoprotein and lipoproteins were measured in 152 RA patients by immunoturbidimetric procedures, electroimmunoassay, and immunoprecipitation. Patients had a coronary artery calcium (CAC) score assessed at baseline and at year 3. Differences in the CAC scores between baseline and year 3 were calculated and dichotomized at 0, where patients with a difference score >0 were denoted as progressors and the rest were denoted as nonprogressors. Differences between means were tested with a 2-sided independent Student's t-test with Satterthwaite's adjustment. Proportion differences were tested with a chi-square test. Multiple logistic regression was performed to assess the relationship between apolipoprotein and lipoprotein levels and the dichotomized CAC score. RESULTS: Progressors accounted for almost 60% of the cohort. Progressors had significantly higher levels of triglycerides, very low-density lipoprotein (VLDL) cholesterol, total cholesterol/high-density lipoprotein (HDL), triglycerides/HDL, Apo B, LpA-II:B:C:D:E, LpB:C, Apo B/Apo A-I, Apo C-III, and Apo C-III-heparin precipitate than the nonprogressors. After adjusting for age, sex, statin use (yes/no), and hypertension (yes/no), significant risk factors of progressors were total cholesterol, triglycerides, VLDL cholesterol, LDL cholesterol, Apo B, LpB:C, Apo C-III, and Apo B/Apo A-I. CONCLUSION: Apo C-III-containing Apo B lipoprotein subclasses were found to be significantly elevated in progressors compared to nonprogressors. Many of these same lipoproteins were found to be associated with an increase in CAC scores among progressors. These lipoproteins may be considered new risk factors for progression of atherosclerosis in RA patients.

Staff perceptions of emergency and home-care telemedicine.

The utilization of a low-bandwidth telemedicine system for emergency and for home-care was studied in a pilot trial. The emergency setting was the emergency department of a small urban hospital and its emergency medical service (EMS); the home-care setting was the home-health agency affiliated to the hospital. Utilization data were obtained through baseline and follow-up interviews with EMS technicians, emergency department and home-health nurses, and the project coordinator. The results indicated that initial enthusiasm for the use of the telemedicine system was not followed by a commitment to the system's utilization during the trial by the relevant administrations. Barriers to optimum utilization were identified, but the actual value of the system to patient care could not be determined. We conclude that the value of a telemedicine system to patient care cannot be realized unless there is an organizational commitment from the top to system utilization.

Experimental infection of chimpanzees with hepatitis G virus and genetic analysis of the virus.

Hepatitis G virus (HGV) was transmitted to 2 chimpanzees by inoculation with human plasma containing approximately 10(8) genome equivalents (GE) of HGV. The infection was characterized by the late appearance (weeks 10 and 11 after inoculation [pi]) of viremia that persisted throughout the 120-week follow-up. Serum HGV titer increased steadily until it plateaued at 10(6)-10(7) GE/mL. However, despite this relatively high titer, neither of the chimpanzees developed hepatitis. The sequence of the viral genome, recovered from each chimpanzee at week 77 pi, differed from that of the inoculum by 5 nt (2 aa) and 27 nt (2 aa). Two more chimpanzees were inoculated with a first-passage plasma pool. The chimpanzee inoculated with approximately 10(6.7) GE of HGV had viremia at week 1 pi. However, the viral titer increased with the same kinetics as observed in the first passage. The second chimpanzee inoculated with approximately 10(4.7) GE of HGV had late appearance (week 7 pi) of viremia.

Short report: evidence of worldwide transmission of hepatitis G virus.

Hepatitis G virus (HGV) has been recently documented in the Americas, Europe, and Australia. Distinct risk populations from North Africa, South America, and Southeast Asia were screened for HGV, in addition to hepatitis B and C viruses. First time recognition of HGV is described from Egypt and Indonesia. Notable is the high proportion of HGV positive individuals among multiply transfused children, ranging from 24% of those sampled from Egypt to 32% in Indonesia. Also, data from Peru suggest the likely association of HGV infection with progressive liver disease. Hepatitis G virus should be considered a world-wide health concern.

Hepatitis G virus and fulminant hepatic failure: evidence for transfusion-related infection.

BACKGROUND/AIMS: In the majority of cases of fulminant "viral" hepatitis in Australia, no known aetiological agent can be isolated. We have examined the possible role of the recently discovered hepatitis G virus (HGV) in such cases. METHODS: An HGV specific reverse transcription polymerase chain reaction (RT-PCR) was performed on pre- and post-liver transplant serum from 14 patients who were referred for transplantation at our unit between 1989 and 1995 for unexplained fulminant hepatic failure. Eleven patients successfully underwent transplantation and three died while waiting for a suitable donor organ. Hepatitis viruses A-E were excluded by standard serological and PCR based testing. HGV RT-PCR was also performed on 21 other, randomly selected, liver transplant recipients ("controls"). RESULTS: The 14 fulminant cases were HGV RT-PCR negative prior to transplantation while five of 21 controls were positive. Post-transplant, eight of the 11 fulminant patients were found to be HGV RT-PCR positive and the same five controls remained HGV RT-PCR positive. In three of the eight fulminant patients the HGV infection resolved. CONCLUSIONS: Our data indicate that HGV infection is unlikely to be responsible for fulminant hepatitis and that it is probably acquired from blood and/or blood products during the transplantation process. Furthermore, long-term carriage of HGV post-transplant is not associated with clinically apparent liver disease.

Sequence variation and phylogenetic analysis of the 5' terminus of hepatitis G virus.

We determined the nucleotide and deduced amino acid sequence of the 5' terminus of the hepatitis G virus (HGV) genome from isolates of varied geographical origins. Our analysis showed that the putative 5' non-coding region (NCR) contains several blocks of highly conserved sequences that may be useful for the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of HGV RNA. Overall, the degree of conservation within the 669-nucleotide (nt) 5'terminal sequence was found to range from 99.5% to 86% sequence identity. We also showed that the HGV NCR from some isolates contained conserved insertions or deletions that altered the translational reading frames at the 5'-end of the genome, resulting in different sizes of predicted polyproteins encoded by genomes of individual isolates. Specifically, the insertions/deletions affected the size of the peptide preceding the putative first envelope (E1) protein. Phylogenetic analysis of the nucleotide sequences suggested that the isolates examined can be classified into distinct groups that may be useful for studying the molecular evolution of HGV and possible relationships between isolate sequence characteristics and infection patterns.

The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease.

BACKGROUND: The role of hepatitis G virus (HGV) in transfusion-associated infection and its relation to liver disease are not well understood. METHODS: Serum samples collected between 1972 and 1995 from 357 transfusion recipients, 157 controls who did not receive transfusions, 500 randomly selected volunteer blood donors, and 230 donors of blood received by HGV-infected patients were tested for HGV RNA by qualitative and quantitative polymerase-chain-reaction assays. Samples obtained before transfusion and serially after transfusion from 79 of the 81 transfusion recipients who had transfusion-associated non-A, non-B hepatitis were available for testing. RESULTS: Of the 79 patients with transfusion-associated hepatitis, 63 (80 percent) had infections related to the hepatitis C virus (HCV) and 3 had preexisting HCV and the cause of their acute hepatitis could not be determined; of the remaining 13 patients, 3 had acute HGV infection, and 10 were infected with unidentified agents. Six of the 63 patients with HCV infection who were tested (10 percent) were also infected with HGV. The three patients infected only with HGV had mild hepatitis (mean peak alanine aminotransferase level, 198 U per liter; none had jaundice); the levels of alanine aminotransferase and HGV RNA were not well correlated. The combined HCV and HGV infections were no more severe than HCV infections alone; the alanine aminotransferase values paralleled the levels of HCV RNA, but not those of HGV RNA. There were 35 HGV infections among the 357 transfusion recipients; only 3 had hepatitis with HGV as the sole viral marker. One of the 157 controls and 7 of the 500 randomly selected blood donors (1.4 percent) had detectable HGV RNA. In all eight instances in which a transfusion recipient had acute HGV infection after transfusion and samples from all donors could be tested, at least one HGV-positive donor was identified. CONCLUSIONS: HGV was common in a group of volunteer blood donors, and it can be transmitted by transfusion. Most HGV infections were not associated with hepatitis. HGV did not worsen the course of concurrent HCV infection. No causal relation between HGV and hepatitis has been established.

Hepatitis G virus infection: clinical characteristics and response to interferon.

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV-C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV-A or GBV-B sequences, two other recently described flaviviruses. Forty-five per cent of the HGV-infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co-infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon-alpha, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV-C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes.

Nucleic acid isolation for amplification-based diagnostics requires techniques that do not co-purify inhibitors of DNA polymerases. Also, other requirements for an ideal sample preparation technology include ease of use, capability for automation, high recovery and the use of nontoxic reagents. Affinity purification techniques provide high purification factor with minimal sample processing. Hybridization is the affinity interaction specific to nucleic acids and thus provides a uniquely advantageous method for purifying DNA or RNA for subsequent manipulation. Nonionic (morpholino) probes (Neu-Probes, AntiVirals, Corvallis, OR, USA) have several unique hybridization properties, including resistance to nucleases and the ability to hybridize independently of salt concentration. Therefore, such probes provide advantages over DNA probes for sample preparation by hybridization capture. Three formats for hybridization-based purification of human immunodeficiency virus (HIV) RNA were evaluated using RNA transcripts spiked into crude lysates of normal human plasma. Indirect capture used streptavidin-coated microparticles to capture hybrids of biotinylated capture probes and HIV RNA. Direct capture used particles precoated with probes. In addition, a novel method for acceleration of sequence-specific hybridization was developed and shown to give consistently high recoveries.

Prevalence and disease association of hepatitis G virus infection in Japan.

UNLABELLED: A reverse transcriptase-polymerase chain reaction procedure (RT-PCR) for the detection of hepatitis G virus (HGV) RNA was used to examine the prevalence of HGV infection and HGV-related disease in Japan. Among 48 patients with acute non-A, B, C, D, E (non-A-E) hepatitis (five transfusion-associated cases and 43 sporadic cases), only one patient (2%), a transfusion recipient, was HGV RNA positive. Similarly, among 50 patients with established chronic non-A-E hepatitis, only two (4%) were positive for HGV RNA. These frequencies were not significantly different from those in 129 voluntary blood donors (0.8%). By contrast, HGV infection was relatively common among patients who were also infected with other hepatitis viruses. HGV co-infection or superinfection was found in seven of 53 (13%) patients with acute hepatitis C, in 15 of 126 (12%) patients with chronic hepatitis C, in three of 21 (14%) patients with acute hepatitis B and in four of 81 (5%) patients with chronic hepatitis B. Among the 29 dually infected patients, 15 (52%) had a history of blood transfusion. HGV was also detected in seven (10%) of 69 haemodialysis patients, of whom only one had a dual infection with hepatitis C virus (HCV) and an elevated aminotransferase level. IN CONCLUSION: HGV RNA was found in only a low percentage of patients with either acute or chronic non-A-E hepatitis: HGV appears to co-infect or superinfect in 10-15% of HCV infections and in 5-15% of HBV infections; the prevalence of HGV infection (0.8%) among voluntary blood donors in Japan is similar to that for HCV infection; a history of blood transfusion was obtained in 22 (55%) of the total 40 HGV-positive subjects; and isolated HGV infection appears to have a low disease burden.

Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Diagnosis of symptomatic visceral leishmaniasis by use of the polymerase chain reaction on patient blood.

To diagnose symptomatic visceral leishmaniasis (kala-azar) using peripheral blood rather than tissue aspirates, a polymerase chain reaction (PCR) technique was developed for which the detection limit is 1 Leishmania-infected macrophage in 8 mL of blood. For Indian, Kenyan, or Brazilian patients with parasitologically confirmed kala-azar, 57 of 63 cases before treatment had blood that was PCR-positive (90% sensitivity). None of 40 clinically healthy persons had PCR-positive blood (100% specificity). Twelve (92%) of 13 clinically cured Indian patients had negative PCR reactions 1-6 months after treatment. This PCR procedure can provide a parasitologic diagnosis for the vast majority of kala-azar cases before therapy, may identify patients who have been successfully treated by chemotherapy, and should substantially reduce the need for invasive tests.

Transfusion-associated human immunodeficiency virus type 1 from screened antibody-negative blood donors.

Cases of human immunodeficiency virus type 1 (HIV-1) infection acquired from transfusion of screened antibody-negative blood have been reported since 1986. Recent reports have proposed new combination antibody assays or the addition of HIV-1 p24 antigen testing to enhance the screening of blood donations further. Since antibody testing for HIV-1 began in 1985, 700,000 donor units have been screened at US Army blood donor centers. The US Army blood donor/recipient "lookback" program recently identified two cases of HIV-1 infection that resulted from a screened negative donation. Samples from the implicated unit, as well as from previous donations from the same donor, were available for testing to assess the performance of current screening methods. Sequential donation samples were assayed by five different Food and Drug Administration-approved HIV-1 screening enzyme-linked immunosorbent assays, a Food and Drug Administration-approved Western blot, a recombinant envelope-based enzyme-linked immunosorbent assay, a p24 antigen capture assay, a radioimmunoprecipitation assay, and a polymerase chain reaction. The HIV-1 p24 antigen and genomic RNA material were detected in a donation that was screened as negative by four of the five Food and Drug Administration-licensed screening enzyme-linked immunosorbent assays. Two recipients of transfusion products from this donation became infected with HIV-1. A sample from a prior donation from this donor was negative for HIV-1 by all assays. The status of blood donors who are in the early stages of HIV-1 infection may not be detected by current screening methods. While this is a rare phenomenon, it highlights the need for technologic developments in screening methods to narrow the time between infection and detection. In addition, it emphasizes the need for more effective education and counseling to enhance the utility of self=deferral.

Accuracy of supplementary serologic testing for human T-lymphotropic virus types I and II in US blood donors. Retrovirus Epidemiology Donor Study.

Blood donations in the United States have been screened for antibody to human T-lymphotropic virus type I (HTLV-I) by HTLV-I enzyme immunoassay (EIA) since November 1988. Specimens repeatedly found to be reactive by EIA undergo confirmation by supplementary serologic tests. We assessed the accuracy of blood center testing of 994 HTLV-I EIA repeat-reactive specimens in five US blood centers between November 1988 and December 1991. Of 410 confirmed HTLV-I/II donations, 407 (99.3%) were infected with HTLV-I/II, as determined by polymerase chain reaction (PCR) (403 cases) and by repeat serologic testing (4 cases). The three false-positive results occurred in the first year of testing. Of 425 HTLV-indeterminate specimens, 6 (1.4%) were found to be infected by PCR (5 with HTLV-II and 1 with HTLV-I). None of 159 confirmatory test-negative donations was PCR positive. Of HTLV-I/II-seropositive specimens, 80.2% to 95.4% could be typed as HTLV-I or HTLV-II by type-specific serologic assays. These results support recommendations that HTLV-I/II-seropositive donors should be advised that they are infected with HTLV-I, HTLV-II, or HTLV-I/II (depending on results of type-specific assays). HTLV-indeterminate donors should be advised that their results only rarely indicate HTLV infection. HTLV confirmatory test-negative donors should be reassured that they are not infected with HTLV-I or HTLV-II.

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction.

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and(32)P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the(32)P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with(32)P-Iabelled probes.

Clinical performance of a polymerase chain reaction testing algorithm for diagnosis of HIV-1 infection in peripheral blood mononuclear cells.

The clinical performance of a modified polymerase chain reaction (PCR) testing algorithm was evaluated for confirming the presence of HIV-1 proviral DNA in peripheral blood mononuclear cells. A whole cell lysate, rather than phenol-purified DNA, was used for PCR amplification, under systematically optimized conditions designed and verified within each PCR run to detect as few as 10 copies of proviral DNA. A sequential testing algorithm was designed requiring reactivity in duplicate (with corresponding non-reactivity in negative controls) with at least two sets of primers, before reporting a specimen as HIV-1-positive. In 196 specimens from patients staged according to the Walter Reed staging system, the PCR test sensitivity and the coculture isolation rate (in parentheses) were found to be: 97% (71%), 100% (85%), and 100% (76%) in stage 1, stage 2, and stage 3 specimens, respectively; and 100% (100%) in stage 4, 5, and 6 specimens. Results were uniformly negative for PCR and coculture isolation from 21 blind negative specimens and 105 (negative) donor leukopacks. These data indicate that this PCR testing algorithm is more accurate than tissue culture isolation methods, especially with early stage patients, and results in detection of HIV-1 in virtually 100% of seropositive individuals, with no false positives.