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C Stephan,
B Dauer,
M Bickel,
A Haberl,
L Locher,
A Müller,
S Klauke, A Berger,
H-W Doerr,
M Stürmer,
S Staszewski
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ABSTRACT: The reverse transcriptase (RT)-mutation K65R limits further therapeutic options and has been selected by unfavorable RT-combinations, e.g. tenofovir in combination with abacavir and/or didanosine.
We identified HIV-1 infected patients from a large treatment cohort who experienced virological failure (HIV-1 RNA >1000 copies/mL) with evidence of resistance mutations including the K65R, but without thymidine analogue mutations (TAMs) in genotypic resistance assay. Phenotype was performed from previously collected frozen plasma. The patients were followed for clinical and resistance outcome after treatment intensification with only zidovudine.
Five patients had experienced antiretroviral treatment failure on various nucleoside analogue combinations, containing abacavir, didanosine, lamivudine, nevirapine, reverset and/or tenofovir. RT-sequence revealed mutations at position K65R in combination with other non-TAMs. The patients' median viral load prior to zidovudine intensification was 3.551 Log10 (range 3.053-4.681) and despite evidence for resistance to the failing drug regimen, all responded within 4 weeks to undetectable levels (<1.699 Log10 or <50 copies/mL) and remained virologically suppressed during follow-up (20 months through 6.5 years).
In virologically failing patients due to K65R- and other non-thymidine-mutations, simple regimen intensification with zidovudine resulted in sustained HIV-1 suppression. The finding of re-sensitized HIV-1 in patients may be clinically relevant.
The Journal of infection 10/2010; 61(4):346-50. · 4.13 Impact Factor
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ABSTRACT: An acute and often severe respiratory illness emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS) came to an end in July 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. Starting in March 2003, the World Health Organization (WHO) organised an unprecedented international effort by leading laboratories working together to find the causative agent. Little more than one week later, three research groups from this WHO-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in SARS patients, using different approaches. After Koch's postulates had been fulfilled, WHO officially declared on 16 April 2003 that this virus never before seen in humans is the cause of SARS. Ever since, progress around SARS-associated coronavirus (SARS-CoV) has been swift. Within weeks of the first isolate being obtained, its complete genome was sequenced. Diagnostic tests based on the detection of SARS-CoV RNA were developed and made available freely and widely; nevertheless the SARS case definition still remains based on clinical and epidemiological criteria. The agent's environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. Despite its grave consequences in humanitarian, political and economic terms, SARS may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more "traditional" techniques carried out to an excellent standard.
Journal of Clinical Virology 02/2004; 29(1):13-22. · 3.97 Impact Factor
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ABSTRACT: In the present study we evaluated the performance of the new LCx HIV RNA quantitative kit (Abbott Laboratories, Delkenheim, Germany) for the quantitative detection of HIV-1 RNA in human plasma in comparison to the Cobas Amplicor HIV-1 Monitor assay (Roche Diagnostics, Branchburg, N.J.), including samples containing a variety of HIV-1 subtypes. LCx and Cobas were compared using archived EDTA plasma samples collected from HIV-infected patients. Considering the lower limit of the linear range of 50 copies/ml, the detection rate of the LCx was 139 out of 174 (79.9%) versus 131 out of 174 (75.3%) of the Cobas. Overall agreement was 95.4% (166/174) at a cut-off of 50 copies. LCx and Cobas results on clinical samples were found linearly associated (r2 = 0.900) and strongly correlated (r = 0.949). The mean viral load in the 174 frozen patient samples was 3.25 log10 copies/ml by LCx compared to 2.71 log10 copies/ml by Cobas. Considering only samples with a viral load > or =50 copies/ml, the average difference was -0.132 log copy/ml. Using a panel consisting of 9 plasma samples spiked with 9 different HIV-1 cultured isolates (A-H, and O) LCx detected the 9 subtypes with a high degree of precision, i.e., 9-33% coefficient of variation. As expected, the Cobas failed to detect the group O isolates. The results of the remaining samples showed a higher degree of variation (when testing four replicates of the subtype panel) than the LCx of 14.2-40.3%. Nevertheless, the results were comparable with the LCx data.
Medical Microbiology and Immunology 12/2001; 190(3):129-34. · 3.83 Impact Factor
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ABSTRACT: Background: The diagnosis of an enterovirus infection may be achieved through direct virus detection from fecal or cerebrospinal fluid
(CSF) samples by virus isolation or PCR. Serologically, a significant rise in antibody titer may be detected and different
enteroviral types can be differentiated using the neutralization assay.
Patients and Methods: We investigated the contribution of these different laboratory parameters to the diagnosis of enterovirus infections occurring
in the Frankfurt am Main area during the years 1997 to 1999, including an echovirus 30 outbreak in a group of children with
aseptic meningitis in 1997. Samples were referred from 1,013 patients; virus isolation was attempted from 579 CSF specimens
and from 400 stool samples. 208 CSF samples were tested by PCR.
Results: During the echorivus 30 outbreak we identified 22.3% of samples as positive, almost exclusively echovirus 30. In 1998 only
7.1% of samples were positive and a rather broad range of agents was isolated. In 1999 10.4% were positive, predominantly
coxsackie B5 and echovirus 11. We could show that in acute enterovirus infections, virus detection by cell culture and PCR
is superior to serological methods (neutralization assay and IgM assay). For virus isolation, there was a higher rate of positives
from stool compared to CSF (1997: 27.8% versus 25%; 1998: 14.4% versus 3%; 1999: 17.9% versus 8.5%). When comparing PCR and
virus isolation from the CSF, the former yielded a higher rate of positive results but was not clearly superior to virus isolation
from CSF.
Conclusion: The recommended method for the diagnosis of acute enterovirus infections is virus isolation from feces. In cases of suspected
aseptic meningitis virus isolation and PCR are valuable for the direct detection of virus in CSF.
Infection 05/2001; 29(3):138-142. · 2.66 Impact Factor
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ABSTRACT: During the last 5 years, considerable scientific and financial efforts have been made in the development of quantitative nucleic acid detection technology. For detection of human immunodeficiency virus (HIV), quantitative culture is time consuming, cumbersome and requires appropriate laboratory safety equipment. Quantitative determination of p24 antigen by enzyme immunoassay is of limited value due to its relatively poor sensitivity. Therefore, quantitative determination of viral load using nucleic acid amplification techniques is the most accurate, prognostic marker for HIV type 1 infection, independently of the CD4+ cell count. Hepatitis B virus (HBV) is not cultivable in vitro. Serological assays allow an accurate diagnosis and follow-up of acute or chronic infection. Quantification of HBV DNA is used for the monitoring of antiviral therapy, determination of infectivity and for resolution of unclear serological profiles, e.g. isolated anti-HBc reactivity, as well as for patients in which HBV mutants are suspected. Hepatitis C virus (HCV) can only be detected by molecular based assays because no cell culture system, which permits a reliable isolation of clinical specimens, is currently available. Furthermore, early diagnosis and follow-up of infection cannot be achieved with antibody serology. The prognostic relevance of quantitative HCV RNA determination is of limited value for the long-term prognosis of chronic hepatitis C. However, viral load may predict the outcome of antiviral therapy. Genetic diversity is another challenge for HCV RNA quantification.
Journal of Clinical Virology 02/2001; 20(1-2):23-30. · 3.97 Impact Factor
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ABSTRACT: Although isolated antibody to hepatitis B core antigen (anti-HBc) is frequently nonspecific or may be the only serological marker of past self-limiting hepatitis B, where antibodies against the surface antigen have disappeared, isolated anti-HBc seropositivity is frequently associated with chronic hepatitis B in HIV- and HCV-infected individuals. Of 5,520 samples that tested positive for anti-HBc (IMx and AxSYM CORE, Abbott, Delkenheim, Germany) at the Institute of Virology, University Clinic Frankfurt during the time interval from January 1994 to February 1996, 643 (11.6%) were isolated anti-HBc-reactive in the IMx and AxSYM CORE assays (inhibition values >90%). There was a statistically significant association between isolated anti-HBc seropositivity and HCV and HIV/HCV coinfection (p < 0.05). A total of 190 samples were available for further testing. Six (3.2%) of 190 isolated anti-HBc-positive samples were considered false-positive since they were only positive in the AxSYM or IMx CORE assay and a linear decrease of the measured signal could not be observed in dilution series. Of 184 serum samples tested with nested PCR using primers of the S genome region, only 6 (3.3%) were HBV DNA-positive. Anti-HBc-IgM antibody could be detected in 3 (1.6 %) of the tested samples using the IMx CORE-M. With the more sensitive VIDAS HBc IgM specific IgM antibody was detected in 15 (8.5%) of 177 samples at concentrations ranging from 10 to >200 Paul Ehrlich Institute U/ml. HIV or HCV coinfection was present in 28.1% and 37.5% of isolated anti-HBc-positive individuals, respectively. We conclude from our observations that only a limited proportion of anti-HBc-isolated individuals are potentially infectious, however anti-HBc-IgM which is detectable in any form of liver disease associated with HBV infection was present in more than 8% of the individuals. Of isolated anti-HBc-positive sera 37% were positive for anti-HCV, suggesting that anti-HCV antibody testing should be performed in isolated anti-HBc-positive individuals.
Intervirology 01/2000; 43(2):71-6. · 2.34 Impact Factor
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ABSTRACT: Quantitative determination of viral load using nucleic acid amplification techniques represents the most accurate prognostic marker for human immunodeficiency virus type 1 (HIV-1) infection, independently of CD4+ cell count. Overall, the different methods for HIV-1 RNA determination (RT-PCR, nucleic acid sequence-based amplification, branched DNA) show a good reproducibility (0.5 log), however for low copy numbers and in HIV-1-infected children the variability may exceed 0.7 log. In non-HIV-1 subtype B infections the copy number is underestimated. While serology permits an accurate follow-up of hepatitis B virus (HBV) infection, HBV DNA quantification is used for monitoring of antiviral therapy, determination of infectiosity and in combination with serological markers for the resolution of unusual profiles, i.e. isolated anti-HBc reactivity. The prognostic relevance of hepatitis C virus (HCV) RNA determination is of limited value for the long-term prognosis of chronic hepatitis C, however the viral load may predict the outcome of antiviral therapy. Genetic diversity represents a challenge for HCV RNA quantification.
Intervirology 02/1998; 41(1):24-34. · 2.34 Impact Factor
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ABSTRACT: Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) account for millions of cases of vertical infections worldwide. Laboratory diagnosis is essential for the detection of potentially infectious carriers. HBeAg represents the best serological marker for HBV replication. Since 10% of HBeAg-negative carriers transmit the virus to their children, determination of viral DNA is more reliable for the assessment of the risk to vertical infection. Risk assessment of vertical HIV transmission and monitoring AZT therapy during pregnancy are achieved by determination of HIV-1 viral load and CD4+ cell count. HIV-1 RNA or cDNA detection permits a nearly 100% sensitive diagnosis of congenital HIV infection already 2 weeks after birth. While qualitative HBV DNA determination should be limited only to anti-HBe carriers in order to assess infectiosity, HIV-1 RNA measurement represents in combination with the CD4+ cell count the best prognostic marker for vertical HIV infection and for the follow-up of infected children.
Intervirology 02/1998; 41(4-5):201-7. · 2.34 Impact Factor
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ABSTRACT: Dengue virus infections have been well known for many years; still dengue virus is regarded as an 'emerging' pathogen, as the disease profile is changing. Its geographical range and overall incidence, and the incidence of the associated complications, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), are on the increase. Modern-day travel and increasing urbanization seem to be the main contributing factors. In order to estimate the risk of infection during long-term stays in dengue-endemic countries, we tested sera obtained from 323 development aid workers and their family members who had spent on average 9.8 years in dengue-endemic regions for the presence of dengue virus antibodies. Dengue virus antibody screening was done by a commercially available immunofluorescence test (IF). Reactive samples were re-tested by an in-house IF and also tested for cross-reactivity to yellow fever virus using yellow fever IF and neutralization test (NT). Evaluation of the results revealed that the screening test has a specificity of at least 63.2%. In 12 of 19 initially positive cases crossreacting antibodies against yellow fever virus could be ruled out. Three cases remained indeterminable, whereas four of the reactive and 10 (out of 12) of the borderline reactive cases showed crossreactivity with yellow fever virus, probably due to previous vaccination. We found seroprevalence rates of 4.3% with no significant differences related to gender or area of upbringing. Seroprevalence rates were evaluated according to region of suspected or confirmed infection. In two cases the dengue infection had taken a classical clinical course; in another three cases an extraordinary febrile illness was reported in the history. None of the other seropositive individuals had a history of an illness possibly attributable to dengue virus infection. Our results show that there definitely is a risk for long-term expatriates to acquire (mostly non- or oligo-symptomatic) dengue infection, which might be important especially in the light of the supposed aetiology of DHF or DSS as a secondary infection with another dengue virus serotype.
Tropical Medicine & International Health 11/1997; 2(10):934-40. · 2.80 Impact Factor
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ABSTRACT: Hepatitis E virus (HEV) is one of the so-called 'emerging' viral pathogens, whose role is increasingly being recognized. To estimate the risk of HEV infection during long-term stays in HEV-endemic countries, 500 serum samples obtained from development aid workers and their family members who had spent on average 9 years in HEV-endemic regions were tested for antibodies against HEV by ELISA and Immunoblot. We found seroprevalence rates of 5-6% with no significant differences related to gender or area of upbringing (raised in an HEV-endemic vs. nonendemic region). Seroprevalence rates did not increase with increasing number of stays or number of expatriate years. None of 77 children and adolescents tested was positive for anti-HEV. The Indian subcontinent showed the highest seropositive rate with 10%. In subjects returning from West and Central Africa, East Africa, South-east Asia and Latin America seroprevalence rates were around 7%. We found a comparatively low seroprevalence rate of 2.1% for the Arab countries and the Middle East. Our results show that there definitely is a risk for long-term expatriates to acquire HEV infection; however, in most of our cases infection seems to have been non- or oligo-symptomatic.
Tropical Medicine & International Health 10/1997; 2(9):885-91. · 2.80 Impact Factor
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ABSTRACT: Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful.
Journal of Virological Methods 06/1996; 59(1-2):141-6. · 2.01 Impact Factor
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ABSTRACT: Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four ‘confirmatory’ assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative ‘core’ region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful.
Journal of Virological Methods.
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ABSTRACT: The diagnosis of an enterovirus infection may be achieved through direct virus detection from fecal or cerebrospinal fluid (CSF) samples by virus isolation or PCR. Serologically, a significant rise in antibody titer may be detected and different enteroviral types can be differentiated using the neutralization assay.
We investigated the contribution of these different laboratory parameters to the diagnosis of enterovirus infections occurring in the Frankfurt am Main area during the years 1997 to 1999, including an echovirus 30 outbreak in a group of children with aseptic meningitis in 1997. Samples were referred from 1,013 patients; virus isolation was attempted from 579 CSF specimens and from 400 stool samples. 208 CSF samples were tested by PCR.
During the echovirus 30 outbreak we identified 22.3% of samples as positive, almost exclusively echovirus 30. In 1998 only 7.1% of samples were positive and a rather broad range of agents was isolated. In 1999 10.4% were positive, predominantly coxsackie B5 and echovirus 11. We could show that in acute enterovirus infections, virus detection by cell cuLture and PCR is superior to serological methods (neutralization assay and IgM assay). For virus isolation, there was a higher rate of positives from stool compared to CSF (1997: 27.8% versus 25%; 1998: 14.4% versus 3%; 1999: 17.9% versus 8.5%). When comparing PCR and virus isolation from the CSF, the former yielded a higher rate of positive results but was not clearly superior to virus isolation from CSF.
The recommended method for the diagnosis of acute enterovirus infections is virus isolation from feces. In cases of suspected aseptic meningitis virus isolation and PCR are valuable for the direct detection of virus in CSF.
Infection 29(3):138-42. · 2.66 Impact Factor
