Elisabeth Puchhammer-Stöckl

Prevalence of Drug-Resistant HIV-1 Variants in Untreated Individuals in Europe: Implications for Clinical Management

BACKGROUND Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis. METHODS We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002. RESULTS In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001. CONCLUSIONS Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

Viral features of lamivudine resistant hepatitis B genotypes A and D

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty‐one patients carrying lamivudine resistant HBV were enrolled. Twenty‐six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P = .006) and rt180M mutants (81% in group A vs 40% in group D, P = .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P = .004). In a multivariate analysis, HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log10 6.99 copies/ml; range 3–9) compared with group D (mean log10 6.1 copies/ml; range 3.3–8; P = .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow‐up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV. (HEPATOLOGY 2004;39:42–50.)

The Calculated Genetic Barrier for Antiretroviral Drug Resistance Substitutions Is Largely Similar for Different HIV-1 Subtypes

Background: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe. Methods: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype. Results: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02_AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes. Conclusions: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

Diagnosis of herpesvirus infections of the central nervous system

BACKGROUND Human herpesviruses may cause infections of the central nervous system (CNS). The early diagnosis of herpesvirus-associated neurological diseases is of high importance. OBJECTIVES The objective of this paper is to summarize the experience gained with the diagnosis of herpesvirus infections of the CNS at our institute by polymerase chain reaction (PCR)-based assays within the past few years. STUDY DESIGN A retrospective analysis of herpesvirus desoxy ribonucleic acid (DNA)-positive cerebrospinal fluid (CSF) samples was performed, with particular emphasis on data obtained by quantification of virus DNA in CSF by newly established real-time quantitative PCR assays. RESULTS Herpesviruses were found in 26.6% of all virus-positive CSF samples detected at our institute between 1995 and 2001. The overall broad testing for different herpesviruses from CSF has led to an increase in the detection rate, especially in relation to varicella zoster virus (VZV)-associated CNS disease. The herpesvirus DNA load in CSF was investigated by TaqMan real-time PCR assays that were established for the individual herpesviruses. The amount of virus varied among the individual diseases, associated with herpes simplex virus type 1, herpes simplex virus type 2, VZV and cytomegalovirus, while for Epstein-Barr virus and human herpesvirus type 6 only low levels of virus were detectable in CSF. CONCLUSIONS A generally broad testing for different herpesviruses in CSF samples is highly recommended. In addition, determination of the virus DNA level in CSF by quantitative assays seems to be of high importance for elucidating aspects concerning the prognosis of disease, the prediction of distinct CNS manifestations, and possibly the differentiation between specific virus-associated disease and unspecific presence of virus in CSF, especially in immunocompromised patients.

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay

BACKGROUND Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. OBJECTIVES (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. STUDY DESIGN (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. RESULTS An RT-nested PCR assay was established with a detection limit of 100-1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. CONCLUSIONS The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.

Recurrent granulocytic aplasia as clinical presentation of a persistent parvovirus B19 infection

Summary. We report a case of persistent infection with human parvovirus B19 (PVB19), manifesting clinically as recurrent agranulocytosis and in bone marrow biopsy as recurrent pure granulocytic aplasia. Persistence of parvovirus infection was documented by the presence of PVB19 DNA and anti‐PVB19‐IgM antibodies in the serum for a period of 19 months. Granulocytic aplasia occurred only when anti‐PVB19‐IgG antibodies were not detectable in the serum and granulopoiesis showed immediate recovery with high dose intravenous immunoglobulin treatment. This case report suggests that the erythroid precursor cell may not be the only target cell of PVB19 infection. We suggest testing for active parvovirus infection in cases of aplasia of any lineage of the haematopoietic system.

Monitoring the Virus Load Can Predict the Emergence of Drug-Resistant Hepatitis B Virus Strains in Renal Transplantation Patients during Lamivudine Therapy

The development of resistant hepatitis B virus (HBV) strains during lamivudine treatment has been described repeatedly. To investigate whether the development of such resistant HBV strains can be predicted in an early phase of therapy, the HBV loads of 11 renal transplantation patients were screened at 3-month intervals by a quantitative HBV polymerase chain reaction (PCR) assay. Lamivudine resistance was detected by sequence analysis. Five patients developed resistance to lamivudine in the 12-15-month follow-up period. In all of them, a virus load of 1x103 HBV DNA copies still was detectable after 3 months of therapy. This was statistically significantly different from those patients who did not develop lamivudine resistance within the observation period, all of whom had no HBV DNA detectable after 3 months of treatment (P=.0022). Thus, virus load testing by use of a sensitive PCR assay allows the early prediction of the emergence of lamivudine-resistant HBV strains.

Detection of Varicella Zoster Virus (VZV) DNA in fetal tissue by polymerase chain reaction

Primary Varicella Zoster Virus (VZV) infection during pregnancy can be associated with severe fetal malformation. The virological diagnosis of congenital VZV syndrome has not yet been successful, however, as VZV could not be isolated from fetal tissue. We have now applied a polymerase chain reaction (PCR) assay to allow the detection of VZV DNA in fetuses whose mothers had acquired chickenpox in the first trimester of pregnancy. In 2 of 3 fetuses investigated viral DNA was identified by PCR independently in 2 laboratories, thus confirming a direct fetal VZV infection.

Association of hepatitis C virus infection with chronic liver disease in paediatric cancer patients

A total of 203 paediatric cancer treatment survivors were tested for serum antibodies against hepatitis-C virus (anti-HCV). Anti-HCV was detected in 41 patients (20.2%) with first generation anti-HCV ELISA. Positive results were confirmed in all samples retested with a second generation ELISA (n=35) and in all but two cases re-analysed by immunoblotting (n=23). Anti-HCV positive children had received significantly more blood product transfusions compared to seronegative patients. In 75 children (32%) chronic liver disease was found. It was defined as an elevation of serum alanine aminotransferase values to a least 2.5 times the upper limit of normal persisting for 6 months or longer. Hepatitis A was never detected, and in 58 children the chronic hepatopathy was unexplained by hepatitits B (non-A non-B chronic liver disease). Of these patients 29 (50%) were seropositive for anti-HCV. Surprisingly, non-A/non-B chronic liver disease was associated with anti-HCV in 14 of 19 solid tumour patients (78.9%), but in no more than 14 of 39 leukaemia and lymphoma patients (35.9%). This phenomenon was not explained by different rates of cytomegalovirus disease and drug toxicity related hepatopathies between the two groups. It may be related to differences of leukaemia/lymphoma compared to solid tumour therapy schedules (differential immuno-suppression and liver toxicity).

Global Dispersal Pattern of HIV Type 1 Subtype CRF01_AE: A Genetic Trace of Human Mobility Related to Heterosexual Sexual Activities Centralized in Southeast Asia

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE originated in Africa and then passed to Thailand, where it established a major epidemic. Despite the global presence of CRF01_AE, little is known about its subsequent dispersal pattern. METHODS We assembled a global data set of 2736 CRF01_AE sequences by pooling sequences from public databases and patient-cohort studies. We estimated viral dispersal patterns, using statistical phylogeographic analysis run over bootstrap trees estimated by the maximum likelihood method. RESULTS We show that Thailand has been the source of viral dispersal to most areas worldwide, including 17 of 20 sampled countries in Europe. Japan, Singapore, Vietnam, and other Asian countries have played a secondary role in the viral dissemination. In contrast, China and Taiwan have mainly imported strains from neighboring Asian countries, North America, and Africa without any significant viral exportation. DISCUSSION The central role of Thailand in the global spread of CRF01_AE can be probably explained by the popularity of Thailand as a vacation destination characterized by sex tourism and by Thai emigration to the Western world. Our study highlights the unique case of CRF01_AE, the only globally distributed non-B clade whose global dispersal did not originate in Africa.