Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

BACKGROUND: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. METHODS: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. RESULTS: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. CONCLUSIONS: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

High CXCL-16 levels correlate with symptomatic disease in lung transplant recipients with human cytomegalovirus replication in the allograft.

Human cytomegalovirus (HCMV) is an important pathogen in lung transplant recipients (LTRs). In LTRs, HCMV may replicate in the transplanted lung, and this is indicated by HCMV DNA detection in the bronchoalveolar lavage fluid (BALF). Local replication may occur without causing clinical symptoms or, in some patients, it may lead to symptomatic HCMV disease. In the present study, we analyzed whether HCMV replication in the allograft induces CXCL-16, a chemokine that may play a key role in the regulation of mucosal immunity, and investigated whether CXCL-16 levels in BALF can be used to differentiate LTRs with asymptomatic HCMV replication from patients who simultaneously develop disease. In total, BALF samples from 57 LTRs, of whom 8 developed HCMV disease, were assessed for CXCL-16 levels using a quantitative enzyme-linked immunosorbent assay. We found that HCMV replication in the lung triggered a significant rise in CXCL-16 levels in the BALF (p < 0.001, Wilcoxon signed-rank test). Furthermore, the CXCL-16 increase, induced by HCMV, was significantly lower in LTRs who did not develop HCMV disease (p < 0.001, Mann-Whitney U-test). Thus, CXCL-16 is a potential marker that may contribute to identify those LTRs in whom local HCMV replication in the lung remains asymptomatic.

Association of human cytomegalovirus DNAaemia and specific granzyme B responses in lung transplant recipients.

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNAaemia could be associated with HCMV disease and reduced allograft survival. In the present study we analysed whether or not HCMV-specific granzyme B (Grz-B) responses indicating CD8(+) T cell cytotoxicity exert an impact on HCMV DNAaemia and relate to specific interferon (IFN)-γ secretion. HCMV-specific Grz-B responses were quantitated by enzyme-linked immunosorbent assay (ELISA) in 70 samples from 39 HCMV seropositive LTRs who were prospectively investigated for HCMV DNA plasma levels and IFN-γ kinetics using a standardized CD8(+) T cell assay (QuantiFERON®-CMV assay). In all LTRs who were protected from HCMV DNAaemia by early and persistent IFN-γ responses, Grz-B responses were also detected. In LTRs who developed episodes of HCMV DNAaemia, the Grz-B responses which were detected prior to viral DNA detection differed significantly in patients who experienced episodes with high (exceeding 1000 copies/ml) and low plasma DNA levels (P = 0·0290, Fisher's exact test). Furthermore, the extent of Grz-B release prior to viral DNAaemia correlated statistically with the detected levels of IFN-γ (P < 0·0001, Spearman's rank test). Of note, simultaneous detection of Grz-B and IFN-γ secretion was associated significantly with protection from high HCMV DNA plasma levels during the subsequent follow-up (P = 0·0057, Fisher's exact test), and this association was stronger than for IFN-γ detection alone. We conclude that, in addition to IFN-γ responses, Grz-B secretion by CD8(+) T cells is essential to control HCMV replication and a simultaneous measurement of IFN-γ and Grz-B could contribute to the immune monitoring of LTRs.

Analysis of plasma surfactant protein D levels in lung transplant recipients.

In lung transplant recipients (LTRs), severe clinical complications, such as microbial infections of the lung or transplant rejection, may occur. Surfactant protein D (SP-D) is a C-type lectin that is mainly produced in alveolar type II cells. Plasma SP-D levels are usually low, but may increase when the lung-blood barrier is impaired. In this study, we analyzed whether plasma SP-D concentrations reflect rejection or infection of the lung allograft. An enzyme-linked immunosorbent assay was used to measure SP-D levels in plasma samples from 58 LTRs during intervals without pathologic respiratory findings and during episodes of acute cellular rejection (ACR), microbial colonization, and microbial pneumonia. Median plasma SP-D levels were significantly increased during episodes of microbial pneumonia, but not in the absence of pathologic respiratory findings, during microbial colonization, or during ACR up to grade A2-A3 (P < 0.05). During pneumonia, an increased plasma SP-D level was detected in 60% of LTRs and this was further associated with a significantly higher risk for the patients to develop stage III bronchiolitis obliterans syndrome (BOS III) or to die within the subsequent 6 months after pneumonia (P = 0.0093). All patients with a plasma SP-D level of >300 ng/mL during pneumonia developed BOS III and/or died within 6 months of follow-up (P = 0.001). The determination of SP-D levels in plasma during pneumonia in LTRs may be of prognostic value and warrants further evaluation.

Prospective analysis of human cytomegalovirus DNAemia and specific CD8+ T cell responses in lung transplant recipients.

In lung transplant recipients (LuTRs), human cytomegalovirus (HCMV) DNAemia may be associated with HCMV disease and reduced survival of the allograft. Because T cells are essential for controlling HCMV replication, we investigated in this prospective study whether the kinetics of plasma HCMV DNA loads in LuTRs are associated with HCMV-specific CD8+ T cell responses, which were longitudinally assessed using a standardized assay. Sixty-seven LuTRs were monitored during the first year posttransplantation, with a mean of 17 HCMV DNA PCR quantifications and 11.5 CD8+ T cell tests performed per patient. HCMV-specific CD8+ T cell responses displayed variable kinetics in different patients, differed significantly before the onset of HCMV DNAemia in LuTRs who subsequently experienced episodes of DNAemia with high (>1000 copies/mL) and low plasma DNA levels (p = 0.0046, Fisher's exact test), and were absent before HCMV disease. In HCMV-seropositive LuTRs, high-level DNAemia requiring preemptive therapy occurred more frequently when HCMV-specific CD8+ T cell responses fluctuated, were detected only after HCMV DNA detection, or remained undetectable (p = 0.0392, Fisher's exact test). Thus, our data indicate that HCMV-specific CD8+ T cells influence the magnitude of HCMV DNAemia episodes, and we propose that a standardized measurement of CD8+ T cell immunity might contribute to monitoring the immune status of LuTRs posttransplantation.

Human cytomegalovirus infection in lung transplant recipients triggers a CXCL-10 response.

Human cytomegalovirus (HCMV) causes significant morbidity in lung transplant recipients (LTRs). The clinical effects of HCMV replication are determined partly by a type 1 T-helper cell (Th1) response. Because the chemokine interferon-inducible protein of 10 kilodaltons (IP-10, CXCL-10) induces a Th1 response, we investigated whether HCMV triggers IP-10 in LTRs. The IP-10 concentration and HCMV DNA load were determined in 107 plasma and 46 bronchoalveolar lavage fluid (BALF) samples from 36 LTRs. Initial HCMV detection posttransplantation was significantly associated with increased plasma IP-10, regardless of whether the patients showed HCMV DNAemia (p = 0.001) or HCMV replication only in the allograft (p < 0.0001). In subsequent episodes of HCMV detection, plasma IP-10 increased regardless of whether HCMV was detected in blood (p = 0.0078) or only in BALF (p < 0.0001) and decreased after successful antiviral therapy (p = 0.0005). Furthermore, levels of HCMV DNA and IP-10 correlated statistically (p = 0.0033). Increased IP-10 levels in HCMV-positive BALF samples were significantly associated with severe airflow obstruction, as indicated by a decrease in forced expiratory volume in one second (FEV1). Our data indicate that HCMV replication in LTRs evokes a plasma IP-10 response and that, when an IP-10 response is observed in BALF, it is associated with inflammatory airway obstruction in the allograft.

First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Twelve months of lamivudine treatment for chronic hepatitis B virus infection in renal transplant recipients.

BACKGROUND: Chronic hepatitis B virus (HBV) infection increases morbidity and mortality in renal transplant recipients (RTR). Lamivudine has shown promising results in patients with chronic hepatitis B, but experience with its use in RTR is limited. METHODS: In a prospective, open labeled, uncontrolled trial, 19 HBsAg(+) RTR were treated with lamivudine for 12 months. HBV-serologic analysis, HBV-DNA quantitation, and HBV genome sequence analysis were performed every 3 months. RESULTS: At baseline 16 patients were HBV DNA(+), 12 patients were HBeAg(+)/Ab (-). After 3 months HBV DNA was negative in 80% of patients. In the 3 patients with elevated liver enzymes, normal values were achieved within 12 weeks. At 12 months 4 of 8 HBeAg(+)/Ab(-) patients on treatment showed HBeAb, two of them with loss of HBeAg. Three patients developed mutations of the HBV polymerase gene associated with lamivudine resistance. CONCLUSIONS: Lamivudine is safe and effective in HB-sAg(+) RTR, the rate of HBe-seroconversion and of lamivudine-resistance is comparable to that of nonimmunosuppressed patients.

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Early diagnosis of perinatally acquired HIV infection by the polymerase chain reaction.

31 children born to HIV-seropositive mothers were tested for the presence of HIV infection by an HIV-polymerase chain reaction (PCR) assay within the first months of life, at a time when the antibody (AB) tests did not yet allow a diagnosis. The follow-up by Western Blot and/or antigen (Ag)-ELISA indicated an HIV infection in 10 of these children. Failure to detect HIV infection by prior PCR occurred in only 1 of these patients. In this case only one sample was obtained within the first week of life and the negative PCR result may have been due to an infection of the child at delivery rather than during pregnancy. No HIV infection was detected in the remaining 21 cases, using both PCR as well as conventional methods. The simultaneously performed Ag-ELISA was clearly inferior to PCR, identifying only 4 of the 9 PCR-positive patients as early as the PCR. The perinatal HIV transmission rate in Austria was shown to be 32%, similar to that described for other European countries.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Resistance--clinical significance and diagnostics.

One of the most important factors, inhibiting the success of antiretroviral treatment of HIV infections is the development of drug resistant HIV strains in the patients during therapy. Detection and identification of such resistant virus strains is therefore becoming increasingly important for the clinical management of the patients, as well as for the detection of the transmission of already resistant virus strains. In the following paper different methods for the genotypic and phenotypic screening for resistant HIV-strains are compared and the problems of resistance testing are discussed.

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.

Detection of varicella-zoster virus DNA by polymerase chain reaction in the cerebrospinal fluid of patients suffering from neurological complications associated with chicken pox or herpes zoster.

The polymerase chain reaction (PCR) was used to detect varicella-zoster virus (VZV) DNA in the cerebrospinal fluid of patients with VZV infection associated with neurological symptoms. Positive results were obtained in three of five children with post-chicken pox cerebellitis and in seven of seven herpes zoster patients with neurological symptoms. The PCR thus provides a useful tool for the early diagnosis of VZV-associated neurological disease.

Establishment of PCR for the early diagnosis of herpes simplex encephalitis.

The early detection of herpes simplex encephalitis (HSE) represents a major problem in viral diagnostics. We have now established a test system based on PCR for the specific amplification of HSV-DNA in cerebrospinal fluid (CSF) samples followed by oligonucleotide hybridization. The test proved to be very sensitive. Five molecules of HSV 1 DNA still yielded positive signals after hybridization. The assay was applied to CSF samples from 6 patients with confirmed HSE. All except one CSF sample obtained from the 2nd to the 8th day after onset of neurological symptoms yielded positive results. The primers used also exhibit a certain degree of crossreactivity with HSV 2, as revealed by testing of a CSF sample from an HSV 2-infected child. No positive signals were obtained with human DNA and with DNA from CMV- and VZV-infected fibroblasts. Also 42 CSF samples of patients suffering from other diseases of the CNS yielded negative results. The present results indicate that the detection of HSV DNA in CSF by PCR represents a valuable tool for the early diagnosis of HSE.

[The effect of high-dose peroral zidovudine treatment in a 4-year-old child with HIV encephalopathy]. / Effekt einer hochdosierten peroralen Zidovudin-Behandlung bei einem vierjährigen Kleinkind mit HIV-Enzephalopathie.

We report a four-year-old boy with HIV encephalopathy after vertical HIV infection. On the first admission to our hospital he showed ataxia, loose of expressive language and interstitial pneumonia. After treatment of the pneumonia the patient was started on oral zidovudine with 6 x 6 mg/kg bw/day, because of persisting neurologic symptoms and deep white matter lesions in the MR tomogram of the brain. Two months later he showed an improvement of the gait and the reappearance of the expressive language. Seven months after the start with zidovudine the MR tomogram of the brain revealed the disappearance of white matter lesions with exception of little areas of demyelinisation. No side effects of treatment were observed. The only persisting pathological clinical signs were developmental delay of about a half year and moderately hyperactive tendon reflexes of the lower extremities. Our case suggests that even oral treatment with zidovudine can have a beneficial effect on the HIV encephalopathy in infants.

Comparison of virtual phenotype and HIV-SEQ program (Stanford) interpretation for predicting drug resistance of HIV strains.

OBJECTIVE: Drug resistant HIV strains can be identified by genotypic analysis. The prediction of resistance from the HIV pol sequence, however, requires expert interpretation which is currently provided by various interpretation programs. In the present study, the comparability of two of these programs was investigated. METHODS: One hundred and six HIV sequences obtained from patient samples were subjected to interpretation by the Stanford HIV-SEQ program (http://hivdb.stanford.edu) and, in parallel, by virtual phenotype analysis (VircoNET). RESULTS: The overall concordance between the two assays was high with respect to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors. Highly discrepant results were obtained from 22 samples (1.5% of all comparisons), and these discrepancies were significantly associated with the interpretation of nucleoside reverse transcriptase inhibitors (NRTIs) (P < 0.01), especially regarding resistance to zalcitabine (ddC), didanosine (ddI) and abacavir (ABC). Nearly all of these discrepant samples showed a sensitive virtual phenotype. Mutations at codons 69 and 74 were associated with a discrepant interpretation for ddC. CONCLUSIONS: The prediction of resistance to certain NRTIs from a given HIV sequence may be contradictory and requires further investigation.

Comparison of sequence analysis and the INNO-LiPA HBV DR line probe assay for detection of lamivudine-resistant hepatitis B virus strains in patients under various clinical conditions.

A line probe assay (INNO-LiPA HBV DR) detecting drug-resistant hepatitis B virus (HBV) strains was evaluated. Results concordant with sequence analysis were obtained with 48 of 56 serum samples from HBV-infected patients undergoing lamivudine therapy. In eight cases, additional minor subpopulations could be identified by the line probe assay.