Prevalence and determinants of resistance mutations in HIV-1-infected patients exposed to integrase inhibitors in a large Italian cohort.

OBJECTIVES: The aim of the study was to analyse the prevalence of integrase resistance mutations in integrase strand transfer inhibitor (INSTI)-experienced HIV-1-infected patients and its predictors. METHODS: We selected HIV-1 integrase sequences from the Antiviral Response Cohort Analysis (ARCA) database, derived from INSTI-experienced patients between 2008 and 2017. Differences in the prevalence of resistance to raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG) were assessed by χ2 test and predictors of resistance were analysed by logistic regression. RESULTS: We included 462 genotypes from INSTI-exposed individuals: 356 'INSTI-failing' patients and 106 'previously INSTI-exposed' patients (obtained a median of 42 weeks after INSTI discontinuation [interquartile range (IQR) 17-110 weeks]). Overall, at least low-level resistance (LLR) to any INSTI (Stanford 8.5 algorithm) was detected in 198 (42.9%) cases. The most frequent INSTI resistance mutation was N155H, followed by Q148H/K/R, G140A/C/S, E138A/K/T and Y143C/H/R. Y143R and E138A were more prevalent in viral subtype B versus non-B [5.2 versus 1.5%, respectively (P = 0.04), and 3.1 versus 0%, respectively (P = 0.02)]. Overall, the Q148H/K/R plus G140A/C/S and/or E138A/K/T pattern, defining an intermediate level of resistance to DTG, was detected in 70 (15%) cases. Independent predictors of at least LLR to any INSTI were current use versus past use of INSTIs, a lower genotypic sensitivity score (GSS) for contemporary antiretroviral drugs used, and having an integrase sequence obtained in calendar year 2016 as compared to 2008-2009. CONCLUSIONS: The results support integrase resistance testing in INSTI-experienced patients. Emergence of INSTI resistance is facilitated by the reduced genetic barrier of the regimen as a consequence of resistance to companion drugs. However, INSTI resistance may become undetectable by standard population sequencing upon INSTI discontinuation.

Pregnancy and neonatal outcomes among a cohort of HIV-infected women in a large Italian teaching hospital: a 30-year retrospective study.

The primary study objective was to investigate three decades from 1985 to 2014 of changes in pregnancies among HIV-infected women. The secondary objective was to assess risk factors associated with preterm delivery and severe small-for-gestational-age (SGA) infants in HIV-infected women. A retrospective review of deliveries among pregnant HIV-infected women at the University of Genoa and IRCCS San Martino-IST in Genoa between 1985 and 2014 was performed. Univariate and multivariable analyses were used to study the variables associated with neonatal outcomes. Overall, 262 deliveries were included in the study. An increase in median age (26 years in 1985-1994 vs. 34 years in 2005-2014), in the proportion of foreigners (none in 1985-1994 vs. 27/70 (38·6%) in 2005-2014), and a decrease in intravenous drug use (75·2% (91/121) in 1985-1994 vs. 12·9% (9/70) in 2005-2014) among pregnant HIV-infected women was observed. Progressively, HIV infections were diagnosed sooner (prior to pregnancy in 80% (56/70) of women in the last decade). An increase in combined antiretroviral therapy (cART) prescription during pregnancy (50% (27/54) in 1995-2004 vs. 92·2% (59/64) in 2005-2014) and in HIV-RNA <50 copies/ml at delivery (19·2% (5/26) in 1995-2004 vs. 82·3% (53/64) in 2005-2014) was observed. The rate of elective caesarean section from 1985 to 1994 was 9·1%, which increased to 92·3% from 2004 to 2015. Twelve (10·1%) mother-to-child transmissions (MTCT) occurred in the first decade, and six (8·3%) cases occurred in the second decade, the last of which was in 2000. Preterm delivery (<37 weeks gestation) was 5% (6/121) from 1985 to 1994 and increased to 17·1% (12/70) from 2005 to 2014. In univariate and multivariable logistic regression analyses, advancing maternal age and previous pregnancies were associated with preterm delivery (odds ratio (OR) 2·7; 95% confidence intervals (CI) 1-7·8 and OR 2·6; 95% CI 1·1-6·7, respectively). In the logistic regression analysis, use of heroin or methadone was found to be the only risk factor for severe SGA (OR 3·1; 95% CI 1·4-6·8). In conclusion, significant changes in demographic, clinical and therapeutic characteristics of HIV-infected pregnant women have occurred over the last 30 years. Since 2000, MTCT has decreased to zero. An increased risk of preterm delivery was found to be associated with advancing maternal age and previous pregnancies but not with cART. The use of heroin or methadone has been confirmed as a risk factor associated with severe SGA.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Rs12979860 and rs8099917 single nucleotide polymorphisms of interleukin-28B gene: simultaneous genotyping in caucasian patients infected with hepatitis C virus.

INTRODUCTION: Recent studies have demonstrated the role of the interleukin 28B (IL28B) polymorphisms in predicting treatment induced and spontaneous clearance from Hepatitis C virus (HCV) infection, suggesting the possibility of tailored therapy in HCV infected patients. Genome-wide association studies have shown that single nucleotide polymorphisms (SNPs) near IL 28B gene on chromosome 19 are strong predictors of sustained virologic response (SVR) to pegylated interferon and ribavirin. This study was aimed at analyzing the co-prevalence of two common and clinically significant SNPs in a cohort of Ligurian patients. METHODS: Two SNPs (rs12979860, rs8099917) were genotyped in the IL28B locus from 175 DNA samples collected from HCV-infected consecutive patients in a Laboratory of Liguria Region, northern Italy. A real-time polymerase chain reaction in a Corbett Research Termocycler (Rotor Gene 3000A) by fluorescent probes (Fast Set IL 28B, Arrow Diagnostics) was used for the detection, according to the manufacturer's instructions. RESULTS: Carriers of rs12979860CT genotype predominated (87/175, 50%), homozygotes for allele C were 68/175 (39%) and the remaining were homozygotes for IFN-resistant allele T (11%). As for the rs8099917 SNP, genotypes were thus distributed: 96/175 (55%) carried the rs8099917 TT genotype, whereas 70/175 (40%) and 9/175 (5%), were heterozygotes or homozygotes for the G allele. The variants rs12979860CC and rs8099917TT were found in 39% and 54% of overall patients with HCV genotype 1, respectively. The combined assessment of examined SNPs resulted in three most prevalent genotypes (rs12979860CC/rs8099917TT, rs12979860CT/rs8099917TG and rs12979860CT/rs8099917TT) with a frequency of 35%, 31% and 18%, respectively. DISCUSSION: Recent findings demonstrated that in carriers of rs12979860CT the determination of additional genotype of rs8099917 SNP could significantly improve the prediction of SVR. In our study cohort carriers of rs12979860CT represented 50% of all patients, who could take advantage with respect to SVR prediction by further determination of the rs8099917 SNP. The simultaneous genotyping of two IL28B SNPs should thus be recommended in HCV infected patients prior to treatment initiation.

Comparative hepatitis A seroepidemiology in 10 European countries.

The WHO recommends hepatitis A virus (HAV) immunization according to level of transmission and disease burden. We aimed to identify susceptible age groups by standardized serosurveys to inform HAV vaccination policy in participating countries: Belgium, Czech Republic, England, Finland, Germany, Italy, Lithuania, Malta, Romania, and Slovakia. Each country tested national serum banks (n = 1854-6748), collected during 1996-2004, for anti-HAV antibodies. Local laboratory results were standardized to common units. Forty-one per cent of those aged <30 years and 6% of those aged ≥30 years were susceptible to HAV in Romania; compared to 70-94% and 26-71%, respectively, elsewhere. Romania reported high HAV incidence in children and young adults. Other countries reported HAV disease primarily in older risk groups. The results suggest low level of HAV transmission in most of Europe. Romania, however, appeared as an area with intermediate transmission. Vaccination of risk groups in countries with high susceptibility of young and middle-aged adults needs to be continued.

Prevalence of mutations and determinants of genotypic resistance to etravirine (TMC125) in a large Italian resistance database (ARCA).

OBJECTIVES: To evaluate whether etravirine (TMC125) might be effective in patients failing therapy with current nonnucleoside reverse transcriptase inhibitors (NNRTIs), we analysed the prevalence of TMC125 mutations and the possible determinants of genotypic resistance to this drug among sequences reported to a large database in Italy [Antiretroviral Resistance Cohort Analysis (ARCA)]. METHODS: We analysed the prevalence of TMC125 resistance-associated mutations (RAMs) and the TMC125 weighted genotypic score (WGS) together with the determinants of genotypic resistance. A total of 5011 sequences from 2955 patients failing NNRTI therapy were evaluated. RESULTS: Among the sequences in ARCA, 68% had at least one and 9.8% at least three TMC125 RAMs, whereas 31% had a WGS>2. Frequent RAMs were Y181C, G190A, K101E and A98G, whereas V179F, Y181V and G190S appeared in <5% of sequences. Multivariate analysis revealed a higher risk of developing at least three TMC125 RAMs associated with both nevirapine and efavirenz exposure, whereas CD4 counts > or = 200 cells/microL retained their protective effect. An increased risk of WGS>2 was linked to higher HIV RNA values (maximum risk at >5 log(10) copies/mL) and nevirapine exposure; CD4 counts > or = 200 cells/microL were protective. CONCLUSIONS: The prevalence of TMC125 resistance mutations in the ARCA cohort was 68%. The DUET studies showed that at least three TMC125-associated mutations were required to impair the efficacy of the drug and Y181C/V, V179F and G190S had the greatest effect on response. The prevalence of these mutations among the patients examined in our study was low. However, WGS>2 was found for one-third of our sequences. Previous nevirapine exposure was associated with an increased risk of having WGS>2 (adjusted odds ratio 1.76).

Efficacy and safety of darunavir and etravirine in an antiretroviral multi-experienced youth with vertically HIV-1 infection.

Multiclass-drug resistance, often caused by poor treatment compliance, is a challenging problem in all categories of HIV-infected patients. Selective pressure is higher in youth for both biological and behavioral reasons. We report the case of a 15-year-old Caucasian male, with vertically acquired HIV-1 infection, who failed several lines of antiretroviral therapy and was successfully treated with darunavir/ritonavir and etravirine.

Detection of influenza A(H1N1)pdm09 virus in a patient travelling from Shanghai to Italy in July 2018: an uncommon clinical presentation in a non-seasonal period.

Influenza is one of the most common infectious diseases in travellers, especially in those returning from subtropical and tropical regions. In late June 2018 an influenza A(H1N1)pdm09 virus infection was diagnosed in a 36-years-old man, returned from a travel in Shanghai and hospitalized at the Ospedale Policlinico San Martino, Genoa, Italy, with a diagnosis of fever and an uncommon clinical presentation characterised by a persistent leukopenia. Phylogenetic analysis revealed a closeness with influenza A(H1N1)pdm09 strains circulating in the US in May-June 2018. Prompt recognition of influenza infection led to a proper case management, demonstrating the crucial role of the continuous influenza surveillance programme.

Unexpected high rate of wild-type HIV-1 genotype among inmates failing antiretroviral therapy.

PURPOSE: Combination antiretroviral therapy (ART) has proven to be effective in treating human immunodeficiency virus (HIV) infection. Chronic administration of antiretrovirals presents significant challenges, including the risk of selecting treatment-resistant viral strains that can determine treatment failure and can be transmitted. In many countries, a large proportion of the HIV-infected population goes through the correctional system at least once. Scarce data are available on circulation of resistant HIV strains in correctional facilities. We evaluated the prevalence of antiretroviral resistance among both naïve and treatment-experienced HIV-infected inmates of a correctional institution in Genoa, Italy. METHOD: The prevalence of antiretroviral resistance among the HIV-infected inmates observed at our institution who underwent genotypic testing from January 2004 to June 2007 was retrospectively reviewed. RESULTS: 45 genotypes from 43 inmates were available. Most of the naïve patients (14/16; 87.5%) showed a wild-type (WT) genotype, as well as most of the ART-experienced patients who had discontinued ART (10/13; 76.9%). A high proportion of WT genotype (6/16; 37.5%) was also observed among the subjects apparently failing HAART. CONCLUSIONS: The prevalence of mutated strains in treatment-naïve individuals of the studied cohort is comparable to what is reported in nonimprisoned naïve subjects of our region. The high prevalence of WT genotypes in ART-failing patients makes it likely that they were not taking their treatments, probably to gain legal benefits from their worsening health conditions. Thus, resistance testing can also be considered as an additional tool for assessing adherence to ART for forensic/medicolegal evaluation. However, further and larger studies are necessary to validate it.

Dolutegravir (DTG)-containing regimens after receiving raltegravir (RAL) or elvitegravir (EVG): Durability and virological response in a large Italian HIV drug resistance network (ARCA).

BACKGROUND: Dolutegravir (DTG) is a next-generation HIV integrase inhibitor (INI) with an increased genetic barrier to resistance with respect to raltegravir (RAL) or elvitegravir (EVG). Few data are available on the durability of DTG-containing regimens. OBJECTIVES: We aimed at investigating the duration of the DTG-containing regimen, the occurrence of an HIV-1 RNA blip, and factors associated with DTG virological response. STUDY DESIGN: From the Antiviral Response Cohort Analysis database, we selected 89 HIV-1-positive four-class-experienced subjects who started DTG after receiving RAL or EVG. Factors associated with durability and virological response were analysed by logistic regression. RESULTS: After a median duration of 18.8 [0.4-76.2] months, 79/89 (88.8%) subjects were still on DTG. All subjects remaining on DTG at the end of follow-up had undetectable HIV-1 RNA, compared to 5/10 subjects who discontinued DTG. DTG discontinuation was less frequent in patients who had experienced ≥10 regimens (HR 0.11, p = 0.040). The probability of having an HIV-1 RNA positive value at the last follow-up significantly increased in patients with non-B HIV-1 subtype (HR 5.77, p < .001) and significantly decreased in patients with CD4 nadir >200/µL (HR 0.29, p = 0.038), with more than 10 previous regimens (HR 0.27, p = 0.040), and who harbored virus with IN mutations (HR 0.12, p = 0.023) at DTG start. CONCLUSIONS: After previous exposure to first-generation INIs, treatment with DTG showed long durability and did not show virological rebound after virological suppression. Subjects infected with a non-B HIV-1 subtype had a greater risk of having detectable HIV-1 RNA at the last observation.

Effectiveness, safety, durability and immune recovery in a retrospective, multicentre, observational cohort of ART-experienced, HIV-1-infected patients receiving maraviroc.

The aim of this retrospective, multicentre, observational study was to assess the durability, safety, immune recovery and effectiveness on viral suppression of antiretroviral therapy (ART) in a maraviroc (MVC)-based cohort. We collected clinical, demographical, immunological and virological parameters of adult HIV patients who were infected by CCR5-tropic virus and started an ART regimen containing MVC from 2005 to 2012. We created a longitudinal mixed model to assess the change over time of data. We enrolled 126 drug-experienced patients; the median duration of MVC treatment was 25 months. The probability of stopping ART at one year was 13.3%, and at three years was 27.3%. Statistically significant changes were observed for CD4+ cell count increase ( p < 0.001), HIV-RNA decrease ( p < 0.001) and total cholesterol decrease ( p = 0.005). Ninety-four patients (79.7%) had CD4 ≥ 200 cells/mm3 at baseline while nine of them reached this threshold at nine months (7.6%), 17 (13%) after nine months and six (5%) remained below 200 cells/mm3 at the end of the study. Overall, 114 patients (90.5%) achieved an HIV-RNA ≤ 50 cp/ml. A majority of patients maintained CD4 cell counts of ≥ 200 cells/mm3 and achieved an undetectable HIV viral load within three months. MVC-containing regimens are safe and appear to be a feasible therapeutic option for ART.

Immunogenicity and tolerability of a trivalent virosomal influenza vaccine in a cohort of HIV-infected children.

Twenty-three children infected with the human immunodeficiency virus (HIV) were vaccinated with a trivalent inactivated virosomal influenza vaccine. Serum haemagglutinin inhibition antibody titres were determined for the three viral strains at the time of vaccination and 1 month later. CD4 cell counts and HIV viral loads were measured to evaluate the effect of vaccination on HIV status. Adverse reactions were monitored during the first hour following vaccination by an investigator and then on a continuous basis by the parents. Seroconversion rates against the three viral strains A/H3N2, A/H1N1 and B were 73.9%, 56.5% and 52.2%, respectively. Geometric mean antibody titres increased after 1 month compared with baseline values (A/H3N2: 70.9 versus 13.5; A/H1N1: 24.7 versus 5.8; B: 34.4 versus 9.1). No significant changes were observed in either HIV viral load or CD4 cell count following vaccination. Vaccination was well tolerated with only a few mild, transient symptoms.

A new assay for hepatitis C virus (HCV) core antigen detection: an alternative to nucleic acid technologies in positive or indeterminate anti-HCV subjects?

Markers of viral replication are fundamental tools for understanding the mode of transmission, diagnosis and management of hepatitis C virus (HCV) infection. A new enzyme-linked immunosorbent assay for quantitative detection of free and complexed HCV core antigen (HCV Ag) has been developed. The aim of this study is to evaluate the clinical performance of the new test and compare it with the most widely used commercially available RT-PCR-based assay. To determine the cut-off value we tested 60 samples from anti-HCV negative samples and selected a qualitative cut-off value of 3 pg/ml. To evaluate the usefulness of the new assay in confirming serologically indeterminate results we collected 62 sera. To evaluate the HCV Ag and HCV-RNA relationship we tested 245 samples from patients with different clinical conditions. The results of 61 out of 62 (98.4%) anti-HCV indeterminate samples were found to agree, whereas only one serum was found to be RT-PCR positive and HCV Ag negative. We also found the results to agree in 77.6% (190/245) of the samples from infected patients, while we observed higher agreement in untreated patients, both with and without evidence of liver damage. The correlation coefficient (r) observed between HCV Ag and HCV-RNA was 0.88. The regression line meets the cut-off value at an HCV-RNA concentration of approximately 40,000 IU/ml. In conclusion, we found that the results from the total HCV Ag test agree with the RT-PCR results and therefore we believe that this test could become a useful tool for the diagnosis and management of HCV infection.

An outbreak of hepatitis C virus in a haemodialysis unit: molecular evidence of patient-to-patient transmission.

Hepatitis C virus (HCV) infection remains a major problem in haemodialysis units despite the risk decrease provided by anti-HCV screening of blood. The exact mode of transmission of HCV in the dialysis units remains unclear. To identify the route of the virus and the mechanisms of transmission an investigation into the outbreak of HCV infection in a haemodialysis unit on a molecular level was held: 12 newly infected patients and 14 already infected were investigated by sequencing the 5' untranslated region of the viral genome. The results showed that 3 strains infected new cases and these strains matched those sequenced in already infected patients. Transmission occurred between patients treated on the same shift and also between different rooms. Console and blood or blood product contamination was excluded. Our study gave molecular evidence of patient-to-patient transmission of HCV in a haemodialysis unit. Our data underline the importance of the strict enforcement of standard precautions to prevent HCV transmission and failure of the isolation of anti-HCV positive patients as preventive measure.

Prevalence of etravirine (ETR)-RAMs at NNRTI failure and predictors of resistance to ETR in a large Italian resistance database (ARCA).

The prevalence of drug resistance associated with the failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and the predictors of resistance to Etravirine (ETR) were assessed in 2854 subjects: 39 < 18 (paediatric) and 2815 ≥ 18 (adult) years old. These subjects failed to respond to their current NNRTI treatment, were three-class experienced and had been exposed to NNRTI for ≥3 months. A total of 1827 adult (64.9%) and 32 paediatric subjects (82.1%) harboured the virus with at least one ETR mutation. V179I, Y181C and G190A were the most frequent mutations in both groups. A significantly increased risk of ETR resistance with all three algorithms (Monogram (MGR) >3, Tibotec (TBT) >2 and enhanced MGR (ENH) ≥4) emerged in the paediatric population. Multivariate analysis revealed an increased risk of developing TBT >2 for NNRTI exposure, ENH ≥4 for NNRTI and EFV exposure in paediatric subjects; NVP exposure and higher (≥3.5 log10) HIV-RNA values for all three algorithms in adult subjects, whereas CD4 ≥ 200/µL appeared to be protective. The risk of being ETR resistant was more than doubled for paediatric vs. adult subjects, probably due to a more extensive use of NNRTI and an incomplete virological control.

Factors associated with virological success with raltegravir-containing regimens and prevalence of raltegravir-resistance-associated mutations at failure in the ARCA database.

Raltegravir (RAL) is the only licensed human immunodeficiency virus (HIV) integrase inhibitor. The factors associated with the virological response to RAL-containing regimens and the prevalence of integrase mutations associated with RAL failure deserve further investigation. From the Antiretroviral Resistance Cohort Analysis database, we selected triple-class-experienced subjects failing their current treatment with complete treatment history available. Selection criteria included HIV-RNA, CD4 count and HIV genotype within 3 months of RAL initiation. Factors associated with 24-week response were analysed; genotypic sensitivity scores (GSS) and weighted-GSS were evaluated. Virological response was achieved in 74.3% of 105 subjects. Mutations associated with RAL failure were detected in 12/24 subjects with an integrase genotype, with the prevalence of Q148H + G140S. Each extra unit of GSS (p 0.05, OR 2.62; 95% CI 1.00-6.87). was found to be a associated with response. Weighted-GSS had borderline statistical significance (p 0.063, OR 2.04; 95% CI 0.96-4.33) When stratifying for different cut-offs (<1 as reference, 1-1.49, ≥1.5), a borderline significant increase in the probability of response appeared for GSS ≥1.5 (p 0.053, OR 4.00; 95% CI 0.98-16.25). GSS ≥1 showed the highest sensitivity, 82.6%. Receiver operating characteristic curves depicted the widest area under the curve (0.663, p 0.054) of GSS ≥1. Unresponsiveness to RAL-containing regimens among triple-class-experienced subjects was low. The activity of the background regimen was strongly associated with response. Although few integrase genotypes were available at failure, half of these were without integrase resistance mutations. The substantial rate of RAL failure in the absence of known RAL-resistance mutations may be associated with adherence issues and this issue warrants further analysis in longer observations.

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010.

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.