Lower Viral Load Levels Research Articles

Truncated chicken MDA5 enhances the immune response to inactivated NDV vaccine

Melanoma Differentiation-Associated protein 5 (MDA5) is a cytoplasmic sensor for viral invasion and plays an important role in regulation of the immune response against Newcastle disease virus (NDV) in chickens. MDA5 was used as an adjuvant to enhance the humoral immune response against influenza virus. In the current study, truncated chicken MDA5 [1–483 aa, chMDA5(483aa)] expressed by recombinant adenovirus was administered to specific-pathogen-free (SPF) chickens to improve the immune response induced by inactivated NDV vaccine. A total of 156 SPF chickens were divided into six groups, and after two rounds of immunization, the humoral immune response, cell-mediated immune (CMI) response and the protective efficacy of the vaccines against NDV challenge were evaluated. The results showed that co-administration of chMDA5(483aa) expressed by adenovirus increased the NDV-specific antibody response by 1.7 times and chickens received chMDA5(483aa) also gained a higher level of CMI response. Consistently, the protective efficacy of the inactivated NDV vaccine against virulent NDV (vNDV) challenge was improved by co-administrate with chMDA5(483aa), as indicated by the reduced morbidity and pathological lesions, lower levels of viral load in organs and reduced virus shedding. Our study demonstrated that chMDA5(433aa) expressed by adenovirus could enhance the immune efficacy of inactivated NDV vaccine in chickens and could be a potential adjuvant candidate in developing chicken NDV vaccines.

Persistence of low levels of plasma viremia and of the latent reservoir in patients under ART: A fractional-order approach

Low levels of viral load are found in HIV-infected patients, after many years under successful suppressive anti-retroviral therapy (ART). The factors leading to this persistence are still under debate, but it is now more or less accepted that the latent reservoir may be crucial to the maintenance of this residual viremia. In this paper, we study the role of the latent reservoir in the persistence of the latent reservoir and of the plasma viremia in a fractional-order (FO) model for HIV infection. Our model assumes that (i) the latently infected cells may undergo bystander proliferation, without active viral production, (ii) the latent cell activation rate decreases with time on ART, (iii) the productively infected cells’ death rate is a function of the infected cell density. The proposed model provides new insights on the role of the latent reservoir in the persistence of the latent reservoir and of the plasma virus. Moreover, the fractional-order derivative distinguishes distinct velocities in the dynamics of the latent reservoir and of plasma virus. The later may be used to better approximations of HIV-infected patients data. To our best knowledge, this is the first FO model that deals with the role of the latent reservoir in the persistence of low levels of viremia and of the latent reservoir.

Sex differences in HIV-1-mediated immunopathology.

The article reviews our current knowledge regarding the role of sex and sex hormones in regulating innate immune responses to viral infections, which may account for the described sex differences in immunity to HIV-1. Prominent sex differences exist in various infectious and autoimmune diseases. Biological mechanisms underlying these differences include the modulation of immunological pathways by sex hormones and gene dosage effects of immunomodulatory genes encoded by the X chromosome. During HIV-1 infections, women have been shown to present with lower viral load levels in primary infection, although their progression to AIDS is faster in comparison with men when accounting for viral load levels in chronic infection. HIV-1-infected women furthermore tend to have higher levels of immune activation and interferon-stimulated gene expression in comparison with men for the same viral load, which has been associated to innate sensing of HIV-1 by Toll-like receptor 7 and the consequent interferon-α production by plasmacytoid dendritic cells. Improvement in understanding the mechanisms associated with sex differences in HIV-1-mediated immunopathology will be critical to take sex differences into consideration when designing experimental and clinical studies in HIV-1-infected populations.

ADVANCED LIVER INJURY IN PATIENTS WITH CHRONIC HEPATITIS B AND VIRAL LOAD BELOW 2,000 IU/mL.

SUMMARYIntroduction:According to the guidelines, the viral load of 2,000 IU/mL is considered the level to differentiate between inactive carriers and HBeAg(-) chronic hepatitis B patients. Even so, liver damage may be present in patients with lower viral load levels, mainly related to regional variations. This study aims to verify the presence of liver injury in patients with viral load below 2,000 IU/mL.Methods:Patients presenting HBsAg(+) for more than six months, Anti-HBe(+)/HBeAg(-), viral load below 2,000 IU/mL and serum ALT levels less than twice the upper limit of normality underwent liver biopsy. Clinical and laboratory characteristics were evaluated in relation to the degree of histologic alteration. Liver injury was considered advanced when F ≥ 2 and/or A ≥ 2 by the METAVIR classification.Results:11/27 (40.7%) patients had advanced liver injury, with a mean viral load of 701.0 (± 653.7) IU/mL versus 482.8 (± 580.0) IU/mL in patients with mild injury. The comparison between the mean values of the two groups did not find a statistical difference (p = 0.37). The average of serum aminotransferases was not able to differentiate light liver injury from advanced injury.Conclusions:In this study, one evaluation of viral load did not exclude the presence of advanced liver damage. Pathologic assessment is an important tool to diagnose advanced liver damage and should be performed in patients with a low viral load to indicate early antiviral treatment.

Systemic Cytokine Levels Do Not Predict CD4(+) T-Cell Recovery After Suppressive Combination Antiretroviral Therapy in Chronic Human Immunodeficiency Virus Infection.

Background. Subjects on suppressive combination antiretroviral therapy (cART) who do not achieve robust reconstitution of CD4+ T cells face higher risk of complications and death. We studied participants in the Women's Interagency HIV Study with good (immunological responder [IR]) or poor (immunological nonresponder [INR]) CD4+ T-cell recovery after suppressive cART (n = 50 per group) to determine whether cytokine levels or low-level viral load correlated with INR status.Methods. A baseline sample prior to viral control and 2 subsequent samples 1 and 2 years after viral control were tested. Serum levels of 30 cytokines were measured at each time point, and low-level human immunodeficiency virus (HIV) viral load and anti-HIV antibody levels were measured 2 years after viral suppression.Results. There were minimal differences in cytokine levels between IR and INR subjects. At baseline, macrophage inflammatory protein-3β levels were higher in IR subjects; after 1 year of suppressive cART, soluble vascular endothelial growth factor-R3 levels were higher in IR subjects; and after 2 years of suppressive cART, interferon gamma-induced protein 10 levels were higher in INR subjects. Very low-level HIV viral load and anti-HIV antibody levels did not differ between IR and INR subjects.Conclusions. These results imply that targeting residual viral replication might not be the optimum therapeutic approach for INR subjects.

PTU-099 Low risk of lamivudine resistance in hbv, hbeag negative patients. time to reconsider?

Introduction Lamivudine has been largely replaced for the treatment of chronic HBV infection by new (and more expensive) antivirals with a high viral resistance barrier. It has been reported that lamivudine resistance can be as high as 20% after one year and up to 80% at five years. We therefore assessed a cohort of our patients for the development of Lamivudine resistance over time. Method 100 chronic HBV hepatitis patients, histologically diagnosed (male 67, female 33), median age 59 years (range 20–75), initially received lamivudine and were followed up for at least 5 years. All were HBeAg negative, HbeAb positive. Genotype D was found in all 23 patients genotyped. All were non-cirrhotic on liver biopsy before treatment (66% Ishak fibrosis stage 2–3, 34% Ishak stage 4–5). Median HBV DNA at the commencement of treatment was 450000 IU/ml. Only 12 patients had a viral load over 1000000 IU/ml. Lamivudine resistance was considered when an increase of 2 logs in the viral load, followed by an aminotransferase increase after an initial response was noted on follow up. Results All patients responded to lamivudine with normalisation of aminotransferases and subsequent reduction of HBV DNA to less than 100 IU/ml within 6 months from treatment initiation. Lamivudine resistance was 5% at one year, 10% at 2 years, 17% at 4 years and 21% at 5 years Conclusion The risk for lamivudine resistance development in our chronic HBV hepatitis patients is well below the reported in the literature. Whether this is due to the relative lower levels of viral load or to the lack of cirrhotics in our cohort remains to be further examined. Nonetheless we believe that in view of the low cost, lamivudine use should possibly be reconsidered as a first line treatment In HBV infection for non-cirrhotic patients with a viral DNA below 1000000 IU/ml. Disclosure of interest None Declared.

Behavioral intervention improves treatment outcomes among HIV-infected individuals who have delayed, declined, or discontinued antiretroviral therapy: a randomized controlled trial of a novel intervention.

Nationally up to 60% of persons living with HIV are neither taking antiretroviral therapy (ART) nor well engaged in HIV care, mainly racial/ethnic minorities. This study examined a new culturally targeted multi-component intervention to address emotional, attitudinal, and social/structural barriers to ART initiation and HIV care. Participants (N=95) were African American/Black and Latino adults with CD4 <500cells/mm(3) not taking ART, randomized 1:1 to intervention or control arms, the latter receiving treatment as usual. Primary endpoints were adherence, evaluated via ART concentrations in hair samples, and HIV viral load suppression. The intervention was feasible and acceptable. Eight months post-baseline, intervention participants tended to be more likely to evidence "good" (that is, 7days/week) adherence (60 vs. 26.7%; p=0.087; OR=3.95), and had lower viral load levels than controls (t(22)=2.29, p=0.032; OR=5.20), both large effect sizes. This highly promising intervention merits further study.

Broad and persistent Gag-specific CD8+ T-cell responses are associated with viral control but rarely drive viral escape during primary HIV-1 infection.

We characterized protein-specific CD8 T-cell immunodominance patterns during the first year of HIV-1 infection, and their impact on viral evolution and immune control. We analyzed CD8 T-cell responses to the full HIV-1 proteome during the first year of infection in 18 antiretroviral-naïve individuals with acute HIV-1 subtype C infection, all identified prior to seroconversion. Ex-vivo and cultured interferon-γ ELISPOT assays were performed and viruses from plasma were sequenced within defined CTL Gag epitopes. Nef-specific CD8 T-cell responses were dominant during the first 4 weeks after infection and made up 40% of the total responses at this time; yet, by 1 year, responses against this region had declined and Gag responses made up to 47% of all T-cell responses measured. An inverse correlation between the breadth of Gag-specific responses and viral load set point was evident at 26 weeks after infection (P = 0.0081, r = -0.60) and beyond. An inverse correlation between the number of persistent responses targeting Gag and viral set point was also identified (P = 0.01, r = -0.58). Gag-specific responses detectable by the cultured ELISPOT assay correlated negatively with viral load set point (P = 0.0013, r = -0.91). Sequence evolution in targeted and nontargeted Gag epitopes in this cohort was infrequent. These data underscore the importance of HIV-specific CD8 T-cell responses, particularly to the Gag protein, in the maintenance of low viral load levels during primary infection, and show that these responses are initially poorly elicited by natural infection. These data have implications for vaccine design strategies.

New dolutegravir resistance pattern identified in a patient failing antiretroviral therapy.

IntroductionThe most recently approved antiretroviral, the integrase inhibitor dolutegravir (DTG), is described to be a very potent drug with a unique resistance profile, but a certain degree of cross-resistance to RAL or EVG induced drug resistance, which is mediated mainly by integrase mutations at positions 140 and 148. The impact of a single N155H mutation to DTG resistance is described to be minor. However, there is only rare data available about the impact of N155H in the context of secondary site integrase mutations. Here, we present a case of virological failure in a DTG treated patient based on N155H mutation background.MethodsTherapy monitoring of an HIV-HCV co-infected patient harbouring already an omni-drug-class resistant HIV-1 in consequence of more than 20 years ART history. Drug susceptibility testing was performed by RT-PCR from plasma and subsequent Sanger sequencing. Tropism testing was done from proviral DNA with FPR cut-offs according to the German recommendations.ResultsIn 2013, the patient harbouring a virus with high level resistance to all RTI and PI received a regimen containing FTC, TDF, DRV/r, RPV, T20, and RAL to handle a viral load of 5000 RNA copies/mL, but never achieved fully suppressed viral load. In June 2013, after S119R, N155H and E157Q mutations in viral integrase were detected, the patient received DTG, and RAL was stopped. One month later, when viral load was undetectable for the first time since 2007, ART was de-escalated by removing T20. Since February 2014, low-level viral load was re-detectable. Two new mutations T97A and S147G in the integrase and no other new resistance associated mutations in protease and reverse transcriptase in comparison to the sample analyzed in June 2013 were detected.ConclusionsHighly resistant HIV-1 strains have been a common problem in the past. Their frequencies were pushed back by highly potent ART, but the virus is still able to become resistant against all available antiretrovirals at once. The here documented strain became resistant to DTG without carrying mutations at the described positions 140 and 148, but in the context of integrase N155H. Since N155H alone is described not to contribute sufficient resistance to DTG, there seems to be a need to re-check viruses with N155H plus minor mutations (like T97A, S119R, S147G and E157Q) potentially contributing to DTG resistance.

The Bounds of Time Lag and Chemotherapeutic Efficacy in the Control of HIV/AIDS

The current use of Highly Active Anti-Retroviral Therapy (HAART) strategy to control Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) is inefficient in eradicating HIV/AIDS due to inadequate understanding of the dynamics relating to interaction between the immune system components and HIV. As a result, a pool of potential transmitters is continuously created and thus HIV has remained a pandemic. In this paper, we formulate a mathematical model using differential equations to study the effects of time lag τ&gt;0 due to cellular latency and pharmacological delays and chemotherapy on the control strategy of AIDS epidemic. Equilibrium points of the model are computed and used to determine the reproductive ratio〖 R〗_0. This important threshold parameter is then used to determine the critical bounds of time lag τ∈[τ_min,τ_min] and therapeutic window C_p∈[MEC,MTC] that is, the bounds; above Minimum Effect Concentration (MEC) and below Minimum Toxic Concentration (MTC), where drug plasma concentration C_p should lie for effective maintenance of low levels of viral load and reduction of drug toxicity. The mathematical model gives qualitative understanding of HIV prognostic information which is a means of rejuvenating the existing Antiretroviral drugs (ARV’s). Numerical simulations show that a stable and persistent endemic equilibrium state of low viral load is achieved when these thresholds τ∈[0,25] and C_p∈[0.79,0.91] are satisfied. This persistent equilibrium state will lead to eventual eradication of HIV/AIDS.

Long-term control of HIV replication with dolutegravir and pegylated interferon alpha-2a in an HIV-infected patient with sixtuple-class resistance

With contemporary antiretroviral therapy (ART), the risk of virological failures leading to multiclass resistance has dramatically diminished. However, there are still patients from the pre highly active ART era with multiple-class resistance for whom designing salvage regimens can be very challenging. We report the use of a novel combination – dolutegravir (DTG) and peg-interferon alfa-2a – in a patient with sixtuple-class resistance. The patient is a 56-year-old man, diagnosed with HIV infection in 1992. After diagnosis, he started treatment with azydothimidine monotherapy and subsequently received dual [nucleoside reverse transcriptase inhibitor NRTI] and triple (NRTI + non-NRTI/protease inhibitor)-drug antiretroviral regimens, as part of routine care and through clinical trials such as TORO (enfuvirtide) [1], DUET (darunavir/ritonavir) [2] (randomized to control arm), and raltegravir expanded access. He had been treated with all of the following: dideoxycytidine dideoxyinosine saquinavir, nelfinavir, nevirapine, abacavir, efavirenz, hydroxyurea, lopinavir/ritonavir, amprenavir, enfuvirtide, tipranavir, tenofovir, emtricitabine (FTC), darunavir/ritonavir, etravirine, maraviroc and raltegravir. Unfortunately, he received many of these antiretovirals as functional monotherapy, without other active drugs. He had never achieved sustained virological suppression. In 2010, while receiving tenofovir difumarate (TDF)/FTC and darunavir/ritonavir and maraviroc, he developed oral candidiasis and wasting. At that time, his CD4+ cell count was 28 cells/μl and the HIV-RNA viral load 1 100 000 copies/ml. Drug resistance genotype testing showed the following as described below. Reverse transcriptase (bold: mutations present in historical genotypes, but not in the last available genotype): M41L, K101E, V108I, V118I, Y181C, M184 V, G190A L210W, T215Y, V179F. According to the Stanford University HIV Drug Resistance Database interpretation algorithm these mutations confer high-level resistance to all nucleos(t)ides and non-nucleoside reverse transcriptase inhibitors. Protease: L10F, V32I, I54L, Q58E, G73S, V82L, I84V, L90M, V11I, M36L, L89V. These mutations confer high-level resistance to all protease inhibitors. Fusion inhibitor resistance: V38M V38A. These mutations reduce enfuvirtide susceptibility by more than 10-fold in site-directed mutants and most clinical isolates. HIV tropism test performed in April 2009 (Trofile): dual/mixed R5/X4 tropic virus. CCR5 inhibitors not active. Integrase: E92Q, N155H, N155I. These mutations confer high-level resistance to raltegravir and elvitegravir and low-level resistance to DTG. On April 2011, the patient initiated DTG (50 mg b.i.d.) through a named patient/compassionate use program, high-dose darunavir/ritonavir (1000/100 mg b.i.d.), peg-interferon alfa-2a (180 μg weekly) and stavudine (d4T), TDF/FTC and enfuvirtide at standard doses (Table 1). This initial regimen produced a rapid decrease in viral load, but the patient did not achieve complete suppression. For this reason, enfuvirtide was switched to foscarnet after 1 month. Subsequently, the patient achieved virological suppression and foscarnet was stopped. We performed one attempt to switch peg-interferon alfa-2a for oral valaciclovir, but due to a low-level viral rebound valaciclovir was discontinued and weekly peg-interferon alfa-2a reintroduced. Subsequently, for the past 18 months, the patient has been receiving DTG, high-dose darunavir/ritonavir, maraviroc and weekly peg-interferon alfa-2a. This regimen has maintained consistent virologic suppression. The patient has experienced substantial immunological reconstitution with increasing CD4+ cell counts and complete resolution of oral candidiasis and wasting.Table 1: HIV-RNA and CD4+ evolution.We believe that peg-interferon alfa-2a contributed significantly to the antiviral efficacy of the regimen. It is a proven antiviral with activity against HIV [3]. In our case, one attempt to stop peg-interferon alfa-2a, when the patient was virologically suppressed was followed by a low-level viral load rebound. Interestingly, Azzoni et al.[4] have recently reported that administration of peg-interferon alfa-2a to individuals receiving suppressive ART resulted in a sustained control of viral replication in almost half of the patients who interrupted antiretrovirals. If we consider peg-interferon alfa-2a as an active antiretroviral in the background regimen, then our patient's response is in accordance with the DTG VIKING 3 clinical trial results. In VIKING 3, if the baseline genotype did not harbor the Q148 integrase resistance mutations, and the background regimen had an overall susceptibility score of 1, then the proportion of patients with HIV-RNA less than 50 copies/ml at week 24 was 88% [5]. We maintain unconventionally high-dose darunavir/ritonavir in light of the high-level resistance, thus expecting residual activity. We also maintain maraviroc with the hope that it retains activity against subpopulations of R5 tropic viruses. These observations remain speculative, as we have not attempted to discontinue each of the components of the regimen. Interestingly, we have been able to stop all inactive nucleos(t)ides. In summary, our case provides a proof of concept that, in the context of sixtuple-class resistance, the combination of DTG and peg-interferon alfa-2a is able to achieve long-term virologic control if there are no Q148 integrase drug-resistance mutations. This combination merits further study in exceptionally difficult-to-treat cases such as ours. Acknowledgements Conflicts of interest There are no conflicts of interest.

APOBEC3G-Augmented Stem Cell Therapy to Modulate HIV Replication: A Computational Study

The interplay between the innate immune system restriction factor APOBEC3G and the HIV protein Vif is a key host-retrovirus interaction. APOBEC3G can counteract HIV infection in at least two ways: by inducing lethal mutations on the viral cDNA; and by blocking steps in reverse transcription and viral integration into the host genome. HIV-Vif blocks these antiviral functions of APOBEC3G by impeding its encapsulation. Nonetheless, it has been shown that overexpression of APOBEC3G, or interfering with APOBEC3G-Vif binding, can efficiently block in vitro HIV replication. Some clinical studies have also suggested that high levels of APOBEC3G expression in HIV patients are correlated with increased CD4+ T cell count and low levels of viral load; however, other studies have reported contradictory results and challenged this observation. Stem cell therapy to replace a patient’s immune cells with cells that are more HIV-resistant is a promising approach. Pre-implantation gene transfection of these stem cells can augment the HIV-resistance of progeny CD4+ T cells. As a protein, APOBEC3G has the advantage that it can be genetically encoded, while small molecules cannot. We have developed a mathematical model to quantitatively study the effects on in vivo HIV replication of therapeutic delivery of CD34+ stem cells transfected to overexpress APOBEC3G. Our model suggests that stem cell therapy resulting in a high fraction of APOBEC3G-overexpressing CD4+ T cells can effectively inhibit in vivo HIV replication. We extended our model to simulate the combination of APOBEC3G therapy with other biological activities, to estimate the likelihood of improved outcomes.

Impact of Latently Infected Cells on Strain Archiving Within HIV Hosts

Latently infected cells are a barrier to HIV eradication on therapy due to long half-lives of between 6 and 44 months. The mechanism behind this long term maintenance is unclear although bystander proliferation and asymmetric division have both been put forward for consideration in mathematical models. The latently infected cell reservoir seems to act as an archive for strains of HIV no longer dominant in the blood, such as wild-type virus when the individual is on therapy. This is particularly significant when patients wish to come off medication and wild-type virus re-emerges. We use a two target cell model capable of producing low-level viral load on therapy and include latent cells and two strains of virus, wild-type and drug resistant, to investigate the impact of two possible mechanisms of latent cell reservoir maintenance on strain archiving. We find that although short term (less than a year) archiving of viral strains is possible in a model with no mechanism for reservoir maintenance, both bystander proliferation and asymmetric division of latent cells allow archiving to occur over much longer timescales (2 or more years). We suggest that regardless of the mechanism involved, latent cell reservoir maintenance allows strain archiving to occur. We interpret our results for clinical consideration.

Biodynamics of Hepatitis C Virus in Haemodialysis Patients in Pahang

Introduction: HCV infection is frequent in patients undergoing maintenance haemodialysis, with prevalence between 8 and 10%. Hepatitis C has an adverse effect on both patient and graft survival in those who get renal transplants. There are relatively scarce reports on the natural fluctuation in viral load level in patients on chronic haemodialysis. Materials and Methods: This is a longitudinal short-term three months study, where 27 chronic haemodialysis patients infected with known HCV genotypes were recruited from seven haemodialysis centres in Pahang. Serum samples were collected monthly, both pre- and post-haemodialysis sessions, over a period of three months. Viral RNA was extracted from serum using QIAamp Viral RNA Extraction kit (Qiagen). The HCV viral load was measured using one-step reverse transcriptase qPCR (Applied Biosystems) targeting the 5`HCV non-coding region (5’UTR). The serum α-IFN level was measured using commercial ELISA kit (Amersham, UK). Six biochemical liver function tests (AST, ALP, TP, albumin, ALT and TB) were also done for all pre-haemodialysis samples. Results: All patients showed persistent low level viral load that varied significantly over the study period (p = 0.001). HCV genotype 1 viral load was significantly higher than that of genotype 3. Conclusion: No apparent correlation could be recognized between the viral loads and the corresponding interferon-alpha levels which were detectable in only a few patients during the period of study.

Reply to Seligman

TO THE EDITOR—We appreciate the suggestions made by Dr Seligman in relation to our work on cerebrospinal fluid (CSF) viral escape. As noted, the detection of low-level viremia in blood samples from patients receiving successful suppressive therapy may result from occasional nonadherence. Because of the limited penetration of some antiretroviral drugs into the central nervous system (CNS), adherence may potentially be even more crucial in the treatment of HIV-1 in the CNS. However, we would argue that the detection of low-level viral load in CSF is indeed of potential importance. Because of limitations in accessibility, residual virus in CSF during suppressive antiretroviral therapy is, if at all present, less well characterized than in blood. However, viral load in the CSF compartment during antiretroviral therapy is usually substantially lower than that in blood in neurologically asymptomatic patients [1, 2]. Previously, detection of selective viral escape in the CSF compartment was rare. Even in patients with systemic treatment failure, the level of HIV-1 RNA is frequently substantially lower in the CSF compartment [2]. However, we fully agree that compartmentalized viral resistance in the CNS remains an important factor to investigate. As suggested by Dr. Seligman, we are currently studying this issue. To increase the sensitivity of the analysis, ultra-centrifugation of CSF samples is implemented for improved HIV-1 RNA yield, followed by polymerase chain reaction amplification, sequencing, and genotypic resistance testing. Using these analyses, we hope to further characterize selective viral resistance as a possible factor in relation to the CSF viral escape found in 10% of patients included in the present study [3].

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study

Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL. A total of 16 511 HIV-1 reverse transcriptase and protease sequences from 11 492 treatment-experienced patients were identified, and linked to clinical data on viral load, CD4 T cell counts and antiretroviral treatment history. Test results from 3162 treatment-naive patients served as controls. Multivariable analysis was employed to identify predictors of reverse transcriptase and protease DRMs. Overall, 2500/16 511 (15.14%) test results were obtained at a viral load <1000 copies/mL. Individuals with viral load levels of 1000-10000 copies/mL showed the highest probability of drug resistance to any drug class. Independently from other measurable confounders, treatment-experienced patients showed a trend for DRMs and other mutations to decrease at viral load levels <500 copies/mL. Genotypic testing at low viral load may identify emerging antiretroviral drug resistance at an early stage, and thus might be successfully employed in guiding prompt management strategies that may reduce the accumulation of resistance and cross-resistance, viral adaptive changes, and larger viral load increases.

Molecular Epidemiology of Epstein-Barr Virus in Adult Kidney Transplant Recipients

In transplant recipients, Epstein-Barr virus (EBV) infection may be related to posttransplantation lymphoproliferative disorders (PTLD), the most important risk factors for which are the patient's serostatus before transplantation and the level of immunosuppression. Herein, we have evaluated the impact of EBV in adult kidney transplant recipients by prospectively investigating its molecular epidemiology in 855 consecutive whole blood samples obtained from 290 patients. EBV-DNA monitoring by real-time polymerase chain reaction (PCR) was performed at 3-month intervals in the first year after transplantation and subsequently once a year. Moreover, we studied 55 healthy, non–transplant recipient, blood donors. PCR for EBV-DNA results were positive in 99/855 samples (11.6%) obtained from 72/290 patients (24.8%) versus none from healthy control subjects ( P < .05). There were no differences in the prevalence of EBV-DNA positivity regarding demographic characteristics, underlying pathology leading to transplantation, renal function, and immunosuppressive protocol. Only 4 patients showed a viral load of >2,000 genome equivalents/mL whole blood at >12 months; no patient developed PTLD. In conclusion, although the occurrence of EBV-DNA positivity was frequent, it was usually at low levels of viral load, confirming that adult kidney graft recipients are at a low risk of developing PTLD.

Biodynamics of HCV infection in haemodialysis patients in Pahang

Hepatitis C is a global disease, WHO has described it as a “viral time bomb”. In Malaysia, the sero-prevalence is 1.6%. HCV infection is frequent in patients undergoing maintenance haemodialysis, with prevalence between 8 and 10%. Hepatitis C has an adverse effect on both patient and graft survival in those who get renal transplants. There are relatively scarce reports on the natural fluctuation in viral load and alpha interferon (α-IFN) level in patients on chronic hemodialysis. Methods A longitudinal short-term three months study where 27 chronic hemodialysis patients infected with known HCV genotypes were recruited from seven hemodialysis centers in Pahang. Serum samples were collected monthly, both pre- and post-hemodialysis sessions, over a period of three months. Viral RNA was extracted from serum using QIAamp Viral RNA Extraction kit (Qiagen). The HCV viral load was measured using one step reverse transcriptase qPCR (Applied Biosystems) targeting the 5`HCV non-coding region. The serum α-IFN level was measured using commercial ELISA kit (Amersham, UK). Six biochemical liver function tests (AST, ALP, TP, albumin, ALT and TB) were also done for all pre-hemodialysis samples. Results All patients showed persistant low level viral load (Fig.1) that varied significantly over the study period (P = 0.001). HCV genotype 1 viral load was significantly higher than that of genotype 3 (Fig.2). The difference between pre- and post-haemodialysis viral load was statistically insignificant. No significant correlation between viral load and liver function status was noted. No correlation was observed between pre-haemodialysis serum α-IFN level and pre-haemodialysis viral load. The difference between pre and post-haemodialysis plasma α-IFN levels was statistically insignificant.

Establishment of an end-point limiting-dilution PCR assay for detection of human immunodeficiency virus type 1 quasispecies

Objective To establish a novel end-point limiting-dilution PCR (EPLD-PCR) assay to detect human immunodeficieney virus type 1 (HIV-1) quasispecies and evaluate its application value. Methods The multiplex limiting-dilution nested PCR base on the second round TaqMan real time PCR ( N-RT PCR) and regular PCR, end-point PCR and sequencing technology were used for detection of the sequences of HIV-1 quasispecies env gene c2-v3-c3 region and gag gene p17 region from the PBMCs of 3 HIV-1 infected patients with low level viral load. All sequences of c2-v3-c3 and p17 were confirmed by sequencing. Phylogenetic tree analysis for nucleetide sequences and alignment analysis for amino acid sequences were performed to evaluate the result of HIV-1 quasispecies assay. The HIV-1 DNA copy number in per million PBMCs was calculated by QUALITY program. Results Total 1 207 N-RT PCRs were carried out for all 3 patients' samples from different visiting time points. Sixty-five sequences of c2-v3-c3 and 101 sequences of p17 were obtained from regular nested second round PCRs; the quantification of HIV-1 DNA in per million PBMCs from 3 patients was between 1.34 copies to 53.76 copies. Phylogenetic tree analysis for all nucleotide sequences showed that the sequences in different patients were separated and they got together specially in the same patient. Amino acid sequences alignment analysis showed that HIV-1 quasispecies sequences were simplex at early stage of infection and became more complex alone with increase of infected time. Conclusions The end-point limiting-dilution real-time PCR technology described in this study is a novel, sensitive, specific and high throughput PCR assay to detect human immunedeficiency virus type 1 quasispecies. This PCR assay could be applied for detection of HIV-1 quasispecies in HIV-1 infected populations with low level viral load. Key words: HIV-1 ; Polymerase chain reaction

Impact of Δ32-CCR5 heterozygosity on HIV-1 genetic evolution and variability—A study of 4 individuals infected with closely related HIV-1 strains

A cluster of four patients acutely infected with a genetically almost identical virus, allowed us to investigate genetic variability and disease progression in early HIV-1 infection with minimal interference of virus specific factors. Two of the patients were heterozygous for the 32-bp deletion in the CCR5 coreceptor gene. Both showed a slower disease progression with lower viral load levels and a reduced rate of genetic evolution compared to the patients with normal CCR5 alleles. During 3 years of treatment-free follow-up, the mean pairwise genetic distance increased with 1.45% and 1.58% in the two patients with a 32-bp deletion allele compared to 3.05% and 3.57% in the two patients with normal CCR5 alleles. The observed relation between slower disease progression and a reduced evolutionary rate illustrates the influence of the virus replicative capacity, here most possibly hampered by the CCR5 heterozygosity in two of the four individuals, on the genetic evolution of the virus in the host.