HIV RNA Polymerase Chain Reaction Research Articles

240.4: Cytomegalovirus-Induced Thrombotic Microangiopathy After Renal Transplant: Case Report and Review of the Literature

Introduction: CMV infection as a triggering factor for de novo post-transplantation TMA has long been suggested. We herein report a case of CMV and BKPyV co-infection in a kidney transplant recipient and CMV-induced TMA manifested by severe AKI and thrombocytopenia. Case report: A 48-year-old man with end stage kidney disease secondary to diabetes mellitus underwent a 4 HLA-mismatched, deceased donor renal transplant from a 40-year-old donor who died of a cerebrovascular accident. Cold ischemia time and warm ischemia time were 31 and 18 minutes respectively. CMV IgG was positive in donor and negative in recipient. Intravenous methylprednisolone and antithymocyte globulin were used for induction for anticipated delayed graft function. He was discharged on postoperative day 6 with a serum creatinine (SCr) of 8.1 mg/dL. His allograft function continued to improve, achieving a SCr of 1.67 mg/dL at 1-month post-transplantation. His SCr had since fluctuated between 1.4-1.7 mg/dL. At 4-month post-transplantation, he was found to have low grade BK DNAemia (900 copies/mL) managed with mycophenolate dose reduction alone. He did well until 14-month post-transplantation when he presented with non-oliguric AKI with a SCr of 15.6 mg/dL (baseline 1.5 mg/dL) requiring dialysis initiation. Peripheral smear demonstrated thrombocytopenia but no schistocytes. A renal allograft biopsy (figure 3) was performed which demonstrated BK nephropathy, TMA, and no evidence of rejection. Labs revelaed CMV DNAemia of >140,000 copies/ml and BK DNAemia of 17,000 copies/ml. Donor-specific antibodies, HIV RNA polymerase chain reaction (PCR), hepatitis C RNA PCR, anticardiolipin antibody, stool shiga toxin PCR, and stool and blood cultures were all negative. Intravenous ganciclovir was initiated with improvement in CMV DNAemia and concomitant improvement in renal function. He was transitioned to oral valganciclovir after 10 days of intravenous therapy with complete resolution of CMV DNAemia and steady improvement in renal function. His SCr continued improve and nadired at 1.7 mg/dL. At 2-year follow-up, his SCr was 1.74 mg/dL. Discussion: Initiation of IV ganciclovir and reduction in CMV DNAemiwith decrease in SCr and a transition of the patient off hemodialysis suggested that CMV is the causative factor for TMA. CMV-induced renal injury can manifest as various histopathological patterns, commonly as a CMV glomerulopathy with viral inclusions usually seen in the glomerular endothelial cells, acute tubulointerstitial nephritis and rarely as TMA.Improvement in renal function despite continuation of tacrolimus in current patient suggested that calcineurin-inhibitor-induced TMA was unlikely.Although the precise mechanism whereby CMV viremia may cause TMA is currently not fully understood, it is speculated that infection can initiate endothelial cell damage and inflammation which triggers the thrombotic cascade leading to TMA.

The optimal time to test for sero-reversion in HIV-exposed uninfected infants: the later the better?

HIV-exposed uninfected (HEU) infants are tested for loss of maternal antibody (sero-reversion) at 18months of age. Highly sensitive fourth-generation antigen/antibody assays can detect very low levels of antibody, leading to retesting. We audited serological screening outcomes in HEU infants at two National Health Service (NHS) Trusts. HEU infants born between January 2013 and August 2016 were identified via case records. Data collected included gestation; age at testing; test results and assay type. One hundred and forty-two infants were identified, of whom 21 were excluded from analysis. One hundred and one (83%) were born at term and 20 (17%) preterm (<37/40 weeks of gestation), and the median age at first serology was 19.1 [interquartile range (IQR) 18.1; 21.4]months. Initial serology was positive in 10 of 121 infants (8.3%), and the median age of these 10 infants was 18.3 (IQR 18.1; 18.8) months, whereas those with negative serology (n=111) had a median age of 19.2 (IQR 18.1; 21.5) months (P=0.12). All infants with positive HIV serology were born at term. Seven of 10 infants had reactive serology on two fourth-generation assays. Subsequent serology was available for eight of 10 infants, with a median age of 21.3months. Five of the eight (63%) were negative. One was reactive but HIV RNA polymerase chain reaction (PCR) was negative, and one was reactive on screening but negative on confirmatory testing. The remaining child was still seropositive at 24.7months but had a non-reactive result at 29.4months. Overall, 8.3% of HEU infants required repeat testing to confirm loss of antibody. Delaying testing until 22months of age reduces retesting to <2%, with associated resource and emotional implications. Positive serology at 22months should prompt an HIV RNA PCR to exclude infection.

Feasibility and safety of extended-release naltrexone treatment of opioid and alcohol use disorder in HIV clinics: a pilot/feasibility randomized trial.

HIV-infected people with substance use disorders are least likely to benefit from advances in HIV treatment. Integration of extended-release naltrexone (XR-NTX) into HIV clinics may increase engagement in the HIV care continuum by decreasing substance use. We aimed to compare (1) XR-NTX treatment initiation, (2) retention and (3) safety of XR-NTX versus treatment as usual (TAU) for treating opioid use disorder (OUD) and/or alcohol use disorder (AUD) in HIV clinics. Non-blinded randomized trial of XR-NTX versus pharmacotherapy TAU. HIV primary care clinics in Vancouver, BC, Canada and Chicago, IL, USA. Fifty-one HIV-infected patients seeking treatment for OUD (n=16), AUD (n=27) or both OUD and AUD (n=8). Primary outcomes were XR-NTX initiation (receipt of first injection within 4weeks of randomization) and retention at 16weeks. Secondary outcomes generated point estimates for change in substance use, HIV viral suppression [HIV RNA polymerase chain reaction (pcr)<200 copies/ml] and safety. Two-thirds (68%) of participants assigned to XR-NTX initiated treatment, and 88% of these were retained on XR-NTX at 16weeks. In comparison, 96% of TAU participants initiated treatment, but only 50% were retained on medication at 16weeks. Mean days of opioid use in past 30 days decreased from 17.3 to 4.1 for TAU and from 20.3 to 7.7 for XR-NTX. Mean heavy drinking days decreased from 15.6 to 5.7 for TAU and 12.5 to 2.8 for XR-NTX. Among those with OUD, HIV suppression improved from 67 to 80% for XR-NTX and 58 to 75% for TAU. XR-NTX was well tolerated, with no precipitated withdrawals and one serious injection-site reaction. Extended-release naltrexone (XR-NTX) is feasible and safe for treatment of opioid use disorder and alcohol use disorder in HIV clinics. Treatment initiation appears to be lower and retention greater for XR-NTX compared with treatment as usual (clinicaltrials.gov NCT01908062).

How Much HIV is Alive? The Challenge of Measuring Replication Competent HIV for HIV Cure Research

How Much HIV is Alive? The Challenge of Measuring Replication Competent HIV for HIV Cure Research

Absence of Detectible HIV-1 Viremia After Treatment Cessation in an Infant.

Until very recently, the only patient “cured” of HIV was the “Berlin patient,” an adult male treated for HIV-associated acute myeloid leukemia with stem cell transplantation from a donor who was intrinsically resistant to HIV. The donor carried homozygous mutations (delta32) in the chemokine receptor 5 gene, which is an essential co-receptor for HIV entry into most target cells. The present study reports on an infant who also may be cured with early, highly active antiretroviral therapy (ART) after documented perinatal infection.A 35-week gestational age infant was born by normal vaginal delivery to an HIV-infected woman who received no prenatal care and who was not taking ART. ART was initiated in the infant at 30 hours of age with zidovudine, lamivudine, and nevirapine. At 1 week of age, this regimen was adjusted to zidovudine, lamivudine, and ritonavir-boosted lopinavir. The infant remained on ART for 18 months and was then lost to follow-up until 23 months of age. ART has been discontinued and, because of results from subsequent studies, has not been restarted.Standard HIV polymerase chain reaction (PCR) assays and HIV antibody testing were used to diagnose and monitor the HIV-exposed infant. Peripheral blood mononuclear cell cultures were analyzed with PCR for virus DNA and with co-culture techniques for replication-competent virus. Lymphocyte subset analysis was conducted with standard flow cytometry, and HIV-specific T-cell responses were evaluated with intracellular cytokine generation assays.During the first month of life and while receiving ART, the infant had elevated quantitative HIV RNA PCR measurements that declined to undetectable levels and remained there. Subsequently, although proviral DNA was detected at the limits of the assay at 26 months of age, plasma viral RNA was undetectable, and replication-competent virus could not be cultured at age 24 months. HIV antibody assays were not detected in the infant at or after 24 months of age, when maternal antibody would not confound the assays, and HIV-specific T-cell responses were not detected at age 28 months. CD4+ T-cell counts remain normal throughout the study.Very early ART therapy in infants born at risk may abort HIV infection.In patients with established HIV infection, effective ART drives the virus to “undetectable” levels, usually defined as <20/copies of viral RNA/mL of plasma. However, viremia persists in such patients at very low levels and as DNA pro-virus in peripheral blood mononuclear cells. Was this infant cured of HIV? According to the Centers for Disease Control and Prevention definition, the infant was “infected”: 2 positive PCR test results on at least 2 different blood specimens. The data presented demonstrated that after unintended discontinuation of therapy, the infant remained free of replication-competent HIV from at least 12 months of age. As the authors conclude, “very early ART in infants may alter the establishment and long term persistence of HIV-infection.” But why might this happen in a newborn when it has not been demonstrated in older patients except under the unique circumstances of the Berlin patient? HIV persistence depends on the establishment of a reservoir of infected cells. Recent studies suggest that this condition involves a specific population of “central memory T cells.” Perhaps very early therapy aborts or inhibits establishment of this reservoir or perhaps these cells have not fully matured to be infected in newborns. In any case, the present report suggests that very early therapy may prevent establishment of HIV in high-risk infants, and certainly this possibility is being studied intensively.

Clinical Versus Rapid Molecular HIV Diagnosis in Hospitalized African Infants

Many African infants fail to receive their diagnostic HIV molecular test results and subsequently, antiretroviral therapy (ART). To determine whether a point-of-care molecular HIV test increases ART access for hospitalized Malawian infants, we simulated a point-of-care test using rapid HIV RNA polymerase chain reaction (Rapid PCR) and compared patient outcomes with an optimized standard care that included assessment with the World Health Organization clinical algorithm for HIV infection plus a DNA PCR with a turnaround time of several weeks (standard care). Randomized controlled trial. Hospitalized HIV-exposed Malawian infants aged <12 months were randomized into Rapid PCR or standard care. Rapid PCR infants obtained molecular test results within 48 hours to facilitate immediate ART, similar to a point-of-care test. Standard care infants meeting clinical criteria were also offered inpatient ART. The primary outcome was appropriate in-hospital ART for DNA or RNA PCR-confirmed HIV-infected infants. Three hundred infants were enrolled. A greater proportion of HIV-infected infants receiving Rapid PCR, versus standard care, started inpatient ART (72.3% vs 47.8%, P = 0.016). Among molecular test-negative infants, 26.9% receiving standard care unnecessarily initiated inpatient ART, versus 0.0% receiving Rapid PCR (P < 0.001). Rapid PCR modestly reduced the median days to ART (3.0 vs 6.5, P = 0.001) but did not influence outpatient follow-up for HIV-infected infants (78.1% vs 82.4%, P = 0.418). Rapid PCR, versus an optimized standard care, increased the proportion of hospitalized HIV-infected infants initiating ART and reduced ART exposure in molecular test-negative infants, without meaningfully impacting time to ART initiation or follow-up rates.

Performance of HIV-1 DNA or HIV-1 RNA Tests for Early Diagnosis of Perinatal HIV-1 Infection During Anti-retroviral Prophylaxis

M Burgard, S Blanche, C Jasseron. J Pediatr. 2012;160(1):60–66.e1 The identification of HIV-derived nucleic acid in the blood of perinatally exposed infants is the most sensitive and specific method for the early detection of HIV infection in this population. HIV DNA is measured in peripheral blood mononuclear cells and HIV RNA in the plasma. This study compares the performance of HIV DNA polymerase chain reaction (PCR) and HIV RNA PCR for the diagnosis of HIV infection in exposed infants. A total of 1567 children, …

False-positive HIV DNA PCR testing of infants: Implications in a changing epidemic

To examine false-positive HIV DNA polymerase chain reaction (PCR) test results in children, and the potential implications for the paediatric HIV epidemic in sub-Saharan Africa. A review was done of records over a 6-year period of children less than 18 months old at an HIV treatment site in South Africa, to evaluate those with an initial 'false'-positive HIV DNA PCR result, but later proven to be HIV-uninfected with HIV DNA PCR and/or quantitative HIV RNA PCR tests. We calculated the influence of changing HIV transmission rates on predictive values (PV) of HIV DNA PCR tests in a hypothetical population of all HIV-exposed infants over a 1-year period. (Positive PV: proportion of individuals with a positive test with disease; negative PV: proportion of individuals with negative test and no disease). Of 718 children, 40 with an initial positive HIV DNA PCR test were subsequently proven to be HIV-uninfected, resulting in a positive PV of 94.4%. Most (75%) uninfected children had PMTCT interventions and were asymptomatic or mildly symptomatic (77.5%). Calculations using a test specificity of 99.4%, as reported previously, show a decrease in positive PV using a single-test strategy from 98.6% at 30% HIV transmission rate, to 94.8% at 10% transmission, to 62.5% at 1% transmission. Reduction in test specificity further decreases positive PV at low transmission rates. Decreasing mother-to-child HIV transmission rates reduce the positive predictive value of a single HIV DNA PCR test result, necessitating adaptations to diagnostic algorithms to avoid misdiagnosis and inappropriate treatment, especially with early initiation of antiretroviral therapy in asymptomatic infants.

The Structure and Outcomes of a HIV Postexposure Prophylaxis Program in a High HIV Prevalence Setup in Western Kenya

In 2001, HIV postexposure prophylaxis (PEP) was initiated in western Kenya. Design, implementation, and evolution of the PEP program are described. Patient data were analyzed for reasons, time to initiation, and PEP outcome. Occupational PEP was initiated first followed by nonoccupational PEP (nPEP). Antiretroviral regimens were based upon national PEP guidelines, affordability and availability, and prevailing HIV prevalence. Emerging side effects data and cost improvements influenced regimen changes. Between November 2001 and December 2006, 446 patients sought PEP. Occupational exposure: 91 patients: 51 males; 72 accepted HIV testing; 48 of 52 source patients were HIV infected; median exposure-PEP time 3 hours (range: 0.3-96 hours). Of 72 HIV-negative patients receiving PEP, 3 discontinued, 69 completed, and 23 performed post-PEP HIV RNA polymerase chain reaction (all negative). Eleven follow-up HIV enzyme-linked immunosorbent assay tests have all turned negative. Nonoccupational exposure: 355 patients; 285 females; 90 children; 300 accepted HIV testing; median exposure-nPEP time 19 hours (range: 1-672 hours). Of 296 HIV-negative patients on nPEP, 1 died, 15 discontinued, 104 are on record having completed PEP, and 129 returned for 6-week HIV RNA polymerase chain reaction (1 patient tested positive). Eighty-seven follow-up HIV enzyme-linked immunosorbent assay tests have all turned negative. It is feasible to provide PEP and nPEP in resource-constrained settings.

Performance Characteristics of HIV-1 Culture and HIV-1 DNA and RNA Amplification Assays for Early Diagnosis of Perinatal HIV-1 Infection

Lambert JS, Harris R, Stiehm R, et al. J Acquir Immune Defic Syndr. 2003;34:512–519The diagnosis of HIV infection in a newborn exposed to HIV in utero is a challenge. In the early years of the epidemic, HIV clinicians monitored the decline of HIV antibody levels for up to 2 years after birth to confirm that a child was not HIV-infected. HIV infection in infants is now typically made by the detection of viral DNA sequences in peripheral blood mononuclear cells by means of a DNA polymerase chain reaction (PCR). Plasma HIV-RNA measurements with PCR may also be valuable but has the theoretic limitation of false-negative reactions resulting from early treatment of the mother and infant. The purpose of this study was to evaluate the performance of HIV DNA PCR, HIV RNA PCR, and HIV culture to identify infected infants exposed to the virus in utero.Infants participating in the Pediatric AIDS Clinical Trials Group protocol 185.Specimens from the infants (24 infected and 100 uninfected) obtained prospectively were studied with standard nucleic acid–amplification assays and peripheral blood mononuclear cell microcultures. The sensitivities, specificities, and positive and negative predictive values were calculated for each of the 3 assay systems.At birth the sensitivity of culture, DNA PCR, and RNA PCR were 21%, 11%, and 27%, respectively. By 6 weeks, the sensitivity had improved to 90%, 83%, and 95%. The specificity was 99% to 100% for all assays at all times.The authors concluded that the diagnostic performance of the RNA PCR assay matched or exceeded that of culture and DNA PCR. Because RNA assays require less blood volume and often can be performed more quickly at reference laboratories, it is suggested that RNA assays may be used for early diagnosis of HIV infection in infants.This study demonstrates that RNA PCR assays are effective for the diagnosis of HIV infection. However, it must be noted that cryopreserved specimens were used for these PCR assays and may have impacted the sensitivity of the DNA PCR. Additionally, we have had 3 false-positive RNA PCR assays in 2 newborns and 1 adolescent exposed to HIV. A negative RNA PCR at or after 6 weeks of age strongly indicates that an infant is not infected.

Dried Blood Spots Improve Access to HIV Diagnosis and Care for Infants in Low-Resource Settings

Effective health care delivery to the majority of perinatally exposed infants worldwide, including those enrolled in prevention of mother-to-child transmission programs, is hampered by lack of access to an HIV diagnosis in infancy. Dried blood spot collection from young infants for centralized HIV polymerase chain reaction (PCR) testing is attainable in low-resource settings, provided PCR methodology suitable for routine laboratory service is available. The accuracy of the Roche Amplicor HIV-1 DNA test version 1.5 (Branchburg, NJ) performed on dried blood spots collected prospectively on ordinary Whatman filter paper from a cohort of 300 6-week-old infants born to HIV-infected women in Johannesburg, South Africa, was assessed. Anonymous analysis of the blood spots using a unique DNA extraction procedure was performed in a routine diagnostic laboratory and the results compared with HIV DNA and RNA PCR liquid blood tests at age 6 weeks, and the HIV status of the infant. Dried blood spots were available for 288 infants (96%) of whom 25 (8.7%) were HIV infected. The Roche Amplicor assay yielded a sensitivity of 100% and a specificity of 99.6%. HIV DNA PCR tests on dried blood spots have the potential to improve health care delivery to HIV-affected children in low-resource settings right now.

CD4 count/viral load in HIV.

CD4 count/viral load in HIV.

Individualisation of HIV therapy based on HIV RNA load: the virologist's perspective

There is increasing evidence that direct quantification of viral load by quantitative HIV RNA polymerase chain reaction (PCR) may be one of the more useful markers of disease status and antiviral treatment efficacy. Given the central role of viral replication in the pathogenesis of HIV infection, it is logical to assume that monitoring levels of cell-free virus in the plasma will be predictive of disease status. Furthermore, since the primary aim of treatment with current antiretroviral therapies is reduction of viral load, changes in plasma viraemia may be expected to be predictive of treatment effects. Preliminary data indeed suggest that viral load, is a useful marker of baseline prognosis, disease status and the effectiveness of antiretroviral therapy in individual patients. These data support the concept that measurement of viral RNA may eventually be of clinical utility in managing therapy of individual patients. However, prospective viral load studies are required to validate such a strategy.