Use of dried-blood-spot samples and in-house assays to identify antiretroviral drug resistance in HIV-infected children in resource-constrained settings.

Monitoring HIV drug resistance is an important component of the World Health Organization's global HIV program. HIV drug resistance testing is optimal with commercially available clinically validated test kits using plasma; however, that type of testing may not be feasible or affordable in resource-constrained settings. HIV genotyping from dried blood spots (DBS) with noncommercial (in-house) assays may facilitate the capture of HIV drug resistance outcomes in resource-constrained settings but has had varying rates of success. With in-house assays for HIV reverse transcriptase, we evaluated the yield of genotyping DBS samples collected from HIV-infected children who were enrolled in two clinical trials conducted in sub-Saharan Africa (median HIV viral load, 5.88 log(10) HIV RNA copies/ml; range, 4.04 to 6.99). Overall, HIV genotypes were obtained for 94 (89.5%) of 105 samples tested (95% and 84% from clinical trials #1 and #2, respectively); however, successful analysis of 15 (16.1%) of the 94 samples required repeat testing using a different set of primers on previously synthesized cDNA. The yield of genotyping was lower on the DBS that were stored suboptimally from clinical trial #2 (56% versus 88% for optimally stored). Concordance with plasma genotypes derived using a clinically validated, commercial kit-based assay (ViroSeq HIV-1 genotyping system) was also assessed in a subset of children with paired testing. For 34 samples with paired DBS and plasma genotypes, there was 100% concordance for major drug resistance mutations. DBS genotyping using in-house assays provides an alternative for antiretroviral drug resistance testing in children in resource-constrained regions but may require region-specific optimization before widespread use.

NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Effect of acyclovir on HIV-1 set point among herpes simplex virus type 2-seropositive persons during early HIV-1 infection.

We evaluated whether acyclovir suppression during human immunodeficiency virus type 1 (HIV-1) acquisition reduces HIV-1 set point, increases CD4 cell counts, and selects reverse-transcriptase mutations among 76 HIV-1 seroconverters identified in a placebo-controlled trial of twice-daily acyclovir (400 mg) for the prevention of HIV acquisition in herpes simplex virus type 2 (HSV-2)-seropositive persons (HIV Prevention Trials Network study 039). We found no significant difference in plasma HIV-1 RNA levels (P =.30) or CD4 cell counts (P =.85) between the acyclovir and placebo recipients. V75I and other mutations in HIV-1 reverse transcriptase reported from in vitro acyclovir studies were not observed. In conclusion, acyclovir suppression during HIV-1 seroconversion and the subsequent 6 months does not affect HIV-1 set point.

Effects of diphencyprone on expression of Bcl-2 protein in patients with alopecia areata.

BACKGROUND: Alopecia areata (AA) development is attributed to a T cell involved autoimmune process. Apoptosis is one of the suspected culprits in pathogenesis of this disorder. This disorder can be treated by contact sensitizers like diphencyprone (DPCP). We investigated the effects of treatment with DPCP on the expression of Bcl-2 protein in hair follicle epithelial cells of AA patients and its relation to clinical response to treatment. MATERIALS AND METHODS: Patients with chronic and extensive AA who had not received any treatment for at least 6 months were included. Furthermore, 3-mm punch biopsies were obtained from the affected areas before starting the treatment, and, six months after DPCP application, punch biopsies of the same size were taken from the following groups of patients: Group 1: six patients with complete hair regrowth, Group 2: six patients with partial regrowth, and Group 3: six patients with no regrowth. The samples were studied by immunohistochemistry to detect and compare the rate of Bcl-2 expression. RESULTS: Level of Bcl-2 expression in respondent patients (Group 1) was significantly higher after DPCP treatment (36.50 +/- 4.23) compared to pretreatment state (3.67 +/- 1.406, P < 0.001). Similar finding was observed in second group with partial regrowth (17.67 +/- 1.745 versus 5.33 +/- 2.076, P < 0.01). Such significant change was not observed in third group (4.75 +/- 1.315 versus 3.50 +/- 0.645, P > 0.05). CONCLUSION: The results of this study indicate the positive effect of DPCP on regulation (inhibition) of apoptotic process in patients with AA.

Evaluation of Glutathione Reductase and Glutathione Peroxidase in the Serum of Iranian Patients with Alopecia Areata: A Case-control Study.

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Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.

The Notch signaling pathway is required in both nonneoplastic neural stem cells and embryonal brain tumors, such as medulloblastoma, which are derived from such cells. We investigated the effects of Notch pathway inhibition on medulloblastoma growth using pharmacologic inhibitors of gamma-secretase. Notch blockade suppressed expression of the pathway target Hes1 and caused cell cycle exit, apoptosis, and differentiation in medulloblastoma cell lines. Interestingly, viable populations of better-differentiated cells continued to grow when Notch activation was inhibited but were unable to efficiently form soft-agar colonies or tumor xenografts, suggesting that a cell fraction required for tumor propagation had been depleted. It has recently been hypothesized that a small population of stem-like cells within brain tumors is required for the long-term propagation of neoplastic growth and that CD133 expression and Hoechst dye exclusion (side population) can be used to prospectively identify such tumor-forming cells. We found that Notch blockade reduced the CD133-positive cell fraction almost 5-fold and totally abolished the side population, suggesting that the loss of tumor-forming capacity could be due to the depletion of stem-like cells. Notch signaling levels were higher in the stem-like cell fraction, providing a potential mechanism for their increased sensitivity to inhibition of this pathway. We also observed that apoptotic rates following Notch blockade were almost 10-fold higher in primitive nestin-positive cells as compared with nestin-negative ones. Stem-like cells in brain tumors thus seem to be selectively vulnerable to agents inhibiting the Notch pathway.

Increased p53 immunopositivity in anaplastic medulloblastoma and supratentorial PNET is not caused by JC virus.

BACKGROUND: p53 mutations are relatively uncommon in medulloblastoma, but abnormalities in this cell cycle pathway have been associated with anaplasia and worse clinical outcomes. We correlated p53 protein expression with pathological subtype and clinical outcome in 75 embryonal brain tumors. The presence of JC virus, which results in p53 protein accumulation, was also examined. METHODS: p53 protein levels were evaluated semi-quantitatively in 64 medulloblastomas, 3 atypical teratoid rhabdoid tumors (ATRT), and 8 supratentorial primitive neuroectodermal tumors (sPNET) using immunohistochemistry. JC viral sequences were analyzed in DNA extracted from 33 frozen medulloblastoma and PNET samples using quantitative polymerase chain reaction. RESULTS: p53 expression was detected in 18% of non-anaplastic medulloblastomas, 45% of anaplastic medulloblastomas, 67% of ATRT, and 88% of sPNET. The increased p53 immunoreactivity in anaplastic medulloblastoma, ATRT, and sPNET was statistically significant. Log rank analysis of clinical outcome revealed significantly shorter survival in patients with p53 immunopositive embryonal tumors. No JC virus was identified in the embryonal brain tumor samples, while an endogenous human retrovirus (ERV-3) was readily detected. CONCLUSION: Immunoreactivity for p53 protein is more common in anaplastic medulloblastomas, ATRT and sPNET than in non-anaplastic tumors, and is associated with worse clinical outcomes. However, JC virus infection is not responsible for increased levels of p53 protein.

A comparison of two measures of HIV diversity in multi-assay algorithms for HIV incidence estimation.

BACKGROUND: Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. METHODS: Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. RESULTS: The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. CONCLUSIONS: MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation.

Association of pol diversity with antiretroviral treatment outcomes among HIV-infected African children.

BACKGROUND: In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6-36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth. METHODS: HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions). RESULTS: In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P<0.001 for all measures). CONCLUSIONS: In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcomes.

Association of HIV diversity and survival in HIV-infected Ugandan infants.

BACKGROUND: The level of viral diversity in an HIV-infected individual can change during the course of HIV infection, reflecting mutagenesis during viral replication and selection of viral variants by immune and other selective pressures. Differences in the level of viral diversity in HIV-infected infants may reflect differences in viral dynamics, immune responses, or other factors that may also influence HIV disease progression. We used a novel high resolution melting (HRM) assay to measure HIV diversity in Ugandan infants and examined the relationship between diversity and survival through 5 years of age. METHODS: Plasma samples were obtained from 31 HIV-infected infants (HIVNET 012 trial). The HRM assay was used to measure diversity in two regions in the gag gene (Gag1 and Gag2) and one region in the pol gene (Pol). RESULTS: HRM scores in all three regions increased with age from 6-8 weeks to 12-18 months (for Gag1: P = 0.005; for Gag2: P = 0.006; for Pol: P = 0.016). Higher HRM scores at 6-8 weeks of age (scores above the 75(th) percentile) were associated with an increased risk of death by 5 years of age (for Pol: P = 0.005; for Gag1/Gag2 (mean of two scores): P = 0.003; for Gag1/Gag2/Pol (mean of three scores): P = 0.002). We did not find an association between HRM scores and other clinical and laboratory variables. CONCLUSIONS: Genetic diversity in HIV gag and pol measured using the HRM assay was typically low near birth and increased over time. Higher HIV diversity in these regions at 6-8 weeks of age was associated with a significantly increased risk of death by 5 years of age.

Use of a high resolution melting (HRM) assay to compare gag, pol, and env diversity in adults with different stages of HIV infection.

BACKGROUND: Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. METHODS: HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14-540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. RESULTS: Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. CONCLUSIONS: The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.

Detection of individuals with acute HIV-1 infection using the ARCHITECT HIV Ag/Ab Combo assay.

BACKGROUND: We evaluated use of the ARCHITECT HIV Ag/Ab Combo assay (HIV Combo; Abbott Diagnostics; available for sale outside the United States only) for detection of acute HIV infection. METHODS: Samples were obtained from a behavioral intervention study (EXPLORE). HIV-uninfected men who have sex with men were enrolled and tested for HIV infection every 6 months. Samples from seroconverters collected at their last seronegative visit (n = 217) were tested individually using 2 HIV RNA assays. Samples with detectable HIV RNA were classified as acute and were tested with HIV Combo. Samples from the enrollment visit (n = 83) and the time of HIV seroconversion (n = 219) were tested with HIV Combo as controls. RESULTS: Twenty-one samples (9.7%) from the last seronegative visit had detectable HIV RNA and were classified as acute. HIV Combo was positive for 13 of the acute samples (61.9%). Samples not detected by HIV Combo had viral loads of 724-15,130 copies per milliliter. Expected results were obtained for positive and negative controls tested with HIV Combo. CONCLUSIONS: HIV Combo detected nearly two thirds of acute HIV infections identified in this high-risk population by non-pooled HIV RNA assays. HIV Combo may be useful for high-throughput screening to identify individuals with acute HIV infection.

Nevirapine resistance in women and infants after first versus repeated use of single-dose nevirapine for prevention of HIV-1 vertical transmission.

Single-dose (SD) nevirapine (NVP) significantly reduces mother-to-child transmission of human immunodeficiency virus (HIV). We analyzed NVP resistance after receipt of SD NVP in 57 previously SD NVP-naive women, in 34 SD NVP-experienced women, and in 17 HIV-infected infants. The proportion of women infected with variants with resistance mutations, the types of mutations detected, and the frequency and level of K103N were similar in the two groups of women at 6 weeks and 6 months post partum. NVP resistance was detected in a similar proportion of infants born to SD NVP-naive versus SD NVP-experienced women. Repeated use of SD NVP to prevent HIV transmission does not appear to influence NVP resistance.