An Efficient, Large-Scale Survey of Hepatitis C Viremia in the Democratic Republic of the Congo Using Dried Blood Spots

Background Efficient viral load testing is needed for hepatitis C (HCV) surveillance and diagnosis. HCV viral load testing using dried blood spots (DBSs), made with a single drop of finger-prick whole blood on filter paper, is a promising alternative to traditional serum-or plasma-based approaches. Methods We adapted the Abbott Molecular m2000 instrument for high-Throughput HCV viremia testing using DBSs with simple specimen processing and applied these methods to estimate the national burden of infection in the Democratic Republic of the Congo (DRC). We tested DBSs collected during the 2013-2014 DRC Demographic and Health Survey, including 1309 adults ≥40 years of age. HCV-positive samples underwent targeted sequencing, genotyping, and phylogenetic analyses. Results This high-Throughput screening approach reliably identified HCV RNA extracted from DBSs prepared using whole blood, with a 95% limit of detection of 1196 (95% confidence interval [CI], 866-2280) IU/mL for individual 6-mm punches and 494 (95% CI, 372-1228) IU/mL for larger 12-mm punches. Fifteen infections were identified among samples from the DRC Demographic and Health Surveythe weighted country-wide prevalence of HCV viremia was 0.9% (95% CI, 0.3%-1.6%) among adults ≥40 years of age and 0.7% (95% CI,.6%-.8%) among human immunodeficiency virus-infected subjects. All successfully genotyped cases were due to genotype 4 infection. Conclusions DBS-based HCV testing represents a useful tool for the diagnosis and surveillance of HCV viremia and can easily be incorporated into specimen referral systems. Among adults ≥40 years of age in the DRC, 100000-200000 may have active infection and be eligible for treatment

The histone deacetylase inhibitor trichostatin A downregulates human MDR1 (ABCB1) gene expression by a transcription-dependent mechanism in a drug-resistant small cell lung carcinoma cell line model

Tumour drug-resistant ABCB1 gene expression is regulated at the chromatin level through epigenetic mechanisms. We examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on ABCB1 gene expression in small cell lung carcinoma (SCLC) drug-sensitive (H69WT) or etoposide-resistant (H69VP) cells. We found that TSA induced an increase in ABCB1 expression in drug-sensitive cells, but strongly decreased it in drug-resistant cells. These up- and downregulations occurred at the transcriptional level. Protein synthesis inhibition reduced these modulations, but did not completely suppress them. Differential temporal patterns of histone acetylation were observed at the ABCB1 promoter: increase in H4 acetylation in both cell lines, but different H3 acetylation with a progressive increase in H69WT cells but a transient one in H69VP cells. ABCB1 regulations were not related with the methylation status of the promoter −50GC, −110GC, and Inr sites, and did not result in further changes to these methylation profiles. Trichostatin A treatment did not modify MBD1 binding to the ABCB1 promoter and similarly increased PCAF binding in both H69 cell lines. Our results suggest that in H69 drug-resistant SCLC cell line TSA induces downregulation of ABCB1 expression through a transcriptional mechanism, independently of promoter methylation, and MBD1 or PCAF recruitment

<i>ABCB1</i> (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells

BACKGROUND: Clinical response to chemotherapy for ovarian cancer is frequently compromised by the development of drug-resistant disease. The underlying molecular mechanisms and implications for prescription of routinely prescribed chemotherapy drugs are poorly understood. METHODS: We created novel A2780-derived ovarian cancer cell lines resistant to paclitaxel and olaparib following continuous incremental drug selection. MTT assays were used to assess chemosensitivity to paclitaxel and olaparib in drug-sensitive and drug-resistant cells±the ABCB1 inhibitors verapamil and elacridar and cross-resistance to cisplatin, carboplatin, doxorubicin, rucaparib, veliparib and AZD2461. ABCB1 expression was assessed by qRT-PCR, copy number, western blotting and immunohistochemical analysis and ABCB1 activity assessed by the Vybrant and P-glycoprotein-Glo assays. RESULTS: Paclitaxel-resistant cells were cross-resistant to olaparib, doxorubicin and rucaparib but not to veliparib or AZD2461. Resistance correlated with increased ABCB1 expression and was reversible following treatment with the ABCB1 inhibitors verapamil and elacridar. Active efflux of paclitaxel, olaparib, doxorubicin and rucaparib was confirmed in drug-resistant cells and in ABCB1-expressing bacterial membranes. CONCLUSIONS: We describe a common ABCB1-mediated mechanism of paclitaxel and olaparib resistance in ovarian cancer cells. Optimal choice of PARP inhibitor may therefore limit the progression of drug-resistant disease, while routine prescription of first-line paclitaxel may significantly limit subsequent chemotherapy options in ovarian cancer patients

Cryptic HBV replicative activity is frequently revealed in anti-HBC-positive/hbsag-negative patients with HIV infection by highly sensitive molecular assays, and can be predicted by integrating classical and novel serological HBV markers

The anti-HBc-positive/HBsAg-negative status is frequent in HIV-infection and correlates with poor survival. Here, by highly-sensitive assays, we evaluate cryptic HBV replication and factors correlated with its detection in 81 anti-HBc-positive/HBsAg-negative HIV-infected patients. Patients were treated for &gt;12 months with HBV-active modern combined antiretroviral-therapy (cART) and had serum HBV-DNA &lt; 20 IU/mL by commercial Real-Time PCR. Serum HBV-DNA was quantified by droplet digital PCR, serum HBV-RNA by an Abbott research assay, and anti-HBc titer (proposed to infer intrahepatic cccDNA) by Lumipulse/Fujirebio. Cryptic serum HBV-DNA was detected in 29.6% of patients (median (IQR): 4(1-15) IU/mL) and serum HBV-RNA in 3.7% of patients despite HBsAg-negativity and HBV-active cART. Notably, cryptic serum HBV-DNA correlated with an advanced CDC-stage (p = 0.01) and a lower anti-HBs titer (p = 0.05), while serum HBV-RNA correlated with lower nadir CD4+ cell-count (p = 0.01). By analyzing serological HBV-markers, the combination of anti-HBs &lt; 50 mIU/mL (indicating lower immune response) plus anti-HBc &gt; 15COI (reflecting higher HBV replicative activity) was predictive of cryptic serum HBV-DNA (OR: 4.7(1.1-21.7), p = 0.046, PPV = 62.5%, and NPV = 72%). In conclusion, cryptic HBV-replication (not detected by classical assays) characterizes a conspicuous set of anti-HBc-positive HIV-infected patients despite HBsAg-negativity and HBV-active combined antiretroviral therapy (cART). The integration of classical and novel markers may help identify patients with cryptic HBV-replication, thus optimizing the monitoring of anti-HBc-positive/HBsAg-negative HIV-infected patients

Functional genetic screening reveals the role of mitochondrial cytochrome b as a mediator of FAS-induced apoptosis

Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b