Biocompatible copper sulfide-based nanocomposites for artery interventional chemo-photothermal therapy of orthotropic hepatocellular carcinoma.

Transcatheter arterial embolization has been considered as a promising targeted delivery approach for hepatocellular carcinoma (HCC). Currently, chemoembolization was the main treatment for unresectable HCC. However, the traditional chemoembolization treatment suffers from undesirable therapeutic effects and serious side-effects. In this study, the doxorubicin (DOX)-encapsulated and near-infrared (NIR)-responsible copper sulfide (CuS)-based nanotherapeutics was developed for magnetic resonance imaging (MRI)-guided chemo-photothermal therapy of HCC tumor in rats. The DOX-loaded CuS nanocomposites (DOX@BSA-CuS) demonstrated distinct NIR-triggered drug release behavior and high photothermal effect. In an orthotopic HCC rat model, DOX@BSA-CuS nanocomposites were selectively delivered to the tumor site via the intra-arterial transcatheter. The proposed DOX@BSA-CuS nanocomposites plus NIR laser irradiation exhibited significant tumor growth suppression performance. Moreover, the treatment progress can be monitored by MRI images. Finally, the preliminary toxicity estimate suggested the negligible side-effect of DOX@BSA-CuS nanocomposites during the therapeutic process. These results suggest the clinical translational potential possibility for imaging-guided arterial embolization with DOX@BSA-CuS nanocomposites for the treatment of HCC.

[Mutation analysis and prenatal diagnosis of MYO7A gene in a case of Usher syndrome type 1].

Objective: To analyze the clinical characteristics and identify the causative gene of a case with congenital deafness. Methods: Detailed medical history and clinical examination of a 4-year-old male child with congenital deafness were conducted in the First Affiliated Hospital of Army Military Medical University in June 2016. He was diagnosed with sensorineural deafness. The venous blood of the child and his parents was drawn, and genomic DNA was extracted. Proband's DNA was performed with targeted capture of high-throughput sequencing, then Sanger sequencing was used to verify the suspected mutation and segregation in this pedigree. According to the genetic diagnosis of the proband's deafness, ophthalmic examinations were performed. Genetic prenatal diagnosis was performed when the proband's mother was pregnant again. Results: The patient was detected with p.Trp1466Ter/p.Tyr2042Ter compound heterozygous mutations of MYO7A gene with targeted high-throughput sequencing. The mutation of p.Trp1466Ter was a reported mutation, while p.Tyr2042Ter has not been reported. In addition to congenital deafness, retinitis pigmentosa was also found by ophthalmologic examination, and the patient was clinically diagnosed with Usher syndrome type 1. Amniocentesis and fetal DNA sequencing were performed on the repregnancy fetus of this family at 18 weeks of gestation. The heterozygous mutation of MYO7A gene p.Tyr2042Ter was found, and the other allele was the wild type, indicating that the child will not exhibit clinical manifestations of Usher syndrome type 1. Indeed, the second child passed neonatal hearing screening. Conclusions: The clinical features and genetic variants were delineated in this family with Usher syndrome type 1. The results of the current study have enriched the phenotype and genotype data of the disease and provided a basis for genetic counseling.

The Mitochondrial tRNAGly T10003C Mutation may not be Associated with Diabetes Mellitus.

Mitochondrial DNA (mtDNA) mutations have long been proposed to play important roles in the pathogenesis of diabetes mellitus (DM). A large proportion of these mutations are localized at the mt-tRNA genes. Owing to its high mutation rate, a growing number of mt-tRNA mutations have been reported; however some of them are neutral genetic polymorphisms and will not result in the alteration of the mitochondrial function responsible for DM. In this study, we reassessed a recent reported "pathogenic" mutation, tRNAGly T10003C, in a clinical manifestation of DM. We first performed the conservation assessment of this mutation between different species. Moreover, the bioinformatics analysis was used to predict the secondary structure of mt-tRNAGly in wild type version and the mutant carrying the T10003C mutation. We also screened the presence of the T10003C mutation in 500 unrelated DM patients and 300 healthy controls. We noticed that the T10003C mutation was not very conserved and did not cause the secondary structure change of mt-tRNAGly. Moreover, this mutation was absent in the 500 unrelated DM patients and controls, suggesting that this mutation may be a rare event in the human population. In conclusion, the current study showed no association between the T10003C mutation and DM in humans.

[Transarterial chemoembolization with bleomycin treatment for moderate-advenced hepatocellular carcinoma].

Objective: To investigate the safety and efficacy of transarterial chemoembolization (TACE) using bleomycin for the treatment of medium-advanced hepatocellular carcinoma (HCC). Methods: Between December 2015 and December 2017, a total of 160 patients from the Chinese PLA General Hospital with moderate-advanced HCC whose diagnoses were confirmed by pathology or clinical imaging and were in accord with the Barcelona Clinic Liver Cancer (BCLC) staging criteria were prospectively analyzed.All patients had shown persistent viable tumor or tumor progression after at least 2 sessions of TACE.All patients included 135 males and 25 females , age 35-74 (57±8)years, were randomly divided into two groups, the treatment group: TACE procedures consisted of bleomycin+ pirarubicin+ oxaliplatin+ fluorouracil, the control group: pirarubicin+ oxaliplatin+ fluorouracil, and according to modified RECIST criteria the tumor response was evaluated once every 4-6 weeks, survival analysis was performed, overall survival and progression free survival were evaluated.the adverse events were recorded. Results: Response rate of the treatment group was 27.5%(22/80), the median progression free survival(mPFS)was 5.8 months, and the median overall survival (mOS) was 8.1 months.Response rate of the control group was 7.5%(6/80), mPFS of 2.9 months, and mOS of 4 months.The differences in mPFS and in mOS between the two groups were statistically significant (P=0.009, 0.002 respectively), and no serious adverse occurred. Conclusion: It is suggested that transarterial chemoembolization with bleomycin is safe and effective for medium-advenced HCC.

FAL1 regulates endothelial cell proliferation in diabetic arteriosclerosis through PTEN/AKT pathway.

OBJECTIVE: This study aims to investigate the role of FAL1 in the occurrence and progression of diabetic arteriosclerosis and its underlying mechanism. PATIENTS AND METHODS: FAL1 expression in coronary artery disease (CAD) tissues, normal artery tissues, and tumor necrosis factor-α (TNF-α)-induced endothelial cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effects of FAL1 on cell proliferation, migration, and cell cycle were examined by cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry, respectively. Western blot was used to detect protein expressions of proliferation-related gene PCNA (proliferating cell nuclear antigen), cell cycle-related genes cyclin D1, PTEN (phosphatase and tensin homolog deleted on chromosome ten) and AKT (protein kinase B) in HUVECs. Subsequently, rescue experiments were performed to assess whether PTEN/AKT signaling pathway is activated during the process of FAL1-regulated proliferation and migration of HUVECs. RESULTS: FAL1 was highly expressed in CAD tissues and TNF-α-induced endothelial cells compared with that of controls. Overexpression of FAL1 in HUVECs promoted cell cycle, proliferation, and migration. FAL1 activated PTEN/AKT pathway in HUVECs, which was partially reversed by PTEN overexpression. CONCLUSIONS: Highly expressed FAL1 can promote proliferation and migration of endothelial cells through activating PTEN/AKT signaling pathway.

[Impact of multiple morphological anomalies of the flagella on the outcomes of intracytoplasmic sperm injection for males with infertility].

Objective: To evalute the clinical outcomes of intracytoplasmic sperm injection(ICSI) for infertility male with severe asthenospermia induced by multiple morphological anomalies of the flagella (MMAF). Methods: The clinical data of 15 patient with MMAF were retrospectively analyzed, who underwent ICSI treatment using hyponotic swelling test the "live" sperm in the Department of Reproductive Medicine of Yantai Yuhuangding Hospital from January 2011 to December 2016. Another 30 obstructive azoospermia (OA)patients are matched strictly who also accepted ICSI in the same treatment time. The two groups were compared in the couples'age, the body mass index(BMI), the duration of infertility, the retrieved oocytes, the number of ICSI oocytes, and the rates of fertilization, cleavage, transferrable embryos, good embryos, embryos implanted, clinical pregncncy, early abortion, singleton and twins. Results: After 27 cycles of ICSI, all of the MMAF patients achieved clinical pregnancy, including 11 cases of live birth, 2 cases of spontaneous abortion, and 2 cases of pregnancy maintenance. There were no significant difference between MMAF and OA groups in the couples'age and BMI, or the numbers of retrieved oocytes and ICSI oocytes(P>0.05), but the differences in the infertility duration had statistical meaning(P<0.001). No statistical differences were observed among groups in ICSI fertility rate(92.0% vs 91.6%), clesvage rate(95.4% vs 96.5%), high-quality embryonic rate(56.5% vs 57.5%), good blastocyst rate(23/61 vs 35/94), embryo implantation rate(20/48 vs 35/75), early abortion rate(4/19 vs 8/36), clinical pregncncy rate(15/27 vs 28/50), singleton rate (10/13 vs 20/25)and twinning rate(3/13 vs 5/25)(P>0.05). Conclusions: MMAF may not affect ICSI treatment outcomes, but genetic defects can be transmitted through ICSI. The affected couples should be informed of the necessity of prenatal genetic diagnosis before embryo implantation and the inevitable vertical transmission of genetic problems to the offspring.

[Clinical effect of ultraselective transcatheter arterial chemoembolization in treatment of hepatocellular carcinoma originating from the caudate lobe: an analysis of 13 cases].

Objective: To investigate the clinical effect of ultraselective transcatheter arterial chemoembolization (TACE) in the treatment of hepatocellular carcinoma (HCC) originating from the caudate lobe. Methods: A retrospective analysis was performed for 13 patients with solitary HCC originating from the caudate lobe who were admitted to Department of Interventional Radiology in PLA General Hospital from March 2013 to December 2016. A 2.6-F microcatheter was used to perform ultraselective TACE, and the embolization material was ultra-liquefied iodinated oil. The number of tumor-feeding arteries, success rate and short-term efficacy of ultraselective technique, and long-term survival were evaluated after surgery. Results: Of all patients, 8 (61.5%) had a single tumor-feeding artery and 5 (38.5%) had multiple tumor-feeding arteries. The success rate of ultraselective technique was 84.6% (11/13). The complete remission rate at 1 month after ultraselective TACE was 63.6% (7/11). During the follow-up period after the expiration date, 10 out of 11 patients who underwent successful ultraselective TACE survived, and one out of two patients who underwent failed ultraselective TACE survived. Conclusion: Ultraselective TACE has good feasibility, clinical effect, and safety in the treatment of HCC originating from the caudate lobe, with an important clinical significance in the prognosis of such disease.

Cloning and functional characterization of the HbSYR1 gene encoding a syntaxin-related protein in Tibetan hulless barley (Hordeum vulgare L. var. nudum HK. f.).

Tibetan barley is a staple food for the natives of Qinghai-Tibet Plateau. Drought causes a reduction in barley production. In this study, the full-length cDNA of a gene encoding a syntaxin-associated protein was cloned from the leaves of a drought-resistant variety of barley, "Himalaya 10"; its expression was evaluated during drought stress and rehydration via real-time PCR. The cloned HbSYR1 cDNA sequence was 1300 bp in length, and included an 840-bp open reading frame that encoded 279 amino acids. Sequence analysis predicted the molecular weight of the encoded protein to be 42.08 kDa, with an isoelectric point of 4.98. ScanProsite analysis showed that the HbSYR1 protein contained a SNARE family characteristic motif, five casein kinase II phosphorylation sites, two N-glycosylation sites, four protein kinase C phosphorylation sites, and two N-myristoylation sites. The TMHMM prediction program indicated that the protein does not contain a transmembrane transfer ribbon. According to the SignalP 3.0 server, this protein does not contain a signal peptide, and is not a secretory protein. Instead, this protein was suggested to be localized in the cytoplasm, as predicted by the protein subcellular localization prediction tool (PSORT). Our results indicated that HbSYR was induced by drought stress and rehydration, and was determined to be a key gene for drought resistance and water retention in barley.

NJ cluster analysis of the SnRK2, PYR/PYL/RCAR, and ABF genes in Tibetan hulless barley.

The abscisic acid (ABA) signaling pathway is known as one of the most important signaling pathways in plants and is mediated by multiple regulators. The genes SnRK2, PYR/PYL/RCAR, and ABF are relevant to both ABA-dependent and -independent signaling pathways. To elucidate the profile of these genes from Tibetan hulless barley (Hordeum vulgare L. var. nudum Hook. f.), we collected available sequences from RNA-Seq data, together with NCBI data from five other model plant species (Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Populus trichocarpa, and Sorghum bicolor). Gene trees of SnRK2, PYR/PYL/RCAR, and ABF were constructed using a neighbor joining (NJ) method. For all genes, we identified a dominant group in which all six species were represented. Three, four, and five groups were found in the NJ trees of SnRK2, PYR/PYL/RCAR, and ABF, respectively. For each gene, Tibetan hulless barley was divided into three groups. Our analyses indicated that Tibetan hulless barley was associated with B. distachyon. The NJ cluster analysis also suggested that Tibetan hulless barley was affiliated with S. bicolor (SnRK2), A. thaliana (PYR/PYL/RCAR), and O. sativa (ABF). These results illustrate a diverse expression of genes SnRK2, PYR/PYL/RCAR, and ABF, and suggest a relationship among the six species studied. Collectively, our characterization of the three components of the ABA signaling pathway may contribute to improve stress tolerance in Tibetan hulless barley.

Cloning and characterization of up-regulated HbSINA4 gene induced by drought stress in Tibetan hulless barley.

Hulless barley is an important crop cereal in Tibetan, China. Drought is a major abiotic stress in barley production. In this study, we cloned the drought-related HbSINA4 gene from the variety 'Himalaya 10' and analyzed its expression patterns under different drought and rehydration conditions. The cDNA of HbSINA4 was 1052 bp long, including an open reading frame of 771 bp that encoded a protein of 256 amino acids. The molecular weight of HbSINA4 protein was predicted to be 29.53 kDa and the theoretical pI was 8.32. Bioinformatic analysis showed that the HbSINA4 gene contained a protein kinase domain profile family signature motif, with high similarity to that of Oryza sativa and Brachypodium distachyon. Real-time polymerase chain reaction (PCR) assays revealed that gene expression declined rapidly with increasing drought stress; in contrast, its expression increased after rehydration treatment. Therefore, the HbSINA4 gene responds to the drought stress and plays an important role in barely drought resistance. Furthermore, our results provide information which may be useful in other temperate crop studies and in aiding resistance to drought.

Correlation analysis of the PNPLA7 gene polymorphism and susceptibility to menstrual disorder.

We examined the correlation between PNPLA7 gene polymorphisms at the rs61754920 and rs11137410 loci and menstrual disorder in women of reproductive age in the Central Plain. Genomic DNA was extracted from peripheral blood; polymerase chain reaction-ligase detection reaction and SNaPshot genotyping were used to detect polymorphisms in the rs61754920 and rs11137410 gene loci, respectively. The results for the 2 loci in individuals of different blood types were statistically analyzed. The proportion of the AA homozygote at the rs61754920 locus in the PNPLA7 gene was the lowest, while the proportion of the CC homozygote at the rs11137410 locus in the PNPLA7 gene was the highest. There were no statistical differences in the frequency distribution of genotypes and alleles at the 2 loci between control and test groups. The frequency of the TT genotype at the rs11137410 locus in women with type O blood was significantly lower in the test group than in the control group. Frequencies of the C and T alleles were significantly different between the 2 groups. There may be an association between the PNPLA7 gene and type O blood or a combined effect of the 2 genes.

Redistribution of body composition in patients with Graves' disease after iodine-131 treatment.

OBJECTIVE: The objective of this study was to investigate body composition redistribution at 3 months after radioactive iodine therapy (RAI). METHODS: Eighty patients with Graves' disease (GD) for RAI and 18 volunteers were recruited. All patients underwent thyroid status test and dual-energy x-ray absorptiometry at baseline and 3 months after RAI. According to the second thyroid status test, patients were divided into the following groups: A, with aggravated hyperthyroidism; B-1, with improved hyperthyroidism; B-2, with euthyroidism; and B-3, with hypothyroidism. RESULTS: Total lean mass (LM) but fat mass (FM) and bone mineral content (BMC) of whole GD patients after RAI recovered to be not different with controls. Compared with baseline, in group A, FM in the left leg increased, and LM in left arm, right arm, trunk and total LM decreased (P<0.05). In B-2, FM in the head increased, and LM in the head, right arm, trunk and total LM increased (P<0.05). In B-3, FM in the right leg and total body fat percentage decreased, but FM in the head, android-to-gynoid fat ratio and body mass index increased (P<0.05); LM of all sites, weight and total mass increased (P<0.05); BMC in lumbar spine and left leg, and total BMC decreased (P<0.05). Body composition of unmentioned sites was retained after RAI in each group (P>0.05). CONCLUSIONS: Replenishment of LM gets priority rather than FM and BMC during the first 3 months after RAI, and the increase in LM starts from the upper body; head is the regional site in which FM recovery occurs first.

Impaired function of CD19(+) CD24(hi) CD38(hi) regulatory B cells in patients with pemphigus.

BACKGROUND: Pemphigus is an organ-specific autoimmune bullous disease. OBJECTIVES: To determine the role of regulatory B cells (Bregs) in patients with pemphigus. METHODS: The frequency of the occurrence of CD19(+) CD24(hi) CD38(hi) Bregs was detected from 34 patients with pemphigus and 20 healthy controls. Interleukin (IL)-10 secretion was processed after stimulating B cells. Specific antidesmoglein antibody (Ab) titres and their subclasses were also measured. Ab response and cytokine production from peripheral blood mononuclear cells (PBMCs) with or without Bregs were analysed. RESULTS: The number of Bregs was significantly increased in patients with pemphigus compared with healthy controls (15 ± 7% vs. 9 ± 3%; P < 0·01) and the proportion of Bregs in the active groups (newly diagnosed and chronic active patients) was significantly higher than in remittent individuals (16 ± 7% vs. 13 ± 8%; P = 0·04). The IL-10-producing B cells were significantly increased upon stimulation both in patients and in healthy controls. However, the increase ratio of IL-10-producing B cells between short- and long-term stimulation was significantly lower in patients with pemphigus (1·0-fold vs. 2·6-fold increase in control group; P < 0·01). Strikingly, Bregs from the controls were able to suppress interferon (IFN)-γ expression and T helper cell 1 (Th1) immune response (26% inhibition rate), while the suppressive function of Bregs from patients with pemphigus was significantly decreased (9% inhibition rate). There was no difference in Ab levels from PBMCs with or without Bregs after stimulation. CONCLUSIONS: Bregs in patients with pemphigus are elevated but with defective regulatory function on Th1 cells.

Serum thyroid hormone reference intervals in the apparently healthy individuals of Zhengzhou area of China.

This study aimed to establish reference intervals for serum thyroid hormones [serum thyroid-stimulating hormone (TSH), triiodothyronine (TT3), thyroxine (TT4), free triiodothyronine (FT3), and free thyroxine (FT4)] in apparently healthy individuals living in Zhengzhou. According to the requirement for laboratory support for the diagnosis and monitoring of thyroid diseases in the National Academy of Clinical Biochemistry (NACB) laboratory medicine practice guidelines, a total of 211 apparently healthy individuals were enrolled (94 men, 117 women, 23-77 years old) from Zhengzhou for measurement of serum levels of TSH, TT3, TT4, FT3, and FT4 by using the Siemens ADVIA Centaur XP analyzer. All markers were analyzed across gender- and age-specific groups by using the t-test and ANOVA. The reference intervals of all markers were determined by P2.5-P97.5. We detected gender-associated statistical significances for TT3, TT4, FT3, and FT4 (t=3.299, 2.141, 5.868, 5.358; P<0.05), but not for TSH (t=-1.776, P>0.05). Correlation analysis showed that all markers were negatively correlated with age (P>0.05). The new reference intervals for TT3, TT4, FT3, FT4, and TSH were established: 0.76-1.38 ng/mL, 5.96-11.27 µg/dL, 3.88-5.59 pM, 11.69-18.84 pM, 0.89-5.93 µIU/mL, respectively. In conclusion, we added a new database of reference intervals of the serum thyroid hormones for the Chinese adult population.

Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel-dependent mechanism.

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.

Validity of the rapid strip assay test for detecting HBsAg in patients admitted to hospital in Uganda.

Commercially available rapid strip assays (RSAs) for hepatitis B surface antigen (HBsAg) are used for most routine clinical testing in sub-Saharan Africa. This study evaluated the validity of RSA and a more sophisticated enzyme immunoassay (EIA) with confirmation by nucleic acid testing (NAT) in hospitalized patients in Uganda. Sera from 380 consecutive patients collected and tested for HBsAg and anti-HIV in Kampala, Uganda by RSA were sent frozen to Dallas for EIA including HBsAg, total anti-hepatitis B core, hepatitis B e antigen, and anti-HIV. NAT was performed on all HBsAg-positives and on a random sample of 102 patients that were HBsAg-negative by both assays. Overall, 31 (8%) were HBsAg positive by RSA while 50 (13%) were HBsAg-positive by EIA; 26 were concordant between the two assays. Of 55 HBsAg-positive patients, nearly all showed detectable serum hepatitis B virus (HBV) DNA by bDNA (46) or PCR (4) assay. The 26 patients who were HBsAg positive by both EIA and RSA had significantly higher median serum HBV DNA levels than the 24 patients who were HBsAg positive by EIA alone. An additional 12/102 (12%) HBsAg negative patients had very low serum HBV DNA levels by NAT. Several differences in expected results of serologic testing were observed in this large series of African patients. RSA HBsAg testing is less sensitive than EIA; even EIA failed to detect all HBV DNA positive sera. A more complex testing protocol than RSA alone will be needed in Africa to improve patient care.

Poor performance of hepatitis C antibody tests in hospital patients in Uganda.

Most hepatitis C testing in Uganda is performed using commercial rapid strip assays (RSA) to detect antibodies to hepatitis C virus (anti-HCV), rather than enzyme immunoassays (EIA). The prevalence of hepatitis C antibodies in a Ugandan hospital population was determined using both methods to test their accuracy using nucleic acid testing (NAT) as a reference. Sera from 380 consecutive hospitalized Ugandan patients were tested for anti-HCV using an RSA in Uganda, with subsequent automated third-generation EIA testing in the United States, followed by NAT. Recombinant immunoblot assays (RIBA) were used as a supplementary test to detect anti-HCV epitopes. Overall, anti-HCV was detected in 48/380 (13%) by one or both antibody tests. Anti-HCV was detected in 19 (5.0%) patients by RSA and in 33 (8.7%) patients by EIA; only four patients were anti-HCV positive by both methods. Fourteen of the 48 anti-HCV positive patients had detectable serum HCV RNA, 7 each by bDNA assay or by PCR. RSA detected only 7 of 14 HCV RNA positive sera. Of 29 RNA negative but anti-HCV positive patients tested by RIBA, only two were anti-HCV positive; 27 were anti-HCV negative or indeterminate. Anti-HCV testing by RSA and/or EIA was neither sensitive nor specific for detection of ongoing HCV infection in hospitalized Ugandan patients. Our findings underscore the importance of confirmatory nucleic acid testing, which, despite its increased cost, appears essential to manage African patients with HCV.

Evolution of hepatitis C virus NS5A region in breakthrough patients during pegylated interferon and ribavirin therapy*.

Investigating the evolution of the hepatitis C viral (HCV) genome in the small number of patients that experience viral breakthrough might shed light on the problem of resistance to interferon therapy. Within the HCV genome, sequence diversity of the viral nonstructural 5A protein-coding region (NS5A) has been linked to interferon responsiveness. We analysed the temporal sequence changes within NS5A in genotype 1a patients: 6 breakthrough (BT), 12 sustained virologic responders (SVR) and 12 non-responders (NR), all of whom had received full dose peg-interferon and ribavirin therapy. The entire NS5A region was amplified by reverse transcription (RT)-PCR followed by direct sequencing of serum samples from baseline and three on-treatment time points for each group. Comparing baseline sequences with week 12 and later time points, BT patients resembled SVR patients in having a higher number of amino acid substitutions at week 12 than NR patients; however, the number of amino acid substitutions in this group decreased at and after BT. Substitutions were focused in the V3 and flanking regions in BT patients but not in SVR patients. The high number of substitutions in NS5A in both BT and SVR groups suggests that selective pressure is associated with viral response to therapy. Our results provide evidence that amino acid substitutions within the NS5A coding region may reflect a host response that drives selective pressure for viral adaptation.