Analysis of sequences of hepatitis C virus NS5A genotype 1 in HIV-coinfected patients with a null response to nitazoxanide or peg-interferon plus ribavirin.

Even though new drugs have been approved for treatment of hepatitis C virus (HCV) infection, the risk of drug-drug interactions and concern about overlapping toxicities has hindered the development of studies in HIV/HCV-coinfected individuals. Traditional treatment with pegylated interferon plus ribavirin (peg-IFN + RBV) is very expensive and has a low rate of sustained virological response in coinfected patients, especially if they are infected with HCV genotype 1. Nitazoxanide (NTZ) is a drug that is being evaluated for the treatment of chronic HCV infection, both in HCV-monoinfected and HIV/HCV-coinfected patients. Understanding the NTZ resistance mechanism could allow the development of resistance to be minimized and would expand the treatment options, mainly in special populations such as HIV/HCV-coinfected patients. Similarly to IFN, NTZ increases the activity of the cellular protein kinase activated by double-stranded RNA (PKR), a key kinase in the innate antiviral response. In order to elucidate whether sequence heterogeneity in the PKR-binding domain of HCV NS5A genotype 1 could influence the antiviral activity of either NTZ monotherapy or peg-IFN + RBV, baseline and end-of-therapy plasma samples from two groups of eleven non-responder HIV/HCV-coinfected patients that had received NTZ or peg-IFN + RBV were studied. Most of the HCV NS5A sequences examined at the end of therapy did not change from the baseline, even after 30 days course of antiviral therapy. An extensive comparison of HCV NS5A genotype 1 and 4 sequences from the database with reported IFN therapy outcome was performed in order to infer their phylogenetic relationships. The HCV genotype 1 NS5A nucleotide sequences from therapy-non-responder patients were intermingled amongst those from the database, irrespective of their IFN-therapy outcome. When comparing NS5A-PKRBD amino acid sequences, significant differences were observed in genotype 4, but not in genotype 1 (p < 0.0001 and p > 0.05, respectively). In conclusion, despite IFN and NTZ sharing the protein kinase activated by double-stranded RNA as their cellular target, the HCV genotype 1 strategy to counteract the IFN action mediated by NS5A ISDR/PKRBD does not explain drug resistance in HIV/HCV-coinfected patients. Other viral factors that are possibly involved are discussed as well.

Comparative analysis of hepatitis B virus genotype a molecular evolution in patients infected with HBV and in patients co-infected with HBV and HIV.

HIV infection has a significant impact on the natural progression of liver disease caused by infection with hepatitis B virus (HBV), but its role in the molecular evolution of HBV is unknown. It is difficult to study the molecular evolution of HBV longitudinally considering its genomic complexity, which implies the analysis of paired samples. This study aimed to analyze the difference in the evolutionary dynamics of HBV among patients with HIV and uninfected individuals. In this study, 17 patients infected chronically with HBV were recruited, 9 of them were co-infected with HIV. Patients were HBe antigen-positive and infected with HBV genotype A. Paired plasma samples were collected from each patient 3 years apart, and they were compared subsequently to each other. The HBV phylogenetic inference among isolates from patients infected with HBV and co-infected with HBV and HIV tends to cluster separately. Likewise, when comparing the HBV evolutionary rate and genetic distances, values were higher in the former in both preC/C and S genomic regions. Intra-host analyses of HBV isolates revealed high diversity and complexity of quasispecies among patients infected with HBV exhibiting high numbers of viral variants and genetic distance. In summary, after studying the HBV molecular evolution among isolates ascribed to genotype A at inter- and intra-host levels, HBV exhibited low quasispecies complexity and diversity as well as low evolutionary rates in the presence of HIV co-infection, suggesting that the co-infection may have an impact on the HBV molecular evolution most likely from the weakened cellular immune response.

Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment.

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

No reduction of HCV viral load in HIV patients co-infected with HCV genotype 1 during a 30 days course of nitazoxanide monotherapy.

There are two new drugs approved and several in development for treatment of chronic HCV; among them nitazoxanide (NTZ). Twelve HIV/HCV genotype 1 co-infected patients were enrolled prospectively to receive a 30 days course of oral NTZ 500 mg bid. This therapy was well tolerated in this group of HIV patients co-infected with HCV genotype 1. Nevertheless no changes in HCV viral load were observed during treatment in none of the patients evaluated. This data suggests that despite the promising results reported for HCV genotype 4 mono-infected patients, NTZ exhibit poor activity as monotherapy in HIV/HCV co-infected patients with genotype 1.

Molecular characterization of hepatitis C virus genotype 4 sequences in HIV-coinfected patients from Argentina.

The prevalence of hepatitis C virus genotype 4 (HCV-4) is increasing in different parts of the World but in Latin America the data are still scarce. We aimed to characterize HCV-4 isolates from 383 HIV-coinfected patients in Argentina. Sequence analyses were based on the non-structural 5B region of HCV. Results from 18 patients indicated a genetic heterogeneity that involved three genotype 4 subtypes. Sequences were ascribed to subtype 4d (67%), 4a (22%), and 4m (11%). In spite of different sources of transmission were defined among patients, no statistical association was found with the genotype 4 subtype. The scenario is also compatible with multiple importation of the epidemic and there is no evidence for transmission-specific clusters or network-like transmission of HCV-4. This HCV-4 does not represent a recent introduction in Argentina, it circulates in all transmission groups and its presence is increasing among HIV-infected patients.

HCV RNA decline in the first 24 h exhibits high negative predictive value of sustained virologic response in HIV/HCV genotype 1 co-infected patients treated with peginterferon and ribavirin.

BACKGROUND: Treatment with Peg-interferon and ribavirin (PEG-IFN/RBV) for HIV patients co-infected with hepatitis C virus (HCV) genotype 1 has suboptimal rates of response. Viral kinetics has emerged as one of the best prognostic factors of treatment outcome. METHODS: Twenty HIV/HCV genotype 1 co-infected patients in treatment with PEG-IFN/RBV, had blood drawn at baseline, 24 h, 4, 12, 24, 48, and 72 weeks. HCV-RNA levels were evaluated at each time point. ROC curves were used to evaluate the log10 HCV-RNA decay at 24 h that exhibits the best predictive value of achieving response. Genomic characterization of HCV NS5A at both interferon sensitivity-determining region (ISDR) and protein-kinase binding (PKRBD) domains were performed in order to evaluate its heterogeneity and association with 24 h HCV-RNA decay and SVR. RESULTS: Non-responder patients exhibited a mean of 0.7 log10 (SD 0.74 log10) HCV-RNA decay at 24 h, whereas responder-patients presented 1.6 log10 (SD 0.28 log10), p = 0.04. A reduction in HCV viral load from baseline to 24 h of < 1.4 had a negative predictive value for achieving SVR of 100% and a positive predictive value of 50%. HCV genotype 1 isolates from patients with a decrease of HCV-RNA at 24 h > 1.4 log10, exhibited 3.1(SD 1.5) amino acids substitutions in ISDR and 4.8(SD 2.3) in PKRBD regions and 1.6(SD 0.7) and 2.4(SD 1.3), respectively, in those patients presenting lower reduction in HCV-RNA. CONCLUSIONS: HIV/HCV genotype 1 co-infected patients with a decrease in HCV-VL at 24 h > 1.4 log10 are more likely to achieve SVR when treated with PEG-IFN/RBV than those with lower levels of HCV-RNA decay. Along with other host-related and viral-related prognostic factors in HIV/HCV co-infected patients, this very early time point of evaluation could be of relevance in the management of HCV-specific treatment.

Short Communication: Hepatitis B Virus Harboring Nucleotide Deletions in the Basal Core Promoter in HBe-Positive HIV-Coinfected Patients Under Lamivudine Therapy.

The presence of HBV genomes with deletions at the basal core promoter (BCP) is associated with more aggressive liver disease. This 3-year longitudinal analysis of two HIV-HBV-coinfected patients allowed identification of three deletions with dissimilar abundance and permanence into the HBV quasispecies composition. These deletions may contribute to HBV pathogenesis in HIV-coinfected individuals.

Preimplantation genetic diagnosis for BRCA1/2--a novel clinical experience.

OBJECTIVE: To describe our 2-year experience with preimplantation genetic diagnosis (PGD) for carriers of mutations in the genes BRCA1 and BRCA2, the dilemmas incurred and the lessons learned. METHODS: We collected data on those carriers of BRCA1/2 mutations who applied for PGD counseling and who decided to proceed. We describe the PGD procedures that were conducted and their outcome. RESULTS: Ten carriers of BRCA1/2 mutations applied for PGD counseling, seven were healthy, and three were BC survivors. Eight women needed in vitro fertilization (IVF) because of coexisting infertility. After counseling, six opted for the procedure and five of them underwent PGD for the BRCA mutation. In one of these PGD, fluorescence in situ hybridization (FISH) analysis for chromosomes 21, X and Y was also performed. Three women conceived, each in the first treatment attempt. One of them gave birth to twins, the second to a singleton and the third is currently pregnant. During the pregnancies, dilemmas concerning PGD confirmation were discussed. CONCLUSIONS: PGD is an acceptable reproductive option for BRCA mutation carriers, especially for those who require IVF due to fertility problems. Discussion of this option should be carried out with sensitivity, taking into account the age of the woman, her health, fertility status and emotional state. Confirmatory prenatal diagnosis may not always be encouraged.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Hepatitis B virus genotype distribution and its lamivudine-resistant mutants in HIV-coinfected patients with chronic and occult hepatitis B.

Hepatitis B virus (HBV) genotypes were examined in HIV-infected patients with chronic and occult HBV infection. From a total population of 593 HIV-infected patients, 22 individuals (prevalence 3.7%) were found to be HBsAg while 72 (12.1%) were found to be anti-HBc alone. From them, 20 and 4 were HBV DNA positive, respectively. These last four patients are therefore considered to be HBV infected in an occult form. The genotypes could be determined in all 24 HBV-infected patients. HBV-A was the most common (20/24; 83.3%), followed by HBV-D (2/24; 8.3%) and HBV-F (1/24; 4.2%). The remaining sample exhibited mixed infection involving genotypes A and D as pure ones, thus also forming part of three intergenotypic recombinant forms exhibiting different mosaic S gene patterns. The sexual route of transmission was predominant among HBV genotype A-infected patients. Among the 24 HBV DNA-positive patients, point mutations related to lamivudine resistance were found in four strains. These viral strains showed a methionine-to-valine substitution at codon 204 (rtM204V) in association with an upstream B-domain change at rtL180M. Additionally, two of them exhibited the additional rtV173L mutation. The value of HBV molecular monitoring including both HBV viral genomic characterization and genotypic resistance profile in HIV-HBV-coinfected individuals is discussed.

Retrospective analysis of 1217 IVF cycles in women aged 40 years and older.

Older women comprise an increasing portion of patients entering assisted reproduction programmes. This study is a retrospective summary of the files of all patients aged 40 years and older at advent of IVF, between 1995 and 2004, in the authors' centre. In all, 381 women underwent 1217 initiated treatment cycles. Embryo transfer was performed in 62.6% of initiated cycles. Success rates declined with each year after age 40; pregnancy and delivery rates were 13.9 and 9.1% at age 40 and 2.8 and 0.7% at age 45. There were no deliveries at an older age. Logistic regression analysis showed the following factors were independently and significantly related to higher pregnancy rates: younger age, lower dose of gonadotrophins, greater number of mature follicles, endometrial thickness, and number of embryos transferred; prior pregnancy did not influence success. Retrieving more than four oocytes increased pregnancy rates in all women over 40. Transferring 3 embryos or more increased pregnancy rates in all ages, but reached statistical significance only in women aged 40-41 (P < 0.000). It is concluded that in women between 40 and 41 years of age, ovarian response is a major determinant of success, but not in women older than that. Unrealistic expectations may be avoided if accurate data are provided regarding delivery rates per year after age 40.

The use of a perinatal internet consultation forum in Israel.

To analyse the use of a free, public, perinatal internet consultation service, 2000 consultations provided by university hospital staff were evaluated over 30 months. Ninety five percent of the questioners were women, and 62% of them were primiparous. The average response rate was 2.3 audience responses per question. Fifty-two percent of the consultations were related to labour and delivery, 23% were related to pregnancy complications, 16% were related to prenatal diagnosis, and 7% were related to the puerperium period. We conclude that medical consultation forums provide an additional way of delivering inexpensive, accessible, fast, and convenient healthcare services.

[Assisted reproduction technologies and the risk of fetal, chromosomal and genetic malformations].

BACKGROUND: Assisted reproduction techniques allowed thousands of otherwise infertile couples to attain pregnancy. As this technology moves into the mainstream of infertility treatment, it has become more critical to reassess its safety. OBJECTIVE: To review the birth outcome of patients undergoing conventional in-vitro fertilization and intracyto- plasmic sperm injection regarding fetal malformations, chromosomal and genetic abnormalities. METHODS: Selective review of the literature. RESULTS: Most of the published data is from observational studies and is not randomized or blinded. Unfortunately, most articles are inherently biased. Chromosomal and genetic abnormalities are increased probably only as a direct corollary to the underlying parental risk and not due to the technology itself. There is a slight increase in the congenital malformations rate, but inspection of these malformations reveal no clustering of any specific abnormality. CONCLUSIONS: Children born after assisted reproduction technologies have an increased risk of a major congenital malformation and chromosomal abnormalities compared with those born after natural conception. The risk is mainly due to paternal and maternal risk factors, which are more prevalent in couples who use assisted reproduction techniques for reproduction. Infertility-linked risk is highly probable for the observed findings. A technique-related risk, however, cannot be ruled out. Intracytoplasmic sperm injection appears to be a safe alternative for couples who otherwise would be unable to achieve pregnancy. The inherent risks associated with these genetically "at risk" couples mandate thorough evaluation and counseling before undertaking ICSI.

Treatment of recurrent IVF failure and human leukocyte antigen similarity by intravenous immunoglobulin.

This study sought to assess the efficacy of intravenous immunoglobulin (IVIg) in improving pregnancy rates and outcome, in a select group of patients with repeated IVF failure and human leukocyte antigen (HLA) similarity. Couples suffering from recurrent IVF failure, defined as at least seven attempts at embryo transfer with no successful implantations, who were found to share at least three HLA loci, and a negative cross-match test, were included in the study. The treatment consisted of two 30 g IVIg doses: one before oocyte retrieval, and a second as soon as a fetal pulse was identified on ultrasound. Ten couples comprised the study group. In total, these couples had undergone 98 IVF cycles with no successful pregnancies prior to initiation of the study. Following a total of 18 IVIg courses, seven women conceived, two women twice. Up to date, five women have delivered at least one live fetus, at 27 weeks or later. One woman is currently in the early third trimester of a twin pregnancy, and one woman had a late abortion at 19 weeks. The results suggest that couples with recurrent IVF failure and HLA similarity, may benefit from IVIg treatment.

In vitro maturation and fertilization of oocytes from an intact ovary of a surgically treated patient with endometrial carcinoma: case report.

Polycystic ovary syndrome (PCOS) with prolonged anovulation had resulted in endometrial carcinoma in a 43-year-old woman. Since she and her husband did not share common biological children, they requested fertility preservation. Due to the woman's age, high dose progesterone and postponing surgery were both considered inappropriate. We therefore proposed oocyte retrieval from the ovaries removed by staging laparotomy followed by in vitro maturation and ICSI. Surrogacy could then enable a future pregnancy. Fourteen of 17 (82%) retrieved oocytes matured in vitro. Following ICSI, eight embryos (two at the pronuclear stage and six cleaved) were cryopreserved. To the best of our knowledge, this is the first report of oocyte aspiration, maturation and fertilization from an ovary removed by laparotomy.

Twin delivery following 12 years of human embryo cryopreservation: case report.

It is uncertain how long IVF units can keep frozen embryos. Few data exist on success of embryo transfer for embryos that have been cryopreserved for many years. We report the delivery of healthy twins following the transfer of embryos cryopreserved for 12 years. To the best of our knowledge, this is the longest reported successful human embryo freezing.

Treatment of obstetric hemorrhage with recombinant activated factor VII (rFVIIa).

Recombinant activated factor VII (rFVIIa, NovoSeven) was used in three patients with massive obstetric hemorrhage due to placenta previa accreta, rupture of the uterus and pre-eclampsia with HELLP. Administration of the drug markedly decreased the bleeding and enabled control of the hemorrhage. rFVIIa seems to be an adjunctive hemostatic measure for the treatment of severe obstetric hemorrhage.

Simplified artificial endometrial preparation, using oral estradiol and novel vaginal progesterone tablets: a prospective randomized study.

There are various successful protocols for artificial endometrial preparation, comprising induction of endometrial proliferation with estrogens and secretory transformation with progestins. The aim of this prospective randomized study was to evaluate a simplified approach for endometrial preparation, comparing two constant doses of oral estradiol combined with a novel low-dose vaginal natural progesterone preparation (100 mg Endometrin tablets). Twenty-nine patients were enrolled in the study and divided randomly into two groups. Both groups received oral estradiol tablets from the beginning of menstruation, group A (15 patients) receiving 4 mg/day divided into two doses of 2 mg each, and group B (14 patients) receiving 6 mg/day divided into three doses. Serum estradiol and progesterone and sonographic thickness of the endometrium were measured on the 1st day of menstruation and on the 6th, 11th, 16th and 21st days of the artificial cycle. Following the first 12 days of estradiol priming, with an endometrial thickness of > or = 8 mm, Endometrin vaginal tablets 100 mg were added twice a day for 10 days. On the 21st cycle day, an endometrial biopsy was taken from all patients using Pipelle. In all 29 patients, appropriate changes in estradiol, progesterone and endometrial thickness were observed. Estradiol levels were significantly higher in the 6 mg/day group on days 6 and 11, but no significant difference was noted in serum progesterone level and endometrial thickess between groups. Histological evaluation of endometrial biopsies, on the 21st day, revealed adequate late-secretory endometrium in 14/15 (93.3%) patients of group A and in 13/14 (92.9%) patients of group B. In conclusion, our results demonstrate that an appropriate endometrial secretory transformation may be induced using an economical regimen of fixed low-dose oral estradiol (4 mg/day) and low-dose vaginal progesterone tablets (Endometrin 100 mg twice daily).

A high predictive value of the first testicular fine needle aspiration in patients with non-obstructive azoospermia for sperm recovery at the subsequent attempt.

BACKGROUND: The objective of this retrospective study, which included 51 men with non-obstructive azoospermia, was to evaluate the predictive value of the results of the first sperm recovery attempt on the probability for sperm recovery in a second attempt. METHODS AND RESULTS: A positive testicular fine needle aspiration (TEFNA) was defined as the recovery of any number of mature sperm. At the first and second TEFNA attempts, mature sperm were recovered in 33 (64.7%) and 25 (49%) of 51 patients respectively. In 23 of the 33 (69.7%) patients with a positive first TEFNA, sperm were recovered at both attempts, whereas in only two of 18 (11.1%) with a negative first TEFNA, sperm were recovered at the second attempt. Our analysis revealed a high predictive value of the first TEFNA for sperm recovery at the subsequent attempt, with a mean positive predictive value of 69.7%, with the highest probability being 90.9% in hypospermatogenesis, 72.7% in Sertoli cell-only pattern, 75% in tubular hyalinization, and the lowest being 28.6% in maturation arrest. The mean negative predictive value was 88.9%, which was high in all categories (80% in Sertoli cell-only pattern and 100% in maturation arrest and tubular hyalinization). CONCLUSION: A second TEFNA attempt should be offered to all non-obstructive azoospermic patients with a positive first TEFNA. Patients with a negative first TEFNA may undergo a repeated attempt, but a donor sperm back-up is strongly advised.