Prevalence of parvovirus B19 antibodies in pregnant women in northern Benin.

OBJECTIVES: Parvovirus B19 (B19V) infection in pregnancy is generally asymptomatic, but in about 3% it can cause complications, including miscarriage, severe foetal anaemia and foetal hydrops. The seroprevalence in pregnancy ranges from 20% to 82% in Africa, but there are no data for Benin. We therefore retrospectively assessed the seroprevalence of B19V in pregnant women attending the Saint Jean de Dieu Hospital in Tanguiéta, a rural district of Atacora, in northern Benin. METHODS: We searched for anti-B19V immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies in 227 sequential sera from as many women (mean age 26.3 years, range: 16-41) of whom 30 were in the first trimester, 66 in the second and 131 in the third. Samples that tested positive for IgM were analysed with an immunoblot test and the viral genome (DNA-B19V) was searched for using a polymerase chain reaction. RESULTS: Of the 227 women, 153 (67.4%) were positive for IgG anti-B19V, 7 (3.1%) for IgM and 73 (32.2%) were non-immune. Six IgM-positive women were also IgG positive. The difference in IgG seroprevalence between trimesters or ages was not statistically significant. Of the seven IgM-positive samples, three were confirmed positive by immunoblot (of which two were DNA-B19V positive), three were indeterminate (DNA-B19V negative) and one was negative (DNA-B19V negative). Of the three women with confirmed positive IgM, two were in the third trimester and one in the second trimester of pregnancy. CONCLUSIONS: The seroprevalence of anti-B19V IgG among pregnant women in Benin is high and in line with those reported in some African countries. IgM seroprevalence is also similar to that described in some African countries in non-epidemic periods. The low viral load observed depicts non-acute infections, but it is difficult to establish the precise time of the infection, especially for women tested in the second or third trimester of pregnancy, when the observed viremia could be a sign of an acute infection that occurred in the previous trimester. Consequently, clinical follow-up and further investigations to highlight possible foetal consequences are indicated.

CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5' LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5' LTR promoter.

Defining stable reference genes in HIV latency reversal experiments.

Quantification of cell associated HIV RNA (ca-RNA) is one of the most important and commonly used methods to evaluate the performance of latency-reversing agents (LRAs). Copies of HIV RNA measured by qPCR, are often normalized to the input RNA or cell number. However, these could be affected by biological variability and/or technical errors, which can be avoided by using an internal reference gene. To obtain reliable data, it is essential to select stable reference genes (RGs) of which the expression is not influenced by biological variability, the type of cells, or the LRAs used. However, to date, no study has carefully evaluated RG stability following LRA exposure. We analyzed the stability of six widely used RGs (GAPDH, TBP, YWHAZ, UBE2D2, HPRT1 and RPL27A) in human PBMC and CD4+ T cells. LRA exposure significantly influenced the stability of these RGs. Overall, TBP, UBE2D2, and RPL27A were the most stable RGs in all tested conditions. TBP was generally the most stable RG whereas GAPDH varied the most. Finally, we evaluated the impact of applying different RG normalizers to host genes and HIV ca-RNA data. Altered results were observed both in host and HIV gene expression when unstable RGs were used. Our data underline the importance of testing the stability of RGs utilized to evaluate LRA-induced HIV ca-RNA expression. To our knowledge, this is the first careful evaluation of the stability of RGs after LRA exposure and will significantly contribute to the quality of data analysis in regard to gene expression.IMPORTANCELatency-reversing agents (LRAs) are ubiquitously used in the "shock-and-kill" HIV cure strategy and their performance is often evaluated by ex-vivo quantification of cell associated HIV RNA. HIV RNA, measured by qPCR, is often normalized to internal reference genes, but the expression of these genes should not be influenced by the experimental settings. We found that treatment of human PBMC and CD4+ T cells with LRAs significantly altered the expression of several commonly used reference genes, such as GAPDH. Finally, we evaluate the impact of different reference genes on normalization of host genes and HIV cell associated RNA expression and demonstrated that using unstable reference genes dramatically altered experimental outcome. Our data highlight the importance of using reference genes that are unaffected by LRAs under study to correctly evaluate host gene and cell associated HIV RNA expression induced by latency-reversing agents.

New Frontiers in Measuring and Characterizing the HIV Reservoir.

A cure for HIV infection remains elusive due to the persistence of replication-competent HIV proviral DNA during suppressive antiretroviral therapy (ART). With the exception of rare elite or post-treatment controllers of viremia, withdrawal of ART invariably results in the rebound of viremia and progression of HIV disease. A thorough understanding of the reservoir is necessary to develop new strategies in order to reduce or eliminate the reservoir. However, there is significant heterogeneity in the sequence composition, genomic location, stability, and expression of the HIV reservoir both within and across individuals, and a majority of proviral sequences are replication-defective. These factors, and the low frequency of persistently infected cells in individuals on suppressive ART, make understanding the reservoir and its response to experimental reservoir reduction interventions challenging. Here, we review the characteristics of the HIV reservoir, state-of-the-art assays to measure and characterize the reservoir, and how these assays can be applied to accurately detect reductions in reservoir during efforts to develop a cure for HIV infection. In particular, we highlight recent advances in the development of direct measures of provirus, including intact proviral DNA assays and full-length HIV DNA sequencing with integration site analysis. We also focus on novel techniques to quantitate persistent and inducible HIV, including RNA sequencing and RNA/gag protein staining techniques, as well as modified viral outgrowth methods that seek to improve upon throughput, sensitivity and dynamic range.

BsmI, ApaI and TaqI Polymorphisms in the Vitamin D Receptor Gene (VDR) and Association with Lumbar Spine Pathologies: An Italian Case-Control Study.

Three adjacent single nucleotide polymorphisms of the vitamin D receptor gene (VDR) BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236) are commonly studied in several pathologies. We aimed to evaluate the distribution of VDR BsmI, ApaI, and TaqI allele, genotype, and haplotype frequencies in an Italian cohort of 266 patients with lumbar spine disorders assessed by Magnetic Resonance Imaging and 252 asymptomatic controls. The exposure to putative risk factors was evaluated by a questionnaire. Polymorphisms were detected by PCR-RFLP and TaqMan® SNP Genotyping Assay. The results were statistically adjusted for the identified conventional risk factors. The three SNPs were in linkage disequilibrium. For all cases BbAaTT was a 3-fold risk factor OR = 3.38), whereas bbAATT (OR = 0.22), and bbaaTT (OR = 0.47) genotypes were found to be protective. Specifically, for patients affected by disc herniation only (n = 88) and all lumbar pathologies excluding stenosis and/or spondylolistesis (n = 215) B allele, Bb, Aa, and BbAaTT genotypes were risky, whereas b allele, bb, aa, and bbaaTT genotypes were protective. In patients affected by osteochondrosis with or without disc hernation (n = 50), T allele, Aa, and bbAaTT genotypes were risky, whereas t allele, AA, tt genotypes were protective. In patients affected by stenosis and/or spondylolistesis (n = 51) no significant associations were found. This is the first study showing an association of the three genetic VDR variants BsmI, ApaI, and TaqI and lumbar spine pathologies. Our study contributes to delineate genetic risk factors for specific subgroups of patients with lumbar spine pathologies highlighting the importance of haplotype analysis, and of detailed clinical evaluation of the patients for identification of genetic biomarkers.

Epidemiology of hepatitis E virus infection during pregnancy in Benin.

OBJECTIVES: Hepatitis E virus (HEV) is the cause of enterically transmitted non-A, non-C hepatitis (an infection that is particularly severe during pregnancy) in tropical and subtropical countries. As there are no published data concerning the prevalence of HEV antibodies in Benin, their presence was investigated in pregnant women undergoing routine HIV screening in a rural area in northern Benin and in pregnant women with acute non-A, non-C hepatitis. METHODS: A total of 278 serum samples were collected from asymptomatic pregnant women in 2011 were tested for HEV and hepatitis A virus (HAV) antibodies, and the HEV IgM-positive samples were further tested for HEV-RNA. A further seven samples of pregnant women with acute non-A, non-C hepatitis collected during episodes of acute hepatitis in 2005 were also analysed. RESULTS: Of the 278 samples collected in 2011, 16.19% were positive for HEV IgG and 1.44% for HEV IgM (none positive for HEV-RNA), and 99.64% were positive for total HAV antibodies (none positive for HAV IgM). Six of the seven samples collected in 2005 were positive for HEV IgG and IgM, and two were also positive for HEV-RNA. CONCLUSIONS: The circulation of HEV infection is significant among pregnant women in Benin, in whom the consequences may be fatal.

Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints.

Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.

In vitro characterization and in vivo behavior of human nucleus pulposus and annulus fibrosus cells in clinical-grade fibrin and collagen-enriched fibrin gels.

The intervertebral disc (IVD) presents a limited self-repair ability and cell-based therapies have been suggested to prevent or treat IVD lesions. Fibrin-based scaffolds as cell carriers are promising candidates in IVD tissue engineering, thanks to their ability to be easily delivered into the defect and to adapt to the lesion shape, to support/retain the injected cells into the implantation site and to favor the production of a suitable extracellular matrix (ECM). We evaluated the in vitro and in vivo behavior of human nucleus pulposus (NP) and annulus fibrosus (AF) cells in a clinical-grade collagen-enriched fibrin that has never been tested before for orthopedic applications, comparing it with clinical-grade fibrin. The survival of IVD cells seeded within fibrin or collagen-enriched fibrin and the ECM synthesis were evaluated by biochemical, immunohistochemical, and transcriptional analyses, prior and after subcutaneous implantation of the gels in nude mice. After 28 days of implantation, NP and AF cells were still detectable within explants, produced tissue-specific ECM, and showed a higher content of glycosaminoglycans (GAGs) and type I and II collagen compared to gels before implantation. Both the fibrin gels, enriched or not with collagen, seemed to be suitable for the culture of AF cells, being able to support the homogeneous synthesis of type I collagen, characteristic of the native fibrocartilaginous AF tissue. Differently, fibrin alone was a more suitable matrix for NP culture, supporting the homogeneous deposition of GAGs and type II collagen. In conclusion, our results suggest to combine AF cells with fibrin, enriched or not with collagen, and NP cells with fibrin alone to maintain the typical features of these cell populations, indicating these clinical-grade materials as viable options in cell-based treatments for IVD lesions.

Fibrin in intervertebral disc tissue engineering.

Fibrin is clinically employed as a versatile, safe, and clinically applicable sealant and cell carrier. It has been able to support disc cell survival, favor extracellular matrix production, and enhance the efficiency of cell transfer in the intervertebral disc (IVD). The aim of this review was to evaluate how fibrin has been used in vitro, in vivo, and in clinical trials for IVD tissue engineering. Within the in vitro studies, disc cells were cultured in fibrin alone or combined with other materials and a difference in the behavior of nucleus pulposus (NP) and annulus fibrosus (AF) cells was sometimes reported, but in general, the formation of fibrocartilaginous matrix was observed. Moreover, data concerning the fibrin long-term stability and its anti-inflammatory properties were found. Disc cells of human origin were never employed in combination with fibrin in vivo or in clinical trials. In vivo, disc degeneration models used to test the fibrin properties essentially involved NP injuries. The addition of cells, in particular if terminally differentiated, to the injected fibrin seemed to promote a more physiological matrix in comparison with fibrin alone. Important aspects should be further investigated in future studies such as the use of fibrin to treat AF lesions as well as the mechanical properties of the fibrin-based biomaterials and of the neoformed tissue. Finally, in vivo studies and clinical trials with in situ injection of fibrin and human disc cells should be performed.

Prevalence of HBV, HDV, HCV, and HIV infection during pregnancy in northern Benin.

Pregnant women are not screened for HBsAg and anti-HCV antibodies in many African countries. As there are few data concerning the prevalence of HBV, HDV, and HCV serological markers in Benin, the aim of this study was to evaluate their 2011 prevalence in pregnant women undergoing HIV screening in a rural area of north Benin, and compare the data with those reported for the same area in 1986. The sera of 283 women were examined for HBsAg, anti-HBs, anti-HBc, anti-HCV, and anti-HIV 1/2 antibodies. In the case of HBsAg positivity, a search was made for the HBeAg, anti-HDV, and HBV genotypes; in the case of anti-HCV positivity, a search was made for the HCV genotypes. HBsAg, anti-HBs, anti-HBc, anti-HCV, and anti-HIV 1/2 were positive in respectively 44 (15.5%), 82 (29.0%), 234 (82.7%), 21 (7.4%), and nine samples (3.2%). Of the HBsAg-positive samples, five (11.4%) were positive for HBeAg, five (11.4%) for anti-HDV, and 19 for HBV genotype E. Of the anti-HCV-positive samples, five were positive for genotype 2a/2c and one for genotype 1a. The prevalence of anti-HBc alone (HBsAg and anti-HBs negative) was very high (41.3%). In comparison with the 1986 data, the prevalence of HBsAg and anti-HBc remained unchanged, that of HBeAg and anti-HDV had decreased, and that of anti-HIV 1/2 had increased. As these data confirm that HBV and HCV are highly endemic in the study area, it may be appropriate to introduce HBsAg and anti-HCV screening for pregnant women. J. Med. Virol. 86:1281-1287, 2014. © 2014 Wiley Periodicals, Inc.

Antenatal screening for Toxoplasma gondii, Cytomegalovirus, rubella and Treponema pallidum infections in northern Benin.

OBJECTIVES: Toxoplasma gondii, cytomegalovirus (HCMV) and rubella virus infections are among the most serious of those contracted during pregnancy in terms of foetal consequences. Toxoplasma, HCMV and rubella antibody screening is unusual in Africa, and there are few published data. The aim of this study was to evaluate the prevalence of these markers among pregnant women in northern Benin on the occasion of routine syphilis screening. METHODS: Toxoplasma, HCMV and rubella IgG and IgM antibodies were determined in the serum of 283 women attending Saint Jean de Dieu de Tanguiéta hospital, using an enzyme immunoassay, and IgM were confirmed using an enzyme-linked fluorescent assay (ELFA). In the case of IgM positivity, the avidity of anti-HCMV and anti-Toxoplasma IgG was measured. Total anti-Treponema pallidum antibodies were determined using an enzyme immunoassay and confirmed by immunoblotting. In the case of positivity, the Venereal Disease Research Laboratory (VDRL) test was used. RESULTS: The prevalence of anti-Toxoplasma, anti-HCMV, anti-rubella IgG and total anti-Treponema antibodies was, respectively, 30.0%, 100%, 94% and 2.5%. The VDRL test was positive in 62.5% of the anti-Treponema-positive samples. The prevalence of anti-Toxoplasma, anti-HCMV and anti-rubella IgM was, respectively, 0.4%, 1.4% and 0%. There were no statistically significant differences in terms of age class or trimester of pregnancy. Anti-Toxoplasma and anti-HCMV IgG avidity was always high. CONCLUSIONS: The prevalence of HCMV and rubella antibodies is high in northern Benin, whereas that of Toxoplasma antibodies is lower. As nearly two-thirds of the pregnant women were anti-Toxoplasma seronegative, antibody screening should be introduced.