Toxoplasma gondii antibody profile in HIV-1-infected and uninfected pregnant women and the impact on congenital toxoplasmosis diagnosis in Rio de Janeiro, Brazil

ObjectiveCompare the anti-T. gondii IgG titer between HIV-1 infected and non HIV-1 infected pregnant women and report three cases of congenital toxoplasmosis resulting from reactivation of infection during pregnancy of HIV-1 infected women.MethodsThis study was conducted among 2,270 pregnant women with chronic Toxoplasma gondii infection (absence of IgM and presence of IgG), including 82 HIV-1 infected and 2,188 non-infected women.ResultsThe average anti-T. gondii IgG titer was 127 for the 2,188 non-HIV-1 infected women, and 227 for the 82 HIV-1-infected women (p = 0,007). These results suggested that higher anti-T. gondii IgG titers in HIV-1-infected pregnant women may not be indicative of an elevated risk for fetal infection. In this study three cases of congenital toxoplasmosis that resulted from infection reactivation during pregnancy of HIV-1-infected women were manifested by fetal death, symptomatic infection, and infant without symptoms, respectively. In two of these women, a ten-fold increase in IgG levels above used cutoff was observed (2,320 UI/mL and 3,613 UI/mL, respectively). In the third pregnant women anti-T. gondii IgG titers during pregnancy did not rise despite the occurrence of congenital toxoplasmosis (204; 198; 172 UI/mL).ConclusionsCongenital toxoplasmosis resulting reactivation of infection during pregnancy in the studied group leads us to believe that it is a public health problem, especially in our population, in which seroprevalence of T. gondii infections is high. These findings also suggest that special attention is necessary during pregnancy, because the serologic diagnosis may not be indicative of toxoplasmosis reactivation.     

How can HIV-type-1-Env immunogenicity be improved to facilitate antibody-based vaccine development?

No vaccine candidate has induced antibodies (Abs) that efficiently neutralize multiple primary isolates of HIV-1. Preexisting high titers of neutralizing antibodies (NAbs) are essential, because the virus establishes infection before anamnestic responses could take effect. HIV-1 infection elicits Abs against Env, Gag, and other viral proteins, but of these only a subset of the anti-Env Abs can neutralize the virus. Whereas the corresponding proteins from other viruses form the basis of successful vaccines, multiple large doses of HIV-1 Env elicit low, transient titers of Abs that are not protective in humans. The inaccessibility of neutralization epitopes hinders NAb induction, but Env may also subvert the immune response by interacting with receptors on T cells, B cells, monocytes, macrophages, and dendritic cells. Here, we discuss evidence from immunizations of different species with various modified Env constructs. We also suggest how the divergent Ab responses to Gag and Env during infection may reflect differences in B cell regulation. Drawing on these analyses, we outline strategies for improving Env as a component of a vaccine aimed at inducing strong and sustained NAb responses.     

Most rhesus macaques infected with the CCR5-tropic SHIVAD8 generate cross-reactive antibodies that neutralize multiple HIV-1 strains

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIV_AD8-EO) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1_DH12 strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIV_AD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIV_AD8-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti–HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41–51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIV_AD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains.A major challenge in HIV vaccine research has been the development of immunogens capable of eliciting potent, broadly acting, neutralizing antibodies (NAbs). It is now appreciated that 10–30% of HIV-1–infected individuals produce cross-reactive NAbs of significant breadth (1–6). Less than 1% of such persons, so-called “elite neutralizers,” produce potent cross-clade–neutralizing activity, but only 2–3 y after virus acquisition (6). Although the emergence of broadly reacting anti–HIV-1 NAbs in elite neutralizers has been associated with multiple rounds of somatic hypermutation (7), little is known about vaccine strategies able to elicit such antibodies. Longitudinal studies of HIV-1– infected persons have suggested that set-point plasma virus loads, CD4⁺ T-cell levels, duration of the infection, or antibody-binding avidity may contribute to the development of cross-reacting NAbs (5, 8, 9). It is also possible that the induction of such antibodies depends on unique gp120 epitopes associated with specific HIV-1 strains and individual B-cell repertoires or is simply a random process (10, 11). A nonhuman primate model capable of generating cross-reactive anti–HIV-1–neutralizing activity could provide answers to some of these questions and contribute to the development of an effective prophylactic vaccine. In this regard, we recently reported that one rhesus monkey, inoculated with an uncloned preparation of the R5-tropic SHIV_AD8, developed broad, potent, and glycan-specific NAbs with cross-reacting activity against virus isolates from different HIV-1 clades similar to that described for “elite” HIV-1 neutralizers (12).In this study, we describe the construction of a pathogenic SHIV_AD8 molecular clone (SHIV_AD8-EO). During its characterization, we discovered that eight of eight SHIV_AD8-EO–infected animals generated cross-reactive NAbs directed against a SHIV carrying an envelope glycoprotein derived from a different HIV-1 isolate (namely HIV-1_DH12). Because this result suggested that a majority of monkeys infected with the SHIV_AD8-EO family of viruses might be able to generate NAbs against heterologous HIV-1 isolates, plasma samples collected from a cohort of 11 rhesus macaques, infected with either uncloned SHIV_AD8 swarm stocks or SHIV_AD8 molecular clones, were tested for their capacity to neutralize a panel of clade A, B, and C HIV-1 isolates. All of the plasmas from this group of 11 animals neutralized several tier 1A and 1B clade B HIV-1 strains. Three of the 11 macaques also generated >1:100 IC₅₀ neutralization titers against some tier 2 clade B HIV-1 isolates, and one of the three produced significant NAb titers against some clade A and C isolates. Taken together, these findings indicate that SHIV_AD8 is uniquely immunogenic during infections of rhesus monkeys and may be a particularly useful reagent for identifying viral determinants that drive B-cell maturation resulting in cross-reactive anti–HIV-1 NAbs.     

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1–infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

The primary objective of a vaccine against a viral infection is to elicit a long-lasting protective neutralizing antibody (NAb) response. The design of an effective vaccine candidate necessitates a high-resolution, residue-level, map of the epitopes on the viral surface that are targeted by NAbs. This information is usually obtained from X-ray crystallography or cryogenic electron microscopy (cryo-EM)–based structural studies of purified complexes of a monoclonal antibody with its target viral protein. These methods provide a detailed high-resolution map of the interaction between the antibody and its target surface and assist in vaccine design. However, apart from being highly labor-intensive and time-consuming, these methods are not suitable for mapping polyclonal antibody responses during a viral infection. Therefore, there is an unmet requirement for a rapid, parallelizable, complementary method to accurately map polyclonal NAb epitopes. We recently described a methodology to decipher epitopes of monoclonal antibody panels using yeast surface display, Cys labeling, and deep sequencing (1, 2). Here, we have combined Cys labeling of viral surface glycoprotein and deep sequencing, with virus neutralization assays to map epitopes of neutralizing monoclonal antibodies and polyclonal sera against the HIV-1 virus. The design of an effective vaccine against HIV-1 has met with limited success so far, because of the inability of immunogens to elicit broadly neutralizing antibodies (bNAbs) targeted to the trimeric envelope (Env) glycoprotein, the sole viral antigen on the surface of the virus (3–5). Most bNAbs isolated from infected individuals are directed to one of the major sites of vulnerability on Env: V1/V2 loop apex, V3 loop, CD4-binding site, center of the gp120 silent face, gp120–gp41 subunit interface, gp41 fusion peptide, and membrane proximal external region (MPER) of gp41 (3, 6, 7).We developed a high-throughput methodology for mapping neutralizing HIV-1 epitopes at single-residue resolution directly on the native viral Env, obviating the need to purify Env glycoprotein. To this end, a library of single-site Cys mutations were engineered at selected solvent-exposed sites in the Env glycoprotein on the surface of the virus. Cys mutants were covalently labeled using a Cys-reactive bulky maleimide label, that masks epitope residues, followed by infection of the labeled mutant viruses in mammalian cells in the presence of NAbs. Subsequently, deep sequencing of the env gene from NAb-resistant viruses was used to delineate epitopes of NAbs (Fig. 1). We focused our epitope mapping efforts to the Env ectodomain of the clade B, CCR5-tropic primary HIV-1 isolate JRFL, which has been extensively characterized both biochemically and structurally, and is used as a model primary virus.Open in a separate windowFig. 1.Approach to rapidly map neutralizing epitopes of HIV-1. (A) A library of single Cys mutants was generated at 81 solvent-exposed sites in the env gene of the HIV-1 molecular clone, pLAI-JRFL. Approximately one-half of these sites are part of known bNAb epitopes, whereas the rest are nonepitopic. The Exposed Cys mutant plasmid library, an equimolar pool of these single Cys env mutants in pLAI-JRFL, was used to transfect HEK293T cells. This led to the production of a library of mutant viruses, having Cys mutations at the selected solvent-exposed residues in Env; henceforth referred to as the ExCysLib. The ExCysLib was covalently labeled with a Cys label, EZ-Link Maleimide-PEG2-Biotin, and then incubated with NAbs (either monoclonal bNAbs or polyclonal sera). At epitope residues, masking by the bulky Cys label prevents bNAbs from binding and neutralizing the epitope mutant viruses. (B) Quantification using infectivity assays of the ExCysLib before and after labeling, in the absence and presence of NAbs. Unlabeled epitope and nonepitope Cys mutant viruses are similarly sensitive to neutralization. However, upon labeling, epitope mutant viruses are resistant to neutralization, unlike the nonepitope counterparts. Infectivity ratio (=infectivity without NAb/infectivity with NAb) was used as a metric to discriminate between epitope versus nonepitope mutant viruses. Upon labeling, infectivity ratio of nonepitope mutant viruses >> 1; however, for epitope mutant viruses, the ratio is ∼1. (C) Deep sequencing of the env gene from antibody/sera-resistant virus pools was used to identify epitope residues at single-residue resolution. The figure was created using BioRender.com.     

噬菌体表达短肽模拟血吸虫致弱尾蚴特异性抗原表位的研究

目的　从噬菌体随机肽库中筛选模拟血吸虫致弱尾蚴特异性抗原表位的噬菌体克隆 ,并探讨其免疫保护效果。方法　用紫外线致弱尾蚴免疫兔血清IgG筛选以融合蛋白形式在丝状噬菌体外壳蛋白Ⅲ表达的随机 7肽库 ,三轮免疫筛选后获得的噬菌体克隆经皮下免疫小鼠 3次 ,攻击感染 45d后剖杀 ,观察减虫和减卵效果。结果　经三轮筛选 ,特异性结合的噬菌体富集增加了 10 0多倍 ,随机挑取 2 4个噬菌体克隆经ELISA测定 ,有 2 2个克隆能与致弱尾蚴免疫兔血清IgG特异性反应。混合噬菌体克隆免疫小鼠试验的减虫率与减卵率分别为 33.5 7%和 5 6 .0 7% (P均 <0 .0 0 1)。结论　采用噬菌体随机肽库技术可获得模拟致弱尾蚴特异性抗原表位的短肽分子 ,这些短肽分子能诱导一定程度的保护性免疫     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV~37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0&#177;1.6) %to (39.0&#177;2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pⅢ in order to get some information on mimotopes.METHODS: Through affinity enrichment and ELISA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times ofenrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6)%to (39.0±2.7)%.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

随机肽库筛选弓形虫特异性抗原表位诱导保护性免疫的研究

目的　从噬菌体随机肽库中筛选出模拟弓形虫特异性抗原表位的短肽分子 ,并探讨其对弓形虫的保护性效果。方法　以纯化的弓形虫免疫兔血清IgG为配基 ,亲和筛选法富集特异性噬菌体 ,随机挑取噬菌体克隆用夹心ELISA法和Dot-ELISA法检测其特异性 ,混合 4个阳性克隆免疫小鼠 ,1月后攻击感染 ,观察小鼠发病时间和死亡时间。结果　经 3轮筛选 ,特异性噬菌体得到了有效富集 ,第 3轮洗脱噬菌体的产量为第 1轮的 16 7倍 ,随机挑取 2 7个噬菌体经Dot -Bloting和夹心ELISA法检测 ,有 2 4个能与弓形虫免疫兔血清及单克隆抗体特异反应。用致死量弓形虫攻击感染经阳性克隆免疫的小鼠 ,存活时间明显长于对照组。结论　免疫筛选噬菌体随机多肽库可获得具有保护性的弓形虫抗原表位 ,为弓形虫疫苗研制提供了新途径。     

Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome

In a cohort study of 56 convalescent patients with severe acute respiratory syndrome (SARS), titers of immunoglobulin G (IgG) antibodies and neutralizing antibodies (NAbs) against SARS-associated coronavirus were assessed at regular intervals (at 1, 4, 7, 10, 16, and 24 months after the onset of disease) by use of enzyme-linked immunosorbent assay and neutralization assay. IgG antibody and NAb titers were highly correlated, peaking at month 4 after the onset of disease and decreasing thereafter. IgG antibodies remained detectable in all patients until month 16, and they became undetectable in 11.8% of patients at month 24. The finding that NAbs remained detectable throughout follow-up is reassuring in terms of protection provided against reinfection; however, NAb titers decreased markedly after month 16.     

Syphilis and HIV-1 among parturient women in Salvador,Brazil: low prevalence of syphilis and high rate of loss to follow-up in HIV-infected women

BackgroundThe occurrence of syphilis and HIV-1 infections during pregnancy are major risks to the fetus due to mother-to-child transmission (MTCT).ObjectivesTo determine peripartum seroprevalence and risk factors of syphilis and HIV-1 infection among pregnant women in Salvador, Brazil, and the rate of HIV-1 MTCT.MethodsCross-sectional study of pregnant women who were admitted for delivery in a reference maternity hospital between May 2008 and March 2009 was conducted. Women were screened for HIV-1 infection and syphilis, and interviewed regarding demographic, behavioral and obstetric data. Newborns to HIV-infected mothers were tested by b-DNA and DNA-PCR to detect HIV-1.ResultsA total 3300/8516 women were evaluated. Mean age was 25.8 ± 7.3 years. HIV-1 and syphilis seroprevalence rates were 0.84% (28/3300) and 0.51% (17/3300), respectively. HIV-1 infection was associated with: low education (p = 0.04), having a partner with known HIV infection (p < 0.0001) or with previous sexually transmitted infection (p < 0.0001), blood transfusion (p = 0.003), or accidental exposure to blood (p = 0.003). Syphilis was associated with being Caucasian (p = 0.02), having no steady partner (p = 0.02), being a housewife (p = 0.01), having an intravenous drug user (IVDU) sexual partner (p = 0.04) or a sexual partner with previous STI (p < 0.001). Higher education (p = 0.04) was protective against HIV-infection. Attending a prenatal care program was protective against syphilis (p = 0.008) and HIV-1 (p = 0.02). No case of HIV-1 MTCT was detected, but 25% of children born to HIV-infected mothers were lost to follow up.ConclusionsIn Salvador, peripartum prevalence of syphilis and HIV-1 infection among pregnant women were low, and associated with classic risk factors for both infections. The great proportion of very late diagnosis of HIV infection, and the high rate of loss of follow-up among positive mothers and their infants are of high concern.     

Prevalence and function of anti-lipoprotein lipase auto-antibodies in type V hyperchylomicronemia

PurposeType V hyperlipidemia (HTG V) characterized by accumulation of both chylomicrons and VLDL results from a complex combination of genetic and environmental factors. However, a large proportion of sporadic cases remains largely unexplained. In a few cases, in a context of autoimmunity, auto-antibodies inhibiting lipoprotein lipase (LPL) activity have been incriminated. To establish their contribution to common type V hyperlipidemia in subjects with no apparent evidence of autoimmune background, we systematically screened the presence of these antibodies and their inhibition properties.MethodsScreening for circulating anti-human LPL immunoglobulin G (anti-hLPL IgG) was carried out by western blotting in 63 subjects with HTG V and 77 controls. Inhibition of lipolytic activity by plasma from these patients was measured ex vivo.ResultsAnti-hLPL IgG was detectable in plasma from both controls and subjects with HTG V. After establishment of a threshold value corresponding to the 95th percentile of the control population, 27% of subjects with HTG V were found to have abnormal antibody levels (P < 0.001). Only plasma obtained from these hyperchylomicronemic subjects with a high level of anti-hLPL IgG inhibited triglyceride hydrolysis whereas plasma from controls or HTG subjects with normal anti-hLPL IgG levels had no inhibitory effect (?13.5 ± 3.4% vs 1.6 ± 3.4%; P = 0.04). However, no correlation was observed between anti-hLPL IgG levels, inhibitory effect and plasma triglyceride concentration.ConclusionHigh levels of anti-hLPL immunoreactivity could be detected in only one out of four adult patients with type V hyperchylomicronemia. Furthermore, only a minority of these subjects (less than 10%) displayed both high anti-hLPL IgG levels and substantial inhibition (>20%) of plasma lipolysis. These auto-antibodies, in this setting only, might contribute to the occurrence of a minority of sporadic type V dyslipidemia cases.     

Longitudinal study of humoral immune responses in HIV type 1 subtype CRF01_AE (E)-infected Thai patients with different rates of disease progression

Identification of immune correlates associated with disease progression will provide information for HIV-1 vaccine design in countries such as Thailand, where the prevalent subtypes (B and CRF01_AE [E]) are characterized. In this study, plasma viral load and humoral immune responses were measured in 20 HIV-1 subtype E-infected Thai patients with different rates of disease progression, based on CD4(+) T cell decline and clinical symptoms. Nine progressors (PRs) and 11 slower progressors (SPs) were evaluated. CD4(+) T cell counts were inversely correlated with viral load (p = 0.004) and positively correlated with p24 Ab (p = 0.022). In progressors, p24 Ab showed a significant decrease (p < 0.001) over time. V3 and gp41 Ab did not change significantly in either group. Both CD4-binding site (CD4/gp120BS) and gp120 titers correlated positively with neutralizing antibody (NAb) against both a subtype E cell line-adapted virus (NP03) and a primary isolate (TH023). However, V3 Ab correlated only with NAb against NP03 (p < 0.001). Increased NAb over time was observed more frequently in SPs as compared with PRs, against both the TH023 (p = 0.004) and NPO3 (p = 0.004) viruses. Cross-clade antibody-dependent cellular cytotoxicity was demonstrated in both groups. These data suggest that in HIV-1 subtype E infection, declining p24 Ab titer is a predictive marker of disease progression, as described for subtype B. Furthermore, in subtype E-infected patients, slower progressors retain the immune competence to develop new antibody responses to Env over time; these evolving responses may contribute to prolonged survival during HIV-1 disease progression.     

Análisis de resultados del Programa de Control de Calidad Externo SEIMC de carga viral del VIH-1, VHC y VHB. Año 2017

BackgroundHuman immunodeficiency virus type 1 (HIV-1) and hepatitis B (HBV) and C virus (HCV) viral load determinations are among the most relevant markers for the follow-up of patients infected with these viruses. External quality control tools are crucial to ensure the accuracy of the results obtained by microbiology laboratories. This article summarised the results obtained from the 2017 SEIMC External Quality Control Programme for HIV-1, HCV, and HBV viral loads and HCV genotyping.Methods and resultsIn the HIV-1 programme, a total of five standards were sent. One standard consisted of seronegative human plasma, while the remaining four contained plasma from three different viremic patients, in the range of 2-5 log₁₀ copies/mL; two of these standards were identical, with the aim of determining repeatability. A significant proportion of the laboratories (35% on average) obtained values outside the accepted range (mean ± 0.25 log₁₀ copies/mL), depending on the standard and on the method used for quantification. Repeatability was good, with up to 94% of laboratories reporting results within the limits (D < 0.5 log₁₀ copies/mL). The HBV and HCV programme consisted of two standards with different viral load contents. Most of the participants, 82% in the case of HCV and 87% in that of HBV, obtained all the results within the accepted range (mean ± 1.96 SD log₁₀ UI/mL).ConclusionsData from this analysis reinforce the utility of proficiency programmes to ensure the quality of the results obtained by a particular laboratory. Due to the marked interlaboratory variability observed, it is advisable to use the same method and laboratory for patient follow-up.     

Análisis de resultados del Programa de Control de Calidad Externo SEIMC de carga viral del VIH-1, VHC y VHB. AÑO 2018

BackgroundHuman immunodeficiency virus type 1 (HIV-1) and hepatitis B (HBV) and C virus (HCV) viral load determinations are among the most relevant markers for the follow-up of patients infected with these viruses. External quality control tools are crucial to ensure the accuracy of the results obtained by microbiology laboratories. This article summarised the results obtained from the 2018 SEIMC External Quality Control Programme for HIV-1, HCV, and HBV viral loads and HCV genotyping.Methods and resultsIn the HIV-1 program, a total of five standards were sent. One standard consisted of seronegative human plasma, while the remaining four contained plasma from three different viremic patients, in the range of 2-5 log₁₀ copies/mL; two of these standards were identical, with the aim of determining repeatability. A significant proportion of the laboratories (28% on average) obtained values outside the accepted range (mean ± 0.25 log₁₀ copies/mL), depending on the standard and on the method used for quantification. Repeatability was good, with most laboratories reporting results within the limits (D < 0.5 log₁₀ copies/mL). The HBV and HCV programme consisted of two standards with different viral load contents. Most of the participants, 87% in the case of HCV and 88% in the HBV, obtained all the results within the accepted range (mean ± 1.96 SD log₁₀ UI/mL).ConclusionsData from this analysis reinforce the utility of proficiency programmes to ensure the quality of the results obtained by a particular laboratory. Due to the marked interlaboratory variability, it is advisable to use the same method and the same laboratory for patient follow-up.     

日本血吸虫雌虫抗原模拟表位的筛选及免疫保护性

目的　筛选日本血吸虫雌虫抗原的模拟表位 ,并探讨其抗日本血吸虫的免疫保护效果。　方法　用日本血吸虫雌虫免疫兔血清IgG作配体对噬菌体随机 12肽库进行 3轮亲和筛选 ,随机挑取 18个噬菌体克隆用Dot ELISA检测其特异性 ,并对其中的 4个阳性克隆进行测序。分别在 0、2、4周用混合噬菌体克隆免疫小鼠 3次 ,第 6周每鼠经腹部感染 40条日本血吸虫尾蚴 ,42d后剖杀冲虫 ,计数虫数和每克肝卵数。　结果　经 3轮筛选 ,特异性噬菌体富集了 2 0 0多倍 ,随机挑取的 18个克隆经Dot ELISA鉴定有 17个能与雌虫抗原免疫兔血清呈阳性反应。DNA自动测序的 4个序列与GenBank的已知序列均无同源性。与对照组相比 ,混合噬菌体克隆免疫小鼠的减虫率为 2 6.5 7% ,减卵率为 65 .3 4%。　结论　采用噬菌体随机肽库技术可获得模拟日本血吸虫雌虫特异性抗原表位的短肽分子 ,这些短肽分子能诱导一定程度的保护性免疫。     

Anti-t-PA antibodies in acute myocardial infarction after thrombolysis with rt-PA

BackgroundThrombolysis with recombinant tissue-type plasminogen activator (rt-PA) is successfully used in acute myocardial infarction with ST elevation (STEMI). Reocclusions follow rt-PA treatment in up to 30% of patients within one year. The infusion of rt-PA may induce the production of anti-t-PA antibodies which could interfere with the function of the native t-PA molecule.MethodsIn order to detect and characterise anti-t-PA antibodies, plasma samples were collected from 30 STEMI patients (20 treated and 10 not treated with rt-PA) at baseline before rt-PA infusion and then 15, 30, 90 and 180 days after STEMI and from 40 healthy subjects at baseline only. Immunoenzymatic, chromatographic and chromogenic methods were employed.ResultsAn increase of anti-t-PA antibodies was observed 15 days (IgM, p = 0.0001) and 30 days (IgG, p = 0.0001) after rt-PA infusion. Six patients had large increases of anti-t-PA IgG which bound the catalytic domain of t-PA (two cases) or kringle 2 domain (four cases), were of IgG1 or IgG3 subclasses and interacted with the t-PA molecule in fluid phase.ConclusionThe infusion of rt-PA may induce the production of specific antibodies that bind active sites of t-PA, thus potentially reducing its in vivo function.     

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals,revealing higher potency than the association of two methyltransferase inhibitors,chaetocin and BIX01294

BackgroundLatent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals.ResultsNAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log₁₀ RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4).ConclusionNAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.     

Elevated immunoglobulin G4 level is associated with reduced colectomy-free survival in patients with primary sclerosing cholangitis and ulcerative colitis

Background and aimPatients with primary sclerosing cholangitis (PSC) and elevated immunoglobulin (Ig) G4 have been shown to have more severe disease with a shorter time to orthotopic liver transplantation (OLT). The aim of the study was to investigate the clinical outcomes of PSC and UC in patients with elevated serum IgG4.MethodsWe analyzed data from 50 patients with PSC and known serum levels of IgG4. They were divided into groups called high IgG4 (> 112 IU/L; n = 10) or normal IgG4 (n = 40). We compared the requirement of OLT and colectomy between groups.ResultsHigh IgG4 was found in 10 PSC patients (20%). UC was associated in 9/10 patients with high IgG4 vs. 32/40 patients with normal IgG4 (p = 0.67). Patients with high IgG4 were younger at PSC diagnosis (28.1 ± 13.9 vs. 37.6 ± 13.4 years, P = 0.04), more likely to have backwash ileitis (7/9 vs. 12/32, P < 0.001) and UC flares (median of 5.5 vs. 1.5, P = 0.02). Kaplan–Meier curve analysis showed that patients with elevated IgG4 had reduced colectomy-free survival than patients with normal IgG4 (Log Rank p < 0.001). The median time to colectomy was 5 years from UC diagnosis in high IgG4 group vs. 12 years in the normal IgG4 group (p = 0.01).ConclusionsElevated IgG4 was seen in a small number of PSC patients. Most of these patients had associated UC, were younger at the time of PSC diagnosis, more likely to have backwash ileitis and had reduced colectomy-free survival than patients with normal IgG4.     

Human Immunodeficiency Virus Infection in Alabama Women: Sociodemographic,Behavioral, and Reproductive Health Characteristics and Factors Associated With Lack of Human Immunodeficiency Virus-1 Viral Control

BackgroundHuman immunodeficiency virus (HIV) infection among women in the southern United States is on the rise. This study examined sociodemographic profile and behavioral risk factors for HIV and sexually transmitted infections and assessed factors associated with HIV-1 viral control in a cohort of 280 HIV-infected Alabama women aged 17 to 66 years.MethodsWomen receiving care for HIV infection at a university outpatient HIV clinic were enrolled in the study. Women completed a self-administered questionnaire on demographics and behavioral risk factors at enrollment. They were followed up with appointments at least every 6 months with Papanicolaou smears, cervicovaginal lavages, cervical and vaginal swabs, and blood specimens collected at each visit.ResultsOf the women in the study, 69% were black, had mean age of 36 years, and ~ three fourths were mothers with annual household income < $20,000. White women were likely to have been HIV infected for a longer period (50.2 versus 36.3 months; P = 0.02) and had significantly lower viral loads at enrollment (P = 0.04) than black women. Factors associated with lack of HIV-1 control (≥ 10,000 RNA copies/mL) at enrollment included black race/ethnicity (odds ratio [OR]: 2.8; 95% confidence interval [CI]: 1.2 ? 6.8), CD4 + T-cell count < 200 cells/μL (OR: 20.1; CI: 8.6 ? 47.0), being diagnosed with HIV < 6 months (OR: 3.5; CI: 1.4 ? 8.9) and not being on any antiretroviral therapy (OR: 2.5; CI: 1.1 ? 5.7).ConclusionsPoorer HIV-1 viral control in black women at enrollment may indicate suboptimal access to HIV testing, delays in receipt of medical care after HIV-1 diagnosis, and/or some underlying biologic or social race-related influence.