RESUMEN
Surveillance of emerging viral variants is critical to ensuring that blood screening and diagnostic tests detect all infections regardless of strain or geographic location. In this study, we conducted serological and molecular surveillance to monitor the prevalence and diversity of HIV, HBV, and HTLV in South Cameroon. The prevalence of HIV was 8.53%, HBV was 10.45%, and HTLV was 1.04% amongst study participants. Molecular characterization of 555 HIV-1 specimens identified incredible diversity, including 7 subtypes, 12 CRFs, 6 unclassified, 24 Group O and 2 Group N infections. Amongst 401 HBV sequences were found a rare HBV AE recombinant and two emerging sub-genotype A strains. In addition to HTLV-1 and HTLV-2 strains, sequencing confirmed the fifth known HTLV-3 infection to date. Continued HIV/HBV/HTLV surveillance and vigilance for newly emerging strains in South Cameroon will be essential to ensure diagnostic tests and research stay a step ahead of these rapidly evolving viruses.
Asunto(s)
Infecciones por VIH/epidemiología , VIH-1/clasificación , Infecciones por HTLV-I/epidemiología , Virus de la Hepatitis B/genética , Hepatitis B Crónica/epidemiología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 3 Humano/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Secuencia de Bases , Camerún/epidemiología , Niño , Preescolar , ADN Viral/genética , Femenino , Genoma Viral/genética , Infecciones por VIH/virología , VIH-1/genética , Infecciones por HTLV-I/virología , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/virología , Virus Linfotrópico T Tipo 1 Humano/clasificación , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 2 Humano/clasificación , Virus Linfotrópico T Tipo 2 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 3 Humano/clasificación , Virus Linfotrópico T Tipo 3 Humano/aislamiento & purificación , Humanos , Lactante , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p=0.03). However, the PCR assay is significantly (p<0.001) more sensitive than any of the serologic assays in the detection of HTLV-II infection. Thus, optimal detection of HTLV-II infection would seem to require both serologic and DNA PCR assays.
Asunto(s)
Anticuerpos Anti-HTLV-I/inmunología , Anticuerpos Anti-HTLV-II/inmunología , Infecciones por HTLV-II/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Reacción en Cadena de la Polimerasa , Estadística como AsuntoRESUMEN
A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.
Asunto(s)
Anticuerpos Anti-VIH/sangre , VIH-1/inmunología , VIH-2/inmunología , Inmunoglobulina G/sangre , Humanos , Técnicas para Inmunoenzimas , Sensibilidad y EspecificidadRESUMEN
Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples (N = 440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.
Asunto(s)
Antígenos VIH/inmunología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-2/clasificación , Péptidos/inmunología , Secuencia de Aminoácidos , Productos del Gen env/inmunología , Productos del Gen gag/inmunología , Productos del Gen pol/inmunología , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , Humanos , Immunoblotting , Datos de Secuencia Molecular , Péptidos/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.
PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.
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Seropositividad para VIH/virología , VIH-1/clasificación , Genotipo , Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Seropositividad para VIH/sangre , Seropositividad para VIH/epidemiología , Seropositividad para VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Filogenia , Uganda/epidemiologíaAsunto(s)
Variación Genética , Proteína p24 del Núcleo del VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Secuencia de Aminoácidos , Camerún , Guinea Ecuatorial , Femenino , VIH-1/clasificación , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Four sera from Equatorial Guinea (EG) suspected to contain antibody against HIV-1 group O-related viruses were identified on the basis of unusual and differential serologic reactivity in selected commercial assays and Western blot. Degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences from the suspect EG sera. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. Analysis (PHYLIP package of programs) of Env amino acid sequences (translated from nucleotide sequences) indicated that the amino acid sequences obtained from EG sera clustered more closely with HIV Env sequences of group O compared to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY (one isolate), RIGPMAWY (two isolates), or GLGPLAVY (one isolate). The V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV (Los Alamos) database, but was present in two of our group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from three of the sera, RLLALETLIQNQQLLNLWGCKGR(K)L(I)VCYTSVK(T)W, whereas sequence from the fourth serum contained three changes as noted in parentheses. IDR sequences derived from EG sera were unique compared to those reported for other HIV-1 group O isolate ANT70, VAU, or MVP5180. Antibody in each EG serum directed against the IDR could be detected using synthetic peptides comprising sequences from the ANT70 or MVP5180 IDRs, but were most reactive against the sequences derived from the samples themselves. Little or no serologic reactivity was detected when EG sera were reacted against peptides comprising the IDR of HIV-1 group M (subtype B consensus) or HIV-2 (consensus).
PIP: The genetic variation and epidemiology of HIV-1 group O isolates are of considerable importance to the design of HIV-1 diagnostic and screening assays, especially since current serologic and genetic methods to detect HIV-1 have been developed mainly on the basis of sequences from isolates belonging to HIV-1 group M. The HIV envelope protein, especially the gp41 immunodominant region, plays a major antigenic role in the detection of HIV infection and for discriminating HIV-1 from HIV-2 antibody. This paper reports upon genetic variation and the serologic characterization of env sequences from 4 people living in Equatorial Guinea (EG) who were infected with HIV-1 group O. Selected commercial assays and Western blot were first used to identify the sera, then degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. The env amino acid sequence analysis found the EG sera sequences to be clustered more closely with the HIV env sequences of group O rather than to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY, RIGPMAWY, or GLGPLAVY. Although the V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV database, it was present in 2 of the group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from 3 of the sera. IDR sequences derived from the EG sera were unique compared to those reported for other HIV-1 group O isolates ANT70, VAU, or MVP5180. Other findings are discussed in detail.
Asunto(s)
Productos del Gen env/genética , Variación Genética , Infecciones por VIH/virología , VIH-1/genética , Secuencia de Aminoácidos , Guinea Ecuatorial , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , VIH-1/clasificación , VIH-1/inmunología , VIH-1/aislamiento & purificación , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Filogenia , Análisis de Secuencia de ADN , SerotipificaciónRESUMEN
PIP: HIV-1 group O is endemic in the west central region of Africa, where the frequency of infection is estimated to be 3-10% of all HIV-1-infected individuals. However, international travel and immigration have led to group O cases being identified in France, Germany, Belgium, Spain, and the US. With the exception of an infected French woman, all reported group O-infected individuals originate from or have a connection to west central Africa. Since most immunoassay reagents are based upon HIV-1 group M, many HIV immunoassays have lower sensitivity for the detection of group O infections. Serum samples were collected from patients at hospitals, tuberculosis (TB) clinics, and STD clinics in endemic regions of Cameroon and Equatorial Guinea in a study of the sequence divergence with group O isolate infections. Screening of the 1086 samples using a range of research and commercial immunoassays found 255 to be HIV-1 seropositive. On the basis of differential reactivity in the various immunoassays, 8 individuals were identified as potentially being infected with group O virus, of which 4 were drawn from TB patients. 7 of the group-O samples were then subjected to polymerase chain reaction (PCR) amplification to verify group O infection. The gp41(env) immunodominant region was successfully amplified and sequenced from 4 of the 7 samples, 2 of which were from the TB patients; 4 of 1086 samples were definitely infected with HIV-1 group O.^ieng
Asunto(s)
Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/inmunología , África Central , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/virología , VIH-1/clasificación , Humanos , Epítopos Inmunodominantes/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.
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Variación Genética , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-2/genética , VIH-2/aislamiento & purificación , Síndrome de Inmunodeficiencia Adquirida/virología , África Occidental , Secuencia de Aminoácidos , Donantes de Sangre , Camerún , Guinea Ecuatorial , Femenino , Productos del Gen env/química , Genes env , VIH-1/clasificación , VIH-2/clasificación , Humanos , Reacción en Cadena de la Polimerasa , Embarazo , ARN Viral/aislamiento & purificación , SerotipificaciónAsunto(s)
Variación Genética/genética , VIH-2/genética , ARN Viral/sangre , Secuencia de Aminoácidos , Côte d'Ivoire , Productos del Gen env/genética , Productos del Gen gag/genética , Anticuerpos Anti-VIH/sangre , VIH-2/inmunología , Humanos , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Antibodies to asialoglycoprotein receptor have diagnostic specificity for autoimmune hepatitis, but it is uncertain if they are complementary or redundant markers of the disease. Our aims were to assess their frequency and significance in type 1 autoimmune hepatitis and determine their contribution to the evaluation of these patients. Sera from 54 well-characterized patients were evaluated for antibodies to asialoglycoprotein receptor by a radioimmunofiltration assay based on rabbit-derived protein. Forty-four patients (82%) were seropositive. Seropositive patients were distinguished from seronegative counterparts by having higher serum gamma globulin (3.7 +/- 0.2 g/dl vs 2.3 +/- 0.3 g/dl, P = 0.0007) and immunoglobulin G levels (3707 +/- 179 mg/dl vs 2203 +/- 263 mg/dl, P = 0.0005) at presentation and a greater frequency of relapse after drug withdrawal (88% vs 33%, P = 0.01). Seropositivity for smooth muscle and/or antinuclear antibodies did not define treatment outcomes and antinuclear antibodies occurred less frequently than the other markers. Concurrent testing for antibodies to asialoglycoprotein receptor and smooth muscle identified all patients. We conclude that antibodies to asialoglycoprotein receptor are common in type 1 autoimmune hepatitis and they identify patients with a high frequency of relapse after corticosteroid withdrawal. Concurrent testing for these antibodies and smooth muscle antibodies has the same diagnostic sensitivity as testing for antinuclear and smooth muscle antibodies but a greater prognostic implication.
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Anticuerpos/sangre , Enfermedades Autoinmunes/inmunología , Hepatitis/inmunología , Receptores de Superficie Celular/inmunología , Corticoesteroides/uso terapéutico , Adulto , Anticuerpos Antinucleares/sangre , Receptor de Asialoglicoproteína , Asialoglicoproteínas/inmunología , Enfermedades Autoinmunes/tratamiento farmacológico , Biomarcadores/sangre , Femenino , Hepatitis/tratamiento farmacológico , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
The ability of rat lenses to import glutamate from the aqueous humor is limited by the activity of the transporter for dicarboxylic acids in this tissue. The principal source of glutamate for rat lens metabolism appears to be glutamine, which enters the lens much more readily than glutamate and is then deamidated by glutaminase. Both soluble and particulate glutaminase activities were obtained from rat lenses by extraction and differential centrifugation. The lens fractions were compared with previously reported isoenzymes of glutaminase from rat kidney and liver. The apparent Km for glutamine of the soluble preparation from lens was in the range from 17- to 24 mM, while that of the particulate lens fraction was 5 mM. Gel filtration on Sepharose 4B demonstrated that the lens soluble glutaminase in Tris buffer is similar in size to the kidney enzyme and, like the glutaminase from kidney, reversibly formed active aggregates in borate buffer. The liver enzyme did not form aggregates under identical conditions. The glutaminase preparations were all dependent on phosphate for complete activation, but the lens particulate fraction is more active than the lens or kidney soluble fractions at low concentrations of inorganic phosphate. Unlike the soluble enzyme, the particulate glutaminase was partially activated by phosphorylcholine. Rat lenses contain 11 mM phosphorylcholine, and this unusually high concentration of phosphorylcholine may be sufficient to partially activate the enzyme. These properties of the particulate fraction suggest that the membrane-bound glutaminase may be physiologically important in the lens in vivo.