Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Vertical transmission of Cryptococcus neoformans from a mother coinfected with human immunodeficiency virus: case report

Disseminated infection with Cryptococcus neoformans was observed in a newborn infant who presented fever and respiratory symptoms since the 52nd day of life. The mother was infected by human immunodeficiency virus and presented pulmonary and meningeal cryptococcal infection. This is a rare case of cryptococcal infection with probable maternal-fetal transmission.

Sexually transmitted diseases in homosexual and bisexual males from a cohort of human immunodeficiency virus negative volunteers (Project Horizonte), Belo Horizonte, Brazil

Sexually transmitted diseases (STD) are very frequent in the whole world. Males who do not use a condom during their sexual relations are at great risk. We report cases of STD during six months of observation, among homosexual/bisexual males who participate in the Project Horizonte. There were 16 cases of genital warts, 6 cases of human immunodeficiency virus infection, 24 cases of unspecific urethritis, 28 cases of herpes simplex virus infection, 30 cases of syphilis, 58 cases of gonorrhea and 84 cases of pediculosis. We concluded that a condom must be used in all sexual relations and new counseling techniques are needed, to avoid this situation.

Les cadres de lectures alternatifs : une approche non-conventionnelle pour le développement de vecteurs vaccinaux

Introduction: Les cadres de lectures alternatifs (CLA) sont utilisés par de multiples virus afin de générer plusieurs protéines à partir d'une seule séquence nucléotidique. Les épitopes dits « cryptiques », c’est-à-dire les épitopes dérivés de protéines codées dans des CLAs, ont étés dernièrement l’objet de différentes études portant sur la réponse immunitaire antivirale et les lymphocytes T cytotoxiques. Méthodologie: Afin de vérifier le potentiel immunogène d'épitopes encodés dans des CLAs programmés, trois cassettes ont été construites pour mener à l'expression de trois épitopes bien caractérisés (épitope GAG77–85 du virus de l'immunodéficience humaine de type 1; épitope NS31406-1415 du virus de l'hépatite C; épitope core18-27 du virus de l'hépatite B) à partir de trois cadres de lectures superposés. La première cassette permet une initiation alternative de la traduction, la deuxième comprend deux signaux bipartites en tandem permettant un frameshift ribosomique et la troisième est une cassette contrôle. Ces éléments ont été introduits dans des vecteurs adénoviraux. Les virions générés ont servi à immuniser des souris C57BL/6 transgéniques pour HLA-A*0201 et HLA-DR1. La réponse immunitaire induite une semaine post-immunisation a été mesurée par essai ELISpot IFN . Résultats: Dans le contexte de cassettes vaccinales, les peptides dérivés d'une initiation alternative de traduction et de changement de cadre de lecture ribosomique ribosomal peuvent être exprimés et détectés par le système immunitaire dans un modèle animal. Conclusion: Ces expériences suggèrent la possibilité de développer de nouvelles stratégies vaccinales dans le but de prévenir ou de guérir certaines maladies associées aux infections virales chroniques telles que celles causées par le virus de l’immunodéficience humaine et le virus de l’hépatite C.

Rates of disease progression according to initial highly active antiretroviral therapy regimen: a collaborative analysis of 12 prospective cohort studies

BACKGROUND: No large clinical end-point trials have been conducted comparing regimens among human immunodeficiency virus type 1-positive persons starting antiretroviral therapy. We examined clinical progression according to initial regimen in the Antiretroviral Therapy Cohort Collaboration, which is based on 12 European and North American cohort studies. METHODS: We analyzed progression to death from any cause and to AIDS or death (AIDS/death), comparing efavirenz (EFV), nevirapine (NVP), nelfinavir, idinavir, ritonavir (RTV), RTV-boosted protease inhibitors (PIs), saquinavir, and abacavir. We also compared nucleoside reverse-transcriptase inhibitor pairs: zidovudine/lamivudine (AZT/3TC), stavudine (D4T)/3TC, D4T/didanosine (DDI), and others. RESULTS: A total of 17,666 treatment-naive patients, 55,622 person-years at risk, 1,617 new AIDS events, and 895 deaths were analyzed. Compared with EFV, the adjusted hazard ratio (HR) for AIDS/death was 1.28 (95% confidence interval [CI], 1.03-1.60) for NVP, 1.31 (95% CI, 1.01-1.71) for RTV, and 1.45 (95% CI, 1.15-1.81) for RTV-boosted PIs. For death, the adjusted HR for NVP was 1.65 (95% CI, 1.16-2.36). The adjusted HR for death for D4T/3TC was 1.35 (95% CI, 1.14-1.59), compared with AZT/3TC. CONCLUSIONS: Outcomes may vary across initial regimens. Results are observational and may have been affected by bias due to unmeasured or residual confounding. There is a need for large, randomized, clinical end-point trials.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIVDH12-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIVDH12 infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4+ T lymphocytes. Although the CD4+ T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIVDH12R, induced marked and rapid CD4+ cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIVDH12R indicated: (i) the input cloned SHIV remained the predominant virus during the first 5–7 months of infection; (ii) variants bearing only a few of the SHIVDH12R consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIVDH12R ever developed.

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo.

Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)adenine on visna virus infection in lambs: a model for in vivo testing of candidate anti-human immunodeficiency virus drugs.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.