Evaluation of bias in HIV seroprevalence estimates from national household surveys.

OBJECTIVES: To evaluate HIV seroprevalence estimates from demographic and health surveys (DHS) and AIDS indicator surveys (AIS) for potential bias because of non-response and exclusion of non-household population groups. METHODS: Data are from 14 DHS/AIS surveys with HIV testing, conducted during 2003-6. Blood samples were collected and analysed for HIV using standard laboratory and quality control procedures. HIV prevalence among non-tested adults was predicted based on multivariate statistical models of HIV for those who were interviewed and tested, using a common set of predictor variables. Estimates of the size of non-household populations in national censuses were used to assess potential bias because of their exclusion in the household surveys under different assumptions about proportion of adults and HIV prevalence in non-household populations. RESULTS: Non-tested men had significantly higher predicted HIV prevalence than those tested in eight of the 14 countries, while non-tested women had significantly higher predicted prevalence than those tested in seven of the 14 countries. Effects of non-response were somewhat stronger in lower-prevalence countries. The overall effect of non-response on observed national HIV estimates was small and insignificant in all countries. Estimated effects of exclusion of non-household population groups were generally small, even in concentrated epidemics in India and Cambodia under the scenario that 75% of the non-household population was adults having 20 times greater HIV prevalence than adults in household surveys. CONCLUSIONS: Non-response and the exclusion of non-household population groups tend to have small, insignificant effects on national HIV seroprevalence estimates obtained from household surveys.

High stability of yellow fever 17D-204 vaccine: a 12-year restrospective analysis of large-scale production.

We have retrospectively analyzed 12 bulk lots of yellow fever vaccine Stamaril, produced between 1990 and 2002 and prepared from the same seed lot that has been in continuous use since 1990. All vaccine batches displayed identical genome sequence. Only four nucleotide substitutions were observed, compared to previously published sequence, with no incidence at amino-acid level. Fine analysis of viral plaque size distribution was used as an additional marker for genetic stability and demonstrated a remarkable homogeneity of the viral population. The total virus load, measured by qRT-PCR, was also homogeneous pointing out reproducibility of the vaccine production process. Mice inoculated intracerebrally with the different bulks exhibited a similar average survival time, and ratio between in vitro potency and mouse LD(50) titers remained constant from batch-to-batch. Taken together, these data demonstrate the genetic stability of the strain at mass production level over a period of 12 years and reinforce the generally admitted idea of the safety of YF17D-based vaccines.

A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Safety and efficacy of chimeric yellow Fever-dengue virus tetravalent vaccine formulations in nonhuman primates.

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

Mechanism of neutralization of influenza virus infectivity by antibodies.

We have determined the mechanism of neutralization of influenza virus infectivity by three antihemagglutinin monoclonal antibodies, the structures of which we have analyzed before as complexes with hemagglutinin. The antibodies differ in their sites of interaction with hemagglutinin and in their abilities to interfere in vitro with its two functions of receptor binding and membrane fusion. We demonstrate that despite these differences all three antibodies neutralize infectivity by preventing virus from binding to cells. Neutralization occurs at an average of one antibody bound per four hemagglutinins, a ratio sufficient to prevent the simultaneous receptor binding of hemagglutinins that is necessary to attach virus to cells.

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'

An antibody which binds to the membrane-proximal end of influenza virus haemagglutinin (H3 subtype) inhibits the low-pH-induced conformational change and cell-cell fusion but does not neutralize virus.

A monoclonal antibody, LMBH6, was derived from mice which had been sequentially immunized with bromelain-cleaved haemagglutinin (BHA) from influenza virus A/Aichi/2/68, A/Victoria/3/75 and A/Philippines/2/82 (all H3N2). LMBH6 recognizes the haemagglutinin (HA) of all H3N2 influenza A strains tested, which were isolated between 1968 and 1989. HA in the low-pH-induced conformation is not recognized, and cleavage of the HA0 precursor to HA1 and HA2 is needed to obtain efficient binding. Compared to other monoclonal antibodies, binding of LMBH6 to virus and to virus-infected cells is weak, while binding to BHA is comparable. Electron microscopy demonstrates binding to the membrane proximal end of the stem structure. The antibody shows no haemagglutination-inhibition activity, but inhibits polykaryon formation and the low-pH-induced conformational change of BHA. However, LMBH6 cannot prevent infection of MDCK cells but slows the growth of virus when included in a plaque assay overlay.

A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture.

The sodium salts of 2-difluoromethyl-phenyl-alpha-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-alpha-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 x 10(-5) M) compared to the low Ki (1' x 1(-10) M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.

A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture.

The sodium salts of 2-difluoromethyl-phenyl-α-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-α-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 × 10-5M) compared to the low Ki (1 × 1-10 M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.

Quantitative tissue sodium concentration mapping of normal rat brain.

A quantitative in vivo method for obtaining maps of tissue sodium concentration (TSC) by MRI is compared to the invasive, global 22Na radionuclide dilutional technique in the normal rat brain. The MR method uses a three-dimensional projectional acquisition scheme to minimize signal losses from transverse relaxation. Internal calibration standards are used to convert the signal intensity into TSC after correction for B1 inhomogeneities by using the ratio of 23Na and 1H images obtained with identical B1 distributions and sensitivities at the two frequencies. Over the biological range of concentrations, the TSC, measured as the ratio of MR signals of 23Na and 1H, gives a linear response with concentration. In the normal rat brain, the mean TSC measured using the MRI method (TSC = 45 +/- 4 mM, animals = 5) is not significantly different from the global 22Na radionuclide method (TSC = 49 +/- 6 mM, animals = 7).

Brain stem energy metabolism response to acute hypoxia in anaesthetized rats: a 31P NMR study.

Mammals react to acute hypoxia with an initial augmentation and a secondary depression of the respiratory rhythm generated by brain stem neuronal networks. To investigate the cytosolic level of energy rich phosphorus metabolites during these responses, we developed 31P nuclear magnetic resonance spectroscopy of the brain stem. Moderate hypoxia (paO2 = 40 mmHg, 2 min) caused a reversible 62 +/- 15% respiratory rhythm depression and decreased cytosolic phosphocreatine levels by 43 +/- 11% (p < 0.01, n = 7) without affecting adenosine triphosphate levels. Cellular metabolic depletion therefore contributes to the brain stem response to hypoxia, and appears to reflect adaptive mechanisms to limited oxygen availability in the brain stem.

Effects of a vigilance-enhancing drug, modafinil, on rat brain metabolism: a 2D COSY 1H-NMR study.

The effects of modafinil, a vigilance-enhancing drug, on brain metabolism were investigated directly in situ by the 2D COSY 1H-NMR spectroscopy in anesthetized rats. Modafinil (600 mg/kg, i.p.) induced significant increases in both aspartate (72% +/- 15%) and glutamate-glutamine pool (28% +/- 8%) simultaneously with increases in inositol (51% +/- 19%) and creatine-phosphocreatine pool (47% +/- 14%) in comparison with control values (P < 0.05; n = 5). These results suggest that the awakening properties of modafinil could be mediated by metabolic activation.

Synthesis of transition-state analogues as potential inhibitors of sialidase from Influenza virus.

Sodium 5-acetamido-2,6-anhydro-3,4,5-trideoxy-D-manno-non-2-enonate (2) has been synthesized from N-acetyl-4-deoxy-neuraminic acid methyl ester (4). Sodium 2,6-anhydro-3-deoxy-L-arabino-hept-2-enonate (3), 4-acetamido-2,6-anhydro-3,4-dideoxy-L-arabino-hept-2-enonic acid (18e), and 4-acetamido-2,6-anhydro-3,4-dideoxy-L-ribo-hept-2-enonic acid (18a) have been prepared from L-arabinose. The above compounds were investigated as inhibitors of sialidase from Influenza virus. Only compound 2 showed a significant inhibitory activity (Ki 8 x 10(-2) mM) against the enzyme.

Effects of kainate-induced seizures on cerebral metabolism: a combined 1H and 31P NMR study in rat.

The cerebral metabolic changes elicited by kainate-induced seizures in the rat were investigated by in vivo combined NMR spectroscopy of 31P and 1H. Systemic injection of kainate induced no significant changes in cerebral ATP or PCr levels during up to 90 min of continuous, generalised seizures, and the cerebral 31P spectra showed only a transient mild cerebral acidosis 30 min after kainate administration. In parallel with the changes in intracellular cerebral pH, the 1H spectra showed a significant increase in lactate, which remained elevated throughout the seizures. These findings indicate that oxidative metabolism does not completely match the increased glycolysis during seizures though the energy homeostasis is maintained. This suggests that oxidative metabolism has a limited capacity to satisfy the brain's energy needs during the kainate-induced seizures, but that the different pathways of energy production in the brain cells can overcome this limitation. Thus the brain damage associated with this experimental model of epilepsy is not due to extended major failure of the energy supply.

The sensitivity of magnetic resonance image signals of a rat brain to changes in the cerebral venous blood oxygenation.

The sensitivity of magnetic resonance image signals of the brain to the change in the cerebral blood oxygenation was measured in gradient echo images of rat brains at a field strength of 7 T. The sensitivity depended on the blood vessel volume relative to the tissue volume within the image voxel, and signal intensities in the cortical area were well correlated with the change in the venous blood de-oxygenation level at the sagittal sinus. Tissue signals in the image (15 ms echo time) showed a sensitivity of 10-20% change for the full range of deoxygenation level from 0-100%. From these observations and image simulations, the extent of the signal response to some neuro-stimulation which induces an increase in regional cerebral blood flow has been estimated for 4 T field strength.

In vivo identification and monitoring of changes in rat brain glucose by two-dimensional shift-correlated 1H NMR spectroscopy.

Intracerebral glucose resonance was directly detected and resolved in vivo by two-dimensional shift-correlated (COSY) 1H NMR spectroscopy in anesthetized rats (n = 4). The relative changes in brain glucose concentration were measured by volume integration of the alpha-D-glucose cross peak in the 2D COSY spectra. This report demonstrates the possibility of monitoring the variations in cerebral glucose following iv injection of glucose.

Two-dimensional 1H spectroscopic imaging for evaluating the local metabolic response to focal ischemia in the conscious rat.

Two-dimensional 1H spectroscopic imaging and magnetic resonance imaging were used to study focal ischemia induced by middle cerebral artery occlusion in rats. A water suppressing spin-echo sequence was used at 4.7 T. Phase encoding during the spin-echo delay (TE = 272 ms) yielded an 8 x 8 array of 35 microL voxels. The injured area of the brain had a higher lactate level and markedly lower N-acetyl aspartate, creatine and choline levels than did the non-ischemic regions. The spectroscopic imaging data clearly showed the localization of the infarct, which agreed well with both magnetic resonance imaging and the histological data obtained post-mortem. This study demonstrates the potential usefulness of combining magnetic resonance imaging and 1H spectroscopic imaging for studying animal models of stroke, and indicates the suitability of the technique for further pharmacological approaches.

Mortality trends in Abidjan, Côte d'Ivoire, 1983-1988.

To assess changes in mortality in Abidjan since the development of the AIDS epidemic, we compared official city mortality statistics and hospital fatality rates in 1983, before AIDS was recognized in Abidjan, with those in 1988. Review of records in the city's major hospitals showed that fatality rates (deaths per 1000 admissions) in adult medical patients increased by 54% between 1983 and 1988, with increases of 106 and 98% in men 20-29 and 30-39 years of age, respectively, and 199 and 42% in women of the same age ranges. Mortality rates in surgical patients showed little change, while in children they declined. Over the same period, official mortality statistics for the city showed reduced mortality rates in children and women 20-29 years of age, but an increase in mortality rates of 54% in men 20 years of age and older, and of 28% in women aged 30 years and older. HIV infection may be a major cause of the increased adult mortality documented in hospital and city records, and jeopardizes improved survival from preventive measures such as maternal and child health services.