Experimental infections in humans-historical and ethical reflections.

Vaccine efficacy and prophylactic treatment of infections are tested best when the vaccinated or treated individual is challenged through deliberate infection with the respective pathogen. However, this trial design calls for particular ethical caution. Awareness of the history of challenge trials is indispensable, including trials that were problematic or even connected to abuse. We briefly introduce historical aspects of experimental infections in humans and the ethical debate around them and give estimates of the numbers of volunteers participating in human experimental infection models. Challenge models can offer a great chance and benefit for the development of medical interventions to fight infectious diseases, but only when they are appropriately controlled and regulated.

L'efficacité des vaccins et le traitement prophylactique des infections sont mieux testés lorsque l'individu vacciné ou traité est exposé par le biais d'une infection délibérée par l'agent pathogène concerné. Cependant, cette conception d'essai appelle à une prudence éthique particulière. Il est indispensable de connaître l'histoire des essais cliniques, y compris des essais qui se sont avérés problématiques ou même liés à des abus. Nous présentons brièvement les aspects historiques des infections expérimentales chez l'homme et le débat éthique autour d'eux et donnons des estimations du nombre de volontaires participant à des modèles d'infection expérimentale humaine. Les modèles d'exposition peuvent offrir une grande chance et un avantage pour le développement d'interventions médicales pour lutter contre les maladies infectieuses, mais uniquement lorsqu'elles sont contrôlées et réglementées de manière appropriée.

High dose artesunate in combination with mefloquine: pharmacovigilance in the Venezuelan Amazon.

Forty-six patients in a remote health post of Amazonas, Venezuela, accidentally received artesunate in a dose of 10 mg/kg/day combined with mefloquine. This corresponds to the upper limit of the therapeutic range recommended by the WHO (2-10 mg/kg/day). Side effects were retrospectively investigated and a pharmacovigilance report was written. The main side effects were vomiting and diarrhea. Four patients developed complications with signs of dehydration. It is suggested to re-assess the therapeutic range of artesunate when given in combination with mefloquine and to establish a worldwide centralized antimalarial toxicity reporting system.

Assessment of routine malaria diagnosis in the Venezuelan Amazon.

The quality of routine malaria diagnosis is a crucial topic of malaria control. The aim of this assessment was to monitor and evaluate the quality of routine malaria diagnosis in Amazonas (Venezuela) and to improve the quality control system. The traditional non-blinded quality control system was found to be overburdened with diagnostic samples. A modified sampling system with fewer samples to be tested was proposed. Expert microscopists blindly double-checked 1000 slides and 550 rapid diagnostic tests (RDT) (OptiMAL-IT) from health posts (HP). For Plasmodium vivax, HP microscopy and OptiMAL-IT showed sensitivies of 86% and 63%, respectively. For P. falciparum, HP microscopy and OptiMAL-IT showed sensitivities of 68% and 89%, respectively. Both methods lost accuracy when fewer parasites occurred in the sample. HP microscopists from different municipalities displayed significant differences in diagnostic quality. Overall, quality of routine malaria diagnosis in the Venezuelan Amazon is good but not optimal. The change from the traditional non-blinded quality control system to blinded cross-checking of a minimal selection of samples is - comparatively - a low cost intervention with possibly high impact on the quality of routine malaria diagnosis. The introduction of RDTs should be discussed carefully in order not to displace an existing network of HP microscopists.

Anaemia and malaria in Yanomami communities with differing access to healthcare.

Inequitable access to healthcare has a profound impact on the health of marginalised groups that typically suffer an excess burden of infectious disease morbidity and mortality. The Yanomami are traditionally semi-nomadic people living in widely dispersed communities in Amazonian Venezuela and Brazil. Only communities living in the vicinity of a health post have relatively constant access to healthcare. To monitor the improvement in the development of Yanomami healthcare a cross-sectional survey of 183 individuals was conducted to investigate malaria and anaemia prevalence in communities with constant and intermittent access to healthcare. Demographic and clinical data were collected. Malaria was diagnosed by microscopy and haemoglobin concentration by HemoCue. Prevalence of malaria, anaemia, splenomegaly, fever and diarrhoea were all significantly higher in communities with intermittent access to healthcare (anaemia 80.8% vs. 53.6%, P<0.001; malaria 18.2% vs. 6.0%, P=0.013; splenomegaly 85.4% vs.12.5%, P<0.001; fever 50.5% vs. 28.6%, P=0.003; diarrhoea 30.3% vs.10.7% P=0.001). Haemoglobin level (10.0 g/dl vs. 11.5 g/dl) was significantly associated with access to healthcare when controlling for age, sex, malaria and splenomegaly (P=0.01). These findings indicate a heavy burden of anaemia in both areas and the need for interventions against anaemia and malaria, along with more frequent medical visits to remote areas.

Malaria diagnosis under field conditions in the Venezuelan Amazon.

To improve practical, accurate diagnosis of malaria in the Amazon rainforest of Venezuela, two rapid diagnostic tests (RDT) (OptiMAL-IT) and FalciVax) and a laboratory light microscope, used in the field with a battery-operated head lamp as an external light source, were evaluated against the standard laboratory microscope procedure for malaria detection. One hundred and thirty-six Yanomami patients were studied for the presence of malaria parasites. Thirty-three patients (24%) were positive for malaria (Plasmodium falciparum, P. vivax, P. malariae). Twenty-one (64%) of the positive patients had <100 parasites/microl. Both RDTs showed poor sensitivity (24.2% for OptiMAL-IT) and 36.4% for FalciVax) but good specificity (99% both for OptiMAL-IT) and FalciVax). Field and laboratory microscopy showed sensitivities of 94% and 91%, respectively. The kappa coefficient was 0.90, indicating a high agreement between field and laboratory microscopy. We conclude that (i) adequate slide reading cannot be substituted by either of the two RDTs in the Venezuelan Amazon and (ii) the use of a light source such as that described above makes slide reading more feasible than hitherto in remote areas without electricity.

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers.

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

Genetic diversity of Plasmodium falciparum and its relationship to parasite density in an area with different malaria endemicities in West Uganda.

Field populations of Plasmodium falciparum can be effectively genotyped by PCR-amplification of selected fragments of the Merozoite Surface Proteins 1 and 2 (MSP1 and MSP2). Genetic diversity of P. falciparum populations in areas with different transmission levels (holo- vs. mesoendemic) was investigated in Kabarole District, West Uganda. 225 samples positive for P. falciparum were analysed by amplification of polymorphic regions and classified according to prevalence of allelic families. A large number of alleles was detected for each locus: 22 for MSP1 block 2 and 24 for MSP2 and, 175 (78%) of MSP1 alleles and 143 (64%) of MSP2 showed multiple infections within a range of 2-8 clones. Significant differences between holoendemic and mesoendemic areas in regards of population structure and number of multiclonal infections of P. falciparum were not apparent. However, a significant correlation between parasite density, selected MSP2 loci and differences between parasite density in monoclonal vs. multiclonal infections occurred. Multiplicity of infection was age-dependent.

Reliability of malaria microscopy in epidemiological studies: results of quality control.

To assess the interrater reproducibility of malaria microscopy in epidemiological studies, 711 thick blood films from population-based surveys were randomly selected and reread by 4 experienced microscopists. Sample estimates of the prevalence of P. falciparum infection, geometric mean parasite density and the proportion of samples above various parasite density cut-off levels were almost identical in the routine and quality control readings. Differences were, however, encountered in the sample estimates for gametocyte ratio, proportion of mixed infection and average density index. In all three cases the quality control result was significantly higher than the routine evaluation. On the level of the individual slide there was good interrater agreement for the presence of P. falciparum infections (Kappa index kappa = 0.79) which was even better when parasite densities between 4 and 100/microl were excluded (kappa = 0.94). With respect to the assessment of parasite density, a high level of disagreement was found. While the mean difference between the two readings was not different from 0, the second reading was between 0.12 and 10 times that of the first. However, the level of disagreement significantly fell with increasing parasite densities. Thus malaria microscopy is very reliable for the estimation of parasite ratios and geometric mean parasite densities within and between studies as long as the same methodology is used, but tends to underestimate the gametocyte ratio and proportion of mixed infections. Care must be taken, however, when individual parasite density is related to other explanatory variables, due to the high degree of variability in the parasite enumeration.

Serological responses of Gambian children to immunization with the malaria vaccine SPf66.

Antibody responses to the malaria vaccine SPf66 and to its constituent peptides were measured over a period of 2 years in Gambian children who had been immunized with SPf66 or with a control vaccine (inactivated polio vaccine). Three hundred and six of 308 children (99%) who had received three doses of SPf66 vaccine had antibodies to SPf66 at a level above that found in European controls who had not been exposed to malaria. Responses to the constituent peptides derived from 35.1, 55.1 and 83.1-kDa proteins were found in 88%, 97% and 97% of children, respectively; 26% had an antibody response to the NANP repeat peptide of circumsporozoite protein which is also included in the SPf66 vaccine. A response to SPf66 was found in 22% of children who had received the control vaccine. Antibody responses to NANP, 35.1, 55.1 and 83.1-kDa peptide were found in 3%, 33%, 49% and 33% of these children. Overall, no significant correlation was found between the level of anti-SPf66 antibody at the beginning of the malaria transmission season following vaccination and the subsequent risk of malaria. However, further analysis showed that among the control children who had acquired antibodies to SPf66 as a result of natural exposure to malaria, those with high levels of anti-SPf66 were less at risk of malaria, perhaps reflecting their greater previous exposure and thus immunity. In contrast, among children who had received three doses of SPf66, those with high antibody levels were at greater risk of have malaria during the subsequent malaria transmission season.

Tumor necrosis factor in Plasmodium falciparum malaria: high plasma level is associated with fever, but high production capacity is associated with rapid fever clearance.

In the present study, we investigated 91 patients with Plasmodium falciparum malaria of different severity in a highly endemic area. Patients were examined at least twice daily until clearance of parasites and fever. Plasma cytokine concentrations without and after ex vivo PHA stimulation of whole blood were determined. On admission we found elevated plasma concentrations of TNF, IFN-gamma, and IL-10 compared to levels during and after chemotherapy. Plasma TNF levels on admission were significantly different between patients with severe and mild malaria (differentiated in schoolchildren and adults). The PHA elicited TNF production capacity of peripheral blood leucocytes was suppressed during the acute phase of malaria. High TNF production capacity was associated with faster fever clearance and parasite clearance and, in patients with severe malaria, with higher blood glucose levels. In conclusion we observed circulating TNF concentrations in malaria patients dependent on the severity of disease, which is itself dependent on age, and an association of a high TNF production capacity with parameters for accelerated cure and good prognosis.

Intravenous or intramuscular anti-HBs immunoglobulin for the prevention of hepatitis B reinfection after orthotopic liver transplantation.

To prevent reinfection with hepatitis B virus after orthotopic liver transplantation, patients receive long-term intravenous anti-HBs immunoprophylaxis. We compared the pharmacokinetics of intravenously and intramuscularly administered commercially available hepatitis B virus immunoglobulins. The study group consisted of 12 patients on immunoprophylaxis after orthotopic liver transplantation, who were Hbs antigen negative; 11 were anti-HBe positive and one was HBe positive. The patients first received intravenous immunoglobulin, and six of them were then transferred to intramuscular immunoglobulin. Our findings show that with fortnightly intramuscular application of 1000 IU of anti-HBs, reproducible and stable antibody titers above 100 IU of anti-HBs can be achieved. Side effects of intramuscular immunoprophylaxis are minimal and the method is safe. The switch from intravenous (1500 IU of anti-HBs) to intramuscular (1000 IU of anti-HBs) reduced the cost of immunoprophylaxis by more than 50%.