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Journal article published in THE LANCET. Health research has a key role in the development of low-income and middle-income countries. There are several current initiatives that have greatly contributed to capacity strengthening of health research in sub-Saharan Africa, including those supported by WHO and Tropical Disease Research (TDR), the Swedish International Development Agency (SIDA) and Department for Research Cooperation (SAREC), the European Union, the Bill & Melinda Gates Foundation, the International Clinical Epidemiology Network (INCLEN), the Fogarty International Centre, the National Institutes of Health (NIH), and the Wellcome Trust. However, enormous challenges remain for sub-Saharan Africa to establish a common framework for sustainable research capacity strengthening. The Global Ministerial Forum on Research for Health in Bamako will give emphasis to the challenges of research for development and health, the need for more health-system research, and a greater intersectoral approach to science, technology, and health. That health research is indispensable for improving health, equity, and development is now widely accepted,1 yet how sub-Saharan African countries can develop their fragile health systems and their own capacity to do health research is rarely discussed.2 A recent African-led initiative—the Initiative to Strengthen Health Research Capacity in Africa (ISHReCA)—has identified nine key requirements to strengthening health-research capacity in Africa (panel 1). We focus on these requirements and suggest practical strategies for sustainable capacity strengthening in African institutions. Health research has a key role in the development of low-income and middle-income countries. There are several current initiatives that have greatly contributed to capacity strengthening of health research in sub-Saharan Africa, including those supported by WHO and Tropical Disease Research (TDR), the Swedish International Development Agency (SIDA) and Department for Research Cooperation (SAREC), the European Union, the Bill & Melinda Gates Foundation, the International Clinical Epidemiology Network (INCLEN), the Fogarty International Centre, the National Institutes of Health (NIH), and the Wellcome Trust. However, enormous challenges remain for sub-Saharan Africa to establish a common framework for sustainable research capacity strengthening. The Global Ministerial Forum on Research for Health in Bamako will give emphasis to the challenges of research for development and health, the need for more health-system research, and a greater intersectoral approach to science, technology, and health. That health research is indispensable for improving health, equity, and development is now widely accepted,1 yet how sub-Saharan African countries can develop their fragile health systems and their own capacity to do health research is rarely discussed.2 A recent African-led initiative—the Initiative to Strengthen Health Research Capacity in Africa (ISHReCA)—has identified nine key requirements to strengthening health-research capacity in Africa (panel 1). We focus on these requirements and suggest practical strategies for sustainable capacity strengthening in African institutions.

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

A novel in vitro whole blood model was developed to study human antimycobacterial immunity. Recombinant reporter mycobacteria were used to enumerate the bacteria, and interactions between host immune cells and mycobacteria were studied using whole blood rather than cell fractions. The ability of healthy tuberculin-positive and tuberculin-negative individuals to restrict mycobacterial growth was compared. Growth of luminescent mycobacteria was significantly lower in blood samples of tuberculin-positive individuals than in blood samples of tuberculin-negative individuals (P=.005). Restricted mycobacterial growth was associated with significantly higher production of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma (P=.01 and.004, respectively). Inhibition of the TNF-alpha and IFN-gamma response pathways by neutralizing monoclonal antibodies increased mycobacterial growth in whole blood. This model is the first functional assay in which individual variations in cell-mediated immunity are shown to correlate with differences in ability to control mycobacterial growth. It provides a new tool for studying human mycobactericidal mechanisms and, potentially, for the evaluation of improved vaccines.

The merozoite surface protein MSP1, which is one of the most promising candidates for a malaria vaccine directed against erythrocytic stages, has been shown to be polymorphic in different malarial species. Characterization of the Plasmodium vivax MSP1 gene (Pv200) in two strains (Belem and Salvador-1) revealed the existence of several polymorphic regions. One of these regions has been examined here in primary parasite isolates obtained from patients in Sri Lanka. Oligonucleotide primers hybridizing to conserved parts of the gene on either side of a polymorphic region were used to amplify DNA from 22 isolates. Sequence analysis of the amplified portion of the MSP1 gene in five patients showed the existence of three types of polymorphic regions. Two were almost identical either to that of the Belem or to that of the Salvador-1 strain. The third polymorphic type appeared to have resulted from recombination between the two others. This recombination event took place inside a repeated part of the sequence.

The degree of polymorphism and the allelic distribution of 2 major Plasmodium falciparum merozoite surface antigens (MSA1 and MSA2) have been analysed in clinical isolates from Colombia. DNA was prepared directly from patients' blood and used in PCR reactions to amplify block 2 of MSA1 and the central region from MSA2. Thirty one samples were analysed and a marked degree of length polymorphism was detected, especially for MSA2. A high proportion of multiple bands was also observed, most probably resulting from mixed infections. Allele-specific oligonucleotides were used to type both alleles. For MSA1, 26 out of 31 clinical isolates were of the RO33 type, 15 were MAD20 and three were typed as KI. When the MSA2 allele was analysed, 7 isolates hybridised with a CAMP specific probe and 6 hybridised strongly with an FC27-derived oligonucleotide. Two samples, which showed multiple bands, hybridised with both probes. Interestingly, in 14 out of 27 isolates the MSA2 allele remained unassigned by the specific probes. Five of these were cloned and their DNA sequenced; these sequences are discussed.

Since the activity of drugs against Mycobacterium tuberculosis grown in microbiological culture can differ from their activity against bacteria present in infected tissues, compounds with optimal activity against in vivo phenotypes may be overlooked in drug-discovery programmes that rely on in vitro screens. The authors have investigated the use of an ex vivo cell-culture model to assess the action of drugs on M. tuberculosis in an environment resembling that encountered during infection. Mycobacterial viability in the ex vivo model was shown to be regulated by the cell-mediated immune system, with growth inhibited by CD4(+) T cells at an early stage of infection in BCG-vaccinated mice, and at a later stage after infection in naive mice. Screening of drugs in the ex vivo model demonstrated a window of pyrazinamide susceptibility that coincides with the onset of the T-cell-mediated immune response in naive or vaccinated mice. It is proposed that pyrazinamide acts on a population of bacteria that are exposed to an acidic environment as a result of immune activation. Clinically, administration of pyrazinamide during the initial phase of treatment reduces the risk of relapse after 6 months, suggesting that the early pyrazinamide-susceptible population may contribute to the later pool of mycobacteria that persist during prolonged chemotherapy.

Publisher Summary Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Recent years have seen the resurgence of TB, particularly where there is a high prevalence of human immunodeficiency virus (HIV) infection. Most mycobacteria are saprophytes in soil or water and are harmless to humans; however, at some point thousands of years ago, M. tuberculosis became a significant human pathogen. The origins of tuberculosis are both ancient and obscure. Mycobacteria are aerobic to microaerophilic Gram-positive members of the actinomycete family. M. tuberculosis occurs as straight rods which, unlike other members of the actinomycete group, produce neither aerial hyphae nor spores. A marked characteristic of mycobacteria is that they behave anomalously during staining with arylmethane dyes. Recent developments have, however, limited the value of sputum-based AFB staining methods. The rise in the numbers of HIV-infected people in particular has posed significant problems. Many of those who are HIV-infected may become co-infected with normally non-pathogenic mycobacteria such as Mycobacterium avium (M. avium) or they may develop extra pulmonary tuberculosis for which sputum smears would be unhelpful.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

International audience; We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

To investigate the genetic basis of drug resistance in human malaria parasites, we have sequenced the entire dihydrofolate reductase thymidylate synthetase DHFR-TS bifunctional gene from the highly pyrimethamine-resistant K1 isolate of Plasmodium falciparum. The protein is predicted to consist of 607 amino acids (aa), (71685 Da), with an N-terminal methionine encoded by the second start codon of the open reading frame. Compared to the sequence from drug-sensitive parasites, there are two nucleotide changes in the coding region which bring about a substitution of Arg for Cys at aa position 59 and Asn for Thr at aa position 108. Both changes occur in regions of the DHFR domain involved in inhibitor and cofactor binding and are hence strongly implicated in drug resistance. The gene is present as a single copy in both K1 and drug-sensitive FCR3 isolates, and is assigned to chromosome 4. Codon usage follows the pattern observed in that of malarial surface antigen genes, with the exception of codons corresponding to Val and Pro. The Asn and Lys contents of the predicted protein are exceptionally high, these residues being particularly concentrated in the DHFR and junction domains.