[How does one treat the osteitis and osteoarthritis of the extremities in older leprosy patients using granulated table sugar?]. / Comment guérir les ostéites et ostéo-arthrites des extrémités des anciens malades de la lèpre par le sucre cristallisé alimentaire?

A common problem of osteitis and septic arthritis is the recurrent bone infection after surgical debridement, a problem frequently encountered in patients with sequela leprosy. In these cases the authors propose the use of an ancient method of post surgical wound care based on the treatment with ordinary granulated sugar. The hyperosmolar climate created this way in the wounds inhibits the bacterial growth, enhances bacterial death and therefore permits the growth of granulation tissue in order to recover the debrided nude bones. At ILAD (Leprosy Institute of Dakar), 36 osteitis and septic arthritis were treated and healed during the last 2 years from March 1995 to March 1997 using this technic. All the wounds healed in the mean-time of 44 days. Only two of them needed a second debridement and healed afterwards. Up to now the method using ordinary sugar was applied in the treatment of infected wounds, eschars and postsurgical infections. Our experience shows that it also can be indicated to treat bone infections. This method is easy to apply also under often difficult field conditions and is very cheap.

[Can plantar ulcers associated with leprosy be treated in the field. Results of experience in Senegal]. / Peut-on soigner les maux perforants plantaires des anciens malades de la lèpre sur le terrain? Résultats d'une expérience au Sénégal.

The introduction of a program for the treatment of plantar ulcers (PU) in field conditions in Senegal was studied. The program was complementary to the Health Education and Protective Footwear to Prevent Disability (POD) initiatives within the Senegalese anti-leprosy program. The wound care given in health centers was coded and simplified. Access to hospitals was made easier for those patients requiring surgery. More than 30% of patients with PU were treated each year, with a mean of 62% cured. An increasing number of leprosy patients have been admitted to regional hospitals for surgery. Never before have patients with signs of leprosy had access to general hospitals. This study emphasizes the need for regular supervision of the individuals treating wounds.

[The impact of curtains impregnated with deltamethrin on the vectors and morbidity of malaria: results in Ankazobe, on the plateaus of Madagascar]. / Impact des rideaux imprégnés de deltaméthrine sur les vecteurs et la morbidité palustre: résultats à Ankazobe, sur les plateaux de Madagascar.

To evaluate the efficacy of deltamethrin impregnated curtains on malaria morbidity in a low transmission area, we studied volunteer families in the village of Ankazobe in the Madagascar Highlands from February 1993 to June 1994. After randomization, we provided 46 houses having 244 inhabitants with impregnated curtains (I) and 45 others having 257 inhabitants with nonimpregnated curtains (NI) as controls. We first estimated the number of mosquito bites in the protected versus nonprotected households. Every month, we captured mosquitos on humans in 6 houses per night for 4 nights. For the I group compared to the NI group, the number of bites by the Anopheles funestus vector per human per night was reduced by 64% in 1993 and 39% in 1994. We also analyzed the malaria morbidity. Malaria morbidity was defined as patients having both temperatures greater than 37.5 degrees C and Plasmodium falciparum parasitemia greater than 1500/microliter with clinical symptoms. From February to July 1993, we observed no significant difference in morbidity: there were 103 cases of malaria among 244 inhabitants of the I group and 117 cases among 257 inhabitants of the NI group. However, during the period of highest transmission from March to May in 1993, there were significantly fewer cases in the I group (68) than in the NI group (94). From January to June 1994, the difference was clear: only 35 malaria cases were observed among the 208 inhabitants of the I group as compared to 65 cases among the 223 inhabitants of the NI group (Chi square = 9.17, p = 0.0024). Inhabitants of the I group could have been contaminated before the curtains were set up. After treatment of the cases and use of curtains during the second year, we observed a reduction in the number of mosquito bites and malaria cases. The small size of the trial made the interpretation of the data difficult. Nonetheless, the results tentatively support the use of impregnated curtains as an antimalaria tool in an integrated control program.

[Simplification and codification of treatment for leprous plantar ulcers]. / Simplification et codification des soins aux maux perforants plantaires lépreux.

Practically leprous plantar ulcers (PU) are difficult to treat and heal under field condition. Considering the important number of patients showing a PU, the directors of national leprosy control programmes are determined, within the programmes on prevention of disabilities (POD), to treat the PU in the field. Therefore it appears to be essential to codify and simplify their treatment thus enabling it to be effective. The healing of PU being the only criteria of effectiveness of the technique. Four clinical stages were defined, each corresponding to a precise way of treatment using only essential and basic products at low cost. During the trainings about the treatment techniques and attitudes much emphasize is given on the discharge of the PU, on the trimming of the wound and on the products to use according to PU's evolutionary stage.

IgG1 and IgG2 antibody responses to Plasmodium falciparum exoantigens correlate inversely and positively, respectively, to the number of malaria attacks.

In Manarintsoa, near Antananarivo, Madagascar, two groups of patients were defined in terms of malaria clinical immune status: Group MA+ consisted of 36 patients who suffered from between one to four malaria attacks (MA) during the 20-week study, and Group MA- who comprised of 48 persons who did not have any malaria attacks during this time. In group MA+, IgM and IgG antibody levels to Plasmodium falciparum exoantigens (E-Ag) were inversely related to the number of malaria attacks. The level of IgM antibodies were significantly higher in group MA+. In contrast, IgG, IgG1, IgG2, IgG3 and IgG4 antibodies to E-Ag were significantly higher in group MA-. The level of IgG1 antibodies was inversely correlated, and IgG2 antibodies were positively correlated to the number of malaria attacks.

A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes.

In the search for subunit vaccines that are able to induce the type of sterile, protective immunity achieved by irradiated sporozoites, there is increasing evidence that defense mechanisms directed at the intrahepatic stage and Ags expressed at this stage are critical. We have initiated a systematic search for such molecules and report here the identification and partial characterization of a novel Plasmodium falciparum gene encoding a 70-kDa protein, expressed in both sporozoite and liver stages (SALSA), with a vaccine potential that stems from its antigenic features. Antigenicity and immunogenicity studies were conducted in individuals exposed to malaria, in immunized mice, and in chimpanzees, using a recombinant protein and two synthetic peptides. Results show that the SALSA nonrepetitive sequence defines 1) major B cell epitopes, as shown by a high prevalence of Abs to each peptide in three African areas differing in their level of endemicity; 2) Th epitopes, as demonstrated by lymphoproliferation and IFN-gamma secretion in cells from the individuals from one of the low transmission areas, as well as helper effect upon Ab secretion in mice; and 3) epitopes for cytolytic lymphocytes, demonstrated in immunized and sporozoite-challenged chimpanzees, and associated with MHC class I leukocyte Ags. The latter are of particular importance, because this is the only part of the malaria life cycle in which the parasite is located in a cell expressing class I Ags and because CD8+ lymphocytes were found to be responsible for protection in experimental models.

[Drug resistance and therapeutic strategy]. / Chimiorésistance et stratégie thérapeutique.

In Madagascar, Plasmodium falciparum resistance to chloroquine was clinically suspected in 1975 by Goasguen and demonstrated in 1981 by Arronson et al. Since then, many studies were conducted throughout the island, in the North, South, East and West, on the high Plateau and on the coasts. Two methods were used, including an in vivo method similar to the therapeutic standard protocol of the WHO, and an in vitro method employing the semi-microtest of Le Bras and Deloron. From 1982 to 1986, the 291 in vivo tests performed showed that 20% of the strains were of the types R1 or R2 (SR1 included). From 1987 to 1994, of the 621 in vivo tests performed, 369 (59.4%) of the cases responded to treatment. The deterioration of the situation observed in 1988 (Lepers et al.) seemed to be stabilized (Ringwald et al.). No strain of the type R3 was found. In conclusion, we report the absence of strain type R3 and also the clinical efficacy of chloroquine. The action of chloroquine was spectacular on the fevers and there was remarkable reduction of the parasitaemia. Thus, for treating outbreaks of simple malaria in Madagascar, chloroquine remains the best choice if it is administered at an efficacious dose.

Antibodies to a Plasmodium falciparum blood-stage antigen as a tool for predicting the protection levels of two malaria-exposed populations.

To evaluate the ability of antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) epitopes to discriminate between individuals well protected or poorly protected against malaria, a receiver operating characteristic analysis was performed in two populations living in Madagascar and Malawi. The definition of protection was based on longitudinal measurements of clinical malarial attacks during the season of high malaria transmission in the Madagascar study, and on a cross-sectional measurement of parasitemia in the Malawi study. Antibodies to peptides reproducing the 4-mer, 8-mer, and 11-mer of the Pf155/RESA were tested for their reactivities using the Falcon assay screening test-enzyme-linked immunosorbent assay. Maximal detection of poorly protected individuals (specificity = 100%) corresponded to high cutoff antibody titers (range = 1.65-3.0 optical density [OD] units in the Madagascar study and 0.67-1.42 OD units in the Malawi study) and a sensitivity less than 50%. For a given sensitivity of 50%, specificity ranged from 55% to 62% in the Madagascar study, and from 67% to 94% in the Malawi study. The antibody cutoff titers corresponding to minimal misclassification rates ranged from 0.24 to 1.73 OD units in the Madagascar study and from 0.15 to 0.55 OD units in the Malawi study. For each antibody, the highest detectability value as measured by the area under the curve was obtained for anti-R11 in the Malawi study (0.838). In demonstrating such qualities, antibodies to Pf155/RESA epitopes could be used for screening poorly protected populations in which malaria control programs have to be implemented.

Lack of correlation between HLA class II alleles and immune responses to Pf155/ring-infected erythrocyte surface antigen (RESA) from Plasmodium falciparum in Madagascar.

To investigate the relationships between predominant HLA class II alleles and immune responses to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), 50 individuals from the highlands of Madagascar were followed-up from 1988 to 1991. The T cell reactivity and antibody responses to synthetic peptides (EENV)4, (EENVEHDA)4, and (DDEHVEEPTVA)3, representing major T and B epitopes of Pf155/RESA antigen, were assessed with an average of five determinations per individual over the four-year follow-up period. The T cell reactivity was investigated by lymphocyte proliferation and assays for interferon-gamma and interleukin-2 release. Antipeptide antibodies were measured using the Falcon assay screening test-enzyme-linked immunosorbent assay. The cumulative prevalence rates of cellular (range for the three peptides = 64-68%) and antibody responders (range = 70-74%) were similar for each peptide. The HLA class II typing was performed using polymerase chain reaction-restriction fragment length polymorphisms. The prevalent alleles or groups of alleles (frequency > 20%) were similar in responders and nonresponders, both for cellular and antibody responses to each peptide. These were HLA-DR 5 group and HLA-DQA1 *0601, *0101-0102-0104, HLA-DQB1 *0301, and HLA-DPB1 *0101-2601 alleles. Allelic distribution was similar in individuals presenting with (74%) or without (26%) a malaria attack during a 20-week follow-up conducted when malaria was hyperendemic (P > 0.05, by Fisher's exact test). Despite repeated immunologic measures that better identify the responders, no relationship was found between HLA class II alleles and the cellular or antibody responses to Pf155/RESA epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Presentation of the Plasmodium falciparum antigen Pf155/RESA to human T cells. Variations in responsiveness induced by antigen presenting cells from different but MHC class II identical donors.

The antibody response in humans naturally primed to a malaria vaccine candidate antigen (Pf155/RESA) is genetically regulated. Here, the impact of antigen presenting cells (APC) on the control of in vitro T-cell responses induced by Pf155/RESA or synthetic peptides corresponding to its major Pf155/RESA epitopes was studied. T cells and APC were from the peripheral blood of monozygotic or dizygotic twins and their age matched siblings, all living in the central highlands of Madagascar. When induced to proliferate (thymidine incorporation) in vitro by antigenic peptides, the T-cell responses varied less within the twin pairs than between them and their siblings or the entire group, implying that they were genetically regulated. Occasional MHC class II associations of some of the responses were weak and did not reflect underlying MHC class II restrictions. When T cells and APC from different but MHC class II identical donors were incubated in various combinations, antigen charged APC from homologous donors induced in vitro T-cell proliferation which differed from that induced by the T-cell donors' own APC. Pretreatment of the APC with either paraformaldehyde or anti-class II antibodies inhibited or abolished this antigen dependent T-cell proliferation. The results suggest that the observed differences in T-cell responses induced by APC from different donors reflect differences at the level of these cells. Whether they reflect differences in the proteases involved in antigen processing, in the costimulatory signals provided by the APC to the T cells or in the secretion of other regulatory factors remains to be elucidated.

Characterization of regulatory T-cell responses in humans induced by the P. Falciparum blood stage antigen Pf155/RESA.

T-cells have a major role both as helper cells for efficient antibody production and as inducers and effector cells in antibody-independent malaria immunity. Thus, antigens to be included into a subunit vaccine must contain T-cell epitopes to become effectively immunogenic. The P. falciparum blood stage vaccine antigen Pf155/RESA has been shown to contain T-helper epitopes inducing T-dependent anti-malarial antibodies in vitro. We have also shown that synthetic peptides representing sequences from the amino-acid repeat regions of Pf155/RESA stimulate T-cells from P. falciparum primed donors to proliferate, to release IFN-gamma and/or IL-4. In individual donors there was no correlation between these different activities. Rather, they were frequently negatively associated. However, IL-4 secretion could be induced in T-cells from donors who had elevated concentrations of serum antibodies to the same peptide as used for T-cell activation. Taken together the results support the occurrence of malaria-specific CD4+ T-cell subsets (e.g. TH1 and TH2) in humans similar to what has been found in mice and suggest the involvement of TH2-type helper cells in the induction of some important P. falciparum specific antibodies. CD4+ T-cells recognize the antigen in the context of MHC class II molecules. However, in human outbred populations no consistent MHC restrictions of anti-Pf155/RESA immune responses could be demonstrated. This is not surprising in view of the extensive polymorphism of the HLA system. Neither were there any obvious MHC class II restrictions seen when antibody- and t-cell responses were measured in naturally primed monozygotic twins.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants.

We have previously identified a Plasmodium falciparum liver stage-specific Ag (LSA-1) found to encode tandem 17 amino acid repeats harboring B cell determinants. Here we extend this study in terms of sequence analysis, protein localization, and immunologic properties. Analysis of the N- and C-terminal regions of LSA-1 from the T9/96 clone reveals high sequence conservation with LSA-1 from NF54. This 200-kDa protein is detected throughout liver schizogony and accumulates in the parasitophorous vacuole space. In our investigation of T and B cell responses to LSA-1, we have focused on both the area of the C-terminal, nonrepetitive "hinge" region and the conserved repetitive region and derived synthetic peptides. These were found to contain major B and T cell determinants. High prevalences and elevated Ab levels to LSA-1, directed primarily, although not exclusively, to the repetitive region, were detected in sera of individuals from one moderately high and two low transmission malaria-endemic areas (prevalences of 97%, 75, and 77%, respectively). In one of these low transmission areas, secretion of the cytokine IFN-gamma, known to inhibit malaria liver stages, and T cell proliferation were detected in PBMC of 22 to 48% and 6 to 20%, respectively, of individuals in response to separate LSA-1 peptides. These results complement the recent finding of conserved CTL epitopes in LSA-1 and support the assertion that immune responses to LS Ag are involved in protection against malaria pre-erythrocytic stages.

Antibodies to the 4-mer repeat of the ring-infected erythrocyte surface antigen (Pf155/RESA) protect against Plasmodium falciparum malaria.

To investigate the protective role of antibodies to the ring-infected erythrocyte surface antigen (Pf155/RESA) epitopes against Plasmodium falciparum clinical malaria, a cohort study was conducted in a Malagasy village over 7 months. In the 304 individuals included, 127 experienced P. falciparum attacks of under 1500 parasites/microliters with no clinical symptoms (protected individuals) and 177 experienced at least one clinical or preclinical P. falciparum attack requiring therapy (unprotected individuals). Antibodies to whole Pf155/RESA, to single epitopes of the 3' terminus, (EENV)4 and EENVEHDA(EENV)2 had higher responses in protected than in unprotected individuals (P = 0.006, P = 0.005, P = 0.05 respectively). Within the whole pattern of antibodies to the Pf155/RESA epitopes, only anti-R4 was related to protection independently of age and anti-wR. The Pf155/RESA 4-mer repeated epitope might be of interest for inclusion in a vaccine against the asexual blood stages of P. falciparum.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies.

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Parasite virulence factors during falciparum malaria: rosetting, cytoadherence, and modulation of cytoadherence by cytokines.

To determine virulence factors of isolates of Plasmodium falciparum and the potential role of cytokines in cerebral malaria, 46 Malagasy patients presenting with cerebral (n = 10), severe (n = 10), and uncomplicated (n = 26) malaria were enrolled in a study. The capacity of 21 of 46 P. falciparum isolates to form rosettes in vitro and to adhere to human umbilical vein endothelial cells (HUVECs) that express intercellular adhesion molecule-1 receptors and to C32 amelanotic melanoma cells that express mainly CD36 receptors was investigated together with the effects of tumor necrosis factor alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 alone and in two-by-two combinations on the cytoadherence of infected erythrocytes to HUVECs. Plasma levels of these cytokines were also measured in the patients at admission. The percentage of rosette formation was higher for the isolates from patients with cerebral (n = 6; 19.5%) and severe (n = 6; 30.5%) malaria than for those from patients with uncomplicated malaria (n = 9; 5%) (P < 0.002). The cytoadherence properties of the isolates did not differ among the three groups whatever the target cell used, but adherence to melanoma cells was systematically higher than that to HUVECs. Adhesion to HUVECs was increased more after TNF-alpha stimulation than after GM-CSF, IL-3, or IL-6 stimulation (P < 0.01). Only the combination of TNF-alpha and IL-3 enhanced cytoadherence more than TNF-alpha used alone (P < 0.02). No difference in the modulation of cytoadherence by cytokines was found in relation to the severity of the disease. TNF-alpha and IL-6 levels in peripheral blood were higher in the patients with cerebral and severe malaria than in the patients with uncomplicated malaria (P < 0.005). Most of the patients' sera contained little or no IL-3 or GM-CSF. Our results challenge the role of intercellular adhesion molecule-1 as the principal receptor mediating the cytoadherence of P. falciparum-infected erythrocytes and contrast with data obtained in the murine model.

Isotypic analysis of Plasmodium falciparum-specific antibodies and their relation to protection in Madagascar.

We previously reported that antibodies to the central repeat (DDEHVEEPTVA) of Pf155/RESA, a major Plasmodium falciparum antigen, were negatively related to antimalarial protection. We measured levels of isotype antibodies to this epitope and to P. falciparum in 76 Madagascan subjects. Immunoglobulin G (IgG) and isotype antibodies specific for P. falciparum had similar levels in individuals who were considered protected and those who were not (as determined during a longitudinal follow-up). The levels of IgG1 antibodies to (DDEHVEEPTVA)3 were higher in nonprotected subjects. The levels of total IgG and of other isotype antibodies had a similar tendency to be higher in nonprotected than in protected individuals.