Population pharmacokinetics of intramuscular artesunate in African children with severe malaria: implications for a practical dosing regimen.

Parenteral artesunate (ARS) is the drug of choice for the treatment of severe malaria. Pharmacokinetics data on intramuscular ARS are limited with respect to the main treatment group that carries the highest mortality, namely, critically ill children with severe malaria. A population pharmacokinetic study of ARS and dihydroartemisinin (DHA) was conducted from sparse sampling in 70 Tanzanian children of ages 6 months to 11 years. All the children had been admitted with severe falciparum malaria and were treated with intramuscular ARS (2.4 mg/kg at 0, 12, and 24 h). Venous plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling (NONMEM). A one-compartment disposition model accurately described first-dose population pharmacokinetics of ARS and DHA. Body weight significantly affected clearance and apparent volume of distribution (P < 0.001), resulting in lower ARS and DHA exposure levels in smaller children. An adapted dosing regimen including a practical dosing table per weight band is proposed for young children based on the pharmacokinetic model.

Inter-observer variation in paediatric clinical signs between different grades of staff examining children admitted to hospital in Tanzania.

BACKGROUND AND OBJECTIVE: Children are often admitted to district hospitals in Africa without an adequate record of clinical examination, a problem that could be reduced by greater involvement of nurses in their assessment. We aimed to ascertain whether hospital nurses in a district hospital could conduct paediatric examinations as reliably as clinical staff, when provided with a short structured training session. METHODS: Hospital nurses (HN), hospital clinical officers (HCO) and research clinical officers (RCO) repeated examinations on children admitted to the paediatric ward shortly after the first examination by an RCO. Kappa scores were used to compare the agreement on the presence or absence of basic clinical signs by different categories of staff. RESULTS: Among 439 paired examinations the agreement between RCOs on clinical signs was slightly higher than for HCOs or HNs; the mean (median) Kappa scores for all signs examined were 0.54 (0.57) for RCO-RCO, 0.49 (0.49) for RCO-HCO and 0.50 (0.49) for RCO-HN. Levels of agreement were lower if children were under the age of 18 months or if they cried during the examination. CONCLUSIONS: Nurses with basic training appear to perform as well as clinically trained staff in eliciting essential signs in acutely ill children. Their role in the initial and ongoing assessment of these children should be reviewed in light of the critical shortages in clinically trained staff in African hospitals.

Attitudes to voluntary counselling and testing prior to the offer of Nevirapine to prevent vertical transmission of HIV in northern Tanzania.

In developed countries much progress has been made in reducing vertical transmission of HIV using antiretroviral therapies. To achieve similar gains in Africa the acceptability of routine HIV testing of pregnant women is becoming increasingly important. Evidence of reluctance of pregnant women to undergo HIV testing has led to suggestions to offer antiretroviral therapy to pregnant women without prior HIV testing. In this study we set out to identify risk factors for preferring to avoid HIV testing among women attending an antenatal clinic in northern Tanzania in the context of a hypothetical offer of Nevirapine and to explore the issues raised in more detail in focus group discussions. Two hundred and fifty women attending an antenatal clinic in late pregnancy were interviewed. Almost half of the women preferred to be offered Nevirapine without HIV testing. In a multiple logistic model having a partner with a history of a sexually transmitted disease (OR 2.72, 95% CI 1.14-6.47, p = 0.02) and having a partner who had another sexual partner in the last year (OR 1.89, 95% CI 1.04-3.45, p = 0.04) were positively associated with a preference to avoid HIV testing; while the presence of a partner living at home or feeling able to ask their partner to go for an HIV test were negatively associated with a preference to avoid HIV testing (OR 0.46, 95% CI 0.24-0.89, p = 0.02 and OR 0.56, 95% CI 0.3-1.05, p = 0.07 respectively). FGDs (focus group discussions) suggested that the major concern of women was for the reaction of their male partners to the possibility of a positive HIV test and low confidence in the confidentiality of HIV testing. This fear may lead to low uptake of antiretroviral programmes and treatment without prior testing should be considered.

Estimating medium- and long-term trends in malaria transmission by using serological markers of malaria exposure.

The implementation and evaluation of malaria control programs would be greatly facilitated by new tools for the rapid assessment of malaria transmission intensity. Because acquisition and maintenance of antimalarial antibodies depend on exposure to malaria infection, such antibodies might be used as proxy measures of transmission intensity. We have compared the prevalence of IgG antibodies with three Plasmodium falciparum asexual stage antigens in individuals of all ages living at varying altitudes encompassing a range of transmission intensities from hyper- to hypoendemic in northeastern Tanzania, with alternative measures of transmission intensity. The prevalence of antibodies to merozoite surface protein-1(19) was significantly more closely correlated with altitude than either point-prevalence malaria parasitemia or single measures of hemoglobin concentration. Analysis of age-specific seroprevalence rates enabled differentiation of recent (seasonal) changes in transmission intensity from longer-term transmission trends and, using a mathematical model of the annual rate of seroconversion, estimation of the longevity of the antibody response. Thus, serological tools allow us to detect variations in malaria transmission over time. Such tools will be invaluable for monitoring trends in malaria endemicity and the effectiveness of malaria control programs.

Prevalence of hospital-acquired infections in a tertiary referral hospital in northern Tanzania.

Hospital-acquired infections (HAI) are a major and largely preventable cause of morbidity and morbidity worldwide. Very few reports on the prevalence of HAI in sub-Saharan Africa have been published and most of those that have appeared in the press have focused on surgical-wound infection. In the present, questionnaire-based, point-prevalence study, in which the doctor on the ward round was used as the primary informant, the prevalences of all HAI among all the inpatients at a tertiary referral hospital in northern Tanzania were estimated. On the day of the study, there were 412 inpatients (in 15 ward areas) and 61 cases of HAI were identified, giving an overall HAI prevalence of 14.8%. The prevalences of HAI were particularly high in the medical intensive-care unit (40%), the surgical (orthopaedic and general surgery) wards (36.7%), and one of the general medical wards (22.2%). Factors associated with a patient having a HAI were hospitalization for >30 days [odds ratio (OR) = 4.07; 95% confidence interval (CI) = 2.07-7.99]; being a patient on the orthopaedic and general surgical ward known as 'Surgical 2' (OR = 2.14; CI = 1.02-4.46); and being referred from another health facility (OR = 1.90; CI = 1.02-3.42). The most commonly identified HAI in the hospital were urinary-tract infections (14 cases), followed by surgical-wound infections (10 cases) and then lower respiratory-tract infections (six cases). Twenty HAI were 'unspecified'. The study was rapid and cheap to carry out. The results not only gave a baseline estimate of HAI in the study setting but also identified key areas for interventions to reduce HAI.

The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins.

The binding of killer cell Ig-like Receptors (KIR) to their Class I MHC ligands was shown previously to be characterized by extremely rapid association and dissociation rate constants. During experiments to investigate the biochemistry of receptor-ligand binding in more detail, the kinetic parameters of the interaction were observed to alter dramatically in the presence of Zn(2+) but not other divalent cations. The basis of this phenomenon is Zn(2+)-induced multimerization of the KIR molecules as demonstrated by BIAcore, analytical ultracentrifugation, and chemical cross-linking experiments. Zn(2+)-dependent multimerization of KIR may be critical for formation of the clusters of KIR and HLA-C molecules, the "natural killer (NK) cell immune synapse," observed at the site of contact between the NK cell and target cell.

A randomized controlled trial of insecticide-treated bednets and chaddars or top sheets, and residual spraying of interior rooms for the prevention of cutaneous leishmaniasis in Kabul, Afghanistan.

Anthroponotic cutaneous leishmaniasis (ACL) is a significant public health problem in many towns and cities of south central Asia and the Middle East, resulting in disfigurement and disability which warrants preventive action. A randomized controlled trial was conducted in 1997/98 amongst a non-immune study population of 3666 people in Kabul, Afghanistan, to compare the efficacy of insecticide-treated nets (ITNs), insecticide-treated Islamic cloth wraps (chaddars) used to sleep in, and residual pyrethroid spraying of individual houses for the prevention of ACL. Dosages of insecticide were: ITNs with permethrin, 0.5 g/m2; chaddars with permethrin, 1 g/m2; rooms with lambdacyhalothrin, 30 mg/m2. Cases of ACL were diagnosed on clinical criteria. At the end of the trial period (15 months) the incidence of ACL amongst controls was 7.2%, amongst ITN users 2.4% (OR 0.31, 95% CI 0.2-0.5), amongst impregnated chaddar users 2.5% (OR 0.33, 95% CI 0.2-0.6) and amongst residents of sprayed houses 4.4% (OR 0.60, 95% CI 0.3-0.95). ITNs and impregnated chaddars were equally effective, providing about 65% protective efficacy, with approximately 40% protective efficacy attributable to individual house spraying. No significant differences for age or sex were found between new cases in the intervention and control groups. No serious side-effects were reported and interventions were generally popular; ITNs were the most popular, followed by residual spraying and then impregnated chaddars.

Expression of the CD80 and CD86 molecules enhances cytotoxicity by human natural killer cells.

In contrast to the inhibitory pathway of NK cell regulation, much less is known about stimulatory or activation signals in NK cells. Both CD80 and CD86 function as costimulatory molecules in T-cell cytotoxicity. Several previous reports, most of them in the murine system, have indirectly or directly indicated the possible role of B7 molecules (CD80 and CD86) triggering NK cell-mediated cytotoxicity in vitro. Nevertheless, only little is known about the role of these molecules on human target cells. Therefore, anti-CD80 and anti-CD86 mAbs were used in blocking experiments and both were shown to inhibit lysis by human NK cells. The degree of inhibition observed was variable. 64% of these NK clones were strongly inhibited by both anti-CD80 and anti-CD86 (Type 1). A small number (19%) were only moderately inhibited by both of these antibodies (Type 2), and 17% of these NK clones were inhibited strongly by anti-CD86 but weakly or not at all by anti-CD80 (Type 3). To further examine the importance of these proteins, B7.1 (CD80) and B7.2 (CD86) genes were transfected into the mouse mastocytoma P815 cell line that could not be killed by the human NK cells. These transfectant cell lines were then tested in cytotoxicity assays using a number of human NK lines. Expression of the CD80 and CD86 molecules resulted in enhanced lysis of P815 by most of the NK lines tested. Thus, both CD80 and CD86 molecules are involved in triggering of human NK cells.

Interaction between the human NK receptors and their ligands.

NK cells are physiologically important in a number of contexts: mediating immunity against viruses, intracellular bacteria and parasites, and in anti-tumour immune responses. Moreover, in addition to these overtly immune protective functions, NK cells also mediate a variety of homeostatic functions, particularly in the regulation of haematopoesis and they may have an important role to play in the maintenance and development of placentation; certainly NK cells are a major component of the lymphocyte population of the decidua. The behaviour of the NK cell in these various situations is regulated by a large number of distinct receptors that transmit positive and negative signals. The balance of these signals determines whether the NK cell does nothing or is activated to proliferate, kill or secrete a wide range of cytokines and chemokines. In this review the structure and function of a number of molecules found on the NK cell surface are discussed, particular emphasis being placed on the molecular details of the recognition of target cell classical class I HLA molecules by Killer cell Immunoglobulin-like Receptors (KIR) and the binding of the non-classical class I molecule HLA-E to the heterodimer formed by the association of CD94 with various members of the NKG2 proteins.

Definition of polymorphic residues on killer Ig-like receptor proteins which contribute to the HLA-C binding site.

Killer cell immunoglobulin-like receptors (KIR) bind HLA class I proteins in an allele- and locus-specific manner. This report describes the use of transfectants expressing recombinant chimeric proteins, comprising the extracellular portions of KIR molecules and the transmembrane and cytoplasmic tails of CD3-zeta, to create an in vitro system in which signaling is readily measured and that preserves the specificity of the KIR / HLA-C interaction. The identity of the amino acid residues on the KIR molecule important for binding to the HLA protein is not well understood; although some KIR2D residues involved in HLA-C recognition have been identified, their relative importance and whether other amino acids contribute to binding was unclear. This novel system was used to study, by site-directed mutagenesis, the role of various amino acids in KIR binding to HLA-C ligand. The data presented here show that while multiple polymorphic residues contribute to the HLA-C binding site on KIR proteins, two clusters of polymorphic residues define the group allotype specificity of HLA-C binding to a KIR2D molecule.

Molecular analyses of the interactions between human NK receptors and their HLA ligands.

NK cell cytotoxicity is regulated by the action of multiple families of receptors. The interactions of these receptors with their ligands control different activating/inhibiting signal pathways and it is the balance of these signals which determines the behavior of the NK cell. The major described inhibitory pathways begin either with the recognition of a target cell classical class I HLA molecule by a killer cell immunologlobulin-like receptor (KIR) or the binding of the non-classical class I molecule HLA-E to the CD94/NKG2-A heterodimer. Activating counterparts to these inhibitory NK receptors have also been described and this review focuses on the molecular details of the binding of the inhibitory and activating receptors to their HLA ligands.

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

An outbreak of cutaneous leishmaniasis in an Afghan refugee settlement in north-west Pakistan.

Cutaneous leishmaniasis (CL) due to Leishmania tropica appears to be an emerging disease in parts of north-east Afghanistan and north-west Pakistan. Timargara, an Afghan refugee camp of 17 years' standing, in the district of Dir, North West Frontier Province of Pakistan, experienced a major outbreak of CL in 1997 for the first time. As part of the investigation, each section of the camp was surveyed for CL. Around 38% of the 9200 inhabitants bore active lesions and a further 13% had scars from earlier attacks. According to interview statements, 99% of earlier infections had healed within the previous 2 years. To confirm the diagnosis, a sample of current CL lesions was examined parasitologically. Amastigotes were detectable by microscopy in only 36% of lesions. However, 48% of slide-negative cases produced positive cultures and some cases negative to both microscopy and culture were positive by PCR. Overall detection rate was about 80%. The sandfly Phlebotomus sergenti, a known vector of L. tropica, was captured within the camp, indicating local transmission. CL has not been reported from this area of Pakistan before. Although the majority of refugees left Afghanistan 2 decades ago, cross-border movement of men is common. The Afghanistan capital, Kabul, is currently experiencing a major epidemic of CL; infected migrant carriers from Kabul are probably the source of the outbreak in Timargara.

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.

Anthroponotic cutaneous leishmaniasis in Kabul, Afghanistan: vertical distribution of cases in apartment blocks.

Owing to the civil war, the inhabitants of Kabul in Afghanistan are suffering a major epidemic of anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica. Surveys conducted among children in 2 high-rise apartment blocks in the city revealed that the prevalence of active lesions was much lower on upper stories: 84% lower in one block (chi 2 = 7.13, d.f. = 1, P = 0.008) and 54% lower in the other (chi 2 = 6.17, d.f. = 1, P = 0.01). Similar trends were apparent with regard to scars from old lesions. These results suggest that in Kabul most transmission of ACL takes place in the home. In addition, the results imply that there must be limited vertical movement of the vector within apartment blocks. Together, these findings suggest that indoor spraying should be an effective means of control and that insecticidal applications could probably be restricted to lower stories.

Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors.

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors.

The recognition of HLA-C molecules by specific inhibitory receptors is a crucial step in the regulation of natural killer (NK) cell function. Using soluble, recombinant HLA-C molecules and NK inhibitory receptors (NKIR, members of the immunoglobulin superfamily), we show that HLA-C binds to NKIR molecules with extremely fast association and dissociation rates, among the fastest of the immune system interactions so far studied. These kinetics may be essential for the biological function of NK cells, i.e., to facilitate the rapid immunosurveillance of cells for absent or diminished expression of class I MHC proteins.

A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan.

A nested PCR was developed to amplify the variable region of the kinetoplast minicircles of all Leishmania species which infect mammals. Each Leishmania parasite contains approximately 10,000 kinetoplast DNA minicircles, which are unequally distributed among approximately 10 minicircle classes. The PCR primers were designed to bind within the 120-bp conserved region which is common to all minicircle classes; the remaining approximately 600 bp of each minicircle is highly conserved within each minicircle class but highly divergent between classes. The nested PCR generated a strong signal from a minimum of 0.1 fg of Leishmania DNA. Restriction digests of the amplicons from the highest dilutions suggested that minicircles from only a limited number of minicircle classes had acted as template in the reaction. One PCR product was directly sequenced and found to be derived from only one minicircle class. Since the primers amplify all minicircle classes, this indicated that as little as 1/10 of one Leishmania parasite was present in the PCR template. This demonstrated that the nested PCR achieved very nearly the maximum theoretically possible sensitivity and is therefore a potentially useful method for diagnosis. The nested PCR was tested for sensitivity on 20 samples from patients from the Timargara refugee camp, Pakistan. Samples were collected by scraping out a small amount of tissue with a scalpel from an incision at the edge of the lesion; the tissue was smeared on one microscope slide and placed in a tube of 4 M guanidine thiocyanate, in which the sample was stable for at least 1 month. DNA for PCR was prepared by being bound to silica in the presence of 6 M guanidine thiocyanate; washed in guanidine thiocyanate, ethanol, and acetone; and eluted with 10 mM Tris-HCl. PCR products of the size expected for Leishmania tropica were obtained from 15 of the 20 samples in at least one of three replicate reactions. The negative samples were from lesions that had been treated with glucantime or were over 6 months old, in which parasites are frequently scanty. This test is now in routine use for the detection and identification of Leishmania parasites in our clinical laboratory. Fingerprints produced by restriction digests of the PCR products were defined as complex or simple. There were no reproducible differences between the complex restriction patterns of the kinetoplast DNA of any of the parasites from Timargara camp with HaeIII and HpaII. The simple fingerprints were very variable and were interpreted as being the product of PCR on a limited subset of minicircle classes, and consequently, it was thought that the variation was determined by the particular minicircle classes that had been represented in the template. The homogeneity of the complex fingerprints suggests that the present epidemic of cutaneous leishmaniasis in Timargara camp may be due to the spread of a single clone of L. tropica.