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The relationship between community prevalence of Plasmodium falciparum and the burden of severe, life-threatening disease remains poorly defined. To examine the three most common severe malaria phenotypes from catchment populations across East Africa, we assembled a dataset of 6506 hospital admissions for malaria in children aged 3 months to 9 years from 2006 to 2020. Admissions were paired with data from community parasite infection surveys. A Bayesian procedure was used to calibrate uncertainties in exposure (parasite prevalence) and outcomes (severe malaria phenotypes). Each 25% increase in prevalence conferred a doubling of severe malaria admission rates. Severe malaria remains a burden predominantly among young children (3 to 59 months) across a wide range of community prevalence typical of East Africa. This study offers a quantitative framework for linking malaria parasite prevalence and severe disease outcomes in children.

Background Pneumonia is among the leading causes of avoidable deaths for young children globally. The main burden of mortality falls on children from poor and rural families who are less likely to obtain the treatment they need, highlighting inequities in access to effective care and treatment. Caretakers’ illness perceptions and care-seeking practices are of major importance for children with pneumonia to receive adequate care. This study qualitatively explores the caretaker concepts of childhood pneumonia in relation to treatment seeking behaviour and health worker management in Moshi urban district, Tanzania. Methods In May - July 2013 data was gathered through different qualitative data collection techniques including five focus group discussions (FGDs) with mothers of children under-five years of age. The FGDs involved free listing of pneumonia symptoms and video presentations of children with respiratory symptoms done, these were triangulated with ten case narratives with mothers of children admitted with pneumonia and eleven in-depth interviews with hospital health workers. Transcripts were coded and analysed using qualitative content analysis. Results Mothers demonstrated good awareness of common childhood illnesses including pneumonia, which was often associated with symptoms such as cough, flu, chest tightness, fever, and difficulty in breathing. Mothers had mixed views on causative factors and treatments options but generally preferred modern medicine for persisting and severe symptoms. However, all respondent reported access to health facilities as a barrier to care, associated with transport, personal safety and economic constraints. Conclusion Local illness concepts and traditional treatment options did not constitute barriers to care for pneumonia symptoms. Poor access to health facilities was the main barrier. Decentralisation of care through community health workers may improve access to care but needs to be combined with strengthened referral systems and accessible hospital care for those in need.

We conducted a genome-wide association study of host resistance to severePlasmodium falciparummalaria in over 17,000 individuals from 11 malaria-endemic countries, undertaking a wide ranging analysis which identifies five replicable associations with genome-wide levels of evidence. Our findings include a newly implicated variant on chromosome 6 associated with risk of cerebral malaria, and the discovery of an erythroid-specific transcription start site underlying the association inATP2B4. Previously reported HLA associations cannot be replicated in this dataset. We estimate substantial heritability of severe malaria (h2~ 23%), of which around 10% is explained by the currently identified associations. Our dataset will provide a major building block for future research on the genetic determinants of disease in these diverse human populations.

Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection, resulting in the acquisition of mechanisms of resistance against severe malarial disease. Many host genetic factors, including sickle cell trait, have been associated with reduced risk of developing severe malaria, but do not account for all of the observed phenotypic variation. Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies (GWAS). We present findings of a GWAS of severe malaria performed in a Tanzanian population (n = 914, 15.2 million SNPs). Beyond the expected association with the sickle cell HbS variant, we identify protective associations within two interleukin receptors (IL-23R and IL-12RBR2) and the kelch-like protein KLHL3 (all P0.3). Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes. The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease., Author summary Malaria, caused by Plasmodium falciparum parasites, is a major cause of mortality and morbidity in endemic countries of sub-Saharan Africa, including Tanzania. Some gene mutations in the human genome, including sickle cell trait, have been associated with reduced risk of developing severe malaria, and have increased in frequency through natural selection over generations. However, new genetic mutations remain to be discovered, and recent advances in human genome research technologies such as genome-wide association studies (GWAS) and fine-scale molecular genotyping tools, are facilitating their identification. Here, we present findings of a GWAS of severe malaria performed in a well characterised Tanzanian population (n = 914). We confirm the expected association with the sickle cell trait, but also identify new gene targets in immunological pathways, some under natural selection. Our approach demonstrates the potential of using GWAS to identify as-yet unknown susceptibility genes in endemic populations with well-characterised malaria phenotypes. The genetic mutations are likely to form potential targets for the design of much needed interventions for preventing or treating malarial disease.

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10(-4) with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

Bats carry a variety of paramyxoviruses that impact human and domestic animal health when spillover occurs. Recent studies have shown a great diversity of paramyxoviruses in an urban-roosting population of straw-colored fruit bats in Ghana. Here, we investigate this further through virus isolation and describe two novel rubulaviruses: Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2). The viruses form a phylogenetic cluster with each other and other bat-derived rubulaviruses, such as Tuhoko viruses, Menangle virus, and Tioman virus. We developed AchPV1- and AchPV2-specific serological assays and found evidence of infection with both viruses in Eidolon helvum across sub-Saharan Africa and on islands in the Gulf of Guinea. Longitudinal sampling of E. helvum indicates virus persistence within fruit bat populations and suggests spread of AchPVs via horizontal transmission. We also detected possible serological evidence of human infection with AchPV2 in Ghana and Tanzania. It is likely that clinically significant zoonotic spillover of chiropteran paramyxoviruses could be missed throughout much of Africa where health surveillance and diagnostics are poor and comorbidities, such as infection with HIV or Plasmodium sp., are common.

Objective Measurement of respiratory rate is an important clinical sign in the diagnosis of pneumonia but suffers from interobserver variation. Here, we assess the use of video recordings as a quality assurance tool that could be useful both in research and in training of staff. Methods Respiratory rates (RR) were recorded in children aged 2–59 months presenting with cough or difficulty breathing at two busy outpatient clinics in Tanzania. Measurements were repeated at 10‐min intervals in a quiet environment with simultaneous video recordings that were independently reviewed by two paediatricians. Results Eight hundred and fifty‐nine videos were sent to two paediatricians; 148 (17.2%) were considered unreadable by one or both. For the 711 (82.8%) videos that were readable by both paediatricians, there was perfect agreement for the presence of raised RR with a kappa value (κ) of 0.85 (P < 0.001); and in 476 (66.9%) cases, both paediatricians agreed on the RR within 2 breaths per minute (±2 bpm). A reported illness of 5 days or more was associated with unreadable video recordings (OR = 3.44, CI: 1.5–6.08; P < 0.001). The multilevel model showed that differences between observers accounted for only 13% of the variability in RR. Conclusion Video recordings are reliable tools for quality assurance of RR measurements in children with suspected pneumonia. Videos with a clear view of respiratory movements may also be useful in training primary healthcare staff.

INTRODUCTION Malaria parasites cause human disease by invading and replicating inside red blood cells. In the case of Plasmodium falciparum , this can lead to severe forms of malaria that are a major cause of childhood mortality in Africa. This species of parasite enters the red blood cell through interactions with surface proteins including the glycophorins GYPA and GYPB, which determine the polymorphic MNS blood group system. In a recent genome-wide association study, we identified alleles associated with protection against severe malaria near the cluster of genes encoding these invasion receptors. RATIONALE Investigation of genetic variants at this locus and their relation to severe malaria is challenging because of the high sequence similarity between the neighboring glycophorin genes and the relative lack of available sequence data capturing the genetic diversity of sub-Saharan Africa. To better assess whether variation in the glycophorin genes could explain the signal of association, we generated additional sequence data from sub-Saharan African populations and developed an analytical approach to characterize structural variation at this complex locus. RESULTS Using 765 newly sequenced human genomes from 10 African ethnic groups along with data from the 1000 Genomes Project, we generated a reference panel of haplotypes across the glycophorin region. In addition to single-nucleotide polymorphisms and short indels, we assayed large copy number variants (CNVs) using sequencing read depth and uncovered extensive structural diversity. By imputing from this reference panel into 4579 severe malaria cases and 5310 controls from three African populations, we found that a complex CNV, here called DUP4, is associated with resistance to severe malaria and fully explains the previously reported signal of association. In our sample, DUP4 is present only in east Africa, and this localization, as well as the extent of similarity between DUP4 haplotypes, suggests that it has recently increased in frequency, presumably under natural selection due to malaria. To evaluate the potential functional consequences of this structural variant, we analyzed high-coverage sequence-read data from multiple individuals to generate a model of the DUP4 chromosome structure. The DUP4 haplotype contains five glycophorin genes, including two hybrid genes that juxtapose the extracellular domain of GYPB with the transmembrane and intracellular domains of GYPA. Noting that these predicted hybrids are characteristic of the Dantu antigen in the MNS blood group system, we sequenced a Dantu positive individual and confirmed that DUP4 is the molecular basis of the Dantu NE blood group variant. CONCLUSION Although a role for GYPA and GYPB in parasite invasion is well known, a direct link between glycophorin polymorphisms and clinical susceptibility to malaria has been elusive. Here we have provided a systematic catalog of CNVs, describing structural diversity that may have functional importance at this locus. Our results identify a specific variant that encodes hybrid glycophorin proteins and is associated with protection against severe malaria. This discovery calls for further work to determine how this particular molecular rearrangement affects parasite invasion and the red blood cell response and may lead us toward new parasite vulnerabilities that can be utilized in future interventions against this deadly disease.

This comparative study in 1898 children in 2 different African population shows that a pfHRP2-based rapid diagnostic test is a reliable diagnostic for diagnosing severe falciparum malaria in these settings and performs better than routine microscopy or a pLDH based test., Background. Rapid diagnostic tests (RDTs) now play an important role in the diagnosis of falciparum malaria in many countries where the disease is endemic. Although these tests have been extensively evaluated in uncomplicated falciparum malaria, reliable data on their performance for diagnosing potentially lethal severe malaria is lacking. Methods. We compared a Plasmodium falciparum histidine-rich-protein2 (PfHRP2)–based RDT and a Plasmodium lactate dehydrogenase (pLDH)–based RDT with routine microscopy of a peripheral blood slide and expert microscopy as a reference standard for the diagnosis of severe malaria in 1898 children who presented with severe febrile illness at 2 centers in Mozambique and Tanzania. Results. The overall sensitivity, specificity, positive predictive value, and negative predictive values of the PfHRP2-based test were 94.0%, 70.9%, 85.4%, and 86.8%, respectively, and for the pLDH-based test, the values were 88.0%, 88.3%, 93.2%, and 80.3%, respectively. At parasite counts

The WHO recommendation for parasitological diagnosis of malaria wherever possible is challenged by evidence of poor-quality microscopy in African hospitals but the reasons are not clear.All 12 of the busier district hospital laboratories from three regions of Tanzania were assessed for quality of the working environment and slide readers read 10 reference slides under exam conditions. Slides that had been routinely read were removed for expert reading.Of 44 slide readers in the study, 39 (88.6%) correctly read90% of the reference slides. Of 206 slides that had been routinely read, 33 (16%) were judged to be unreadable, 104 (51%) were readable with difficulty, and 69 (34%) were easily readable. Compared to expert reading of the same slide, the sensitivity of routine slide results of easily readable slides was 85.7% (95% confidence interval: 77.4-94.0), falling to 44.4% (95% confidence interval: 34.5-54.4) for slides that were 'readable with difficulty'.The commonest cause of inaccurate results was the quality of the slide itself, correction of which is likely to be achievable within existing resources. A minority of slide readers were unable to read slides even under ideal conditions, suggesting the need for a 'slide reading licence' scheme.

(See the editorial commentary by Levine and Farag, on pages 349-351.) Background. Few studies describe patterns of human immunodeficiency virus (HIV) co-infections in African hospitals in the antiretroviral therapy (ART) era. Methods. We enrolled consecutive admitted patients aged ≥13 years with oral temperature of ≥38.0°C during 1 year in Moshi, Tanzania. A standardized clinical history and physical examination was done and hospital outcome recorded. HIV antibody testing, aerobic and mycobacterial blood cultures, and malaria film were performed. HIV-infected patients also received serum cryptococcal antigen testing and CD4+ T lymphocyte count (CD4 cell count). Results. Of 403 patients enrolled, the median age was 38 years (range, 14–96 years), 217 (53.8%) were female, and 157 (39.0%) were HIV-infected. Of HIV-infected patients, the median CD4 cell count was 98 cells/μL (range, 1–1,105 cells/ μL), 20 (12.7%) were receiving ART, and 29 (18.5%) were receiving trimethoprim-sulfamethoxazole prophylaxis. There were 112 (27.7%) patients who had evidence of invasive disease, including 26 (23.2%) with Salmonella serotype Typhi infection, 24 (21.4%) with Streptococcus pneumoniae infection, 17 (15.2%) with Cryptococcus neoformans infection, 12 (10.7%) with Mycobacterium tuberculosis complex infection, 8 (7.1%) with Plasmodium falciparum infection, and 7 (6.3%) with Escherichia coli infection. HIV infection was associated with M. tuberculosis and C. neoformans bloodstream infection but not with E. coli, S. pneumoniae, or P. falciparum infection. HIV infection appeared to be protective against Salmonella. Typhi bloodstream infection (odds ratio, .12; P = .001). Conclusions. While Salmonella Typhi and S. pneumoniae were the most common causes of invasive infection overall, M. tuberculosis and C. neoformans were the leading causes of bloodstream infection among HIV-infected inpatients in Tanzania in the ART era. We demonstrate a protective effect of HIV against Salmonella. Typhi bloodstream infection in this setting. HIV co-infections continue to account for a large proportion of febrile admissions in Tanzania.

Participation rates in epidemiologic studies in African communities are historically high. We recently performed a pilot prevalence study for neurologic morbidity in Mungushi, Tanzania, where the participation rate was 645/657 subjects (98.2%).1 We questioned whether this high rate was due to a sincere motivation to participate or a fear of refusal of participation. As Western investigators are increasingly performing neuroepidemiologic work in Africa, it is imperative to distinguish between the 2 explanations. Investigators need to tailor the consent process to the local community's needs, and be sensitive to the motivation of the participants whose culture may differ from their own.

We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.

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.

OBJECTIVE In Africa antimalarials are often prescribed when malaria is unlikely, a problem that is becoming critical as more expensive antimalarials replace established drugs. However, little is known about what drives the overuse of antimalarials. We conducted this study to explore to what extent current prescribing behaviour in hospitals is driven by patient demand. METHODS Consultations were observed followed by exit interviews with patients or caretakers. Five district hospitals where microscopy was routinely available were selected in areas of low (n = 3) and high (n = 2) malaria transmission in north-eastern Tanzania. All outpatient consultations during the study period were observed (n = 669). Those sent for a malaria blood slide or treated with antimalarials presumptively were interviewed (n = 326). At the end of the study, clinicians were interviewed for their opinions on the use of antimalarials. Findings Patients were not observed to demand antimalarials from clinicians, but occasionally asked for a malaria slide. Patient satisfaction on exit was similar between those prescribed antimalarials and those not prescribed antimalarials, but more patients or carers expressed satisfaction when the patient had been tested than when not. Clinicians rarely reported perceiving patient demand for antimalarials and asserted that such demand for medication would not affect their prescribing behaviour. CONCLUSIONS Patient demand was not found to be driving the over-prescription of antimalarials found in the hospitals in our setting. To the contrary, the involvement of patients may provide an opportunity to improve prescribing practice if their expectations for testing and treatment in line with test results can be effectively communicated to clinicians.

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; alpha-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions.

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers., Author Summary Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Numerous genetic variants of G6PD, residing in the X chromosome, are found among African populations: mutations causing A- deficiency can lead to serious clinical outcomes (including hemolytic anemia) but also confer protection against severe malaria. Epidemiological studies have used some of the genetic markers that cause A- deficiency to establish who is protected from severe malaria, with differing results. Whether females, with one or two copies of mutant genes, males with one copy, or both genders are protected is uncertain. This uncertainty is due to G6PD and malaria phenotypic complexity and misclassification, and to genetic differences between populations and the limited numbers of genetic markers (usually 2) considered. In this study we analysed more than 30 G6PD genetic markers in 506 Tanzanian children with severe malaria and 477 without malaria. We found that only females with one normal and one mutant copy of the gene (heterozygotes) were protected from severe malaria. Further, we established that the G6PD gene is under evolutionary pressure with the likely mechanism being selection by malaria. Our work demonstrates that studies of severe malaria and G6PD enzymatic function across African populations require, in addition to complete and accurate G6PD phenotypic classification, the identification and analysis of the full repertoire of G6PD genetic markers.

Previous analyses have suggested that immunity to non-cerebral severe malaria due to Plasmodium falciparum is acquired after only a few infections, whereas longitudinal studies show that some children experience multiple episodes of severe disease, suggesting that immunity may not be acquired so quickly. We fitted a mathematical model for the acquisition and loss of immunity to severe disease to the age distribution of severe malaria cases stratified by symptoms from a range of transmission settings in Tanzania, combined with data from several African countries on the age distribution and overall incidence of severe malaria. We found that immunity to severe disease was acquired more gradually with exposure than previously thought. The model also suggests that physiological changes, rather than exposure, may alter the symptoms of disease with increasing age, suggesting that a later age at infection would be associated with a higher proportion of cases presenting with cerebral malaria regardless of exposure. This has consequences for the expected pattern of severe disease as transmission changes. Careful monitoring of the decline in immunity associated with reduced transmission will therefore be needed to ensure rebound epidemics of severe and fatal malaria are avoided.

Malaria clinical trials need precise endpoints to measure efficacy. In endemic areas where asymptomatic parasitaemia is common, 'fever plus parasitaemia' may not differentiate between malaria cases and non-cases. Case definitions based on parasite cut-off densities may be more appropriate but may vary with age and transmission intensity. This study examines appropriate case definitions from parasitological surveys conducted over a broad range of transmission intensities, using altitude as a proxy for transmission intensity.Cross-sectional data collected from 24 villages at different altitudes in an endemic area of northeastern Tanzania were used to calculate malaria-attributable fractions using a modified Poisson regression method. We modelled fever as a function of parasite density and determined the optimum cut-off densities of parasites to cause fever using sensitivity and specificity analyses.The optimum cut-off density varied by altitude in children aged under 5 years: a case definition of 4,000 parasites per mul at altitudes600 m (high transmission intensity) was most appropriate, compared with 1,000 parasites per mul at altitudes600 m (low transmission intensity). In children aged over 5 years and adults, there was little variation by altitude and a case definition of any parasites plus fever was the most appropriate.Locally appropriate case definitions of malaria should be used for research purposes. In our setting, these varied independently with age and transmission intensity.

The isotype/subclass of immunoglobulin determines antibody function, but rather little is known about factors that direct class switching in vivo. To evaluate factors that might influence the maturation of the antibody response during infection, we conducted a seroepidemiological study of the immunoglobulin G (IgG) subclass response to four merozoite-associated antigens ofPlasmodium falciparumin a mountainous region of northeastern Tanzania, where malaria endemicity declines with increasing altitudes. We found that IgG1/IgG3 class switching is independently affected by the nature of the antigen, cumulative exposure to the antigen, and the maturity of the immune system (i.e., the age of the individual). These observations provide insights into the effects of immune system maturity, the duration and intensity of antigen exposure, and inherent characteristics of individual antigens on the process of class switching in human B cells. Our data also throw light on the consequences of class switch decisions on the gradual acquisition of antimalarial immunity.

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.

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 threePlasmodium falciparumasexual 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-119was 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.

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.

Background In 2006, the first-line anti-malarial drug treatment in Tanzania was changed from sulphadoxine-pyrimethamine (SP) to artemether-lumefantrine (ALu), an artemisinin-based combination (ACT), since when the use of SP has been restricted for intermittent preventive treatment in pregnancy (IPTp). A number of Plasmodium falciparum mutations are known to be associated with resistance to SP, but it is not known if the prevalence of these mutations is increasing or decreasing under the conditions of reduced levels of SP use. This study reports on the current SP resistant quintuple Pfdhfr-Pfdhps mutations in six regions of Tanzania. Methods Finger-prick blood on filter paper and rapid diagnostic test strips from P. falciparum-positive individuals of all age groups attending health facilities in six regions of Tanzania between June 2010 and August 2011 were obtained. Using chelex-100 extracted DNA, genotyping was done for mutations on codons 51, 59 and 108 of Pfdhfr and 437 and 540 of Pfdhps genes using PCR-RFLP technique. Results A total of 802 malaria-positive samples were screened and genotyped. The prevalence of Pfdhfr 51I, Pfdhps 437G and 540E varied between the regions (p

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.

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 Hae III and Hpa II. 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 .

Background Plasmodium falciparum resistance to anti-malarial drugs remains a major obstacle to the control of malaria. In 2001 Tanzania replaced chloroquine (CQ) with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2006. SP has however, continued to be used in intermittent preventive treatment of malaria in pregnancy (IPTp) despite reports of high levels of resistance to SP due to the lack of alternatives to SP for IPTp. Recent reports have indicated recovery of CQ-susceptibility in Malawi, Kenya, Mozambique, and Tanzania based on the prevalence of wild types at codon 76 of the Pfcrt gene in indigenous P. falciparum populations. The current prevalence of this Pfcrt- 76 CQ resistance marker from six regions of Tanzania mainland is hereby reported. Methods DNA extracted from filter-paper dried blood spots and rapid diagnostics kit strips collected from finger-prick blood were used to genotype the Pfcrt-76 resistance marker using PCR-RFLP. Data from previously published studies were used to generate CQ susceptibility recovery trends using logistic regression model. Results Seven hundred and forty one (741) samples were genotyped. The current frequency of the CQ-susceptible Pfcrt-K76 was above 92% and did not differ between regions in Tanzania (χ 2 = 2.37; p = 0.795). The K76 allelic prevalence was between 85.7 and 93% in regions (χ 2 = 7.88, p = 0.163). The CQ resistance recovery trends showed regional variability that may be caused by differences in malaria transmission intensity, but overall the trends converge as the susceptibility levels in all regions approach >90%. Conclusions CQ withdrawal in Tanzania has resulted into >90% recovery of susceptibility in ten years of withdrawal. These findings are in support of the search for CQ-based combination drugs as a possible future alternative to SP for IPTp in places where full recovery of CQ-susceptibility will be evident.

Data from a prospective study of 3,319 children ages 2 months to 5 years admitted with febrile illness to a Tanzanian district hospital were analyzed to determine the relationship of blood glucose and mortality. Hypoglycemia (blood sugar < 2.5 mmol/L and < 45 mg/dL) was found in 105 of 3,319 (3.2%) children at admission, and low-normal blood glucose (2.5-5 mmol/L and 45-90 mg/dL) was found in 773 of 3,319 (23.3%) children. Mortality was inversely related to admission blood sugar; compared with children with an admission blood glucose of > 5 mmol/L, the adjusted odds of dying were 3.3 (95% confidence interval = 2.1-5.2) and 9.8 (95% confidence interval = 5.1-19.0) among children with admission blood glucose 2.5-5 and < 2.5 mmol/L, respectively. Receiver operating characteristic (ROC) analysis suggested an optimal cutoff for admission blood sugar of < 5 mmol/L in predicting mortality (sensitivity = 57.7%, specificity = 75.2%). A cutoff for admission blood glucose of < 5 mmol/L represents a simple and clinically useful predictor of mortality in children admitted with severe febrile illness to hospital in resource-poor settings.

Background: In malaria-endemic settings, asymptomatic parasitemia complicates the diagnosis of malaria. Histidine-rich protein 2 (HRP2) is produced by Plasmodium falciparum, and its plasma concentration reflects the total body parasite burden. We aimed to define the malaria-attributable fraction of severe febrile illness, using the distributions of plasma P. falciparum HRP2 (PfHRP2) concentrations from parasitemic children with different clinical presentations. Methods: Plasma samples were collected from and peripheral blood slides prepared for 1435 children aged 6−60 months in communities and a nearby hospital in northeastern Tanzania. The study population included children with severe or uncomplicated malaria, asymptomatic carriers, and healthy control subjects who had negative results of rapid diagnostic tests. The distributions of plasma PfHRP2 concentrations among the different groups were used to model severe malaria-attributable disease. Results:The plasma PfHRP2 concentration showed a close correlation with the severity of infection. PfHRP2 concentrations of >1000 ng/mL denoted a malaria-attributable fraction of severe disease of 99% (95% credible interval [CI], 96%–100%), with a sensitivity of 74% (95% CI, 72%–77%), whereas a concentration of 10% (95% CI, 3%–27%) of patients. Bacteremia was more common among patients in the lowest and highest PfHRP2 concentration quintiles. Conclusions: The plasma PfHRP2 concentration defines malaria-attributable disease and distinguishes severe malaria from coincidental parasitemia in African children in a moderate-to-high transmission setting.

Human genetic background strongly influences susceptibility to malaria infection and progression to severe disease and death. Classical genetic studies identified haemoglobinopathies and erythrocyte-associated polymorphisms, as protective against severe disease. High throughput genotyping by mass spectrometry allows multiple single nucleotide polymorphisms (SNPs) to be examined simultaneously. We compared the prevalence of 65 human SNP's, previously associated with altered risk of malaria, between Tanzanian children with and without severe malaria. Five hundred children, aged 1-10 years, with severe malaria were recruited from those admitted to hospital in Muheza, Tanzania and compared with matched controls. Genotyping was performed by Sequenom MassArray, and conventional PCR was used to detect deletions in the alpha-thalassaemia gene. SNPs in two X-linked genes were associated with altered risk of severe malaria in females but not in males: heterozygosity for one or other of two SNPs in the G6PD gene was associated with protection from all forms of severe disease whilst two SNPs in the gene encoding CD40L were associated with respiratory distress. A SNP in the adenyl cyclase 9 (ADCY9) gene was associated with protection from acidosis whilst a polymorphism in the IL-1α gene (IL1A) was associated with an increased risk of acidosis. SNPs in the genes encoding IL-13 and reticulon-3 (RTN3) were associated with increased risk of cerebral malaria. This study confirms previously known genetic associations with protection from severe malaria (HbS, G6PD). It identifies two X-linked genes associated with altered risk of severe malaria in females, identifies mutations in ADCY9, IL1A and CD40L as being associated with altered risk of severe respiratory distress and acidosis, both of which are characterised by high serum lactate levels, and also identifies novel genetic associations with severe malaria (TRIM5) and cerebral malaria(IL-13 and RTN3). Further studies are required to test the generality of these associations and to understand their functional consequences.

Excluding HIV infection among infants and young children in resource-poor settings where nucleic acid amplification tests (NAAT) are not routinely available remains a considerable challenge.To assess the performance of two rapid HIV antibody tests (RT) used alone and in parallel for excluding HIV infection among acutely ill infants and children18 months in comparison to NAAT in a region where maternal HIV prevalence was approximately 7%.Infants and children ≥218 months admitted to hospital with an acute febrile illness had two rapid antibody tests in parallel, with single and parallel results subsequently compared against NAAT.HIV prevalence among 1602 enrolled infants was 3.4%. All 1526 infants with 2 negative RT were HIV negative by NAAT. All 46 infants with 2 positive RT were HIV positive by NAAT. The overall specificity of two rapid tests for excluding HIV infection was 99.5%. Sensitivity and specificity were ≥99% and98%, respectively, across all age brackets ≥218 months. Overall sensitivity and specificity for a single RT was 98.2% and 99%, respectively, for Determine, and 85.5% and 99.6%, respectively, for Capillus.In a setting with a maternal HIV prevalence rate of10%, a single negative RT had excellent specificity and two negative RT performed in parallel had a perfect negative predictive value for HIV infection among acutely ill patients18 months of age. In this and similar settings, RT could assist with excluding HIV infection with much lower complexity and cost than NAAT.

Background While child mortality is declining in Africa there has been no evidence of a comparable reduction in neonatal mortality. The quality of inpatient neonatal care is likely a contributing factor but data from resource limited settings are few. The objective of this study was to assess the quality of neonatal care in the district hospitals of the Kilimanjaro region of Tanzania. Methods Clinical records were reviewed for ill or premature neonates admitted to 13 inpatient health facilities in the Kilimanjaro region; staffing and equipment levels were also assessed. Results Among the 82 neonates reviewed, key health information was missing from a substantial proportion of records: on maternal antenatal cards, blood group was recorded for 52 (63.4%) mothers, Rhesus (Rh) factor for 39 (47.6%), VDRL for 59 (71.9%) and HIV status for 77 (93.1%). From neonatal clinical records, heart rate was recorded for3 (3.7%) neonates, respiratory rate in 14, (17.1%) and temperature in 33 (40.2%). None of 13 facilities had a functioning premature unit despite calculated gestational age Conclusion Key aspects of neonatal care were found to be poorly documented or incorrectly implemented in this appraisal of neonatal care in Kilimanjaro. Efforts towards quality assurance and enhanced motivation of staff may improve outcomes for this vulnerable group.

The distribution of T cells using the V beta 2 gene was studied in a group of 99 humans. The distribution of V beta 2+CD4+ levels was bimodal. Twelve individuals had levels of V beta 2+CD4+ less than 2% and 86 others had values greater than 5%. Only one individual had a value between 2% and 5%. The V beta 2 low (mean 1.3 +/- 0.49) and V beta 2 high (mean 8.2 +/- 1.65) phenotypes were stable. The V beta 2 low phenotype is inherited and not limited to HLA or T cell receptor V beta gene complexes. The CD8V beta 2 levels of CD4V beta 2 low individuals are also low. The residual V beta 2+ T cells in V beta 2 low individuals were not anergic to V beta 2-specific stimulation. These data are compatible with the effects of an endogenous superantigen.

Background In malaria endemic areas, individuals are frequently asymptomatic and may be undetected by conventional microscopy or newer, rapid diagnostic tests. Molecular techniques allow a more accurate assessment of this asymptomatic parasite burden, the extent of which is important for malaria control. This study examines the relative prevalence of sub-microscopic level parasite carriage and clonal complexity of infections (multiplicity of infection) over a range of endemicities in a region of north-eastern Tanzania where altitude is an established proxy of malaria transmission. The PCR prevalence was then compared against other measures of transmission intensity collected in the same area. Methods This study used 1,121 blood samples collected from a previously conducted cross-sectional malario-metric survey during the short rainy season in 2001 from 13 villages (three at < 600 m, four at 600-1,200 m and six at > 1,200 m in altitude above sea level). Samples were analysed by PCR for carriage of parasites and multiplicity of infection. These data were compared with other measures of transmission intensity collected from the same area. Results Parasite prevalence was 34.7% by PCR and 13.6% by microscopy; a 2.5-fold difference in line with other recent observations. This fold difference was relatively consistent at the different altitude bands despite a marked decrease in parasite prevalence with altitude: < 600 m 70.9 vs 28.6, 600-1,200 m 35.5 vs 9.9, > 1,200 m 15.8 vs 5.9. The difference between parasite prevalence by PCR was 3.2 in individuals aged between 15 and 45 years (34.5 vs 10.9) compared with 2.5 in those aged 1-5 (34.0 vs 13.5) though this was not statistically significant. Multiplicity of infection (MOI) ranged from 1.2 to 3.7 and was positively associated with parasite prevalence assessed by both PCR and microscopy. There was no association of MOI and age. Village level PCR parasite prevalence was strongly correlated with altitude, sero-conversion rate and predicted entomological inoculation rate. Conclusions Asymptomatic, low density, multi-clone malaria infection was common in this study area. These infections are important as potential contributors to the infectious reservoir of parasites and need to be identified by control programmes especially in this era where malaria elimination is a focus. High throughput standardized PCR approaches are needed to identify individuals who are malaria carriers.

Severe febrile illness is a major cause of adult hospital admission in Africa. Studies of non-malarial fever come largely from children or from high HIV prevalence settings. This prospective study of adult admissions with severe febrile illness in a malaria-endemic area with moderate/low HIV prevalence investigated admission diagnosis as well as final diagnosis based on results of investigations. Severe malaria was the admission diagnosis in 148/198 (74.7%) cases. Plasmodium falciparum was identified in 38/188 (20.2%) admissions and 26/198 (13.1%) were bacteraemic, with 13/25 (52%) prescribed empirical antibiotics. HIV was equally common among those with (16/37; 43.2%) and without P. falciparum (50/138; 36.2%) (p=0.44). In 6/22 (27.3%) deaths, blood cultures were positive for a pathogen, with Streptococcus pneumoniae, Escherichia coli and non-Typhi Salmonella predominating. Chest radiography was suspicious for bacterial/mycobacterial disease in 5/22 additional deaths. Systemic inflammatory response syndrome criteria were more sensitive but less specific than WHO severe malaria criteria for predicting mortality. Malaria is overdiagnosed in adults with severe febrile illness and was not associated with mortality in the absence of co-infection in this high-incidence setting. Adults with severe febrile illness should be tested for malaria and HIV using rapid, sensitive tests. Early antibiotic use should be promoted. Improved diagnostics for invasive bacterial disease are needed.

Background WHO guidelines for the treatment of young children with suspected malaria have recently changed from presumptive treatment to anti-malarial treatment guided by a blood slide or malaria rapid diagnostic test (RDT). However, there is limited evidence of the safety of this policy in routine outpatient settings in Africa. Methods Children 3-59 months of age with a non-severe febrile illness and no obvious cause were enrolled over a period of one year in a malaria endemic area of Tanzania. Treatment was determined by the results of a clinical examination and RDT result, and blood culture and serum lactate were also collected. RDT-negative children were followed up over 14 days. Results Over the course of one year, 965 children were enrolled; 158 (16.4%) were RDT-positive and treated with artemether-lumefantrine and 807 (83.4%) were RDT-negative and treated with non-anti-malarial medicines. Compared with RDT-positives, RDT-negative children were on average younger with a lower axillary temperature and more likely to have a history of cough or difficulty in breathing. Six (0.6%) children became RDT-positive after enrolment, all of whom were PCR-negative for Plasmodium falciparum DNA at enrolment. In addition, 12 (1.2%) children were admitted to hospital, one with possible malaria, none of whom died. A bacterial pathogen was identified in 9/965 (0.9%) children, eight of whom were RDT-negative and one was RDT-positive, but slide-negative. Excluding three children with Salmonella typhi, all of the children with bacteraemia were ≤12 months of age. Compared to double-read research slide results RDTs had a sensitivity of 97.8% (95%CI 96.9-98.7) and specificity of 96.3% (95%CI 96.3-98.4). Conclusions Use of RDTs to direct the use of anti-malarial drugs in young children did not result in any missed diagnoses of malaria although new infections soon after a consultation with a negative RDT result may undermine confidence in results. Invasive bacterial disease is uncommon in children with non-severe illness and most cases occurred in infants with a current fever.

Summary objective To compare the performance of the Paracheck rapid diagnostic test (RDT) withmicroscopy for diagnosing malaria in hospitalised children.methods Children aged between 2 months and 13 years with fever were enrolled in the study over1 year. A standard clinical history and examination were recorded and blood drawn for culture,complete blood count, Paracheck RDT and double-read blood slide.results Of 3639 children enrolled, 2195 (60.3%) were slide positive. The sensitivity and specificityof Paracheck were 97.5% (95% CI 96.9–98.0) and 65.3% (95% CI 63.8–66.9), respectively. There wasan inverse relationship between age-specific prevalence of parasitaemia and Paracheck specificity. Inlogistic regression model, false-positive Paracheck results were significantly associated with pre-admission use of antimalarial drug (OR 1.44, 95% CI 1.16–1.78), absence of current fever (OR 0.64,95% CI 0.52–0.79) and non-typhi Salmonella bacteraemia (OR 3.89. 95% CI 2.27–6.63). In spite ofhigh sensitivity, 56⁄2195 (2.6%) of true infections were Paracheck negative and 8⁄56 (14.3%) were inpatients with >50 000 parasites⁄ll.conclusions Paracheck had poor specificity in diagnosing malaria in severely ill children; this waslikely to be due to HRP2 persistence following recent parasite clearance. The combination of positiveParacheck and negative blood slide results identified a group of children at high risk of non-typhiSalmonella infection. While Paracheck was highly sensitive, some high-density infections were missed.For children with severe febrile illness, at least two reliable negative parasitological test results should beavailable to justify withholding antimalarial treatment; the optimal choice of these has yet to beidentified.keywords malaria, severe, rapid diagnostic test, hospitalIntroductionThe use of rapid immunochromatographic tests for malaria(RDTs) in primary care facilities has been studied over anumber of years, and these tests are now being rolled outon a large scale in Africa as a means to restrict antimalarialdrug use to parasitologically confirmed cases, a policy nowsupported by WHO (WHO 2010). However, the WHOcriteria for malaria diagnosis apply irrespective of severity,and it is therefore surprising that as far as we are aware,only one small study has so far been published on the use ofRDTs in patients admitted to hospital (Birku et al. 1999).The wider use of RDTs in patients hospitalised withsevere febrile illness seems inevitable given the increasingavailability of RDTs. Given current evidence of the lowaccuracy of routinely read slide results, this may representan improvement over current practice (Reyburn et al.2004; Zurovac et al. 2006). However, the performance ofHRP2-based RDTs may differ when used for severely illchildren compared to use in non-severe illness. First,specificity may be reduced by the persistence of HRP2 forup to 5 weeks following clearance of parasites and this

OBJECTIVE: Tanzania has a policy of free provision of inpatient care for young children in order to promote timely access and thus reduce the current levels of mortality. However, little is known about out-of-pocket costs that may be incurred by families in seeking care for sick children. We conducted this study to identify the magnitude of these costs in relation to family income. METHODS: Five hundred and ten caretakers were interviewed on the day of discharge of their child from 11 hospitals in north-east Tanzania. Caretakers were asked to report expenditure related to hospitalization in various categories and family wealth was assessed through reported expenditure in the previous month. RESULTS: Food (mean US$2.2, median US$1.6), transport (mean US$1.7, median US$0) and medicines (mean US$1.0, median US$0.4) were the leading categories of expenditure, and overall the mean out-of-pocket expenditure was US$5.5 (median US$3.7) per admission. Mean out-of-pocket expenditure was more than 1.5 times higher for households in the highest monthly expenditure quintile compared with those in the lowest. However, this differential was reversed when expenditure was considered as a proportion of family expenditure in the previous month; for the lowest quintile, families spent more than three-quarters of their total monthly expenditure on a single paediatric admission. CONCLUSION: Out-of-pocket expenditure on child hospitalization places a considerable burden on poor families. Our findings justify a closer scrutiny of how this expenditure could be reduced, particularly through the provision of adequate food for both children and caretakers and through reducing stock-outs of essential medicines.

OBJECTIVES: To assess the performance of WHO's "Guidelines for care at the first-referral level in developing countries" in an area of intense malaria transmission and identify bacterial infections in children with and without malaria. DESIGN: Prospective study. SETTING: District hospital in Muheza, northeast Tanzania. PARTICIPANTS: Children aged 2 months to 13 years admitted to hospital for febrile illness. MAIN OUTCOME MEASURES: Sensitivity and specificity of WHO guidelines in diagnosing invasive bacterial disease; susceptibility of isolated organisms to recommended antimicrobials. RESULTS: Over one year, 3639 children were enrolled and 184 (5.1%) died; 2195 (60.3%) were blood slide positive for Plasmodium falciparum, 341 (9.4%) had invasive bacterial disease, and 142 (3.9%) were seropositive for HIV. The prevalence of invasive bacterial disease was lower in slide positive children (100/2195, 4.6%) than in slide negative children (241/1444, 16.7%). Non-typhi Salmonella was the most frequently isolated organism (52/100 (52%) of organisms in slide positive children and 108/241 (45%) in slide negative children). Mortality among children with invasive bacterial disease was significantly higher (58/341, 17%) than in children without invasive bacterial disease (126/3298, 3.8%) (P

Background Sero-epidemiological methods are being developed as a tool for rapid assessment of malaria transmission intensity. Simple blood collection methods for use in field settings will make this more feasible. This paper describes validation of such a method, by analysing immunoglobulins from blood retained within immunophoretic rapid diagnostic tests (RDTs) for Plasmodium falciparum. RDTs are now widely used for the diagnosis of malaria and estimation of parasite rates, and this method represents a further use for these devices in malaria control. Methods Immunoglobulins eluted from RDTs, designed to detect parasite histidine rich protein-2 (HRP-2), were analysed by indirect ELISA for IgG recognizing the P. falciparum blood stage antigens merozoite surface protein-119 (MSP-119) and apical membrane antigen-1 (AMA-1). Optimal storage conditions for RDTs were evaluated by comparing antibody responses from RDTs stored in dry or humid conditions at 4°C or at ambient temperature (with or without air-conditioning) for 7, 31 or 70 days. Antibody levels estimated using 3,700 RDT samples from attendees at health facilities in North-eastern Tanzania were compared with contemporaneously collected filter paper blood spots (FPBS) and used to estimate seroconversion rates. Results Storage of RDTs at 4°C was optimal for immunoglobulin recovery but short-term storage at ambient temperatures did not substantially affect anti-malarial IgG levels. Results from RDTs were comparable with those from FPBSs, for both antigens. RDT-generated titres tended to be slightly higher than those generated from FPBSs, possibly due to greater recovery of immunoglobulins from RDTs compared to filter paper. Importantly, however, RDT-based seroconversion rates, and hence serological estimates of malaria transmission intensity, agreed closely with those from FPBSs. Conclusion RDTs represent a practical option for collecting blood for sero-epidemiological surveys, with potential cost and logistical advantages over filter paper and other blood collection methods. RDT-based seroepidemiology can be incorporated into routine monitoring of malaria endemicity, providing information to supplement parasite prevalence rates and generating rapid, robust assessment of malaria transmission intensity at minimal extra cost.

Contains fulltext : 80249.pdf (Publisher’s version ) (Closed access) Disturbances in iron homeostasis are frequently observed in individuals with malaria. To study the effect of malaria and its treatment on iron homeostasis and to provide a mechanistic explanation for observed alterations in iron distribution, we studied the course of the iron regulatory hormone hepcidin in anemic Tanzanian children with febrile Plasmodium falciparum malaria. Before initiation of antimalarial treatment, urinary concentrations of hepcidin were strongly elevated and were associated with iron maldistribution, as was suggested by the presence of hypoferremia and high serum concentrations of ferritin. Antimalarial treatment resulted in a rapid decrease in urinary concentrations of hepcidin and reversal of the hypoferremia. Exploration of regulatory pathways of hepcidin production by analysis of iron, erythropoietic, and inflammatory indices suggested that reduced erythropoietic activity and inflammation stimulated hepcidin production. We conclude that high concentrations of hepcidin explain the observed disturbances in host iron homeostasis associated with malaria and may contribute to malarial anemia and an impaired erythropoietic response to iron supplementation.

Background There is no clear consensus on the most sustainable and effective distribution strategy for insecticide treated bed nets (ITNs). Tanzania has been a leader in social marketing but it is still not clear if this can result in high and equitable levels of coverage. Methods A cluster-randomized survey of ITN and bed net ownership and use was conducted in a rural area exposed to intense Plasmodium falciparum transmission in NE Tanzania where ITN distribution had been subject to routine delivery of national strategies and episodic free distribution through local clinics. Data were collected on household assets to assess equity of ITN coverage and a rapid diagnostic test for malaria (RDT) was performed in all ages. Results Among 598 households in four villages the use of any or insecticidal bed nets in children less than five years of age was 71% and 54% respectively. However there was a 19.8% increase in the number of bed nets per person (p < 0.001) and a 13.4% increase in the number of insecticidal nets per person (p < 0.001) for each quintile increase in household asset score. The odds of being RDT-positive were reduced by more than half in the least poor compared to the poorest households (OR 0.49, 95% CI 0.35–0.70). Poorer households had paid less for their nets and acquired them more recently, particularly from non-commercial sources, and bed nets in the least poor households were less likely to be insecticidal compared to nets in the poorest households (OR 0.44, 95% CI 0.26–0.74). Conclusion Marked inequity persists with the poorest households still experiencing the highest risk of malaria and the lowest ITN coverage. Abolition of this inequity within the foreseeable future is likely to require mass or targeted free distribution, but risks damaging what is otherwise an effective commercial market.

Large-scale studies of genomic variation could assist efforts to eliminate malaria. But there are scientific, ethical and practical challenges to carrying out such studies in developing countries, where the burden of disease is greatest. The Malaria Genomic Epidemiology Network (MalariaGEN) is now working to overcome these obstacles, using a consortial approach that brings together researchers from 21 countries. © 2008 Macmillan Publishers Limited. All rights reserved.

OBJECTIVE: To gain a better understanding of the decision-making context in the diagnosis of malaria in order to inform behaviour change strategies, using quantitative methods. METHODS: We observed hospital outpatient and inpatient consultations in northeast Tanzania where malaria testing was routinely available, recording potential influences on testing and prescribing decisions. We analysed the effects of variables at patient, clinical context and clinician levels on three key decisions in malaria diagnosis and treatment: decision to test for malaria, presumptive treatment and treatment of test-negative patients with antimalarials. RESULTS: Observation of 2082 consultations took place during 120 clinics (different shifts on different days and in different departments) with 34 clinicians. Malaria tests were requested for 16.9% of patients. This decision was driven primarily by clinical symptoms. Of patients not tested for malaria, 36.0% were prescribed antimalarials, this decision being associated with both clinical and non-clinical factors. In outpatients fever was a strong predictor of presumptive treatment [adjusted odds ratio (AOR): 45.9, 95% CI: 30-73], in inpatients this was less so (AOR: 2.7, 95% CI: 0.98-7.7). Outpatient clinicians who were working alone or who had attended

Background Intermittent preventive treatment (IPTp) is efficacious in reducing the adverse outcomes associated with pregnancy-associated malaria, however uptake of the recommended two doses is low in Tanzania, and little is known of the timepoint during pregnancy at which it is delivered. This study investigated the timing of delivery of IPTp to pregnant women attending antenatal clinics (ANC), and the potential determinants of timely uptake. Methods Structured interviews were conducted with staff and pregnant women at antenatal clinics in northeast Tanzania, and antenatal consultations were observed. Facility-based and individual factors were analysed for any correlation with timing of IPTp uptake. Results Almost half the women interviewed first attended ANC during or before the fourth month of gestation, however 86% of these early attendees did not receive IPTp on their first visit. The timing of IPTp delivery complied closely with the national guidelines which stipulate giving the first dose at 20–24 weeks gestation. Uptake of at least one dose of IPTp among women who had reached this gestation age was 67%, although this varied considerably between clinics. At one facility, IPTp was not delivered because SP was out of stock. Conclusion Early uptake of IPTp was found to be hampered by factors external to health worker performance or women's individual preferences. These include insufficient drug stocks and an apparent lack of information to health workers on the reasoning for continued use of SP for IPTp when it has been replaced as a first-line treatment. In addition, an unexpectedly high proportion of women attend antenatal clinics before 20 weeks of pregnancy. While current policy denies the use of IPTp at this time, there is emerging, but incomplete, evidence that malaria in early pregnancy may contribute considerably to the burden of pregnancy-related malaria. Current policy may thus result in a missed opportunity for maximising the benefit of this intervention, and efforts to encourage earlier attendance at ANC alone are unlikely to improve uptake of IPTp. More evidence is needed to weigh the benefits of early IPTp use against theoretical risks of antifolate drugs in early pregnancy.

Background Malaria over-diagnosis in Africa is widespread and costly both financially and in terms of morbidity and mortality from missed diagnoses. An understanding of the reasons behind malaria over-diagnosis is urgently needed to inform strategies for better targeting of antimalarials. Methods In an ethnographic study of clinical practice in two hospitals in Tanzania, 2,082 patient consultations with 34 clinicians were observed over a period of three months at each hospital. All clinicians were also interviewed individually as well as being observed during routine working activities with colleagues. Interviews with five tutors and 10 clinical officer students at a nearby clinical officer training college were subsequently conducted. Results Four, primarily social, spheres of influence on malaria over-diagnosis were identified. Firstly, the influence of initial training within a context where the importance of malaria is strongly promoted. Secondly, the influence of peers, conforming to perceived expectations from colleagues. Thirdly, pressure to conform with perceived patient preferences. Lastly, quality of diagnostic support, involving resource management, motivation and supervision. Rather than following national guidelines for the diagnosis of febrile illness, clinician behaviour appeared to follow 'mindlines': shared rationales constructed from these different spheres of influence. Three mindlines were identified in this setting: malaria is easier to diagnose than alternative diseases; malaria is a more acceptable diagnosis; and missing malaria is indefensible. These mindlines were apparent during the training stages as well as throughout clinical careers. Conclusion Clinicians were found to follow mindlines as well as or rather than guidelines, which incorporated multiple social influences operating in the immediate and the wider context of decision making. Interventions to move mindlines closer to guidelines need to take the variety of social influences into account.

User and provider perceptions of quality of care are likely to affect both use and provision of services. However, little is known about how health workers and mothers perceive the delivery of care in hospital paediatric wards in Africa. Paediatric staff and mothers of paediatric inpatients were interviewed to explore their opinions and experience of the admission process and conditions on the ward. Overcrowding, unsanitary conditions and lack of food were major concerns for mothers on the ward, who were deterred from seeking treatment earlier due to fears that hospital admission posed a significant risk of exposure to infection. While most staff were seen as being sympathetic and supportive to mothers, a minority were reported to be judgemental and authoritarian. Health workers identified lack of trained staff, overwork and low pay as major concerns. Staff shortages, lack of effective training and equipment are established problems but our findings also highlight a need for wards to become more parent-friendly, particularly with regard to food, hygiene and space. Training programmes focused on professional conduct and awareness of the problems that mothers face in seeking and receiving care may result in a more supportive and cooperative attitude between staff and mothers.

OBJECTIVE: To compare rapid diagnostic tests (RDTs) for malaria with routine microscopy in guiding treatment decisions for febrile patients. DESIGN: Randomised trial. SETTING: Outpatient departments in northeast Tanzania at varying levels of malaria transmission. PARTICIPANTS: 2416 patients for whom a malaria test was requested. INTERVENTION: Staff received training on rapid diagnostic tests; patients sent for malaria tests were randomised to rapid diagnostic test or routine microscopy MAIN OUTCOME MEASURE: Proportion of patients with a negative test prescribed an antimalarial drug. RESULTS: Of 7589 outpatient consultations, 2425 (32%) had a malaria test requested. Of 1204 patients randomised to microscopy, 1030 (86%) tested negative for malaria; 523 (51%) of these were treated with an antimalarial drug. Of 1193 patients randomised to rapid diagnostic test, 1005 (84%) tested negative; 540 (54%) of these were treated for malaria (odds ratio 1.13, 95% confidence interval 0.95 to 1.34; P=0.18). Children aged under 5 with negative rapid diagnostic tests were more likely to be prescribed an antimalarial drug than were those with negative slides (P=0.003). Patients with a negative test by any method were more likely to be prescribed an antibiotic (odds ratio 6.42, 4.72 to 8.75; P

Background There is a need for improved targeting of antimalarial treatment if artemisinin combination therapy is to be successfully introduced in Africa. This study aimed to explore why malaria slides are requested and how their results guide treatment decisions in an area of low transmission of P. falciparum. Methods Outpatients attending a district hospital in a highland area of Tanzania were studied over a 3-week period. Clinical and social data were collected from patients who had been prescribed an antimalarial or sent for a malaria slide. Hospital slides were re-read later by research methods. Results Of 1,273 consultations 132(10%) were treated presumptively for malaria and 214(17%) were sent for a malaria slide; only 13(6%) of these were reported positive for P. falciparum but 96(48%) of the 201 slide-negative cases were treated for malaria anyway. In a logistic regression model, adults (OR 3.86, P < 0.01), a history of fever (OR1.72, P = 0.03) and a longer travel time to the clinic (OR 1.77 per hour travelled, P < 0.01) independently predicted the request for a malaria slide. Only a history of a cough predicted (negatively) the prescription of an antimalarial with a negative slide result (OR 0.44, P < 0.01). The sensitivity and specificity of hospital slide results were 50% and 96% respectively. Conclusion Progress in targeting of antimalarials in low malaria transmission settings is likely to depend on consistent use of malaria microscopy and on the willingness of health workers to be guided by negative slide results. Further studies are needed to identify how this can be achieved.

Effective malaria control requires information about intensity of transmission across large areas and populations. Estimates based on entomological factors lack precision and are not cost-effective to obtain. We tested altitude and rainfall measurements as correlates of transmission intensity in different ecological settings. We conducted 2 cross-sectional surveys of approximately 12,000 people (1-45 years old) in 6 altitude transects (150-1800 m) in the Kilimanjaro and Tanga regions of Tanzania. Data were analyzed for associations with altitude and rainfall estimates by use of appropriate regression models. Plasmodium falciparum prevalence showed a negative relationship with altitude (19% and 21% decrease/100-m altitude increase, respectively, in children in Kilimanjaro and Tanga) and rainfall during the 3 months before the survey (46% decrease/100-mm rainfall increase in children in Kilimanjaro). Mean hemoglobin concentrations increased with altitude (0.05 and 0.09 g/dL/100-m altitude increase, respectively, in children in Kilimanjaro and Tanga) and rainfall (0.17 g/dL/100-mm rainfall increase in children and adults in Kilimanjaro). Altitude and rainfall were correlated with parasite prevalence and mean hemoglobin concentration; however, the relationship varied according to ecological setting. Climatological variables alone cannot predict malarial outcomes. Local variations in seasonality of malaria transmission--together with vector species composition, topography, host and parasite genetics, and socioeconomic factors--may influence malaria prevalence.