acase@tulane.edu / The Zambian Ministry of Health (MOH) National Malaria Control center (NMCC) adopted artemisinin combination therapy (ACT) as a first-line antimalarial drug for uncomplicated malaria in 2003, and included rapid diagnostic testing (RDT) in its case management guidelines to reduce over-diagnosis of malaria and over-prescription of antimalarials. Prior research has highlighted gaps in the malaria case management process in Zambia, especially in diagnosis and treatment. The first paper of this study aimed to build quality indices or indicators for the four components of malaria case management: assessment, diagnosis, treatment and counseling. The Zambia MOH/NMCC conducted a nationally representative health facility survey in 2011 with the Malaria Control and Evaluation Partnership in Africa. The mean assessment quality (percentage of assessment items correctly completed) rate was 49.9%. The diagnostic quality (concordance with gold standard diagnosis) rate was 82.4%, with 86.9% sensitivity and 79.4% specificity. The treatment quality rate (correct treatment for those needing antimalarials and no treatment for patients not needing it) was 89.6%, and the mean counseling quality (percentage of counseling items correctly completed) rate was 48.6%. The second paper investigated factors association with each of the four components of malaria case management. Supervision was significantly associated with assessment and counseling but not diagnosis and treatment. Health facility managing authority was associated with assessment and diagnosis. Availability of blood tests was associated with correct diagnosis, and diagnosis was strongly associated with treatment. Malaria endemicity and availability of IMCI guidelines were associated with counseling quality. The third paper investigated the associated between counseling and patient recall of treatment regimen, and found that they were associated as hypothesized. The Zambia NMCC has improved the quality of malaria case management over previous years, although it is recommended that more health facility surveys are conducted in order to study the change in health worker performance over time. / 1 / Louie Rosencrans

Abstract not available

El objetivo del presente trabajo fue identificar antígenos relevantes de valor diagnóstico de aislados de P. vivax y P. falciparum provenientes del departamento de Loreto, mediante la técnica de inmunoblot. Se seleccionaron pacientes entre 3 y 64 años con diagnóstico de malaria, gota gruesa positiva, procedentes de centros de salud en el departamento de Loreto. Fueron analizadas 4 mezclas de antígenos, una de P. falciparum (PF1) y tres de P. vivax (PV1, PV4 y PV5), preparadas a partir de 36 muestras de pacientes con alta parasitemia por P. vivax (2 700 – 69 000 parásitos/μL) y P. falciparum (2 750 – 10 000 parásitos/μL). Las mezclas de antígenos fueron enfrentadas a 39 sueros (12 de P. falciparum y 27 de P. vivax) mediante ensayos de inmunoblot.

Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

Opportunistic feeding behaviour and partial exophily make An. arabiensis much more difficult to control by indoor residual spraying than any other vector in the Afro-tropical region. The persistent malaria outbreaks in Zimbabwe despite decades of indoor house spraying prompted this investigation into the role of An. arabiensis in malaria transmission and assessment of the possible impact of this control measure. The study was conducted in the malaria endemic districts of Binga and Gokwe. An. gambiae complex mosquitoes were collected from artificial outdoor resting sites, and from human dwellings by i) daytime hut searches, ii) pyrethrum spray catches and iii) exit window traps. Mosquito components were processed to enable: i) the distinction of An. arabiensis from An. quadriannulatus and An. merus on the basis of the pale band at the junction of the hind leg 3/4 tarsomeres; ii) species identification and scoring of inversion polymorphism on the basis of the X chromosome and autosomes respectively; iii) the determination of blood meal sources using the Ouchterlony precipitin test; and iv) identification of An. gambiae s.l. using polymerase chain reaction (PCR) and enzyme electrophoresis techniques. Entomological assessment of residual spraying included determining: the vector resting densities indoors and outdoors, bioassay and insecticides susceptibility tests. Data were also collected on hut profiles, knowledge-attitudes-practices surveys, and household malaria prevalence . surveys. An. arabiensis and An. quadriannulatus were found in sympatry in Binga and Gokwe, and in addition, An. merus was found in Gokwe. Most species identifications were made using PCR; which was found to have 7.5% and 41.6% levels of error for An. arabiensis and An. quadriannulatus respectively, using the cytogenetic technique as benchmark. The pale band technique yielded > 80% correct identification for An. arabiensis but the extent of overlap in the pale band lengths between An. arabiensis and An. quadriannulatus renders the method unsuitable for distinguishing these two species. Inversions 2Rb and 3Ra were found floating in An. arabiensis, with 60% frequency in the former. The Wright's F statistic value of -0.0416 indicated an excess of heterozygotes, and a state of panmixis in the vector population. No significant differences were observed between 2Rb karyotypes in host choice. Human blood indices among indoor (0.82), exit trap (0.98) and outdoor resting (0.30) specimens suggested exophilic behaviour. This was corroborated by the high fed:gravid ratios of 6.8: 1 and 11.6: 1 in sprayed and non-sprayed dwellings respectively. This was worsened by a high feeder-survivor index (FSI) of 93 % among exit trap specimens. The susceptibility to deltamethrin coupled with residual efficacy nine weeks post-spray indicated the suitability of the insecticide. Rural dwellings were suitably built for spraying but had no mosquito proofing. Personal protective measures are hardly known; sleeping outdoors occurs in Siabuwa. While An. arabiensis bites humans indoors the partial exophily it exhibits is a threat to indoor residual insecticide spraying. An integrated malaria control approach is recommended. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Plasmodium vivax agente etiológico de la malaria, exhibe una gran variabilidad genética durante episodios recurrentes de la enfermedad. Esta recurrencia es informada como de baja prevalencia asociada con la malaria asintomática. Así mismo los episodios recurrentes (reinfecciones o relapsos) a menudo pueden ser confundidos por resistencia a fármacos como la cloroquina. Por lo tanto el objetivo principal de este estudio fue relacionar los patrones de recurrencia y la resistencia con la variabilidad genética de P. vivax. En este estudio se evaluaron las muestras secuenciales de individuos provenientes de una región endémica del Perú (Mazán-Iquitos), diagnosticados previamente con malaria, por microscopía, durante seguimientos activos y sometidos a un régimen de tratamiento estándar con cloroquina. La genotipificación realizada en base al gen pvmsp3-α, utilizando el Nested PCR y la digestión enzimática, permitió identificar una alta variabilidad genética de P. vivax, a partir de la cual, se identificaron los patrones de recurrencia, establecidos como relapsos, a partir de estadios latentes o hipnozoitos homólogos (con haplotipos idénticos) y reinfecciones (con haplotipos diferentes). Los rangos de tiempo permitieron una identificación más precisa, observándose mayores frecuencias de relapsos por hipnozoitos homólogos antes de los 90 días post-primera evaluación y mayores frecuencias de reinfecciones después de este periodo. Así mismo las recurrencias en el primer periodo de tiempo, por haplotipos diferentes, pueden deberse también a hipnozoitos heterólogos. Complementando el estudio, el análisis de secuenciamiento del gen pvmdr1, permitió identificar SNPs, codificantes de mutaciones no sinónimas, relacionadas con resistencia a cloroquina. Estos SNPs, a través del software U-Melt (análisis in sílico), presentaron variaciones en las temperaturas de fusión. Finalmente los resultados de cuantificación relativa con qPCR Real Time no mostraron diferencias significativas en el número de copias del gen pvmdr1. Palabras clave: Cloroquina, Genotipificación, Haplotipos, Hipnozoito, Malaria Asintomática, Recurrencia, Variabilidad. / --- Plasmodium vivax etiologic agent of malaria has a large genetic variability during recurrent episodes of the disease. This recurrence is reported as low prevalence associated with asymptomatic malaria. Also recurrent episodes (reinfection or relapse) can often be mistaken for drug resistance as chloroquine. Therefore the main objective of this study was to correlate the patterns of recurrence and resistance to the genetic variability of P. vivax. In this study, we evaluated the sequential samples of individuals from an endemic region of Peru (Mazán-Iquitos), previously diagnosed with malaria microscopy during active follows and subjected to a standard treatment regimen with chloroquine. Genotyping based on the pvmsp3-α gene, using Nested PCR and enzymatic digestion, identified high genetic variability of P. vivax, from which were identified recurrence patterns established as relapse, from latent stages or homologous hypnozoites (with identical haplotypes) and reinfections (with different haplotypes). The time ranges allow more accurate identification, with higher frequency of relapses by homologous hypnozoites before 90 days post-first evaluation and higher frequencies of reinfection after this period. Also recurrences in the first period of time, for different haplotypes may also be due to heterologous hypnozoites. Complementing the study, the sequencing analysis of the gene pvmdr1, identified SNPs, encoding nonsynonymous mutations related to resistance to chloroquine. These SNPs, through U-Melt software (in sílico analysis), showed variations in the melting temperatures. Finally the results of relative cuantification with Real Time qPCR no showed significant differences in copy number of the pvmdr1 gene. Keywords: Chloroquine, Genotyping, Haplotypes, Hipnozoite, Recurrence, Asymptomatic Malaria, Variability.

Malaria disease poses a serious global health burden as recent reports have indicated that nearly half of the world’s population is at risk (WHO 2008). The World Health Organization (WHO) Expert Consultative Team has reported that 90% of all malaria deaths occur in Sub Saharan Africa. (WHO, 2008). Despite the numerous global efforts to control and manage the disease, through various ways, use of chemotherapeutic agents continues to be the major intervention strategy in the control of malaria. The WHO recommended use of Artermisinin combination therapy (ACT) has been hampered by an imbalance between demand and supply in the poor socioeconomically challenged rural populations of Sub Saharan Africa, the epicenter of malaria infection. Chloroquine (CHQ), therefore, continues to be used in most malaria endemic areas in developing countries despite development of P. falciparum resistance to the drug (WHO, 2006). Oral administration is the major delivery route for CHQ. However, CHQ is a bitter drug, with an inconvenient dosing schedule leading to incomplete courses of therapy by most malaria patients. Oral CHQ administration is also associated with adverse effects in various organ systems resulting from deposition of CHQ in these organs to elicit impairment of glucose homeostasis, renal and cardiovascular function. Alternative methods of CHQ administration such as transdermal delivery have, therefore, been suggested, in an effort not only to avoid the bitter taste, but also to modify the dosing schedule, which may improve patient comfort and compliance. Transdermal delivery of CHQ via an amidated matrix patch, which is envisaged to ensure a slow, controlled and sustained release of therapeutic concentrations of CHQ, may circumvent the previously reported adverse effects of oral CHQ. It is against this background that the current study compared the effects of transdermal CHQ patch and oral chloroquine in the management of malaria as assessed by the ability to clear parasites of P. berghei infected rats. The other aims were to investigate and distinguish between the patho physiological effects of malaria and CHQ treatments on blood glucose and plasma insulin concentration, renal and cardiovascular function in male Sprague-Dawley rats. To investigate and distinguish between the pathophysiological effects malaria infection and CHQ treatments on blood glucose homeostasis, renal and cardiovascular function markers, separate groups of non infected and P. berghei infected male Sprague Dawley rats (90g-150g) were used. The animals were treated twice daily with oral CHQ (60mg/kg) and a once off transdermal delivery of CHQ via topical application of pectin CHQ matrix patch (53mg/kg) in a 21 day study divided into pre treatment, (days 0-7) treatment (days 8-12) and post treatment (days 13-21) periods. The animals were housed individually in metabolic cages for the duration of the study. Treatment was for 5 consecutive days. Measurements of body weight, food and water intake, mean arterial pressure (MAP), blood glucose concentration, % parasitaemia, haematocrit, and 24 hour urine volume, Na+, K+, urea and creatinine outputs were done every day during the treatment period, and every third day during the pre and post treatment periods. Separate groups of non fasted conscious animals (n=6) were sacrificed on days 0, 7, 8, 9, 10, 12, 14 and 21, at 24 hours after the last treatment for oral CHQ administration and after a once off patch application on the first day of treatment. The plasma obtained was assayed for plasma insulin, lipid profile parameters and plasma Na+, K+, urea and creatinine. The harvested liver and gastrocnemius muscle were used for determination of glycogen concentration. The current study has demonstrated the sustained controlled release of CHQ from the pectin matrix patch, demonstrating the therapeutic ability to clear P. berghei malaria parasites from systemic circulation. Malaria infection and oral CHQ treatment exhibited blood glucose lowering effects which were circumvented by topical application of the pectin CHQ matrix patch. Oral CHQ elevated hepatic glycogen concentration through mechanisms that are still to be elucidated. Topical application of CHQ via pectin matrix patch did not alter hepatic and gastrocnemius muscle glycogen concentrations. Malaria infection and oral CHQ delivery reduced food intake, water intake and % body weight changes of the animals as well as inducing natriuresis, reduced urine output and increased urinary creatinine outputs. Malaria infection was also shown to elicit hyperkalaemia and kaliuresis in experimental animals. Hypotensive effects of malaria infection and oral CHQ delivery were also demonstrated in the current study. Malaria infection and oral CHQ delivery elevated plasma total cholesterol and LDL-c as well as reduction in the cardio protective particle, plasma HDL-c, concentrations. Topically delivered CHQ via pectin CHQ matrix patch did not evoke any such alterations, suggestive of its ability to circumvent the observed adverse effects of oral CHQ delivery due to sustained, controlled release of therapeutic concentrations of CHQ from the transdermal formulation. To the best of our knowledge, the results of the present study provides the first evidence of the release of therapeautic CHQ concentrations from pectin CHQ matrix patch that cleared the malaria parasites from systemic circulation as well as demonstrating the ability of the transdermal formulation to circumvent the adverse effects of oral CHQ delivery in glucose homeostasis, renal and cardiovascular function markers. This is clinically relevant as it provides a feasible and novel alternative method of CHQ delivery that could play a major role in the effective management of malaria. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Malaria is the one of most important diseases in the world, especially in sub-Sahara Africa. This dissertation outlines the enormous burden of the disease in terms of social and economic costs in southern Africa. This dissertation assessed the range and quality of the cost-effectiveness of malaria prevention in sub-Sahara Africa. Six studies published from 1999 to 2003 are reviewed, covering insecticide treated nets, residual spraying, chemoprophylaxis for infants and environmental management. For infants, ITNs cost from US$ 2019 - $2879 per death averted and cost $ III per DALY; chemoprophylaxis cost $ 4.1 per DALY and chemoprophylaxis plus iron cost $ 5.0 per DALY. For children, ITNs cost $ 1559 per death averted, $ 57 per DALY and $ 61 per sick child averted. For non-specific age group, ITNs cost $ 29 per infection averted, and RHS $ 9. Generally all interventions assessed are cost effective use of resources. The chemoprophylaxis is the least expensive malaria prevention among cost effective malaria prevention interventions, followed by residual spraying one round a year, residual spraying two rounds a year, insecticide treated nets with net treatment only and insecticide treated nets with net provision and treatment. There are operational, managerial and financial challenges faced these most cost-effective malaria interventions. Particularly, chemoprophylaxis is faced the tremendous drug resistance potential and is not being recommended to wide use; financial constraints and the potential delaying of children's immunity acquisition lowers the cost-effectiveness of insecticide treated nets; residual spraying is a relatively simpler, faster and cheaper method, but faces political and economic pressure of concerning environmental issues, especially the use of DDT. The integrated approach of environmental management plus residual spraying could be the most cost-effective method of malaria prevention with least adverse environment effects. However, policy makers should apply their knowledge to local conditions. Further, comprehensive education programmes are needed to gain support and understanding from local communities. This would raise the cost-effectiveness of all interventions. / Thesis (M.B.A.)-University of Natal, Durban, 2003.

Malaria is a life-threatening parasitic disease endemic throughout the world. Control methods for malaria are becoming less reliable; thus, efforts to develop a safe and effective vaccine are critical. Immunity to malaria requires both cell- and humoral-mediated immunity, CMI and HMI, respectively. CD4+ T cells play a central role in protection against blood stage Plasmodium infection. Given that clinical features of malaria are caused by blood stages, a vaccine against this stage will be very effective in reducing morbidity and mortality. During the blood stage, purine nucleotides, which are essential for parasites’ survival and proliferation, are in high demand. The inability of the parasite to engage in de novo synthesis of purine nucleotides makes the enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) an essential nutrient salvage enzyme. HGXPRT is located in electron-dense regions in merozoites and in vesicles in the red cell cytoplasm. In contrast to other blood stage antigens, those located on the merozoite surface are targets of HMI. To advance HGXPRT as a malaria vaccine candidate, fermentation and purification of the protein from Plasmodium falciparum (PfHGXPRT) was performed using facilities at Q-Gen, the Queensland Institute of Medical Research (QIMR). Escherichia coli carrying PfHGXPRT gene were a gift in-kind from the University of Queensland (UQ). Recombinant PfHGXPRT expressed in E.coli was purified using anion exchange liquid chromatography and gel filtration techniques. Three methods were used to confirm the Q-Gen PfHGXPRT identity: (1) Western blotting showing identical bands of UQ PfHGXPRT and Q-Gen PfHGXPRT at 26 kDa; (2) N terminal sequencing was compatible with the PfHGXPRT sequence; and (3) mass spectrometry showed homogeneity by giving a subunit molecular mass of 26,231 Da. The purification method used is reproducible and affordable, yielding reasonably pure protein for animal experimentation. Following purification of PfHGXPRT, its efficacy as a subunit vaccine candidate in a rodent model of infection was examined. Multiple rodent models of malaria infection were assessed and it was determined that Plasmodium chabaudi AS (P. chabaudi AS) exhibited the highest cross-reactivity against PfHGXPRT in mice. Hence, P. chabaudi AS was chosen as the appropriate rodent model for study in this thesis. Natural immunity against PfHGXPRT during a blood stage P. chabaudi AS infection was assessed by testing sera and splenocyte responses to PfHGXPRT. IFN- and IL-4, as well as antibodies specific for PfHGXPRT, could be detected after infection, suggesting that PfHGXPRT is a target of natural immunity during the blood stage infection. Therefore, further studies of protective immunity generated by immunisation with PfHGXPRT were conducted, specifically to determine their protective efficacy and to determine immune mechanisms elicited by immunisation. Mice immunised with PfHGXPRT and challenged with P. chabaudi AS developed a slightly reduced parasitaemia. T-cell proliferation, but not antibody responses, was detected after immunisation. Protective mechanism(s) were assessed by adoptively transferring immune CD4+ T cells, B cells or sera to naïve SCID mice followed by parasite challenge. Only recipients of immune CD4+ T cells showed extended survival. Nevertheless, immunisation with PfHGXPRT followed by sub-patent infection induced better protection than immunisation with PfHGXPRT alone, which appeared to be related to CD4+ T cells. Reduction of parasitaemia, as well as augmentation of T cell proliferation and IFN-γ production, was evident in PfHGXPRT and sub-patent infected immunised mice. Recipients of CD4+ T cells from PfHGXPRT and sub-patent infection immune mice also showed some degree of protective immunity. PfHGXPRT was shown to induce natural and acquired immunity to P. chabaudi AS. HGXPRT is highly conserved in parasites and humans; therefore, it is essential to define minimal protective epitopes that could be included in a vaccine. Hence, 22 overlapping peptides (termed P1 P22) corresponding to the entire P. chabaudi AS HGPRT sequence were used to define minimal protective epitopes. Following immunisation of mice with seven pools of peptides (P1 P3, P4 P6, P7 P9, P10 P12, P13 P15, P16 P18 and P19 P22), three immunogenic peptides (P11, P13, and P17), which stimulated significant proliferative and IFN-γ responses were chosen for immunisation studies. Peptide P9 (position 76-95 from N-terminal), which induced the highest IFN- levels during P. chabaudi AS infection was also included in the pool of peptides. Mice immunised with P9, P11, P13 and P17 had significantly decreased parasitaemia. Antibody mediated immunity had a partial effect on suppressing parasite growth. CMI, on the contrary, played a central role in adoptively transferred protection by significantly reducing parasitaemia and prolonging survival of recipient SCID mice. Strong T cell proliferation and IFN- secretion were also detected after stimulation of splenocytes from immune mice with P. chabaudi AS antigen. CMI response was significantly increased after immunisation with the peptides followed by sub-patent infection. The findings that four short epitopes of HG(X)PRT confer strong CMI protection suggest that homologues of such epitopes could be included in a multi-component malaria vaccine.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 5 uppsatser.