Laboratories of Tropical Diseases
1 University of New Mexico
Albuquerque, NM 87131
Phone: (505) 272-8441
1 University of New Mexico
Albuquerque, NM 87131
Phone: (505) 272-8441
Human malaria, transmitted by female Anophelesmosquitoes, is a protozoan disease typically caused by one of four members of the genus Plasmodium: P. falciparum, P. vivax, P. ovale, and P. malariae. Malaria is one of the leading causes of morbidity and mortality of infectious disease origin throughout the world, resulting in 300-500 million clinical cases per annum and approximately 2 million deaths. Over 90% of the malaria cases occur in sub-Saharan Africa, with immune-naive individuals such as children less than five years of age bearing the highest disease burden.
The majority of malaria-related morbidity and mortality in African children is due to infection with P. falciparum. The severe clinical manifestations of falciparum malaria are defined by one or more of the following: hypoglycemia, hyperparasitemia, cerebral malaria, malarial anemia, and respiratory distress. Among these disease sequelae, malarial anemia is responsible for the greatest amount of malaria-related morbidity and mortality worldwide. When hemoglobin levels become profoundly reduced, life-threatening severe malarial anemia can result. Although several definitions of severe malarial anemia exist, the World Health Organization defines severe malarial anemia as hemoglobin levels less than or equal to 5.0 g/dL in the presence of a peripheral parasitemia that is 10,0000 (/μL) or greater.
Since anemia in African children is often a multi-factorial disease due to the presence of a number of anemia-provoking factors, such as nutritional deficits and co-infection with other pathogens (e.g., hookworm, bacteremia, and HIV), the etiologic basis of malarial anemia is complex. As such, when investigating malarial anemia it is important to carefully define the diverse factors that can contribute to reduced hemoglobin levels. We are currently performing comprehensive hematological investigations in infants and young children with malaria in western Kenya. In addition, we are also examining the presence and severity of malarial anemia in children with HIV/AIDS.
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Perhaps one of the most intriguing aspects of falciparum malaria is the diverse clinical presentations of severe disease. In areas of holoendemic P. falciparum transmission, the clinical presentation of severe malaria is predominantly severe malarial anemia. This condition most commonly occurs in infants and young children. The prevalence of cerebral malaria in areas with high rates of falciparum malaria is typically very low. Conversely, in areas with seasonal and/or low levels of P. falciparumtransmission, severe malaria can present as a combination of clinical features, including malarial anemia, cerebral malaria, and respiratory distress.
Because bacteremia (blood borne bacterial infection) is common in sub-Saharan Africa, it represents an important source of morbidity and mortality in infants and children. In western Kenya, there is a high rate of co-infection with bacteremia and malaria that influences the clinical outcomes of these common diseases. Since there was previously no ability to perform blood culture in children at SDH, we established a diagnostic microbiology laboratory to isolate and identify common pathogenic bacteria, and to determine the most suitable antimicrobial agents for treatment of such infections. Our goal is to provide laboratory support services to the pediatric population and to train individuals from western Kenya on the appropriate diagnosis and treatment of microbial pathogens.
The isolation of pathogenic bacteria is currently being performed on small quantities of blood drawn from infants and children using Pediatric Isolator Tubes (Wampole, Inc.). Routine analysis of blood specimens from children is being performed to identify known and novel pathogenic bacteria that cause bacteremia in African children.
We have developed an identification and characterization scheme for pathogenic bacteria that aids in clinical management of the children and also allows for sustainability in resource-limited settings. The scheme for identification and characterization of bacteria includes, Gram staining, differentiation of various bacterial spp. based on colony and growth characteristics, and the use of rapid biochemical tests (e.g. API 20e, oxidase, catalase, indole, etc.).
Since the ultimate goal of any investigative process is to improve the clinical care of patients, we established specific protocols for susceptibility testing of various bacterial isolates with commonly available antimicrobial agents. We are currently performing antimicrobial susceptibility testing using the Disk Diffusion Method.
Malaria continues to represent a global health concern as evidenced by the 300-500 million infections each year that result in 1.5-2.7 million deaths, with greater than 90% of the severe complications occurring in children less than 5 years of age. In areas of intense Plasmodium falciparum exposure, such as western Kenya, the primary clinical manifestation of severe malaria occurs in children less than two years of age and presents as severe malarial anemia (SMA). Since there is presently no effective vaccine for malaria, development of a vaccine that protects against SMA is an important step in preventing the vast morbidity and mortality associated with malaria. Development and testing of a malaria vaccine would be dramatically facilitated by several essential intermediate steps including: 1) identifying soluble mediators such as, cytokines and effector molecules that are responsible for promoting SMA; and 2) determining the critical genes and their patterns of expression that underlie disease susceptibility to SMA. Accomplishment of these tasks that take into account the complex genetic, inflammatory, and clinical factors associated with SMA could be used to predict disease outcomes. Our ongoing studies in western Kenya are focused on defining the genetic factors that condition the outcomes and severity of malarial anemia.
Since P. falciparum is an intracellular pathogen, cell-mediated immunity is important for providing protection against severe disease. Our research, therefore, focuses on cytokines, chemokines, and effector molecules that regulate cell-mediated immunity. We aim to determine how dysregulation of these inflammatory molecules promote SMA in children with falciparum malaria. To accomplish this task, we are currently determining how variation in innate immune response genes condition clinical outcomes of childhood malaria. Selection of candidate innate immune response genes for our genetic investigation are based on several parameters including: 1) targeting of genes that we and others have previously shown to be important regulators of cell-mediated immunity in children with severe malaria; 2) targeting of genes that are thought to be crucial mechanistic determinants of anemia in a variety of acute and chronic inflammatory diseases; and 3) targeting of polymorphic variability that we and others have previously identified as important functional variants for altering gene expression profiles that can potentially impact on clinical outcomes of malaria.
The majority of our genetic-based investigations are examining the role of single-nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs) in altering gene expression and ultimately, the manifestation of severe malarial anemia. SNPs are sequence variations that occur in DNA when a single nucleotide (A,T,C,or G) in the DNA sequence is replaced by a different nucleotide. In the three-billion-base human genome, SNPs occur every 100 to 300 bases, and represent approximately 90% of the genetic variation in the human genome. Although many SNPs in the genome have no effect on gene function, a number of SNPs have been found to have important determinants of disease susceptibility and responses to pharmacological interventions. SNPs can occur in both coding and noncoding regions of the genome with those in the coding region typically having the greatest impact on gene function (more information at the Human Genome Program
A VNTR is a location in a genome where there is a short nucleotide sequence that is organized into a tandem repeat. The number of repeats within the genetic sequence often differ between individuals, particularly in innate immune response genes, and can influence gene expression. By examining patterns of VNTRs, we aim to identify important sequence variation that alters susceptibility to SMA.
Although most of our previous genetic investigations focused on individuals SNPs and VNTRs, our current research is based on the the role of haplotypes in conditioning clinical outcomes of childhood malaria.
Although some of the earlier research findings suggested that the interaction between malaria and HIV was minimal, more recent findings from our laboratories and others, illustrate that malaria and HIV co-infection exacerbates both diseases. Upon implementation of the project in western Kenya (2002), it became apparent that there was a high prevalence of HIV/AIDS in the pediatric population. This, along with the fact that the hallmark clinical sign of pediatric HIV is anemia, prompted our laboratories to investigate the important influence of HIV as a co-factor in the etiology of childhood anemia. Malaria and HIV co-infection investigations in western Kenya have offered important information about the interactions between these two pathogens. These studies are also demonstrating that interactions between these two pathogens have a much greater impact on severe malarial anemia than was originally recognized in pediatric populations (please refer to our publication list in the website).
Identification of HIV in infants and children differs from that in adults (particularly in children less than 18 months), due to the transfer of maternal HIV antibodies to the child from those mothers that are infected with HIV. To diagnose HIV in the pediatric population in western Kenya, we perform HIV-1 serology tests to identify those children that had in utero exposure to HIV. Those children that have either one or both of the serological tests showing a positive result are then diagnosed with qualitative HIV-1 DNA-PCR.
We have validated the use of two rapid serological tests for HIV-1 diagnosis commonly used in adults. The strategy involves parallel testing of whole blood specimens with Determine™ HIV-1/2 ( Abbott Laboratories, Dainabot Co. Japan) and Uni-Gold™ (Trinity Biotech, Inc, USA). These assays have been evaluated and found to be highly specific and sensitive for HIV-1 variants predominant in Kenyan pediatric population.
In order to diagnose HIV-1 infection in children, we have optimized detection of HIV-1 proviral DNA in whole blood specimens collected on specialized collection filter papers (Dried Blood Spots). To identify HIV-1 viral particles, DNA is extracted from dried blood spots, followed by PCR amplification and detection. HIV-1 DNA PCR is performed at birth, and at the first and third month of postnatal life. A child with a positive DNA test at two consecutive time points is considered to be definitively infected with HIV-1 in utero or intrapartum. Additional PCR analyses are performed annually until two years of age to assess the proportion of transmission of HIV-1 through breast feeding.
As part of our investigations on the immunologic basis of severe malarial anemia, the laboratories examine phagocytosis of hemozoin (malarial pigment) by peripheral blood mononuclear cells (PBMCs) as an in vitromodel of host-parasite interactions. Hemozoin is an insoluble aggregated polymer of heme subunits formed during the erythrocytic stage of malaria when parasites digest host red blood cell (RBC) hemoglobin. Parasites obtain essential amino acids from the globin portion of hemoglobin, however, the heme subunit is highly toxic to the parasites. To avoid these toxic effects, parasites polymerize heme into a non-toxic insoluble compound, hemozoin (1). Upon rupture of infected erythrocytes, hemozoin is released into the blood stream and avidly phagocytosed by circulating monocytes and tissue macrophages (2). Hemozoin is also acquired by monocytes/macrophages through phagocytosis of parasitized erythrocytes (3). Experiments in cultured blood mononuclear cells illustrate that hemozoin augments the release of both pro-inflammatory cytokines such as TNF-α (4), and anti-inflammatory cytokines such as IL-10 (5). Additional effects of hemozoin on blood mononuclear cells include; inhibition of oxidative burst and protein kinase C activity (6), reduction of microbicidal and anti-tumor capabilities (7), impaired expression of MHC class II and CD54 expression in monocytes (2), and an inability of monocytes to undergo repeated phagocytosis (8).
Our initial investigations on this in vitro model were focused on the altered regulation of effector molecules in response to PBMC phagocytosis of hemozoin. These studies illustrated that hemozoin suppresses cyclooxygenase (COX)-2-derived prostaglandin (PG) E2 and augments NOS2-dependent NO production in cultured human PBMCs from malaria-naïve donors (9, 10). As part of this work, we have shown that hemozoin-induced suppression of COX-2-derived PGE2 production allows over-expression of tumor necrosis factor (TNF)-α (11). Studies in non-human primate models of malaria illustrated that hemozoin stage-specifically augments TNF-α production in cultured PBMCs from SIV-infected rhesus macaques, and that hemozoin-induced TNF-α levels increased SIV viral replication (12). Furthermore, hemozoin augments interleukin (IL)-10 production, which is responsible for decreased levels of IL-12 (13). Our more recent investigations have shown that hemozoin elicits dysregulation of β-chemokines [i.e., monocyte inhibitory protein (MIP)-1α, MIP-1β and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES)] in cultured PBMCs (14). Ongoing activities in this area of research involve further examination of the regulation and interactions of innate inflammatory molecules (i.e., cytokines, chemokines, and effector molecules) in response to hemozoin. We are also determining the effects of hemozoin on cultured CD34+ stem cell proliferation and differentiation.
Since there is presently no effective vaccine for malaria, development of a vaccine that protects against the development of malaria is an important step in preventing the vast morbidity and mortality associated with the disease.
A comprehensive understanding of malaria that takes into account the complex genetic, inflammatory, and clinical factors associated with malaria could be used to predict disease outcomes. This information would be extremely valuable for designing novel vaccines, evaluating the immune response in vaccine trials, and identifying at risk groups that would be the most appropriate target for vaccine intervention. The primary goal of our studies in western Kenya is to gain a better understanding of the genetic factors that condition the diverse outcomes and severity of malarial anemia.
While much effort has been devoted to the development of malaria vaccines, to date, no effective vaccine(s) have been developed that provide long-lived protection against malaria. Information now available from recent success in sequencing the human genome, Anopheles gambiae, and P. falciparum may facilitate malaria vaccine efforts.
The primary treatment interventions for the management of severe malarial anemia are the use of antimalarial drugs, transfusion, and fluid replacement. The alarming rate of antimalarial drug resistance and the lack of rapid development of new antimalarial drugs are providing challenging treatment problems throughout much of sub-Saharan Africa. An additional challenge for treatment of malarial anemia is the frequent lack of an adequate blood supply for transfusion. This problem has been dramatically exacerbated by the high rates of HIV in many of the malaria endemic areas.