The Risk of and Associated Demographic and Laboratory Variables for Amputations for Inpatients with Diabetic Foot Ulcers.

OBJECTIVE: Diabetic foot ulcers (DFUs) are a leading cause of morbidity and mortality, which disproportionately impacts underserved populations. This study aimed to provide data regarding the rates and outcomes of amputation in patients admitted with DFU in our health system, which cares for an ethnically diverse and underserved population. METHODS: This retrospective study examined the electronic medical records of adult patients hospitalized with DFU at 3 hospitals in our health system between June 1, 2016, and May 31, 2021. RESULTS: Among 650 patients admitted with DFU, 88% self-identified as non-White race. Male sex (odds ratio [OR], 0.62), low body mass index (OR, 0.98), and history of smoking (OR, 1.45) were significantly associated with amputation during the study period. A higher erythrocyte sedimentation rate (OR, 1.01), C-reactive protein level (OR, 1.05), and white blood cell count (OR, 1.11) and low albumin level (OR, 0.41) were found to be significantly associated with amputation versus no amputation during admission. The amputation risk during the index admission for DFU was 44%. CONCLUSION: Our study identified a high DFU-related amputation risk (44%) among adult patients who were mostly Black and/or Hispanic. The significant risk factors associated with DFU amputation included male sex, low body mass index, smoking, and high levels inflammation or low levels of albumin during admission. Many of these patients required multidisciplinary care and intravenous antibiotic therapy, necessitating a longer length of stay and high readmission rate.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Pipecolic Acid, a Putative Mediator of the Encephalopathy of Cerebral Malaria and the Experimental Model of Cerebral Malaria.

BACKGROUND: We explored a metabolic etiology of cerebral malaria (CM) coma. METHODS: Plasma metabolites were compared between Malawian children with CM and mild Plasmodium falciparum malaria. A candidate molecule was further studied in animal models of malaria. RESULTS: Clinically abnormal concentrations of pipecolic acid (PA) were present in CM plasma, and nearly normal in mild malaria samples. PA is renally cleared and the elevated PA blood levels were associated with renal insufficiency, which was present only in CM subjects. Prior studies demonstrate that PA has neuromodulatory effects and is generated by malaria parasites. PA brain levels in Plasmodium berghei ANKA-infected animals in the experimental cerebral malaria (ECM) model inversely correlated with normal behavior and correlated with blood-brain barrier (BBB) permeability. Mice infected with malaria species that do not induce neurological abnormalities or manifest BBB permeability had elevated plasma PA levels similar to ECM plasma at 7 days postinfection; however, they had low PA levels in the brain compared to ECM mice brains at 7 days postinfection. CONCLUSIONS: Our model suggests that malaria-generated PA induces coma in CM and in ECM. The role of BBB permeability and the mechanisms of PA neuromodulation in CM will require additional investigation.

Update on pathogenesis, management, and control of Plasmodium vivax.

PURPOSE OF REVIEW: This is a review of Plasmodium vivax epidemiology, pathogenesis, disease presentation, treatment and innovations in control and elimination. Here, we examine the recent literature and summarize new advances and ongoing challenges in the management of P. vivax . RECENT FINDINGS: P. vivax has a complex life cycle in the human host which impacts disease severity and treatment regimens. There is increasing data for the presence of cryptic reservoirs in the spleen and bone marrow which may contribute to chronic vivax infections and possibly disease severity. Methods to map the geospatial epidemiology of P. vivax chloroquine resistance are advancing, and they will inform local treatment guidelines. P. vivax treatment requires an 8-aminoquinoline to eradicate the dormant liver stage. Evidence suggests that higher doses of 8-aminoquinolines may be needed for radical cure of tropical frequent-relapsing strains. SUMMARY: P. vivax is a significant global health problem. There have been recent developments in understanding the complexity of P. vivax biology and optimization of antimalarial therapy. Studies toward the development of best practices for P. vivax control and elimination programs are ongoing.

Diagnosis, Treatment, and Prevention of Malaria in the US: A Review.

Importance: Malaria is caused by protozoa parasites of the genus Plasmodium and is diagnosed in approximately 2000 people in the US each year who have returned from visiting regions with endemic malaria. The mortality rate from malaria is approximately 0.3% in the US and 0.26% worldwide. Observations: In the US, most malaria is diagnosed in people who traveled to an endemic region. More than 80% of people diagnosed with malaria in the US acquired the infection in Africa. Of the approximately 2000 people diagnosed with malaria in the US in 2017, an estimated 82.4% were adults and about 78.6% were Black or African American. Among US residents diagnosed with malaria, 71.7% had not taken malaria chemoprophylaxis during travel. In 2017 in the US, P falciparum was the species diagnosed in approximately 79% of patients, whereas P vivax was diagnosed in an estimated 11.2% of patients. In 2017 in the US, severe malaria, defined as vital organ involvement including shock, pulmonary edema, significant bleeding, seizures, impaired consciousness, and laboratory abnormalities such as kidney impairment, acidosis, anemia, or high parasitemia, occurred in approximately 14% of patients, and an estimated 0.3% of those receiving a diagnosis of malaria in the US died. P falciparum has developed resistance to chloroquine in most regions of the world, including Africa. First-line therapy for P falciparum malaria in the US is combination therapy that includes artemisinin. If P falciparum was acquired in a known chloroquine-sensitive region such as Haiti, chloroquine remains an alternative option. When artemisinin-based combination therapies are not available, atovaquone-proguanil or quinine plus clindamycin is used for chloroquine-resistant malaria. P vivax, P ovale, P malariae, and P knowlesi are typically chloroquine sensitive, and treatment with either artemisinin-based combination therapy or chloroquine for regions with chloroquine-susceptible infections for uncomplicated malaria is recommended. For severe malaria, intravenous artesunate is first-line therapy. Treatment of mild malaria due to a chloroquine-resistant parasite consists of a combination therapy that includes artemisinin or chloroquine for chloroquine-sensitive malaria. P vivax and P ovale require additional therapy with an 8-aminoquinoline to eradicate the liver stage. Several options exist for chemoprophylaxis and selection should be based on patient characteristics and preferences. Conclusions and Relevance: Approximately 2000 cases of malaria are diagnosed each year in the US, most commonly in travelers returning from visiting endemic areas. Prevention and treatment of malaria depend on the species and the drug sensitivity of parasites from the region of acquisition. Intravenous artesunate is first-line therapy for severe malaria.

Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum.

Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine nucleoside phosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased PfPNP gene copy number and protein expression. At increased drug pressure, additional PfPNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant PfPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PfPNP. Crystal structures of native and mutated PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2136 clonal selection). Transition-state analog inhibitors against PfPNP are slow to induce resistance and may have promise in malaria therapy.

Dimethyl fumarate reduces TNF and Plasmodium falciparum induced brain endothelium activation in vitro.

BACKGROUND: Cerebral malaria (CM) is associated with morbidity and mortality despite the use of potent anti-malarial agents. Brain endothelial cell activation and dysfunction from oxidative and inflammatory host responses and products released by Plasmodium falciparum-infected erythrocytes (IE), are likely the major contributors to the encephalopathy, seizures, and brain swelling that are associated with CM. The development of adjunctive therapy to reduce the pathological consequences of host response pathways could improve outcomes. A potentially protective role of the nuclear factor E2-related factor 2 (NRF2) pathway, which serves as a therapeutic target in brain microvascular diseases and central nervous system (CNS) inflammatory diseases such as multiple sclerosis was tested to protect endothelial cells in an in vitro culture system subjected to tumour necrosis factor (TNF) or infected red blood cell exposure. NRF2 is a transcription factor that mediates anti-oxidant and anti-inflammatory responses. METHODS: To accurately reflect clinically relevant parasite biology a unique panel of parasite isolates derived from patients with stringently defined CM was developed. The effect of TNF and these parasite lines on primary human brain microvascular endothelial cell (HBMVEC) activation in an in vitro co-culture model was tested. HBMVEC activation was measured by cellular release of IL6 and nuclear translocation of NFκB. The transcriptional and functional effects of dimethyl fumarate (DMF), an FDA approved drug which induces the NRF2 pathway, on host and parasite induced HBMVEC activation was characterized. In addition, the effect of DMF on parasite binding to TNF stimulated HBMVEC in a semi-static binding assay was examined. RESULTS: Transcriptional profiling demonstrates that DMF upregulates the NRF2-Mediated Oxidative Stress Response, ErbB4 Signaling Pathway, Peroxisome Proliferator-activated Receptor (PPAR) Signaling and downregulates iNOS Signaling and the Neuroinflammation Signaling Pathway on TNF activated HBMVEC. The parasite lines derived from eight paediatric CM patients demonstrated increased binding to TNF activated HBMVEC and varied in their binding and activation of HBMVEC. Overall DMF reduced both TNF and CM derived parasite activation of HBMVEC. CONCLUSIONS: These findings provide evidence that targeting the NRF2 pathway in TNF and parasite activated HBMVEC mediates multiple protective pathways and may represent a novel adjunctive therapy to improve infection outcomes in CM.

Tafenoquine: the new kid on the block.

PURPOSE OF REVIEW: This is a review of tafenoquine, a new antimalarial drug. Here we examine the recent literature supporting the use of tafenoquine and summarize the opportunities and challenges for its well tolerated use worldwide. RECENT FINDINGS: Tafenoquine was recently approved by the US Food and Drug Administration for the treatment of dormant liver stage (hypnozoite) in Plasmodium vivax and for malaria prophylaxis. Single-dose tafenoquine provides equivalent efficacy to 14 days of primaquine for radical cure in P. vivax, and it can be dosed weekly to prevent malaria. However, tafenoquine can only be used in patients with normal G6PD activity and is contraindicated in children and during pregnancy or in lactating mothers with infants of deficient or unknown G6PD status. SUMMARY: Tafenoquine's long half-life allows a single dose to achieve radical cure, and weekly dosing for chemoprophylaxis to provide an exciting therapeutic option for patient care and as a new weapon for malaria control/eradication programs. Global implementation of tafenoquine will require the development and validation of a robust, low-cost diagnostic to reliably identify G6PD-deficient individuals. In addition, studies on tafenoquine safety in children are needed.

A Capsular Polysaccharide-Specific Antibody Alters Streptococcus pneumoniae Gene Expression during Nasopharyngeal Colonization of Mice.

Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included Streptococcus pneumoniae serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression in vitro Here, we determined whether 1E2 affects ST3 gene expression in vivo during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the piuBCDA operon, which encodes an iron uptake system, and decreased the expression of dpr, which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 in vivo required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. In vitro, 1E2 induced dose-dependent ST3 growth arrest and altered piuB and dpr expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding in vitro, suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.

STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe Plasmodium yoelii Malaria.

Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.

Higher Rates of Misdiagnosis in Pediatric Patients Versus Adults Hospitalized With Imported Malaria.

OBJECTIVES: Despite the availability of effective antimalarial prophylaxis, imported adult and pediatric malaria occurs in the United States, and this can pose diagnostic issues. We examined the clinical characteristics and diagnostic challenges of imported malaria requiring adult or pediatric inpatient admission at Montefiore Medical Center in the Bronx which provides care for a large population of immigrants from malaria endemic areas. STUDY DESIGN: We conducted a retrospective single center review of patients admitted with a diagnosis of malaria at Montefiore Medical Center from 2005 through 2012. We extracted historical, clinical, and laboratory values from the electronic medical record and patient charts. RESULTS: We identified 95 patients who were diagnosed and hospitalized with malaria from 2005 to 2012, 33 (35%) of them were children and 17 (18%) were with severe malaria. Most patients contracted malaria while visiting friends and relatives in West Africa. Only 38% of travelers took prophylaxis, and fewer than half reported taking it as prescribed. Misdiagnosis by emergency room or primary care doctors was observed in almost one quarter of all of the patients. Misdiagnosis occurred significantly more frequently in children (43%) compared to adults (13%) (P = 0.002). Pediatric patients were more likely to present with abdominal pain (42% vs. 15%; P = 0.005). CONCLUSIONS: Pediatric patients admitted for imported malaria at Montefiore Medical Center had a higher rate of misdiagnosis and presented with more gastrointestinal symptoms than hospitalized adults. By describing the clinical characteristics of patients with imported malaria, we hope to improve diagnostic accuracy by health care workers and raise awareness that friends and relatives may require more intensive pretravel counseling.

A genome-wide map of diversity in Plasmodium falciparum.

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.

The T-Cell Inhibitory Molecule Butyrophilin-Like 2 Is Up-regulated in Mild Plasmodium falciparum Infection and Is Protective During Experimental Cerebral Malaria.

Plasmodium falciparum infection can result in severe disease that is associated with elevated inflammation and vital organ dysfunction; however, malaria-endemic residents gain protection from lethal outcomes and manifest only mild symptoms during infection. To characterize host responses associated with this more effective antimalarial response, we characterized whole-blood transcriptional profiles in Rwandan adults during a mild malaria episode and compared them with findings from a convalescence sample. We observed transcriptional up-regulation in many pathways, including type I interferon, interferon γ, complement activation, and nitric oxide during malaria infection, which provide benchmarks of mild disease physiology. Transcripts encoding negative regulators of T-cell activation, such as programmed death ligand 1 (PD-L1), programmed death 1 ligand 2 (PD-L2), and the butyrophilin family member butyrophilin-like 2 (BTNL2) were also increased. To support an important functional role for BTNL2 during malaria infection, we studied chimeric mice reconstituted with BTNL2(-/-) or wild-type hematopoietic cells that were inoculated with Plasmodium berghei ANKA, a murine model of cerebral malaria. We found that BTNL2(-/-) chimeric mice had a significant decrease in survival compared with wild-type counterparts. Collectively these data characterize the immune responses associated with mild malaria and uncover a novel role for BTNL2 in the host response to malaria.

Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria.

BACKGROUND: Cerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies. METHODS: To examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume. RESULTS: The metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability. CONCLUSIONS: Taken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.

Inferring developmental stage composition from gene expression in human malaria.

In the current era of malaria eradication, reducing transmission is critical. Assessment of transmissibility requires tools that can accurately identify the various developmental stages of the malaria parasite, particularly those required for transmission (sexual stages). Here, we present a method for estimating relative amounts of Plasmodium falciparum asexual and sexual stages from gene expression measurements. These are modeled using constrained linear regression to characterize stage-specific expression profiles within mixed-stage populations. The resulting profiles were analyzed functionally by gene set enrichment analysis (GSEA), confirming differentially active pathways such as increased mitochondrial activity and lipid metabolism during sexual development. We validated model predictions both from microarrays and from quantitative RT-PCR (qRT-PCR) measurements, based on the expression of a small set of key transcriptional markers. This sufficient marker set was identified by backward selection from the whole genome as available from expression arrays, targeting one sentinel marker per stage. The model as learned can be applied to any new microarray or qRT-PCR transcriptional measurement. We illustrate its use in vitro in inferring changes in stage distribution following stress and drug treatment and in vivo in identifying immature and mature sexual stage carriers within patient cohorts. We believe this approach will be a valuable resource for staging lab and field samples alike and will have wide applicability in epidemiological studies of malaria transmission.