COVID-19 infection and transmission includes complex sequence diversity.

SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report sequence variation in all samples tested. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern.

SARS-CoV-2 seroprevalence among blood donors in Uganda: 2019-2022.

BACKGROUND: The true burden of COVID-19 in low- and middle-income countries remains poorly characterized, especially in Africa. Even prior to the availability of SARS-CoV-2 vaccines, countries in Africa had lower numbers of reported COVID-19 related hospitalizations and deaths than other regions globally. METHODS: Ugandan blood donors were evaluated between October 2019 and April 2022 for IgG antibodies to SARS-CoV-2 nucleocapsid (N), spike (S), and five variants of the S protein using multiplexed electrochemiluminescence immunoassays (MesoScale Diagnostics, Rockville, MD). Seropositivity for N and S was assigned using manufacturer-provided cutoffs and trends in seroprevalence were estimated by quarter. Statistically significant associations between N and S antibody seropositivity and donor characteristics in November-December 2021 were assessed by chi-square tests. RESULTS: A total of 5393 blood unit samples from donors were evaluated. N and S seropositivity increased throughout the pandemic to 82.6% in January-April 2022. Among seropositive individuals, N and S antibody levels increased ≥9-fold over the study period. In November-December 2021, seropositivity to N and S antibody was higher among repeat donors (61.3%) compared with new donors (55.1%; p = .043) and among donors from Kampala (capital city of Uganda) compared with rural regions (p = .007). Seropositivity to S antibody was significantly lower among HIV-seropositive individuals (58.8% vs. 84.9%; p = .009). CONCLUSIONS: Despite previously reported low numbers of COVID-19 cases and related deaths in Uganda, high SARS-CoV-2 seroprevalence and increasing antibody levels among blood donors indicated that the country experienced high levels of infection over the course of the pandemic.

Human monoclonal antibodies inhibit invasion of transgenic Plasmodium knowlesi expressing Plasmodium vivax Duffy binding protein.

BACKGROUND: Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). METHODS: Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). RESULTS: The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. CONCLUSION: The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Patient Transport Van.

We report 2 episodes of potential SARS-CoV-2 transmission from infected van drivers to passengers despite masking and physical distancing. Whole-genome sequencing confirmed relatedness of driver and passenger SARS-CoV-2. With the heater operating, fluorescent microspheres were transported by airflow >3 meters from the front to the back of the van.

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti.

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, Brugia timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti (Wb). Here we use population genomics to reconstruct the probable route and timing of migration of Wb strains that currently infect Africa, Haiti, and Papua New Guinea (PNG). We used selective whole genome amplification to sequence 42 whole genomes of single Wb worms from populations in Haiti, Mali, Kenya, and PNG. Our results are consistent with a hypothesis of an Island Southeast Asia or East Asian origin of Wb. Our demographic models support divergence times that correlate with the migration of human populations. We hypothesize that PNG was infected at two separate times, first by the Melanesians and later by the migrating Austronesians. The migrating Austronesians also likely introduced Wb to Madagascar where later migrations spread it to continental Africa. From Africa, Wb spread to the New World during the transatlantic slave trade. Genome scans identified 17 genes that were highly differentiated among Wb populations. Among these are genes associated with human immune suppression, insecticide sensitivity, and proposed drug targets. Identifying the distribution of genetic diversity in Wb populations and selection forces acting on the genome will build a foundation to test future hypotheses and help predict response to current eradication efforts.

Cytomegalovirus immunoglobulin G titers do not predict reactivation risk in immunocompetent hosts.

Cytomegalovirus (CMV) reactivation occurs in roughly one-third of immunocompetent patients during critical illness, and is associated with worse outcomes. These outcomes have prompted consideration of early antiviral prophylaxis, but two-third of patients would receive unnecessary treatment. Tissue viral load has been associated with risk of reactivation in murine models, and recent work has suggested a relationship between immune responses to CMV and underlying viral load. We, therefore, sought to confirm the hypothesis that serum CMV-specific immunoglobulin G (IgG) correlates with tissue viral load, and might be used to predict the risk of reactivation during critical illness. We confirm that there is a good correlation between tissue viral load and serum CMV-specific IgG after laboratory infection of inbred mice. Further, we show that naturally infected outbred hosts have variable tissue viral DNA loads that do not correlate well with serum IgG. Perhaps as a consequence, CMV-specific IgG was not predictive of reactivation events in immunocompetent humans. When reactivation did occur, those with the lowest IgG levels had longer durations of reactivation, but IgG quartiles were not associated with differing peak DNAemia. Together our data suggest that CMV-specific IgG titers diverge from tissue viral loads in outbred immunocompetent hosts, and their importance for the control of reactivation events remains unclear.

Ensemble survival tree models to reveal pairwise interactions of variables with time-to-events outcomes in low-dimensional setting.

Unraveling interactions among variables such as genetic, clinical, demographic and environmental factors is essential to understand the development of common and complex diseases. To increase the power to detect such variables interactions associated with clinical time-to-events outcomes, we borrowed established concepts from random survival forest (RSF) models. We introduce a novel RSF-based pairwise interaction estimator and derive a randomization method with bootstrap confidence intervals for inferring interaction significance. Using various linear and nonlinear time-to-events survival models in simulation studies, we first show the efficiency of our approach: true pairwise interaction-effects between variables are uncovered, while they may not be accompanied with their corresponding main-effects, and may not be detected by standard semi-parametric regression modeling and test statistics used in survival analysis. Moreover, using a RSF-based cross-validation scheme for generating prediction estimators, we show that informative predictors may be inferred. We applied our approach to an HIV cohort study recording key host gene polymorphisms and their association with HIV change of tropism or AIDS progression. Altogether, this shows how linear or nonlinear pairwise statistical interactions of variables may be efficiently detected with a predictive value in observational studies with time-to-event outcomes.

Spatio-temporal mapping of Madagascar's Malaria Indicator Survey results to assess Plasmodium falciparum endemicity trends between 2011 and 2016.

BACKGROUND: Reliable measures of disease burden over time are necessary to evaluate the impact of interventions and assess sub-national trends in the distribution of infection. Three Malaria Indicator Surveys (MISs) have been conducted in Madagascar since 2011. They provide a valuable resource to assess changes in burden that is complementary to the country's routine case reporting system. METHODS: A Bayesian geostatistical spatio-temporal model was developed in an integrated nested Laplace approximation framework to map the prevalence of Plasmodium falciparum malaria infection among children from 6 to 59 months in age across Madagascar for 2011, 2013 and 2016 based on the MIS datasets. The model was informed by a suite of environmental and socio-demographic covariates known to influence infection prevalence. Spatio-temporal trends were quantified across the country. RESULTS: Despite a relatively small decrease between 2013 and 2016, the prevalence of malaria infection has increased substantially in all areas of Madagascar since 2011. In 2011, almost half (42.3%) of the country's population lived in areas of very low malaria risk (<1% parasite prevalence), but by 2016, this had dropped to only 26.7% of the population. Meanwhile, the population in high transmission areas (prevalence >20%) increased from only 2.2% in 2011 to 9.2% in 2016. A comparison of the model-based estimates with the raw MIS results indicates there was an underestimation of the situation in 2016, since the raw figures likely associated with survey timings were delayed until after the peak transmission season. CONCLUSIONS: Malaria remains an important health problem in Madagascar. The monthly and annual prevalence maps developed here provide a way to evaluate the magnitude of change over time, taking into account variability in survey input data. These methods can contribute to monitoring sub-national trends of malaria prevalence in Madagascar as the country aims for geographically progressive elimination.

Metabolism of primaquine in normal human volunteers: investigation of phase I and phase II metabolites from plasma and urine using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

BACKGROUND: Primaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism. METHODS: These methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC-MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards. RESULTS: Besides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations. CONCLUSION: This approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.

Single-cell genomics for dissection of complex malaria infections.

Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.

Prevalence and genetic variants of G6PD deficiency among two Malagasy populations living in Plasmodium vivax-endemic areas.

BACKGROUND: The prevalence and variants of G6PD deficiency in the Plasmodium vivax-endemic zones of Madagascar remain unknown. The admixed African-Austronesian origins of the Malagasy population make it probable that a heterogeneous mix of genetic variants with a spectrum of clinical severity will be circulating. This would have implications for the widespread use of P. vivax radical cure therapy. Two study populations in the P. vivax-endemic western foothills region of Madagascar were selected for G6PD screening. Both the qualitative fluorescent spot test and G6PD genotyping were used to screen all participants. RESULTS: A total of 365 unrelated male volunteers from the Tsiroanomandidy, Mandoto, and Miandrivazo districts of Madagascar were screened and 12.9% were found to be phenotypically G6PD deficient. Full gene sequencing of 95 samples identified 16 single nucleotide polymorphisms, which were integrated into a genotyping assay. Genotyping (n = 291) found one individual diagnosed with the severe G6PD Mediterranean C563T mutation, while the remaining G6PD deficient samples had mutations of African origin, G6PD A- and G6PD A. CONCLUSIONS: Deployment of P. vivax radical cure in Madagascar must be considerate of the risks presented by the observed prevalence of G6PDd prevalence. The potential morbidity associated with cumulative episodes of P. vivax clinical relapses requires a strategy for increasing access to safe radical cure. The observed dominance of African G6PDd haplotypes is surprising given the known mixed African-Austronesian origins of the Malagasy population; more widespread surveying of G6PDd epidemiology across the island would be required to characterize the distribution of G6PD haplotypes across Madagascar.

Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.

BACKGROUND: Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite's red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation. METHODS AND RESULTS: Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country's National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166-961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture. CONCLUSIONS/SIGNIFICANCE: Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite.

A preliminary assessment of Toll-like receptor and ß-defensin gene polymorphisms in Papua New Guinea - what does it mean for HIV/AIDS?

Polymorphisms in Toll-like receptor (TLR) and human ß-defensin (hBD, encoded by DEFB) genes have been evaluated for their associations with HIV infection and disease outcomes. Those studies, conducted in various populations under a variety of study designs, generally revealed that specific single nucleotide polymorphisms (SNPs) in TLR1, 2, 3, 4, 6, 7, 8, and 9 genes, and copy number variation (CNV) in DEFB4 (encoding hBD-2), DEFB103A (encoding hBD-3), and DEFB104A (encoding hBD-4) genes are among potential genetic factors that can affect susceptibility to HIV infection and/or disease progression. The information regarding their prevalence in Papua New Guinea (PNG) is very limited for TLR SNPs, and not available for DEFB CNV. The present study provides a preliminary assessment of these genetic polymorphisms in samples collected from the Wosera (East Sepik Province, n = 29) and Liksul (Madang Province, n = 23) areas. Wosera samples were analyzed for a total of 41 SNPs in 8 TLR genes (TLR1, 2, 3, 4, 6, 7, 8, and 9), and both sample sets were analyzed for CNV in DEFB4/103A/104A genes. A number of TLR SNPs were not detected, and many other SNPs were present at low frequencies (minor allele frequencies ≤0.05) in the Wosera samples. The DEFB4/103A/104A copy numbers were significantly different between the two sample sets (p = 0.024). Validation of these results, using larger sample sizes as well as samples from other areas of PNG, is warranted. In addition, genetic association studies are needed to estimate the effects of these polymorphisms on HIV infection and disease progression in PNG.

Insecticidal bed nets and filariasis transmission in Papua New Guinea.

BACKGROUND: Global efforts to eliminate lymphatic filariasis are based on the annual mass administration of antifilarial drugs to reduce the microfilaria reservoir available to the mosquito vector. Insecticide-treated bed nets are being widely used in areas in which filariasis and malaria are coendemic. METHODS: We studied five villages in which five annual mass administrations of antifilarial drugs, which were completed in 1998, reduced the transmission of Wuchereria bancrofti, one of the nematodes that cause lymphatic filariasis. A total of 21,899 anopheles mosquitoes were collected for 26 months before and 11 to 36 months after bed nets treated with long-lasting insecticide were distributed in 2009. We evaluated the status of filarial infection and the presence of W. bancrofti DNA in anopheline mosquitoes before and after the introduction of insecticide-treated bed nets. We then used a model of population dynamics to estimate the probabilities of transmission cessation. RESULTS: Village-specific rates of bites from anopheline mosquitoes ranged from 6.4 to 61.3 bites per person per day before the bed-net distribution and from 1.1 to 9.4 bites for 11 months after distribution (P<0.001). During the same period, the rate of detection of W. bancrofti in anopheline mosquitoes decreased from 1.8% to 0.4% (P=0.005), and the rate of detection of filarial DNA decreased from 19.4% to 14.9% (P=0.13). The annual transmission potential was 5 to 325 infective larvae inoculated per person per year before the bed-net distribution and 0 after the distribution. Among all five villages with a prevalence of microfilariae of 2 to 38%, the probability of transmission cessation increased from less than 1.0% before the bed-net distribution to a range of 4.9 to 95% in the 11 months after distribution. CONCLUSIONS: Vector control with insecticide-treated bed nets is a valuable tool for W. bancrofti elimination in areas in which anopheline mosquitoes transmit the parasite. (Funded by the U.S. Public Health Service and the National Institutes of Health.).

Population genomics of the filarial nematode parasite Wuchereria bancrofti from mosquitoes.

Wuchereria bancrofti is a parasitic nematode and the primary cause of lymphatic filariasis--a disease specific to humans. W. bancrofti currently infects over 90 million people throughout the tropics and has been acknowledged by the world health organization as a vulnerable parasite. Current research has focused primarily on the clinical manifestations of disease and little is known about the evolutionary history of W. bancrofti. To improve upon knowledge of the evolutionary history of W. bancrofti, we whole genome sequenced 13 W. bancrofti larvae. We circumvent many of the difficulties of multiple infections by sampling larvae directly from mosquitoes that were experimentally inoculated with infected blood. To begin, we used whole genome data to reconstruct the historical population size. Our results support a history of fluctuating population sizes that can be correlated with human migration and fluctuating mosquito abundances. Next, we reconstructed the putative pedigree of W. bancrofti worms within an infection using the kinship coefficient. We deduced that there are full-sib and half-sib relationships residing within the same larval cohort. Through combined analysis of the mitochondrial and nuclear genomes we concluded that this is likely a results of polyandrous mating, the first time reported for W. bancrofti. Lastly, we scanned the genomes for signatures of natural selection. Annotation of putative selected regions identified proteins that may have aided in a parasitic life style or may have evolved to protect against current drug treatments. We discuss our results in the greater context of understanding the biology of an animal with a unique life history and ecology.

Malaria transmission dynamics surrounding the first nationwide long-lasting insecticidal net distribution in Papua New Guinea.

BACKGROUND: The major malaria vectors of Papua New Guinea exhibit heterogeneities in distribution, biting behaviour and malaria infection levels. Long-lasting, insecticide-treated nets (LLINs), distributed as part of the National Malaria Control Programme, are the primary intervention targeting malaria transmission. This study evaluated the impact of LLINs on anopheline density, species composition, feeding behaviour, and malaria transmission. METHODS: Mosquitoes were collected by human landing catch in 11 villages from East Sepik Province and Madang Province. Mosquitoes were collected for 3 years (1 year before distribution and 2 years after), and assayed to determine mosquito species and Plasmodium spp. infection prevalence. The influence of weather conditions and the presence of people and animals on biting density was determined. Determinants of biting density and sporozoite prevalence were analysed by generalized estimating equations (GEE). RESULTS: Mosquito biting rates and entomological inoculation rates decreased significantly after the distribution. Plasmodium falciparum and P. vivax sporozoite prevalence decreased in year 2, but increased in year 3, suggesting the likelihood of resurgence in transmission if low biting rates are not maintained. An earlier shift in the median biting time of Anopheles punctulatus and An. farauti s.s. was observed. However, this was not accompanied by an increase in the proportion of infective bites occurring before 2200 hours. A change in species composition was observed, which resulted in dominance of An. punctulatus in Dreikikir region, but a decrease in An. punctulatus in the Madang region. When controlling for village and study year, An. farauti s.s., An. koliensis and An. punctulatus were equally likely to carry P. vivax sporozoites. However, An. punctulatus was significantly more likely than An. farauti s.s. (OR 0.14; p = 0.007) or An. koliensis (OR 0.27; p < 0.001) to carry P. falciparum sporozoites. CONCLUSIONS: LLINs had a significant impact on malaria transmission, despite exophagic and crepuscular feeding behaviours of dominant vectors. Changes in species composition and feeding behaviour were observed, but their epidemiological significance will depend on their durability over time.

Contemporary epidemiological overview of malaria in Madagascar: operational utility of reported routine case data for malaria control planning.

BACKGROUND: Malaria remains a major public health problem in Madagascar. Widespread scale-up of intervention coverage has led to substantial reductions in case numbers since 2000. However, political instability since 2009 has disrupted these efforts, and a resurgence of malaria has since followed. This paper re-visits the sub-national stratification of malaria transmission across Madagascar to propose a contemporary update, and evaluates the reported routine case data reported at this sub-national scale. METHODS: Two independent malariometrics were evaluated to re-examine the status of malaria across Madagascar. First, modelled maps of Plasmodium falciparum infection prevalence (PfPR) from the Malaria Atlas Project were used to update the sub-national stratification into 'ecozones' based on transmission intensity. Second, routine reports of case data from health facilities were synthesized from 2010 to 2015 to compare the sub-national epidemiology across the updated ecozones over time. Proxy indicators of data completeness are investigated. RESULTS: The epidemiology of malaria is highly diverse across the island's ecological regions, with eight contiguous ecozones emerging from the transmission intensity PfPR map. East and west coastal areas have highest transmission year-round, contrasting with the central highlands and desert south where trends appear more closely associated with epidemic outbreak events. Ecozones have shown steady increases in reported malaria cases since 2010, with a near doubling of raw reported case numbers from 2014 to 2015. Gauges of data completeness suggest that interpretation of raw reported case numbers will underestimate true caseload as only approximately 60-75 % of health facility data are reported to the central level each month. DISCUSSION: A sub-national perspective is essential when monitoring the epidemiology of malaria in Madagascar and assessing local control needs. A robust assessment of the status of malaria at a time when intervention coverage efforts are being scaled up provides a platform from which to guide intervention preparedness and assess change in future periods of transmission.

Automatic Classification of Ultrasound Screening Examinations of the Abdominal Aorta.

Our work facilitates the identification of veterans who may be at risk for abdominal aortic aneurysms (AAA) based on the 2007 mandate to screen all veteran patients that meet the screening criteria. The main research objective is to automatically index three clinical conditions: pertinent negative AAA, pertinent positive AAA, and visually unacceptable image exams. We developed and evaluated a ConText-based algorithm with the GATE (General Architecture for Text Engineering) development system to automatically classify 1402 ultrasound radiology reports for AAA screening. Using the results from JAPE (Java Annotation Pattern Engine) transducer rules, we developed a feature vector to classify the radiology reports with a decision table classifier. We found that ConText performed optimally on precision and recall for pertinent negative (0.99 (0.98-0.99), 0.99 (0.99-1.00)) and pertinent positive AAA detection (0.98 (0.95-1.00), 0.97 (0.92-1.00)), and respectably for determination of non-diagnostic image studies (0.85 (0.77-0.91), 0.96 (0.91-0.99)). In addition, our algorithm can determine the AAA size measurements for further characterization of abnormality. We developed and evaluated a regular expression based algorithm using GATE for determining the three contextual conditions: pertinent negative, pertinent positive, and non-diagnostic from radiology reports obtained for evaluating the presence or absence of abdominal aortic aneurysm. ConText performed very well at identifying the contextual features. Our study also discovered contextual trigger terms to detect sub-standard ultrasound image quality. Limitations of performance included unknown dictionary terms, complex sentences, and vague findings that were difficult to classify and properly code.

Acquisition of antibodies against Plasmodium falciparum merozoites and malaria immunity in young children and the influence of age, force of infection, and magnitude of response.

Individuals in areas of Plasmodium falciparum endemicity develop immunity to malaria after repeated exposure. Knowledge of the acquisition and nature of protective immune responses to P. falciparum is presently limited, particularly for young children. We examined antibodies (IgM, IgG, and IgG subclasses) to merozoite antigens and their relationship to the prospective risk of malaria in children 1 to 4 years of age in a region of malaria endemicity in Papua New Guinea. IgG, IgG1, and IgG3 responses generally increased with age, were higher in children with active infection, and reflected geographic heterogeneity in malaria transmission. Antigenic properties, rather than host factors, appeared to be the main determinant of the type of IgG subclass produced. High antibody levels were not associated with protection from malaria; in contrast, they were typically associated with an increased risk of malaria. Adjustment for malaria exposure, using a novel molecular measure of the force of infection by P. falciparum, accounted for much of the increased risk, suggesting that the antibodies were markers of higher exposure to P. falciparum. Comparisons between antibodies in this cohort of young children and in a longitudinal cohort of older children suggested that the lack of protective association was explained by lower antibody levels among young children and that there is a threshold level of antibodies required for protection from malaria. Our results suggest that in populations with low immunity, such as young children, antibodies to merozoite antigens may act as biomarkers of malaria exposure and that, with increasing exposure and responses of higher magnitude, antibodies may act as biomarkers of protective immunity.

Malaria diagnosis for malaria elimination.

PURPOSE OF REVIEW: Limitations of blood smear microscopy contributed to failure of the 1950-1960s WHO Global Programme to Eliminate Malaria. All diagnostic methods encounter limits of detection (LOD) beyond which it will not be possible to identify infected individuals. When this occurs, it becomes difficult to continue evaluating progress of malaria elimination. The purpose of this review is to compare available diagnostic technologies, factors that underlie their LOD, and their potential roles related to the goal of elimination. RECENT FINDINGS: Parasite-containing cells, parasite proteins, hemozoin, nucleic acids, and parasite-specific human antibodies are targets of diagnosis. Many studies report advantages of technologies to detect these diagnostic targets. Nucleic acid amplification tests and strategies for enriching capture of malaria diagnostic targets have consistently identified a parasite reservoir not detected by methods focused on the other biological targets. Exploiting magnetic properties of hemozoin may open new strategies for noninvasive malaria diagnosis. SUMMARY: Microscopy and rapid diagnostic tests provide effective surveillance for malaria control. Strategies that detect a reservoir of submicroscopic infection must be developed and standardized to guide malaria elimination.