Michael P. Fay

Permutation tests for joinpoint regression with applications to cancer rates

The identification of changes in the recent trend is an important issue in the analysis of cancer mortality and incidence data. We apply a joinpoint regression model to describe such continuous changes and use the grid-search method to fit the regression function with unknown joinpoints assuming constant variance and uncorrelated errors. We find the number of significant joinpoints by performing several permutation tests, each of which has a correct significance level asymptotically. Each p-value is found using Monte Carlo methods, and the overall asymptotic significance level is maintained through a Bonferroni correction. These tests are extended to the situation with non-constant variance to handle rates with Poisson variation and possibly autocorrelated errors. The performance of these tests are studied via simulations and the tests are applied to U.S. prostate cancer incidence and mortality rates.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

Wilcoxon-Mann-Whitney or t-test? On assumptions for hypothesis tests and multiple interpretations of decision rules

In a mathematical approach to hypothesis tests, we start with a clearly defined set of hypotheses and choose the test with the best properties for those hypotheses. In practice, we often start with less precise hypotheses. For example, often a researcher wants to know which of two groups generally has the larger responses, and either a t-test or a Wilcoxon-Mann-Whitney (WMW) test could be acceptable. Although both t-tests and WMW tests are usually associated with quite different hypotheses, the decision rule and p-value from either test could be associated with many different sets of assumptions, which we call perspectives. It is useful to have many of the different perspectives to which a decision rule may be applied collected in one place, since each perspective allows a different interpretation of the associated p-value. Here we collect many such perspectives for the two-sample t-test, the WMW test and other related tests. We discuss validity and consistency under each perspective and discuss recommendations between the tests in light of these many different perspectives. Finally, we briefly discuss a decision rule for testing genetic neutrality where knowledge of the many perspectives is vital to the proper interpretation of the decision rule.

Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study

BACKGROUND Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. METHODS Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10,000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003. FINDINGS We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). INTERPRETATION Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. FUNDING Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America.

Dihydroartemisinin–piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study

BACKGROUND Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin-piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. METHODS In this prospective cohort study, we enrolled patients aged 2-65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin-piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov, number NCT01736319. FINDINGS Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. INTERPRETATION Dihydroartemisinin-piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin-piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin-piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin-piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. FUNDING National Institute of Allergy and Infectious Diseases.

A West Nile Virus DNA Vaccine Induces Neutralizing Antibody in Healthy Adults during a Phase 1 Clinical Trial

BACKGROUND West Nile virus (WNV) is a mosquito-borne flavivirus that can cause severe meningitis and encephalitis in infected individuals. We report the safety and immunogenicity of a WNV DNA vaccine in its first phase 1 human study. METHODS A single-plasmid DNA vaccine encoding the premembrane and the envelope glycoproteins of the NY99 strain of WNV was evaluated in an open-label study in 15 healthy adults. Twelve subjects completed the 3-dose vaccination schedule, and all subjects completed 32 weeks of evaluation for safety and immunogenicity. The development of a vaccine-induced immune response was assessed by enzyme-linked immunosorbant assay, neutralization assays, intracelluar cytokine staining, and enzyme-linked immunospot assay. RESULTS The vaccine was safe and well tolerated, with no significant adverse events. Vaccine-induced T cell and antibody responses were detected in the majority of subjects. Neutralizing antibody to WNV was detected in all subjects who completed the 3-dose vaccination schedule, at levels shown to be protective in studies of horses, an incidental natural host for WNV. CONCLUSIONS Further assessment of this DNA platform for human immunization against WNV is warranted. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00106769 .

A Randomized Controlled Phase 2 Trial of the Blood Stage AMA1-C1/Alhydrogel Malaria Vaccine in Children in Mali

A double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1), adjuvanted with Alhydrogel. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000/microL/day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin <8.5 g/dL, in the direction of lower hemoglobin in the children who received AMA1-C1, although these differences were not significant after correction for multiple tests. These differences were not seen in the second year of transmission.

Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies

Leishmaniasis is transmitted between mammalian hosts by the bites of bloodsucking vector sand flies. The dose of parasites transmitted to the mammalian host has never been directly determined. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by individual infected flies (Phlebotomus duboscqi). The number of parasites transmitted varied over a wide range in the 58 ears in which Leishmania were detected and demonstrated a clear bimodal distribution. Most of the infected mice were inoculated with a low dose of <600 parasites. One in four received a higher dose of >1,000 and up to 100,000 cells. High-dose transmission was associated with a heavy midgut infection of >30,000 parasites, incomplete blood feeding, and transmission of a high percentage of the parasite load in the fly. To test the impact of inoculum size on infection outcome, we compared representative high- (5,000) and low- (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly-derived metacyclic forms of L. major and preexposed the injection site to the bites of uninfected flies. Large lesions developed rapidly in the ears of mice receiving the high-dose inoculum. The low dose resulted in only minor pathology but a higher parasite titer in the chronic phase, and it established the host as an efficient long-term reservoir of infection back to vector sand flies.

Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.