Statistical analysis plan for the LAKANA trial: a cluster-randomized, placebo-controlled, double-blinded, parallel group, three-arm clinical trial testing the effects of mass drug administration of azithromycin on mortality and other outcomes among 1-11-month-old infants in Mali.

BACKGROUND: The Large-scale Assessment of the Key health-promoting Activities of two New mass drug administration regimens with Azithromycin (LAKANA) trial in Mali aims to evaluate the efficacy and safety of azithromycin (AZI) mass drug administration (MDA) to 1-11-month-old infants as well as the impact of the intervention on antimicrobial resistance (AMR) and mechanisms of action of azithromycin. To improve the transparency and quality of this clinical trial, we prepared this statistical analysis plan (SAP). METHODS/DESIGN: LAKANA is a cluster randomized trial that aims to address the mortality and health impacts of biannual and quarterly AZI MDA. AZI is given to 1-11-month-old infants in a high-mortality setting where a seasonal malaria chemoprevention (SMC) program is in place. The participating villages are randomly assigned to placebo (control), two-dose AZI (biannual azithromycin-MDA), and four-dose AZI (quarterly azithromycin-MDA) in a 3:4:2 ratio. The primary outcome of the study is mortality among the intention-to-treat population of 1-11-month-old infants. We will evaluate relative risk reduction between the study arms using a mixed-effects Poisson model with random intercepts for villages, using log link function with person-years as an offset variable. We will model outcomes related to secondary objectives of the study using generalized linear models with considerations on clustering. CONCLUSION: The SAP written prior to data collection completion will help avoid reporting bias and data-driven analysis for the primary and secondary aims of the trial. If there are deviations from the analysis methods described here, they will be described and justified in the publications of the trial results. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT04424511 . Registered on 11 June 2020.

Testing the effects of mass drug administration of azithromycin on mortality and other outcomes among 1-11-month-old infants in Mali (LAKANA): study protocol for a cluster-randomized, placebo-controlled, double-blinded, parallel-group, three-arm clinical trial.

BACKGROUND: Mass drug administration (MDA) of azithromycin (AZI) has been shown to reduce under-5 mortality in some but not all sub-Saharan African settings. A large-scale cluster-randomized trial conducted in Malawi, Niger, and Tanzania suggested that the effect differs by country, may be stronger in infants, and may be concentrated within the first 3 months after treatment. Another study found no effect when azithromycin was given concomitantly with seasonal malaria chemoprevention (SMC). Given the observed heterogeneity and possible effect modification by other co-interventions, further trials are needed to determine the efficacy in additional settings and to determine the most effective treatment regimen. METHODS: LAKANA stands for Large-scale Assessment of the Key health-promoting Activities of two New mass drug administration regimens with Azithromycin. The LAKANA trial is designed to address the mortality and health impacts of 4 or 2 annual rounds of azithromycin MDA delivered to 1-11-month-old (29-364 days) infants, in a high-mortality and malaria holoendemic Malian setting where there is a national SMC program. Participating villages (clusters) are randomly allocated in a ratio of 3:2:4 to three groups: placebo (control):4-dose AZI:2-dose AZI. The primary outcome measured is mortality. Antimicrobial resistance (AMR) will be monitored closely before, during, and after the intervention and both among those receiving and those not receiving MDA with the study drugs. Other outcomes, from a subset of villages, comprise efficacy outcomes related to morbidity, growth and nutritional status, outcomes related to the mechanism of azithromycin activity through measures of malaria parasitemia and inflammation, safety outcomes (AMR, adverse and serious adverse events), and outcomes related to the implementation of the intervention documenting feasibility, acceptability, and economic aspects. The enrolment commenced in October 2020 and is planned to be completed by the end of 2022. The expected date of study completion is December 2024. DISCUSSION: If LAKANA provides evidence in support of a positive mortality benefit resulting from azithromycin MDA, it will significantly contribute to the options for successfully promoting child survival in Mali, and elsewhere in sub-Saharan Africa. TRIAL REGISTRATION: ClinicalTrials.gov NCT04424511. Registered on 11 June 2020.

Associations Between Eight Earth Observation-Derived Climate Variables and Enteropathogen Infection: An Independent Participant Data Meta-Analysis of Surveillance Studies With Broad Spectrum Nucleic Acid Diagnostics.

Diarrheal disease, still a major cause of childhood illness, is caused by numerous, diverse infectious microorganisms, which are differentially sensitive to environmental conditions. Enteropathogen-specific impacts of climate remain underexplored. Results from 15 studies that diagnosed enteropathogens in 64,788 stool samples from 20,760 children in 19 countries were combined. Infection status for 10 common enteropathogens-adenovirus, astrovirus, norovirus, rotavirus, sapovirus, Campylobacter, ETEC, Shigella, Cryptosporidium and Giardia-was matched by date with hydrometeorological variables from a global Earth observation dataset-precipitation and runoff volume, humidity, soil moisture, solar radiation, air pressure, temperature, and wind speed. Models were fitted for each pathogen, accounting for lags, nonlinearity, confounders, and threshold effects. Different variables showed complex, non-linear associations with infection risk varying in magnitude and direction depending on pathogen species. Rotavirus infection decreased markedly following increasing 7-day average temperatures-a relative risk of 0.76 (95% confidence interval: 0.69-0.85) above 28°C-while ETEC risk increased by almost half, 1.43 (1.36-1.50), in the 20-35°C range. Risk for all pathogens was highest following soil moistures in the upper range. Humidity was associated with increases in bacterial infections and decreases in most viral infections. Several virus species' risk increased following lower-than-average rainfall, while rotavirus and ETEC increased with heavier runoff. Temperature, soil moisture, and humidity are particularly influential parameters across all enteropathogens, likely impacting pathogen survival outside the host. Precipitation and runoff have divergent associations with different enteric viruses. These effects may engender shifts in the relative burden of diarrhea-causing agents as the global climate changes.

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.

Consumption of multiple micronutrients or small-quantity lipid-based nutrient supplements containing iodine at the recommended dose during pregnancy, compared with iron and folic acid, does not affect women's urinary iodine concentration in rural Malawi: a secondary outcome analysis of the iLiNS DYAD trial.

OBJECTIVES: Inadequate iodine intake during pregnancy increases the risk of neonatal morbidity and mortality. We aimed to evaluate whether prenatal supplements containing iodine affect urinary iodine concentrations (UIC) of pregnant women in Malawi. DESIGN: A randomised controlled trial. Pregnant women (n 1391) were assigned to consume 60 mg/d Fe and 400 µg/d folic acid (IFA) or 18 vitamins and minerals including 250 µg/d iodine (MMN) or 20 g/d small-quantity lipid-based nutrient supplements (SQ-LNS) with similar nutrient contents as MMN group, plus macronutrients (LNS) until childbirth. In a sub-study (n 317), we evaluated group geometric mean urinary iodine concentration (UIC) (µg/L) at 36 weeks of gestation controlling for baseline UIC and compared median (baseline) and geometric mean (36 weeks) UIC with WHO cut-offs: UIC < 150, 150-249, 250-499 and ≥500 reflecting insufficient, adequate, above requirements and excessive iodine intakes, respectively. SETTING: Mangochi District, Malawi. PARTICIPANTS: Women ≤20 weeks pregnant. RESULTS: Groups had comparable background characteristics. At baseline, overall median (Q1, Q3) UIC (319 (167, 559)) suggested iodine intakes above requirements. At 36 weeks, the geometric mean (95 % CI) UIC of the IFA (197 (171, 226)), MMN (212 (185, 243)) and LNS (220 (192, 253)) groups did not differ (P = 0·53) and reflected adequate intakes. CONCLUSIONS: In this setting, provision of supplements containing iodine at the recommended dose to pregnant women with relatively high iodine intakes at baseline, presumably from iodised salt, has no impact on the women's UIC. Regular monitoring of the iodine status of pregnant women in such settings is advisable. Clinicaltrials.gov identifier: NCT01239693.

Age-dependent association between hepatic lipase gene C-480T polymorphism and the risk of pre-hospital sudden cardiac death: the Helsinki Sudden Death Study.

OBJECTIVE: We investigated the association between hepatic lipase (HL) C-480T polymorphism and the risk of acute myocardial infarction (AMI) as well as pre-hospital sudden cardiac death (SCD). METHODS: Seven hundred sudden or unnatural pre-hospital deaths of middle-aged (33-70 years, mean 53 years) Caucasian Finnish men were subjected to detailed autopsy (Helsinki Sudden Death Study). Genotype data were obtained for 682 men. RESULTS: In logistic regression analysis with age, body mass index, hypertension, diabetes, smoking and alcohol consumption as covariates, men with the TT genotype had an increased risk for SCD and AMI compared to CC carriers (OR=3.0, P=0.011; and OR=3.7, P=0.003). There was a significant age-by-genotype interaction (P<0.05) on the risk of SCD. Compared to CC genotype carriers, the association between the TT genotype and SCD was particularly strong (P=0.001) among men <53 years of age, but this association was non-significant among older men. This was mainly due to a strong association between the TT genotype and AMI due to severe coronary disease in the absence of thrombosis. Carriers of the TT genotype were more likely to have severe coronary stenoses (> or =50%) than men with the CT or CC genotype (P=0.019). CONCLUSIONS: The results suggest that HL C-480T polymorphism is a strong age-dependent risk factor of SCD in early middle-aged men.

The influence of hepatic lipase C-480T polymorphism on coronary flow reserve in young men is independent of the plasma cholesterol level.

BACKGROUND: The hepatic lipase (HL) gene C-480T promoter polymorphism affects gene transcription and enzyme activity and leads to CC, CT, and TT genotypes. Recently, HL expression was detected in macrophages. It has been postulated that HL might have a direct role in the pathogenesis of atherosclerosis without changes in the plasma profile. We hypothesized that the difference of plasma cholesterol level may not influence the effect of HL genotype on coronary reactivity. METHODS: A total of 108 young men (aged 34+/-5 years) were genotyped and divided into three groups. These groups contained 45, 49 and 14 men having either normal (4.9+/-1.2 mmol/L), mildly (5.5+/-0.8 mmol/L) or severely (7.8+/-1.9 mmol/L, subjects with familial hypercholesterolemia) elevated mean plasma cholesterol level, respectively. Myocardial blood flow (MBF) was measured at rest and during adenosine or dipyridamole-induced hyperemia with positron emission tomography using [(15)O] H(2)O. RESULTS: The effect of HL genotype on the indices of MBF was parallel within all cholesterol groups and therefore they were combined. In all subjects, basal flow did not differ between the genotypes. However, men with CC genotype had a significantly higher hyperemic blood flow (3.86+/-1.26 mLg(-1)min(-1) versus 3.20+/-1.38 mLg(-1)min(-1), p=0.007), higher coronary flow reserve (CFR, 4.80+/-1.77 versus 3.77+/-1.43, p=0.001) and lower coronary resistance during hyperemia (25.63+/-9.98 mmHg min g mL(-1) versus 35.00+/-23.95 mmHg min g mL(-1), p=0.003) than T allele carriers. In multivariate regression analysis, after adjustment for age, body mass index, serum lipids, blood pressure, adenosine or dipyridamole administration, and study group, HL polymorphism was an independent predictor of blood flow during hyperemia (p=0.016), coronary resistance (p=0.014), and CFR (p=0.005), respectively. CONCLUSIONS: The HL C-480 T polymorphism is associated with CFR, which is an early indicator of atherosclerosis, independently of the level of plasma cholesterol in young men.

The effect of mannan-binding lectin variant alleles on coronary artery reactivity in healthy young men.

Mannan-binding lectin (MBL) is a serum acute-phase protein and a complement component secreted by the liver. Its deficiency caused by point mutations in the MBL gene has recently been associated with severe atherosclerosis. In this study, we investigated the effect of MBL variant alleles on coronary artery reactivity, which is an early marker of coronary dysfunction and predicts the development of atherosclerosis and coronary artery disease. The study population consisted of 51 apparently healthy, normo- or mildly hypercholesterolemic young men. Myocardial blood flow was measured at baseline and during adenosine-induced hyperemia with positron emission tomography (PET), and MBL genotyping was performed using restriction fragment-length polymorphism. As a result, MBL variant alleles had no effect on coronary artery reactivity. This finding suggests that MBL deficiency is not an independent risk factor for coronary dysfunction and early atherogenic changes but rather a co-factor in the development of atherosclerosis. Thus, the connection of MBL variant alleles with environmental risk factors in atherosclerosis should further be assessed.

The effect of hormone replacement therapy on atherosclerotic severity in relation to ESR1 genotype in postmenopausal women.

OBJECTIVE: The atheroprotective action of estrogen is mediated by estrogen receptors (ESR) 1 and 2, expressed in atherosclerotic lesions. The effects of hormone replacement therapy (HRT) and ESR1 PvuII genotypes on atherosclerosis have not previously been studied prospectively in postmenopausal women. METHODS: We investigated the effect of HRT on the progression of atherosclerosis in a 5-year follow-up study of 88 postmenopausal women aged 45-71 years at baseline allocated into three groups based on the use of HRT. The HRT-EVP group (n=26) used sequential estradiol valerate (EV) plus progestin (P), the HRT-EV group EV alone (n=32), and a control group (n=30) was without HRT. The atherosclerosis severity score (AS) of the abdominal aorta and carotid arteries were determined by sonography and the ESR1 PvuII genotypes (P/P, P/p and p/p) by PCR. RESULTS: HRT, time and ESR1 PvuII genotype had a statistically significant or borderline significant main effect on AS during 5-year follow-up (P=0.004, P<0.001 and P=0.090, respectively), when analyzed by repeated measures analysis of variance. There was a significant genotype-by-treatment (HRT-EVP and control groups) interaction for AS (P=0.034). In response to HRT-EVP, subjects with P/P, compared with those with P/p and p/p genotypes, had a less increase in AS (1.61+/-1.14 vs. 1.71+/-1.27 vs. 2.43+/-1.27). Baseline AS as covariate in similar model does not change the significant interaction effect between HRT-EVP and control groups (P=0.036). But this effect was not found between HRT-EV and control groups. CONCLUSIONS: Our results suggest that the effect of HRT-EVP in postmenopausal women on progression of AS may be determined in part by the genotype of ESR1 PvuII.