The Zambian Preterm Birth Prevention Study (ZAPPS): Cohort characteristics at enrollment [version 2; referees: 2 approved]

Background:Sub-Saharan Africa bears a disproportionate burden of preterm birth and other adverse outcomes. A better understanding of the demographic, clinical, and biologic underpinnings of these adverse outcomes is urgently needed to plan interventions and inform new discovery.  Methods:The Zambian Preterm Birth Prevention Study (ZAPPS) is a prospective observational cohort established at the Women and Newborn Hospital (WNH) in Lusaka, Zambia. We recruit pregnant women from district health centers and the WNH and offer ultrasound examination to determine eligibility. Participants receive routine obstetrical care, lab testing, midtrimester cervical length measurement, and serial fetal growth monitoring. At delivery, we assess gestational age, birthweight, vital status, and sex and assign a delivery phenotype. We collect blood, urine, and vaginal swab specimens at scheduled visits and store them in an on-site biorepository. In September 2017, enrollment of the ZAPPS Phase 1 – the subject of this report – was completed. Phase 2 – which is limited to HIV-uninfected women – reopened in January 2018.  Results:Between August 2015 and September 2017, we screened 1784 women, of whom 1450 (81.2%) met inclusion criteria and were enrolled. The median age at enrollment was 27 years (IQR 23–32) and thee median gestational age was 16 weeks (IQR 13–18). Among parous women (N=866; 64%), 21% (N=182) reported a prior miscarriage, 49% (N=424) reported a prior preterm birth, and 13% (N=116) reported a prior stillbirth. The HIV seroprevalence was 24%. Discussion:We have established a large cohort of pregnant women and newborns at the WHN to characterize the determinants of adverse birth outcomes in Lusaka, Zambia. Our overarching goal is to elucidate biological mechanisms in an effort to identify new strategies for early detection and prevention of adverse outcomes. We hope that findings from this cohort will help guide future studies, clinical care, and policy

TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in <i>Drosophila</i>

<div><p>The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using <i>Drosophila</i> genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs.</p></div

High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.

<p>(A) Levels of dTSPO mRNA in the heads of elav-GS/+; TSPO-IR/+ male and female flies with or without RU486 dsRNA induction, n = 3 groups of flies tested. Data presented as mean ± SEM. *** p < 0.001. (B-D) Gene switch and control flies with or without RU486 (elav-GS/+ = elav-GeneSwitch and TSPO-IR/+ = UAS-dTSPO-RNAi). To induce gene switch, the flies were raised on regular food with 50 μl of 4 mg/ml RU486 added on the surface of the food in vials for three days. (B) Sensitivity of elav-GS/+;TSPO-IR/+ flies to 44% ethanol vapor with and without RU486, half sedation time with RU486 was 16.0±0.6 min and without RU486 was 23.3±1.5, p < 0.001, n = 10. (C) Sensitivity of flies harboring elav-GS/+ with or without RU486 exposed to 44% ethanol vapor, half sedation time with RU486 was 14.0±0.5 min and without RU486 was 15.3±0.9, p > 0.05, n = 13, vials tested. (D) Sensitivity of flies harboring only TSPO-IR/+ exposed to 44% ethanol and with and without RU486, half sedation time with RU486 was 25.0±2.0 min and without RU486 was 22.5±1.0, p > 0.05, n = 10.</p

Systemic loss of dTSPO inhibits the development of ethanol tolerance.

<p>Tolerance is revealed as a longer period required for sedation at a second exposure to 54% ethanol solution vapor. (A) Differential sedation from first versus second ethanol exposure of <i>tspo</i> +/+ versus <i>tspo</i>-/- flies: For <i>tspo</i> +/+ flies half sedation time for first exposure was 12.0±0.5 and for second exposure was 26.4±1.9, p < 0.001. For <i>tspo</i>-/- flies half sedation time for first exposure was 12.3±0.3 and for second exposure was 15.0±1.6, p = 0.11. Difference between second exposure sedation of <i>tspo</i> +/+ and <i>tspo</i>-/- flies, p < 0.001, n = 8, vials tested. (B) Differential sedation from first versus second exposure of elav-GS/+; TSPO-IR/+ flies with or without RU486 induction, n = 12. Gene switch was induced as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005366#pgen.1005366.g002" target="_blank">Fig 2</a>. (C) dTSPO mRNA levels in the heads of tspo+/+ male flies after exposure to 54% ethanol vapor showing progressive loss of dTSPO mRNA for the first 4 hours after exposure followed by partial recover to original levels, n = 3 groups of flies tested. (D) dTSPO mRNA levels in the bodies of male <i>tspo</i>+/+ flies after exposure to 54% ethanol vapor showing a progressive increase up to 4 hours after exposure followed by a decline (n = 3). Data presented as mean ± SEM.</p

Comparable ethanol sensitivity in female <i>tspo</i>-/- and <i>tspo</i> +/+ flies.

<p>(A) With 34% ethanol vapor, half sedation time for <i>tspo</i>+/+ was 23.0±2.5 min and for <i>tspo</i>-/- was 20.0±1.7 min, p > 0.05, n = 14 vials tested. (B) With 44% ethanol vapor half sedation time for <i>tspo</i>+/+ was 11.0±0.5 min and for <i>tspo</i>-/- was 11.0±0.5 min, p > 0.05, n = 10. (C) Recovery after withdraw from exposure to 44% ethanol vapor, half recovery rate for <i>tspo</i>+/+ was 16.0±1.2 min and for <i>tspo</i>-/- was 18.0±1.2 min, p > 0.05, n = 8. Data presented as mean ± SEM.</p

Depletion of dTSPO in neurons suppresses caspase activity which is sufficient to increase ethanol sensitivity.

<p>Gene switch was accomplished as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005366#pgen.1005366.g002" target="_blank">Fig 2</a>. (A) Caspase 3/7 activity was moderately reduced in heads of elav-GS/+; TSPO-IR/+ or elav-GS/+; p35/+ (elav-GeneSwitch plus UAS-p35) flies when the dTSPO dsRNA or p35 were induced with RU486. All groups were measured twice, and data presented as mean. Since the TSPO-IR and P53 products are only expressed in neurons, but the caspase activity was assayed in whole heads, the ~20% decrease in caspase 3/7 underrepresents the extent of caspase reduction in neurons. This is demonstrated by whole body knockout of TSPO in which the relative whole body caspase 3/7 activity of <i>tspo</i> +/+ flies was 1.000±0.008 and of <i>tspo</i>-/- flies was 0.078±0.015, p < 0.001, n = 4. (B) Induction of caspase inhibitor p35 with RU486 significantly increased sensitivity to 34% ethanol vapor, Chi Square log rank test, p = 0.0006, n = 8 vials tested. (C) Flies harboring only UAS-p35 (p35/+) exposed to 34% ethanol vapor with or without RU486 were not different, Chi Square log rank test, p = 0.846, n = 12. Data presented as mean ± SEM.</p

Increased ethanol sensitivity in male <i>tspo</i> mutant flies.

<p>Sensitivity to acute ethanol sedation was increased in <i>tspo</i>-/- flies compared with <i>tspo</i>+/+ flies (A-C). (A) With 34% ethanol solution, half sedation time for <i>tspo</i>+/+ was 30.6±1.9 min and for <i>tspo</i>-/- was 17.8±1.5 min, p = 0.01, n = 13 vials tested. (B) With 44% ethanol solution, half sedation time for <i>tspo</i>+/+ was 14.3±2.2 min and for <i>tspo</i>-/- was 13.8±0.6 min, p > 0.05, n = 8 vials tested. (C) With 54% ethanol solution, half sedation time for <i>tspo</i>+/+ was 11.3±0.8 min and for <i>tspo</i>-/- was 11.3±0.3 min, p > 0.05, n = 8. (D). The rate for recovery after ethanol withdraw was slower in <i>tspo</i>-/- than <i>tspo</i>+/+ flies, half recovery time for <i>tspo</i>+/+ was 20.4±2.1 min and for <i>tspo</i>-/- was 28.1±3.6 min, p > 0.05, n = 8. Data presented as mean ± SEM.</p

Improving preterm newborn identification in low-resource settings with machine learning.

BACKGROUND:Globally, preterm birth is the leading cause of neonatal death with estimated prevalence and associated mortality highest in low- and middle-income countries (LMICs). Accurate identification of preterm infants is important at the individual level for appropriate clinical intervention as well as at the population level for informed policy decisions and resource allocation. As early prenatal ultrasound is commonly not available in these settings, gestational age (GA) is often estimated using newborn assessment at birth. This approach assumes last menstrual period to be unreliable and birthweight to be unable to distinguish preterm infants from those that are small for gestational age (SGA). We sought to leverage machine learning algorithms incorporating maternal factors associated with SGA to improve accuracy of preterm newborn identification in LMIC settings. METHODS AND FINDINGS:This study uses data from an ongoing obstetrical cohort in Lusaka, Zambia that uses early pregnancy ultrasound to estimate GA. Our intent was to identify the best set of parameters commonly available at delivery to correctly categorize births as either preterm (94% of newborns and achieved an area under the curve (AUC) of 0.9796. CONCLUSIONS:We identified a parsimonious list of variables that can be used by machine learning approaches to improve accuracy of preterm newborn identification. Our best-performing model included LMP, birth weight, twin delivery, HIV serostatus, and maternal factors associated with SGA. These variables are all easily collected at delivery, reducing the skill and time required by the frontline health worker to assess GA. TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT02738892

Highly diverse anaerobe-predominant vaginal microbiota among HIV-infected pregnant women in Zambia.

Vaginal dysbiosis has been shown to increase the risk of some adverse birth outcomes. HIV infection may be associated with shifts in the vaginal microbiome. We characterized microbial communities in vaginal swabs collected between 16-20 gestational weeks in the Zambian Preterm Birth Prevention Study to investigate whether HIV and its treatment alter the microbiome in pregnancy. We quantified relative abundance and diversity of bacterial taxa by whole-genome shotgun sequencing and identified community state types (CST) by hierarchical clustering. Associations between exposures-HIV serostatus (HIV+ vs HIV-) and preconceptional ART (ART+ vs ART-)-and microbiome characteristics were tested with rank-sum, and by linear and logistic regression, accounting for sampling by inverse-probability weighting. Of 261 vaginal swabs, 256 (98%) had evaluable sequences; 98 (38%) were from HIV+ participants, 55 (56%) of whom had preconceptional ART exposure. Major CSTs were dominated by: L. crispatus (CST 1; 17%), L.] iners (CST 3; 32%), Gardnerella vaginalis (CST 4-I; 37%), G. vaginalis & Atopobium vaginae (CST 4-II; 5%), and other mixed anaerobes (CST 4-III; 9%). G. vaginalis was present in 95%; mean relative abundance was higher in HIV+ (0.46±0.29) compared to HIV- participants (0.35±0.33; rank-sum p = .01). Shannon diversity was higher in HIV+/ART+ (coeff 0.17; 95%CI (0.01,0.33), p = .04) and HIV+/ART- (coeff 0.37; 95%CI (0.19,0.55), p < .001) participants compared to HIV-. Anaerobe-dominant CSTs were more prevalent in HIV+/ART+ (63%, AOR 3.11; 95%CI: 1.48,6.55, p = .003) and HIV+/ART- (85%, AOR 7.59; 95%CI (2.80,20.6), p < .001) compared to HIV- (45%). Restricting the comparison to 111 women in either CST 3 (L. iners dominance) or CST 1 (L. crispatus dominance), CST 3 frequency was similar in HIV- (63%) and HIV+/ART- participants (67%, AOR 1.31; 95%CI: (0.25,6.90), p = .7), but higher in HIV+/ART+ (89%, AOR 6.44; 95%CI: (1.12,37.0), p = .04). Pregnant women in Zambia, particularly those with HIV, had diverse anaerobe-dominant vaginal microbiota