Maternal Subpopulation Variances in Vaginal and Cesarean Section Delivery Methods Predicts

Excess Infant Mortality of Blacks in United States: Linked Birth/Infant Death Records, 2007-2016

O’Neill, L. 1,2 , Enwere, M. 1,3 , Pelaez, L.1, Dabney K. Dr. 1, Holmes, L. Jr. 1,4 Nemours Healthcare System for Children, Wilmington, DE 198031, Christopher Newport University, Newport News, Virginia2, Walden University, Minneapolis, Minnesota3, University of Delaware, Newark, Delaware, Biological Sciences Department4

Introduction

Background

• Racial disparities have been observed in infant mortality but the risk factors are not fully understood.

• In 2017, there were more vaginal deliveries (n = 2,621,010) relative to cesarean deliveries (n = 1,232,339) with the rates of vaginal deliveries declining and cesarean section delivery rates increasing.1

• In comparison to vaginal deliveries (VD), cesarean deliveries (CD) come with the increased risk of maternal and fetal complications.2

• Infant mortality is the death of an infant before their first birthday and is expressed as the rate of deaths per 1,000 live births.3

• In 2015, the Center for Disease Control and Prevention (CDC) reported that sudden unexpected infant death (SUID) represented 15% of all infant deaths in the United States (US).3

• There are 3 reported types of SUID cases: sudden infant death syndrome (SIDS), unknown cases, and accidental suffocation/strangulation in bed.1

• Available literature identified risk factors of both the mother (such as education, income, social stress and racial discrimination) and infant (such as birth weight and sex).

• Non-Hispanic black women have a higher rate of cesarean delivery (36%) compared to non-Hispanic white women (30.9%).4

• In 2017, the CDC reported that infant mortality rates were higher among non-Hispanic black women (11.4 deaths per 1,000 live births) relative to non-Hispanic white women (4.9 deaths per 1,000 live births).3

• The disproportion of infant mortality rates between black and white women within the US has more than doubled in the past decade.5

Study Aims

• The current study aimed to assess the exposure function of labor and delivery procedures as potential explanations for excess black/African American (B/AA) infant mortality relative to white infant mortality in the US.

Methods

• After an institutional review board (IRB) approval we conducted a study to assess the relationship between infant mortality and method of delivery depending on their racial heterogeneity.

Study Design

• A cross sectional ecologic non-experimental design was used to determine the dependence of labor and delivery methods with infant mortality, factors associated with infant mortality, and the period of prevalence.

Data Source

• Data from linked birth/infant death records from 2007 to 2016 were acquired from the National Health Statistics Center (NHSC) of the CDC.

Study Variables

• Variables assessed were maternal bridged race, maternal education, infant gender, infant year of death, and infant birth weight.

Study Analysis

• Chi squared statistics, incidence rate ratio and descriptive statistics were performed utilizing STATA 14.0.

• A standardized formula was used in computation of the period prevalence to determine whether or not the method of delivery, with respect to mortality, remained stable or changed.

Results

Study Limitations

• There was the potential for misclassification of the exposure variable (mortality) and unmeasured confounding

• Other limitations include misclassification of infant race and too small of a sample size in regard to certain racial groups, such as American Indian/Alaskan Native and Asian/Pacific Islander

Conclusion

• The cumulative infant mortality rate during 2007-2016 was 6.16 per 1,000.

• Rates varied by the method of delivery; infant mortality related to c-section (8.49 per 1,000) was higher relative to vaginal delivery (6.75 per 1,000), indicating a 1.74 per 1,000 infant mortality rate difference.

• Racial differences were observed highest among black/African American infants (11.41 per 1,000), intermediate among American Indian/Alaskan Native (8.32 per 1,000) and whites (5.19 per 1,000), and lowest among Asian/Pacific Islander (4.24 per 1,000).

• There was a 6% decreased risk in infant mortality among B/AA mothers with vaginal delivery.

• In comparison, infant mortality following vaginal delivery among white mothers were associated with a 32% decreased risk, incidence rate ratio (IRR) = 0.68, 95% confidence interval (CI) 0.67-0.69, p<0.001.

• Infant mortality varied by mother’s education regardless of the method of delivery.

• Among B/AA, either vaginal (9.8 per 1,000) or c-section (13.0 per 1,000) infant mortality was highest among mothers with less than a high school education, while mothers with a post college degree experienced lower vaginal (7.46 per 1,000) and cesarean (6.45 per 1,000) infant mortality rates.

• White mothers with less than a high school education experienced lower vaginal (4.19 per 1,000) or cesarean section (10.93 per 1,000) infant mortality rates. While mothers with a post college degree were associated with the lowest rate of vaginal (2.5 per 1,000) delivery, and cesarean section (3.9 per 1,000)

• Regardless of the method of delivery, race or education, the risk of dying as an infant was higher for males compared to their female counterpart.

• Overall, the findings from this research would further support the racial disparities that exist between labor and delivery methods and infant mortality outcomes.

References

1. “Births - Method of Delivery.” Centers for Disease Control and Prevention, U.S. Department of Health & Human Services, 20 Jan. 2017, www.cdc.gov/nchs/fastats/delivery.htm

2. “Complications of Cesarean Deliveries.” Medscape, WebMD, www.medscape.org/viewarticle/512946_4

3. “Reproductive Health.” Centers for Disease Control and Prevention, U.S. Department of Health & Human Services, 27 Mar. 2019, www.cdc.gov/reproductivehealth/maternalinfanthealth/infantmortality.htm

4. Martin, J.A., M.P.H., Hamilton, B.E., Ph.D., Osterman M.J.K., M.H.S., Driscoll, A.K., Ph.D., Drake, P., M.S. “Births: Final Data for 2017.” National Vital Statistics Reports, vol. 67, no. 8, 7 Nov. 2018.

5. Matthews, T. J., MacDorman, M.F., PhD, Thomas, M.E., PhD. “Infant Mortality Statistics from the 1999 Period: Linked Birth/Infant Death Data Set.” National Vital Statistics Reports, vol. 64, no. 9, 6 Aug. 2015, doi:10.1037/e558952006-001.

Detection of bacterial contamination by nitroreductase

Ryan Saal, Zoe Stauffer, and Shreya Ganta

Faculty Sponsor: Dr. Todd

Gruber, Department

of Molecular Biology and Chemistry

Abstract

Nitroreductase is a bacterial enzyme not present in mammalian cells. In previous research, we engineered a nitroreductase to release dyes, indicators and drugs in a cellspecific manner. Furthermore, we are extending this work into the area of bacterial detection. Because nitroreductase is absent in mammalian cells, this is a unique activity to identify contamination in clinical samples. With further development, we aim to apply this method to the medical field in terms of detecting bacteria in bodily fluids.

We find that E. coli nitroreductase, as natively expressed in the clinically-isolated E. coli K-12 strain, reacts with masked bis(2-nitro-N-methyl imidazolyl)-Oregon Green (NMOG) to release the fluorophore Oregon Green. This method successfully detects E. coli K-12 strain using a fluorescence plate reader. Additionally, qualitative analysis using blue light supports our findings. Limits of detection have also been analyzed for this process.

Ryan Saal is a second year student at Christopher Newport University and is majoring in Biochemistry with a minor in Leadership Studies. He is a Presidential Scholar while also being a member of both the Honors College and the President’s Leadership Program. On campus, Ryan is involved in CNU Residence Life as a Resident’s Assistant and the Center for Academic Success as an Organic Chemistry tutor. In his free time, he enjoys undergraduate research and singing in an Acapella group called University Sounds. He hopes to one day attend medical school and become a physician.

Zoe Stauffer is a junior at Christopher Newport University. She is majoring in Cellular, Molecular, and Physiological Biology with minors in Leadership and Spanish. She is a member of both the Presidential Leadership program and Pre-Med Scholars program on campus. Zoe is also a captain on the track and field team and competes in the heptathlon. In her free time, she enjoys anything outdoors such as skiing, scuba diving, and hiking. Her career aspiration is to be a physician.

Shreya Ganta is a second year student at Christopher Newport University and is majoring in Cellular, Molecular and Physiological biology with a minor in Leadership Studies. She is a Presidential and Riverside Medical Group Scholar while also being a member of both the Honors College and President’s Leadership Program. Shreya is an active brother of Alpha Phi Omega and is also the Social Media Chair for the bone marrow registry awareness club, Be The Match. She is a nationally ranked Division III golfer. In her free time, Shreya enjoys undergraduate research, volunteering, and going to the beach. She hopes to attend medical school and become a physician.

Abstract

Detection of bacterial contamination by nitroreductase

Zoe Stauffer, Ryan Saal, Shreya Ganta, Todd D. Gruber Department of Molecular Biology and Chemistry, Christopher Newport University, Newport News, VA 23606

Nitroreductase is a bacterial enzyme not present in mammalian cells. In previous research, we engineered a nitroreductase to release dyes, indicators and drugs in a cellspecific manner. We are extending this work into the area of bacterial detection. Because nitroreductase is absent in mammalian cells, this is a unique activity to identify contamination in clinical samples. With further development, we aim to apply this method to the medical field in terms of detecting bacteria in bodily fluids. We find that E. coli nitroreductase, as natively expressed in the clinically-isolated E. coli K-12 strain, reacts with masked bis(2-nitro-N-methyl imidazolyl)-Oregon Green (NMOG) to release the fluorophore Oregon Green. This method successfully detects E. coli K-12 strain using a fluorescence plate reader. Additionally, qualitative analysis using blue light supports our findings. We are in the process of analyzing limits of detection for this process.

Background

Bacteria make up the vast microbial world that carry out tasks ranging from producing oxygen to causing disease. Bacterial contamination is a recurrent problem in both clinical and laboratory settings. Thus, there is a need for an efficient and adequate detection process. We recently showed that transfected nitroreductase, an enzyme natively found in bacteria but not mammalian cells, could be used as a unique enzymatic activity in mammalian culture to deliver various small molecules (Gruber 2018). Here, we examine whether a nitroreductase activated masked fluorophore can be used to detect bacteria.

Bis(2-nitro-N-methyl imidazolyl)-Oregon Green (NMOG) is non-fluorescent, and nitroreductase converts it to the fluorescent dye Oregon Green.

Standard Fluorescent Analysis

A standard linear progression was calculated to show the relationship between OG concentration and relative fluorescent units. The data was used to determine the amount of NMOG required for bacterial analysis. RFU of bacterial assays using 5 µM NMOG did not exceed 2000. More NMOG may be required for greater detection.

NMOG Interacts with Nitroreductase in

Bacteria

Red and Blue: 5 µM NMOG, 100 mM HEPES 100 µM NADH Green and Purple : 5 µM NMOG, 100 mM HEPES 100 µM NADH, 10 µL K-12 sample

Qualitative Analysis with Blue Light

We find that E. coli K-12 samples can be distinguished from samples without bacteria after addition of NMOG using both quantitative and qualitative methods. Analysis reveals that in the absence of bacteria, the RFU is negligible, seen as the red and blue lines. Upon the addition of bacteria, a robust signal is emitted. After three hours, the RFU reaches 1200, seen as the purple and green lines. Qualitative analysis was carried out using excitatory blue light. An amber emission filter revealed the green fluorescence in wells that correlate with the purple and green lines. No fluorescence is seen in wells that correlate with the red and blue lines.

Limits of Detection

Red: 5 µM NMOG, 100 mM HEPES, 1 µM NADH, 10 µL k-12 Blue: 5 µM NMOG, 100 mM HEPES, 1 µM NADH, 1/10th k-12 Green: 5 µM NMOG, 100 mM HEPES, 1 µM NADH, 1/100th k12

An analysis of nitroreductase limits of detection reveals that the method efficiently detects 10 µL k-12 sample, with an RFU of 3000. The solutions were diluted. The 1/10th dilution was detected, though the RFU did not surpass 250. The RFU for dilutions of 1/100th the sample or less was not sufficiently detected. The normal 10 µL k-12 sample (red line) plateaus after 2 hours. This data suggests that the NMOG or NADH (reaction catalyst) was depleted. Optical density was used to estimate the amount of k-12 bacterial cells in the sample as a benchmark. The next step would be getting a more accurate quantification of bacterial cells for analysis.

Summary

• The nitroreductase activated masked fluorophore was successfully used in the detection of k-12 bacteria

• Quantitative analysis showed that samples containing the bacteria emitted an RFU around 1200 after three hours

• The detection technique was examined qualitatively using blue light which adequately distinguished wells with bacteria from wells without bacteria

• Limits of detection for the technique are underway

References

Gruber, T. D., Krishnamurthy, C., Grimm, J. B., Tadross, M. R., Wysocki, L. M., Gartner, Z. J., Lavis, L. D. ACS Chemical Biology 13(10), 2888-2896 (2018).

Shiferaw, T., Beyene, G., Kassa, T., Sewunet, T. Annals of Clinical Microbiology and Antimicrobials 12(39), (2013).

Acknowledgments

This work was supported by startup funds to TDG from CNU and a 2018 Small Project Research Grant from the Virginia Academy of Sciences.