Tuberculosis Infection in Women of Reproductive Age and Their Infants

Objective The overall objective of this project is to investigate longitudinal patterns and consequences of tuberculosis (TB) infection in a prospective cohort of women recruited during pregnancy. The relation between maternal TB infection and pregnancy outcomes, as well as infant health and development, will be evaluated over time.

Specific aims

1. To determine whether maternal TB infection is associated with adverse pregnancy outcomes

2. To determine the excess risk of incident active TB during pregnancy and post-partum, with regard to the prevalence of latent TB infection at registration in antenatal care

3. To investigate long-term health and survival in infants in relation to maternal TB and adverse pregnancy outcomes

Methods Study setting and participants This project will be conducted in public health facilities in the city of Adama, Central Ethiopia. Participants will be recruited and followed at antenatal care (ANC) clinics until delivery. After delivery their children will be followed at clinics for extended program for immunization (EPI); continued follow-up of the mothers after the first post-natal visit will also take place in these clinics.

All study procedures, including interviews, physical examination, and collection of informed consent, will be performed by regular health facility staff (nurses and midwives) in the respective clinics. Biological samples including blood and sputum will be collected at the respective health facility laboratory. Study samples will be transported to the Adama Regional Laboratory. At this laboratory, additional analyses and storage of plasma will be done.

For supervision and monitoring of data collection, as well as for data entry, the project will use staff and infrastructure in a field research station which has been established in Adama since 2010 by the research group.

Study procedure Both scheduled and unscheduled study visits can occur for participants in this project. Scheduled study visits coincide with routine antenatal and postnatal visits and visits at the EPI clinics for the infant's immunization following National guidelines. Unscheduled visits will occur in case of symptoms suggestive of active TB (according to the study definitions) in the pregnant/postnatal/non-pregnant woman, or in the child, at any time in between these routine visits.

Antenatal care visits At inclusion, structured information on socio-demographic conditions, education, occupation, and poverty indicators will be collected; as well as medical history (in particular for obstetric, gynecological and TB-related details). Physical and obstetric examination will be performed. Apart from the study procedures and investigations, participants will receive care according to current Ethiopian ANC guidelines.

At the inclusion visit blood and urine samples will be obtained for routine care and for study specific investigations from all participants. At the health facility laboratory, the following analyses will be performed: HIV rapid test, malaria microscopy and urine dipstick. Ten mL of venous blood will be collected. This sample will be used for analysis of complete blood count, blood/Rh group, syphilis serology, creatinine and fasting glucose. The remaining plasma will be aliquoted and stored at -80° C for research analyses that will be performed at the completion of inclusion. Blood will be collected separately for QuantiFERON TB Plus (QFT) testing, in order to determine the prevalence of LTBI. For the last 500 women included in the study, QFT testing will be repeated at the 3rd antenatal care visit. From HIV positive women blood for viral load and CD4 count will be drawn at each scheduled visit pre- and post-delivery except from 2nd and 3rd antenatal care visit.

Participants will be followed up four times in accordance with WHO recommended focused antenatal visits until delivery or term of pregnancy. They will also be instructed to contact the study clinic in between these visits in case of health-related events. In case of delivery (or other unintended termination of pregnancy) in another place than any of the study health facilities, they will be asked to communicate this to their study site for further follow-up examinations.

All participants with suspected active TB according to the study definition (irrespective of Quantiferon results) at enrolment and/or during follow up will be asked to submit samples for bacteriological TB investigations. These will encompass liquid culture, smear microscopy and PCR (using Xpert MTB/RIF technique). All these tests will be performed on two consecutive morning sputum samples. Lymph node aspirates will be obtained from participants with enlarged peripheral lymph nodes. Moreover, acquisition of blood for QuantiFERON testing will be repeated for all women investigated for suspected active TB.

Stored aliquots of plasma will be analyzed at the end of inclusion for potential blood biomarkers of TB infection during pregnancy. These analyses will be performed using ELISA and/or Luminex technique, and they will include the following markers of immune activation and inflammation: C-reactive protein (CRP), neopterin, soluble urokinase-plasminogen activator receptor (suPAR), interferon-inducible protein 10 (IP-10).

Postpartum follow-up visits for women Further follow-up visits for women will be scheduled for 6 weeks and 9 months after delivery or termination of pregnancy, coinciding with the routine child visits for immunization. The first postnatal visit will take place at the ANC clinic, whereas all subsequent visits will take place at the respective EPI clinics (where infant follow-up will also occur). At the 6 week postnatal visit details on delivery outcome will be recorded.

For investigation of long-term health effects after delivery, participants will be asked to come for further annual visits with similar assessments until 54 months after termination of pregnancy. At all follow-up visits symptoms and signs on physical examination will be recorded using structured questionnaires.

At inclusion, and repeatedly on scheduled follow-up visits, participants will be informed about symptoms and signs that may indicate active TB. Study subjects will be instructed to contact the study site clinic (ANC during antenatal period, EPI during postnatal period) directly if they develop such symptoms for unscheduled study visits (in order to assess incident active TB during follow-up).

At all visits, whether scheduled or unscheduled, bacteriological testing (smear microscopy, Xpert MTB/RIF assay, and liquid culture) will be performed according to the study protocol in case study criteria for suspected active TB are met. These tests will be performed on two morning sputum samples for women; in addition, lymph node aspirates may be sent for TB analysis in case of suspected lymphadenitis.

In case study participants do not come for their scheduled study visits they will be contacted by phone by the study site staff and/or the research study team. If the participant cannot be reached, her contact person will be contacted by phone. At mid-time in between the scheduled visits, women will be contacted by phone by a member of the local research team to assess whether any health events or symptoms compatible with TB have occurred.

Follow-up visits for infants If consent has been provided by the mother, infants will be registered and included in a separate infant follow-up cohort as soon as possible after birth. Birth details, including delivery method, weight, length, APGAR score, and head circumference will be registered at inclusion of the infant. Follow-up of included infants will take place in the EPI clinic at the respective study health facilities. Follow-up visits will be scheduled at the same time interval as the mother, i.e. at 6 weeks and 9 months and subsequently at annual intervals congruent with the mothers until four and a half year after birth. The two first visits coincide with routine immunization visits. On all study visits, parameters of growth, nutritional and neurological development will be recorded using structured questionnaires. In addition, data on socio-demographic characteristics, health-related events and feeding details will be recorded, as well as data on known or suspected exposure to TB.

At inclusion, and repeatedly on scheduled follow-up visits, caretakers will be informed about symptoms and signs that may indicate active pediatric TB. Study infant caretakers will be instructed to contact the study site EPI clinic directly if the infants develop such symptoms for unscheduled study visits (in order to assess incident active TB during follow-up).

If criteria for suspected TB are met at any time during follow-up bacteriological investigations (liquid culture, PCR and microscopy) will be performed on gastric lavage specimens (or cerebrospinal fluid in case of suspected meningitis; or fine-needle lymph node aspirates in case of peripheral lymphadenopathy). These investigations will be performed by trained health care staff at the Department of Pediatrics, Adama Regional Hospital.

Stored aliquots of plasma will be analyzed at the end of inclusion for potential blood biomarkers of TB in young children. These analyses will be performed using ELISA and Luminex technique, and they will include the following markers of immune activation and inflammation: C-reactive protein (CRP), neopterin, soluble urokinase-plasminogen activator receptor (suPAR).

In case infant participants do not come for their scheduled study visits their caretakers will be contacted by phone by the study site staff and/or the research study team. If the caretaker cannot be reached, the contact person will be contacted by phone. At mid-time in between the scheduled visits, caretakers will be contacted by phone from a study investigator to assess whether any health events or symptoms compatible with TB have occurred in the infant.

Study definitions Suspected adult active TB is defined as any of the following symptoms or findings: Cough, fever, night sweating, weight loss (except following delivery), chest pain, reduced Karnofsky performance score (after pregnancy), peripheral lymph node enlargement, abdominal distension and/or abdominal mass (during study periods without pregnancy), pyuria and/or hematuria.

Suspected pediatric active TB is defined as any of the following symptoms or findings: Cough, fever, night sweating, chest pain, peripheral lymph node enlargement, abdominal distension and/or mass, weight loss, poor growth development, failure to thrive, acute or chronic malnutrition, neck stiffness, seizures, and loss of consciousness Latent TB infection is defined as a positive QuantiFERON® TB Gold Plus result (according to the manufacturer's reference range), in the absence of confirmed active TB and/or national TB guideline criteria for clinically diagnosed TB.

Confirmed active TB is defined as any subject with a positive bacteriological result (as per the national TB guideline), irrespective of clinical manifestations.

Clinically diagnosed active TB is defined as any subject without positive bacteriological results for TB, but who fulfils national TB guideline criteria for clinically diagnosed TB, and who are referred for anti-TB treatment.

Adverse pregnancy outcomes are defined as maternal or infant death following inclusion until 3 months after unintended termination of pregnancy; preterm birth (before 37 gestational weeks); spontaneous abortion; PE (as defined by the International Society for the Study of Hypertension in Pregnancy; 25); IUGR; peri-partum hemorrhage.

Intra uterine growth restriction is defined as growth deviation of weight below 2 SD (-22% or below 3rd quartile) at birth.

Post-partum TB is defined as confirmed or clinically diagnosed active TB within the 6 months period following termination of pregnancy.