The Aetiology of Neonatal Infection in South Asia (ANISA) uses standard and molecular diagnostic tests for detecting the etiology of community-acquired infections. ANISA staff also collect pre- and postpregnancy information for identifying associated risk factors for possible serious bacterial infection (pSBI) in neonates. These data are collected alongside bimonthly pregnancy surveillance, birth surveillance and surveillance of newborns for pSBI. Young infants are followed up to 59 days of age. Blood and nasopharyngeal-oropharyngeal (NP-OP) swabs are collected from physician-confirmed pSBI cases. Detailed study activities have been described elsewhere in this supplement.1–4 Matiari, Pakistan, was chosen as a study site for ANISA due to its high neonatal mortality rate and the presence of an established community surveillance system.5–8
STUDY SITE AND POPULATION
ANISA is conducted in 10 union councils (UCs) in Matiari District, located 185 km north of Karachi (Fig. 1). The study area covers a catchment population of 350,000 with approximately 55,000 married women of reproductive age (MWRA), aged 13–49 years and an annual birth cohort of 8500. One field office is located in Matiari city and another in Hala city, 35 km from Matiari. The site data management unit and site laboratory are located in Matiari city.
Matiari is mainly a rural area, and the primary economic activity of its inhabitants is agriculture. The existing health care infrastructure includes primary, secondary and tertiary care facilities.The Matiari study site has 3 rural health centers, 6 basic health units, 1 dispensary and around 220 lady health workers (LHWs). The rural health centers are the primary and secondary care facilities, whereas Taluka headquarters and district headquarters are the referral/tertiary care hospitals. LHWs are community-based females employed by Pakistan’s Ministry of Health who deliver maternal and child health services at the household level. LHWs also serve as a link between communities and tertiary level health facilities.
ANISA pilot activities in Matiari were initiated in July 2011 and the site became incorporated into the main study in March 2012 after fulfilling set criteria.4 These criteria included employment and activation of a minimum of 80% of community health workers (CHWs) and their supervisors, active bimonthly MWRA surveillance in place with 5% of CHW visits observed by a supervisor, 80% of CHW referrals validated by study physicians, successful clinical specimen collection from more than 80% of physician-confirmed pSBI cases and blood collection volume of >1 mL in 80% of phlebotomies conducted.
Staff recruitment started in May 2011. The study team includes 60 CHWs, 18 team leaders, 8 study physicians, 8 phlebotomists and 2 field supervisors. The study area originally was composed of 8 UCs; 2 more UCs were added at the time of graduation. With the expansion of the study area, an additional 60 CHWs, 2 physicians and 2 phlebotomists were recruited in July 2012. Each team is led by a research associate with either a master’s degree or a minimum of 14 years of education and with experience of working in the community. The team leader is responsible for supervision of surveillance activities and counseling of caregivers who refuse to provide clinical specimens from selected young infants.
The CHWs have a minimum of 12 years of education, are residents of the study area and are fluent in Sindhi, the local language. They are responsible for registration of pregnant women and follow-up until birth, 10 subsequent postnatal visits to registered newborns, newborn screening for pSBI and referral of cases to study physicians. The study physicians are registered practitioners with at least 1 year of clinical experience. They are responsible for confirmation of clinical signs of pSBI, enrollment of cases and collection of clinical specimens from confirmed pSBI cases. Our phlebotomists are proficient in drawing blood from young infants in hospital settings and have been successfully collecting blood in the field. The 2 field supervisors are responsible for ensuring data quality and supervision of overall surveillance activities. The project workflow at Matari site is detailed in Figure 2.
The ANISA study coordination team trained 2 master trainers for CHWs and 2 for study physicians from each ANISA site. These master trainers trained the CHWs and team leaders of the Matiari site through a 12-day workshop. The training focused on communication skills, antenatal and newborn care, identifying signs of pSBI in young infants, clinical assessment of newborns and understanding the data capture forms. The World Health Organization’s Integrated Management of Childhood Illness curriculum,9 role-plays and video aids were used for explaining different study activities to the CHWs. Refresher training is conducted every 2 months to reinforce proper and timely text message notification of newborn registration and control enrollment. A senior anthropologist from The Aga Khan University (AKU), Pakistan, conducted a 1-day training course for study physicians on communication and counseling in the consent process. We also conducted a 3-day training course for the physicians on study protocol and surveillance activities, and they received quarterly refresher training from AKU faculty. The phlebotomists are trained on collection and storage of blood and NP-OP specimens every 2 months.
Ethical approval was obtained from World Health Organization, and site-specific approval has been obtained from the AKU Ethical Review Committee in 2011 and in subsequent years.
CHALLENGES IN THE IMPLEMENTATION OF THE ANISA PROTOCOL AND REMEDIAL ACTIONS
Low Rate of Newborn Registration Within 24 hours of Birth
During the pilot phase, the rate of newborn registration within 24 hours of birth was low (17%) in Matiari. To facilitate early identification of pregnancy outcomes, ANISA’s Technical Advisory Group revised the target to at least 80% newborn registration within 24 hours of birth.
In response, the Matiari site team made adjustments to its field teams and reallocated 32 CHWs along with 8 vehicles to birth registration activities. Additional staff were hired after the approval of a supplementary budget. These adjustments made additional human resources available for birth registration. To further improve the early birth registration rate, MWRA surveillance teams also provide information on pregnancy outcomes (Fig. 3).
At the Matiari site, about 65% of births take place at health care facilities, and approximately one third of these facilities are situated outside the study area. About 30%–40% of pregnant women in the study area travel to these facilities for delivery and newborn care. It is not feasible for the birth registration teams to visit newborns both within and outside the study area. To overcome this problem, 2 mobile birth registration teams were formed and tasked with registering births that took place at health facilities outside the study area. Simultaneously, staff at the facilities were requested to notify the project office of every live birth as soon as possible through mobile phones.
Liaison between study staff and families of pregnant women, LHWs and traditional birth attendants (TBAs) is well established, and 2 staff members are stationed at the study office to receive their birth notification calls. In addition, LHWs, TBAs and the families of pregnant women receive prepaid phone cards worth 100 Pakistani Rupees (~US$ 1.00) for every birth notification. In areas where LHWs or TBAs are not available, residents of the villages with access to mobile phones are requested to notify study staff of births, and the cost of calling is reimbursed. The prepaid phone card is also used by parents to inform the study staff of any illness among enrolled young infants. Office staff, in turn, relay information to the mobile surveillance team to organize a home visit. Study staff also make calls to pregnant women and inquire about pregnancy outcomes. Of 13,753 calls received so far, 71% were made by parents, 11% by TBAs and 8% by LHWs. Improving early birth registration has a positive impact on early enrollment of pSBI cases and facilitates tracking of the incidence of illness among young infants. We also have instituted mandatory documentation of reasons for failure (approximately 25%–30%) to register newborns within 24 hours of birth, which has led to accountability and improved performance of study teams (Fig. 4A).
One challenge that remains is the limited working hours for field staff, from 9:00 am to 5:00 pm. Field travel after sunset is not advisable because of security concerns. Cultural barriers in rural areas, such as unwillingness to remain outdoors after sunset, also serve as major obstacles in extending working hours. Therefore, teams are unable to capture infants born after working hours until the next morning.
About 12%–14% of births can be missed due to holidays. To overcome this problem, 8 birth registration teams work on holidays, resulting in a 10% increase in birth registration. The Matiari site team has managed to register 62% of births within 24 hours (Fig. 4B) against the targeted 80%.
Blood and NP-OP Specimen Collection From Enrolled pSBI Cases
CHWs screen the young infants on each of the 10 follow-up visits for signs of pSBI.1 If a CHW detects any one of the signs, she refers the infant to study physicians. The study physician validates the identified case and refers the infant to the nearest health care facility for treatment as soon as possible. In cases where the family is unable to reach the health facility due to lack of transport, the study team assists. Before referral to a health care facility, a study physician collects respiratory specimens and a phlebotomist collects blood from the young infant. One of the difficulties faced in collection of specimens is refusal to provide consent. In these cases, the study physicians visit the families for counseling to overcome refusal. Despite the challenges, of 1857 enrolled pSBI cases, blood specimens have been collected for 1499 (81%) and NP-OP specimens for 1604 (86%).
Second Blood Culture Collection
To interpret blood culture outcomes, we collect a second blood specimen from enrolled pSBI cases whose condition does not improve or deteriorates by the time their blood culture bottles indicate bacterial growth. In these cases, CHWs make additional household visits to reassess the young infants. Upon confirmation by the CHW and study physician, the phlebotomists collect a second blood specimen. In cases where blood has been drawn recently and the newborn is still unwell, significant time and effort are required to counsel caregivers regarding the need for a second blood specimen.
Healthy Control Specimen Collection
To understand the normal flora of young infants, potential healthy controls are selected using the ANISA database software.3 A newborn can be selected as a healthy control on any of the 10 postnatal follow-up visits. On the day of healthy control selection, if the selected newborn does not manifest any sign or symptom of pSBI, blood and respiratory specimens are collected. Obtaining caregiver consent for collection of specimens from healthy young infants, especially on day 0, is problematic. To overcome this challenge, CHWs initiate counseling and sensitizing of caregivers from the day of newborn registration, followed by study physician counseling. Because of this approach and liaison with the community, only 11% of the 800 potential healthy controls have refused to provide specimens (Fig. 5).
Blood Culture Collection Technique
The Matiari site does not have a fixed specimen collection point where procedures can be performed under optimal aseptic conditions. Collection of blood and NP-OP specimens in a home setting makes it challenging to achieve the project’s target of a contamination rate of less than 5%. This rate was 8% in the pilot phase, and several interventions were introduced to reduce it. Clean plastic sheets are placed under an infant during collection. Hand sanitizers are provided to study physicians and phlebotomists because water for hand washing is not available at most households. Phlebotomists also wear disposable gloves during specimen collection. Because of the dusty environment, all laboratory consumables are kept in resealable plastic bags. The skin of a newborn is thoroughly cleaned with antiseptic swabs before blood collection. We initially used povidone-iodine and later replaced it with 4% chlorhexidine spray to ease cleansing of the puncture site. Cleaning of the puncture site with alcohol followed by 4% chlorhexidine spray and again with alcohol is enforced if a vein is palpated again. A checklist for blood collection technique is available for the study physicians to ensure the completeness of all steps performed by the phlebotomists. After the implementation of abovementioned measures, the contamination rate decreased to 4% (Fig. 6).
Interruptions in Electricity Supply of Field Laboratory
Ensuring uninterrupted power supply to the BACTEC (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD), blood culture machine is challenging in Matiari as this area suffers from frequent power outages. Interruptions in power supply may result in beep-positive culture-negative (BPCN) blood specimens. BPCN is a false alarm for the possible presence of organisms in the blood culture bottle. A heavy-duty diesel generator and voltage stabilizers have been installed at the site laboratory to maintain an uninterrupted power supply to the BACTEC machine. Despite these efforts, the BPCN rate has remained at 4%.
During the initial period of the study, pathogen isolation from blood culture was lower than the expected 5%. Insufficient blood volume and prior antibiotic administration in 25% of cases are considered possible reasons for this low yield of pathogens. Therefore, phlebotomists are encouraged to collect the maximum recommended volume of blood (3 mL). Separate mobile medical teams are tasked with collecting blood from pSBI cases on public holidays. However, the pathogen isolation rate remains low at 2%, although it has improved from 1% in the initial 8 months of the study period.
Specimen and Reagent Transport
Blood culture bottles, NP-OP specimens and blood in ethylenediaminetetraacetic acid (EDTA) tubes must be transported from the field site to the field laboratory within 2 hours of collection. There is a dedicated vehicle for this purpose. Average specimen transportation time from collection site to laboratory is 1 hour and 48 minutes. Blood culture bottles are transported at 25–28°C and NP-OP and EDTA tubes at 2–8°C in coolers. A thermometer is placed in the cooler, and phlebotomists monitor the temperature during transportation. If the temperature rises above the specified levels, phlebotomists add more ice packs to bring it down. The temperature inside the cooler is also recorded by personnel at the time of arrival at the field laboratory. Blood culture bottles are put inside the BACTEC machine, whereas EDTA and NP-OP specimens are transported daily to the Infectious Diseases Research Laboratory at AKU in Karachi.
SUMMARY AND CONCLUSION
ANISA protocol implementation in a large cohort under surveillance in a rural setting with specimen collection and newborn assessment occurring at household level has offered a novel experience to the Matiari site team.
The success of the ANISA protocol implementation has been largely due to cohesive team work and able leadership. Some of the strategies implemented include creation and sustenance of community liaison, comprehensive training, focused tasks for each field staff member, extensive supervision and monitoring of field activities. The interventions adopted to reduce blood culture contamination in the community offer opportunities for replication in settings where the contamination rate is high.
One of the challenges that we have not been able to overcome entirely is the late registration of newborns, predominantly due to accessibility and time. More young infants could be saved if we adopted the model of village-based female volunteers who can reach newborns soon after birth and promptly treat or refer them in case of illness.
This study would not be a success without the commitment and dedication of the field and laboratory staff and the sustained cooperation of the study community. The authors thank the ANISA study coordination team at the Child Health Research Foundation, Dhaka, for their support, especially Mr. Nicholas Connor. They are also indebted to Dr. Shamim Qazi and Dr. Salim Saddruddin for their invaluable suggestions given during monitoring visits. Special thanks go to the administrative support and data management staff from AKU.
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