Effect of scheduled antimicrobial and nicotinamide treatment on linear growth in children in rural Tanzania: A factorial randomized, double-blind, placebo-controlled trial

Background Stunting among children in low-resource settings is associated with enteric pathogen carriage and micronutrient deficiencies. Our goal was to test whether administration of scheduled antimicrobials and daily nicotinamide improved linear growth in a region with a high prevalence of stunting and enteric pathogen carriage. Methods and findings We performed a randomized, 2 × 2 factorial, double-blind, placebo-controlled trial in the area around Haydom, Tanzania. Mother–child dyads were enrolled by age 14 days and followed with monthly home visits and every 3-month anthropometry assessments through 18 months. Those randomized to the antimicrobial arm received 2 medications (versus corresponding placebos): azithromycin (single dose of 20 mg/kg) at months 6, 9, 12, and 15 and nitazoxanide (3-day course of 100 mg twice daily) at months 12 and 15. Those randomized to nicotinamide arm received daily nicotinamide to the mother (250 mg pills months 0 to 6) and to the child (100 mg sachets months 6 to 18). Primary outcome was length-for-age z-score (LAZ) at 18 months in the modified intention-to-treat group. Between September 5, 2017 and August 31, 2018, 1,188 children were randomized, of whom 1,084 (n = 277 placebo/placebo, 273 antimicrobial/placebo, 274 placebo/nicotinamide, and 260 antimicrobial/nicotinamide) were included in the modified intention-to-treat analysis. The study was suspended for a 3-month period by the Tanzanian National Institute for Medical Research (NIMR) because of concerns related to the timing of laboratory testing and the total number of serious adverse events (SAEs); this resulted in some participants receiving their final study assessment late. There was a high prevalence of stunting overall (533/1,084, 49.2%). Mean 18-month LAZ did not differ between groups for either intervention (mean LAZ with 95% confidence interval [CI]: antimicrobial: −2.05 CI −2.13, −1.96, placebo: −2.05 CI −2.14, −1.97; mean difference: 0.01 CI −0.13, 0.11, p = 0.91; nicotinamide: −2.06 CI −2.13, −1.95, placebo: −2.04 CI −2.14, −1.98, mean difference 0.03 CI −0.15, 0.09, p = 0.66). There was no difference in LAZ for either intervention after adjusting for possible confounders (baseline LAZ, age in days at 18-month measurement, ward, hospital birth, birth month, years of maternal education, socioeconomic status (SES) quartile category, sex, whether the mother was a member of the Datoga tribe, and mother’s height). Adverse events (AEs) and SAEs were overall similar between treatment groups for both the nicotinamide and antimicrobial interventions. Key limitations include the absence of laboratory measures of pathogen carriage and nicotinamide metabolism to provide context for the negative findings. Conclusions In this study, we observed that neither scheduled administration of azithromycin and nitazoxanide nor daily provision of nicotinamide was associated with improved growth in this resource-poor setting with a high force of enteric infections. Further research remains critical to identify interventions toward improved early childhood growth in challenging conditions. Trial registration ClinicalTrials.gov NCT03268902.

RE: Responses to suspension of a clinical trial titled" Early Life Interventions for Childhood Growth and Development in Tanzania"(ELICIT) 1. There were three points of critique stated under point 1; we will address these individually: a. All samples were collected from participants and stored at -80 degrees until testing.
RESPONSE: Stool testing aimed for antimicrobial-related outcomes largely toward the end of the study. We now see that the wording in our protocol was unclear, and we apologize for the initial lack of clarity. Our protocol ((Protocol Version 5.0 (attached) on page 17, as is referenced in the Visit Report)) addresses laboratory testing of stool samples in point 2.a.: Point i. states the timing of collection ("Stool will be collected monthly and evaluated for pathogens and microbiota at months 3, 6, 6+14 days, 9, 12, 12+14 days, 15 and 18 and yearly thereafter") Point ii. states the immediate handling of samples ("Samples will be batched and stored at -80 degrees") Point iii. describes the various tests involved ("testing will include Taqman array cards, culture and microbiota analysis").
We now appreciate how the reviewer may have interpreted point i. to suggest that the evaluation of stool was performed immediately upon collection. A further explanation; the testings are not being performed immediately because of the labor-intensive the testing procedures. Again some of these samples are being tested in Haydom (using Taqman Array Cards, TAC), while other tests will be performed at the University of Maryland (approved MTA attached). The results from these stool tests are for research purposes only and the data obtained will be analyzed at the end of the study in the context of other factors (e.g. final anthropometry measures, intervention sub-group, etc.). (The same is true for other types of testing, with point 1.f.iii [at the top of page 17], the protocol states, "Serum will be batched and stored at -80 until time of testing.") If it is desirable by the Committee, we will clarify this point further by revising the wording of the protocol point 2.a.i. to state: "Stool will be collected at months 3, 6, 6+14 days, 9, 12, 12+14 days, 15 and 18 and yearly thereafter, with testing performed when possible." b. Some stool samples were taken to KCRI for testing; however, there was no approval for Material Transfer Agreement issued by NatHREC.

RESPONSE:
We deeply apologize for this. Our understanding was that the requirement for MTA was related to samples that are being shipped oversee (out of the country), as documented in the MRCC _form2 "item number 9" which state that "… Please note that if samples are to be shipped outside Tanzania MTA clearance is required." We had shipped some stool to KCRI (in Moshi, TZ) because of the large amount of work needed to test more than 7000 stool samples. The lack of contract/MoU was pointed out by the TFDA during their last year inspection (2-3 July 2018), and we have established a MoU between Haydom Lutheran Hospital and Kilimanjaro Clinical Research Centre (attached). However, that lack of MTA was also pointed out by TFDA, who performed a site visit at the same time as the NatHREC visit. In response to the TFDA comments, we suspended shipment of stool samples to KCRI and we are applying NIMR for MTA to transfer stool to KCRI; this solution was accepted by the TFDA. Shipment of stool samples to KCRI will only continue after getting MTA approval.
c. PI is absent from the Research site: He is doing PhD study in Norway without notifying the NatHREC secretariat.
RESPONSE: We agree with the Monitor observation on the absence of the PI during the visit. We again apologize for this as it was a coincidence that the monitoring visit coincide with the PI travel and it was difficult to postpone as was a long-time planned travel. We tried to ask a favor for the TFDA reschedule their visit but was not possible. The absence of the PI during the GCP inspection visit was communicated with TFDA team prior to the visit and arrangements were made to all sections/departments of the study to ensure the inspection visit is successful even in the absence of the PI.
PhD: The PI started work toward the PhD in 2012. This is not a full-time course, and most of the PhD-related activities take place in Haydom and consume minimum time (about 30% which is out of the time effort required to oversee the trial). Additionally, the PI occasionally travels for one or two weeks for the purpose related to the study and his work on his PhD. The PI is continuously in contact with the study team while away from Haydom, via phone and emails. In the PI's absence, the chain of command for the next responsible person in the study is Samwel Jatosh (Research Coordinator), Paschal Mdoe MD (Investigator), Joshua Gideon MD (Investigator), and Justine Museveni MD (Investigator). In rare cases when one of these is away, it is communicated to the next in this line and to the PI who is in charge on site. The PI maintains contact with the person in charge to receive updates on a daily basis and to help problem solve issues that arise as needed.
We apologize for not alerting the NatHREC secretariat of this arrangement as we were not informed that the TFDA inspectors were coming together with the NatHREC member, and in the future, as directed, we will notify NatHREC in the event of prolonged travel out of country.
RESPONSE: We apologize for this. This point is further addressed under 1.b. above.
(Please note, points of critique for numbers 5 to 8 were not listed in the letter; the report proceeded from point number 4 directly to point number 9, to which we reply below.) 9. There were 237 SAEs reported of which majority were Gastroenteritis and Anaemia according to the DSMB report of the meeting held on 9 th August, 2018 the same was discussed and PI instructed to conduct ad hoc analysis of the samples collected for the participants in both study groups (Intervention and Control arms) by December, 2018. The aim was to investigate if there will be significance difference of the results between the two groups in order for the DSMB to decide whether to un-blind or continue blinding the study. RESPONSE: Our interim analysis came about from two influences: 1. As a study team, we had been following for differences in AE's and SAE's by nicotinamide intervention group (Nic-A vs. Nic-B, where we are blinded which group is the active drug and which is the placebo) and wished to investigate the potential that one study arm had more diarrhea AE's than the other. 2) The Bill & Melinda Gates Foundation (the funder of the study) was interested in whether there were potential differences in anthropometry measures between intervention groups at the 6-month point in the study. The Gates Foundation had requested that we perform an interim analysis to assess these 6-month outcomes and had initially identified mid-December as a date for this. When we had our DSMB meeting on 9 October (we did not have a DSMB meeting in August, so this must have been the DSMB meeting in question), we informed the DSMB about our concerns about the possibility of differential AE's between nicotinamide intervention groups, and said that we would perform a more formal analysis of this when we did the outcomes testing in December. However, the Gates Foundation then requested that we perform this analysis later to allow a larger number of participants to reach 6 months; we informed the DSMB chair of the change in timing on 18 December 2018. We performed the interim analysis on 25 January 2019 and informed the DSMB of the results on 28 February. These results showed no difference between nicotinamide intervention group for AE's or SAE's, either all together or AE's and SAE's specific to gastroenteritis or pneumonia. This was acceptable to the DSMB (please see attached report for interim analysis), and no un-blinding was requested. We have also determined that there was no difference in SAE anemia by nicotinamide group. 10. Many protocol deviations which related to not adhering to the administration of IMP schedule were observed. RESPONSE: We have carefully re-reviewed our data regarding this. Out of 9840 drug administration forms, we found 70 that were given outside the planned time window. While our goal is 0%, the doses outside the timing window represent less than 1% of doses of study medications-and this error rate has decreased over time, with only 6 incidents happening since 1 January 2019. To further improve this rate, we will hold a re-training session with the study team on the anticipation and adherence to the medication time windows.
(Please note, we did not receive any indication of a point of critique numbered 11; the report proceeded from point number 10 to point number 12.) participants files reviewed example PID TZ1C1031, TZ1C1032, TZ1C1041, TZ1C1191,  TZ1C1192, TZ1C1162, TZ1C1222, TZ1C1172, TZ1C1151, TZ1C1052, TZ1C1071, TZ1C1072, TZ1C1142, TZ1C1011, Including Child monthly form, drug administration form, specimen collection form, pill/pack counting form, breast milk collection form were not signed and dated by first and second data entry personnel and some were signed by first of second data entry personnel only. RESPONSE: We apologize for these omissions of signatures. To clarify more on this matter, this was also raised during the FHI trial-monitoring visit (shared with NIMR in the past), which took place on the 12-16 March 2018. The site amended the data management SOP (attached) and implemented the procedure to "sign and stamp" all the CRFs after data entry (both 1 st and 2 nd entry) as from 3 rd April 2018, and a note to file was completed to cover the CRFs before the date and stated the reason to not have signature and stamp. What was now observed in the resent inspection (on this discussion), 02 out of the 14 PIDs are involved and the rest (12) were before implementation of the SOP. However we will also hold a re-training session with field workers and data entry personnel on the importance of signing forms upon completion of data entry, to avoid such omissions in the future.

CRF's in the several
Conclusion: We are grateful for the NatHREC's review of our study and are eager to respond to any further points of critique the committee might have. The ELICIT study aims to reveal important relationships between intervention with antimicrobials and nicotinamide and child thriving (growth and cognitive development) in a resource-limited setting. We are hopeful that NatHREC will permit us to complete this study as planned. Please do not hesitate to contact us with any future questions. Finally, as the PI of this project, I will be happy to appear before the NatHREC Clinical trial subcommittee for more conversations as early as possible for their convenience.