The genetic hallmark of CML is known as the appearance of t (BCR-ABL1) which is present in more than 95% of cases. Here, we demonstrated practical laboratory tools for monitoring of BCR-ABL1 transcripts in chronic myeloid leukemia patients undergoing TK inhibitor therapy. Real time quantitative PCR and direct sequencing were performed for monitoring of BCR-ABL1 transcripts in 245 treated CML. BCR-ABL1 TKD mutations. Interestingly, one third (17 of 49) of TKD mutated cases were positive for compound/polyclonal mutation patterns. While major molecular response were observed in the majority of patients without TKD mutation, resistant to TKI were detected in patients with T315I mutation , compound/polyclonal mutations with T315I , and other TKD mutations , respectively. At month 3 after first time point of monitoring, we found that 89% (218/245), 2% (5/245), and 9% (22/245) of patients are determined as optimal, warning, and failure response, respectively. The responses to TKI were slightly decreased at months 6 as following 73% optimal (180/245), 18% warning (43/245), and 9% failure response (22/245). Additionally, responses to TKI were gradually decreased at month 12 after first time point of monitoring as following 65% optimal (160/245), 13% warning (31/245), and 22% failure (54/245). We could detect 20% (49/245) of patients positive for These practical laboratory techniques provided a more comprehensive understanding of CML progression during drug therapy and could be of benefit in earlier prognosis. BCR-ABL1 fusion gene, and BCR-ABL1 mRNA) is very important during a treatment to prevent disease relapse as well as to establish the end-point of therapy . Recently, the European LeukemiaNet recommendations for the management of chronic myeloid leukemia suggested that bone marrow studies including cytogenetic analysis should be performed at 3, 6, and 12 months after starting therapy . Additionally, the improvement of clinical outcomes in treated CML patient are strongly associated with the deep and rapid molecular response. Hence, highly specific and sensitive techniques such as RT-PCR and real-time quantitative PCR (RQ-PCR) are important for routine monitoring of minimal residual disease (MRD) in treated patients . Although IM has been proved for the effective treatment of CML, approximately to 20-30% of treated patients become IM resistance . Those patients become predominantly increasing in the BCR-ABL1 level. The resistance to IM treatment in CML patients could be categorized into two main mechanisms. Firstly, a BCR-ABL1 dependent mechanism which is characterized by aberrations on the BCR-ABL1 fusion gene, including BCR-ABL1 kinase domain mutations (the most common cause of IM resistance), BCR-ABL1 amplification, and clonal evolution. Secondly, a BCR-ABL1 independent mechanism, which includes the impairment in function of specific IM transporters (influx and efflux transporters), drug concentration, the dysregulation of alternative signaling pathways, and epigenetics . At present, it is clear that the mutation in ABL1 tyrosine kinase domain (TKD) is the major cause of IM resistance . Common TKD mutations are including the highly resistant mutations  which are recommended to alternative treatment options, the mildly resistant mutations  which permit the improvement of clinical outcomes by increasing the IM dose to 600 or 800 mg/day . Thus, molecular methods that can be able to early detect BCR-ABL1 kinase domain mutations are very important for monitoring of CML patients after treatment with tyrosine kinase inhibitors (TKIs). In this report, we described our routine RQ-PCR for the measurement of BCR-ABL1 mRNA and PCR sequencing technique for the detection of BCR-ABL1 TKD mutations in CML patients during TKIs treatments. Finally, we reported the frequency of BCR-ABL1 TKD mutations and the impact of clinical important TKD mutations on molecular response to TKIs treatment in Thai patients with CML.Chronic myeloid leukaemia is a clonal haematopoietic stem cells disorder characterized by the proliferation of malignant myeloid cells at all stages of differentiation. The genetic hallmark of CML is known as the appearance of the Philadelphia chromosome (Ph chromosome); t which is present in more than 90% of cases. The Ph chromosome is associated with the production of BCR-ABL1 chimeric protein with high ABL tyrosine kinase activity and recognized as a pathogenesis of the disease . Patients with CML frequently have three phases of disease progression including the initial chronic phase, the transitional accelerated phase, and the progressive blast crisis phase . Since the discovery of imatinib (IM) , the drug has been shown to be the highly effective therapy in Ph positive CML. IM specifically blocks the ATP-binding site of BCR-ABL1 protein in leukemic cells resulting in inhibiting downstream signaling pathways. The first application of IM was to cure CML patients who were resistant to interferon-alpha (IFNα) . The drug can induce a rapid haematological response and a major cytogenetic response in a chronic phase CML patients . In addition, IM could rise the overall survival and the event-free survival in the accelerated phase CML . Moreover, adverse effects of IM are generally mild when compared with interferon-alpha. IM could dramatically improve the overall survival of CML patient from about 20-80% to about 80-90%. Presently, treated CML patients are expected to live longer as well as have very well quality of life. Therefore, monitoring of the persistence specific surrogate disease biomarkers in CML .This study was a retrospective analysis on a total of 245 CML patients during treatment at the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand during January 2011 to December 2016. Cytogenetic study, RQ-PCR, and BCR-ABL1 kinase domain mutations screening were performed by the request from hematologists. The management of CML were based on the recommendations of European LeukemiaNet . Bone marrow and peripheral blood specimens were collected from patients at the time of diagnosis as well as during treatment. Standard karyotyping was mainly performed using bone marrow samples. The quantification of BCR-ABL1 mRNA levels were conducted by following the EAC protocol . Real-time quantitative PCRTaqMan-based real-time quantitative PCR (RQ-PCR) was performed according to a Europe Against Cancer Program (EAC) protocol to measure the BCR-ABL1 mRNA level during treatment . Briefly, total RNA was isolated from peripheral blood/bone marrow samples of each patient by using TRIzol® Reagent  according to the manufacturer’s instructions and subsequently measured the concentration by using the Nanodrop 2000 spectrophotometer . cDNA was generated from a total of 1 μg RNA using SuperScript™ VILO™ cDNA Synthesis Kit  with random oligonucleotide primers. Real time quantitative PCR was performed on ABI 7500 Fast Real-Time PCR System . ABL1 was used as a house-keeping gene. BCR-ABL1/ABL1 copy ratio and % IS  of individual patient during treatment (every 3 months) were reported. The major molecular response (MMR) was defined as ≥ 3-log reduction (% IS ≤ 0.01) in BCR-ABL1/ABL1 ratio. The laboratory has achieved ISO15189 and 15190 accreditations and participated in the College of American Pathology (CAP) External Quality Assurance (EQA)/Proficiency Testing program. The definition of response to TKIsThe definition of response to any TKIs was followed the ENL 2013 recommendation guideline for the management of CML . The response to TKIs of individual patient could be assessed by both cytogenetic and molecular (RQ-PCR) techniques. The specific cut-off values at diverse time points of monitoring were clearly written in the guideline. The optimal response was associated with the achievement of finest long-term outcomes. Therefore, in this case, it was not necessary to change the treatment. In contrast, failure response meant that patient should change the treatment strategies to avoid the risk of disease progression and death. The middle between optimal and failure responses is called warning . In this group, patients required more frequent monitoring to allow appropriate changes for new treatments in a case of failure response. The guideline was able to be used for the definition of response to TKIs in all phases of CML  and could be applied for the definition of response in second-line TKIs treatments. BCR-ABL1 kinase domain mutation analysisBCR-ABL1 kinase domain mutations was adapted from the previous published protocol by Branford and Hughes, (2006) and Gabert et al., (2003). Briefly, semi-nested RT-PCR was performed to initially amplify BCR-ABL1 fusion transcripts (ENF501 and ABLR1) and subsequently amplify the ABL1 kinase domain in a second round of PCR (ABLF1 and ABLR1). The PCR primers used in both semi-nested PCR and sequencing reaction were list in oC for 10 minutes, 40 cycles of 94oC for 1 minute, 68oC for 1 minute, 72oC for 2 minutes, and final extension at 72oC for 10 minutes. 5 µl of the first round PCR product was used as a template in the second round PCR. PCR product sizes of 1,464 or 1,539 base pairs for the first round PCR and 863 base pairs for the second round PCR were determined by 2 % agarose gel electrophoresis. PCR product (863 bp) was subsequently purified by using ExoSAP-IT™ PCR Product Cleanup Reagent  prior to cycle sequencing process. Sequencing reaction was performed by using BigDye® Terminator v1.1 Cycle Sequencing Kit  and 1.6 pmol of each sequencing primer (ABLF1 and ABLR1). The amplified condition was following 96oC for 1 minute, 25 cycles of 96oC for 10 second, 50oC for 5 seconds, 60oC for 4 minutes. Sequencing product was purified using DyeEx 2.0 Spin Kit  and subsequently sequenced with ABI 3130 Genetic Analyzer  according to the instruction protocol. Finally, sequence was compared to the wild-type ABL sequence (GenBank accession number X16416.1). PCR sequencing analysis for the detection of Statistical analysisStatistical analysis was performed using SPSS version 24.0 software . Mean and median percent IS values of patients at 12 months of monitoring were analysed using independent T-test and Mann-Whitney test respectively. Statistical significance is accepted at p-values <0.05.The overall molecular response of 245 CML patients to TKI treatmentBCR-ABL1 TKD mutations analysis was subsequently confirmed in cDNA samples of patients who exhibited signs of treatment failure. Using quantitative RT-PCR, we could determine the molecular response of CML patients after TKI treatment. We performed both cytogenetic analysis and real-time quantitative PCR on sequential samples collected from different time points of individual patients during treatment. The definition of response to tyrosine kinase inhibitors as a first-line treatment was based on the European LeukemiaNet recommendations for the management of chronic myeloid leukemia; 2013 . At month 3 of monitoring, we found that 89% (218/245), 2% (5/245), and 9% (22/245) of patients were determined as optimal, warning, and failure response, respectively. The overall responses to TKI treatment were slightly decreased at the month 6 of disease monitoring as following 73% of patients with optimal response (180/245), 18% of patients with warning (43/245), and 9% of patients with failure response (22/245). Additionally, we observed that the overall responses to TKI treatment were gradually decreased at month 12 of monitoring as following 65% of patients with optimal response (160/245), 13% of patients with warning (31/245), and 22% of patients with failure response (54/245) . BCR-ABLMutation spanning of BCR-ABL1 TKD in treated CML patients BCR-ABL1 TKD mutations analysis by Sanger sequencing was performed in cDNA samples of patients with TKIs treatment failure. We could detect 20 % (49/245) of patients with positive results for BCR-ABL1 TKD mutations. Interestingly, we found that one third (17 of 49) of TKD mutated cases were positive for compound/polyclonal mutation patterns. An example of electropherogram from patient with compound/polyclonal mutation was presented in BCR-ABL1 TKD was the clinical relevant, T315I which was positive in 57% (28/49) of all mutated cases. Interestingly, compound/polyclonal mutations of T315I with other BCR-ABL1 TKD mutations were observed in nearly half of T315I mutated cases . Other point mutation types observed in this study were following 20% of E255K/V , 10% of F359V/I , 8% of F317L/I , 6% of Y253H , 4% of D276G , 4% of E459V/K (2/49), 2% of Q252H (1/49), 2% of F311I (1/49), 2% of A350T (1/49), 2% of E455K (1/49), 2% of F486S (1/49), respectively. Interestingly, G250E mutation was restricted positive as compound/polyclonal mutation patterns. There was no single mutation of G250E mutation observed in this study  to TKIs at month 12 of monitoring. 142 patients without BCR-ABL1 TKD mutation were analysed. We observed that the median % IS of patients at month 12 is 0.0150 (< -4 log). This data suggested the majority of patients was achieved major molecular response after TKI treatment. However, there were few patients with a sign of molecular resistance at one year after treatment without BCR-ABL1 TKD mutation. Because of T315I is well-recognized as a clinical significant point mutation that contributes to imatinib resistant in CML and it was predominantly observed in this study , we classified the treated patients into 4 main groups including patients without BCR-ABL1 TKD mutation, T315I mutation, compound/polyclonal mutations with T315I, and other TKD mutations . While major molecular response was observed in the majority of patients without BCR-ABL1 TKD mutation, resistance to TKI treatments was detected in patients with T315I mutation , compound mutations with T315I , and other TKD mutations , respectively. Additionally, an increase of % IS to upper than 10 (> -1 log) was observed in nearly half of patients (44.44%) with both T315I single mutation and compound/polyclonal T315I mutations at 12 months of monitoring. Moreover, statistical analysis indicated that significant different in molecular response was observed in patients without BCR-ABL1 TKD mutation compared with patients who carried BCR-ABL1 TKD mutations with T315I (p = 0.000395), compound/polyconal with T315I (p = 0.000009), and other type of BCR-ABL1 mutations (p = 0.000325), respectively. However, it was not statistical different among patients with T315I, compound/polyconal with T315I, and other BCR-ABL1 mutations observed in this study  as well as participated in the College of American Pathology (CAP) External Quality Assurance (EQA)/Proficiency Testing program. During the past two decades, treatment of BCR-ABL1 positive CML has dramatically improved since the development of imatinib and other tyrosine kinase inhibitors. While the majority of patients remain in prolonged complete molecular response, nearly one third of patient develops an acquired Perspective of cytogenetic analysis for the diagnosis and monitoring of CML is still limited used and routinely service in Thailand and nearby countries. We use the International System for Human Chromosome Nomenclature (ISCN2016) as a guideline for the description of individual CML karyotype. The main advantages of conventional cytogenetic analysis in CML are including; it is still a gold-standard method for the detection of Ph chromosome and it could detect several additional chromosomes in both newly diagnosed (baseline) and treated patients. Therefore, the assay is still mandatory for the monitoring of clonal evolution during treatment. However, the technique has some drawbacks such as low resolution, low sensitivity, time consumable, and laborious technique. The molecular cytogenetic, FISH is not frequently used in our laboratory for both diagnosis and monitoring of CML due to the assay is not provide much clinical relevant data compared with conventional cytogenetic analysis. However, we use FISH in special scenarios such as for the monitoring of Ph positive CML/AML with rare BCR-ABL1 transcriptional variants. Since the announcement of European Against Cancer (EAC) procedure for standardization and quality control of real-time quantitative PCR for minimal residual disease detection in leukemia , we had followed this valuable protocol for the detection of several fusion transcripts including the BCR-ABL1 major (p210) transcript. This protocol have been proved to be practically used for monitoring CML patient during treatment because it has several benefits such as high specificity and sensitivity, easy to perform, economy, and short turn-around time. More importantly, the result from RQ-PCR is very informative and representing to disease progression/response in individual patient during the treatment. For the international standardization, we could achieve the conversion factor for international scale calculation in 2014 by the support from the Division of Genetics & Molecular Pathology, Royal Adelaide Hospital, Australia. Here, based-on the ELN 2013 guideline , we serially monitored (every 3 months) BCR-ABL1 mRNA by using RQ-PCR in 245 CML patients after treatment. We observed that the overall response to TKI treatment in patients were gradually decreased at the month 12 after first time point of monitoring . Moreover, we found that 22% of patients were categorized as failure to TKI at month 12 by monitoring with RQ-PCR. Those patients were successively analyzed the BCR-ABL1 TKD profiles using PCR sequencing. While we did not have much clinical therapeutic data of individual patients, we could perform the robust RQ-PCR method for effective monitoring of BCR-ABL1 mRNA in CML during treatment. BCR-ABL1 TKD mutations in treated patients, we found that 20% of patients are positive for BCR-ABL1 TKD mutations by mean of using direct sequencing technique. Our data was similar to some of previous publications regarding to the overall frequency of BCR-ABL1 TKD mutations in treated CML. Those were including in the Australian study (19%) , Argentinean study (23%) , and Malaysian study (22.4%) (using dHPLC and direct sequencing) . However, there were some publications reported that the observed in frequency of BCR-ABL1 TKD mutations are range from 30% to 65% . The overall frequency of BCR-ABL1 TKD mutation in treated CML patients from various studies were summarised in BCR-ABL1 TKD mutations from various studies could be explained by the variation in number and inclusion criteria of selected case, treatment protocol, phase of disease, and the method of used for the detection of BCR-ABL1 TKD mutations. Similar to previous reports , the most frequents BCR-ABL1 TKD mutations involving in the establishment of TKI resistance by mean of RQ-PCR was the T315I which was accounted for 57% of all mutated cases. Interestingly, we found that 35% of all mutated cases persisted compound/polyclonal mutation patterns with dominantly harbour co-mutation of T315I. Additionally, patients with T315I compound/polyclonal mutations showed higher %IS and might had phenotypic resistance than those with T315I alone and other TKD mutation patterns at month 12 of monitoring. Although we had no high performance technology such as digital droplet PCR and next generation sequencing to deeply and massively monitor BCR-ABL1 TKD mutation profile, we could perform the effective laboratory developed test (direct sequencing) for the detection of BCR-ABL1 TKD mutations in our tested samples. Based on this observation data, we are going to further investigate the impact of compound/polyclonal mutations in the treatment of CML in the context of precision medicine in recent future. Perspective of the detection of BCR-ABL1 TKD mutations in patients who failed to TKI therapy. Patients with T315I compound/polyclonal mutations had high level of %IS than those with T315I alone, others TKD mutations, and without TKD mutation. Regarding to ELN 2013 guideline, the integration of laboratory tools including cytogenetic analysis, iFISH, RQ-PCR and direct sequencing is critical for the comprehensive monitoring of Ph positive CML during treatment. In summary, we described the practical laboratory tools for routinely monitoring of CML patients during treatment. Serially measurement of BCR-ABL mRNA using RQ-PCR was able to effectively monitor the dynamic change of residual disease in treated patients. Moreover, we could identify several


Older adults are less prepared for disaster situations than younger adults due to expenses, complicated preparation processes, and lack of support. We previously developed and tested a disaster preparedness program for older adults and showed its impact on improving preparedness and personal emergency support network. Using a 5-step community-engaged approach and in collaboration with our Stakeholder Advisory Board (SAB), we adapted this Disaster PrepWise program to an online platform and developed implementation plans within existing community-based service infrastructure. Community engagement processes allowed us to create an online tool that was considered “important” and “useful” by older adults and program deliverers. In terms of reach, 73 potential participants attended an introductory presentation and 47 participated. Those who participated were similar to those who did not in terms of gender and race. Twenty-seven participants completed surveys before and one-month after the program and reported engaging in an average of 2.9 additional recommended behaviors after intervention (t(26)=2.85, p=0.00). The major organizational barrier to adoption was limited staff time. Participating older adults reported adopting what they learned through the program in their daily life. Implementation process data showed multiple modifications made during delivery including delivery of the program in one instead of two sessions. The implementing organization reported their likelihood of maintaining this program within their practice as “extremely likely.” Community-engaged approaches and process data led to an acceptable and useful program and implementation strategies that helped enhance preparedness behaviors among older adults. Future work will focus on program dissemination and larger scale evaluation.


Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis.Human parvovirus B19, human cytomegalovirus, and  Toxoplasma gondii infections during pregnancy can lead to severe complications. While traditional diagnosis of infections is mostly confined to one pathogen at a time, a multiplex array is a feasible alternative to improve diagnostic management and cost-efficiency. In the present study, for these three pathogens, we developed microsphere-based suspension immunoassays (SIAs) in multiplex and monoplex formats for the detection of antimicrobial IgM antibodies as well as corresponding chaotrope-based IgG avidity SIAs. We determined the diagnostic performances of the SIAs versus in-house and commercial reference assays using a panel of 318 serum samples from well-characterized clinical cohorts. All the newly developed assays exhibited excellent performance compared to the corresponding high-quality reference methods. The positive and negative percent agreements of the IgM SIAs in comparison with reference methods were 95 to 100% and 98 to 100%, and those of the IgG avidity SIAs were 92 to 100% and 95 to 100%, respectively. Kappa efficiency values between the SIAs and the corresponding reference assays were 0.91 to 1. Furthermore, with another panel comprising 391 clinical samples from individuals with primary infection by B19, HCMV, or T. gondii, the IgM SIAs were highly sensitive for the detection of acute infections, and the IgG avidity SIAs were highly specific for the separation of primary infections from past immunity. Altogether, the strategy of IgM multiplex screening followed by IgG avidity reflex testing can provide high-throughput and accurate means for the detection and stage determination of B19, HCMV, and T. gondii infections.Human parvovirus B19 (here B19), human cytomegalovirus (HCMV), and IMPORTANCE Human parvovirus B19, human cytomegalovirus, and Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis. Toxoplasma gondii cause infections worldwide. Although these infections are usually asymptomatic in immunocompetent individuals, they can lead to severe complications during pregnancy. Maternal B19 infection can cause spontaneous abortion, fetal hydrops, and intrauterine death , human cytomegalovirus (HCMV), and ne death , whereasectively , 4. As actively 46. With Bctively 4, and witestation –10, wherstation –, 12.T. gondii secondary infections. An approach for the dating of primary infection, e.g., with each of these pathogens, is the measurement of the antigen-binding avidity  of antimicrobial IgG. Upon initial antigenic challenge, IgG matures from low to high avidity due to antigen-driven B-cell selection and narrowing of the range of epitopes. This feature has been used in the clinical laboratory setting to distinguish between acute and past immunity via the implementation of either of two approaches: the use of a chaotropic agent to disrupt weak antigen-antibody interactions  and IgM antibodies. Specific IgM is a sensitive indicator of recent primary infection with all these microbes, yet the antibody can persist in the blood for a long time –15 and c–ractions  or the aractions .Current serodiagnosis is mostly confined to the detection of one pathogen at a time. A convenient approach for high-throughput antibody detection is a suspension immunoassay (SIA) employing flow cytometric analysis of fluorescent bead sets. In a previous study, we developed SIAs for the simultaneous detection and identification of IgGs against these three pathogens . In the T. gondii, or B19. The antimicrobial IgM SIAs were run in a multiplex format, and the IgG avidity SIAs were run in a singleplex format.The diagnostic performances of the assays were evaluated using different sample types that have been stratified here into two panels . Panel 110.1128/mSphere.00905-19.4TABLE S1Table S1, DOCX file, 0.02 MB.Characteristics of all the reference immunoassays used in this study. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (a) HCMV. Altogether, 97 samples were studied for IgM as well as for IgG avidity, and results were compared to those of Abbott’s Architect assays. The IgM SIA had an overall agreement of 97.9% (94/96 samples) (samples) , and thesamples) . Among t(b)T. gondii. IgM and IgG avidity were examined in 94 serum samples, and the results showed full concordance with the corresponding Vidas results ( results  and 3.(c) B19. IgM was tested in 40 clinical samples and 87 samples from students, and the results were compared to those of a reference IgM test (IgM test . The oveIgM test .(a) HCMV. In total, 108 samples from 39 patients with symptomatic primary infection were analyzed T. gondii. Subgroup A, comprising 48 samples from 9 patients with primary infection, was examined. In the SIA, a significant increase in IgG avidity was observed in 88.9% (8/9) of patients. All 11 samples collected within 90 days showed positive or borderline IgM SIA results  of patients were IgM positive, and 76.9% (10/13) exhibited a significant IgG avidity increase at follow-up. Among the 29 samples collected within 90 days after T. gondii seroconversion, 82.8% (24/29) were IgM SIA positive or borderline (n = 20) or borderline (n = 4) avidity by the SIA  B19. We tested 126 serum samples from 66 children or adults with symptomatic B19 infection. In the SIA, B19 IgM was found in all 82 samples collected within 90 days of onset. Of the 44 samples obtained after day 90, 72.7% (32/44) were IgM SIA negative (negative . All 61 negative . Beyond negative .T. gondii primary infection). Among the 25 samples, 13 presented with homologous (cf. IgM) IgG. To identify the origin of the heterologous IgM reactivity, we performed multiplex IgG avidity SIAs and found that 11 samples showed high avidity against the homologous antigen, excluding recent primary infection by that pathogen  of the samples in this study. Of these, 8 samples belonged to panel 1 (6 HCMV IgM positive with low IgG avidity and 2 HCMV IgM negative with high IgG avidity), and 17 belonged to panel 2  of HCMV, T. gondii infections  and for IgG avidity . In clarifying the B19 infection time, the new VP1u IgG avidity SIA also agreed well with the established EIA for the “conformation dependence” or “epitope type specificity” (ETS) of VP2 IgG, divergent qualitative determinants of antimicrobial IgG maturing progressively during the months since the first antigenic challenge , 24, 25.Of note, the three HCMV IgG avidity tests used in the current study  are technologically distinct: (i) while the SIA and Vidas are based on protein denaturation, Architect utilizes antigen competition , and  were correctly identified (high avidity) as past infection. Likewise, the T. gondii IgG avidity SIA could effectively distinguish acute from latent/chronic T. gondii infections; however, low-avidity IgG was seen in five patients beyond 200 days of primary infection. Persistence of low-avidity IgG after T. gondii infection has been seen in many studies, especially among pregnant women and in medicated patients  serum samples sent to the Helsinki University Central Hospital Laboratory Service (HUSLAB) for diagnostic evaluation between 2003 and 2013 as well as 87 samples collected from constitutionally healthy medical students . The mai(a) HCMV. Ninety-seven serum samples were examined for HCMV IgM antibodies and IgG antibody avidity using the respective Architect assays as reference tests (Abbott). In the Architect HCMV IgM assay, 44 serum samples were positive, 1 was borderline, and 52 were negative. In the Architect HCMV IgG avidity assay, 38 serum samples displayed low avidity, and 59 displayed high avidity. IgG avidity discordances between the SIA and Architect were assessed with the Vidas assay (bioMérieux).(b)T. gondii. Ninety-four serum samples were analyzed for T. gondii IgM antibodies and IgG avidity using the Vidas Toxo IgM and IgG avidity assays as reference tests (bioMérieux). In the Vidas Toxo IgM assay, 46 samples were positive, 47 were negative, and 1 was borderline. In the Vidas Toxo IgG avidity assay, 47 serum samples were of low avidity, and 47 were of high avidity.(c) B19. Forty serum samples were tested for B19 IgM antibodies by Biotrin’s B19 IgM assay  as well as for VP2 IgG epitope type specificity (ETS) by an in-house ETS EIA  HCMV. A total of 108 samples originated from 39 patients with HCMV primary infection T. gondii. A total of 116 samples were obtained from 22 pregnant women with T. gondii primary infection (n = 48 samples). These patients had been monitored serologically for a year (or more), except for one, who was monitored for 64 days. The other 13 patients were IgG seroconverters monitored for up to 503 days, constituting subgroup B (n = 68 samples). These serum samples had been collected between 1989 and 1990  B19. A total of 126 serum samples were obtained from 66 children or adults  with symptomatic B19 infection. The patients had been monitored serologically for up to 700 days. Collected between 1992 and 2001, there were 1 to 4 serum samples per patient  HCMV. The 39 patients with HCMV primary infection presented with seroconversion of HCMV IgG and a low avidity of HCMV IgG in the first positive sample, and 7 patients also had HCMV IgM seroconversion, whereas 32 patients were IgM positive or borderline with the first sample. The 13 patients with a profile of HCMV secondary infection had a 4-fold (or higher) increase of the HCMV IgG level, from the existing presence in the first sample of high-avidity HCMV IgG. Nine of these patients showed IgM seroconversion, 2 were IgM borderline, and 2 remained IgM negative.(b)T. gondii. The 9 patients of T. gondii subgroup A exhibited T. gondii IgM as well as low-avidity IgG in the first sample, and the 13 patients in subgroup B showed seroconversion of T. gondii IgG. The seroconverters showed IgM seroconversion (n = 3) or were IgM positive or borderline with the first sample (n = 9), while a single patient lacked IgM. All IgG seroconverters were of low or borderline avidity with the first seropositive sample.(c) B19. The 66 patients with recent B19 infection had B19 IgM (n = 66) as well as seroconversion (n = 25) or a significant rise (n = 41) in the level of B19 IgG and had low (<15%) avidity or a low (<10) ETS EIA ratio in the first seropositive sample.The Helsinki University Hospital Ethics Committee accepted the use of clinical samples in this study . The serum samples from medical students were obtained with informed consent. All other samples in this study were taken as part of standard care and were analyzed anonymously.6 microspheres). The conditions for each assay are presented in The coupling of antigens to carboxylated fluorescent microspheres  was performed according to the manufacturer’s protocol and as described previously by Wang et al. . The cou(a) Naked microspheres. For specificity, each test run included control uncoupled (“naked”) microspheres stored in SG buffer (G buffer .(b) Rheumatoid factor (RF) control. In the IgM test, each sample was also tested with human IgG -coated microspheres to monitor the effectiveness of IgG removal  control. In the IgM SIA, each sample was tested with human IgG -coupled microspheres. RF in serum can be semiquantified when RF bridges between human IgG-coated microspheres and biotinylated anti-human IgM. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 1 min to remove IgG precipitates. The supernatant was further diluted 4-fold. Next, 50 μl of this (1:80) IgG-depleted serum was incubated with 1.75 × 103 antigen-coated (or control) microspheres/analyte/well for 45 min. After washes, 50 μl of biotinylated anti-human IgM  at 3 μg/ml was added for 30 min. After washes, 50 μl of 6 μg/ml streptavidin-conjugated phycoerythrin  in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST) was applied for 20 min. After final washes, each well was resuspended in 120 μl of PBST and read on a Bio-Plex 200 instrument (Bio-Rad). The median fluorescence intensity (MFI) values were determined.The multiplex IgM SIA included the removal of IgG with GullSORB . According to our previous determination, described in 10.1128/mSphere.00905-19.1TEXT S1Text S1, DOCX file, 0.01 MB.IgG depletion in IgM SIAs. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00905-19.3FIG S2y axis shows MFI values, and the x axis shows series of serum dilutions. The IgM response from each serum dilution is indicated: blue squares, IgM-positive pool without IgG depletion; red upward triangles, IgM-positive pool with IgG depletion; blue downward triangles, IgM-negative pool without IgG depletion; red diamonds, IgM-negative pool with IgG depletion. Download FIG S2, DOCX file, 0.1 MB.IgG depletion in IgM SIAs. The pools containing or lacking specific HCMV IgM were used to compare the IgM SIA results before (blue) and after (red) IgG depletion, respectively. The Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Heterologous IgM reactivity among the three microbes was observed in 25 samples of this entire study. To identify the original immunoactivity, we employed a multiplex IgG avidity SIA (see below) for the simultaneous determination of infection stages of the three pathogens. In addition, two samples showing SIA IgG and IgM responses against B19 VP2 but not VP1u were resolved by IgM and VP2 ETS EIAs.3 antigen-coated microspheres/well/analyte for 45 min. Next, series 1 samples were washed three times for 5 min each with 6 M freshly prepared urea  in PBS, as opposed to series 2, with PBS only. Subsequently, biotinylated protein G  and SA-PE were added, and the MFIs were measured, as for the IgG SIAs  over those of series 2 (non-urea treated), calculated by the curve-fitting software Avidity 1.2 .T. gondii, the cutoffs were defined with separate sets of 60 serum samples shown to lack the respective antibodies by the corresponding Architect IgG and IgM tests (The IgM SIA cutoffs were set at the mean MFIs plus 2 to 5 standard deviations (SDs) of negative controls. For B19, the cutoff was determined with 86 serum samples lacking specific antibodies according to Biotrin’s B19 IgG and IgM EIAs , 39. ForgM tests . The IgMT. gondii are presented in T. gondii, samples were taken within 3 months after seroconversion. As defined previously (The cutoff values for low and high avidities of IgG for B19, HCMV, and The intra-assay variability for the IgM SIA was calculated with 8 replicates in the same run, and interassay variability was calculated with 6 distinct runs, employing serum pools containing or lacking the respective IgM. Interassay variability for the IgG avidity SIA was evaluated with 6 to 10 distinct runs during 3 months, using pools containing acute-phase or past-infection serum samples.The positive percent agreement, negative percent agreement, and kappa values between SIAs and the reference assays were calculated with serum panel 1. For the statistical calculations, borderline values in IgM SIAs were considered positive, given the primary role of IgM assays in screening. In IgG avidity SIAs, in turn, borderline-avidity values were considered high-avidity values, due to the important role of these assays in ruling out recent primary infections . All the


In 2014, the government of Togo implemented a pilot unconditional cash transfer (UCT) program in rural villages that aimed at improving children’s nutrition, health, and protection. It combined monthly UCTs (approximately US$8.40 /month) with a package of community activities  delivered to mother–child pairs during the first “1,000 days” of life. We primarily investigated program impact at population level on children’s height-for-age z-scores (HAZs) and secondarily on stunting (HAZ < −2) and intermediary outcomes including household’s food insecurity, mother–child pairs’ diet and health, delivery in a health facility and low birth weight (LBW), women’s knowledge, and physical intimate partner violence (IPV).n = 82) or a control arm . Two different representative samples of children aged 6–29 months and their mothers were surveyed in each arm, one before the intervention in 2014 , the other 2 years afterwards in 2016 . Difference-in-differences (DD) estimates of impact were calculated, adjusting for clustering. Children’s average age was 17.4 (± 0.24 SE) months in the control arm and 17.6 (± 0.19 SE) months in the intervention arm at baseline. UCTs had a protective effect on HAZ , which deteriorated in the control arm while remaining stable in the intervention arm, but had no impact on stunting . UCTs positively impacted both mothers’ and children’s (18–23 months) consumption of animal source foods (ASFs)  and household food insecurity . UCTs did not impact on reported child morbidity 2 week’s prior to report  but reduced the financial barrier to seeking healthcare for sick children . Women who received cash had higher odds of delivering in a health facility  and lower odds of giving birth to babies with birth weights (BWs) <2,500 g . Positive effects were also found on women’s knowledge  and physical IPV . Study limitations included the short evaluation period (24 months) and the low coverage of UCTs, which might have reduced the program’s impact.We implemented a parallel-cluster–randomized controlled trial, in which 162 villages were randomized into either an intervention arm  programs, which consist of direct cash payments to eligible beneficiaries, are among the promising nutrition-sensitive interventions, having demonstrated  positive impacts on poverty, food security, attendance at health services, and, to a lesser extent, children’s health and nutritional status.Despite their great potential, CTs’ impact on child linear growth is not conclusive, and their pathways of impact are not clearly understood. Studies thoroughly examining CTs’ mechanisms are needed, especially in West Africa, where evidence of CTs’ impact is scarce and where the successes seen in Latin America may not be replicable because of specific constraints that may affect the West African family environment.The researchers randomly allocated 162 villages from 5 northern districts in Togo to either an intervention arm or a control arm. Although both study arms benefited from the integrated community case management of childhood illnesses and acute malnutrition (ICCM-Nut) and behavior change communication (BCC) activities related to children’s nutrition, health, and protection, only women from the intervention arm received in addition monthly unconditional cash transfers (UCTs) (US$8.40/month) during their child’s “first 1,000 days” of life (from pregnancy to their second birthday).n = 2,658) and 2 years later .Surveys were conducted at 2 points in time among different samples of mothers and their children, 6–29 months old, both before the intervention began  that may hinder or enhance impact. In addition, theory-driven evaluations, which precisely estimate the contribution of a wide range of mediating factors to the final impact on linear growth  are also needed to identify the most effective levers of actions and help design future programs. Although the number of stunted children under age 5 has fallen in many countries over the past decades, global figures remain alarming, and stunting is declining too slowly to meet the target set by the World Health Assembly for 2025 . In WestPromising nutrition-sensitive interventions include cash transfer (CT) programs. Originating in Latin America, these programs proved to be effective in reducing poverty and food insecurity and in increasing school enrollment and use of health services . DespiteOptimizing the nutritional impact of future programs will require a better understanding of how CT programs work . Very feWe hypothesized that the program would improve children’s growth through a synergy effect between the community activities, which would raise mothers’ awareness and provide them with essential knowledge in health and nutrition, and the cash that would allow them to put into practice what they have learned through those community activities. This would result in improved health and nutrition of mother–child pairs , which in turn would help improve children’s growth. Providing women with better knowledge and financial means would also contribute to their empowerment, which has been recognized as an important factor in their own health and that of their children.m + 1 sensitization meetings, where m is the mean number of times women of a given village took part in sensitization meetings.The pilot CT program was administered for 30 months in 2014 by the government of Togo, with financial and technical support from the World Bank and UNICEF. It consisted of monthly cash distributions to women during their children’s first 1,000 days of life  combined with behavior change communication (BCC) activities (including home visits and community sensitization meetings) and with the integrated community case management of childhood illnesses and acute malnutrition (ICCM-Nut). The program was implemented in the 5 districts  of the Kara and Savanes regions, where the highest rates of acute and chronic malnutrition in children under 5 exist. In 2014, 33.7% and 32.1% of children under 5 years of age were stunted in the Kara and Savanes regions, respectively, versus 27.5% at the national level. The same regions also saw rates of wasting in children of 7.2% and 11.2% versus 6% at the national level. Within the 5 aforementioned districts, the program was implemented in the villages where the “ICCM-Nut program” was running. Carried out since 2011 by the Ministry of Health and UNICEF, this program targeted rural landlocked villages that had poor access to health facilities. In those villages, community health workers (CHWs) were trained and equipped to screen and treat childhood malaria, diarrhea, pneumonia, and acute malnutrition and to lead sensitization meetings on essential family practices . On top of those preexisting activities, BCC sessions focusing on child protection issues  were organized by specifically trained community child protection workers (CCPWs) together with the CTs . CHWs anWomen who were at least 3 months pregnant and mothers of children aged 0–23 months were eligible to receive the CT. No minimum age limit was set up. If eligible, adolescent women could also benefit from the CT. Women with several eligible children received the intervention for the youngest one only. However, mothers of twins and mothers with both an eligible biological child and an eligible fostered child received the CTs for both children. Mothers with a child aged 24–59 months suffering from severe acute malnutrition were also eligible to receive the CTs. Beneficiary women could enter the program anytime between their second trimester of pregnancy and their child’s second birthday throughout the program duration (knowing that the beneficiaries’ list would be updated every 2 months). Regardless of when they entered, they received the CTs for a minimum of 12 months. Those who entered early in pregnancy took full advantage of the program, benefiting from the CTs for the maximum duration of 30 months.n = 80 villages) or into an intervention arm benefiting from the ICCM-Nut program, CCPWs’ package of activities, and CTs (n = 82 villages). Initially, 81 villages were allocated to each arm, but 1 control village was accidentally allocated to the intervention arm while disseminating the randomization results to the communities; it was decided to maintain this village in the intervention arm for ethical reasons. The randomization was stratified by district and carried out by the government of Togo and the World Bank using a random real number, generated with the RAND function in Excel.A nonblinded parallel-cluster–randomized controlled trial was carried out to assess the impact of the cash component of the program. A total of 162 villages were randomized into either a control arm benefiting from the ICCM-Nut program  and from CCPWs’ package of activities (sensitization meetings and home visits around child protection issues)  and the other after 24 months of program operation  . Represez-score in height-for-age (HAZ) among 6- to 29-month–old children while considering the following parameters: 5% significance level, 90% of power, 10% of missing/invalid data, 162 villages to be randomized in 2 arms, and an intraclass correlation coefficient of 0.02. To detect a 0.20 HAZ difference between the intervention and control arms at endline (assuming a variance of 1.3 determined from previous studies), a sample size of 1,020 children per arm and per survey was required. This sample size also allowed detecting a difference in the prevalence of stunting of 9% between the 2 arms. This calculation was conservative because it did not account for the district stratification used for randomization. An average of 13 mother–child pairs were surveyed in each village (n = 128), ranging from 5 pairs in small villages (n = 16) to 17 pairs in large villages (n = 18).The sample size was calculated to detect a change of 0.20 Data were collected using the same standardized questionnaire at baseline and endline. The questionnaire was administered face-to-face to women and heads of households during home visits by experienced enumerators speaking the local languages of the study area. These enumerators were identified through a highly selective process and extensively trained over 2 weeks. Their training mainly consisted of the exhaustive review, translation, and pretest of the questionnaire; each question was standardized across the different vernacular languages spoken in the study area. Enumerators also learned how to perform anthropometric measurements and how to collect data through tablets. Built upon the program’s theory of change, the questionnaire covered a wide range of intermediary outcomes meant to document the program’s theoretical impact pathways, which are presented in their simplified version in The impact was primarily evaluated on HAZ and secondarily on stunting (HAZ < −2 SD) among 6- to 29-month–old children. Children’s height measurement was standardized according to WHO recommendations  and carrWe identified 2 major pathways whereby the program may impact children’s growth and several enabling factors that may facilitate the impact achievement .Indicators along this pathway included the Infant and Young Child Feeding (IYCF) practices indicators, namely the Minimum Dietary Diversity (MDD), the Minimum Meal Frequency (MMF), and the Minimum Acceptable Diet (MAD), which were computed among 6- to 23-month–old children following WHO guidance . We alsoAt the child level, we collected data on overall health since birth (as perceived by the mother), morbid episodes over the previous 15 days, and medical monitoring since birth. At the mother level, we collected information on antenatal follow-up, delivery, and postnatal care. In children aged less than 20 months, who had a chance to be exposed to the program in utero, we collected birth weights (BWs) from health documents when available and calculated the proportion of children with low birth weight (LBW) . The hygiene of individuals and their houses was assessed using spot-check observations ,34. In tSee We collected information on household composition and size, on the quality of the house , and on the head of household’s sex, level of education, religion, and primary occupation. A youth ratio, defined as the number of household members <15 years of age over the number of persons ≥15 years, and a dependency ratio, defined as the number of people not contributing to household income over the number of people contributing to household income, were computed to account for household structure. Mothers’ ages, pregnancy statuses, education, and marital statuses were collected, as well as the child’s sex and age.Data were collected using Android tablets (through ODK Collect at baseline and Survey CTO at endline), ensuring quality controls at data entry. Collected data were regularly sent to an online server, allowing additional quality checks and feedback for fieldwork improvement. Data quality was also guaranteed by the close field supervision of enumerators throughout the study. Data management was performed with R 3.3.2., and data analysis was performed using Stata 14.2. HAZs and stunting were computed using WHO’s multicenter growth reference standards macro for R . All anaComparability of the trial arms at baseline was tested on sociodemographic characteristics and anthropometric outcomes using linear regression models for continuous variables and logistic regression models for categorical variables. Using the same method, we also compared at endline the coverage of the BCC component between arms and provided a few descriptive statistics on the coverage of the CT component in the intervention arm.Yi is the outcome of interest, phase indicates the time of the survey (baseline/endline), arm indicates the intervention (CT + package of community activities/package of community activities only), phase × arm is the interaction term between the phase and the trial arm, Xi is a matrix containing a set of control variables, μi is the random unobserved error term, β1 represents the time trend common to intervention and control arms β2 accounts for the time-invariant differences between the intervention and control arms, β4 is the vector of coefficients corresponding to the matrix of control variables, and β3 is the DD estimator, which estimates the average program’s effect. For HAZ, the DD estimate is equal to the regression coefficient (β3); for stunting, the DD estimate is based on predicted adjusted values obtained from the regression model by arm and survey. It is reported in percentage points (pp) along with the relative odds ratio (ROR) arising from the logistic regression. Beta and ROR are presented along with their 95% confidence interval (95% CI) and p-value.The program’s impact on primary and secondary outcomes  was estimated using a difference-in-differences (DD) analysis. DD estimates were computed using linear regressions for HAZ and logistic regressions for stunting. The DD model can be specified in regression form asThe program’s impacts on intermediary outcomes within the “food and nutrient” and “health and hygiene” pathways and on enabling environment variables were estimated via the exact same procedures used for HAZ for continuous outcomes and stunting for categorical outcomes. For several outcomes, we restricted the analyses to a subsample according to the child’s age at endline and his/her theoretical exposure to the program ; e.g., tTo increase the precision of our estimates, we systematically included in our analyses the following covariates: the district in all impact analyses, the child’s age and sex when examining the impact of the program on HAZ and stunting, the child’s age when examining the impact of the program on health outcomes, the child’s sex and maternal height when examining the impact of the program on BW and LBW, and verification (yes/no) of the interviewee responses in a health document when examining the impact of the program on pregnancy-related outcomes.n = 400). All these analyses have been prespecified in the study protocol  approach . Becauseprotocol .Ethical clearance was provided by the Ministry of Health of Togo and by the consultative ethic committee of the Institut de Recherche pour le Développement in France. Written informed consent was obtained from all mothers who took part in the study. The trial was registered in the ISRCTN registry under the reference 83330970.The number of households and mother–child pairs surveyed at baseline and endline is presented in n = 635) (n = 400), 235 recalled how many times and claimed that they received it 13 times on average , 58% of eligible women from our sample said they never received any cash (n = 635) . The mai average . The medp < 0.001). CCPWs from intervention villages were also more likely to organize sensitization meetings than those from control villages . The same was true for CHWs . In turn, women from the intervention villages had higher odds of always attending sensitization meetings organized by CCPWs  and CHWs  than women from the control villages.In comparison to CTs, CCPWs’ BCC activities and ICCM-Nut activities were implemented without major issues. The ICCM-Nut activities were already well orchestrated, and CHWs were able to help less experienced CCPW’s on a daily basis. Moreover, those program components were less multisectoral than the CT component and only relied upon 2 ministries, the Ministry of Health for the ICCM-Nut component and the Ministry of Social Action for the CCPWs’ activities. Although the implementation of those components was smoother and did not lead to substantial issues with program fidelity, they were nonetheless better implemented in the intervention arm than the control arm. Both CCPWs’ and CHWs’ BCC activities were more frequent in the intervention villages than in control villages . For insp = 0.039) (p < 0.01), whereas HAZ remained stable in the control arm . The impact found on HAZ did not apply to child stunting  . Howeverp = 0.031). Children aged 18–23 months, who were still benefiting from the program at endline, also had higher odds of eating at least 2 types of ASF . The program also had a positive impact on the proportion of women who had at least 3 meals on the day prior to the survey  and on the proportion of women who consumed at least 2 types of ASF . No significant impacts were found on WDDS-10 or MDD-W. At the household level, the program had a positive impact on food security. Households from the intervention arm had lower odds of experiencing severe food insecurity than those from the control arm .See p = 0.024). The program had no impact on child morbidity . However, sick children from the intervention arm were less likely to forgo visits to the health center because of financial constraints than their control counterparts . The program had a positive impact on several antenatal care variables, including the proportion of women who received iron  and intermittent preventive treatment (IPT) for malaria . Cash-recipient women also had higher odds of delivering in a health facility as compared to nonrecipients . The program had a protective effect on LBW . Regarding postnatal care, beneficiary women were more likely to attend the postdelivery visit  and to receive postnatal iron supplementation  than nonbeneficiary women. The program had a positive impact on overall hygiene , which was mainly driven by the positive impact observed on women’s hygiene .See p < 0.001) but a rather negative impact on their decision-making power . This result is mainly driven by a negative impact on their decision-making pertaining to children . Regarding IPV, women receiving the CTs had lower odds of having experienced physical violence in the last 12 months than nonbeneficiaries . We did not find any impact on controlling behavior or emotional violence. In line with the consumption of ASFs among women and children, the program had a positive impact on the amount that households spent at market to buy ASFs .See Our study showed UCTs associated with community activities  and implemented over the “first 1,000 days” in Togo had a protective effect on linear growth among 6- to 29-month–old children when compared with community activities only. The program also had positive impacts on several intermediary outcomes along the “food and nutrient” pathways, those being household food security and consumption of ASFs by women and older children (18–23 months); along the “health and hygiene” pathway, positive impacts were seen on prenatal and postnatal iron supplementation, the proportion of women receiving IPT for malaria and sleeping under an impregnated mosquito net during pregnancy, the percentage of deliveries at health services, the proportion of LBW (among 6- to 19-month–old children), the proportion of women attending at least 1 postnatal consultation, and the hygiene of the mother. In the enabling environment, the program had a negative impact on women’s decision-making power but positive impacts on women’s knowledge, physical IPV experienced by women over the past 12 months, and household purchases of ASFs at markets. All of these impacts were found despite a suboptimal coverage of the CT component of the program.p < 0.001) and control arms . In the intervention arm, however, households were less likely to report a high impact of those losses on their economic situation at endline than at baseline , pointing to a protective effect of the CTs. Moreover, given the low coverage of the program, the effect of the CTs in the intervention arm may also have been mitigated by children who never received it. PP analysis, even if exploratory, shows that CTs not only prevented a deterioration in children’s HAZ but actually improved it , as reviewed in De Groot and colleagues , and 1 fWithin the “health and hygiene” pathway, the program had no impact on children’s health or on children’s medical follow-ups, which were already quite good in both groups at baseline. In the literature, the effects of CTs on children’s health status are mixed, with some studies showing reduction in the prevalence of diarrhea or reduction in episodes of illness and other studies showing no impact at all . Neverthp = 0.031). Finally, the impact found on women’s knowledge could be explained not only by a better implementation of the BCC activities in the intervention arm, as already mentioned, but also by the strong motivations of beneficiaries themselves. They were incentivized both by the bonus and by the untrue belief that they needed to attend sensitization meetings to continue receiving the CTs.Regarding the enabling variables, our findings showed, contrary to what could have been expected, that the program rather had a negative impact on women’s decision-making, at least when considering a score based on sole decisions by women. Taking a closer look at data, we showed that this result was mainly due to a decrease in beneficiaries’ decision-making power regarding decisions relative to children . Interviews with beneficiary women and their husbands, conducted as part of the process evaluation, indeed showed that fathers interfered more in decisions on children since the start of the program because they wanted to ensure "proper" use of the CTs. However, this does not mean that women had no say. Besides, the validity of decision-making measures have been criticized ; for exaNevertheless, improving women’s awareness and lifting financial constraints may not always be sufficient. Demand-side interventions such as CTs should also invest in the supply side to be fully efficient. If the cash incentive allows women access to health services and food markets, then both quality healthcare and nutrient-rich foods should also be available to enhance their health and diet. To improve their impact, CT programs should also tackle issues that may optimize their implementation and coverage, such as communication and work sharing between stakeholders, staff organization and management, and planning and delivery of activities. Our study indeed showed that in remote and resource-poor settings, implementing such complex multisectoral programs according to protocol and ensuring their full coverage was challenging. This was especially true here given that the program was a pilot and that most of its operators were inexperienced. The program’s results could have been maximized through further experience, but although it was temporarily extended to other districts based on its positive results, the program did not continue in its current configuration. Nevertheless, it opened the road for CTs in Togo, as evidenced by the recent creation of a governmental CT program to support those who have lost their income because of the adoption of response measures against COVID-19. Moving beyond pilot and emergency programs to implement long-lasting and large-scale social safety nets will require more time and experience, but Togo now seems to be on the right track.One major strength of our study lies in the use of a well-designed randomized controlled trial, in which we documented the program impact on a wide range of intermediary outcomes along the hypothesized program impact pathways, in addition to the impact on mean HAZ. Providing a global picture of the program impact rather than fragmented results helped in understanding the results found on linear growth. As foreseen in the theory of change of the program, our analyses confirmed that the intervention had an impact on multiple factors acting at different times over the “1,000 days” course to influence children’s growth. Examining the relative effect of each factor over the entire course of the intervention would have required performing a mediation analysis on the subsample of children aged 18–19 months, i.e., children who took full advantage of the intervention from early pregnancy to age 2. Unfortunately, our study was not powered for this. Besides, using a longitudinal design rather than repeated cross-sectional surveys would have allowed examining the change in children’s growth charts between groups to document the incidence of stunting rather than its prevalence. Because stunting results from a cumulative process, a longitudinal design would have provided more insight on children who were in the process of becoming stunted. Longitudinal designs may also offer opportunities for dose–response analyses. On the other hand, the usual delays in program roll-out and full implementation can have negative effects on an evaluation study cohort , making it unusable for assessing impact on age-sensitive outcomes such as stunting. Additional constraints of longitudinal designs include the logistical complexity and related additional cost of tracking individual children over time, with high risk of loss to follow-up. Finally, an information bias may also occur because individuals from a cohort are followed more closely than the whole population, with possible consequences on representativeness and external validity, whereas cross-sectional design better preserves real-life conditions.This impact evaluation had some limitations. Although the program was running for 30 months, the impact study had to be conducted over a relatively short period of time—24 months—to avoid any seasonal effect between baseline and endline surveys. Theoretically, this time lapse was largely manageable in terms of the program’s correct functioning; nevertheless, there were still major implementation issues at the time of the endline survey, which probably weakened the impact of the program. This encouraged us to use PP analyses to discuss our results on children’s linear growth and stunting. Although this helped to confirm some ITT trends, we are aware that PP analysis is not without bias, especially because our PP analyses only concerned the CT component of the program and did not deal with participation in BCC activities. Because monitoring data were not made available to us, our analysis of program coverage is only based on retrospective declarative data collected from interviewees at endline. Such data are subject to recall bias. Beneficiary women may have been more likely than nonbeneficiaries to remember the number of times frontline workers organized sensitization meetings in their village or visited them. Moreover, we did not collect data on the functioning of health facilities, which could have helped explain certain results. Another limitation lies in the fact that the program itself may have influenced some interviewee’s responses, either because they wanted to stay in or because they wanted to make a good impression as beneficiaries. We attempted to mitigate such social desirability bias through the extensive training of enumerators. We are confident in the fact that our enumerators were trained enough to minimize this bias, however. Lastly, the external validity of our study is limited to rural at-risk areas of Western African countries.To conclude, we demonstrated that CTs implemented during the “first 1,000 days,” in combination with community activities , had a protective effect on child’s growth among young children living in a vulnerable area of northern Togo. Our study contributes to the existing literature on CTs by providing new evidence from a West African country where data are missing and fills a gap by examining the effect of CTs on multiple intermediary outcomes along the theoretical impact pathways. Besides, the impacts found on various factors along those pathways confirm that to be efficient in the fight against stunting, interventions should address several determinants at a time and give particular attention to the conception and preconception periods, as evidenced by the positive effects observed on pregnancy- and birth-related outcomes. Our study also suggests that to impact growth, CT program should meet certain conditions including articulating clear nutrition objectives, providing sufficient amounts of cash, targeting the most at-risk populations over the first 1,000 days, and combining CTs with supply-side investments and educational components. It indeed supports the view that moving toward “cash plus” programs—i.e., programs that bundle CTs with additional components in an effort to improve the effect of the money—is key to making social protection truly transformative . To be fS1 FigCT cluster-randomized controlled trial, Northern Togo, 2014–2016. CT, cash transfer.(TIFF)Click here for additional data file.S2 FigCT cluster randomized controlled trial, Northern Togo, 2014–2016. CT, cash transfer; ITT, intention to treat; PP, per protocol.(TIFF)Click here for additional data file.S3 FigCT cluster-randomized controlled trial, Northern Togo, 2014–2016. CT, cash transfer; HAZ, height-for-age z-score; PP, per protocol; SE, standard error.(TIFF)Click here for additional data file.S1 TableCT cluster-randomized controlled trial, Northern Togo, 2014–2016. CT, cash transfer.(DOCX)Click here for additional data file.S2 TableCT cluster-randomized controlled trial, Northern Togo, 2014–2016. CT, cash transfer.(DOCX)Click here for additional data file.S1 ProtocolCT, cash transfer.(DOCX)Click here for additional data file.S1 CONSORT ChecklistCT, cash transfer.(DOC)Click here for additional data file.


Dear Editor,First of all, we would like to thank the authors for the criticism of and contributions to our recent article about idiopathic granulomatous mastitis (IGM) [1]. IGM is a rare disease; however, arousing interest in it is seen in recent years.In the current study, our IGM treatment experience was presented from a rheumatology perspective. The patients, who had a histopathological appearance of granulomatous inflammation in breast specimens, were referred to our rheumatology outpatient clinic for differential diagnosis of inflammatory disease of the breast. The patients who participated in our study were referred to our clinic from general surgery clinics of our hospital or other centers. All the patients had already been treated with antibiotics or surgical interventions, such as drainage, at the referring centers. These procedures are not the routine practice of our rheumatology clinic. The unresponsive patients were referred to our clinic and were reevaluated primarily for infectious and noninfectious causes of granulomatous diseases. To us, it is more rational to make the differential diagnosis by a rheumatologist since the other granulomatous diseases mimicking IGM are somewhat familiar to the rheumatology practice. Making the differential diagnosis is more complicated than ordering “autoimmune markers”. We have diagnosed one case of eosinophilic granulomatosis with polyangiitis, two cases of sarcoidosis, and one case of tuberculosis in the patients referred to us as IGM. After excluding these diseases, the patients were followed up with the diagnosis of IGM in our rheumatology outpatient clinic.There is no consensus on the treatment algorithm for IGM. Antibiotics, surgical intervention, glucocorticoids (GCs), and immunosuppressive therapy are options. In the current study, we reported our experience, especially about methotrexate (MTX). In the literature, the recurrence rate of IGM has been reported as high as 50%. Therefore, it is obvious that the need for alternative approaches is mandatory. Although follow-up period is relatively short in our study, the recurrence rate is quite low in comparison to the reported literature data. Based on our MTX experience, which is incomparable to the surgeons, we initiate MTX for almost all IGM patients concurrently with GCs. Although MTX is also known as an antineoplastic agent and has potential side effects, it is quite safe in the long term in doses used in rheumatology practice with appropriate precautions. MTX is used and recommended to decrease or prevent the serious adverse effects of GCs in patients with other inflammatory diseases, which constitutes our rationale behind initiating this agent as early as possible . Besides, the initiating of MTX as low as 5 mg/week is quite far from its therapeutic dosage. If the patient has no concomitant diseases, such as renal or hepatic disease, MTX should be initiated at a minimum dose of 10 mg/ week. The efficacy of MTX treatment appears after 2–4 weeks of treatment. Appropriate dose titration can be made with an increase of 2.5–5 mg/week every 4–6 weeks. Generally, doses of 10–15 mg weekly are sufficient in most of the patients. Doses may be increased to 25 mg/week [3]. In the case of remission, firstly, GC dose should be reduced and discontinued if possible. There is no standard approach to MTX dose reduction, which can be applied for those who have been in remission for at least 6 months. In our clinical practice, the dose is reduced 2.5 mg every 3 months to the minimum effective dose (7.5 mg/week). The cessation should be considered in patients who have been in remission for at least 6 months with the lowest effective dose of MTX. If relapse occurs during tapering MTX or GC, the dose is increased to the previous effective dose immediately. If remission cannot be achieved, either increasing the dose or switching to another immunosuppressive treatment alone or in combination are options for the treatment.Classifying IGM as a benign disease only because it does not have malignant features should be taken as controversial. According to this approach, we need to consider other inflammatory disorders, such as rheumatoid arthritis or vasculitis as benign diseases, which should be questioned in terms of the indication of MTX and GCs. IGM may potentially cause cosmetic, physiological, and psychological undesirable results. For this reason, to prevent these unfavourable outcomes, and to protect patients from short and long term adverse events of GC, initiating MTX early with GC seems feasible, which corresponds to our therapeutic approach.Finally, since there is a growing body of evidence about imaging modalities, such as USG and MRI, that can be used in the diagnosis of IGM, we needed to emphasize the indispensable role of biopsy .The rheumatology follow-up of the IGM patients who were presented to our rheumatology outpatient clinic was evaluated in the current study. We extend our thanks to the authors for their interest and allowing us to emphasize our contributions.