In pediatric practice it is common for infants under 2 months of age to undergo evaluation for sepsis when they are ill, often including lumbar puncture to assess for central nervous system (CNS) infection. The FilmArray Meningitis/Encephalitis (ME) panel is a newly approved test for rapid identification of CNS pathogens. Our objective was to study the epidemiology of CNS infection in young infants and the potential impact of rapid multiplex PCR on their care. A performance evaluation of the FilmArray ME panel was conducted from February 2014 to September 2014 at 11 sites. FilmArray ME panel results were compared to reference standards but not shared with providers. In our study, medical records for infants (aged 1 to 60 days) enrolled at three sites were reviewed for clinical, laboratory, and outcome data. A total of 145 infants were reviewed. The median age was 25 days. Most of the infants were hospitalized (134/145 [92%]) and received antibiotics (123/145 [85%]), and almost half (71/145 [49%]) received acyclovir. One infant had a bacterial pathogen, likely false positive, identified by the FilmArray ME panel. Thirty-six infants (25%) had a viral pathogen detected, including 21 enteroviruses. All infants with enteroviral meningitis detected by the FilmArray ME panel and conventional PCR were hospitalized, but 20% were discharged in less than 24 h when conventional PCR results became available. The FilmArray ME panel may play a role in the evaluation of young infants for CNS infection. Results may be used to guide management, possibly resulting in a decreased length of stay and less antimicrobial exposure for infants with low-risk viral infection detected. Infants in the first 2 months of life are at high risk for invasive bacterial infection, and it is the standard of care for them to undergo an evaluation for sepsis when they have fever or other concerning signs, such as lethargy or poor feeding . As partBacterial meningitis and herpes simplex virus (HSV) encephalitis are severe infections that require prompt treatment with parenteral therapy , 3. OtheThe introduction of molecular testing for viral illness into the care process for evaluating young infants for sepsis has led to a number of changes in management –7. Altho–We performed a retrospective evaluation of clinical and outcome data for 145 infants 1 to 60 days old enrolled in the performance evaluation for the FilmArray  Meningitis/Encephalitis (ME) panel  at threeThis study was approved by the Institutional Review Boards of the University of Utah and Primary Children's Hospital , Children's Hospital Los Angeles , and Nationwide Children's Hospital .Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae), seven viruses , and one yeast group (Cryptococcus neoformans/C. gattii).A prospectively enrolled performance study for the FilmArray ME panel was conducted between February and September of 2014 at 11 U.S. sites . The totFor the present study, medical records for infants aged 1 to 60 days enrolled in the clinical performance study at 3 of the 11 sites were retrospectively interrogated for demographic, clinical, and outcome data. Clinical and outcome data included laboratory data, clinical presentation on admission, length of hospital stay (LOS), administered therapies and discharge diagnoses.PCR results and clinical data were available for review for 145 infants 1 to 60 days old. Demographics, clinical characteristics and results of the FilmArray ME panel and conventional testing are shown in Streptococcus pneumoniae) identified by the FilmArray ME panel, and one had a fungal pathogen (Cryptococcusneoformans/C. gattii) identified. Neither child had abnormal CSF studies or positive conventional testing, and neither was diagnosed or treated for CNS infection. Secondary testing during the parent study could not confirm these detections, suggesting that these were both false-positive findings (findings .Thirty-six infants (25%) had 37 viral pathogens detected from CSF. One infant had two pathogens (EV and HPeV) detected. Of the 37 pathogens, 36 (97%) were detected by the FilmArray ME panel, and 21 (57%) were detected by conventional CSF testing. Of these, 14 infants (39%) had CSF pleocytosis, defined as a CSF white blood cell (WBC) count of >14  (Ninety-two percent (33/36) of the infants with a virus detected from the CSF were admitted to the hospital; one was admitted to the ICU. The median LOS for admitted infants was 44 h . Three infants (8%) with virus-positive CSF  had a concomitant bacterial infection diagnosed, including one bacterial meningitis (mentioned above) and two UTIs. Twenty-nine of the infants with a viral detection from CSF (81%) received antibiotics, and 20 (56%) received acyclovir. Fifty percent received both antibiotics and acyclovir.E. coli) and sixteen with UTIs. Most  received antibiotics, and almost half  received acyclovir.One hundred seven infants (74%) had no pathogen detected in CSF by either the FilmArray ME panel or conventional tests. The mean CSF WBC count was 4 , but 17 infants (16%) with negative CSF testing had CSF pleocytosis . Ninety-nine infants (93%) with no CNS pathogen detected were admitted to the hospital, with a median LOS of 70 h . Two had blood PCR results positive for EV. Eighteen (17%) were diagnosed with a bacterial infection, including two infants with bacteremia (one group B streptococcus and one n = 4) positive for EV by the FilmArray ME panel who did not receive a diagnosis of EV by CSF or blood PCR for statistical comparisons. There were too few infants , while conventional testing identified a CNS pathogen in 21 (14%). In two cases, the FilmArray ME panel results were likely false positives. This was suggested by secondary PCR testing in the original study , and thepathogen , was detted here . Of noteThirty-six (25%) infants in this study had CSF positive for a viral pathogen. EV was the most common virus detected in our cohort, followed by HPeV. Infants with viruses other than EV detected infrequently had CSF pleocytosis, including those with HPeV and HHV-6, a finding consistent with previous reports –18. A nu–Many hospitals have introduced institutional guidelines for the management of infants diagnosed with EV , 10, andHPeV is less well studied, but it has been suggested that with further data these infants may be able to be managed similarly to those who are positive for EV , 18. IncFurther studies will need to be performed to guide recommendations for evaluation and management of infants in whom herpesviruses other than HSV are detected. In our study, one of the infants with CSF positive by the FilmArray ME panel for HHV-6 was diagnosed with bacterial meningitis based on a positive CSF Gram stain, suggesting that detection of HHV-6 from CSF may not exclude other CNS infections. This has also been shown in other studies . HHV-6 iDetection of CMV in the CSF of young infants with suspected sepsis is also complex. While detection of CMV may not explain their presentation, it suggests the possibility of congenital CMV infection and the need for additional testing and possibly treatment . This isNo infant in this cohort had CSF positive for HSV, which is consistent with the low incidence of HSV encephalitis in this age group , 28. ThiE. coli K1, only six bacterial pathogens are represented, and early neonatal pathogens such as other E. coli serotypes and Citrobacter spp. are not included. Thus, in infants with a high suspicion for meningitis, antibiotics should not be stopped based on a negative test (Drawing conclusions about potential management changes for infants with negative CSF rapid molecular testing is difficult. While the FilmArray ME panel detects many of the common pathogens of neonatal meningitis , includiive test , 29. SevOur study has several limitations. While this is the largest study to date examining the use of multiplex molecular testing for CNS pathogens in young infants, there were a limited number of positive detections, and sample sizes were too small for statistical comparisons. Most infants with pathogens detected were positive for viruses, and we could not evaluate the impact of molecular testing on the detection and management of bacterial meningitis. No infant had HSV detected, and the impact on HSV infection also could not be evaluated. Our study was performed retrospectively, and the results of FilmArray ME panel testing were not shared with providers. Thus, we can only postulate how management may have changed if results were available. Granular data on hospital costs were not available such that conclusions could be made regarding the potential effect of rapid testing; however, this is a critical measure of the impact on care.Young infants are often evaluated for sepsis and LP is frequently performed. The FilmArray ME panel can rapidly detect a number of the most common pathogens causing meningitis/encephalitis in young infants and may play a role in their evaluation. Clinical judgment should drive both the choice to use this test and interpretation of the results with regard to further management. The results of rapid multiplex PCR testing may be used to guide further work up and antimicrobial therapy, possibly resulting in decreased LOS and antimicrobial exposure for infants with viruses such as EV that are rapidly detected.


A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform’s utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient’s response to therapy and show the patient’s achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring. Digital PCR offers clinicians the necessary tool for absolute quantification and enhanced precision tracking of ultra-rare (<1%) mutations.It is becoming increasingly accepted that while Next Generation Sequencing (NGS) is an invaluable tool for broad based sequence characterization, its precision in tracking rare mutant events is low. In a recent study, Stetson et al. in 1988 highlighting a Poisson distribution of individual PCR target sequences2 with Volgelstein et al., later coining the term “Digital PCR”3. Unlike the popular quantitative PCR (qPCR) where PCR templates are quantified by comparing the rate of PCR amplification of an unknown sample to the rate of a set of known qPCR standards, dPCR has been proven to exhibit higher sensitivity4, higher precision5 and improved reproducibility6. The advent of microfluidics has greatly aided the advancement of dPCR; however, current available partitioning technologies suffer from limitations in three critical areas – low partition volume consistency, low partition number consistency and high dead volume of loaded sample. Droplet-based dPCR technologies9 have innate issues with droplet-to-droplet volume uniformity that can affect the accuracy of quantification, impact reproducibility and hinder conformance to clinical regulatory requirements. Reagent partitioning that requires user-controlled moving parts or user-applied thin films on the consumable12 can create non uniformity in partition size. Partition and volume non-uniformity, as well as the potential for instrument contamination will likely be obstacles preventing other dPCR platforms from transitioning from research-use to the clinic. Furthermore, variable partition volumes result in variable distribution of targets inside the partitions, thus undermining the mathematical basis of dPCR and downstream analysis. Fluctuating partition numbers underscore the instability of the partitioning process and introduce unnecessary variables into the assay and limit sensitivity. Unanalyzed sample via a high dead volume in the consumable introduces subsampling error and reduces the overall accuracy of the results. Subsampling error is a function of both the expected target concentration as well as the degree of subsampling. The combination of rare targets with high dead volume synergistically lowers dPCR accuracy and broader based applicability for clinical applications.The concept of Digital Polymerase Chain Reaction (dPCR) was first published by Saiki 13. This means that users are only analyzing between 55% and 80% of their loaded sample.Here we present a novel Microfluidic Array Partitioning (MAP) consumable device that utilizes precisely manufactured high volume injection molded arrays combined with precise fluidic control to overcome current dPCR limitations. The high-volume injection molding process ensures that all partitions have a consistent and well-defined sample volume. The partition size is defined by the physical limitation of the microarray wells and not by a stochastic process. The partition array consists of a fixed geometry and ensures a consistent number of partitions for all samples across each experimental run. The number of partitions is defined by the array geometry and is minimally susceptible to the variation in the partitioning process. Coupled with a novel precise fluidic loading process, the MAP consumable device utilizes >95% of input sample and significantly reduces subsampling error compared to other currently available technologies that utilize <80% of the loaded sample. For example, in the BioRad QX200 ddPCR system users routinely only generate between 11,000 and 16,000 analyzable droplets per sample out of a possible 20,000To address the needs of adopting dPCR as a routine tool for personalized medicine, a complete clinic-friendly single instrument dPCR integrated platform was developed. This dPCR platform was designed for simplified workflows, a reduction in contamination potential and rapid time-to-answer. Using three unique cancer-specific applications we have highlighted the platform’s versatility to perform precision, personalized cancer biomarker monitoring.et al.14 and Farrar et al.15. The combination of active heating and low thermal mass synergistically enables faster rates of temperature ramp while still maintaining uniform temperature profiles across the MAP consumable. These combined features allow this novel platform to achieve sample-to-answer in <90 minutes with no change to reagent composition.The novel dPCR platform consists of a micro-molded plastic Microfluidic Array Partitioning (MAP) consumable device Fig.  and a fuThe MAP consumable partitions each sample into 20,000 micromolded array wells without the need for valves, user-applied films or any moving parts. After the user pipettes 10.5 µL of bulk PCR reagent into a MAP consumable inlet, the user next overlays 5 µL of silicone oil into the same inlet well. After these two liquid handling steps (which can be automated), the inlet wells are capped (and hence closed off) with specialized caps that allow for positive pressure to pass through. The consumable is then loaded into the instrument where the automated loading protocol is initiated. Figure The mechanism by which the MAP consumable is loaded relies upon the 40 µm thin Cyclo Olefin Polymer (COP) film to which the micromolded features are bonded. When positive pressure is applied to a reagent-containing inlet well this 40 µm thin film becomes gas permeable. As the reagent enters the microfluidic features, air is outgassed through the thin film. This allows reagent to completely fill dead-ended features such as the dPCR partitions and also removes all bubbles from inside of the microfluidic features Fig. . First tThe loading of the MAP consumable and the reagent partitioning is accomplished in one instrument by applying positive air pressure to the inlet well with a pneumatic pump . The cap applied on the inlet well prevents the instrument from contacting any potentially contaminated surfaces. Positive displacement pressure of 50 psi directed into the MAP consumable throughout the workflow ensures that no aerosolized contaminants will enter the instrument. This novel fully integrated instrument is dry, contamination-free and requires no routine maintenance to allow for the potential to transition to routine clinical use.Fully automated software controls the sample digitization into the MAP partitions, thermal cycling and imaging. The partition arrays are imaged before and after PCR thermal cycling in each of the fluorescent dye colors enabling the analysis software to subtract the background fluorescence and correct for non-uniform excitation. A ROX quality control dye that is added to the PCR reaction mix is visible in all partitions and used to confirm the sample was loaded completely and to locate each partition. Partitions without a ROX signal are rejected to reduce the number of false negatives. For each PCR probe dye color, partitions with a significant increase in brightness after PCR thermal cycling are considered positive. The ratio of positive partitions relative to the total number of partitions is used to calculate the sample concentration by applying a Poisson distribution analysis. To initially verify the capabilities of our system, pre-quantified standards from a Next Generation Sequencing (NGS) library quantification kit were run (Takara Cat No. 638324). Quantitative results from the novel platform showed great linearity across dilutions and also small coefficients of variation across replicates  and those achieved on the novel dPCR platform (e.g.: 0.13%) could be explained by the fragmentation process used to generate the synthetic samples. These results show the novel dPCR platform’s capability to precisely quantify samples containing <1.0% T790M EGFR in a background of WT EGFR with high reproducibility, high accuracy and high specificity.EGFR is an important drug target for the treatment of non-small cell lung carcinoma (NSCLC). During the treatment of NSCLC with tyrosine kinase inhibitors (TKIs), there is typically an early response, followed by relapse upon outgrowth of a pre-existing subclonal mutation or the acquisition of a de-novo mutation that confers resistance. Early detection of resistance mutations can better inform patient treatment and guide appropriate more efficacious drug selection. One key EGFR mutation that leads to TKI resistance is the T790M mutation. There are only a few clinical assays that have been approved as companion diagnostics in patient biopsies (FFPE or plasma), while a larger number of laboratory developed tests (LDTs) under CLIA/CAP guidance are finding routine use in cancer disease diagnosis or cancer treatment monitoring. EGFR T790M mock cell-free DNA (cfDNA) standards  in combination with a commercially available EGFR T790M dPCR assay  were used to validate the novel platform presented in this study. The cfDNA standards tested were synthetically manufactured, fragmented and then blended with well-characterized background material to fully mimic cfDNA that would be purified from a patient blood sample. Premixed PCR reagent containing mastermix, the EGFR T790M dPCR assay and the following cfDNA standards were run in triplicate units on the platform: 100% wild-type(WT) EGFR, 1.0% EGFR T790M and 0.1% EGFR T790M Fig. . All repet al.16 to a duplex dPCR assay. Using this dPCR assay, synthetic gBlocks (double stranded DNA fragments) for both ABL1 and BCR-ABL1 were quantified to determine their nominal concentrations  cases are characterized by the presence of the BCR-ABL1 fusion gene. Wild type (WT) ABL1 is a tyrosine kinase that plays a role in cell cycle regulation and cellular differentiation. The expression of the BCR-ABL1 fusion oncogene generates a constitutively active tyrosine kinase leading to uncontrolled cellular proliferation and eventually cancer. The advent of tyrosine kinase inhibitor (TKI) therapy has had a transformative effect on CML patient outcomes. Regular monitoring involving precise quantification of BCR-ABL1 transcripts is integral to successful TKI treatment, occasionally leading to drug discontinuation upon achievement of deep remissions. The current gold standard for clinical BCR-ABL1 transcript quantification is Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). A well-characterized single-plex RT-qPCR BCR-ABL1 assay was previously modified by Alikian ons Fig. . These sons Fig. . Thresho18. A seven-week-old boy diagnosed with JMML was shown to have WT status for all known JMML mutations. Both an institutional DNASeq panel and RNASeq revealed an inframe CCDC88C-FLT3 fusion19. Prior to this discovery, FLT3 fusions had not been reported in any pediatric malignancy. Given the novelty of this CCDC88C-FLT3 fusion, no commercial assays were available to identify fusion transcripts below the sensitivity of cytogenetics. As such, the oncologist designed a specific ddPCR assay to analyze the abundance of this CCDC88C-FLT3 fusion in peripheral blood RNA over serial time points18. This ddPCR-based method for monitoring the patient’s response to Sorafenib (Nexavar) enabled the patient to proceed safely to hematopoietic stem cell transplantation (HSCT) and eventually achieve a complete molecular remission.To demonstrate the platform’s potential utility in personalized medicine, a droplet digital PCR (ddPCR) assay that was previously designed for precision monitoring of a specific patient was replicated on the novel digital PCR platform. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood with nearly half of all patients relapsing within three yearsCCDC88C-FLT3 fusion positive dPCR partitions were set using the no template control. All measurements post-HSCT were true negatives on the novel dPCR platform, showing zero positive partitions containing the CCDC88C-FLT3 fusion target. These results replicated the trends achieved with this assay and samples from this patient using a commercially available ddPCR platform19 indicating a high level of concordance. This personalized medicine approach to precision monitoring of patient-specific biomarkers showcases the potential for dPCR as a powerful tool for companion diagnostics.Replicating this assay on the prototype dPCR platform with bio-banked samples from the same patient revealed that the fusion transcript levels in the patient’s peripheral blood did not decrease following cytotoxic chemotherapy but then dropped following Sorafenib monotherapy and reached undetectable levels following HSCT Fig. . ThreshoHere we have presented a novel fully integrated digital PCR platform that was designed to facilitate the adoption of dPCR for precision oncology-based diagnostics. We have showcased the platform’s utility for quantifying <1.0% mutant burden using two clinically relevant applications (EGFR T790M and BCR-ABL1). Furthermore, we demonstrated the utility of this platform for a personalized medicine application by replicating a published clinical study that tracked a patient’s transition to complete molecular remission using a custom, patient-tailored dPCR assay.20. Future integration of our dPCR platform with artificial intelligence (AI) and machine learning could enable unparalleled dPCR multiplexing capabilities.  Real-time monitoring is also critical for improving assay development and can be used to improve partition classification over traditional endpoint dPCR alone. Real time measurement of dPCR partitions can potentially remove the need for arbitrary thresholding entirely. These features combined with dPCR’s innate ability to quantify ultra-rare (<1.0%) genetic events will unlock the power of dPCR for precision cancer monitoring and for improving patient outcomes to advance human health.Finally, the MAP consumable and fully integrated single system dPCR platform presented is the first of its kind that has potential to allow for real-time and melt-based analysis of digital PCR partitions. Melt-based multiplexing is the key to unlocking the potential for dPCR to produce higher plex quantitative assays, allowing clinicians to precisely quantify panels of 10 or more nucleic acid biomarkers per sample run. In fact, it has previously been shown that a computer can be trained to recognize 89 unique, amplicon-specific PCR melt-signaturesMAP consumables were micro-injection molded with high optical clarity Cyclo-Olefin Polymer (COP)  and sealed with a thermoplastic thin film. Using con-focal laser interferometry, the devices have shown dimensional variability within ±0.1 µm in planar and ±1.0 µm in height, which translated to ±1.5% total volume variation. In addition, a lamination strength study was conducted to show that the devices could withstand over 100 Psi pressure without delamination for over 24 hours.Seraseq® ctDNA EGFR T790M reference standards with nominal (company provided) values were acquired from SeraCare . These reference standards were used as the template in dPCR reactions containing a commercially available EGFR T790M dPCRassay . The probe for EGFR WT fluoresces in the HEX channel, whereas the probe for T790M fluoresces in the FAM channel. Primers, probes and Seraseq template were combined with the following dPCR master mix and ROX loading control dye: QuantStudio 3D Digital PCR Master Mix v2 , ROX Reference Dye . Once mixed, 10.5 µL of the complete dPCR reagent was loaded into an inlet well on the consumable and overlaid with 5 µL of silicone oil . In the final reagent mix the mastermix was diluted 2X, the ROX dye 100X, the primer/probe assay mix 20X, the template 10X, then brought up to volume with water. After the instrument had completed the reagent digitization, the consumable was thermal-cycled using the following program: 1 cycle of 95 °C for 1 minute, 40 cycles of 95 °C for 4 seconds followed by 60 °C for 18 seconds. After running this program the instrument captured images of the various partition arrays using 3 filter sets: ROX, FAM and HEX.et al. (2017). Primers, probes and full BCR-ABL1 and ABL1 template sequences (as synthetic double stranded DNA gBlocks) were custom ordered from Integrated DNA Technologies . The probe for ABL1 fluoresces in the HEX channel, whereas the probe for BCR-ABL1 fluoresces in the FAM channel. Primers, probes and gBlock template were combined with the following dPCR master mix and ROX loading control dye: QuantStudio 3D Digital PCR Master Mix v2 , ROX Reference Dye . In the final reagent mix the mastermix was diluted 2X, the ROX dye 100X, the primer/probe assay mix 20X, the template 10X, then brought up to volume with water. Once mixed, 10.5 µL of the complete dPCR reagent was loaded into an inlet well on the consumable and overlaid with 5 µL of silicone oil . After the instrument had completed the reagent digitization, the consumable was thermal-cycled using the following program: 1 cycle of 95 °C for 1 minute, 40 cycles of 95 °C for 4 seconds followed by 60 °C for 18 seconds.. After running this program the instrument captured images of the various partition arrays using 3 filter sets: ROX, FAM and HEX.Primer, probe and full amplicon sequences for both the BCR-ABL1 and ABL1 dPCR targets were derived from the study by Alikian et al. (2019). RNA was extracted from peripheral blood and bone marrow using the RNeasy kit  and 100 ng RNA was reverse transcribed using the Maxima H Minus RT kit  and used as a template for dPCR. To prepare the dPCR reactions, a mastermix was prepared using equivalent to 10 ng RNA of cDNA per 10.5 µL reaction. Primers, probes and cDNA template were combined with the following dPCR master mix and ROX loading control dye: QuantStudio 3D Digital PCR Master Mix v2 , ROX Reference Dye . In the final reagent mix the mastermix was diluted 2X, the ROX dye 100X, the primer/probe assay mix 20X, the template 10X, then brought up to volume with water. Once mixed, 10.5 µL of the complete dPCR reagent was loaded into an inlet well on the consumable and overlaid with 5 µL of silicone oil . After the instrument has completed the reagent digitization, the consumable was thermal-cycled using the following program: 1 cycle of 95 °C for 1 minute, 40 cycles of 95 °C for 4 seconds followed by 60 °C for 18 seconds. After running this program the instrument captured images of the various partition arrays using 2 filter sets: ROX and FAM.Primers and FAM-labeled probes were designed to amplify and detect CCDC88C-FLT3 fusion cDNA templates according to the methods described by Chao Supplementary Information


It was found that both Cr@IrO2 and Ni@IrO2 NPs, fabricated under basic conditions (pH = 13.5) denoted as Cr@IrO2-B and Ni@IrO2-B, respectively, were the best catalysts among the eight tested ones. Moreover, it was confirmed that variation in pH resulted in the changes in the surface area (defect structure), which were considered to be responsible for the changes in the catalytic properties of these NPs. For the oxygen evolution reaction, these NPs exhibited relatively smaller overpotential (η) values than other tested Cr@IrO2- and Ni@IrO2-containing NPs. Furthermore, methylene blue degradation analysis and OH radical formation experiments by benzoic acid showed the same trend. Thus, we confirmed that the catalytic activity of transition metals doped IrO2 NPs fabricated under basic conditions can be improved.The effects of pH variation on the catalytic activity of IrO MO NPs such as those of TiO2, SnO2, and CeO2 are particularly stable catalysts and have hence been synthesized in various forms 10. Since MO NPs are also used in batteries, many researchers are still studying them particularly to enhance their effectiveness13. Iridium oxide nanoparticles (IrO2 NPs) also constitute smart materials having impressive applications as catalysts17, components in fuel cells20, and others23. They have even attracted the attention as catalysts for the oxygen evolution reactions (OER) in batteries27.Metal oxide nanoparticles (MO NPs) have various advantages such as low cost, high stability, and better catalytic properties than those of metals, and are thus being used as efficient catalysts since several decades32. In other words, the defect structure on the IrO2 NPs has been proposed as the active sites of electrochemical oxidation and photocatalytic reactions through the excitation from the O-p band to the Ir-d band33. Hence, many research groups have been tried via inserting dopants such as transition metals into these NPs to enhance their catalytic activities, because the doped transition metals can increase the defect structure by the charge transfer through reaction with the IrO2 NPs35. In case of IrO2 NPs, transition metal dopants can access various oxidation states and then consequentially change their electronic states of IrO2 NPs. Although the active sites of the IrO2 NPs can be different depending on the catalytic reactions due to the different chemicals and reaction pathways, similar strategies such as doping have been successfully demonstrated to enhance the catalytic activity of IrO2 NPs.Meanwhile, several studies have identified the catalytic efficiency of MO NPs to be closely correlated with the concentration of defect structures formed on their surface, and in particular, dependent on their surface area2 NPs, as well as those doped with metallic Cr  or Ni , display relatively poor catalytic properties, a modification of their surface affects their catalytic activities. These studies also showed that the changes in pH levels of the environment of NPs may modify their activities38.Various other studies showed that although undoped IrO2 NPs doped with Cr and Ni, denoted as Cr@IrO2 and Ni@IrO2 NPs, analyzed the effect of pH variation on their catalytic properties, and then systematically compared their catalytic activities with those of NPs which pH levels were not adjusted. We characterized the electronic structures and morphologies of Cr@IrO2 and Ni@IrO2 NPs by using high-resolution photoemission spectroscopy (HRPES) and scanning electron microscopy (SEM), respectively. We specifically aimed to compare the catalytic properties of these NPs and to determine the driving force of each catalyst, with the ultimate purpose of deriving the pH condition that would yield the highest catalytic efficiency for eight tested NPs. To figure out NPs that provide the best catalytic efficiency, we tested their catalytic activity with the change of overpotential by OERs, methylene blue (MB) degradation, and OH radical formation of benzoic acid in aqueous solutions.In the current work, we synthesized recently well-studied IrOf and O 1s) of the eight IrO2-containing samples  were obtained by HRPES  and 63.4 eV , and O 1s peaks at 532.4 eV (IrO2) and 529.6 eV . When comparing the spectra of different samples, we focused on the variation in the intensities of the peaks induced by the defect structures . The intensities of these peaks obtained from both TM@IrO2-B NPs  were observed to be larger than those of other samples  were evaluated in the alkaline 1 M KOH electrolyte condition  displayed greater OER activities than did the other Cr- and Ni@IrO2-containing NPs, with the overpotential (η) values of 258 mV and 305 mV, respectively .In order to compare the electrocatalytic activities of the investigated samples, we recorded the electrochemistry measurementsion Fig. . As showely Fig. . It is n2-B and Ni@IrO2-B samples showed only a modest decrease, to 93.5 and 94.3%, respectively, of their initial values. Thus, all synthesized NPs can be considered to be relatively stable catalysts. The modest decrease observed for the Cr@IrO2-B and Ni@IrO2-B samples may be due to some sample desorption or deformation that possibly occurred during the oxidation reaction. Our results showed that we were able to improve  the overpotential of TM@IrO2 NPs, especially Ni@IrO2 NPs, by appropriately changing pH of these NPs, and suggested a general way for improving the catalytic efficiency of such NPs.In addition, the ability of eight tested NPs to maintain an efficient catalytic activity in the oxidation reaction for many cycles is an important factor that determines their practical applicability. To test this, the oxidation reaction for each sample was run for 100 cycles, and the results are shown in Fig. 2, TM@IrO2-A, TM@IrO2-N, and TM@IrO2-B NPs; TM = Cr or Ni) present a negligible extent of dye degradation. While slow degradation occurred, approximately 30–60% of MB degraded in 180 minutes under the same conditions for all samples. As shown in the Fig. 2-B is better than those of other samples, as expected; in case of Cr@IrO2-B NPs, MB degraded by nearly 35% in 180 minutes.MB degradation analysis was carried out to compare the catalytic efficiencies of the as-prepared samples. As shown in Fig. 2 NPs, Ni@IrO2-B NPs (55%) shows the best catalytic activity, as expected  of NPs. Table In addition, among the four differently treated Ni@IrOsee Fig. . These rC and C0 are absorbance of the M at time t and at time 0. k is the rate constant (min−1) and t is the time (min.).As shown in the plot Fig. , the degC/C0) was plotted against time, a linear relationship was obtained. As can be seen from the first-order reaction plot shown in Fig. 2-B and Ni@IrO2-B NPs are remarkably higher than those of other NPs. This indicates that the photocatalytic activity of the two NPs is better than that of the other NPs. It can therefore be confirmed that the photocatalytic property of TM@IrO2 NPs can be enhanced under basic condition (pH = 13.5).When ln(k) for the change in concentration (ln(C/C0)) with time (min.) for the eight tested samples. In detail, the apparent rate constant for the decay of the reactant for Cr@IrO2 NPs was 5.73 × 10−3 min−1 when using Cr@IrO2-B NPs, greater than the cases using other NPs . In case of Ni@IrO2 NPs, we can confirm the same trends; 3.13 × 10−3 min−1 for Ni@IrO2, 2.07 × 10−3 min−1 for Ni@IrO2-A, 2.67 × 10−3 min−1 for Ni@IrO2-N, and 3.33 × 10−3 min−1 for Ni@IrO2-B. In other word, the significantly higher rate constant for the degradation reaction in the presence of Cr@IrO2-B or Ni@IrO2-B NPs would be attributed to the higher surface area of the NPs compared with those of other NPs.We also calculated the rate constant value (2 NPs has previously been shown to be closely related to the concentration of defect structures (or increased surface area) in the samples. In addition, we found that the intensities of the HRPES defect-induced peaks, i.e., those corresponding to Irx+ or IrOx defect-induced structures, differed for NPs with different pH treatments , and the oxidation reactions catalyzed by them, we found that Cr@IrO2-B and Ni@IrO2-B NPs showed better OER  and MB degradation performances than did the other Cr@IrO2- and Ni@IrO2-containing NPs , respectively.As shown in Tables 2-B and Ni@IrO2-B) than in other six TM@IrO2 NPs (TM = Cr or Ni), and the analysis of the SEM and BET results  is formed. By comparing the amount of p-HBA formed with time, the above-mentioned influence of the ·OH radicals on the catalytic reaction can be comparatively analyzed49.To confirm that the OH radicals of eight tested NPs synthesized by their treatment under basic conditions (pH = 13.5) were generated in large amounts, the radical reaction of benzoic acid was performed.2 NPs  synthesized in the basic conditions were the most reactive regardless of the doping metal. In particular, Cr@IrO2-B NPs exhibited remarkably good reactivity. The results are also consistent with the results of the OER electrochemical experiment and the MB degradation experiment. In addition, it was confirmed that the tendency was similar to the change in the surface area, confirmed by the BET experiment, which provides a direct proof that the concentration of ·OH radicals existed on the surface of TM@IrO2 NPs are proportional to that of the defect structures.As can be seen in Fig. 2 NPs could be the most effective way to improve the efficiency of the catalyst.Therefore, our results proved that the concentration of defect structures  is the most important factor that influences the quality of the catalyst, which can be enhanced by the pH treatment under basic conditions. Specifically, maximizing the number of defect structures while maintaining the characteristics of IrO2 NPs. We found that Cr@IrO2 and Ni@IrO2 NPs fabricated under basic conditions, i.e., Cr@IrO2-B and Ni@IrO2-B NPs, to be the best catalysts among those tested. We specifically found that varying pH resulted in changes in the surface structure, which in turn was considered to be responsible for the changes in the catalytic properties. High-performance OER catalysts were achieved by doping the transition metals (TM = Cr or Ni) into IrO2 NPs, with Cr@IrO2-B, and Ni@IrO2-B NPs exhibiting the relatively small overpotential values of 258 mV and 305 mV, respectively, while other tested NPs showed poorer activities. In particular, the relatively high overpotential value and poor MB degradation of Ni@IrO2 NPs could be overcome by treating them with base (pH = 13.5); this pH change was found to affect the concentration of defect structures. In addition, we also found that the catalytic activity of TM@IrO2-B NPs is better than those of other samples by evaluating their MB degradation performances . We also provided a direct evidence with surface morphological change and the variations of ·OH radical formations on the surface of eight tested TM@IrO2 NPs when the concentration of the defect structures is increased. In conclusion, we found it necessary to modulate the pH levels of the NPs during their fabrication in order to endow them with high catalytic activities.Our experimental analysis showed a clear effect of pH treatment on the catalytic properties of IrO2 NPs were prepared from potassium hexachloroiridate (K2IrCl6) and sodium hydroxide (NaOH) by following a modified version of Wohler’s method51. Upon heating, the initially dark green solution became increasingly transparent, and then turned dark blue at 90 °C, as desired. An increasing quantity of particles was seen over time after the heating was completed. After being stored at room temperature, the upper layers of the heated solution separated from the lower level of the upper stream, and a sediment eventually formed over this separation. No such sediment was found when the solution was heated to 90 °C. After preparing 1 mole % doped Cr@IrO2 and Ni@IrO2 solutions 2∙xH2O and Ni(NO3)2∙6H2O, respectively), we transferred the solutions into a Teflon-lined autoclave, which was then placed for 7 h in a convection oven preheated to 220 °C, to result in the corresponding TM@IrO2 NP (TM = Cr or Ni) solutions, which had a gel-like appearance. We changed the pH levels of some of the samples of these solutions with HNO3 (acid) and of others with KOH (base), and maintained them at the target pH for 6 h. After approximately 20 min, the synthetic gel solutions became transparent. These transparent solutions were transferred into the Teflon-lined autoclaves that were then sealed and heated to 220 °C for 7 h in a convection oven. Resulting TM@IrO2 NPs  were filtered and washed with double-distilled water (DDW) to remove any residue. Using this procedure, we prepared eight different IrO2 NP-containing samples.Aqueous solutions of IrO29. Methylene blue (MB: 0.15 mM) degradation of Cr@IrO2 and Ni@IrO2 NPs was measured at regular intervals, mixed immediately with 1 mL of methanol as inhibitor, and then filtered. Real-time concentration variation was measured using an UV–Vis spectrophotometer  at 484 nm. Electrocatalytic activities of prepared Cr@IrO2 and Ni@IrO2 NPs were compared by carrying out the OER in 1 M KOH electrolyte. Each of the eight samples (10 mg) was dispersed in a solution containing either Nafion  or absolute ethanol  by ultra-sonicating the mixture for 30 min. The electrochemical OER activities were then measured in 1 M KOH  by using a potentiostat . The results of the repeated measurements for each sample varied by ± 4.5%. The radical formation of benzoic acid (20 µM) for the eight tested samples (0.4 g/L) was measured using a 300-W Xe arc lamp (NEWPORT 300 W Xenon Light Source) with a 320 nm cut-off filter.Surface morphologies of the prepared samples were measured by using a field-emission scanning electron microscope  operating at 15 kV. Surface area analyses were performed by the Brunauer-Emmett-Teller (BET) method using an Autosorb-iQ 2ST/MP apparatus (Quanta chrome). Electronic structures of the samples were analyzed using the high-resolution photoemission spectroscopy (HRPES) at the 8A1 beamline (the Pohang Accelerator Laboratory)


To qualitatively assess the effects of a multi-modal school-based water, sanitation and hygiene (WASH) intervention on handwashing behaviour among primary students in North Western (NW) Tanzania.The study was a qualitative assessment of barriers and facilitators to handwashing among students attending primary schools participating in the Mikono Safi Trial (Kiswahili for ‘Clean Hands), a cluster-randomised trial assessing the impact of a school-based WASH intervention on selected soil transmitted helminth infections. Data collection methods included in-depth interviews with teachers, focus group discussions and friendship pair interviews with students collected between April and October 2018. The Capability-Opportunity-Motivation and Behaviour model was used to inform data collection and analysis.The study was conducted in four purposively selected intervention schools in three districts of Kagera region, NW Tanzania .Participants comprised 16 purposively selected teachers aged between 23 and 52 years and 100 students aged 7–15 yearsThe Mikono Safi intervention increased students’ reported capability and motivation to wash their hands with soap at key times, particularly after visiting the toilet. Improvements in students’ handwashing knowledge and skills were reported by both teachers and students, and motivation for handwashing was enhanced by emotional drivers such as disgust, fear and nurture. Newly established handwashing stations improved the physical opportunity to wash hands, although the availability of water and the provision of soap was not always consistent . Students and teachers were actively engaged in intervention implementation which created a school community that valued and supported improved hand hygiene.The intervention was successful in improving capability and motivation for handwashing. Handwashing opportunity was also greatly improved, although the supply with water and soap was sometimes interrupted, calling for much stronger multi-sectoral collaboration to improve access to water at schools.ISRCTN45013173; Pre-results. In-depth interviews of teachers and focus group discussions and in-depth friendship pair interviews proved to be suitable methods to examine barriers to and facilitators of appropriate handwashing behaviour.These methods generated consistent results suggesting that the Mikono Safi intervention was effective in improving handwashing behaviourThis was a qualitative study involving a small purposively selected sample of schools, teachers and pupils and may therefore not be representative for all eight intervention schools participating in the trial or for other primary schools in Tanzania.Soil-transmitted helminth (STH) infections are among the most prevalent infections worldwide, affecting over 1.5 billion people.The Mikono Safi (Kiswahili for ‘clean hands’) intervention was designed to increase handwashing with soap (HWWS) among school-aged children in an attempt to sustain the effects of routine MDA. It is a multi-modal intervention, combining health education, games and stories targeting key motivational drivers, improvements in school handwashing infrastructure and training and support to school staff. Training for teachers in all the intervention school was done by a qualified public health specialist and a social scientist with a Master’s degree in sociology. Further details are provided below. The impact of the Mikono Safi intervention is being assessed in an ongoing cluster-randomised trial. As part of the trial, the STH prevalence in eight randomly selected schools in the Kagera region receiving the Mikono Safi intervention will be compared with eight schools in a randomly selected control arm.intervention fidelity and compliance—the extent to which schools and/or implementing organisations have provided WASH services in schools routinely and in a manner consistent with intervention protocols.While school-based WASH interventions in low-income and middle-income countries have been examined with regards to a wide range of health and educational outcomes, including: diarrhoea, respiratory infection, school attendance and health outcomes in the domestic environment,The goal of our study was to explore the mechanisms by which and the extent to which the Mikono Safi intervention influenced the determinants of children’s handwashing behaviour. Our primary research question was: to what extent has the Mikono Safi intervention influenced the capability, opportunity and motivation for HWWS among pupils attending intervention schools? Findings from this research will be used to modify current intervention approaches, to further understand the extent to which school-based interventions influence individual drivers of behaviour, and to provide contextual information for interpreting forthcoming trial results.et al, in review)The Mikono Safi intervention was informed by the Behaviour Centred Design processThere are multiple models and frameworks that have been proposed to understand WASH behaviours.This was a qualitative study where we completed a range of qualitative data collection activities with students and teachers at four schools receiving the Mikono Safi intervention over a 6-month period. We used purposive sampling to select schools reflecting the range of schools participating in the Mikono Safi Trial. This included selected two urban and two rural schools for participation in our qualitative assessment from the four urban and four rural schools participating in the Mikono Safi Trial. We purposively sampled schools with both high and low pretrial STH infection prevalence. At the school-level, selection of teachers ensured inclusion of school administration, teachers in-charge of sanitation and other teachers who were involved in implementing hand hygiene education. Selection of students ensured inclusion of boys and girls, both younger and older children.In each school, we completed two rounds of data collection. The first round of data was collected in April 2018; 2 months after the Mikono Safi intervention had been launched in the four schools. The second round was collected in the last week of October 2018; . The purIn the first round of data collection, we completed two focus group discussions (FGDs) in each school, resulting in a total of eight FGDs with a total of 60 students. The number of FGD participants ranged between seven and 10. The FGDs were age and gender segregated—resulting in two FGDs each for older boys, older girls, younger boys and younger girls. During the FGDs, we explored students’ shared perceptions about the influence of the intervention on handwashing. We also assessed students’ handwashing skills: at the end of each FGD, the field researcher walked with the participating students to the handwashing stations for students to demonstrate how they normally washed hands. Handwashing education sessions had a practical component that took students through five handwashing steps which included: (i) wetting hands, (ii) putting soaps, (iii) scrubbing the palms together, (iv) scrubbing between the fingers and nails while counting up to 10 and (v) rinsing hands with running water. Handwashing skills for each student were assessed during observation by noting how many of the five recommended steps of handwashing were completed.In addition to FGDs, we conducted four in-depth interviews (IDIs) with teachers to collect information on their perceptions about students’ knowledge regarding the relationship between HWWS and STH infection; experience with implementing handwashing lessons; availability and accessibility of handwashing materials at school, and perceived effectiveness of the intervention in targeting motivational and emotional drivers for handwashing.For the second round of data collection (October 2018), we completed five friendship pair interviews at each school, for a total of 10 respondents per school. FPI is a method used to collect data from children where the target respondent is encouraged to come to the interview with a friend in order to create a conducive, non-threatening environment for the children.Data was collected using pilot tested interview guide  . All in10.1136/bmjopen-2019-030947.supp1Supplementary dataThe data collection team comprised three Tanzanian researchers  at PhD or MSc level  and two research assistants, one male and one female with experience in qualitative research methods. Prior to starting fieldwork the research assistants received additional 1 week’s training in qualitative research methods. The first author (ESO), participated in actual data collection at the beginning of each data collection round and coordinated the study. Before each round of data collection the study team visited the schools, explained the purpose of the study, discussed study activities with the school administration and made appointments for interviews and FGDs.This qualitative study is assessing an intervention that was preceded by rigorous formative research. During the formative research there was extensive involvement of students, teachers and regional and district education officers and school WASH coordinators in developing, testing and modifying the various components of the intervention. The feedback received from these stakeholders informed the design and implementation of the intervention. Similarly, the interview and FGD guides used in the qualitative study were pilot-tested and revised to include feedback from the participants. After the end of the research, the findings from the main trial and from this qualitative study will be disseminated through stakeholders meetings.Audio recorded interviews and FGDs were transcribed in Kiswahili and translated into English by the lead author and combined with field notes ready for analysis. Data were analysed using Atlas.ti V.7, a qualitative data analysis and research softwareThe COM-B conceptual framework served as a guide for data analysis and was used to define themes in analysis. A thematic analysis approach was used to facilitate the structuring, description and interpretation of results.Data from both rounds were analysed together using the same methods. Data from each of the two rounds of data collection were then directly compared.All participants provided informed assent (students) and consent (teachers); and parental consent was obtained for all participating students. Permission to interview students was sought from parents/guardians, while permission to interview teachers and school staff was sought from the school authorities. Personal data was anonymised, using ID numbers and stored data was stripped of names, and password protected for use by named research staff only.A total of 60 students participated in eight FGDs and another group of 40 students participated in 20 FPIs. On average a student’s FGD/FPI lasted for 1 hour 40 min while teachers interview was about 40 min. In addition, IDIs were held with 16 teachers who were interviewed 2 months after the start of intervention and again 8 months after intervention had been rolled out. . All teacapability comprised knowledge and understanding of the relationship between handwashing and STH, knowledge of correct handwashing; practical skills regarding handwashing following practical demonstrations of handwashing. Factors related to social and physical opportunity included the successful development of a school community of students and teachers who supported and encouraged handwashing; and the improved quality, number and location of handwashing stations with access to both water and soap. Reflective and automatic factors related to increased motivation for handwashing comprised an increased understanding of the link between handwashing and good health that encourages positive feeling about handwashing, disgust caused by realising that STH infection is linked to ingesting faecal matter; the use of relatable child characters that triggered a feeling of nurture. The main barrier identified was the inconsistent availability of water and soap.Factors that study participants identified as facilitators of good handwashing behaviour were classified as according to the COM-B conceptual framework. Factors related to Two significant themes emerged from the analysis related to handwashing capability: perceived improvements in handwashing skills and techniques and reported improvements in knowledge about when and how to wash hands.Handwashing skills: During handwashing demonstrations, all volunteers were able to follow correct handwashing steps. Participants reported to have mastered handwashing because they had participated in practical handwashing sessions.The teacher took us to the handwashing stations and showed us how to wash hands. We were told that when you come from the toilet you wet your hands, apply soap, then scrub .Both teachers and students believed that the acquired skills were important in facilitating handwashing.Handwashing lessons and educational messaging: Both children and teachers indicated that teaching about handwashing and the use of educational messaging had greatly improved children’s knowledge about how and when to wash hands. All children in both the FGDs and FPIs recalled receiving at least two lessons specific to handwashing in addition to the general sanitation and hygiene education provided within their normal school curriculum. During the interviews and FGDs we asked students the following question: What happens if you don’t wash your hands? Students could explain the link between hand hygiene and infections. The quote below is an example of common stories throughout the students’ interviews.If I do not wash my hands, I get diseases especially worms. For example if I eat even a fruit without washing my hands that means I am eating dirt and I will get worms and stomach ache. So whenever you eat without washing hands you get worms Students also discussed their own behaviour related to the key times to wash hands with soap (after using the toilet and before eating), as illustrated in the following quote.Before the project came … We did not always wash hands after using the toilet. But now were are motivated because we have been educated on the importance of handwashing so we now wash our hands always Knowledge about handwashing and key times to wash hands with soap were consistent across all interviews and FGDs. We noted no meaningful differences between students in rural and urban areas or differences between older and younger children regarding handwashing knowledge.The Mikono Safi intervention influenced opportunity for handwashing in two important ways—changing the physical opportunity for handwashing and altering the social environment related to handwashing at schools.Physical Opportunity: Teachers and students alike indicated that availability and location of the handwashing facilities were important facilitators of handwashing. Teachers in particular noted that every child had to pass handwashing stations on their way to and from the latrine. Handwashing stations were visible and difficult to ignore.Given the location of the handwashing stations… on the way out of the toilet, everybody who uses the toilet can see them on their way out…but you know children like to play with water, so making water and soap available for children makes handwashing part of a game …We wash our hands at the handwashing stations near the toilet. Before they installed the handwashing tanks we never used to wash hands because we did not have water to wash hands but these days we wash our hands because they have put the tanks. But in the past we never used to wash hands Children in the FGDs indicated that the improved handwashing facilities were not only easier and more convenient to use when compared with the tippy-taps that the school was using before but also the tanks contained much more water allowing a larger number of students to have access to water for washing hands.…Before the installation of the handwashing tanks we were using tippy-taps to wash hands and we had only few of them, the water would get finished quickly. But they were even difficult to use. With the current handwashing tanks you go there and open the tap and wash hands. The tanks are big and many students are able to wash hands using water in the tanks. Beyond the physical availability of handwashing facilities, the most important facilitator to handwashing was the consistent provision of supplies (water and soap). In some schools, especially schools with a large student population, it was not uncommon for water to get used up early in the day—particularly during break time when the majority of the children used the toilet. Schools were able to mitigate these interruptions in water availability in various ways including creating rosters for fetching water. In schools that had well supervised and well implemented class rosters for fetching water more children indicated that water was consistently available at the handwashing station.…there are times when water gets finished, but in our school we have a teacher and students responsible for health and hygiene. So when water is finished from the tanks we inform the teacher or student leader and they send a group of students to bring water Water availability, however, was often determined by factors outside of the school’s direct control. All schools experienced disruptions in water supply to varying degrees. In three of the four schools in the study, this was a problem that occurred during the morning break when the majority of the students used the toilets and during the dry season in which the majority of schools in the region experience water shortages. In one school this problem occurred multiple times during a week. During periods of limited availability, teachers needed to interrupt classes for certain groups of students in order for them to fill the tanks. Often, teachers were unwilling to interrupt classes, especially in schools that did not have a well-established class roster for refilling water tanks. Lack of water had a negative influence on handwashing behaviours in general, particularly if water was unavailable for more than 1 day in a week.In general, both students and teachers reported that soap was not consistently provided for washing hands. This was primarily an issue of distribution rather than of availability. At one school, for example, there was no soap available for students at the handwashing station when FGD participants were asked to demonstrate behaviours. Students noted that they had reported this to the teacher responsible earlier but in vain. However, when the moderator sent a student to the head teacher’s office, the soap was provided. Similarly, discussions with school administrators indicated that, when provision of soap was inconsistent, it was usually available at the store.There are a number of times during the week when soap is not available at the handwashing station. This can happen like three times in a week. If the soap is not at the handwashing station we usually go to the Head teacher’s office to get the soap There are times when the soap is not available because the teacher responsible did not check or the students have not reported. This happens sometimes but that usually is for a few hours in a day, not the whole day Social opportunity: Data suggest that the Mikono Safi intervention had created school communities that valued handwashing and that teachers were willing to support handwashing activities through teaching and reminding children to wash hands, supervising children to fill the water tanks, and ensuring that soap was available. Students were also willing to participate in activities that ensured water and soap were available at the handwashing stations:If I came out of the toilet and there is no water I will pick a bucket get water from the main reserve tank and fill the handwashing tanks so that other children and I can wash our hands Today I went to the toilet in the morning but there was no soap at the handwashing station at that time. I reported to the student in charge of sanitation and s/he promised to inform the teacher that there was not soap at the handwashing station Students, especially the youngest ones, were keen to report colleagues that did not wash hands after using the toilet. Teachers described experiences where students would report to them other students who did not wash hands.Certainly, there are changes in children’s handwashing behaviour. I am a teacher for the pre-primary class (the youngest group in school). What I have observed is that these days these young ones report colleagues that do not wash hands after using the toilet. If one of them used a toilet and did not wash hands the others will come running to you to report him or her; they will come and tell you ‘teacher, so and so did not wash his/her hands after using the toilet’; they give you such reports Automatic motivation/Emotional triggers: Data suggest that the intervention was successful in reaching targeted emotional drivers of disgust, fear and nurture in children. In particular, many children described Muta—the boy character depicted in the educational materials as a boy who did not know how to wash his hands—as repulsive and disgusting. On the other hand children found Koku (portrayed as a hygiene loving child) attractive and every child interviewed indicated that he or she would like to be like Koku. Koku was described as clean and smart, but also kind and willing to teach a fellow student who may not have knowledge of handwashing yet.I feel bad about Muta’s behaviour; the fact that he did not wash his hand makes me feel disgusted…. but we like Koku because she was clean and she knew how to wash hands in order to protect herself from disease. Everyone would like to be like Koku, she is clean, smart and she likes helping others … the teachers told us that if you use the toilet but you do not wash your hands and you go and eat anything you may be eating feces. So when you think about that you remember to wash your hands and you will remind all your friends to wash hands Reflective motivation: Although introducing class rosters for fetching water was not part of the original intervention package, two of the four schools used rosters to ensure water was available at the handwashing facilities. For some schools this activity was led by students lead and for others by a teacher. For example, in schools that had active students’ health clubs, members of these clubs were charged with the responsibility for supervising all health related activities in school. Hence, schools which had active student health clubs developed the roster for health activities for each day of the week.Students in schools that had student leaders involved in taking decisions about replenishing water and soap were more motivated to take action to ensure they have materials to wash hands. When children believed that they had power to influence their own handwashing practices—they were willing to take an active role in activities that would facilitate handwashing among themselves and others.The students have received the handwashing lessons very well. They wash hand but they also encourage others to wash hands. For example if they saw a colleague who is dirty they encourage him/her to wash hand in order to avoid being labeled Muta, the character depicted in some of the education materials of a dirty boy… Results from the two rounds of data collection were compared to detect possible differences in terms of perceived barriers and facilitators related to capability, opportunity and motivation for handwashing. We did not find any differences. In both round one and two, students demonstrated good knowledge and skills for handwashing. Similarly, the opportunity for handwashing remained largely unchanged between the data collection points at round one and two. The installed handwashing facilities remained well maintained in all four schools. The schools continued to experience disruptions in water supply, and as before success in mitigating such disruptions varied from school to school.Our study set out to describe the barriers and facilitators to handwashing behaviour from the perspectives of teachers and students participating in the Mikono Safi intervention trial. Our results show that participants associated the intervention with improved skills and knowledge. Children’s description of the intervention matched with the targeted emotional drivers of behaviours. Both students and teachers described changes in the social environment of the school that fostered better HWWS behaviours. However, the physical opportunity for handwashing was mixed. When both water and soap were available, the new handwashing stations increased the ease and convenience of handwashing among students. On the other hand, we noted that schools reported on-going challenges with water availability and that the provision of soap at handwashing stations—even when available at the school—remained inconsistent.Our data suggest that children receiving the Mikono Safi intervention had high capability for HWWS. Students could both describe and demonstrate proper hand hygiene and reported knowing when and why hands should be washed with soap. Although insufficient on its own, knowledge may be a necessary precursor for HWWS.The qualitative data from students and teachers suggest that the Mikono Safi intervention was associated with the targeted emotional drivers of nurture, fear and disgust. In the student data, there were rich stories of children describing Muta’s poor hygiene as repulsive and disgusting. Students noted that they often washed hands to avoid being labelled ‘Muta’ by their colleagues. Studies have shown how motivational drivers can be successful in changing hygiene behaviours,Our findings suggest that the nurture motive was particularly salient to students. Students spoke fondly of the fact that Koku  helped her friends out; and both teachers and students discussed the ways that the school had provided a supportive environment for handwashing. The salience of the nurture motive in our study population is consistent with other studies of students handwashing. For example, a study in Bangladesh that used covert video cameras to record handwashing behaviour found numerous instances of older children helping younger children washing hands and modelling positive behaviours for one another.Making handwashing facilities visible, difficult to ignore but also convenient, were among the attributes mentioned as important by both teachers and students. However, beyond the physical positioning and convenience, the most important facilitator to handwashing was consistent provision of water and soap. Although all schools experienced disruptions in water supply especially at peak hours, some schools that were able to implement a well supervised class roster for fetching water succeeded in avoiding interruptions in water availability at handwashing stations. Similarly students and teachers suggested that inconsistency in the availability of soap at the handwashing stations was largely an issue of internal distribution rather than of external logistics. Our data suggests that while the Mikono Safi intervention was successful in changing capability and motivation, the opportunity for HWWS may not have been sufficiently improved in some schools, either due to external factors such as the dry season, or to internal factors such as the lack of class duty rosters for the collection of water or soap.Our study has some limitations : first, this was a qualitative study involving a small sample of schools, teachers and pupils, and therefore our results may not be generalisable to other intervention schools. However, our schools were purposively selected to reflect the general diversity of schools included in the Mikono Safi study and are similar to most schools in the Kagera region. Second, findings are primarily based on individual interviews and FGDs. Data from direct observation of students’ behaviour was not collected during the qualitative study. However, FGDs, FPIs with students and IDIs with teachers proved to be well suited methods to examine barriers and facilitators to appropriate handwashing behaviour, and produced consistent results. We also do not have information on drivers of behaviour before the intervention was implemented and were not able to interview students in enrolled control groups and therefore can only draw associations between the intervention and students reported determinants of handwashing. Students and teachers were aware of our affiliation with the larger Mikono Safi intervention trial and courtesy bias may have influenced both teachers and students to provide answers they assumed we wanted to hear. Reports on changes in behaviour should therefore be interpreted with caution. However, our study explored the drivers of behaviour rather than the behaviours themselves. We also found high consistency in responses from both students and teachers, and respondents were forthcoming about challenges related to sustained soap provision at schools.The COM-B Framework provided a prudent way to explore the success of the Mikono Safi intervention in addressing the barriers and facilitators for handwashing in our study population. Our analysis was based on drivers and facilitators among students. Viewed from an institutional perspective, several of our key findings could relate to other COM-B determinants. For example, consistent provision of soap from the institution’s perspective may be a factor of reflective motivation—schools may not have prioritised soap distribution or they could have made conscious decisions to withhold materials from students out of budget or supply concerns. Given the important role in intervention compliance in several school-based WASH interventions,In conclusion, the Mikono Safi intervention was associated with high motivation and capability for handwashing, but ensuring the opportunity for handwashing remained a challenge at some schools, especially with respect to a consistent availability of water. For such schools a much stronger multi-sectoral collaboration between the departments for education and water may be required. Results from the forthcoming trial will allow us to understand the impact this intervention had on child health outcomes. Future research focussed on hygiene and hygiene behaviour change should further explore the ways in which motivational and educational messaging can change specific determinants of behaviour; new intervention modalities that promote school-level adherence to routine provision of basic supplies need development and testing in resource-scarce environments like Tanzania


The micro-groove structure on the planar surface has been widely used in the tribology field for improving the lubrication performance, thereby reducing the friction coefficient and wear. However, in the conventional cutting (CC) process, the high-quality, high-precision machining of the micro-groove on titanium alloy has always been a challenge, because considerable problems including poor surface integrity and a high level of the material swelling and springback remain unresolved. In this study, the ultrasonic elliptical vibration assisted cutting (UEVC) technology was employed, which aimed to minimize the level of the material swelling and springback and improve the machining quality. A series of comparative investigations on the surface defect, surface roughness, and material swelling and springback under the CC and UEVC processes were performed. The experimental results certified that the material swelling and springback significantly reduced and the surface integrity obviously improved in the UEVC process in comparison to that in the CC process. Furthermore, for all the predetermined depths of the cut, when the TSR  was equal to one of twenty four or one of forty eight, the accuracy of the machined micro-groove depth, width and the profile radius reached satisfactorily to 98%, and the roughness values were approximately 0.1 μm. The experimental results demonstrate that the UEVC technology is a feasible method for the high-quality and high-precision processing of the micro-groove on Ti-6Al-4V alloy.  Titanium alloys have been increasingly used in aerospace, aviation, shipbuilding and biomedical fields because of their excellent properties such as high yield stress, high toughness, high strength to weight ratio, high creep and corrosion resistivity and good biocompatibility . HoweverTitanium alloys have been considered to be typical hard-to-cut materials owing to their inherent properties such as the high chemical reactivity, high strain hardening (work hardening), low thermal conductivity and small deformation coefficient . Hence, More remarkably, the effect of the material swelling and springback of titanium alloy was enhanced due to its low elastic modulus and thermal conductivity . FurtherThe ultrasonic elliptical vibration assisted cutting (UEVC) is a promising cutting technique which shows particular advantages over CC, like lower machining forces, higher machining stability, less tool wear and a better surface finish ,31,32. FTherefore, as discussed above, the UEVC process is effective to reduce the cutting forces, lower the surface roughness and attain better machining accuracy in micro-groove machining of easy-to-cut materials , brittle materials as well as steel materials. However, the UEVC technology is yet to be used in the machining of micro-grooves on titanium alloy. Moreover, a few studies have been conducted to assess the effect of material swelling and springback in micro-groove machining, which is the dominant factor to deteriorate the form accuracy and surface integrity of micro-grooves, especially for titanium alloy with the high level of material swelling and springback ,38. In txoz plane, which is formed by the nominal cutting direction  and the chip flow direction . Relative to the workpiece, the transient position of the cutting tool can be described as follows:As shown in a and b are the vibration amplitudes in x-direction and z-direction, respectively. θ is the phase shift of the 3rd longitudinal resonant and 6th bending resonant, CV is the nominal cutting speed and f is the vibration frequency. In addition, it should be noted that the ratio of the nominal cutting speed to the peak horizontal vibration speed is an important parameter in the UEVC process. In this study, this ratio is named as TSR. And TSR can be written as:Thus, the tool velocity relative to workpiece can be written as:It should be noted that the intermittent cutting only occurs when TSR < 1. Nath et al. have demonstrated that the machined surface quality improved with the decrease of the value of TSR, and the TSR was usually set to be less than one-twelfth in the ultra-precision machining process . Hence, 0, and the cutting tool separates from workpiece at time t4. Thus, in each cycle of vibration, the contact time between the tool and workpiece is only (t4–t0). The cooling medium was more likely to enter the cutting area, and the cooling effect was enhanced [2. The maximal instantaneous uncut chip thickness (imaxa) is also smaller than the nominal one (pa). This means that the cutting forces and cutting temperature during the UEVC process are smaller than in CC process. At time t3, the velocity of the cutting tool in the z direction is equal to the velocity of the chip flow. This can be represented by the following equation:pV is the velocity of the chip flow and is considered as a constant. According to the vibration equations of the tool (Equations (3) and (4)), the velocity of the tool in the z direction is increased in time period (t4–t3). Therefore, during the time period of (t4–t3), the friction force between the chip and tool is reversed. That means the friction force and the velocity of the chip flow have the same direction, which is conducive to break the chip and pull the chip away from the workpiece, and the results in a remarkable decrease in the cutting force, the suppression of the tool chatter, and an increase in the nominal shear angle [In the UEVC process, the three most important features are the intermittent cutting, the reduced instantaneous uncut chip thickness and the reversal of friction force between the tool and the chip . As showenhanced . As a rear angle ,42. In tz-axis, and the high-precision adjustment platform was used to achieve the height adjustment of the UEVC device. It should be noted that the UEVC device could be considered as a traditional tool holder when it was not powered. The samples were round pie with the diameter of 50 mm and the height of 20 mm. The pre-turning was first completed with the CC process. Following this, a series of micro-groove machining experiments under different machining conditions were carried out. The experimental conditions and cutting parameters are listed in In the experimental setup, a typical titanium alloy Ti-6Al-4V alloy, was chosen. The physical properties of Ti-6Al-4V alloy are listed in dL) is equal to the predetermined depth of cut. The theoretical value of the micro-groove width is wL, and it can be expressed as: dL is the theoretical value of the micro-groove depth. Thus, the theoretical value of the micro-groove width can be obtained from Equation (7). In the micro-groove machining of titanium alloy, the suppression of material swelling and springback is crucial. To evaluate whether the UEVC technology, especially at different TSR, meets to suppress the material swelling and springback, the machining quality assessment of the machined micro-grooves is respectively performed by ultra-depth 3D microscopy system  and a white light interferometer . The machining quality of the micro-grooves with the predetermined depth of cut of 8 μm produced by different processing methods was first analyzed. The reduction in the level of material swelling and springback was directly reflected in the dimensional parameters of the generated micro-groove. As displayed in For further investigating the effect of the predetermined depth of cut on material swelling and springback under different machining conditions, a series of experiments about micro-groove machining of titanium alloy were carried out. The measured parameters of the generated micro-grooves are presented in As can be seen from Surface integrity is an important indicator for evaluating the machined surface quality and has a large impact on the reliability and service life of the part. In contrast, the machined micro-grooves produced by the UEVC process displayed nearly no surface defects on the bottom and side surfaces, as shown in During the processing of the micro-groove on titanium alloy using the CC process, the clear, obvious and straight swelling marks appeared on the bottom and the side of the generated micro-groove. These facts implied that the material swelling and springback effect was very obvious. Further, with the increase of the predetermined depth of the cut, the deviation of the micro-groove width did not change significantly, while the deviation of the micro-groove depth gradually increased. Moreover, the deviation of the profile radius of the micro-groove rapidly increased with the increase of the predetermined depth of the cut. Remarkably, the profile shape of the generated micro-groove was distorted, and deviated largely from the tool shape. Thus, the actual parameters of the micro-groove machined by the CC process greatly strayed from the designed parameters.During the processing of the micro-groove on titanium alloy using the UEVC process, no surface defect and no swelling mark appeared on the machined surfaces of the generated micro-grooves. The profile shape of the generated micro-grooves was smooth, and the profile became smoother with the decrease of TSR. This indicated that the material swelling and springback effect was obviously suppressed. As the predetermined depth of the cut increased, the deviations between the designed parameters and experimental results of the micro-groove width, depth and the profile radius did not significantly change. It is noticeable that, for all TSR, the percentage errors of the generated micro-groove parameters were smaller than 5%, and were smaller than 2% when TSR = 1/48 or TSR = 1/24. This indicated that the accuracy of the machined micro-groove improved significantly by using the UEVC technology.A series of surface defects including irregular ridges, cavities and tearing marks appeared on the micro-groove machined by using the CC process, and these defects vastly degraded the surface integrity and the accuracy of the generated micro-groove. In contrast, the generated micro-grooves produced by the UEVC process displayed nearly no surface defects. Moreover, the bottom surface roughness of the machined micro-grooves increased quickly with the increase of the predetermined depth of the cut during the CC process. However, the bottom surfaces roughness of the machined micro-grooves did not obviously change with the increase of the predetermined depth of the cut during the UEVC process. The roughness values were approximately 0.1 μm, when TSR = 1/48 or TSR = 1/24. Therefore, the application of the UEVC technology led to an obvious improvement of the machined micro-groove in surface integrity.The experimental results certified that the reduction of the material swelling and springback and the improvement of surface integrity can be achieved simultaneously by using the UEVC technology. The value of TSR has an obvious effect on the action mechanism of the UEVC technology, and the decrease of TSR can further promote the superiority of the UEVC technology. In this study, TSR = 1/24 is recommended. Further investigation should be made to obtain the preferable machined surface by optimizing the combination of processing parameters with the consideration of tool wear.In this work, the UEVC technology is firstly introduced into the micro-groove machining of Ti-6Al-4V alloy to improve the machining performance and reduce the level of material swelling and springback, thereby realizing the high-quality and high-precision processing of micro-groove. Based on the theoretical analysis and the experiment results, the main conclusions can be drawn as follows: