Retinol-binding protein-4 (RBP4) has been reported to be potentially involved in the pathogenesis of gestational diabetes mellitus (GDM); however, the findings are inconsistent. Our aims were to review the studies that investigated the association of serum levels and polymorphisms of RBP4 with GDM risk, and to provide recommendations for future research.The databases PubMed, EBSCO, ScienceDirect, and Web of Knowledge were searched up to October 2015 to find out studies evaluating the relationship between serum RBP4 level/ RBP4 polymorphisms and GDM risk. In the meta-analysis of serum RBP4 levels the key inclusion was that studies were designed as BMI-matched studies or had observed non-significant differences in BMI between cases and controls.RBP4 polymorphisms and GDM risk were involved. Our results showed that high serum RBP4 levels represent a risk factor for GDM . The results of subgroup analyses based on “gestational age at blood sampling” or “diagnostic criteria” are consistent with the overall results. However, the postpartum subgroup and “before 24 weeks” subgroup both only include one article and indicate no association between serum RBP4 level and GDM risk. The meta-analysis on the association between rs3758539 polymorphism and GDM risk shows that RBP4 rs3758539 polymorphism is not associated with the development of GDM.Fourteen case–control studies (647 cases and 620 controls) reporting the association between serum RBP4 level and GDM risk, and three studies (1012 cases and 1605 controls) investigating the association between The results of this meta-analysis support the hypothesis that RBP4 is a modest independent risk factor for GDM . The serum RBP4 level is associated with the risk of GDM. However, the association in the first-trimester and postpartum period should be validated by further research. The association between RBP4 rs3758539 polymorphism and GDM risk was not confirmed.The online version of this article (doi:10.1186/s12884-016-0838-7) contains supplementary material, which is available to authorized users. Gestational diabetes mellitus (GDM) is defined as varying degrees of glucose intolerance that is first detected during pregnancy . The preRetinol-Binding Protein-4 (RBP4) is mainly synthesized by hepatocytes and adipose tissues. It was identified in 2005 as an adipocytokine with potential capability in reducing insulin sensitivity and enhancing hepatic gluconeogenesis . A multiIn patients with a history of GDM, insulin resistance exists before pregnancy but worsens during gestation. Insulin secretion is insufficient to compensate for the insulin resistance and this leads to hyperglycemia, which can be detected through routine glucose screening during pregnancy. Thus, chronic insulin resistance is a central component of the pathophysiology of GDM . FurtherRBP4 expression levels  have been investigated for their potential association with the risk of GDM, but the reported findings are inconsistent [RBP4 genetic variants examined in the studies to date, only the rs3758539 variant was analyzed more than once, in the USA, China, and Mexico, and therefore we only analyzed the association between RBP4 genetic variants (rs3758539) and GDM in the present study.To date, several genetic variants that affect nsistent –13. Falsnsistent , 15. AmoThe association between serum RBP4 levels and GDM risk has also been investigated, and, the reported results are also inconsistent , 16–33. RBP4 polymorphisms with GDM risk, and to provide recommendations for future research.Our aims were to review previous studies that investigated the association of serum RBP4 levels and First, the databases PubMed, EBSCO, ScienceDirect, and Web of Knowledge were searched up to October 2015 to find out studies evaluating the relationship between serum RBP4 level and GDM risk. The following keywords were used:  AND (“retinol-binding protein-4” OR “retinol binding protein-4” OR “retinol binding protein 4” OR “retinol-binding protein 4” OR RBP4 OR RBP-4). Next, we systematically searched the same databases for studies that examined RBP4 polymorphisms in patients with GDM. The following keywords were used in this step:  AND (“polymorphism” OR “variant”) AND (“retinol-binding protein-4” OR “retinol binding protein-4” OR “retinol binding protein 4” OR “retinol-binding protein 4” OR “RBP4” OR “RBP-4”); furthermore, the names of specific polymorphisms were combined with “gestational diabetes.” All reference lists of the resulting primary research reports and relevant reviews were manually searched to identify additional eligible studies.RBP4; (2) included a case group of patients with GDM and a control group; (3) diagnosed GDM according to the oral glucose-tolerance test (OGTT); (4) provided data with mean and standard deviation (SD) or median and interquartile range ; (5) were not animal studies; (6) were designed as BMI-matched studies or had observed non-significant differences in BMI between cases and controls . Studies with overlapping data were excluded from our analysis. We identified only one study that required additional information: Ping et al. [P value of the difference in BMI between cases and controls in their analysis of serum RBP4 levels. However, we obtained Ping’s PhD dissertation, written in Chinese, and extracted this information from it. Data were extracted independently by two reviewers in consultation with a third, from each study included. The information regarding the first author, year of publication, study population (country and ethnicity), number of patients and controls, and diagnostic criteria were extracted. For genetic-association studies, information was extracted on the frequency of genotypes, the Hardy-Weinberg equilibrium (HWE) status, and the genotyping method used. For studies investigated serum RBP4 levels, information was extracted on serum RBP4 measurements, the mean and SD of serum RBP4 levels, and the gestational age at the time of blood sampling. When the standard error of the mean (SEM) was reported, SD was calculated as 1 + n2, Eligible studies included in this meta-analysis: (1) investigated the relationship between serum RBP4 levels and GDM risk, or between GDM and at least 1 genetic variant of g et al.  had not 2n1+n2−1 . When a 2n1+n2−1 . If the 2n1+n2−1 . When a RBP4 rs3758539 polymorphism with GDM risk was assessed, by calculating the pooled odds ratio (OR) and 95 % CI, according to general, dominant, and recessive genetic models, and an allelic model [p < 0.05.The association between serum RBP4 level and GDM risk was estimated by calculating the pooled standardized mean difference (SMD) and 95 % confidence interval (CI). To analyze the potential influences of gestational age at blood sampling and diagnostic criteria, we performed subgroup analysis. Based on the gestational age at blood sampling, the subjects were divided into 4 subgroups: (1) before 24 weeks, (2) 24–28 weeks, (3) after 28 weeks, and (4) postpartum; For two studies , 28, theic model , 41. TheQ test and the I2 statistic [P < 0.1 in the Q test, and I2 > 50 %), a random effects model was used for pooling data from the primary studies; if the heterogeneity was not significant, a fixed effects model was used. The HWE compliance of the controls in each study was assessed using the Chi-square test. Sensitivity analysis was performed by sequentially excluding individual studies to assess the stability of the results. Funnel-plot asymmetry was assessed using Egger’s linear regression test, P < 0.05 representing significant publication bias. If asymmetry was observed, contour-enhanced meta-analysis funnel plots were used to distinguish publication bias from other causes of asymmetry [p values were 2-tailed.Heterogeneity among studies was assessed using the tatistic , 43. Whesymmetry , 45. AllP < 0.05), ③ and three papers with the plasma RBP4 concentrations or unavailable data. Final selected 14 case–control studies included 647 cases and 620 controls were included in our meta-analysis  with a total of 647 cases and 620 controls. The results show that serum RBP4 level and GDM risk are significantly associated  Table , Fig. 1.In the sensitivity analysis, 1 eligible study was excluded at a time in order to assess the influence of each dataset on the pooled SMD. We observed no changes in the corresponding pooled SMD or in the significance of the results . In order to analyze the potential causes to the funnel plot asymmetry, we treated the contour-enhanced funnel plots with the trim-and-fill method. This treatment resulted in 4 filled studies, which were all in the region of p < 0.05 . The following results were obtained only including two studies [RBP4 rs3758539 polymorphisms may not be associated with GDM risk.Our meta-analysis shows that higher prenatal serum RBP4 level is related to GDM risk, but this association cannot be shown in the first-trimester and postpartum periods. The GDM diagnostic criteria affected the strength of association between RBP4 level and GDM risk. Adopting a higher threshold of OGTT would result in a larger difference of serum RBP4 level between GDM women and controls. We pooled 17 studies (647 cases and 620 controls) in which the BMI-matched strategy was used, and then analyzed the relationship between serum RBP4 levels and GDM risk. Our results indicated that high serum RBP4 levels were related to GDM risk. We performed subgroup analyses of the effects of gestational age at blood sampling and diagnostic criteria. The studies included in the meta-analysis covered all periods during the pregnancy and only one study concerned the postpartum situation. The pooled results of second- and third-trimester subgroups were consistent with the overall result, GDM gravidas have higher serum RBP4 level than the controls. No statistically significant association was found between serum RBP4 levels and GDM risk in the postpartum subgroup and first-trimester subgroup. In the postpartum study blood samples were collected immediately after delivery. High exertion at delivery, which requires high output of energy, might temporarily enhance insulin sensitivity , and speTwo articles reported the odds ratio about serum RBP4 level between women with and without GDM estimated from logistic regression models. The study by Abetew et al.  revealedRBP4 is located at chromosome 10q23–24 in humans, contains 5 exons and 6 introns, and has been linked to increased risk for type 2 diabetes in various populations [RBP4 rs3758539 polymorphism and susceptibility to GDM, but did not find any significant association. Among the five races included in the meta-analysis, significant results were only observed in Han Chinese. However, given the low frequency of the minor allele of rs3758539 in these ethnic groups [The coding region of ulations , 47. Numulations –56. Someulations , 55, 57.c groups , 13 and c groups , non-sigRBP4 and the risk of GDM. However, the study has certain limitations. First, the subgroup analysis performed based on gestational age at blood sampling included only one postpartum study, which makes the stratified analysis unreliable. Moreover, the meta-analysis of RBP4 polymorphisms involved a small number of samples and studies. Therefore, the results must be interpreted with caution. Second, funnel-plot asymmetry can confound the interpretation of meta-analyses. Here, the observed funnel-plot asymmetry might have resulted from poor methodological quality related to the small size and heterogeneity of the studies, in addition to publication bias [To the best of our knowledge, this is the first meta-analysis that has evaluated the relationship between genetic variants of ion bias , 44, 45.RBP4 rs3758539 polymorphism and GDM risk was not confirmed.The results of this meta-analysis support the hypothesis that RBP4 is a modest independent risk factor for GDM . The serum RBP4 level is associated with the risk of GDM. However, the association in the first-trimester and postpartum period should be validated by further research. The association between Additional file 1: Table S1.Adjusted covariates of all eligible studies for the association with serum RBP4 levels and GDM. (DOCX 18 kb)Additional file 2: Figure S1.PRISMA flow diagram of study selection process (a) genetic variants and (b) serum concentration. (TIFF 4988 kb)Additional file 3: Figure S2.The results of sensitivity analysis of serum RBP4 level with GDM risk. (TIFF 8567 kb)Additional file 4: Figure S3.The results of sensitivity analysis of rs3758539 (GG vs. GA + AA) with GDM risk. (TIFF 5448 kb)


Corticobasal ganglia networks coursing through the striatum are key structures for reward-guided behaviors. The ventral striatum (nucleus accumbens (nAc)) and its reciprocal connection with the ventral tegmental area (VTA) represent a primary component of the reward system, but reward-guided learning also involves the dorsal striatum and dopaminergic inputs from the substantia nigra. The majority of neurons in the striatum (>90%) are GABAergic medium spiny neurons (MSNs), but both the input to and the output from these neurons are dynamically controlled by striatal interneurons. Dopamine is a key neurotransmitter in reward and reward-guided learning, and the physiological activity of GABAergic and cholinergic interneurons is regulated by dopaminergic transmission in a complex manner. Here we review the role of striatal interneurons in modulating striatal output during drug reward, with special emphasis on alcohol. The striatum collects inputs from the entire neocortex and projects to other nuclei in the basal ganglia, ultimately reaching cortical areas implicated in motor planning and execution . DespiteThe majority of neurons (>90%) in the rodent striatum are GABAergic medium spiny neurons (MSNs). MSNs can be activated by motor behaviors triggered by both memory-encoded and environmental cues and exhibit highly context-dependent firing patterns . Local mμm in diameter and have moderately branched aspiny dendrites. The local axonal plexus is extremely dense and heavily invested with presynaptic boutons [ in vitro [The striatal nucleus contains different subtypes of GABAergic interneurons that produce a strong inhibitory postsynaptic potential in MSNs , 20–22.  boutons . FSIs co boutons , 24, 25,in vitro . The prein vitro , where tin vitro , 29. Thein vitro , 29.FSIs play a complex role in coordinating the activity of MSNs, and a deficit in these cells has been observed in dystonia and Tourette syndrome , 30. FSIμm in diameter and possess few, aspiny dendrites. These cells distribute in a rostrocaudal gradient, with a greater density in the rostral parts of the striatum [A second class of GABAergic interneuron comprises the neuropeptide Y (NPY), nitric oxide synthetase, and somatostatin expressing interneurons. These cells have the least dense axonal arborization of all cells in the striatum and receive both cholinergic and dopaminergic inputs . Electrostriatum . Little striatum , 41. SomμM in diameter. In rodent they comprise approximately 1-2% of cells in the striatum [Cholinergic interneurons are large aspiny cells, which can exceed 40 striatum , 42 but striatum . Althougstriatum . Axon costriatum , 21, 44.striatum , 45–47. striatum . The synstriatum –51.Cholinergic transmission shapes both inhibitory and excitatory influences in the striatum, depending on receptor type and projection target. Striatal cholinergic interneurons modulate striatal activity through the corelease of glutamate and acetylcholine, mediating both glutamate- and acetylcholine-mediated currents in striatal FSIs , 53. In Although multiple neurocircuitries are implicated in the rewarding effects of drugs of abuse, the mesocorticolimbic dopamine system is considered the major neurochemical pathway for reward , 60. TheReward associated behavior, however, is an integrative function of corticobasal ganglia networks , 69. Rep in vivo microdialysis conducted on awake and freely moving rodents has repeatedly shown that ethanol increases extrasynaptic dopamine levels in the nAc. This occurs regardless of whether the drug is ingested [Even though fast-scan cyclic voltammetry studies performed in brain slices or anesthetised rodents suggest that ethanol depresses evoked terminal dopamine release in the nAc –80, in vingested , adminisingested , 82, 83,ingested , 84. Posingested , 86. Preingested –90, suggingested , 91, 92.ingested , supportingested , 94.Dopamine is a crucial regulator of striatal microcircuitry. Midbrain dopaminergic neurons project in a topographical pattern, with VTA dopaminergic neurons preferentially innervating the nAc, while dopaminergic neurons in the substantia nigra mainly project to the dorsal striatum , 3. DopaA receptor antagonists exerting a stronger influence over dopamine output in nAc as compared to the DLS [Dopamine influences striatal interneuron activity via presynaptic and postsynaptic actions and distinct receptor subtypes . Overall the DLS , 125.Dopaminergic and cholinergic systems dynamically interact with gate and potentiate sensory inputs to the striatum in a manner that includes presynaptic regulation of neurotransmitter release and postsynaptic effects in target cells . The intIn the dorsal striatum, stimulation of dopaminergic axons evokes several ionic conductances in cholinergic interneurons, suggesting that dopamine dynamically controls cholinergic tone . Phasic Similarly, the control of extracellular dopamine levels by endogenous cholinergic activity results from a complex convergence of neurotransmitter/neuromodulator systems. Tonically active cholinergic interneurons modulate dopamine output through activation of both muscarinic and nicotinic cholinergic receptors on dopamine terminals , 139–141 in vivo, indicating that firing of striatal interneurons is tightly regulated by afferents from other brain regions. Interestingly, electrophysiological recordings performed in vivo show a robust decrease in firing frequency in the majority of FSIs in the nAc, indicating subregion specific effects by ethanol [ in vivo and in vitro recordings support the idea that ethanol affects FSIs directly, while MSN activity is indirectly influenced to a greater degree, such as though modulation by the cholinergic tone [The effects of ethanol on striatal interneurons appear to be highly cell-type specific and region-specific, where decreases in firing rates are shown in cholinergic interneurons and LTSs, while the excitability of FSIs in acutely isolated brain slices is increased in the dorsal striatum  Figure . Slice r ethanol . Importagic tone . The chogic tone , 149. Algic tone , and thegic tone . At the gic tone . Systemigic tone , 154. Elgic tone , 155, bugic tone . Importagic tone , 157–162gic tone , 163.Even though they comprise less than 5% of the neuronal population in the rodent striatum, interneurons play a major role in regulating the response to drugs of abuse, including ethanol. Both cholinergic interneurons and GABAergic interneurons express receptors that are crucial for the rewarding properties of drugs of abuse, and altered activity of striatal interneurons might be a crucial component during the formation of behavioral adaptations associated with addiction and the motivation to consume the drug. Defining the regulation of striatal interneurons is thus an important step in understanding the physiology of the basal ganglia and in developing pharmacological compounds that are able to selectively target a specific neuronal function.


Batrachedra nuciferae Hodges (Lepidoptera: Batrachedridae) was the first phytophagous insect to be reported from inflorescences of coconut, Cocos nucifera L. , in Trinidad, West Indies. At that time, it was suggested to be an introduced species contributing to decreasing coconut yields on the island and potentially a threat to other palms. In this preliminary study, inflorescences of coconut, seven indigenous palms, and six exotic ornamental palms were surveyed in several areas of Trinidad. Caterpillars of more than 10 species of Lepidoptera were found and reared through to the adult stage. Batrachedra nuciferae was positively identified. It was concluded that the caterpillars of B. nuciferae feed on pollen in the male flowers of coconut and palmiste or royal palm, Roystonea oleracea (Jacquin) O.F. Cook. There was no evidence that B. nuciferae bred on any of the other palms surveyed, but it is not conclusive that they do not do so. A parasitoid, Apanteles (sensu lato) sp. (Hymenoptera: Braconidae), of B. nuciferae was reared. On available information, B. nuciferae is more likely to be an indigenous species that has hitherto been overlooked than an introduced species. In view of what is known about damage-yield relationships and biological control agents, B. nuciferae is unlikely to cause yield losses to coconut, so control measures are not justified.In 2006,  Batrachedra nuciferae Hodges (Lepidoptera: Batrachedridae) is a newly reported pest of coconut, Cocos nucifera L. , on the island of Trinidad apparently introduced at some time prior to 2006. Since B. nuciferae was already known as a pest of coconut and other palms in Brazil, concerns have been raised locally about its potential impact onHistory, distribution, and biology of Bactrachedra nuciferae coconuts and indigenous palms in Trinidad and Tobago, and the threat it may present in the wider Caribbean.Bactrachedra nuciferae, which is referred to as the coconut moth in Trinidad and Tobago , S. vagans (= C. vagans), S. schizophylla (= C. schizophylla), Attalea funifera, and A. piassabossu. Bondar considered that the damage to male flowers reduced the probability of fertilization of female flowers and hence could adversely affect nut production, but presented no evidence for this conclusion. He gives brief descriptions of the caterpillar, pupa, and cocoon.Bondar , b studiB. nuciferae, until the work of S. Sanchéz-Soto this century. The moth was the subject of his research thesis , Centeno , and Waller Field (a mixed plantation east of Arima), which produced material for identification and confirmed the biology and damage symptoms of M. flexuosa at Aripo Savannah. Each sample was a whole inflorescence and was placed in prepared sample bags . Where possible, the inflorescence was cut from the palm with minimum vibration  using secateurs or long handled clippers, and the detached inflorescence was placed directly into a sample bag. This process was not always practical due to the difficulty of access or the toughness of the rachis, so in some cases a sample bag was placed over the whole inflorescence before cutting the stem with a machete. In the case of the samples from M. flexuosa and Sabal mauritiiformis (Karsten) Grisebach and Wendland, the inflorescences were too big for the sample bags and so they were cut and lowered to the ground as gently as possible, and then cut up and placed in the sample bag. In one case, a Euterpe precatoria Martius was felled to obtain a sample. The samples were labelled and placed in a chest cool box for transport back to the laboratory. When samples exceeded the capacity of the cool box, they were kept in the shade and then taken to the laboratory in an air-conditioned vehicle. At each site, pictures were taken of the palm, its inflorescence and other diagnostic features, and any other features considered worth recording. Where there was any doubt about the identity of the palm, specimens of the leaves, inflorescence, and fruit were taken to facilitate later identification. A GPS was used to record the position, elevation, and time of each sample. The palms and sample inflorescences obtained are given in At each site, the procedure was as follows. A search was made on foot or from a vehicle to locate flowering palms in the survey area. The palms were identified from Comeau et al.  and locaOn return to the laboratory, the sample bags containing inflorescence samples were placed in an air-conditioned room overnight and processed the next day. For each sample, the following steps were followed. The stage of flowering was recorded, and a photograph was taken of the entire inflorescence and any parts that were considered of interest, e.g., details of inflorescence and damage symptoms. Part or all of the inflorescence was examined by eye and under a binocular microscope to look for symptoms of Lepidoptera . Samples were taken of any living caterpillars or cocoons and either placed in individual vials (30–45 mL) or in groups in containers (14 diameter × 8.5 cm height round containers or 16.5 × 12.5 × 7.5 cm height rectangular boxes). Notes were made on what was found, and observations were quantified as practical, e.g., results per primary, secondary, or tertiary branch, number per 10 flowers, etc.B. nuciferae and other species. On 22 December, only a very small number of moths had emerged in the previous week from two of the emergence boxes, so the boxes were discarded.The remains of the inflorescence were placed in an emergence box and monitored for emergence of moths over more than two months. An emergence box is a cardboard box (up to 57 × 40 × 64 cm in size) with the seams sealed with masking tape to exclude ants and prevent escape of insects. Two round holes are cut in one side exactly large enough to fit a clear plastic or glass vial, which is then inserted with the open end just fitting into the hole and the closed end projecting. The box is placed in a room lit with fluorescent lighting and kept at ambient temperatures. Moths and most other insects are attracted to light, and so insects in the inflorescence or insects that complete their development there are attracted into the transparent vials from where they can be collected. The emergence boxes were checked several times a day, and when moths were found in the transparent vials, the vial was carefully removed, capped, and labelled with the collection and date of emergence. A record sheet on each box was used to record the dates of emergence and provisional division of the moths into The subsamples of living material from the first examination of the inflorescence were either placed in individual vials or in ventilated plastic boxes. The former did not prove satisfactory, perhaps because ventilation was inadequate, and most caterpillars thus treated did not complete development. The ventilated plastic boxes were more successful, and moths were successfully reared in these.The reared moths were placed in a freezer (approximately -18° C) at least overnight before pinning, spreading, and staging using normal techniques for very small moths e.g., . A selecCocos nucifera  to confirm the identification. The size, shape, and surface structure of the signum were found to be diagnostic. This identification was confirmed by a USDA Lepidoptera taxonomist at the United States National Museum based on an examination of the male and female genitalia and comparison with reference material in the museum.The adult moths were as characterised by Sánchez Soto  and SáncB. nuciferae were reared from coconut at all localities at which coconut was surveyed , but were not found at the Royal Botanic Gardens, where coconut could not be surveyed. The numbers obtained were variable .Adults of variable . Only 1 Most adult moths emerged within 15 days of collection of the inflorescence  top, andApanteles (sensu lato) sp. parasitic wasp (Hymenoptera: Braconidae) was reared from B. nuciferae, but was not identified. It is referred to as Apanteles (sensu lato) sp. because although the genus Apanteles has been divided into a large number of genera, it is still easily recognisable and familiar, whereas the new genera are not. The parasitoids attack the caterpillars before maturity, and the mature parasitoid larva only emerges once the caterpillar has spun its cocoon and developed to the prepupa stage. The parasitoid larva then spins a white cocoon within the B. nuciferae cocoon and emerges at about the same time as the adult moth would have. When cocoons were formed on a translucent rearing container, the parasitoid cocoons were readily visible through the container and host cocoon. Apanteles sp. were thus obtained from the Centeno coconut sample, direct from the B. nuciferae cocoons. Of 16 cocoons reared from this sample, 4 were parasitized.Ants and predatory wasps were common in the inflorescences sampled in the field. An Roystonea oleracea (Jacquin) O.F. Cook at Kernahan was included in the survey. Each inflorescence contains both male and female flowers, and an unusual feature of the genus is that the inflorescence is packed with millions of very small, fluffy, branched hairs before the spathe opens  Martius is common at Bush Bush, but only an unopened spathe, an old dead male inflorescence, and a long-dead female inflorescence from a fallen palm could be included in the survey. The contents of the unopened spathe were unblemished, with no signs of insect damage. The very old female inflorescence showed no recent sign of Lepidoptera damage and was discarded after examination in the laboratory on the basis that any Lepidoptera still associated with it would be saprophytic. The old male inflorescence, in which the flower parts, including the long pollen- bearing stamens , rattan palm, Calamus sp., mangrove fan palm, Licuala spinosa Roxburgh, Fiji fan palm, Pritchardia pacifica, hurricane palm, Ptychosperma macarthurii (H. Wendland ex H.J. Veitch) H. Wendland ex Hook.f., and queen palm, Syagrus romanzoffiana (Chamisso) Glassman.The remaining palms are all introduced ornamentals in Trinidad: Manila palm, B. nuciferae caterpillars were found on coconut inflorescences about 25 m away from the A. merrillii surveyed at Curepe, no sign of Lepidoptera feeding could be found in the inflorescence of A. merrillii, and none were obtained by emergence box.Although Calamus sp. was 2 old, dry, male inflorescences, comprising a densely packed mass of dry dead flowers. Examination showed some webbing and caterpillar frass in most parts of the inflorescence, and caterpillars with a dark head and dark purplish body were associated with this. Two cocoons that could have been Batrachedra sp. were found, but nothing emerged from these and nothing emerged from the emergence box, so it cannot be established whether or not this species is a suitable food plant for a Batrachedra sp. or any of the Lepidoptera found in this survey, but certainly it is host to at least 1 species of Lepidoptera.The survey sample from L. spinosa from which the male flowers had been dropped showed no sign of Lepidoptera feeding, webbing of male flowers, or cocoons.An old inflorescence of Pritchardia pacifica, but no caterpillars or cocoons matching those of Batrachedra spp. were found, and 19 adults of 2 other species of moths were obtained by emergence box.There was light webbing and frass among dropped male flowers in an inflorescence of Ptychosperma macarthurii are not synchronised, hence there were small numbers of scattered male flowers on the inflorescences included in the survey at both Curepe and the Royal Botanic Gardens. These and some dropped flowers caught up between the inflorescence branches at the Royal Botanic Gardens were sampled. There was no trace of webbing or frass on fresh or dropped male flowers, and no Lepidoptera were obtained by emergence box.The male flowers of S. romanzoffiana inflorescence sampled, the male flowers had already mostly dropped. The inflorescence seemed completely healthy, except that the ends of many apical secondary branches were caught up in the apex of the spathe together with many male flowers, which had started to go moldy. This seemed ideal for attack by Lepidoptera, but there was no trace of caterpillar damage. A small collection of dropped male flowers was also made from the base of the palm and set up separately. No Lepidoptera emerged from either sample.In the B. nuciferae to be widespread in Trinidad. Information has been generated on the feeding damage, phenology, and natural enemies of B. nuciferae. Coconut has been confirmed as a food plant, palmiste is a new food plant record, and several other palms have been provisionally shown to be unsuitable food plants. This information was used to assess the threat that B. nuciferae presents to coconut and other palms and the need for management of the moth.A preliminary spot survey of the incidence of Lepidoptera in the flowers of coconuts and selected other palms in Trinidad was carried out. The results have enabled confirmation of the presence of R. oleracea were suitable hosts for B. nuciferae in Trinidad. No evidence was found of Batrachedra spp. attacking B. major, B. simplicifrons, A. maripa, M. flexuosa, E. precatoria, or 4 introduced palms. Sabal mauritiiformis was attacked by another Batrachedra sp., but not by B. nuciferae. However, while proof of attack was easily demonstrated, proof of non-attack cannot be demonstrated without more samples taken at a suitable stage. In the specific case of B. nuciferae, these samples should be taken in places where B. nuciferae is known to be present on other nearby hosts.The results show that both coconut and B. nuciferae feeding abundantly in the inflorescences of Attalea spp. in Brazil, the indigenous A. maripa were expected to be suitable hosts in Trinidad. However, in spite of the large amount of old damage on the old male inflorescence, this did not appear to be the case. There are several possible explanations: (1) Bondar saw similar damage due to other Lepidoptera and assumed it was due to B. nuciferae without rearing out specimens (he appears to have sent only specimens from coconut for identification); (2) the species of Attalea that Bondar observed are suitable food plants, but A. maripa is not ; (3) further samples may show that A. maripa in Trinidad is a suitable food plant for B. nuciferae. The last would be the easiest to test, especially if fresh flowering male inflorescences could be sampled. It can be concluded provisionally that A. maripa is not a suitable food plant for B. nuciferae. The male flowers of A. maripa consist of little more than a bunch of pollen-bearing stamens , and there was no evidence from this survey that B. nuciferae attacks A. maripa. Note however, that contrary to the observations reported here, Bondar , but the ones sampled, Bactris spp. (Bactridinae) and Euterpe precatoria (Euterpeae), were not attacked by B. nuciferae. However, R. oleracea, which is in a different tribe (Roystoneae) of the same subfamily as Cocos (Arecoideae), was attacked. It seems likely that because of the great diversity of pollination systems in Arecaceae  standardising the stage of development of inflorescence sampled, i.e., to pollen producing; (2) separating pollen producing flowers from old dropped flowers and holding each separately for moth emergence; (3) establishing that B. nuciferae is present in a local area before sampling other nearby palm species to assess host specificity.As a measure of population density, taking a sample at a single time point as done here unavoidably makes no allowance for eggs that would have been laid in the inflorescence after collection, nor for moths that had already emerged before the sample was taken. By making a careful examination of the material before setting it up in the emergence box, there is a qualitative control for the latter aspect, but the former could not be controlled. In some cases, Batrachedra nuciferae was first found in Trinidad in 2006. However, there are two possible interpretations of this: either it is an indigenous species that had not previously been noticed and identified, or it is an alien species introduced some time before 2006. There is not definitive evidence to address this issue, but there are several points that should be considered.B. nuciferae. However, the lists include very few species of the socalled Microlepidoptera families (which include Batrachedridae), and those mentioned are mostly important pests recorded through studies by the Department of Agriculture (Trinidad and Tobago) entomologists and staff or students of the former Imperial College of Tropical Agriculture . Batrachedra nuciferae is a small, undistinguished moth, most unlikely to be found by casual observation and collecting. It would have to be located through a study or survey of coconut pests that examined the insects associated with the inflorescence. Similarly, although at least 12 other species of Lepidoptera were reared in this study from coconut or other palm inflorescences, only 1 had previously been recorded from Trinidad. There is no evidence that any entomologists have studied what insects affect coconut inflorescence in Trinidad prior to 2006.The available lists of moths from Trinidad  do not mA. subrufella was a damaging indigenous moth pest of coconut inflorescence  sp. on coconut. Apanteles (sensu lato) spp. are usually specialist parasitoids, so this evidence supports an indigenous origin rather than an exotic one. Finally, if B. nuciferae were recently introduced in Trinidad, its distribution might be restricted, whereas it seems to occur throughout the island .If B. nuciferae is more likely to be an indigenous species in Trinidad than an alien species. However, none of these points are conclusive. To resolve this issue, a study on the molecular genetics of B. nuciferae in Trinidad (and elsewhere) would be needed. If B. nuciferae is a recently introduced species in Trinidad, it should have a much-reduced genetic diversity there compared to areas where it is indigenous.Thus, the balance of evidence does suggest that B. nuciferae should be a priority compared to other adverse factors affecting coconut and other palms in Trinidad, e.g., red palm mite (Raoiella indica), red ring disease (caused by the nematode Bursaphelenchus cocophilus), and other diseases known in the region. The limited observations in this study did not suggest that B. nuciferae causes direct damage or occurred at high enough densities to do so. The observations also showed that the caterpillars are attacked by an Apanteles (sensu lato) sp. parasitoid, which could be quite common. Ants were observed carrying away B. nuciferae caterpillars in the laboratory. Other natural enemies may well attack the eggs or pupae, but would not have been detected in this survey. Until demonstrated otherwise, it is anticipated that the action of natural enemies  would increase when the density of B. nuciferae increased, bringing any incipient outbreak under control. In extreme cases, for example if damage is observed early in the cropping cycle of newly planted dwarf varieties of coconut, further observations should be made, but as long as the inflorescences are being visited by pollinators, action to control flower pests such as Batrachedra spp. that do not attack the female flowers are not likely to be useful.At the moment, the evidence available does not suggest that control of


In vitro studies have demonstrated that granulocyte colony-stimulating factor/receptor exhibits a high affinity binding and this biological axis increases proliferation of the carcinoma. Urothelial carcinoma of the bladder is rarely associated with a leukemoid reaction and autocrine growth induced by paraneoplastic production of granulocyte colony-stimulating factor. In the world literature, there have been less than 35 cases reported in the last 35 years. The clinicopathological aspects, biology, prognosis and management of granulocyte colony-stimulating factor-secreting bladder cancers are poorly understood.Granulocyte colony-stimulating factor produced by nonhematopoietic malignant cells is able to induce a leukemoid reaction by excessive stimulation of leukocyte production. Expression of granulocyte colony-stimulating factor and its functional receptors have been confirmed in bladder cancer cells. A 39-year-old Caucasian woman with an invasive high-grade urothelial carcinoma presented with hematuria and low-grade fevers. Laboratory tests revealed an elevated white blood cell count and absolute neutrophil count and an elevated 24-hour urine protein. Upon further evaluation she was found to have locally advanced high-grade urothelial carcinoma without nodal or distant metastasis. Her serum granulocyte colony-stimulating factor level was 10 times the normal limit. This led to the diagnosis of a paraneoplastic leukemoid reaction. Her white blood cell count immediately normalized after cystectomy but increased in concordance with recurrence of her disease. Unfortunately, she rapidly progressed and expired within 10 months from the time of first diagnosis.This is one of the few cases reported that illustrates the existence of a distinct and highly aggressive subtype of bladder cancer which secretes granulocyte colony-stimulating factor. Patients presenting with a leukemoid reaction should be tested for granulocyte colony-stimulating factor/receptor biological axis. Moreover, granulocyte colony-stimulating factor could be a potential neoplastic marker as it can follow the clinical course of the underlying tumor and thus be useful for monitoring its evolution. Neoadjuvant chemotherapy should be considered in these patients due to the aggressive nature of these tumors. With a better understanding of the biology, this autocrine growth signal could be a potential target for therapy in future. In vitro studies have demonstrated that G-CSF/G-CSFR exhibit high affinity [Paraneoplastic leukemoid reaction is a type of paraneoplastic syndrome in which there is an elevation in the white blood cell count, predominantly neutrophils. Granulocyte colony-stimulating factor (G-CSF) secreted by nonhematopoietic malignant cells has been reported to be able to induce a leukemoid reaction through excessive stimulation of leukocyte production -3. Urothaffinity  binding affinity ,9-11. Thaffinity . Here, wAerococcus urinae for which she was treated with a course of ceftriaxone. Repeat urine and blood cultures were negative but she had continued elevation of her WBC count. The results of anti-double stranded deoxyribonucleic acid (DNA) antibody, rheumatic factor, anti-SSA, anti-SSB, anti-glomerular basement membrane, myeloperoxidase, proteinase 3, cytoplasmic antineutrophil cytoplasmic antibody, perinuclear antineutrophil cytoplasmic antibody, hepatitis panel, and human immunodeficiency virus antibody tests were negative. Serum protein electrophoresis, creatine phosphokinase, quantitative immunoglobulin assay, complement levels and chest radiograph were all normal. Hence, symptoms, signs and laboratory studies were negative for an infectious etiology (including a tagged WBC scan) and autoimmune/rheumatological disease. A bone scan and positron emission tomography scan ruled out metastasis , pulse 110 per minute, respiratory rate 20 per minute, and blood pressure 150/83mmHg. Her examination was only remarkable for progressive anasarca. Laboratory studies revealed elevated white blood cell (WBC) count and absolute neutrophil count , leukocyte alkaline phosphatase score 295, erythrocyte sedimentation rate (ESR) >140mm/hour, C-reactive protein (CRP) 29.5mg/dL, and antineutrophilic antibody (ANA) titer 320. She also had nephrotic range proteinuria with 24-hour urine protein of 14.65g and a serum creatinine of 3.27mg/dL  and serum creatinine 1.90. A CT scan showed multiple pelvic masses with abdominal lymphadenopathy and a large right pelvic mass in close approximation to the area of the sigmoid colon causing obstruction. Her serum G-CSF level was 406.6pg/mL  confirming the diagnosis of paraneoplastic leukemoid reaction. She underwent a diverting colostomy to relieve symptoms of bowel obstruction. However due to poor performance status, renal failure and the development of a colovaginal fistula, she was placed on palliative care and expired soon thereafter.in vitro studies [This rare case of urothelial carcinoma of the bladder had several unusual features including young age at presentation, marked leukemoid reaction, elevated G-CSF levels , nephrotic range proteinuria and very rapid progression of disease to death within 6 months of the onset of the leukemoid reaction. Urothelial carcinoma in this case showed squamous differentiation which has been reported to have elevated G-CSF levels -6. A pos studies ,9,11. Co studies ,13. Thre studies ,15. HoweIn the present case, as the tumor recurred and grew in her pelvic cavity, the concentration of serum G-CSF was elevated with an associated increase in her WBC count. These findings support the hypothesis that the bladder cancer cells producing G-CSF may grow more rapidly or that the G-CSF production exhibited by the tumor cells may promote the malignant progression. We thus undertook a comprehensive review of all studies and similar cases reported to date to explore the above hypothesis. It is also probable that the patient had micrometastatic disease, undetectable by currently existing diagnostic modalities, prior to surgery. Rapid development of metastases within 3 months after surgery may have been invigorated by G-CSF secretion and activation of the G-CSF/G-CSFR biological axis stimulating tumor growth.et al. showed that the average age of these patients was 70.1 years and the male to female ratio was 3:2. The WBC was as high as 181,800/mL  and there was an average G-CSF level of 203pg/mL with a mean survival of 5.9 months following surgical therapy in these patients. However, those who received adjuvant chemotherapy reported survivals of >20 months [et al. in 1995 showed that 9.2% of bladder tumors had elevated G-CSF levels (mean G-CSF of 328), which positively correlated with an increase in grade and progression of stage of cancer, more so in patients with distant metastasis. Furthermore, patients with bladder cancer without elevated G-CSF had better disease-specific survival rates at 5 years follow-up when compared with those with an elevated level of serum G-CSF [A review by Turalic 0 months . An obsein vitro bladder cancer cell line study. This capability to respond to various stimuli implies that numerous regulatory pathways may be involved in the production of cytokines [et al. conducted a series of in vitro studies which have shown that G-CSFR specifically signals for beta-1 integrin expression, adhesion and invasion of bladder cancer cells which could promote metastasis [The mechanism responsible for overexpression of G-CSF in bladder cancer is yet to be clearly elucidated. Possibilities include rearrangement of the G-CSF gene occurring within one of the alleles  and intrytokines . Althougtastasis . Furthertastasis . The histastasis . In additastasis . The fretastasis . Hematoptastasis . Hence, et al. reported a case of rapid clinical deterioration and leukemoid reaction after treatment of urothelial carcinoma with G-CSF along with chemotherapy [in vitro. It may stimulate the growth of the residual tumor cells after chemotherapy while it is being used clinically to combat myelosuppression induced by antitumor chemotherapy in bladder cancer [G-CSF administration has been in vogue as an adjunct to the management of granulocytopenias, either with or without prior chemotherapy -26. Subsotherapy . Recombir cancer . Due to On the basis of this case experience and review of literature we conclude that there is a distinct and highly aggressive subtype of bladder cancer which secretes G-CSF and expresses G-CSFR inducing an autocrine growth cycle. Hence such patients presenting with leukemoid reaction should be tested for G-CSF/G-CSFR biological axis. Moreover, G-CSF could be a potential neoplastic marker as it can follow the clinical course of the underlying tumor and thus be useful for monitoring its evolution. Neoadjuvant chemotherapy should be considered as the aggressive nature of these tumors appears to overwhelm these patients before they receive adjuvant treatments. With a better understanding of the biology, this autocrine growth signal could be a potential target for therapy in future.We are currently planning to conduct a preliminary study to identify patients with such bladder cancer, not only to estimate its prevalence but also to assess the variation in G-CSF levels in response to various therapies . In addition we will evaluate G-CSF as a prognostic marker and as a tool to predict a more aggressive biological phenotype of invasive bladder cancer. Future studies will be necessary to determine the regulatory effects of G-CSF on bladder cancer cells.Written informed consent was obtained from the patient’s next of kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ANA: Antineutrophilic antibody; ANC: Absolute neutrophil count; CRP: C-reactive protein; CT: Computed tomography; ESR: Erythrocyte sedimentation rate; G-CSF: Granulocyte colony-stimulating factor; G-CSFR: Granulocyte colony-stimulating factor receptor; WBC: white blood cell.The authors declare that we have no competing interests.AKLK drafted the manuscript and prepared the figures. PVV, MM and JH reviewed and amended the manuscript. All authors read and approved the final manuscript.


The combination of decreased amyloid β42 (Aβ42) and increased total tau proteins (T-Tau) and phosphorylated tau (P-Tau) in cerebrospinal fluid (CSF) has recently been considered as a biological diagnostic criterion of Alzheimer’s disease (AD). Previous studies showed significant heterogeneity in CSF Aβ42 levels to discriminate AD from non-AD patients. It was also suggested that the CSF amyloid peptide β42/β40 ratio has better diagnostic performance than Aβ42 alone. The objective of the present study was to investigate the potential added value of determining CSF amyloid β40 peptide (Aβ40) for biological diagnosis of AD when CSF Aβ42 levels failed. CSF AD biomarkers were run in 2,171 samples from 1,499 AD and 672 non-AD patients. The following pathologic thresholds were used to define an AD-positive CSF biomarker profile: T-Tau ≥ 400 ng/L, P-Tau181 ≥ 60 ng/L, and Aβ42 ≤ 700 ng/L. CSF Aβ40 was assayed in AD patients with CSF Aβ42 levels above 700 ng/L and non-AD patients with CSF Aβ42 levels below 700 ng/L. CSF Aβ40 levels were higher in AD than non-AD patients. The receiver operator characteristic curves of CSF Aβ40 and the Aβ42/Aβ40 ratio defined AD cut-off values at 12,644 ng/L and 0.06, respectively. In AD patients with non-pathological CSF Aβ42, CSF Aβ40 concentration was able to correct 76.2% of cases when expressed as CSF Aβ42/Aβ40 ratio and 94.7% of cases when used alone. Using CSF Aβ42 and then CSF Aβ40, the percentage of misinterpreted AD patients fell to 1.0%. CSF Aβ40 concentration improved interpretation of Aβ42 level for the diagnosis of AD. CSF Aβ40 alone showed better diagnostic performance than the amyloid peptide Aβ42/Aβ40 ratio. The added value of determining CSF Aβ40 in AD diagnosis now needs confirming in a cohort of definite AD patients and to be completed with novel amyloid cascade biomarkers. According to the revised criteria for Alzheimer’s disease (AD), definite diagnosis is founded on neuropathology as gold standard, when patients meet the clinical and cognitive criteria for AD dementia . DiagnosOne of the crucial challenges to improve screening in clinical trials is to identify an accurate CSF biomarker reflecting amyloid pathology. There is now strong evidence that CSF Aβ42 levels depend not only on impaired brain clearance in Alzheimer’s pathophysiology, but also on the total load of amyloid peptides, which shows large interindividual variability –14. GammCerebrospinal fluid samples were collected between October 2010 and January 2013 from 2,171 patients who underwent lumbar puncture (LP) for routine clinical diagnosis of AD in the Neurochemistry Unit and Biochemistry Department of the University Hospital of Lyon (France). Patients were included in a multicenter memory clinic and had at least 2 years’ follow-up. They were classified into two groups: 1,499 AD and 672 non-AD patients. The non-AD group consisted of 259 patients with probable frontotemporal lobar degeneration (FTLD), 119 with probable dementia with Lewy bodies (DLB), 159 with normal pressure hydrocephalus (NPH), and 135 with psychiatric disorders.The patients’ age, gender, and mini mental state evaluation (MMSE) score were recorded when the LP was performed. At that time, initial diagnosis was based on medical history, caregiver interviews, neurologic examination, neuropsychological battery evaluation, and brain imaging. Clinical diagnosis was made in multidisciplinary team meeting, comprising neurologists, neuropsychologists, and radiologists, and confirmed on follow-up. Dementia was defined according to DSM IV-TR criteria , and allCommission Nationale de l’Informatique et des Libertés (CNIL)].This study, based on routine biological analyses, was not considered as “biomedical research” under French regulations, and therefore did not require informed consent. Samples were, however, stored in a biobank with authorization from the French Ministry of Health (Declaration number DC-2008-304). Authorization for handling personal data was granted by the French data protection commission  in AD patients with CSF Aβ42 levels above 700 ng/L and in non-AD patients with CSF Aβ42 levels below 700 ng/L.U test, Kruskal–Wallis test, and receiver operator characteristic (ROC) analyses were performed using MedCalc version 11.3.1.0 . Differences were considered statistically significant at p < 0.05. ROC curves were applied to define optimal biomarker cut-off values to discriminate between AD and non-AD groups. The cut-off value was defined as the value corresponding to the highest average for sensitivity and specificity. Accuracy was calculated as the sum of true positives and true negatives in the total number of patients  fulfilled the pathological CSF Aβ42 criteria; the remaining 18.7%  presented CSF Aβ42 levels above cut-off (>700 ng/L). 63.7% of non-AD patients (428/672) presented CSF Aβ42 levels above 700 ng/L; 36.3% (244/672) had CSF Aβ42 levels below 700 ng/L , sensitivity and specificity for AD diagnosis were 76.2 and 58.2%, respectively  using the CSF Aβ42/Aβ40 ratio, and 94.7 and 91.0%, respectively  using CSF Aβ40 determination.In the overall population, the percentage of patients in whom amyloid pathology was misinterpreted fell from 24.2%  using CSF Aβ42 alone to 7.8%  when it was followed by CSF Aβ42/Aβ40 ratio, and to 1.7%  when followed by CSF Aβ40  had CSF Aβ42/Aβ40 ratios higher than 0.06 and 91.0% (222/244) had CSF Aβ40 levels below 12,644 ng/L.About 76.2% of AD patients (214/281) had CSF Aβ42/Aβ40 ratios below 0.06 and 94.7% (266/281) had CSF Aβ40 levels higher than 12,644 ng/L. In the overall AD population, percentage misinterpretation fell from 18.7%  with CSF Aβ42 alone to 4.5%  using CSF Aβ42 and then CSF Aβ42/Aβ40 ratio and 1.0%  using CSF Aβ42 and then CSF Aβ40 . CSF Aβ42 was previously reported to be less effective for differential diagnosis of the main neurodegenerative dementia than CSF Tau proteins –41. To dThe diagnostic performance of CSF Aβ42 is increasingly questioned. It should be noted that biological diagnosis as performed in specialized memory clinics is also founded on the second pathway of AD pathophysiology, reflected by CSF Tau protein levels. Nevertheless, a more accurate evaluation of CSF amyloid biomarkers is important to include patients in therapeutic trials involving the amyloid cascade, using added Aβ peptides or other amyloid cascade biomarkers. For example, the soluble peptide APPβ (sAPPβ) and CSF Aβ40 come from the same enzymatic digestion of APP, and it would be interesting to assess sAPPβ to complete this study. Increased CSF sAPPβ levels were already reported in AD patients as compared to non-AD demented patients  and FTD In conclusion, the present study offers an improvement in biological diagnosis of AD focusing on the amyloid pathway. In the misinterpretation using CSF Aβ42 levels, classification based on the CSF Aβ42/Aβ40 ratio gives good results. More interestingly, CSF Aβ40 assay alone also provides better results: the misinterpretation rate using CSF Aβ42 and then CSF Aβ40 alone falls to 1.7%. Sequential assessment of CSF Aβ40 would also provide a better cost-effectiveness ratio than systematic determination of the CSF Aβ42/Aβ40 ratio. Finally, these results need to be confirmed in a prospective study including autopsy-proven AD patients, and completed with novel amyloid cascade biomarkers.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diagnostic coding has several potential benefits, including improving the feasibility of data collection for research and clinical audits and providing a common language to improve interdisciplinary collaboration. The primary aim of this study was to determine the views and perspectives of chiropractors about diagnostic coding and explore the use of it in a chiropractic setting. A secondary aim was to compare the diagnostic coding undertaken by chiropractors and an independent coder.A codin exercise based on the International Classification of Primary Care version 2, PLUS extension (ICPC-2 PLUS) provided the 14 chiropractors with some experience in diagnostic coding, followed by an interview on the topic. The interviews were analysed thematically. The participating chiropractors and an independent coder applied ICPC-2 PLUS terms to the diagnoses of 10 patients. Then the level of agreement between the chiropractors and the coder was determined and Cohen’s Kappa was used to determine the agreement beyond that expected by chance.1) Advantages and disadvantages of using a clinical coding system in chiropractic practice, 2) ICPC-2 PLUS terminology issues for chiropractic practice and 3) Implementation of a coding system into chiropractic practice. The participating chiropractors did not uniformly support or condemn the idea of using diagnostic coding. However there was a strong agreement that the terminology in ICPC-2 PLUS would not be applicable or desirable for all practice types. In the coding exercise the chiropractors in total coded 202 diagnoses for 135 patients. The overall percentage agreement between the chiropractors and the coder was 52% (17% expected by chance) with a Kappa score of 0.4 (95% CI 0.3-0.7). Agreement was lower for more detailed coding ).From the interviews the three emerging themes were: It appears that implementation of diagnostic coding would be possible in the majority of the chiropractic practices that participated in this study. However for those chiropractors who do not focus on symptoms in their approach to clinical care, it could be challenging to use the ICPC-2 PLUS coding system, since ICPC-2 PLUS is a symptom-based classification.The online version of this article (doi:10.1186/s12998-015-0051-1) contains supplementary material, which is available to authorized users. Diagnostic coding in chiropractic practice has several potential benefits. These benefits include improving the feasibility of data collection for research and clinical audits, improving the clinical applicability of research, and providing a common clinical language to help improve interdisciplinary collaboration. Diagnostic coding allows for systematic classification of clinical information in clinical practice and assists with the conduct of clinical audits and data collection for research purposes. Use of the same classification system by different clinical groups creates a common language between different healthcare professions and sectors and could enhance interdisciplinary collaboration. This could lead to better communication, improved continuity of care as well as simplifying the process of referring patients .The International Classification of Primary Care (ICPC) is an example of a diagnostic coding system that could be relevant for chiropractic practice . The ICPDiagnostic coding is not commonly undertaken in chiropractic practice, and the choice of an appropriate coding system is important as the profession moves towards this. A study in Danish general medical practice showed that the ICPC has a good reliability and validity at the chapter level for musculoskeletal conditions . Thus, irubric .“The minute I start doing this [coding exercise] with trying to match their reported reason for encounter that then assumes that what you are doing on that day is a treatment for that reason for encounter right? And I can understand why you would have this if you are in medicine because that is what they do right? But that’s not what we do. So someone might have a presenting complaint: “okay let’s check you, let’s see if you are subluxated, if you are then I’ll adjust you, whether or not you’ve got that presenting complaint, it’s irrelevant okay.. So I don’t know how you could fit this [ICPC-2 PLUS] into the way I have just explained it, if there was a way that would be great.”.“If it’s going to create a language that helps through - not just the chiropractic profession communicating well with each other, but chiropractic profession communicating with the rest of the medical industry - medical - well, rather, the health world. Then that’s good for everybody. So, that’s a great role and if we’re talking about chiropractic - getting a larger level of research towards it and integrating itself into a mainstream system more. I think that would be actually essential. So, yeah there’s certainly a role for it.”.However, some of the chiropractors did not see the relevance or purpose of ICPC-2 PLUS in chiropractic practice. An issue mentioned by many was that the terminology would not fit into what they called the “wellness-based approach” of some chiropractors. Some chiropractors suggested that the use of ICPC-2 PLUS in chiropractic practice could “medicalise” the profession. On the other hand many chiropractors discussed how diagnostic coding could create a common language that could facilitate better communication between the different musculoskeletal health professions. Some chiropractors also mentioned how this could lead to integration into the mainstream healthcare system. Some illustrative quotes demonstrating these issues follows:terms to choose from, but also that the terms were not specific enough to reflect chiropractic clinical practice. They believed that there were not enough relevant terms for the stage of the problem/diagnosis, for example the limited possibilities to describe if a problem/diagnoses was acute or chronic or somewhere between the two. Some illustrative quotes demonstrating these issues follows:“Obviously they are not specific enough, it doesn’t tell you the state of whether it’s acute, chronic or what have you. Again it might tell you the region you are looking at but not the anything specific of what you are looking at” “It wasn’t easy cause 27000 things for the same thing (laughs) almost, yes too many choices and not specific enough or they are trying to be too specific which then makes it a bit harder to find exactly what is going on.” The terminology in ICPC-2 PLUS was a topic mentioned frequently by the chiropractors. Many of the chiropractors mentioned that there were too many It got easier. I guess part of it is just being familiar with what your options are. “ When you familiarize yourself with it, it would be great.”“I really think it’s gotta start at the undergraduate level. To start teaching people this is the way of the future. And through training sessions and seminars disseminating it to the field practitioners.”“Many chiropractors thought that implementation of ICPC-2 PLUS into chiropractic clinical practice would be feasible after some practice. Some of the issues mentioned were getting ICPC-2 PLUS into the existing electronic clinical record systems and that not all chiropractors use electronic clinical practice records. Some chiropractors mentioned that implementing a system like ICPC-2 PLUS would have to start at the undergraduate level. One chiropractor stated that the idea was good but the motivation for clinicians was not great enough to make implementation of ICPC-2 PLUS possible. Some illustrative quotes demonstrating these issues follows:The 135 included patients provided 202 reasons for encounter. The chiropractors spent between 30 seconds to 20 minutes choosing a code for a particular diagnosis/problem.Each of the 14 participating chiropractors recorded 10 patient encounters. For each of these patient encounters, the chiropractor could list up to 3 diagnoses. The total number of diagnostic codes provided by the chiropractors, and used in the agreement analysis, was 167 (Table Rubric level (three-character code) was just over 50% agreement. On ICPC-2 PLUS term level (six-character code) the agreement was just over 30%. The level of agreement beyond chance was moderate with a Kappa score 0.4 (95% CI 0.3-0.7) on the ICPC-2 Rubric level, and fair with a Kappa score 0.3 (95% CI 0.2-0.5) on the ICPC-2 PLUS term level.As presented in Table terms for chiropractic practice is under development ICPC-2 PLUS (Chiro) [This paper provides a first insight into views and perspectives of chiropractors about diagnostic coding in chiropractic practice. ICPC-2 PLUS was chosen as the example of a diagnostic coding system in this study because it has been proven to have a good reliability and validity for musculoskeletal diagnosis, which is the most common conditions treated in chiropractic practice. Also, it is already used in hospital settings/ secondary sector, the COAST data was coded with this particular classification and alongside the process a set of  (Chiro) .wellness chiropractors typically expressed the view that the terms available were not adequate to describe their clinical practice. The main issue here was the fact that they did not have a particular focus on specific symptoms. They said that it was obvious that ICPC-2 PLUS terms were made for medical practice, and that it would be better to make a new system for chiropractic practice with more relevant terms. Coding with the current terms in ICPC-2 PLUS may therefore not be relevant for all practice types. The terminology issue was also addressed alongside the conduct of COAST and resulted in a chiropractic-specific coding system being developed, ICPC-2 PLUS (Chiro) [Most chiropractors were positive toward using a diagnostic coding system in their daily practice but some of those chiropractors did not see the relevance of the ICPC-2 PLUS for their practice. The chiropractors who described themselves as  (Chiro) . Many hoFor the coding exercise if agreement between the chiropractor and coder was high, this would provide some support that ICPC-2 PLUS was potentially a good choice for diagnostic coding in chiropractic practice. If the agreement was low it would tell us that something needed improvement. Either ICPC-2 PLUS was not a good fit for chiropractic practice, or the encounter forms the chiropractors completed did not provide enough information for the coder to agree with the chiropractors’ choice of code.Rubric level and the ICPC-2 PLUS term level. However, the agreement was only fair to moderate and this could be due to a number of reasons. First, none of the chiropractors were familiar with the terms available for coding when they completed the encounter forms. The amount of time spent to get familiar with the terms available before the coding exercise differed considerably. The practitioners spent between 30 seconds to 20 minutes choosing a term for a particular diagnosis/problem. Some of the participating chiropractors mentioned that they would have preferred some prior knowledge about the available terms before filling out the encounter forms. To avoid adding to the burden for the participating chiropractors, we decided not to include the list of terms in the study materials they received prior to the interviews. We do not know whether this would have improved the level of agreement or had an influence on their overall view on the idea of diagnostic coding in chiropractic practice.The agreement between the coder and the chiropractors was higher than agreement expected by chance at both the ICPC-2 rubric level two chiropractors had 100% agreement with the coder, eight chiropractors had over 50% agreement with the coder. But the two chiropractors who achieved 100% agreement with the coder had only chosen one term for all the diagnoses. Despite a variation in their patients’ reasons for encounter, the diagnoses, written by the chiropractor on the encounter form, were all the same. All other chiropractors had used at least three different terms when coding. In order to conduct the agreement analysis the participating chiropractors had to choose a term for every one of their diagnoses. They were asked to choose the term that came closest to describing the diagnosis, rather than being given an option of not choosing a term. This might have resulted in these two chiropractors choosing the same term for all the diagnoses.For the agreement on ICPC-2 terms chosen was positive to using ICPC-2 PLUS in practice. Another chiropractor had an 82% agreement rate with the coder but was negative to the idea of using ICPC-2 PLUS in practice and emphasized a lack of relevance of the coding system to their clinical work and therefore lacked of motivation to implement the system. This chiropractor did not have a wellness practice.Even the chiropractor who did not agree with the coder on any of the The sample size for both chiropractors and patient participants were smaller than planned. We did not reach our intended number of practitioners (14 instead of 15) and therefore also less patient participants. This meant one interview less than expected and less data for the agreement analysis. However, this study being the first of its kind and therefore exploratory we do not know what effects this would have had on the results. Qualitative research is not meant to be representative, it is meant to provide in-depth information and this study offers some insight to what possibilities and challenges diagnostic coding could bring to chiropractic practice.The qualitative interviews, as well as the analysis, were completed with an awareness of the first author’s (CT) personal background as a chiropractic student with minimal clinical experience, and a preconception that diagnostic coding could potentially improve patient care. Being aware of this a neutral approach was emphasized when interviewing and when conducting the analysis.This study was the first to examine the role of diagnostic coding using an established primary care coding system in chiropractic practice. To determine the applicability of ICPC-2 PLUS or another diagnostic coding system in chiropractic practice, more research is needed. A study with a bigger sample would provide further information on this topic.In Denmark an introduction of the Danish version of ICPC-2, ICPC-2-DK to chiropractic practice is currently underway. ICPC-2-DK has been part of the electronic medical records in Danish general medical practice since 2009 . A similMost of the chiropractors in this study found the use of diagnostic coding both feasible and applicable in their practices but recommended specific training for chiropractors in the use of the coding system. However, there was a strong agreement that the terminology in ICPC-2 PLUS would not be applicable or desirable for those members of the chiropractic profession who do not focus on symptoms in their approach to clinical care. Clinically relevant research opportunities and further integration in the mainstream health care system were mentioned as some of the future perspectives of diagnostic coding.Thus, this first insight into the use of diagnostic coding in chiropractic practice is encouraging, but more research is needed to determine if these results are applicable to chiropractic practice in other countries and to investigate possibilities for improving the chiropractic relevance of the terminology.


Handwashing practices among caretakers of case and control children < 5 years of age enrolled in the Global Enteric Multicenter Study in Mirzapur, Bangladesh were characterized and analyzed for association with moderate-to-severe diarrhea. Soap or detergent ownership was common, yet 48% of case and 47.7% of control caretakers also kept ashes for handwashing, including 36.8% of the wealthiest households. Soap, detergent, and ash were used for multiple hygiene purposes and were kept together at handwashing areas. Caretakers preferred soap for handwashing, but frequently relied on ash, or a detergent/ash mixture, as a low-cost alternative. Moderate-to-severe diarrhea was equally likely for children of caretakers who kept soap versus those who kept ash . Contact with ash and water reduced concentrations of bacterial enteropathogens, without mechanical scrubbing. Thus, washing hands with ash is a prevalent behavior in Mirzapur and may help diminish transmission of diarrheal pathogens to children. Diarrheal disease is estimated to be responsible for approximately one in ten deaths among children < 5 years of age globally, or a total of ∼800,000 fatalities annually.10The cost of soap may be a particularly important impediment in achieving long-term improvements in handwashing. Poor households commonly report that soap is unaffordable, and allocate few resources for its purchase.21There is a paucity of data comparing health outcomes in households that use soap versus other types of low-cost, locally resourced materials. Our primary objective was to address this knowledge gap by analyzing whether the use of ashes for handwashing, instead of soap, by caretakers of children < 5 years of age enrolled in the Global Enteric Multicenter Study (GEMS)Finally, we explored the biological plausibility that exposure to ash and water for actual handwashing times could effectively reduce concentrations of enteric diarrheal pathogens that are common in this population.Cases and controls were selected from among participants in GEMS.36 Epidemiological data were analyzed using SAS version 9.3 . Descriptive statistics were reported as proportions, means, and ranges. Means were compared using a two-sample t test. Categorical variables were compared using χ2. Reported P values are two-tailed; P < 0.05 was considered significant. A wealth index quintile (WIQ) was generated using principal component analysis of 13 household characteristics.Supplemental Table 1. Conditional logistic regression was performed using data collected from matched case-control pairs to test for associations  between handwashing indicators and MSD, while adjusting for wealth index quintile.39Forms were scanned and sent electronically to the data coordinating center  where the database was maintained.We used qualitative data collection methods to gather more detail on hygiene practices of caretakers in our study, and to explore the perceptions and determinants related to the selective use of different types of handwashing materials. Qualitative approaches included focus group discussions (FGDs) conducted in the community, semi-structured in-depth interviews (IDIs) with mothers or caretakers, and key informant interviews (KIIs) of grandmothers, conducted in the home of young children enrolled in GEMS . CaretakA manual for FGD, IDIs, and KIIs was developed based upon research objectives, translated into Bengali, back-translated, pilot-tested, and refined before data collection. Topics that were explored included types of personal and household hygiene materials that caretakers use, where they were acquired and stored, when and how they use them, and the motivations for using various types of cleaning materials. The FGD sessions were conducted in the community, with each session lasting 40–45 minutes. We conducted nine FGD sessions (group size of 6–8 participants), which included 26 case caretakers and 25 control caretakers. Twelve caretakers  underwent IDIs and 12 grandmothers  who had not previously participated in a FGD took part in a KII; these sessions lasted for about 30–40 minutes. All interviews and group discussions were conducted in Bengali. Two research officers (authors FDF and FF) with an educational background in Nutrition and Public Health and professional training and experience in qualitative research methodology carried out these sessions and interviews. FGDs, IDIs, and KIIs were recorded using audio-tape, which was then transcribed verbatim and translated into English.Data analysis of transcribed audiotapes involved extensive memoing to identify core inductive themes that were then used to manually code the entire data set according to our research objectives, and generate code-specific reports for detailed analysis. After coding, we translated these data into English and compared for consistency. The KIIs, IDIs, and FGD results were analyzed individually to verify consistency in the content of caretakers' responses in group and private interviews, and then compared as one body of information for drawing inferences for descriptive reporting. We performed thematic content analysis to provide a descriptive narrative of our results. No attempt was made to quantitatively compare study findings.To document the process that caretakers use for handwashing, the first 10 case and control caretakers who were being visited for a 60-day follow-up visit were asked to demonstrate their technique. An observer recorded the steps used by each caretaker, and estimated the amount of ash and water used. A stop watch was used to time how long the caretaker scrubbed her hands after wetting hands with water or a handwashing material.Aeromonas hydrophila was cultured on Ryan medium, Vibrio cholerae O1 on thiosulfate citrate biosalts (TCBS) agar and Shigella flexneri 2a, enteraggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) on MacConkey's agar. Survival of each bacterial strain in ash and water was determined by spiking concentrations of each bacteria type in triplicate into slurries of sterile water and ash collected from one of three different Mirzapur households chosen randomly from the 10 samples. Slurries of ash and water were mixed at a 1 gram autoclaved ash per 5 mL autoclaved water ratio to replicate crude observed amounts demonstrated by Mirzapur caretakers. The pH for each ash and water slurry was tested using pH test strips .The caretakers who performed handwashing demonstrations were asked to describe where they obtained the ash used for handwashing and to provide a sample of ash from their designated container in the handwashing area, which was then sent to the Center for Vaccine Development (CVD), University of Maryland, Baltimore, MD. A series of experiments was conducted to assess the ability of specific bacteria to survive in ash as follows. Before the laboratory experiments, ashes were autoclave-sterilized. Fresh bacteria for spiking experiments were isolated by streaking swabs from glycerol stocks maintained in the CVD repository onto selective media and culturing plates overnight at 37°C. 4 colony-forming units (CFU) were spiked into each of the three separate tubes with 2 grams ash and 10 mL water, and mixed by rotating the tube five times (spiking inoculum concentration of 103/100 uL). One tube of each of the three ash samples was spiked with 100 μL of water as a negative control. After 30 seconds and 1 minute time intervals, 100 μL of spiked ash slurry were plated in duplicate upon an appropriate culture medium. After overnight incubation at 37°C, the total number of recoverable CFU on duplicate plates were counted and averaged. The concentration of replicate ash samples was consistent, therefore the results for each of the three ash samples types were averaged to report percent reduction and log reductions in bacteria. Percent reduction is reported as CFU remaining per original spiking concentration. The role of pH in bacterial killing was explored by preparing ash and water slurries, and adding HCl to neutralize the pH to 7.0. Spiking experiments were repeated and recovered CFU quantified.Bacterial inocula  were prepared by collecting swabs of bacteria from fresh overnight cultures into phosphate buffered saline. Concentrations of inoculating solutions were confirmed by plating 10-fold serial dilutions on appropriate selective media. For each experiment, 10Written informed consent was obtained from adult caretakers of children enrolled in the GEMS case-control study.A total of 1,394 case and 2,465 control children were enrolled between December 2, 2007 and December 5, 2010 in the GEMS case-control study in Mirzapur, Bangladesh.Household spot checks were used to observe and record all indicators of hygienic behavior in a total of 1,374 case and 2,429 control households. A designated handwashing area with water was observed in 99.2% of case and control households, and a handwashing material such as soap, detergent, or ash was observed in almost all households at that location . Soap orP < 0.0001, P < 0.0001). Having a container of only ash for handwashing, with no soap or detergent, has decreased with wealth quintile from 18.8% of the poorest households to 5.7% of the wealthiest (P < 0.0001). Although soap and ash ownership showed significant patterns of association with wealth of a household, there was no difference between case and control caretakers in ownership of any soap (P = 0.85), soap only (P = 0.98), any ash (P = 0.50), or ash only (P = 0.10)  .Qualitative studies were used to understand how and when caretakers choose to use specific types of hygiene materials, especially for handwashing, how those materials are obtained and stored, and the motivations for using non-soap materials for handwashing.Pucca toilet users (of both shared and privately owned facilities) stated that cleaning the toilet with ashes prevents bad odor. Ash, along with a broom, is considered to be a suitable substitute for toilet cleaner products. However, ash has additional uses for caretakers using a pit latrine and practicing open defecation. Caretakers using a pit latrine reported that facilities frequently look and smell bad, so they spread ash in the latrine pit every morning to minimize the smell. They also frequently observe insects on the feces; chickens and hens roam around the household and eat the insects (and feces). Therefore, ash is also spread in the latrine to prevent “growing” these insects. Caretakers who reported having no access to a facility, or who had a facility but couldn't conveniently use it at that time (such as in an emergency), would go to an open area (jungle or river side) for open defecation and use ash to cover their feces to prevent the smell.Caretakers use a variety of materials for multiple personal and household purposes. Ash and detergent serve many household needs, such as for laundry (detergent only), dishwashing, and cleaning and sanitizing the latrine (both) . The Puc“We have to maintain our children and feed him; it's better if our hands are clean at that time.”“It (handwashing) matters a lot; it affects our body. It causes diarrhea, dysentery, etc.”“After using toilet, our hands might be contaminated with germs and soap can clean those germs; for that cleaning purpose, we use soap.”Caretakers have a high degree of personal hygiene awareness, and understand that lack of hygiene is related to diseases like diarrhea. The action of handwashing at specific times with any material is considered a priority.“Sometimes, I rub my hands on the soil.”“Soap sometimes; ash occasionally.”“I use soil (for hands) after cleaning the baby's bottom; sometimes soap is used.”They reported that soap is the only material used for bathing one's self or a child, and is the primary material for handwashing after defecation, cleaning a child, before feeding a child, and at any point where she felt she had contacted a dirty thing . However“As I use ash first (after defecation), I think, it might not clean my hand properly, for that reason I use two agents (soap later) to clean my hands properly.”Furthermore, handwashing is not always performed with one single material or mixture of materials. Some caretakers indicated a multistep process where a second material was used sequentially to wash if the hands were still perceived to be dirty or malodorous.Caretakers indicated that they purchase soap and detergent using household income from a local vendor, and store these materials beside the household tube well or outside the latrine where hands are usually washed . SometimCaretakers participating in handwashing demonstrations first grabbed a handful of ash, or an ash and detergent mixture (10–15 grams), from a pot in their designated handwashing area. They wet their hands and the ash with water from a pitcher or faucet (mean volume ∼50 mL), scrubbed their hands for 15 to 30 seconds, and then rinsed with 0.75–1.0 L of water.To understand whether caretakers made distinctions between the need to wash the left and right hand, we asked the mothers to describe when they felt that they needed to wash both of their hands. One mother reported washing her (right) hand with water only before feeding the child. Caretakers listed times when both hands were washed as during cleaning utensils, handling cow dung, after defecation, after completion of household tasks, before cooking, before eating, and after cleaning the bottom of the child (that defecated).Quantitative and qualitative studies clearly show that caretakers perceived soap as the best way for washing hands, so we asked them to describe the situations where they might not wash at all, or wash with ash, soil, or water only instead of soap. The primary factors that influenced their practices included: perceived necessity, stress, convenience, affordability, and environment.“I use detergent powder to clean my hand; but if I have to clean my utensils after defecation, that time I do not wash my hands because I wash crockeries and utensils with soap; so, I do not need to clean my hands.”Perceived necessity influenced whether a caretaker washed at all with any material. Some caretakers did not think washing hands after defecation was necessary if they were going to perform some other task involving detergent or detergent/ash.“We have to work a lot, sometimes we get time to clean our hands and sometimes not: usually we clean our hand with water before eating and feeding our child; all of us do the same practice.”“Sometimes it happens that we couldn't apply soap due to time constraint; I just wash my hands with water or with ash sometimes.”Convenience of access influenced the type of material caretakers used for washing. Ash and detergent seemed to be more consistently available within the household. Caretakers pointed out that sometimes a bar soap was finished before she noticed, and was not available when needed. If she was busy with household chores, usually perceived to be the case, she would collect detergent or ash from close to the household, instead of going to purchase another bar at that time. Convenience of time influenced whether caretakers washed at all, and the type of material they washed with. Caretakers felt that they were frequently too busy to wash their hands, and suggested that washing with water only or ash was quicker and easier. Some critical times, such as feeding a child, were prioritized as important times to wash, regardless of time constraints.“Soil is also used; who can use so many soaps?”Caretakers emphasized that the cost of soap was a major limitation, and that they could not afford to use it at all times for all purposes. Thus, they used alternative materials that performed equally well and come at lower cost.Environmental factors also played an influence on material use. Caretakers informed us that this area had iron in the water supply, so ash and detergent are mixed to produce a better cleaning product than with either material alone.V. cholerae were detected using selective media (S. flexneri (95% decrease in concentration), −0.8 for EAEC (83.8%), −1.2 for ETEC (93.9%), and −0.8 for A. hydrophila (82.4%) (S. flexneri (98.9%), −2.0 for EAEC (99.0%), −1.2 for ETEC (93.9%), and −2.6 for A. hydrophila (99.7%)  for set time intervals. After 30 seconds, no ve media . After 3 (82.4%) . After 1 (99.7%) . No reduThe primary objective of this study was to analyze whether hygiene practices of GEMS case-control caretakers were associated with moderate-to-severe diarrhea in children. We used household spot checks to efficiently quantify the presence of handwashing materials in GEMS households. Spot checks of handwashing stations with water and soap are commonly used as a proxy for handwashing practices in demographic health surveys and studies,We used a mixed-method approach of quantitative household spot checks for hygiene indicators and qualitative handwashing demonstrations, interviews, and focus group discussions to characterize handwashing practices among GEMS caretakers and confirm that spot check indicators were valid proxies for handwashing practices. Household spot checks documented that nearly all households with young children maintained a designated handwashing area equipped with a source of water and a type of cleansing material. Ash, soap, and detergent were all common handwashing and household cleansing materials, and were frequently stored together at handwashing stations. The use of ash for post-defecation handwashing has been previously reported as a common practice in rural and urban slum areas of Bangladesh, and in urban Kolkata, India.There are few studies that have assessed the efficacy of washing with ash on risk of diarrhea. In one instance, a lower risk of diarrhea was observed for children whose mothers washed their hands with soap after defecation versus with water only, but was not observed for children of mothers who washed with ash.We tested whether simple contact with ash and water for typical handwashing times could reduce the risk of hand-transmitted disease by decreasing concentrations of enteric pathogens that were common causes of MSD in our Bangladesh study.frequency of handwashing behavior among caretakers in this population. Studies employing motion-tracking sensors embedded in soap have found poor correlation between soap use and proxies like the presence of a handwashing area, water, and soap.One caveat of this study is that household spot checks for handwashing materials were conducted roughly 2 months after the clinically confirmed episode of diarrhea in a child. It is possible that case and control children might have experienced diarrhea in that window of time or that caretakers switched handwashing materials. Additionally, we did not use methods to assess whether the spot checks were a valid proxy for Ultimately, promoting handwashing at critical times with any locally available resource may be effective for achieving sustainable improvements in handwashing behavior.Although the poorest households are the most likely to use ash, 36.2% of the wealthiest households in Mirzapur still keep ash at a handwashing area.Supplemental Table.