We investigated stimulus–response (S–R) memory links during object priming using a binary associative size judgement paradigm. At study, participants decided which of two objects was bigger in real life and, at test, made the same or the reverse judgement. We examined the effects of response congruence on item S–R priming in the associative paradigm. In Experiment The online version of this article (10.3758/s13421-020-01021-9) contains supplementary material, which is available to authorized users. Priming is a kind of implicit memory in which the processing of a stimulus during a learning phase leads to more accurate or faster response times (RTs) to that stimulus when the same response is made during a subsequent test phase , the direct retrieval of which, in an appropriate test situation, speeds responding or makes it more accurate  or incongruent (mismatches) with the response at study, by changing the test task  or changing the reference object . By using congruence manipulations like these, it was shown that incongruence at some or all response levels reduced RT item S–R priming , some pairs consist of a recombination of items that appeared in different pairs at study (recombined condition), and some are novel pairs (new condition). A significant performance advantage in the intact relative to the recombined condition indicates associative priming, whereas a similar advantage for the recombined relative to the novel pair condition indicates item priming . A power analysis . These pictures were scaled down to fit in a box of 400 × 400 pixels, so as not to create a response bias for larger images. The objects were carefully selected to have an unambiguous size in real life and similar line complexity. The 240 study–test pictures were split into 30 groups, each containing eight pictures. The pictures in each group were further divided into two subgroups of four pictures with the restriction that the pictures in a subgroup were unrelated to the pictures in the other subgroup. Two different word association norms  and pair type  as within-subject factors, and task order  and prime level  as between-subject factors. This paper focused on item priming as measured in a novel associative task, so we omitted intact pairs from all analyses (the results from the intact condition will be presented elsewhere). In addition, task order did not systematically interact with the other factors, so, to simplify statistical analyses, we do not report it further.d and partial eta-squared (η2p) where appropriate.In order to ensure that comparisons of conditions across tasks were not biased due to, for example, the “reverse” task being more difficult to execute, which might increase RTs or errors, we computed baseline-corrected scores  toolbox Cogent (http://www.vislab.ucl.ac.uk/cogent.php) was used to present stimuli and record participants’ responses.Figure SDs) above or below the mean value of each condition, were removed from subsequent analyses.In order to obtain a representative index of S–R binding, and to conform to other studies of this kind  = 2.30, p > .10, η2p = .05, or an interaction, F = .03, p > .10, η2p = .001. Despite the absence of a main effect of test task, we decided to test single effects, given that previous studies have reported significant differences between “same” and “reverse” tasks. Accuracy priming was indeed significant in the “same” task in both prime level conditions , whereas it was nonsignificant in the “reverse” task .Difference error rate scores (accuracy priming) were computed by subtracting recombined from new pairs see Fig. , top. A ts > 6.57, ps < .001). Despite a numerical advantage for “same” (.17) versus “reverse” (.14) item priming, a 2 (prime level) × 2 (test task) mixed repeated-measures ANOVA, revealed a main effect only of prime level, F = 15.80, p < .001, η2p = .26, which, unsurprisingly, indicated greater priming for the high than low primed condition. Neither the main effect of test task, F = 2.64, p > .10, η2p = .05, nor the interaction, F = .28, p > .10, η2p = .01, were significant, suggesting that RT item priming in this experiment was not affected by a task reversal. Indeed, the difference between “same” and “reverse” priming was not significant when tested separately for each prime level condition .Next, proportional RT item priming scores were computed to account for the different baseline RTs within each test task see Fig. , bottom.The above interpretation, however, was based on a null finding, so, we decided to conduct a Bayes factor (BF) analysis to determine the strength of evidence favouring the alternative hypothesis of a genuine difference between “same” and “reverse” tasks. A BF value smaller than 1/3 or greater than 3 is commonly interpreted as substantial evidence, whereas anything in between is only anecdotal evidence. The BF for the comparison between the alternative (difference between “same” and “reverse”) and the null (no difference between the tasks) hypothesis was 0.67, suggesting that, even though there is not much evidence to distinguish between the hypotheses, the data do not preferentially favour the alternative hypothesis.Contrary to what has been found with the single-item paradigm, RT item priming, as measured during an associative paradigm, was not significantly reduced in the “reverse” relative to the “same” task. Although this conclusion rests on a null result and should, therefore, be interpreted with caution, it is consistent with the results of a very similar study  but not in the “reverse” task . This finding indicates that the influence of incongruent decision/action levels of response on recombinations may be affecting accuracy to a greater extent than it is influencing RTs. We should note, however, that the main effect of test task was nonsignificant, so caution must be exercised interpreting these simple effects. Experiment When accuracy was used as a measure, however, item priming appeared to be present in the “same” .Two-hundred and sixty-eight (256 for the experiment and 12 for practice) coloured clip-art images were selected and went through the same selection procedures and relatedness checks as those described in the CONINC in Fig. CON and R2INC were matched in terms of object-level response congruence as well as size-judgement difficulty  were presented in the test phase.To construct the two types of recombined pairs, for each group, the pictures in Subgroups A and D were randomly recombined with the pictures of Subgroups C and B, respectively. Thus, the objects in Subgroups A and D , as well as proportional RT scores for each recombined type . Thus, the experimental design for this experiment consisted of congruence  and test task  as within-subject factors and prime level  as a between-subjects factor. The statistical analysis performed on these data followed a similar pattern to that of Experiment We computed difference scores between each recombined type and new pairs  split by test task (same and reverse) and prime level .Figure CON, R2INC) × 2  repeated-measures ANOVA. There was a significant Congruence × Test Task interaction, F = 7.80, p < .01, η2p = .15, as the result of greater accuracy priming for R2CON/REV (.02) relative to R2CON/SAME (.001), but greater accuracy priming for R2INC/SAME (.02) relative to R2INC/REV (.002). The three-way interaction also reached significance, F = 6.68, p < .05, η2p = .13, which suggested that the differences above were present for the high primed condition but not for the low primed condition  were submitted to a 2  × 2  = 58.31, p < .001, η2p = .56, indicating that recombinations with classification congruence for the selected object alone (R2CON) were judged faster than recombinations with classification incongruence for the selected object alone (R2INC) regardless of test task . Note that the main effect of classification congruence was properly matched for both test task and decision/action congruence—R2CON and R2INC differed only in that the classification was congruent for the selected object and incongruent for the nonselected object for R2CON, whereas it was the other way round for R2INC  × 2 (congruence) × 2 (test task) repeated-measures ANOVA revealed a significant main effect only of congruence, F1, 6 = 58.31CON/SAME versus R2INC/REV RT priming, and (2) R2CON/REV versus R2INC/SAME RT priming. These contrasts can be thought of as pure tests of the interaction between classification congruence and object selection. This is because, for R2CON/SAME (and R2CON/REV), the classification was congruent for the selected object, but incongruent for the nonselected object, whereas for R2INC/REV (and R2INC/SAME), it was the other way round. Critically, responses at the decision/action levels between the two types of recombination were matched in both Contrast 1 and 2 (see Table CON/SAME vs. R2INC/REV), t(47) = 4.24, p < .001, d = .63; Contrast 2 (R2CON/REV vs. R2INC/SAME), t(47) = 2.21, p < .05, d = .29. This result not only indicates that the congruence effect was due to classification-specific incongruence, but also suggests that the congruence effect was not a consequence of reinstating the same task at test, because, in Contrast 2, “reverse” task recombinations showed speedup relative to “same” task recombinations.Even though the interaction between congruence and test task was not significant, we decided to directly perform two separate contrasts (collapsed across prime level): 1) R2 R2CON/SACON/SAME with R1SAME because these two types of recombination only differed for the nonselected object (compare these two conditions in Table t(94) = .48, p > .10, d = .10, which suggests that classification incongruence of nonselected objects on their own does not disrupt item RT priming.Given that item priming declined more when classification incongruence was for the selected object, we wondered how important response congruence for the nonselected object was in our associative task. For that purpose, we compared R2CON (congruent classification for the selected item and incongruent classification for the nonselected), than R2INC (incongruent classification for the selected item and congruent classification for the nonselected item). Nevertheless, RTs for both kinds of recombination were faster than those for new pairs in both test tasks, revealing some item priming.The results of the present experiment indicated that classification incongruence of the selected item has a much bigger effect on RT S–R priming than incongruence of the nonselected object. This was indicated by faster RTs for R2CON and R2INC cannot be simply explained by changes in item-level classification status: Both R2CON and R2INC contained one item with a congruent classification  did not show additional RT priming than R2CON/SAME (which had congruent S–R bindings for the selected item but incongruent S–R bindings for the nonselected item). This result could explain why RT priming was similar between R1SAME and R1REV in Experiment Another important finding was the observation that incongruence for the nonselected object had a negligible impact on the RT item priming effect, since R1CON/SAME) relative to when it occurred for the selected object , although this effect emerged only in the high primed condition. Some studies have failed to observed S–R priming effects when only stimulus–action bindings changed between study and test . From this finding, we concluded that S–R bindings of the nonselected item may have played a minor role in item S–R priming detected in Experiment In the previous experiment, we did not find a difference in the magnitude of RT priming between recombinations in which all levels of response representation for both the selected and nonselected object were congruent R1) relative to recombinations in which all levels of response representation for the nonselected object alone were incongruent (R2 relativeForty-six undergraduate students were recruited in exchange for monetary compensation or course credits (age range: 19–35 years). The materials were largely taken from Experiment To construct the two types of recombined pairs, for each group, the pictures in Subgroups A and C were randomly recombined with the pictures of Subgroups D and B, respectively. Thus, both objects in A–D associations  = 4.60, p < .05, η2p = .09, with lower accuracy priming for R3 in the “same” (−.09) than in the “reverse” (−.05) task . Collapsed across prime level, a negative accuracy priming effect was observed for both R3SAME, t(47) = −5.86, p < .001, d = .81, and R3REV, t(47) = −3.87, p < .001, d = .56.A 2 (prime level) × 2 (test task) mixed repeated-measures ANOVA on accuracy priming only yielded a significant main effect of test task, Fs < 2.28, ps > .10). In stark contrast to the previous two experiments, recombined pairs appeared to have been judged slower than baseline pairs during both test tasks  = −.80, p > .10, d = .11, and there was even a modest negative priming for the “same” task, t(47) = −1.64, p = .05, d = .24 (one-tailed). BF analysis revealed that, in both tasks, the null hypothesis of no priming was favoured relative to the alternative hypothesis of positive priming (BFs < 0.15).For the proportional RT item priming analysis, a 2 (prime level) × 2 (test task) mixed repeated-measures ANOVA did not reveal any significant main effects or interaction  = 4.77, p < .001, d = .96, indicating greater proportional RT priming for R2INC/SAME than R3SAME. One could argue that the lack of priming in the present experiment resulted from the fact that the present items were more difficult to prime than those in Experiment t(94) = 1.05, p > .29, d = .22, so it is highly unlikely that this result could be explained by this kind of difficulty confound. Finally, we also replicated the outcome congruence effect reported by Dennis and colleagues  relative to when both the selected and nonselected objects had incongruent responses (R3). This result provides strong evidence that the retrieval of S–R links associated with the nonselected object does contribute to the general item priming effect.facilitation for completely incongruent recombined pairs relative to new pairs.Interestingly, our results showed that R3 did not significantly differ from new item pairs in either test task, indicating that item RT priming in the present experiment was not obtained. In fact, there was some evidence of negative RT priming occurring for the “same” task, as well as negative accuracy priming in both tasks see Fig. . These rThere are several possible reasons for this discrepancy. First, the present experiment used pairs of pictures of objects, whereas Dennis and colleagues , with worse performance for pairs with decision/action incongruence of both selected and nonselected items.Item S–R priming was investigated in three experiments using a novel associative priming paradigm. Experiment At first glance, our results appear to challenge current theories of S–R priming. First, the lack of sensitivity of RT priming in our associative priming paradigm after a task reversal  made to each recombined stimulus in the previous study episodes. It involves relatively automatic and fast activation of the three levels of response that were made at study to each re-paired object picture. It works in a nonhierarchical way, so each level of response may be directly activated in about the same time, rapidly and automatically. With response incongruence, there is likely to be competition/interference with the slower effortful stream at each affected level of response. In contrast, with congruence, the slower effortful stream may receive some facilitatory energisation at each affected level of response  than for congruent decision/action responses for both the selected and nonselected items (R3REV), and (2) why the fast stream disrupted accuracy more for incongruent decision/action responses for the nonselected item only (R2CON/SAME and R2INC/REV) than incongruent decision/action responses for the selected item only (R2CON/REV and R2INC/SAME).The two-stream priming account must explain the accuracy item priming effects by indicating (1) why the fast stream disrupted accuracy more for incongruent decision/action responses for both the selected and nonselected items  showed worse accuracy relative to R3REV— R3REV had congruent decision/action study–test responses for both the selected (appropriate activation) and nonselected (appropriate inhibition) test items , the fast stream should have correctly activated the item that should have been selected, but also incorrectly activated the item that should not have been selected . Contrastingly, for congruent decision/action responses for the nonselected item alone (R2CON/REV and R2INC/SAME), the fast stream should have incorrectly inhibited the item that should have been selected but also correctly inhibited the item that should not have been selected . If inhibition and activation effects of the fast stream have equivalent biasing effects on response selection, the effects should cancel out in each condition, and there should be no difference between the conditions. This obviously did not happen as recombinations with “activation-incongruent” decision/action responses  were less accurate than recombinations with “inhibition-incongruent” decision/action responses . One explanation could be that inappropriate fast-stream activation of the nonselected item response  is very effective, whereas fast-stream inhibition of the selected item response  is much less effective. This makes intuitive sense: Inhibition of an item will never bias the fast stream to choose that item (since it is associated with a “do not select” response), whereas activation of an item may bias the fast stream to choose that item (since it is associated with a “select” response).Regarding the second point, for recombinations with incongruent decision/action responses for the nonselected item alone (R2CON/SAME and R2INC/REV) than for recombinations with appropriate decision/action response inhibition for the nonselected item (R2CON/REV and R2INC/SAME). There was indeed such a tendency (see The above hypothesis indicates that the fast stream should sometimes cause rapid and wrong selection of the nonselected items that the slow stream would (appropriately) not have selected. It should, therefore, predict faster inaccurate responses for recombinations with inappropriate decision/action response activation for the nonselected item (R2ency see .REV) were less accurate than new pairs. The two-stream account, however, would predict no difference in error rates between these two conditions, since fast and slow streams would be in accord. Given that decision/action congruence for both items in R3REV was accompanied by classification incongruence, our finding suggests that decision/action congruence must have somehow interacted with classification incongruence, although the exact mechanism underlying this interaction is unclear.In Experiment Finally, we should note that because accuracy priming effects appeared meaningful (effect sizes were medium to large), we decided to interpret these results. Nevertheless, we acknoweledge that the small magnitude of the accuracy priming effects in Experiments CON), showed more priming than recombinations with incongruent classification for the selected object, but congruent for the nonselected object  showed more proportional RT priming than recombined pairs in the “same” task that had incongruent classification for the selected item alone (R2INC/SAME), which means that the pairs in the “reverse” task showed a speed-up in relation to the pairs in the “same” task.Two pieces of evidence suggest that the longer RTs were not partially or totally due to the reversal of the test task. First, we used proportional priming measures, which effectively take into account the baseline levels of each test task. Second, and more importantly, recombined pairs in the “reverse” task that had congruent classification for the selected item alone (R2CON) suggests that the fast stream can activate the correct classification response of the selected item , whereas inappropriate activation of the wrong classification response of the nonselected item  does not seem to occur. In contrast, slower RTs for recombinations with incongruent classification for the selected item alone (R2INC) suggests that the fast stream can only activate the incorrect classification response of the selected item , whereas activation of the correct classification for the nonselected item  does not seem to occur. This seems consistent with the finding that the effect of incongruence for the nonselected item alone was negligible—RT priming was equivalent between recombinations with incongruent classification for the nonselected item only (R2CON/SAME), and recombinations with congruent classifications for both selected and nonselected objects (R1SAME). This suggests that incongruence for the nonselected object alone played at most a minimal role in RT priming, and that the RT difference between recombinations with congruent classification only for the selected item (R2CON) and recombinations with congruent classification only for the nonselected item (R2INC) resulted exclusively from incongruence of the selected item.The faster correct RTs for recombinations with congruent classification for the selected item alone , item priming was eliminated, suggesting that nonselected classification incongruence has an additive effect with selected classification incongruence. One explanation for this result could be that there was some form of interaction between the fast and slow streams. For example, when presented with “train–deer”, fast-stream retrieval of the classification “bigger” for the “train” would match the response generated by the slow stream . This should produce RT facilitation even if “deer” has an incongruent classification, because the system has sufficient information to generate an adequate response . Presumably, the classification of the associate item might even be ignored (producing no RT costs), which may explain why RT priming did not differ between recombinations with incongruent classification only for the nonselected item (R2CON/SAME), and recombinations with congruent classifications for both selected and nonselected objects (R1SAME). However, when both items have incongruent classifications


Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tβ4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tβ4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tβ4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tβ4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tβ4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.Prior work has indicated that thymosin beta 4 (Tβ4) administered with ciprofloxacin markedly improves disease outcome for  Bacterial keratitis is a deleterious infection of the cornea that causes corneal scarring, opacification, and perforation. While this disease is commonly associated with contact lens wear, risk factors also include refractive corneal surgery and immunocompromised conditions ,2. Curre−5 to 5.6 × 10−1 M [Pseudomonas aeruginosa (PA)-induced bacterial keratitis has shown that when used as an adjunct to ciprofloxacin, disease outcome was significantly improved [Thymosin beta 4 (Tβ4) was first isolated and identified from calf thymus 30 years ago, by Goldstein et al. . This 43× 10−1 M . Initial× 10−1 M ,9. Priorimproved , howeverWe also assessed the indirect influence of Tβ4 on bacterial killing via the modulation of the host response. Antimicrobial peptides (AMPs) are small positively charged molecules that function to defend against invading pathogens. Human ocular surface epithelia secrete peptides from the defensin and cathelicidin families, which are thought to exert protective broad-spectrum activity against a sundry of microorganisms. AMP production is also enhanced through the activation of pro-resolution circuits . TherefoOverall, our objective was to elucidate the mechanism(s) by which Tβ4 affects bacterial killing, host response, and ciprofloxacin activity in the setting of bacterial keratitis. Our findings indicate that Tβ4 does in fact play an indirect role in bacterial killing, while synergistically enhancing the inhibitory effects of antibiotics.2 incubator, at 37 °C. Before treatment, cells were seeded at a density of 0.2 × 106/mL  into 6-well culture plates that were precoated with fibronectin collagen  and cultured in defined keratinocyte SFM for 2 days. Upon confluency, cells were treated as described below. HUCLs were used between passages 3–5 for all experiments.Human telomerase-immortalized corneal epithelial cells (HUCLs), kindly provided by the laboratory of Fu-Shin Yu, were used for the AMP studies. These cells, previously infected with a retroviral vector encoding human telomerase reverse transcriptase to generate an immortalized cell line, have been characterized and validated as a viable model for corneal epithelial cell investigation ,14. The Minimum inhibitory concentration (MIC) assays were executed using a 96-well microdilution technique using the following four treatment groups: media only (control), Tβ4, ciprofloxacin, and Tβ4 + ciprofloxacin. Serial dilutions were carried out in PTSB with a final total volume of 200 µL/well. Final concentrations of Tβ4 ranged from 0.8 to 0.0015%; ciprofloxacin ranged from 0.3 to 0.0006%. For the Tβ4 + ciprofloxacin combination treatment group, Tβ4 was used at a final concentration of 0.1% and varying concentrations of ciprofloxacin ranged from 0.3 to 0.0006%.5 or 106 CFU/mL, then, incubated for 18–20 h in 5% CO2, at 37 °C. Optical density (OD) readings were collected by spectrophotometry. Results are reported as OD600 ± SEM.Bacterial inoculum was prepared, as previously described . Each we® Green Master Mix , per the manufacturer’s instruction, with the reaction mixture previously described [Cultured HUCLs were divided into the following four treatment groups: media (control), Tβ4 (0.1%), ciprofloxacin (0.3%), and Tβ4 + ciprofloxacin. Dosing of both Tβ4 and ciprofloxacin have been previously established ; furtherescribed . All priescribed . Results+ SD unless indicated otherwise. Results were obtained in a blinded fashion and analyzed by a one-way ANOVA followed by Bonferroni’s multiple comparison test  with data considered significant at p < 0.05.All assays were carried out from a minimum of three independent experiments with representative data from a typical experiment shown. Data are presented as mean 5 as compared with 106 CFU/mL of bacteria. In contrast, all concentrations of Tβ4 remained similar to untreated controls for both concentrations of bacteria, thus, indicating a lack of bactericidal effect. Combination treatment showed similar trends to that observed for ciprofloxacin alone at both concentrations of bacteria.Bacterial killing for each treatment group was assessed using a MIC assay, the results of which are shown in Pseudomonas keratitis. Select AMPs and related molecules  remained unchanged for all treatment groups at 6 h after LPS stimulation, yet was significantly elevated at 24 h after Tβ4, ciprofloxacin, and combination treatments. DRV2/GPR18 expression (D) was elevated at 6 h following Tβ4 treatment, but significantly decreased in cells that received ciprofloxacin and the combination treatment. In fact, while DRV2/GPR18 expression was restored with the combination treatment at 24 h, this receptor remained significantly downregulated after ciprofloxacin only treatment as compared to all three groups .Bacterial keratitis is an extremely debilitating and rapidly progressive infection of the cornea which can advance into sight-threatening consequences including endophthalmitis and corneal opacification. Although the criticality of treating this condition is evident, our current standard of care for bacterial keratitis topical antibiotics  with judicious use of topical steroids (loteprednol and prednisolone) comes with a number of drawbacks that include antibiotic resistance, toxicity issues, steroid induced glaucoma, and increased risk for infection ,4,5. FurStaphylococcus aureus, and Staphylococcus epidermis, although this effect was inhibited in the presence of NaCl and tears [While it has been suggested that Tβ4 exhibits bactericidal activity, results, herein, indicate that Tβ4 does not appear to play a direct or synergistic role with antibiotic in bacterial killing. These results were somewhat surprising in light of previous work, including a report that Tβ4 showed moderate bactericidal activity against PA, nd tears . In addind tears . Howevernd tears .Pseudomonas-induced ocular infection [Next, we investigated the impact of Tβ4 on bacterial killing through indirect mechanisms, specifically the production of AMPs and related molecules in human corneal epithelial cells. Cell membrane receptors that bind Tβ4 remain largely unknown, although work carried out by Zetter has revealed an extracellular pathway for Tβ4-induced regenerative properties that involved purinergic signaling . In addinfection . S100A8 nfection . The sigTo complement the AMPs and related molecules, we also explored the impact of Tβ4 on pro-resolution enzymes, 12-LOX and 15-LOX. Prior work carried out by our lab and others has established the significance of lipoxygenase activity and activation of SPM pathways in promoting resolution and tissue restoration during ocular infection . ActivatWhile a synergistic relationship between Tβ4 and ciprofloxacin was expected, our findings regarding ciprofloxacin alone were particularly notable. Significant bacterial killing was detected at all tested concentrations, even those that were markedly lower than what is currently used in the clinical setting (0.0006% vs. 0.3%). These results suggest the potential for lower dosing of antibiotics, which may help to alleviate toxicity issues and the development of antibiotic resistance. Furthermore, it has been historically accepted that antibiotics are predominantly limited to bactericidal and bacteriostatic activity. Our findings suggest off-target effects on the host response, as indicated by an upregulation in AMP transcript levels. Additionally, it was observed that ciprofloxacin may have a regulatory role on SPM receptors as indicated by decreased expression of DRV2/GPR18 in the human corneal epithelial cells. These findings establish a basis for further studies on the functional activities of ciprofloxacin, as well as other antibiotics that are widely used in the clinical setting.In light of these findings, Tβ4 shows promise as an adjunctive therapy to ciprofloxacin, in the context of corneal infections. By helping to establish its regulatory role in AMP production and activation of SPM pathways, the current work has further explicated mechanisms by which Tβ4 communicates intracellularly to improve disease outcomes as an adjunct therapy. This novel combination treatment provides an alternative to topical steroid use, and may alleviate the drawbacks associated with current standard of care.


Ascariasis, Trichuriasis and Hookworm infections poses a considerable public health burden in Sub-Saharan Africa, and a sound understanding of their spatial distribution facilitates to better target control interventions. This study, therefore, assessed the prevalence of the trio, and mapped their spatial distribution in the 20 administrative regions of Ogun State, Nigeria.Parasitological surveys were carried out in 1,499 households across 33 spatially selected communities. Fresh stool samples were collected from 1,027 consenting participants and processed using ether concentration method. The locations of the communities were georeferenced using a GPS device while demographic data were obtained using a standardized form. Data were analysed using SPSS software and visualizations and plotting maps were made in ArcGIS software.Findings showed that 19 of the 20 regions were endemic for one or more kind of the three infections, with an aggregated prevalence of 17.2%. Ascariasis was the most frequently observed parasitic infection in 28 communities with a prevalence of 13.6%, followed by hookworm infections with a prevalence of 4.6% while Trichuriasis was the least encountered with a prevalence of 1.7%. The spatial distribution of infections ranges between 5.3–49.2% across the regions. The highest and lowest distribution of overall helminth infections was recorded in Egbado South and Egbado North respectively. Nine regions had infection status between 20.0%-49.2%, while 10 regions had infection status between 5.3%-15.8%.This study provides epidemiological data on the prevalence and spatial distribution of ascariasis, trichuriasis and hookworm infections which will add to the baseline data and guide the public health officers in providing appropriate control strategies in the endemic communities. Ascaris lumbricoides (Ascariasis), Trichuris trichiura (Trichuriasis) and hookworms (Ancyclostomiasis / Necatoriasis) are widely distributed in tropical and subtropical areas, with the greatest numbers occurring in sub-Saharan Africa, the Americas, China and East Asia  in your submission contain [map] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . 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If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful: http://viewer.nationalmap.gov/viewer/USGS National Map Viewer (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/The Gateway to Astronaut Photography of Earth (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.htmlMaps at the CIA (public domain): http://earthobservatory.nasa.gov/NASA Earth Observatory (public domain): http://landsat.visibleearth.nasa.gov/Landsat: http://eros.usgs.gov/#USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://www.naturalearthdata.com/Natural Earth (public domain): Additional Editor Comments (if provided):The study results provided some useful information, the spatial distributions of STH could be evaluated in a better/clearer way. I suggest the author modify the spatial analysis and map presentations and re-submit the revised version.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript entitled “Spatial Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria” investigated STH infections in Ogun State and try to mapped the spatial distribution of STH infection. Though the study results provided some useful information, the spatial distributions of STH could be evaluated in a better/clearer way. I suggest the author modify the spatial analysis and map presentations and re-submit the revised version. Below are some comments:Major issues:1. It seems that the georeferenced household’s information was not analyzed in the study because the figure only showed the locations of the selected communities. Since you have more than thousand georeferenced households participated in your study, why don’t you apply interpolation techniques (e.g. kriging or kernel density) to evaluate the prevalence and intensity?2. The range of different age groups are different , any reason for the grouping criteria? The age distributions could be biased.3. The investigation has been done at community level; however, the results were still presented at LGA level, and it’s hard to identify where is the most prevalent area. For instance, I have no idea where is “Yewa South” on the map . Please modify the map presentation in a better way.4. This is a cross-sectional survey which might be affected by many other factors. For example, does the infection prevalence influence by any intervention activities? Such limitation should be discussed in the manuscript.5. Though the overall prevalence is not very high in the study area, the transmission still varied by locations. Can you explain why “Yewa South” has the highest prevalence?Minor issue:1. The scale bar in Figure 1 is not correct. It should not be “millimeters.” Please check whether you apply a correct projection coordination system.2. Table 1 can be moved to the supplementary materials.3. Figure 5. What does “Ogun State” mean in the legend? For the legend indicated the LGA with no case, please add the border.Reviewer #2: Specific commentsAbstractResult sectionLine 2-4: The sentences “Ascariasis was the most spatially distributed found in 28 communities, with 140(13.6%) infected subjects, followed byHookworm infection in 19 communities, with 47(4.6%) infected subjects and Trichuriasis in 9 communities, with 17(1.7%) infected subjects.”should be reconstructed to read “ Ascariasis was the most frequently observed parasite in 28 communities with a prevalence of 13.6%, followed by hookworm with a prevalence of 4.6% while Trichuris was the least encountered with a prevalence of 1.7%.Line 6-7: The sentence is incomplete “The highest and lowest distribution of what (?) was recorded in Yewa South and Yewa North respectively.Line 8: Delete the phrase “By WHO preventive chemotherapy thresholds”ConclusionThis study provides information on the prevalence and spatial risk of Ascariasis, Trichuriasisand Hookworm infection that will serve as decision-support tool to help facilitate targeting ofcontrol interventions.The conclusion should be re-written as “ This study provides epidemiological data on the prevalence and spatial distribution of ascariasis , trichuriasis and hookworm infection which will add to the baseline data and guide the public health officers in providing appropriate control strategies in the endemic communities.BackgroundLine 7-8: Replace the phrases in parentheses (children between age 2 and 5) and (children between age 5 and 14) with (age 2-5 years) and (age 5-14 years).Line 15: The sentence “there is an established morbidity control programme targeted at school-aged children due to their high level of contact with soils” should be re-written as “there is an established morbidity control programme targeted at school-aged children due to high worm burden as a result of their frequent contact with contaminated soils, ……………………….MethodsStudy areaLine 10: The phrase “The major occupation of the population is……………… should be re-phrased as “Major occupations of the population are………………Study design and sampling techniqueLine 1: The sentence “This study was community-based and cross-sectional in design” should be re-written as “This is a cross-sectional and community-based study”.Selection of households for surveyLine 2: The sentence “communities were visited and sensitized about the study proceduresand were openly invited to participate in the research through community meetings and townannouncers” should be replaced with “selected communities were visited and with the permission of the community leaders, meetings were held with the members of the community to intimate them about the purpose of the study and the procedures to be adopted”.The sub title “Collection of faecal samples and parasitological diagnosis” be replaced with “ Faecal samples collections and processing”.Line 3-4: The sentence “All participants were asked to provide a sufficiently large stool sample (at least 5 g)” should be replaced with “Individuals who consented to take part in the study were requested to provide stool up to half (about 5gm) into the labelled plastic bottles provided.Line 6: Delete the word “Precisely”.Line 7-8: The sentence “The bottle was covered and agitated vigorously to suspend the stool efficiently in the solution” be replaced with “The bottle was covered and shaken vigorously to form a cloudy suspension”.Line 9: Delete the word “further”Line 11: The sentence “The supernatant was also discarded after centrifuging” be replaced with “The supernatant was decanted”.Line 12-14: The sentence “7 ml of normal saline was later added to the sediment, after which 3 ml of ether was finally added to the suspension”. . The deposit or residue obtained after decanting the supernatant should be resuspended in distilled water or saline of a known vol and shaking vigorously. Thereafter, an equal vol of ether will then be added . The authors should correct this procedure.ResultsSex and age distribution of study participantsThe sentence “A total of 1,499 respondents were subsequently enrolled after consent across the 33 communities and 20 LGAs. However, only 1,027 (68.5%) of them consented to the provisionof adequate stool samples (~5g) for laboratory analysis for parasites’ ova or larva. Bydemography, 899(60.0%) were females and 600(40.0%) were males” should be rewritten as “ A total of 1499 participants comprising of 899 (60%) females and 600 (40%) males were enrolled across the 33 communities in 20LGAs out of which 1024 (68.5%) returned stool samples for laboratory analysis.The sentence “The sex ratio of females to males was 3:2. In addition, the highest number of respondents were within the age range 41-70yrs (40.6%), followed by 26-40yrs (30.3%), 5-15yrs (10.7%), 16-25yrs (10.4%) and >70yrs (7.0%) (Table 2)” should be re-written as “The ages of the enrolled participants are provided in table 2.Co-distribution patterns of Ascaris, Trichuris and Hookworm infections in Ogun StateLine1: Replace the words “mono infection” with single infectionLine 2: Delete the word “only”Line 3: Replace the word “co-distribution” with the word “combination”Line 5: Begin with combination of Ascaris and Trichuris and delete the word “co-distribution”Line 6 & 7: Rewrite “while Trichuris and Hookworm co-distribution was recorded in 6 LGAs;” as “while combination of Trichuris and hookworm was……………………….”Line 8-10: Rewrite “However, for triple infections, the three species  were co-distributed in 6 LGAs; Ado-Odo, Ipokia, Obafemi-Owode, Ogun waterside, Yewa North and Yewa South” as “ Triple infection comprising Ascaris, Trichuris and hookworm was recorded in 6 LGAs (table 3)”.Aggregated prevalence of Ascaris, Trichuris and Hookworm infections in Ogun StateThe sentences “The aggregated prevalence of 17.2% was recorded for at least one infection of Ascaris lumbricoides, Trichuris trichiura or hookworm. The prevalence range was between 5.3 –49.2% across the LGAs . Infections were highest in Yewa south, lowest in Yewanorth and no infection was recorded in Ijebu-Ode. Of the 19 endemic LGAs, 9 had prevalencestatus ranging between 20.0%-49.2% .Specific prevalence estimates for Ascaris, Trichuris and Hookworm infections in OgunState.The contents under this section is too wordy. The authors should highlight significant findings in terms of the most frequently encountered parasite in each LGA, the range of other parasites and if there is significant differences in prevalence of infection across the communities.Aggregated and specific mean intensity estimates for Ascaris, Trichuris and Hookworminfections in Ogun State.The authors should express the intensity as eggs per gram (epg) of faeces i.e epg should be put in front of the mean intensity values in all cases.The authors stated under this section about the single, doubleand triple infections, however there was no dat presented to show the prevalences of this infection. The table should be provided.Discussion.The authors should use simple grammar to discuss their findings.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentComments on the reviewed MS.docxSubmitted filename: Click here for additional data file. 2 Apr 2020PONE-D-20-06434Spatial Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria.PLOS ONEACADEMIC EDITOR: The study results provided some useful information, the spatial distributions of STH could be evaluated in a better/clearer way. I suggest the author modify the spatial analysis and map presentations and re-submit the revised version. Attached, 2 independent reviewers have some suggestions for your consideration.Response: Thank you very much for this observation. We will like to inform you that the objective of this present study is to provide epidemiological data on STH morbidity in the state, using household data, rather than school-based data. These data are useful for assessing the impact of school-based STH control on community prevalence. Households data were grouped into community data, and then aggregated at LGA level. The LGA is the implementation unit for school-based preventive chemotherapy programme in the state. Spatial analysis of the household data are still under analysis, and it not part of this report. As such, not to mislead the reviewers, we have removed the word spatial from the title to reflect the current analysis.Reviewer 1:Reviewer #1: The manuscript entitled “Spatial Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria” investigated STH infections in Ogun State and try to mapped the spatial distribution of STH infection. Though the study results provided some useful information, the spatial distributions of STH could be evaluated in a better/clearer way. I suggest the author modify the spatial analysis and map presentations and re-submit the revised version. Below are some comments:Major issues:C1. It seems that the georeferenced household’s information was not analyzed in the study because the figure only showed the locations of the selected communities. Since you have more than thousand georeferenced households participated in your study, why don’t you apply interpolation techniques (e.g. kriging or kernel density) to evaluate the prevalence and intensity?R1: Thank you very much for this observation. We will like to inform you that the objective of this present study is to provide epidemiological data on STH morbidity in the state, using household data, rather than school-based data. These data are useful for assessing the impact of school-based STH control on community prevalence. Households data were grouped into community data, and then aggregated at LGA level. The LGA is the implementation unit for school-based preventive chemotherapy programme in the state. Spatial analysis of the household data are still under analysis, and it not part of this report. As such, not to mislead the reviewers, we have removed the word spatial from the title to reflect the current analysis.C2. The range of different age groups are different , any reason for the grouping criteria? The age distributions could be biased.R2: We intended to understand the dynamics of STH morbidity that may exist among the different age classes, i.e. preschoolers (<5yrs), School-aged children (5-15years), 16-25years (post-primary), youth (26-40 years), 40-70 (Adults), and aged (>70 years). Only the age classes 5-15 years are under preventive chemotherapy in the State.C3. The investigation has been done at community level; however, the results were still presented at LGA level, and it’s hard to identify where is the most prevalent area. For instance, I have no idea where is “Yewa South” on the map . Please modify the map presentation in a better way.R3: Yes, the investigations were done at household level in the selected communities. However, results were presented both at the community level and LGA level. For instance, the maps created  show the spatial distribution of the parasites at the community level (i.e. the points on the map refers to communities)However, the map  shows the aggregated prevalence of STH infection by LGA. To avoid the presentation of cumbersome tables, results presented in tables were summarized at the LGA level. The LGA level is quite important as it remains the focal point for planning/delivery of MDA in the state. In addition, we realized that Yewa-north and Yewa south are not reflected in the maps. This is because these LGAs are also known as Egbado North and Egbado South respectively. We have thus adjusted our text to reflect Egbado North and Egbado South.C4. This is a cross-sectional survey which might be affected by many other factors. For example, does the infection prevalence influence by any intervention activities? Such limitation should be discussed in the manuscript.R4: Thank you very much. We have included this limitation in our discussion (Line 273-275)This study therefore adds to the wealth of existing epidemiological data on the prevalence and distribution of these infections in Ogun State, Nigeria. However, no attempt was made to analyse for risk factors.C5. Though the overall prevalence is not very high in the study area, the transmission still varied by locations. Can you explain why “Yewa South” has the highest prevalence?R5: The southern region of the state has been characterized to have favorable environmental conditions that allow STH parasites to thrive. This coupled with other socio-economic factors may have contributed to the high prevalence recorded. Minor issue:C6: The scale bar in Figure 1 is not correct. It should not be “millimeters.” Please check whether you apply a correct projection coordination system.R6: We have to removed Figure 1 since it is less informative. And more importantly we have four figures showing the study area already. Thank you.C7: Table 1 can be moved to the supplementary materials.R7: Thank you very much. We have moved it to supplementary materials as suggested.C8: Figure 5. What does “Ogun State” mean in the legend? For the legend indicated the LGA with no case, please add the border.R8: Thank you very much. We have removed Ogun State from the legend. We have also included the border for the LGA with zero prevalence. Reviewer 2 Comments:Reviewer #2: Specific commentsAbstractResult sectionC1: Line 2-4: The sentences “Ascariasis was the most spatially distributed found in 28 communities, with 140(13.6%) infected subjects, followed by Hookworm infection in 19 communities, with 47(4.6%) infected subjects and Trichuriasis in 9 communities, with 17(1.7%) infected subjects.” should be reconstructed to read “ Ascariasis was the most frequently observed parasite in 28 communities with a prevalence of 13.6%, followed by hookworm with a prevalence of 4.6% while Trichuris was the least encountered with a prevalence of 1.7%.R1: We have modified our text as suggested. Please find the improved text in between lines 45-48. Thanks C2: Line 6-7: The sentence is incomplete “The highest and lowest distribution of what (?) was recorded in Yewa South and Yewa North respectively.R2: We have improved our text.Line 49: The highest and lowest distribution of overall helminth infections was recorded in Egbado South and Egbado North respectivelyC3: Line 8: Delete the phrase “By WHO preventive chemotherapy thresholds”R3: The phrase has been deleted as suggested. Line 50-51: Nine regions had infection status between 20.0%-49.2%, while 10 regions had infection status between 5.3%-15.8%. ConclusionC4: This study provides information on the prevalence and spatial risk of Ascariasis, Trichuriasis and Hookworm infection that will serve as decision-support tool to help facilitate targeting of control interventions.The conclusion should be re-written as “ This study provides epidemiological data on the prevalence and spatial distribution of ascariasis , trichuriasis and hookworm infection which will add to the baseline data and guide the public health officers in providing appropriate control strategies in the endemic communities.R4: We have modified our text as suggested. Please find the improved text in between lines 53-56. ThanksLine 53-56: This study provides epidemiological data on the prevalence and spatial distribution of ascariasis , trichuriasis and hookworm infections which will add to the baseline data and guide the public health officers in providing appropriate control strategies in the endemic communities.BackgroundC5: Line 7-8: Replace the phrases in parentheses (children between age 2 and 5) and (children between age 5 and 14) with (age 2-5 years) and (age 5-14 years).R5: We have modified our text as suggested. Please find the improved text in between lines 66-67. ThanksLine 66-67: Besides, about 267 million pre-schoolers (age 2-5 years) and 568 million school-aged children (age 5-14 years) who live in areas where theseC6: Line 15: The sentence “there is an established morbidity control programme targeted at school-aged children due to their high level of contact with soils” should be re-written as “there is an established morbidity control programme targeted at school-aged children due to high worm burden as a result of their frequent contact with contaminated soils, ……………………….R6: We have modified our text as suggested. Please find the improved text in between lines 72-74. ThanksLines 72-74: Nonetheless, in most endemic settings, there is an established morbidity control programme targeted at school-aged children due to high worm burden as a result of their frequent contact with contaminated soilsMethodsStudy areaC7: Line 10: The phrase “The major occupation of the population is……………… should be re-phrased as “Major occupations of the population are………………R7: We have modified our text as suggested. Please find the improved text in between lines 96. ThanksLine 96: Major occupations of the population are farming, timber logging, and trading.Study design and sampling techniqueC8: Line 1: The sentence “This study was community-based and cross-sectional in design” should be re-written as “This is a cross-sectional and community-based study”.R8: We have modified our text as suggested. Please find the improved text in between lines 101-102. ThanksLine 101-102: This is a cross-sectional and community-based study, conducted between July 2016 and November 2018.Selection of households for surveyC9: Line 2: The sentence “communities were visited and sensitized about the study procedures and were openly invited to participate in the research through community meetings and town announcers” should be replaced with “selected communities were visited and with the permission of the community leaders, meetings were held with the members of the community to intimate them about the purpose of the study and the procedures to be adopted”.R9: We have modified our text as suggested. Please find the improved text in between lines 113-115. Thank you so much.Line 113-115: Prior to data collection, selected communities were visited and with the permission of the community leaders, meetings were held with the members of the community to intimate them about the purpose of the study and the procedures to be adopted.C10: The sub title “Collection of faecal samples and parasitological diagnosis” be replaced with “ Faecal samples collections and processing”.R10: We have modified our text as suggested. Please find the improved text in line 122. Thank youLine 122: Faecal samples collections and processing C11: Line 3-4: The sentence “All participants were asked to provide a sufficiently large stool sample (at least 5 g)” should be replaced with “Individuals who consented to take part in the study were requested to provide stool up to half (about 5gm) into the labelled plastic bottles provided.R11: We have modified our text as suggested. Please find the improved text in between lines 125-127. Thank you so much.Lines 125-127: Individuals who consented to take part in the study were requested to provide stool up to half (about 5gm) into the labelled plastic bottles provided.C12: Line 6: Delete the word “Precisely”.R12: Deleted as suggested. Please check Line 129C13: Line 7-8: The sentence “The bottle was covered and agitated vigorously to suspend the stool efficiently in the solution” be replaced with “The bottle was covered and shaken vigorously to form a cloudy suspension”.R13: We have modified our text as suggested. Please find the improved text in between lines 130-131. Thank you so much.Line 130-131: The bottle was covered and shaken vigorously to form a cloudy suspension. The stool suspension……C14: Line 9: Delete the word “further”R14: Deleted as suggested.Line 131: The stool suspension was strained into…..C15: Line 11: The sentence “The supernatant was also discarded after centrifuging” be replaced with “The supernatant was decanted”.R15: We have modified our text as suggested. Please find the improved text in line 133. Thank youLine 133: The supernatant was decantedC16: Line 12-14: The sentence “7 ml of normal saline was later added to the sediment, after which 3 ml of ether was finally added to the suspension”. . The deposit or residue obtained after decanting the supernatant should be resuspended in distilled water or saline of a known vol and shaking vigorously. Thereafter, an equal vol of ether will then be added . The authors should correct this procedure.R16: Thank you for this explanation. However, we followed the procedure describe by Endriss et al. 2005. We have included the reference immediately after the text. ResultsSex and age distribution of study participantsC17: The sentence “A total of 1,499 respondents were subsequently enrolled after consent across the 33 communities and 20 LGAs. However, only 1,027 (68.5%) of them consented to the provision of adequate stool samples (~5g) for laboratory analysis for parasites’ ova or larva. By demography, 899(60.0%) were females and 600(40.0%) were males” should be rewritten as “ A total of 1499 participants comprising of 899 (60%) females and 600 (40%) males were enrolled across the 33 communities in 20LGAs out of which 1024 (68.5%) returned stool samples for laboratory analysis.R17: We have modified our text as suggested. Please find the improved text in between lines 161-163. Thank you so much.Line 161-163: A total of 1499 participants comprising of 899 (60%) females and 600 (40%) males were enrolled across the 33 communities in 20LGAs out of which 1024 (68.5%) returned stool samples for laboratory analysis.C18: The sentence “The sex ratio of females to males was 3:2. In addition, the highest number of respondents were within the age range 41-70yrs (40.6%), followed by 26-40yrs (30.3%), 5-15yrs (10.7%), 16-25yrs (10.4%) and >70yrs (7.0%) (Table 2)” should be re-written as “The ages of the enrolled participants are provided in table 2.R18: We have modified our text as suggested. Please find the improved text in between lines 130-131. Thank you so much.Line 163-164: The ages of the enrolled participants are provided in table 1.C19-24: Co-distribution patterns of Ascaris, Trichuris and Hookworm infections in Ogun StateC19: Line1: Replace the words “mono infection” with single infectionC20: Line 2: Delete the word “only”C21: Line 3: Replace the word “co-distribution” with the word “combination”C22: Line 5: Begin with combination of Ascaris and Trichuris and delete the word “co-distribution”C23: Line 6 & 7: Rewrite “while Trichuris and Hookworm co-distribution was recorded in 6 LGAs;” as “while combination of Trichuris and hookworm was……………………….”C24: Line 8-10: Rewrite “However, for triple infections, the three species  were co-distributed in 6 LGAs; Ado-Odo, Ipokia, Obafemi-Owode, Ogun waterside, Yewa North and Yewa South” as “ Triple infection comprising Ascaris, Trichuris and hookworm was recorded in 6 LGAs (table 3)”.R19-24: We have considered all the corrections (C19-24) and modified our text accordingly to reflect them. Please find the improved text in between lines 173-182. Thank you so muchLine 173-182:Co-distribution patterns of Ascaris, Trichuris and Hookworm infections in Ogun StateFigure 3 shows the co-distribution patterns of STH infections in the State. Single infection was recorded for Ascaris lumbricoides in 3 LGAs; Ewekoro, Ikenne and Ijebu northeast. For double infections, combination of Ascaris and Hookworm were the most predominant, observed in 15 LGAs except Ewekoro, Ijebu east, Ijebu northeast, Ikenne and Ijebu ode. Combination of Ascaris and Trichuris co-distribution was recorded in 7 LGAs; Yewa north, Yewa south, Ado-odo ota, Ijebu east, Ipokia, Obafemi owode and Ogun waterside, while combination of Trichuris and hookworm was recorded in 6 LGAs; Ado-odo ota, Ipokia, Obafemi owode, Ogun waterside, Yewa north and Yewa south. Triple infections comprising Ascaris, Trichuris and hookworm was recorded in 6 LGAs (Table 3). Aggregated prevalence of Ascaris, Trichuris and Hookworm infections in Ogun StateC25: The sentences “The aggregated prevalence of 17.2% was recorded for at least one infection of Ascaris lumbricoides, Trichuris trichiura or hookworm. The prevalence range was between 5.3 – 49.2% across the LGAs . Infections were highest in Yewa south, lowest in Yewa north and no infection was recorded in Ijebu-Ode. Of the 19 endemic LGAs, 9 had prevalencestatus ranging between 20.0%-49.2% . C26: Delete this sentence “However, 10 LGAs haprevalence status ranging between 5.3%-15.8% which is below the WHO recommended thresholds for preventive chemotherapy .R26: We have considered the comments raised. We believe deleting this sentence will mask the pattern we are trying to showcase. Therefore we have modified our text accordingly to reflect them. Please find the improved text in between lines 188-191.Of the 19 endemic LGAs, 9 had prevalence status ranging between 20.0%-49.2% and 10 LGAs had prevalence status ranging between 5.3%-15.8%. There were significant differences in the prevalence record for any STH species across the 19 endemic LGAs (P=0.000) .Specific prevalence estimates for Ascaris, Trichuris and Hookworm infections in Ogun State.C27: The contents under this section is too wordy. The authors should highlight significant findings in terms of the most frequently encountered parasite in each LGA, the range of other parasites and if there is significant differences in prevalence of infection across the communities.R27: We have considered the suggestions raised and modified our text accordingly to reflect them. Please find the improved text in between lines 197-205Specific prevalence estimates for Ascaris, Trichuris and Hookworm infections in Ogun State.By species’ prevalence, an overall prevalence of 13.6% was recorded for Ascaris lumbricoides, followed by hookworm with 4.6% and Trichuris trichiura with 1.7% (Table 3). The lowest prevalence for the three infections were observed in Yewa north., while Abeokuta south, Obafemi owode and Ipokia recorded the highest prevalence for ascariasis, hookworm infections and trichuriasis respectively (Table 3). Spatial-wise, Ascaris lumbricoides was the most predominant, found in 28(84.8%) communities , followed by hookworm in 19 (57.6%) communities  and Trichuris trichiura in 9(27.3%) communities. There were significant differences in the prevalence record for each parasitic infection across the endemic LGAs (P=0.000). .Aggregated and specific mean intensity estimates for Ascaris, Trichuris and Hookworm infections in Ogun State.C28: The authors should express the intensity as eggs per gram (epg) of faeces i.e epg should be put in front of the mean intensity values in all cases.R28: We have considered the suggestions raised and modified our text accordingly to reflect them. Please find the improved text in between lines 221-234Line 221-234Aggregated and specific mean intensity estimates for Ascaris, Trichuris and Hookworm infections in Ogun State.Table 4 shows the intensity estimates for Ascaris, Trichuris and hookworm infections in Ogun state. The aggregated geometric mean intensity of infections was 0.14±0.01epg with mean intensity ranging from 0.03±0.01epg to 0.43±0.06epg across the LGAs. The aggregated intensity shows that worm loads were highest in Obafemi owode and lowest in Imeko-afon. By species intensities, Ascaris lumbricoides infection intensity was the highest with, 0.11±0.01epg, followed by hookworm with 0.03±0.01epg and Trichuris trichiura with 0.01±0.00epg. Ascaris mean intensities range between 0.01±0.01epg and 0.32±0.05epg, with the lowest in Imeko afon and highest in Ifo and Yewa south. For Hookworm infection, intensities range between 0.01±0.00epg and 0.13±0.04epg, with the lowest in Ijebu north, Yewa north, Yewa south, Ogun waterside, Imeko afon and highest in Obafemi-owode. Trichuris mean intensities range between 0.01±0.01epg and 0.10±0.05epg with the highest load in Ipokia and the lowest in Ogun waterside, Ijebu east and Yewa north. C31: The authors stated under this section about the single, double and triple infections, however there was no dat presented to show the prevalences of this infection. The table should be provided.R31: A venn diagram was used to present the number of LGAs where single, double and triple infections could be found. Please see figure 3. We didn’t report specific combination prevalences in our text to reduce the number of tables.Discussion.C32: The authors should use simple grammar to discuss their findings.R32: We have re-worked our discussion to enhance clarity. Please check Line 273-286AttachmentResponse to Reviewers_2.docxSubmitted filename: Click here for additional data file. 27 Apr 2020PONE-D-20-06434R1Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria.PLOS ONEDear Dr Mogaji,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.ACADEMIC EDITOR: Please revise the ms according the reviewer's suggestions to polish your ms.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Jun 11 2020 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Chia Kwung Fan, LL.M, PhDAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: Comments on the revised version of the MSGeneral comments: The authors revised the MS in line with the suggestions provided, however, there are still some minor corrections that needs to be effected in the discussion section.Specific commentsDiscussionLine 283-284: The prevalence estimates also corroborates ………………………………….due to chemotherapy programmes in SSA countries (The statement is not clear). However, I would suggest that the sentence should be re-written to read “The prevalence values for the 3STHs recorded in this study is comparable ………………………………. .Line 287: Replace the words “Sub-state level” with LGAs and communitiesLine 292: Replace the following words “reported” with “recorded”; “corroborates” with “comparable”; “recent” with “previous”.Line 293-295: The sentence “Ascaris lumbricoides infections were observed in …………………………………..by Oluwole et al. (12) should be recast to read “The increase in prevalence of A. lumbricoides infection as observed in 19 of the 20 LGAs studied is similar to the findings of Oluwole et al (12) with respect to spatial pattern of A. lumbricoides. This may be due to similar ecological factors (mention the factors) which favours the transmission of STHs in the areas of study”.Line 296-299: Please delete the sentences “where moderate……………………………………… of STH parasites in the state”.Line 300-302: The sentences “However, none of the LGAs studied has……………………. Population in the report of Oluwole et al. (12)” should be recast to read “The moderate prevalence of less than 20% recorded in various communities in contrast with prevalence of more than 50% reported by Oluwole et al. (12) may be due to the difference in the composition of population studied.Line 313: Please delete the word “aggregate”Line 316: Your assertion that hookworms egg can withstand extreme temp (I guess you meant extreme heat). Remember hookworm eggs have thin shells compared with thick shell of Ascaris eggs. It is only Ascaris eggs which can withstand adverse environmental conditions.Line 317: Please replace the word ‘commonalities” with the word “similarities”Line 326: replace the word “observed” with “reported”Line 332: Replace the word “study” with “studied”Table 3: The title should be rephrased to read “Intensity (Mean ± SEM) of Ascaris, hookworm and Trichuris infection in selected population in Ogun State”.In table 3: Re-write the abbreviation “SE” as “SEM”. Define SEM as Standard Error of MeanTable 4: The title should be rephrased to read "Prevalence of STH infections in relation to sex and age in selected populationStill in Table 4: Under Age group insert the word "Years" and delete the word "Years" from each age group.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  28 Apr 2020Reviewer #2: Comments on the revised version of the MSGeneral comments: The authors revised the MS in line with the suggestions provided, however, there are still some minor corrections that needs to be effected in the discussion section.Specific commentsDiscussionC1: Line 283-284: The prevalence estimates also corroborates ………………………………….due to chemotherapy programmes in SSA countries (The statement is not clear). However, I would suggest that the sentence should be re-written to read “The prevalence values for the 3STHs recorded in this study is comparable ………………………………. .R1: The corrections have been made as suggested. We have compared our prevalence values with three reports  Please find our improved text between Line 283-287The prevalence values of the three soil transmitted helminth (STH) infections recorded in this study is comparable with the findings of Mogaji et al. [2], Oluwole et al. [12] and Gemechu et al. [14]. The similarities observed supports existing presumptions on decreasing trend of STH infections due to on-going chemotherapy programmes in most endemic SSA countries .C2: Line 287: Replace the words “Sub-state level” with LGAs and communitiesR2: This correction has been made (Line 289). Thank you C3: Line 292: Replace the following words “reported” with “recorded”; “corroborates” with “comparable”; “recent” with “previous”.R3: This correction has been made.Line 293-294: The prevalence recorded for Ascaris lumbricoides infections in this study is comparable with findings from previous epidemiological surveys .C4: Line 293-295: The sentence “Ascaris lumbricoides infections were observed in …………………………………..by Oluwole et al. (12) should be recast to read “The increase in prevalence of A. lumbricoides infection as observed in 19 of the 20 LGAs studied is similar to the findings of Oluwole et al (12) with respect to spatial pattern of A. lumbricoides. This may be due to similar ecological factors (mention the factors) which favours the transmission of STHs in the areas of study”.R4: The corrections have been made as suggested (Line 294-298)The increase in prevalence of A. lumbricoides infection as observed in 19 of the 20 LGAs studied is similar to the findings of Oluwole et al [12] with respect to spatial pattern of A. lumbricoides. This may be due to similar ecological factors such as soil moisture, pH or temperature, which favours the transmission of STHs in the areas of study.C5: Line 296-299: Please delete the sentences “where moderate……………………………………… of STH parasites in the state”.R5: The sentences have been deleted. Thank youC6: Line 300-302: The sentences “However, none of the LGAs studied has……………………. Population in the report of Oluwole et al. (12)” should be recast to read “The moderate prevalence of less than 20% recorded in various communities in contrast with prevalence of more than 50% reported by Oluwole et al. (12) may be due to the difference in the composition of population studied.R6: The corrections have been made as suggested (Line 298-300)The moderate prevalence of less than 20% recorded in various communities in contrast with prevalence of more than 50% reported by Oluwole et al. [12] may be due to the difference in the composition of population studied.C7: Line 313: Please delete the word “aggregate”R7: The word has been deleted. Thank youC8: Line 316: Your assertion that hookworms egg can withstand extreme temp (I guess you meant extreme heat). Remember hookworm eggs have thin shells compared with thick shell of Ascaris eggs. It is only Ascaris eggs which can withstand adverse environmental conditions.R8: Thank you for this comment. We have removed the line entirely to improve on clarity C9: Line 317: Please replace the word ‘commonalities” with the word “similarities”R9: The corrections have been made as suggested (Line 313)C10: Line 326: replace the word “observed” with “reported”R10: The corrections have been made as suggested (Line 317)C11: Line 332: Replace the word “study” with “studied”R11: The corrections have been made as suggestedC12: Table 3: The title should be rephrased to read “Intensity (Mean ± SEM) of Ascaris, hookworm and Trichuris infection in selected population in Ogun State”.R12: The corrections have been made as suggested (Line 253)C13: In table 3: Re-write the abbreviation “SE” as “SEM”. Define SEM as Standard Error of MeanR13: The corrections have been made as suggested (Line 253-256)C14: Table 4: The title should be rephrased to read "Prevalence of STH infections in relation to sex and age in selected populationR14: The corrections have been made as suggested (Line 264-266)C15: Still in Table 4: Under Age group insert the word "Years" and delete the word "Years" from each age group.R15: The corrections have been made as suggested (Line 264-266)AttachmentResponse to Reviewers comments.docxSubmitted filename: Click here for additional data file. 6 May 2020Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria.PONE-D-20-06434R2Dear Dr. Mogaji,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Chia Kwung Fan, LL.M, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 13 May 2020PONE-D-20-06434R2 Distribution of Ascariasis, Trichuriasis and Hookworm Infections in Ogun State, Southwestern Nigeria. Dear Dr. Mogaji:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Chia Kwung Fan Academic EditorPLOS ONE