Diagnostic value of the molecular detection of Sarcoptes scabiei from a skin scraping in patients with suspected scabies.

Scabies is a highly contagious parasitic disease associated with long-term residence in nursing homes, and it is a public health burden worldwide. However, atypical skin manifestations are frequent and the widely used diagnostic test based on microscopic examinations has limited sensitivity. We evaluated the diagnostic value of polymerase chain reaction (PCR) from skin scraping in patients with suspected scabies. Adult patients with suspected scabies, unrelated diseases or healthy volunteers were enrolled at a tertiary hospital, in Seoul, South Korea, from December 2017 through October 2018. We classified participants based on the consensus criteria established by the International Alliance for the Control of Scabies in 2018; confirmed (microscopic mite detection), clinical (scabies burrow or typical lesions with two history features including itch and close contact with scabies patients), suspected scabies (typical lesion with one history feature or atypical lesion with two history features), or no scabies. PCR was performed on the skin scrapings to target the cytochrome c oxidase subunit 1 (cox1) gene of Sarcoptes scabiei. A total of 47 participants, 33 with suspected scabies, 10 with unrelated diseases, and 4 healthy volunteers were enrolled. Of the 33 patients, 22 were classified as confirmed scabies, 2 as clinical scabies, 6 as suspected scabies, and 3 as no scabies. The sensitivities of the microscopic examination were 100%, 92%, and 73% in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.006). The sensitivities of PCR were 86%, 83%, and 80% in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.59). The specificity of the scabies PCR in the no scabies control was 100% (95% CI = 80-100).PCR testing for scabies may be helpful in the improvement of sensitivity for the diagnosis of scabies by clinical criteria.

Introduction

Scabies caused by Sarcoptes scabiei mites, is a highly contagious parasitic disease characterized by intense itching which is aggravated at night. Infections by scabies mite result in various skin eruptions consisting of papules, nodules, vesicles, and excoriated eczematous lesions due to scratching. The skin lesions involve intertriginous areas including the finger webs, the wrists, axillae, buttocks, genitals, and the breasts (in females only) [1]. The clinical features of scabies in the elderly may differ from those in younger individuals [2]. Transmission of scabies occurs predominantly through close and prolonged contact. Nosocomial outbreaks are usually associated with long-term residence in nursing homes and represent a tremendously increasing problem in high-income countries [3]. One of the most important risk factors for nosocomial outbreaks is the failure to recognize scabies in patients by attending clinicians. The crusted scabies has particularly atypical presentation making it difficult to diagnose and high risk of transmission, therefore it has been the index case in most recent outbreaks [4]. The misdiagnosis of scabies is common due to its multifarious manifestation and the absence of available and appropriate diagnostic laboratory tests [5]. Recently, clinical consensus criteria have been proposed by the International Alliance for the Control of Scabies (IACS) to overcome the low sensitivities of conventional diagnostic tests for scabies [6]. However, few studies have evaluated the diagnostic performance of these tests.

Microscopic examination of skin scrapings is widely used for the diagnosis of scabies, but it has a suboptimal sensitivity of only 50% [7]. Dermoscopy has been widely used, but it has a disadvantage in that it has low specificity and is affected by the dermatologist’s experience [7]. More sensitive and specific diagnostic tests are urgently required. Polymerase chain reaction (PCR) has been studied for the diagnosis of scabies, offering higher sensitivity than conventional microscopy [8-12]. We developed a novel real-time PCR assay for cytochrome c oxidase subunit 1 (cox1) gene, which is relatively conserved and has no known homology with other common human ectoparasites. We evaluated the diagnostic capability of this in-house real-time PCR for the diagnosis of scabies from skin scraping in subjects with suspected scabies and with unrelated disease according to the previously used criteria of the IACS.

Method

Patients and clinical diagnosis of scabies

Adult patients with suspected scabies or other unrelated diseases, such as onychomycosis, or control healthy volunteers were enrolled between December 2017 and October 2018. This prospective cross-sectional study was performed at a 2700-bed tertiary-care teaching hospital in Seoul, Republic of Korea. As a first step, an experienced dermatologist delineated clinically suspicious lesions. Skin scrapings for patients with suspected scabies were obtained from clinically suspected lesions by an experienced nurse specialist for microscopic examination and scabies PCR simultaneously [13]. The specimens were collected at 10~15 sites for all delineated lesions. Skin scrapings for healthy volunteers were obtained from extensor sites of extremities, which were appropriate places in getting keratinous layer without skin lesions. Decision regarding the appropriate anti-scabies treatment was made based on the clinical features and microscopic results and the blinded results of the scabies PCR by the attending dermatologist. Only the results of scabies PCR and microscopic examination for the specimens obtained on the first day of diagnosis with scabies were included in the analysis.

We classified participants based on the consensus criteria established by the IACS in 2018 [6]: confirmed (mites, eggs, or feces on microscopic examination of skin scrapings); clinical (scabies burrow, typical lesions affecting the male genitalia, or lesions in a typical distribution with two history features including itch and close contact with an individual who had clinical scabies); suspected scabies (typical lesion with one history feature or atypical lesion with two history features); or no scabies. Imaging devices such as dermoscopes were not used. Typical lesions were defined as multiple small papules, nodules, vesicles, or excoriation, and a typical distribution of lesions was defined on observation of the lesions on the finger-webs, wrists, hands, axillae, gluteus, genitals, or breasts in the case of female patients [1].

DNA extraction and real-time PCR

Samples obtained via skin scraping were collected into and stored at −80°C until PCR analysis was performed. DNA extraction was performed using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions, with some modifications. In brief, skin samples were placed in a sterile microcentrifuge tube and digested with 40 μL of 1 M dithiothreitol (DTT, Sigma-Aldrich, Inc, St. Louis, Missouri, USA), 300 μL of ATL buffer, and 40 μL of proteinase K (Qiagen) at 56°C for 2 h. Lysates were incubated with 380 μL of AL buffer (Qiagen) at 70°C for 10 min. After incubation, 380 μL of 100% ethanol (Sigma-Aldrich) were added and further extraction and purification procedures were followed according to the manufacturer’s protocols. Purified DNA was eluted in 100 μL of AE buffer (Qiagen) and used for PCR amplification.

To detect scabies, primers and probe were designed for the cox1 gene of Sarcoptes scabiei (GenBank accession number: KX827306.1). A 196 fragment of the highly conserved region was amplified using the specific primers cox1F (5’- ATGATTTCTATTGCAACTTTAGG-3’) and cox1R (5’- TTGCTCAATACATAGAGGGGTTA-3’). Taqman probe cox1P2 (5’-FAM- AATATTAGGGGGAAAATTAGATTTTAACCC-BHQ1-3’) was used for real-time detection. The specificity of each primer and probe was checked using BLAST search against the NCBI database, and no homologies between the designed primers and probe for S. scabiei and other mite species such as Demodex sp., Dermatophagoides sp., Cheyletus sp., and Thyrophagus puterescentiae were found. The internal control was amplified and detected using ACBT_F (5’- ACTAACACTGGCTCGTGTGA-3’), ACBT_R (5’- CTTGGGATGGGGAGTCTGTT-3’), and ACBT_P (5’-HEX- AGGCTGGTGTAAAGCGGCCTTGG-BHQ1-3’).

Multiplex real-time PCR assays to detect S. scabiei were performed using the LightCycler FastStart Essential DNA Probe Master (Roche, Basel, Switzerland) in 20 μL of reaction mixtures containing 10 μL of 2X master mix, 3.1 μL of primer and probe mix consisting of 500 nM cox1 primers, 150 nM cox1 probe, 150 nM ACBT primers and 100 nM ACBT probe, and 5 μL of extracted DNA. Real-time PCR amplification was performed with the LightCycler 96 system (Roche) in the following conditions: pre-incubation at 95°C for 5 m, followed by 45 cycles of a 2-step amplification (95°C for 5 s and 56°C for 30 s). Each assay included a positive and a negative control. The PCR product was electrophoresed and purified using the QIAquick gel extraction kit (Qiagen) (Fig 1). The purified PCR product was sequenced and the DNA sequences confirmed their identity through a BLASTn search in NCBI.

Fig 1

Detection of Sarcoptes scabiei DNA by polymerase chain reaction (PCR).

DNA was purified from skin scraping samples from suspected scabies patients (lanes 1–5), and run by electrophoresis with a positive control (lane 6), and a negative control (lane 7). Lane M is a 100-bp DNA ladder marker.

Generation of calibration curves and confirmation of specificity

PCR products identified as the cox1 gene of S. scabiei were inserted into a plasmid vector (T-Blunt PCR cloning kit, Solgent, Daejeon, Korea) and used as standards for quantification and positive controls. The standard plasmids were quantified with a Nanodrop spectrophotometer (Thermo Scientific) and 7 serial 10-fold dilutions of the plasmid, ranging from 106 to 100 copies, were amplified using multiple real-time PCR. The correlation curves were determined by plotting the Ct values against the log of the copy number (Fig 2). The analytical sensitivity of the real-time PCR assay was one copy per reaction.

Fig 2

Correlation curves for real-time PCR detection of scabies.

(A) Amplification curves and (B) correlation curves of standard DNA ranging from 106 to 100 copies.

We further evaluated the potential risks of cross-reaction with this PCR. As a negative control, DNA extracted from ground bodies of house dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was also used to test the analytic specificity of the novel PCR. All DNA samples from specimens with Dermatophagoides pteronyssinus and Dermatophagoides farinae were negative for the PCR using the cox1 gene primer and probe set.

Statistical analysis

To investigate the diagnostic value of the PCR testing, we calculated the 95% confidence interval (CI) of the sensitivity and specificity of both PCR and microscopy assays for each clinical category according to the level of diagnostic certainty: confirmed; confirmed and clinical; and confirmed, clinical, and suspected scabies. The sensitivity of the scabies PCR was compared with those of microscopic examination in each clinical category group. Data are expressed as percent and Clopper-Pearson exact 95% CI. The linear-by-linear association test was used to examine the linear association between the level of diagnostic certainty and the sensitivity of the microscopic examination or the scabies PCR test. The software used for statistical analyses was SPSS v. 24.0 (IBM, Armonk, New York, USA).

Ethics statement

Written informed consent was obtained from all patients. The study’s approval was obtained from the Institutional Review Board of the Asan Medical Center (2018–0640).

Results

A total of 47 participants including 33 patients with suspected scabies and 14 participants with an unrelated disease (10 patients with onychomycosis and 4 healthy volunteers) were enrolled in this study. Of the 33 patients with suspected scabies, 22 were classified as confirmed scabies, 2 as clinical scabies, 6 as suspected scabies, and 3 as no scabies (2 as drug eruption, 1 as another superficial fungal infection). A total of 17 subjects including 3 with no scabies, 10 with onychomycosis, and 4 healthy volunteers were classified as no scabies control.

The median age of the patients at the time of diagnosis in the 30 scabies patients was 60±17 years (95% CI = 54–67) and 57% were male. Thirteen of these patients (43%) had a history of hospitalizations and 10 (33%) had a history of hospital visits associated with family or friends. Seven patients (23%) were bedridden resulting from other debilitating medical conditions and 1 patient (3%) did not bring the itching symptoms to the physician’s attention due to other previously diagnosed neurological diseases. Overall, the median time from symptoms onset to diagnosis of scabies was 77 days (95% CI = 47–107). The most common skin manifestation was papules in 23 patients (77%), followed by nodules in 2 (7%) and excoriated patches in 2 (7%). There was no case reported of crusted scabies. The torso was the most common location in 27 patients (90%), followed by the extremities in 22 (73%), and the genitalia in 7 (23%) of the patients. Approximately 43% of patients with scabies had the skin lesion in the hands (S1 Fig).

The diagnostic performances of PCR and microscopy are shown in Table 1. The sensitivity of the microscopic examination was 100% (95% CI = 85–100; 22/22), 92% (95% CI = 73–99; 22/24), and 73% (95% CI = 54–88; 22/30) in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.006). As the level of diagnostic certainty decreased, the sensitivity of microscopy decreased. The microscopic examination specificity was 100% (95% CI = 80–100; 17/17). The sensitivity of the scabies PCR test was 86% (95% CI = 65–97; 19/22), 83% (95% CI = 63–95; 20/24), and 80% (95% CI = 61–92; 24/30) in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.59). As the level of diagnostic certainty decreased, the sensitivity of the scabies PCR also decreased, but there was no statistically significant difference between the groups. The sensitivity of scabies PCR test was slightly higher than that of the microscopic examination but there was no statistically significant difference between these tests in patients with confirmed, clinical, and suspected scabies (80 vs 73%, p = 0.54). Of the 30 patients with scabies, 5 (17%) revealed negative results for microscopic examination, but positive results for scabies PCR. They were composed of 1 patient with clinical scabies and 4 patients with suspected scabies. On the other hand, 3 patients (10%) exhibited positive results for microscopic examination, but negative results for scabies PCR. Skin scraping specimens from 10 patients with onychomycosis, 4 healthy donors without any other dermatologic condition, and 3 classified as no scabies by the IACS criteria were all negative for the PCR test. Thus, the specificity of the scabies PCR in no scabies controls was of 100% (95% CI = 80–100; 17/17). There was no statistically significant difference in the PCR sensitivity according to gender, age, location of skin lesion, and type of skin lesion.

Table 1

Comparison of diagnostic performance of the scabies PCR and microscopy in confirmed, clinical, and suspected scabies patients.

		Confirmed scabies(n = 22)	Confirmed + clinicalscabies (n = 24)	Confirmed + clinical+ suspected scabies(n = 30)	No scabies(n = 17)a
PCR	Positive (n)	19	20	24	0
	Negative (n)	3	4	6	17
	Sensitivity (%, 95% CI)b	86 (65–97)d	83 (63–95)e	80 (61–92)f
	Specificity (%, 95% CI)				100 (80–100)
Microscopy	Positive (n)	22	22	22	0
	Negative (n)	0	2	8	17
	Sensitivity (%, 95% CI)c	100 (85–100)d	92 (73–99)e	73 (54–88)f
	Specificity (%, 95% CI)				100 (80–100)

a“No scabies” included 13 skin scrap specimens from patients with alternative diagnosis and four skin scrap specimens from healthy volunteers.

bDifference in the sensitivity of the PCR test between the three groups was not statistically significant (p = 0.59).

cDifference in the sensitivity of the microscopic examination between the three groups was statistically significant (p = 0.006).

dDifference in the sensitivity between PCR and the microscopic examination in the patients with confirmed scabies was not statistically significant (p = 0.23).

eDifference in the sensitivity between PCR and the microscopic examination in the patients with confirmed and clinical scabies was not statistically significant (p = 0.67).

fDifference in the sensitivity between PCR and the microscopic examination in the patients with confirmed, clinical, and suspected scabies was not statistically significant (p = 0.54).

Discussion

According to previous reports, the sensitivity of the scabies PCR ranged between 30% and 60% based on clinically suspected scabies [8-12]. However, there was no appropriate reference standard test, thus microscopic examination used as a reference standard in these studies, which limits the diagnostic usefulness of the scabies PCR test. To overcome this limitation, we applied the consensus criteria for the diagnosis of scabies developed by the IACS [6] in our study. We categorized each group according to the level of diagnostic certainty and compared the sensitivity of the microscopic examination and the scabies PCR test for each group. Consequently, we found that the sensitivity of the microscopic examination and the scabies PCR test tended to decrease as the level of diagnostic certainty decreased, although the decreasing sensitivity of the microscopic examination was more prominent than that of the scabies PCR. Possible explanations for this observation are that patients with clinical or suspected scabies by the IACS criteria did not have scabies or revealed false-negative results for the microscopic examination due to its low sensitivity. However, given the high specificity of the scabies PCR in our study and previous consistent reports on the low sensitivity of the microscopic examination, the scabies PCR was considered to have an ability to identify cases that could not be confirmed by microscopic examination. Our findings support this hypothesis and showed that the scabies PCR test detected five additional patients (17%) that were not initially diagnosed by microscopic examination. However, in three patients, the microscopic examination results were positive and the scabies PCR results were negative. This discrepancy could have been due to the genetic diversity of S. scabiei. In a recent study, it was suggested that S. scabiei mites in humans were mainly distributed into three genetically distinct clades [14]. The authors performed sequencing of cox1 gene obtained in mites from 60 humans and showed that mites could belong to different clades genetically even if they were collected in the same area. Taken together, despite no significant difference, the detection rate of scabies by PCR tended to be higher than that of the microscopy examination, suggesting the high sensitivity of scabies PCR.

In this study, the topographic distribution of skin lesions in patients with scabies presented similarly in a recent study [2]. The torso was the most common location involving scabies. The proportion of hand (43%) and genitalia (23%) was relatively higher than that in the recent study (36% and 7%) [2]. The possible explanation for this observation might be that this study mainly included symptomatic patients in the non-outbreak situation, unlike the recent study based on the nosocomial outbreaks [2]. It is worth to note that there are limited data on whether the sensitivity of the microscopic examination depends on the location of skin lesions. We found that there was no statistically significant difference in the scabies PCR sensitivity according to the locations of skin lesions. However, further studies are needed on this area.

There are some potential limitations to our study. First, the consensus criteria for the diagnosis of scabies [6] was still reliant upon expert opinion. Further studies will be required to understand if these criteria can be used to evaluate the clinical potential of new diagnostic tests. Second, skin scraping for scabies PCR was required and depended on the expertise of an experienced specialist for obtaining good-quality skin samples. Further studies are needed on the diagnostic performance of various tests for scabies depending on the expertise of skin scraping. Third, this study included a small number of patients, which potentially accounts for the lack of difference in the sensitivity between the microscopy examination and the scabies PCR test.

In conclusion, scabies PCR was shown to offer an improvement in assay sensitivity compared to that of microscopy examination for the diagnosis of scabies by clinical criteria. This technique can, therefore, be considered as an adjunct method for the diagnosis of scabies, particularly in microscopy-negative suspected cases. Further larger-scale studies will be needed to evaluate the diagnostic performance of the scabies PCR and to validate the new IACS criteria by using more sensitive diagnostic tests.

Distribution of skin lesions.

Percentage in the specific locations referred to the proportion of patients with scabies who presented skin lesion at that location.

(TIF)

Click here for additional data file.

11 Jan 2020

Dear Dr. Kim,

Thank you very much for submitting your manuscript "Diagnostic value of the molecular detection of Sarcoptes scabiei from a skin scraping in patients with suspected scabies" (#PNTD-D-19-01821) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

When you are ready to resubmit, please be prepared to upload the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt.

(5) To enhance the reproducibility of your results, we recommend that 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. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

We hope to receive your revised manuscript by Mar 11 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there.

Sincerely,

Kosta Y. Mumcuoglu, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Paul J. Brindley, PhD

Editor-in-Chief

PLOS Neglected Tropical Diseases

***********************

Line 36: Do not use abbreviations such as "exam"

Line 38: Place a full stop at the end of the sentence

Line 62: scabiei rather than scabies

Line 63-65: The sum of the patients mentioned is not 43 (see also lines 195-197)

Line 83: Mention also the crusted scabies

LIne 90: Mention also the dermoscope

Line 232: Mention the number as 13.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Please add no and date of ethical approval

Reviewer #2: The study design was appropriate to address the stated objectives, with the novel use of IACS consensus criteria as ‘gold standard’, allowing sensitivity for PCR and microscopy to be calculated independently of each other. Additional information, and correction of some details is required, as follows:

1) Please double check the numbers in each of the categories throughout the study, as follows:

Line 62-65: The numbers of participants in each group is inconsistent with the total number of participants, as well as differing from the description given later in the paper. I.e. 43 suspected +14 other diseases+4 healthy = 61, not 57; 22 confirmed + 2 clinical + 6 suspected + 3 no scabies=33 suspected scabies cases, not 43.

Line 193-194: Here, the 14 participants without suspected scabies is broken down into 10 with onychomychosis and 4 healthy, inconsistent with the abstract in which the 4 healthy were stated as additional to the 14.

Line 195-196: As per the abstract, the numbers in each category of scabies do not add to the total stated with suspected scabies.

Line 198-200: With 17 controls (10 with onychomychosis, 3 with no scabies, 4 healthy volunteers) and 30 scabies diagnoses, it appears as though the total number of participants was 47 rather than 57.

2) Where were the skin scrapings taken from for the healthy volunteers with no scabies? To ensure comparability with the scrapings taken from suspected cases, the sampling sites should be similar. Please state the sampling strategy for the controls in the methods.

3) Line 182-183: Could you please state that you have calculated ‘exact’ confidence intervals (your results in Table 1 appear to be consistent with this).

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Are there any differences for the results according to gender or age?

-Are there any differences for the results according to the distribution of lesions (finger-webs, wrists, hands, axillae, gluteus, genitals, or breasts or may be mixed?)?

-Are there any differences for the results according to lesions (multiple small papules, nodules, vesicles, or

excoriation or others ???)?

Please add your results (Could be the reason for getting different results).

-Samples collected for microscopy and PCR were from the same place? Give details (Could be the reason for getting different results).

Do you search only one sample or you repeated??

New statistical analyses could be required for lesion distribution and type..

For 222-225

"Of the 30 patients with scabies, 5 (17%) revealed negative results for microscopic examination, but positive results for scabies PCR". Which type of scabies? Confirmed, clinical, or suspected?

"On the other contrary, 3 patients (10%) exhibited positive results for microscopic examination, but negative results for scabies PCR". Which type of scabies? Confirmed, clinical, or suspected?

Reviewer #2: The results presented follow the outlined methods, with tables and figures clearly presented. There are minor corrections required to the numbers, with details provided under the methods section.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Additional evaluation mentioned in the result section that should be discussed in discussion section

Reviewer #2: The conclusions are supported by the experimental results and the limitations of the study are clearly described. Further work required is described. A couple of sentences should be clarified, as follows:

1) Line 254-255: ‘clinical or suspected cases is more likely to be detected by the scabies PCR’ – this needs to be reworded. By design, clinical or suspected cases cannot be positive for microscopy – because if they were, then they would be confirmed cases. Therefore, it doesn’t make sense to compare the ability of the scabies PCR and microscopy to identify such cases. It would be better to discuss in terms of PCR having an ability to identify cases that could not be confirmed by microscopy.

2) Line 274-275: Did you statistically test whether there was a difference in the sensitivity for PCR and for microscopy? From what I can see, you tested whether there was a difference for each of these methods across the different IACS categories, but didn’t test the methods against each other. Please change the wording to ensure it is clear whether or not a formal statistical test was undertaken.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Writing of the references should be checked

Reviewer #2: Minor corrections and suggested re-phrasing:

1) Line 28: ‘Sarcoptes’, not ‘Scarcoptes’

2) Line 30: ‘proposed by the International…’

3) Line 31: ‘tests’ not ‘test’

4) Line 32: as no specific PCR test has yet been mentioned, ‘this in-house…’ does not make sense

5) Line 62: ‘scabiei’, not ‘scabies’

6) Line 74: delete ‘is’

7) Line 76: ‘result in various skin eruptions…’

8) Line 87: ‘tests’, not ‘test’

9) Line 145: ‘were performed’

10) Line 158: ‘scraping’, not ‘scrapping’

11) Line 197-198 seems to simply repeat the preceding sentence.

12) Line 203: 10/30 = 33%, not 30%

13) Line 214: for confirmed, clinical and suspected, it should be 22/30 (73% is correct), not 24/30

14) Line 224: ‘on the other contrary’ sounds strange – how about ‘on the other hand’?

15) Line 261: ‘The authors’, not ‘they’

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: --

Reviewer #2: Thank you for the opportunity to review this manuscript in which the authors aim to compare the sensitivity and specificity of a new PCR diagnosis tool for scabies to another commonly used method, microscopic confirmation. To do this, the authors use as the ‘gold standard’ the consensus criteria recently established by IACS, which appears to be a novel method, as previous studies treat either PCR or microscopy as the ‘gold standard’ and compare the other method to it. The authors claims that PCR testing may be useful in the diagnosis of scabies, due to its increased sensitivity over microscopy, are justified by the research study. Any improvement in the ability to diagnose scabies will be an important and relevant advance to both researchers and practitioners. There is some work to be done to ensure accuracy of numbers used throughout the paper, however, the conclusions would not change. While the paper is generally well written, I have indicated some typographical errors I noticed during review.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Aysegul TAYLAN OZKAN

Reviewer #2: No

5 Mar 2020

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

17 Mar 2020

Dear Dr. Kim,

We are pleased to inform you that your manuscript 'Diagnostic value of the molecular detection of Sarcoptes scabiei from a skin scraping in patients with suspected scabies' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Kosta Y. Mumcuoglu, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Paul J Brindley, PhD

Editor-in-Chief

PLOS Neglected Tropical Diseases

***********************************************************

None

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The author made all necessary changes

Reviewer #2: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The author made all necessary changes

Reviewer #2: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The author made all necessary changes

Reviewer #2: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The author made all necessary changes

Reviewer #2: There is a small number of typographical errors to be corrected (line numbers are from the marked-up word version of the manuscript):

Line 8: 'Dermatology' not 'Dermartology'

Lines 51-52: either 'tests...have' or 'test...has'

Line 76: 'result' (to match with 'Infections')

Line 242: ...'according to gender...'

Line 285-287: sentence is incomplete

References 4 & 10 have inconsistent capitalisation of the title

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The author made all necessary changes

Reviewer #2: Thank you for the opportunity to re-review this manuscript. I am satisfied that all of my comments have been addressed, and I thank the authors for the clear manner in which they have presented the changes, which saved time in re-review.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Aysegul TAYLAN-OZKAN

Reviewer #2: No

1 Apr 2020

Dear Dr. Kim,

We are delighted to inform you that your manuscript, "Diagnostic value of the molecular detection of Sarcoptes scabiei from a skin scraping in patients with suspected scabies," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases