Antimicrobial susceptibility profile of Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implications for national syndromic treatment guideline.

BACKGROUND: Neisseria gonorrhoeae (gonococcus) is the etiologic agent for the sexually transmitted Infection gonorrhea, a disease with a significant global public health impact. The treatment regimen for gonorrhea has been changed frequently over the past few decades due to the organism's propensity for developing antibiotic resistance. This study investigated antimicrobial susceptibility patterns of quinolones, third-generation cephalosporin, and other relevant antimicrobials found in N. gonorrhoeae isolated from men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia, with the aim of revising the national treatment regimen based on the information generated from this study.
METHODS: A total of 599 male patients presenting with urethral discharge were included in the current study. Urethral discharge specimens were cultured on Modified Thayer Martín media and suspected gonococcal colonies were confirmed using Oxidase and Superoxol tests followed by identification through a commercial kit (API-NHR). Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method using ciprofloxacin (5μg), ceftriaxone (30μg), cefixime (5μg), cefoxitin (30 μg), penicillin (10μg) and spectinomycin (100 μg) on enriched GC agar. Minimum Inhibitory Concentration (MIC) was also carried out using concentration gradient strips (E-tests) of the same antimicrobial agents.
RESULTS: The prevalence of gonococcal isolates in the current study was 69%. Out of the 361 gonococcal isolates, close to 68% were fluoroquinolone non-susceptible, with 60% resistant and 7% having an intermediate status. However, all tested isolates were susceptible to ceftriaxone. In addition, all of the isolates have shown reduced non-susceptibility to spectinomycin and cefoxitin.
CONCLUSION: The prevalence of gonococcal isolates in men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia was found to be high. The high level of fluoroquinolone resistance observed in gonococcal isolates recovered in this study necessitates revision of the national syndromic treatment guideline.

Background

Neisseria gonorrhoeae (gonococcus) is the second most common etiologic agent known to cause sexually transmitted <span class="Disease">infection (STI) with a significant global public health impact [1]. In uncomplicated infections, the disease manifests as urethritis in men and mucopurulent cervicitis in women. Because gonococcal infections are often asymptomatic in women, the lack of noticeable symptoms may result in serious complications such as pelvic inflammatory disease, ectopic pregnancy, and infertility. Infants born to a mother with the infection could develop conjunctivitis which may eventually lead to blindness if the infection is left untreated. In men, if left untreated, the disease could result in problems such as epididymitis, urethral stricture, and infertility. The transmission of the disease occurs by direct contact with secretions of infected mucosal surfaces and the incubation period can range from 1 to 10 days [1]. Gonorrhea, as with any STI, can work as a gateway to HIV and other infections [2, 3]. Globally, more than millions of people are affected by curable STIs. According to WHO, in 2012 alone, globally, there were an estimated 78 million new case of gonococcal diseases [1]. Public health control of gonorrhea requires both treatment of patients with appropriate antimicrobials as well as generalized and targeted prevention efforts [1, 4, 5].

Treatment regi<span class="Species">mens for gonorrhea have been changed frequently over the past few decades due to the organism’s propensity for developing antibiotic resistance [1]. Over the past few years, gonococcus have become less susceptible to previously used antibiotics such as ciprofloxacin or tetracycline [1]. Until recently, quinolones have been used as an alternative to treat gonococcal infections. However, the emergence and spread of gonococci resistant to the quinolone group and reduced susceptibility to third generation cephalosporin antibacterial was reported from different corners of the world [1]. This trend is concerning considering no alternative antibiotic treatment options or combinations have been proven to be effective against the organism [6-10].

The treatment for <span class="Disease">gonococcal infection in sub-Saharan Africa countries, including Ethiopia, is based on a syndromic approach using single dose fluoroquinolone treatment. The basis for this regimen was under the assumption that resistance to fluoroquinolones is considered to be low in Africa. However, with the occurrence of resistance to commonly prescribed antibiotics in both developed and developing countries, it is imperative to investigate the resistance pattern of gonococcal isolates periodically. Therefore, updated knowledge of the prevailing susceptibility patterns of gonococcal isolates in Ethiopia is important for the proper selection and use of antimicrobial drugs as well as for the development of an appropriate prescription policy. Therefore, this study aimed to investigate the susceptibility patterns of quinolones and third-generation cephalosporin found in N. gonorrhoeae isolated from urethral discharge of male patients seen in selected Addis Ababa city health centers.

Methods

Study sites and design

This study was conducted within the Addis Ababa, Ethiopia City Administration. Addis Ababa, the capital city of the Democratic Republic of Ethiopia, is geographically located in the central part of the country. A cross-sectional, facility-based study was conducted in eight healthcare centers of Addis Ababa. The selected healthcare facilities consisted of the Arada, Tekalehaimanot, Addis-Ketema, Kirkos, Kotebe, Akaki-Kaliti, Shiromeda, and Kassanchis health centers. These healthcare facilities were selected based on a high flow of STI <span class="Species">patients determined from a previous assessment. The study team collected samples from visiting patients over a span of twelve months at each study site following training on study protocols, procedures, and research ethics.

Source population and study participants

The source population consisted of <span class="Species">patients visiti<span class="Species">ng the selected healthcare facilities within Addis Ababa, Ethiopia with symptoms of urethral discharge who also gave consent to participate in the study. All urethral discharge specimens analyzed between August 2013 and August 2014 were included in this study.

Laboratory methods

Specimen collection

Men presenti<span class="Species">ng to the selected healthcare facilities with urethral discharge syndrome were recruited in the study following their consent. Afterward, a sterile Dacron swab-tipped applicator was used to collect urethral secretions. The swabs were then inoculated on Modified Thayer Martin Agar plates made of Gonococcal agar base supplemented with isovitalex (vitox); vancomycin, colistin, nystatin, and trimethoprim (VCNT); and synthetic hemoglobin (Oxoid and BBL) prepared in-house. The inoculated plates were incubated on site using a candle jar and then transported to the Ethiopian Public Health Institute (EPHI), Clinical Bacteriology and Mycology Reference Laboratory within the same day of collection. Swabs were rolled onto a microscopy slide, labeled, heat fixed, placed in a slide box, and sent to EPHI for Gram-stain analysis.

Culture and identification

In the clinical bacteriology laboratory at EPHI, inoculated plates were incubated at 35°C in a carbon dioxide enriched environ<span class="Species">ment (5–8% CO2) for 72-hours. Plates were inspected every day for the growth of small, translucent, and non-pigmented colonies. Plates that were gram-negative, diplococcic, convex, glistening, elevated, had mucoid colony characteristics, and were oxidase, catalase, and supercool (30% H2O2) positive were considered as probable N. gonorrhoeae and further confirmed by carbohydrate and enzymatic tests using API-NHR. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method using ciprofloxacin (5 μg), ceftriaxone (30 μg), cefixime (30 μg), cefoxitin (30 μg), penicillin (10 μg) and spectinomycin (100 μg) on enriched GC agar (Oxoid Ltd) plus 1% BBL Isovitalex Enrichment. Minimum Inhibitory Concentration (MIC) was done using concentration gradient strips (E-test) of the same antibiotics. The range of inhibition zones and MIC for each type of antibiotic disk were interpreted according to Clinical Laboratory Standard Institute (CLSI) guidelines [11]. Neisseria gonorrhoeae reference strain ATCC 49226 was used as a positive control.

Data extraction methods

A structured checklist was used to collect information on socio-demographics, clinical history, <span class="Disease">sexual behaviors, pro-antibiotics taken, and laboratory data such as the antibiotic susceptibility results. All data were double entered to Cespro 8 software by two individuals and data analysis was done usi<span class="Species">ng SPSS version 20.

Operational definitions

Non-susceptible <span class="Species">N. gonorrhoeae isolates were defined as those that are not sensitive to the antibiotic tested for susceptibility, i.e., those isolates exhibiti<span class="Species">ng resistance or intermediate resistance.

Dual non-susceptibility was defined as lack of susceptibility to any two of the antibiotics tested for susceptibility. Multi Drug Non-susceptibility was defined as combined non-susceptibility to an injectable cephalosporin and any two of either <span class="Chemical">quinolones, penicillins, or tetracyclines.

Ethics and consent to participate

This study was ethically cleared by the Scientific and Ethical Review Office (SERO) of the Ethiopian Public Health Institute and the Institutional Review Board (IRB) of CDC-Atlanta. At the enrollment visit, all <span class="Species">men with urethral discharge provided written consent after being diagnosed according to the syndromic treatment guidelines approved in Ethiopia. Those who were eligible (> 18 years of age) and willing to participate in the study were recruited using a structured questionnaire for their demographic and behavioral data. All data were kept confidential and anonymous. Brief counseling on the importance of adherence to STI medications, not having sex while taking medications, HIV/STI prevention, and recommendations to use condoms to reduce STI/HIV acquisition and transmission was also given.

Results and discussion

Between August 2013 and August 2014, a total of 599 urethral discharge specimens were collected from male <span class="Species">patients visiting one of the eight selected healthcare centers for routine clinical care and the collected specimens were microbiologically analyzed. The mean age of the study participants was 27 years (SD ± 7.2), with all being male. Observation of the urethral discharge specimens revealed that over 90% of them were profuse/thick discharge (Table 1).

Table 1

Clinical presentations of urethral discharge from patients visiting health centers in Addis Ababa.

Clinical feature	Category	N (%)
Fluid coming out of penis	Yes	597(99.7)
	No	2 (0.3)
	Total	599 (100)
Nature of Urethral discharge	Profuse/Thick	547 (91.3)
	Watery	44 (7.3)
	Other	8 (1.3)
	Total	599 (100)

Proportion of gonococcal isolates recovered

Of all the specimens analyzed, 415 (69.3%) <span class="Disease">gonococcal isolates were identified through culture methods. Compared to culture, the proportion of presumptive gonorrhea-positive samples was higher (75%) by gram stain (Table 2). This is not surprising as considerable proportion of the patients (20%) were on antibiotics when the specimens were collected which would affect culture results (Table 3). The prevalence of gonococcus in this study was relatively higher than other studies conducted in Ethiopia [12-14]. The difference may be due to the nature of the participants in the current study considering all of them were males and also showing clinical manifestation of the disease. The general notion is that naturally, males tend to be more symptomatic for gonococcal infection and hence can have increased level of healthcare seeking behavior which in turn makes them statistically overrepresented [15].

Table 2

Comparison of gram stains and culture methods for the detection of gonococcus isolates from urethral discharge specimens.

GC confirmation method	Result	N (%)
Gram stain	Positive	449 (75)
	Negative	150 (25)
Total		599(100)
Culture	Positive	415 (69)
	Negative	184 (31)
Total		599 (100)

Table 3

Medical treatment history of patients with urethral discharge from health centers in Addis Ababa, August 2013–August 2014.

Medication history	Response	N (%)
Taking medication	Yes	123 (20.5)
	No	476 (79.5)
	Total	599 (100)
Know the type of medication	Yes	107 (87)
	No	16 (13)
	Total	123 (100)
Ciprofloxacin	Yes	91 (85)
	No	16 (15)
	Total	107 (100)
Doxycycline	Yes	93 (86.9)
	No	14 (13.1)
	Total	107 (100)
Metronidazole	Yes	12 (11.2)
	No	95 (88.8)
	Total	107 (100)

Antimicrobial resistance profile

In sub-Saharan Africa, gonococcal treat<span class="Species">ment practice is based on a syndromic approach using a single dose fluoroquinolone treatment. It is hypothesized that resistance to fluoroquinolones is low in Africa, but there has been limited systematic data collection and analysis to verify this notion. A multicounty antimicrobial resistance study on gonococcal strains isolated in 2004–2006 indicated low rates of fluoroquinolone resistance with 0%, 1.3% and 4.0% in the Central African Republic, Cameroon, and Madagascar, respectively [16]. Similarly, a study conducted in Maputo and Mozambique in 2005 suggested that there was no resistance to fluoroquinolone by gonococcal isolates [17].

In contrast, the findings from several other countries in sub-Saharan Africa suggested increasi<span class="Species">ng levels of fluoroquinolone resistance in gonococcal isolates. According to a study done in South Africa in 2004, 7% of the gonococcal isolates from the Pretoria region, 8% from the Western Cape, and 17% from Johannesburg were found to be resistant to antibiotics from the class of fluoroquinolone. In addition, another study conducted in same country and the same study populations in 2007 indicated that 27% of the gonococcal isolates from Cape Town and 32% of isolates from Johannesburg were found to be resistant to ciprofloxacin [18]. This represents a 2.9 fold and 1.9 fold increases, respectively, within a 3-year time period. Similarly, a two-year prospective study carried out among STI patients from 2004 to 2006 in Johannesburg indicated an increase in ciprofloxacin resistance from 13% in the first year to 26.3% in the second year [19]. Another study conducted in Kenya and Uganda also showed that gonococcal resistance level to fluoroquinolone has reached up to 53% and 83%, respectively [20, 21].

The present study has revealed that N. gonorrhoeae isolates recovered from urethral discharge of male <span class="Species">patients in Addis Ababa, Ethiopia have shown a high level of resistance to the commonly prescribed fluoroquinolone class of antibiotics in Ethiopia (60%). This finding is in agreement with other studies which reported resistance levels between 53% and 83% in the East African region including Kenya and Uganda. Reports from South Africa also indicate that the resistance level has reached up to 32% [19-22]. The proportion of ciprofloxacin-resistant gonococcal isolates in the United States has also reached more than 30% [3]. The high proportion of quinolone resistance in this study might be due to prior treatment using ciprofloxacin, as indicated in Table 3. Gonococcal syndromic treatment using oral fluoroquinolone has become very problematic due to the emergence of a high proportion of resistant isolates, as witnessed from the current study. The good news, however, is that non-susceptibility to ceftriaxone has not been detected in any of the isolates tested during the study period. This finding is not in agreement with other studies conducted in different part of Ethiopia [12-14]. This may be due to exposure of participants to a specific group of antimicrobial agents during the study period. However, our finding was in agreement with studies conducted elsewhere [23-27]. As indicated in Fig 1, majority of the isolates have shown MIC value of 0.016 μg/ml for ceftriaxone with all of them having MIC values well below the cut-off point (0.25 μg/ml). However, the existence of certain segments of the isolate population with MIC values close to the cut-off point may indicate potential for a minority non-susceptible bacterial population to potentially replace the susceptible majority population. Therefore, investigating the molecular mechanism of resistance in these group of isolates may be imperative to fully understand the epidemiology [26].

Fig 1

Minimum Inhibitory Concentration (MIC) of ceftriaxone against N. gonorrhoeae.

The figure shows the MIC levels of ceftriaxone for <span class="Disease">gonococcal isolates recovered from urethral discharge specimens that were collected from male patients. All of the isolates tested using concentration gradient strips (E-test) were well below the cut-off point (0.25 μg/ml) for ceftriaxone MIC, with majority of them having MIC value of 0.016 μg/ml and none of them being non-susceptible. A small proportion of the isolates had MIC value at the cut-off point.

In our study, the Penicillinase test was carried out by a chromogenic test showi<span class="Species">ng almost more than half of the isolates to be positive for beta lactamase. Most of the isolates in the current study were resistant to Benzyl penicillin even though the antibiotic is not used for the national gonococcal treatment algorithm (Table 4). This finding from our study was also in line with other studies [24, 25, 27–29].

Table 4

Percent of antimicrobial susceptibility pattern of gonococcal isolates recovered from urethral discharge of patients (N = 361).

Antimicrobial agent	Class	Susceptibility profile
		Resistant, n (%)	Intermediate, n (%)		Susceptible, n (%)	Non susceptible, n (%)
P	Penicillins	191(52.9)		0(0)	170 (47.1)	-
Sp	Aminocyclitols	11 (3)		4 (1.1)	346 (95.9)	-
Cip	Flouroquinolone	217 (60.2)		26 (7.1)	118 (32. 7)	-
CRO	Cephalosposrin	0 (0)		0(0)	361 (100)	-
CFX	Cephalosposrin	-		0(0)	307 (85)	54 (15)
CTX	Cephalosporin	4 (1.1)		0(0)	357 (98.9)	-
AZ	Macrolides	36 (10)		0(0)	325 (90)	-

AZ = Azithromycin, CIP = Ciprofloxacin, CFX = Cefixime, CRO = Ceftriaxone, CTX = Cefoxitin, P = Penicilin, SP = Spectinomycin.

AZ = Azithromycin, <span class="Disease">CIP = Ciprofloxacin, CFX = Cefixime, CRO = Ceftriaxone, CTX = Cefoxitin, P = Penicilin, SP = Spectinomycin.

According to WHO, dual therapy is the preferred option for treat<span class="Species">ment of gonococcal infection instead of single therapy [1]. In the present study, non-susceptibility for the combination of ciprofloxacin and penicillin was observed at a rate of 6.9% (25/361), while for ciprofloxacin and spectinomycin were at a rate of 0.8% (3/361). Institutionalizing a surveillance system in the country might help track the resistance level of the isolates.

Formerly, the STI treatment practice in Ethiopia was based on the syndromic approach (treati<span class="Species">ng individuals immediately for possible causes of STI syndromes based on symptom). The syndromic treatment guideline was produced in 2006 and has been in use for years [10]. However, because of the findings from this study and other studies in the country, the national guideline for treatment has been changed [30]. In the former guideline, ciprofloxacin was recommended to treat gonococcal infections because resistance to it was not then documented. The current guideline recommends ceftriaxone instead of ciprofloxacin [30]. The etiologic approach to diagnosis of gonorrhea is important, especially for revisiting clients, in order to identify non-susceptible isolates to serve as candidates for antimicrobial agents in practice.

Conclusions

In the current study, the proportion of N. gonorrhoeae isolates in males with urethral discharge in Addis Ababa, Ethiopia, was found to be high. Of greatest concern was the findi<span class="Species">ng that these gonococcal isolates were highly resistant to the new generation of antibiotics, fluoroquinolones (ciprofloxacin), which has been indicated as the treatment of choice according to previous national guidelines. Results generated from this study were used as input to revise the national syndromic guidelines for management of patients presenting with urethral discharge due to gonorrhea. As a result, ciprofloxacin was replaced by ceftriaxone which was found to be effective in terms of in vitro susceptibility results [30]. In conclusion, the syndromic-based diagnostic approach needs to be periodically validated and modified based on determination of susceptibility patterns of N. gonorrhoeae isolates in the region. Since this study was done in 2014, additional studies are warranted to understand the current antimicrobial resistance status of gonorrhoeae isolates in Ethiopia.

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31 Dec 2019

<span class="Chemical">PONE-D-19-32111

Antimicrobial Susceptibility Profile of <span class="Disease">Gonococcal Isolates Obtained from Men Presenting with Urethral Discharge: Implication for National Syndromic Treatment guideline

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Reviewer #1: The manuscript describes a study of urethral discharges from male patients in Addis Ababa, Ethiopia. It is motivated by the growi<span class="Species">ng problem of antibiotic resistance in Neisseria Gonorrhoea. Its main findings are that gonococcal isolates are detected in 69% of those discharges. Of these isolates, a significant proportion were non-susceptible to fluoroquinolone, which is part of the basis of gonorrhoea management in sub-Saharan African countries including Ethiopia. The scientific work is sound but considerable improvement is required in its presentation.

1) The manuscript appears to be technically sound. The study is described with an appropriate level of detail and its design was clearly suitable for the topic of interest so that one would expect its data to be capable of supporti<span class="Species">ng its conclusions. However the actual data are not supplied.

2) The statistical analysis is appropriate to the question at hand and done correctly.

3) The authors have not made their data available. It is not in the manuscript, nor is there a link provided, and there is no supple<span class="Species">mentary section.

4) The English in this manuscript does not meet the standards expected for a credible, reviewed scientific journal. It is intelligible most of the time but replete with grammatical errors and poorly worded phrases, too many to outline here. An exception is paragraph five in "Results and Discussion" in which figure one is referenced and discussed. The authors presumably mean to say that <span class="Chemical">ceftriaxone non-susceptibility was not found in any isolates. The rest of this paragraph is incomprehensible. The authors are advised to rewrite the manuscript with assistance from a native speaker of English.

Additional Com<span class="Species">ments:

A) The presentation of percentages throughout the manuscript is inconsistent, most notably in the third paragraph of the "Results and Discussion" section and in table 1. It is indicated that percentages are bei<span class="Species">ng presented but some figures have "%" signs while others do not. The "%" should be used in text while the convention used in table 3 should be used consistently in all tables.

b) Table 2 is not referenced in the text although it is apparently relevant to the fourth paragraph in "Results and Discussion". Also, most of its data appears to be missi<span class="Species">ng which renders the table confusi<span class="Species">ng. As far as I can make out, each row should add up to 100 (%) but they do not. This table should be either corrected and properly referenced, or deleted.

c) The page with table 2 has what appears to be a footnote entitled "MIC Ra<span class="Species">nges for Resistance and Intermediate Susceptibility as defined by CLSI Guidelines". This useful and relevant information should be clearly presented in its own table and appropriately referenced from within the text.

d) The bars in table 1 have no apparent order. They should be plotted in order and positioned to scale on the x-axis since these categories are defined by the quantifiably measurable MIC.

e) It is stated in the final paragraph of the "Results and Discussion" section and again in the "Conclusions" that this work has already led to a revision of Ethiopia's national guidelines for managi<span class="Species">ng <span class="Species">patients with urethral discharge, but no description or reference to a description is given. The revisions should be described and ideally a paper or relevant government document should be referenced.

f) References to tables and figures should use a capital letter, "Table 3" instead of "table 3". This was sometimes neglected, particularly for Table 3 and Figure 1.

Reviewer #2: Antimicrobial-resistant <span class="Disease">gonorrhea is a major public health threat. Therefore, manuscript addressing the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) isolates are important. However, this manuscript, as written, lacks the necessary level of detail and scientific rigor to be published in PLOS one Medicine.

Major issues.

Introduction.

1) Does not describe the current WHO recommended treat<span class="Species">ment for gonorrhea. This is important in order to provide context for the recommended treatment in Ethiopia.

2) There are several recent papers describing the susceptibility of <span class="Species">NG isolates in Africa which have found that most NG isolates from African countries are resistant to Ciprofloxacin.

3) The references cited are older. A quick search on Pubmed could yield a list of recent publications on this topic.

4) Overall, the introduction is too short.

Results/discussion

1) Although table 1 describes the gram stain results, a discussion of gram stain results in comparison to culture results should be included.

2) The authors need to be careful in attributing the high level of <span class="Species">NG infections to the fact that the samples were collected from men. The reason for the high positivity rates is because the samples were collected from symptomatic subjects.

3) The discussion on the implication for National syndromic treatment guideline need to be expanded. It is currently limited to one line. “Antimi<span class="Chemical">crobial Susceptibility Profile of Gonococcal Isolates Obtained from Men Presenting with Urethral Discharge: Implication for National Syndromic Treatment guideline”

The title should also include Ethiopia to better guide the reader.

Overall, this manuscript is suitable for a journal like STD which is more related to public health

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28 Feb 2020

Dear Editor

Thank you for your constructive com<span class="Species">ments. We have tried to address all the com<span class="Species">ments accordingly

Submitted filename: Repsonse to reviewers.docx

Click here for additional data file.

24 Mar 2020

<span class="Chemical">PONE-D-19-32111R1

Antimicrobial susceptibility profile of <span class="Disease">Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implication for national syndromic treatment guideline

PLOS ONE

Dear Mr Dinku,

Thank you for submitti<span class="Species">ng 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 duri<span class="Species">ng the review process.

We would appreciate receivi<span class="Species">ng your revised manuscript by May 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needi<span class="Species">ng Revision' folder to locate your manuscript file.

If you would like to make cha<span class="Species">nges to your financial disclosure, please include your updated state<span class="Species">ment in your cover letter.

To enhance the reproducibility of your results, we recom<span class="Species">mend 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the followi<span class="Species">ng items when submitti<span class="Species">ng your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights cha<span class="Species">nges made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Cha<span class="Species">nges'.

An unmarked version of your revised paper without tracked cha<span class="Species">nges. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while formi<span class="Species">ng 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 com<span class="Species">ments. If eligible, we will contact you to opt in or out.

We look forward to receivi<span class="Species">ng your revised manuscript.

Kind regards,

David Gabriel Regan, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Com<span class="Species">ments (if provided):

The reviewers agree that the revised version of the manuscript is is substantially improved but the language and grammatical issues have not been adequately addressed. It was recom<span class="Species">mended that the authors seek editorial assistance in addressing these issues and it seems this recommendation has not been taken on board. The reviewers agree that the manuscript is scientifically sound on the whole but the language and presentation is not up to the standard required for publication in PLoS ONE. I feel the authors should have a final opportunity to address the language and presentation issues highlighted by the reviewers but the manuscript will need to be substantially improved for me to be able to recommend publication in PLoS ONE. The authors also need to provide a consistent statement that aligns with PLoS ONE publication policy in regard to availability of data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' com<span class="Species">ments:

Reviewer's Responses to Questions

Com<span class="Species">ments to the Author

1. If the authors have adequately addressed your com<span class="Species">ments 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 “Com<span class="Species">ments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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. Experi<span class="Species">ments 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: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlyi<span class="Species">ng the findi<span class="Species">ngs in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findi<span class="Species">ngs 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.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard E<span class="Species">nglish?

PLOS ONE does not copyedit accepted manuscripts, so the la<span class="Species">nguage 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: No

Reviewer #2: No

**********

6. Review Com<span class="Species">ments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional com<span class="Species">ments for the author, includi<span class="Species">ng concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been considerably improved in both the discussion of content and in the quality of its writing. Unfortunately it still falls well short of the standard expected for a credible scientific journal. There is still a large number of spelli<span class="Species">ng and basic grammatical errors, clearly indicating that a native speaker was not consulted as advised. Other sentences were either badly worded, or too long and needing to be broken into smaller sentences. Examples are the final sentence of the abstract and "Globally, more than millions of curable STD are affecting people every day" from the Background section.

The caption for Figure 1 inexplicably appears in isolation in the middle of page nine with no figure. This may be an artifact of separati<span class="Species">ng figures and tables from the main body of the text in the draft copy but the same has not happened for the tables so care must be taken with this.

Table 3 (previously Table 2) is now much clearer but the percentages in the bottom two rows still do not add to 100%. My best guess is that the remaini<span class="Species">ng samples were non-susceptible, but this should be indicated explicitly.

Tables 3 and 4 are referenced out-of-order and their numberi<span class="Species">ng should be reversed.

The y-axis of the figure is labelled "Proportion of <span class="Disease">Gonococcal isolates" but the bars clearly do not add up to 100%. Either it is mislabelled or the scale is wro<span class="Species">ng. If the axis is a proportion then the ticks should be labelled 20%, 40%, ... , 100% .

The authors are inconsistent in their responses to PLOS ONE's data sharing require<span class="Species">ment. They have claimed under their Data Availability statement that "data are fully available without restriction" and "included in the manuscript" and that they "can give the raw data", but stated in their response to reviewers that they are restricted from doing so from privacy concerns. I suspect that they do not properly understand this requirement. The PLOS Data policy allows exceptions if the data "compromise the privacy or confidentiality of human research subjects" but the de-identification of data in a study such as this has long been standard practice and there is no discussion of these restrictions in the Materials and Methods.

While the scientific core of this work appears to be sound, the manuscript and data-shari<span class="Species">ng still do not approach the standards expected for a high quality scientific journal. I suggest after attendi<span class="Species">ng to these comments that the authors submit their work to a journal more specific to public health.

Reviewer #2: Thanks to the authors for maki<span class="Species">ng the required cha<span class="Species">nges.

Please consider maki<span class="Species">ng the followi<span class="Species">ng revisions.

1) Abstract. Please note that gonorrhea is the disease/<span class="Disease">infection. No need to see gonorrhea disease.

2) Introduction. Please consider editi<span class="Species">ng the followi<span class="Species">ng sentence "Globally, more than millions of curable STD are

affecting <span class="Species">people every day. According to WHO, in 2012 alone, there were an estimated 78 million new

case of <span class="Disease">gonococcal diseases [1].

3) Introduction: The followi<span class="Species">ng section of the introduction could use some editi<span class="Species">ng. "Over the past few years, gonococcus have

become less susceptible to previously used antibiotics such as sulfonamides or <span class="Chemical">tetracycline. Until recently, quinolones and third-generation cephalosporins are alternative to treat gonococcal infections". Please note that sulfonamides have not been recently prescribed and that third generation celaphosporins are currently the recommended treatment option.

4) Methods. STI needs to be spelled out the first time that is used in the document. Overall, this reviewer recom<span class="Species">mends changing STD to STI.

5) Methods. The followi<span class="Species">ng two sentences are confusi<span class="Species">ng. "A sterile cotton-tipped swab were used to obtain a swab specimens. Then sterile Dacron swabs tipped applicator were used to collect urethral secretions". It sounds like two different swabs were used for sample collection.

6) Operating definitions. Penicillin is mentioned here, but in the methods section it is not clear that the isolates were tested for penicillin susceptibility.

7) Ehtics and Consent. Please clarify the following sentence. "At the enroll<span class="Species">ment visit, all men with urethral discharge (UD) were given written consent diagnosed according to the syndromic treatment guidelines approved in Ethiopia". It is not clear to this reviewer what "written consent diagnosed" means.

8) Results. This reviewer does not understand the relevance of the followi<span class="Species">ng sentence. "Observation of the speci<span class="Species">mens revealed that over 90% of them were profuse /thick discharge (Table-1).

9) Proportion of gonococcal isolates recovered. The sentence "Compared to culture, the proportion of the identified isolates was higher (75%) when gram stain method was used (Table-2)" requires editi<span class="Species">ng. It is not possible to isolate NG by gram stain. Please edit sentence as follows "Compared to culture, the proportion of presumptive gonorrhea-positive samples was higher (75%) by gram stain.

10) Figure 1 header. Please change N. <span class="Disease">gonorrhea to N. gonorrhoeae.

11) Conclusion. Please <span class="Disease">change gonorrhea disease to <span class="Disease">gonorrhea.

12) Conclusion. The authors should add a sentence regarding the limitation of usi<span class="Species">ng samples that were collected in 2013-2014. The epidemiology of resistance is probably different now. They can also say that additional studies are warranted to understand the current (2020) epidemiology of antimicrobial resistance in NG in Ethiopia.

**********

7. 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, includi<span class="Species">ng consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Michael Luke Walker

Reviewer #2: No

[NOTE: If reviewer com<span class="Species">ments were submitted as an attach<span class="Species">ment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion E<span class="Species">ngine (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. Registration is free. 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. Please note that Supporting Information files do not need this step.

2 May 2020

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. Experi<span class="Species">ments 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: Yes

Reviewer #2: Yes

Response to reviewers:

Dear reviewers, thank you so much.

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Response to reviewers:

Dear reviewers, thank you so much.

________________________________________

4. Have the authors made all data underlyi<span class="Species">ng the findi<span class="Species">ngs in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findi<span class="Species">ngs 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.

Reviewer #1: No

Reviewer #2: Yes

Response to reviewers:

Dear reviewers, thank you so much. As to the availability of Data, we have requested our institute for de-identifying the data set and are able to share it, as supple<span class="Species">ment file, during this current submission. Thank you.

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard E<span class="Species">nglish?

PLOS ONE does not copyedit accepted manuscripts, so the la<span class="Species">nguage 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: No

Reviewer #2: No

Response to reviewers:

Dear reviewers, thank you. We have tried to address the major issue raised accordingly. We sought an editorial assistance from native English speaker, Dr. Laura Binkley (binkley.69@osu.edu) from the Ohio State Unviersity, USA. And we thank Dr. Laura for the English language edits and overall comments that, we believe, has substantially improved the manuscript. We have checked the spelling and over all neatness of the manuscript in as much as we can.

________________________________________

6. Review Com<span class="Species">ments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional com<span class="Species">ments for the author, includi<span class="Species">ng concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been considerably improved in both the discussion of content and in the quality of its writing. Unfortunately it still falls well short of the standard expected for a credible scientific journal. There is still a large number of spelli<span class="Species">ng and basic grammatical errors, clearly indicating that a native speaker was not consulted as advised. Other sentences were either badly worded, or too long and needing to be broken into smaller sentences. Examples are the final sentence of the abstract and "Globally, more than millions of curable STD are affecting people every day" from the Background section.

Response to reviewer:

Dear reviewer, thank you for the priceless com<span class="Species">ments. We have tried to address your com<span class="Species">ments and have consulted a native English speaker to address the language issues. Thank you once again.

The caption for Figure 1 inexplicably appears in isolation in the middle of page nine with no figure. This may be an artifact of separati<span class="Species">ng figures and tables from the main body of the text in the draft copy but the same has not happened for the tables so care must be taken with this.

Response to reviewer:

Dear reviewer, thank you for the comment; as you rightly put it, it was an artifact left duri<span class="Species">ng the process of separating the figure from the main body of the text. We are very sorry for killing your precious time. We have made the correction. We have also tried to check to avoid such mistakes all over the manuscript. Thank you.

Table 3 (previously Table 2) is now much clearer but the percentages in the bottom two rows still do not add to 100%. My best guess is that the remaini<span class="Species">ng samples were non-susceptible, but this should be indicated explicitly. Tables 3 and 4 are referenced out-of-order and their numberi<span class="Species">ng should be reversed.

Response to reviewer:

Dear reviewer, thank you for the comments and positive words. The numbers were simply counted for those sample how have <span class="Disease">CIP + Spectinomycin combined resistance level. To avoid confusion we have omitted the bottom two rows that do not add up to 100. We have also revised the position of table-3 and 4. Thank you.

The y-axis of the figure is labelled "Proportion of <span class="Disease">Gonococcal isolates" but the bars clearly do not add up to 100%. Either it is mislabelled or the scale is wro<span class="Species">ng. If the axis is a proportion then the ticks should be labelled 20%, 40%, ... , 100% .

Response to reviewer:

Thank you. Y-axis is proportion but we did not indicate in percentage. We have corrected this by addi<span class="Species">ng % on the y-axis label.

The authors are inconsistent in their responses to PLOS ONE's data sharing require<span class="Species">ment. They have claimed under their Data Availability statement that "data are fully available without restriction" and "included in the manuscript" and that they "can give the raw data", but stated in their response to reviewers that they are restricted from doing so from privacy concerns. I suspect that they do not properly understand this requirement. The PLOS Data policy allows exceptions if the data "compromise the privacy or confidentiality of human research subjects" but the de-identification of data in a study such as this has long been standard practice and there is no discussion of these restrictions in the Materials and Methods.

Response to reviewer:

Dear Reviewer, You are right we did not fully understood the policy but after the com<span class="Species">ments, this issue has been addressed by provision of the de-identified data set as a supple<span class="Species">ment file with this submission.

While the scientific core of this work appears to be sound, the manuscript and data-shari<span class="Species">ng still do not approach the standards expected for a high quality scientific journal. I suggest after attendi<span class="Species">ng to these comments that the authors submit their work to a journal more specific to public health.

Response to reviewers:

Dear reviewer, thank you so much for the very constructive comments you gave us and definitely that have significantly improved our manuscript in terms of la<span class="Species">nguage usage and data presentation. Thank you for your precious time and patience in reviewing our manuscript.

Reviewer #2: Thanks to the authors for maki<span class="Species">ng the required cha<span class="Species">nges.

Please consider maki<span class="Species">ng the followi<span class="Species">ng revisions.

1) Abstract. Please note that gonorrhea is the disease/<span class="Disease">infection. No need to see gonorrhea disease.

Response to reviewers:

Dear reviewer, thank you so much for the very constructive com<span class="Species">ments. The correction have been made as per the com<span class="Species">ment.

2) Introduction. Please consider editi<span class="Species">ng the followi<span class="Species">ng sentence "Globally, more than millions of curable STD are

affecting <span class="Species">people every day. According to WHO, in 2012 alone, there were an estimated 78 million new

case of <span class="Disease">gonococcal diseases [1].

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. The correction have been made as per the com<span class="Species">ment.

3) Introduction: The followi<span class="Species">ng section of the introduction could use some editi<span class="Species">ng. "Over the past few years, gonococcus have

become less susceptible to previously used antibiotics such as sulfonamides or <span class="Chemical">tetracycline. Until recently, quinolones and third-generation cephalosporins are alternative to treat gonococcal infections". Please note that sulfonamides have not been recently prescribed and that third generation celaphosporins are currently the recommended treatment option.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. The correction have been made as per the com<span class="Species">ment.

4) Methods. STI needs to be spelled out the first time that is used in the document. Overall, this reviewer recom<span class="Species">mends changing STD to STI.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. The correction have been made as per the com<span class="Species">ment. And STD was replaced with STI.

5) Methods. The followi<span class="Species">ng two sentences are confusi<span class="Species">ng. "A sterile cotton-tipped swab were used to obtain a swab specimens. Then sterile Dacron swabs tipped applicator were used to collect urethral secretions". It sounds like two different swabs were used for sample collection.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ment. It was Da<span class="Chemical">cron swab that was used. Correction has been made accordingly. Thank you once again.

6) Operating definitions. Penicillin is mentioned here, but in the methods section it is not clear that the isolates were tested for penicillin susceptibility.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. <span class="Chemical">Penicillin was unintentionally omitted. The correction have been made as per the comment.

7) Ehtics and Consent. Please clarify the following sentence. "At the enroll<span class="Species">ment visit, all men with urethral discharge (UD) were given written consent diagnosed according to the syndromic treatment guidelines approved in Ethiopia". It is not clear to this reviewer what "written consent diagnosed" means.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. The sentence has been clarified per the com<span class="Species">ment given.

8) Results. This reviewer does not understand the relevance of the followi<span class="Species">ng sentence. "Observation of the speci<span class="Species">mens revealed that over 90% of them were profuse /thick discharge (Table-1).

Response to reviewers:

Dear reviewer, thank you so much for the comments. All the urethral discharge speci<span class="Species">mens were observed visually for checking the clinical manifestation.

9) Proportion of gonococcal isolates recovered. The sentence "Compared to culture, the proportion of the identified isolates was higher (75%) when gram stain method was used (Table-2)" requires editi<span class="Species">ng. It is not possible to isolate NG by gram stain. Please edit sentence as follows "Compared to culture, the proportion of presumptive gonorrhea-positive samples was higher (75%) by gram stain.

Response to reviewers:

Dear reviewer, thank you so much for the comments. The sentence was not meant to mean that we isolated usi<span class="Species">ng gram stain. Since gram stain, in resource limiting setting like ours, can be using to guide the treatment of patients (male), we felt to compare both. We have amended the sentence as commented. Thanks you for your helpful comment.

10) Figure 1 header. Please change N. <span class="Disease">gonorrhea to N. gonorrhoeae.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. Corrections have been made per the com<span class="Species">ment.

11) Conclusion. Please <span class="Disease">change gonorrhea disease to <span class="Disease">gonorrhea.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. Corrections have been made per the com<span class="Species">ment.

12) Conclusion. The authors should add a sentence regarding the limitation of usi<span class="Species">ng samples that were collected in 2013-2014. The epidemiology of resistance is probably different now. They can also say that additional studies are warranted to understand the current (2020) epidemiology of antimicrobial resistance in NG in Ethiopia.

Response to reviewers:

Dear reviewer, thank you so much for the com<span class="Species">ments. The issue has been addressed as per the given com<span class="Species">ment.

Submitted filename: Repsonse to reviewers.docx

Click here for additional data file.

7 May 2020

<span class="Chemical">PONE-D-19-32111R2

Antimicrobial susceptibility profile of <span class="Disease">Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implications for national syndromic treatment guideline

PLOS ONE

Dear Mr Dinku,

Thank you for submitti<span class="Species">ng 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 duri<span class="Species">ng the review process.

We would appreciate receivi<span class="Species">ng your revised manuscript by Jun 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needi<span class="Species">ng Revision' folder to locate your manuscript file.

If you would like to make cha<span class="Species">nges to your financial disclosure, please include your updated state<span class="Species">ment in your cover letter.

To enhance the reproducibility of your results, we recom<span class="Species">mend 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the followi<span class="Species">ng items when submitti<span class="Species">ng your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights cha<span class="Species">nges made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Cha<span class="Species">nges'.

An unmarked version of your revised paper without tracked cha<span class="Species">nges. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while formi<span class="Species">ng 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 com<span class="Species">ments. If eligible, we will contact you to opt in or out.

We look forward to receivi<span class="Species">ng your revised manuscript.

Kind regards,

David Gabriel Regan, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Com<span class="Species">ments (if provided):

The authors have, on the whole, addressed the concerns expressed by the reviewers and the manuscript is much improved. There remain a couple of minor issues that need to be addressed before I can recom<span class="Species">mend this manuscript for publication;

1) the frequency columns in tables 1-3 are redundant and should be removed because the identical information in provided in the 'N (%)' column

2) the total for Gram stain is missi<span class="Species">ng in Table 2

3) Table 4 does not contain information regardi<span class="Species">ng multi-drug non-susceptibility as stated on page 9. This information needs to be added to the table or presented in a separate table.

4) The results presented in Figure 1 are not adequately described in the results on page 9 and the cut-off MIC is <span class="Species">not specified so this can not be interpreted. There is also no figure legend for Figure 1, only a figure title.

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer com<span class="Species">ments were submitted as an attach<span class="Species">ment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion E<span class="Species">ngine (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. Registration is free. 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. Please note that Supporting Information files do not need this step.

11 May 2020

The authors have, on the whole, addressed the concerns expressed by the reviewers and the manuscript is much improved. There remain a couple of minor issues that need to be addressed before I can recom<span class="Species">mend this manuscript for publication;

Response to the Editor:

Dear editor, thank you so much for all the constructive com<span class="Species">ments. And thank you also for this last opportunity, again, for us to be able to publish our findi<span class="Species">ng. We have tried to address the major issue raised accordingly.

1) the frequency columns in tables 1-3 are redundant and should be removed because the identical information in provided in the 'N (%)' column

Response to the Editor:

The correction has been made. The frequency columns in all tables has been removed. Thank you so much for due diligence.

2) the total for Gram stain is missi<span class="Species">ng in Table 2

Response to the Editor

The correction has been made. The total for gram stain has been added. Thank you so much for due diligence.

3) Table 4 does not contain information regardi<span class="Species">ng multi-drug non-susceptibility as stated on page 9. This information needs to be added to the table or presented in a separate table.

Response to the Editor:

Correction has been made to the proportion of isolates with non-susceptibility to ciprofloxacin and <span class="Chemical">pencillin and ciprofloxacilin and spectinomycin (In combination) and the reference made to table-4 was removed because it is presented in a narrative form in page 9. In the previous submissions, we added the information in the bottom 2 rows of table -4 and later omitted it because that has caused a confusion to the reviewers as the numbers in these rows did not add up to hundred. Since we only had the combined non-susceptibility for the two agents so did not feel to put the information in a table of its own rather we have indicated in the narrative on page 9.

4) The results presented in Figure 1 are not adequately described in the results on page 9 and the cut-off MIC is <span class="Species">not specified so this cannot be interpreted. There is also no figure legend for Figure 1, only a figure title.

Response to the Editor:

Thank you for the com<span class="Species">ment. We have attempted to describe the result presented in the figure on page 9 and also have included figure legend as com<span class="Species">mented on page 9 right immediately after the paragraph where the figure was referenced.

Submitted filename: Response to reviewers May 11 2020.docx

Click here for additional data file.

13 May 2020

Antimicrobial susceptibility profile of <span class="Disease">Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implications for national syndromic treatment guideline

<span class="Chemical">PONE-D-19-32111R3

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Additional Editor Com<span class="Species">ments (optional):

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20 May 2020

<span class="Chemical">PONE-D-19-32111R3

Antimicrobial susceptibility profile of <span class="Disease">Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implications for national syndromic treatment guideline

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