Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women.

There is a scarcity of studies on the prevalence of Trichomonas vaginalis (TV) in indigenous populations of Brazil. We conducted a cross-sectional study between January and December 2018, on indigenous women living nearby an urban center of the Midwest region of Brazil and determined the prevalence of TV. Factors associated with TV infection and a comparison of molecular and direct microscopy diagnoses were determined. 241 indigenous women aged above 18 years participated in the study. Cervical and vaginal brush samples were collected to diagnose TV through polymerase chain reaction (PCR). Direct microscopy for detection of TV, and cellular changes was performed. A sociodemographic and behavioral questionnaire was applied at the beginning of the study. All the data were analyzed using Statistical Package for the Social Sciences. The result obtained showed that 27.8% [95% CI: 22.2-33.9] were positive for TV on PCR, while 7.41% [95% CI: 4.1-11] showed positive on direct microscopy. Direct microcopy also found 21 (8.71%) and 8 (3.31%) women infected with Gardnerella vaginalis and Candida albicans, respectively. In addition, 10 women presented atypical squamous cells of unknown significance and 14 lesions suggestive of HPV. Single women, under the age of 30 and who do not use condoms, were found to have a greater chance of getting TV infection. The high prevalence TV found in this population is comparable to highly vulnerable populations, as prisoners, sex workers and women in regions with low socioeconomic levels, moreover, seems to be an underdiagnosis of this infection. Therefore, a routine test program, as well as a review of the diagnostic method used, is encouraged for proper management.

Introduction

Developing countries are mainly threatened by infectious diseases of the reproductive tract and sexually transmitted infections (STIs), which are considered a major public health problem. The main and most prevalent STIs described in the literature are caused by Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis (TV), and Treponema pallidum [1, 2]. These pathogens can cause acute urogenital conditions, such as cervicitis, urethritis, vaginitis, and genital ulceration [1, 2]. Trichomoniasis, caused by TV, is the non-viral STI most common in the world [3, 4]. It is a flagellated protozoan affecting both sexes, and women are more affected comparatively. Approximately 85% of women are asymptomatic carriers of TV. However, once the symptoms arise, it affects the region of the vulva, vagina, and cervix, being associated with pelvic inflammation, neoplasms, premature birth, and the promotion of HIV infection [4].

The diagnosis of TV in the public health network is basically performed through direct microscopy of a sample obtained in the Pap smear test. Although the Pap smear has specificity for T. vaginalis, there are sensitivity limitations that prevent the use of this technique for the diagnosis of infection [3, 5]. The main focus of this technique is on the search for cellular changes such as atypical granular cells and squamous lesions, other findings such as Gardnerella vaginallis infection or Candida albicans, end up being in the background, which can also be related with problems in the female reproductive tract, especially the imbalance of bacterial flora, increasing the risk of infection by other STIs [6-8].

In countries like Brazil, trichomoniasis remains unreported, making it difficult to measure the extent of the disease [9]. In 2012, the World Health Organization (WHO) reported about 357 million new cases of non-viral STIs worldwide, of which 142 million were caused by TV [4]. Studies conducted in Brazil have reported TV infection prevalence rates between 3.7% and 30% [4, 10]. Many populations related to low socioeconomic conditions, along with other factors such as age, gender, and sexual choice, have shown above-average prevalence rates for STIs [4, 11]. T. vaginalis mainly affects the poorest sections of the population and people in vulnerable situations [12].

An estimated indigenous population of 817 thousand people from 305 different ethnicities lives in Brazil. This population is divided into 5,614 villages, occupying approximately 12.6% of the Brazilian territory, most of them living in lower socioeconomic conditions than the rest of the country [13-15]. Although Brazil has specific legislation for indigenous health, only little is applied. Research on this population is still primarily related to sociocultural issues, and very little has been published on health issues [14]. In this context, the prevalence of the TV was determined in indigenous women living in the Midwest region of Brazil. We determined the factors associated with TV infectious as well as performed a comparison of molecular and direct microscopy diagnoses used in primary care.

Methods

Type of study and sample size calculation

A cross-sectional study was conducted to determine the prevalence of TV in indigenous women from two Indigenous Reserves of Dourados—Mato Grosso do Sul, in the Midwest region of Brazil. We obtained approval from the National Research Ethics Commission (number 2.000.496) for this study. Based on the population of 8,000 women aged above 18 years, considering an average prevalence of T. vaginallis of 15% (4–27%) and a 5% margin of error, a minimal sample of 192 women was estimated [12].

Study participants and epidemiological questionnaire

The study was performed between January 2018 and December 2018. A total of 241 women, over the age of 18, agreed to participate in the study and signed an informed consent form. Data and samples were obtained during their visit to the Basic Health Units of the two indigenous reserves evaluated: Bororó and Jaguapiru village, where Guarani (Kaiowá and Nhandeva) and Terena ethnicities predominate.

A questionnaire addressing demographic was given to the participants contemplating the following variables: village (Bororó or Jaguapirú), and ethnicities (Guarani-Kaiowa, Guarani Nhandeva, Terena, and others not predominant in the region); socioeconomic data (age, employment status, income, government benefits, and years of schooling); clinical data: history of cancer, symptoms, and other STIs diagnoses; and behavioral factors: marital status, number of partners in the last year, alcohol use, tobacco use, illicit drug use, share objects (syringes and personal hygiene supplies), condom use. Categorical variables were represented as “yes” or “no”, and numerical variables were categorized later.

All possible actions were taken to reduce bias during data collection. For easy comparisons and identification of errors and inconsistencies, questionnaires were filled out in Redcap with double data entry. Questionnaires were redone to resolve inconsistencies. Incomplete questionnaires were discarded before laboratory analysis.

The exams results were submitted to the Health Unit, where the necessary treatment measures could be taken for the patients in whom an infection was detected.

Sampling and DNA extraction

Vaginal samples were collected using a VAGISPEC® cervical and vaginal brush and placed in a tube containing 1 mL of extraction buffer (EB; 10 mM Tris-HCl pH 8.5, 1 mM EDTA). A total of 300 μL of samples was added to 500 μL of lysis buffer (15 mM sodium citrate, 450 mM NaCl, 0.2% SDS, and 2 mg/mL lysozyme). Phenol-chloroform was used for DNA extraction, and 95% ethanol and 100 μL of 3 M sodium acetate (pH 5.2) were used for precipitation. The precipitate was oven-dried, eluted in 30 μL of TE buffer (10 mM Tris HCl [pH 7.6], 1 mM EDTA), and then frozen at –80°C. DNA was quantified using the UV-Vis Biodrop μLite Spectrophotometer (Biochrom, USA). DNA integrity was evaluated by agarose gel and by amplification of β-globin gene (268 base pairs) representing the internal control of the reaction.

Molecular diagnosis, and direct microscopy of T. vaginalis

All procedures for polymerase chain reaction (PCR), including primers, were performed according to the one described by Gatti et al. (2017). The primer set TVK3/7 (5’AT TGT CGA ACA TTG GTC TTA CCC TC-3’/5`-TCT GTC CCG TCT TCA AGT ATG C-3`), which amplifies a 260 bp DNA fragment, along with a commercial kit: Master Mix (LUDWIG)® was used. The samples were resolved by 1.5% agarose gel electrophoresis and detected by ethidium bromide staining under ultraviolet light for the confirmation of the results. For all PCR assays, T. vaginalis DNA extracted from the culture of ATCC 30236 strain was used as a positive control. Sterile water was used as a negative control to monitor crossover contaminations. The DNA extracted from a PCR-negative sample was used as a negative control [4]. PCR-positive samples were sequenced to confirm the molecular diagnosis in the CREBIO-Jaboticabal/SP (Centro de Biotecnologia, Jaboticabal, SP, Brazil). These sequences were analyzed using Bio Edit software and aligned on BLAST to obtain sequence homology with T. vaginalis.

The samples were smeared directly on the slide using a cervical and vaginal brush. The slides were heat-fixed, Giemsa stained, and then examined under 10x and 40x magnification in a microscope. Trained and experienced personnel processed the readings of the slides. The microscopy also evaluated, besides TV infection, cellular changes, that may indicate HPV infection, as well as the presence of other microorganisms such as C. albicans and G. vaginalis.

Statistical analysis

All the data were analyzed using Statistical Package for the Social Sciences (SPSS) version 22.0 (IBM, Armonk, USA). Descriptive statistics were performed to examine the categorical variables and the results presented in proportions (%). A chi-squared test was used to compare categorical variables. The p < 0.05 was considered statistically significant. A one-tailed Z test was done to test the differences in proportions. The Clopper–Pearson exact test was done to calculate the binomial confidence interval. The independent contribution of each variable toward the chance of trichomoniasis being present was determined by multivariate analysis. All variables of the bivariate analysis were tested to construct the model. After controlling for all potential confounders, binary logistic regression was used to evaluate the association between TV infection and sociodemographic factors at p <0.05, and they were presented as odds ratio (ORs) together with confidence intervals (95% CI).

Results

Sample profile and prevalence of TV

Of 283 samples, 42 (23: questionnaire failure; 19: sampling failure) were excluded. Therefore, 241 indigenous women were screened for the study (Fig 1). The baseline characteristics showed that 81 (33.6%) were aged 18 to 29 years, 200 (83%) were less educated (less than eight years in school), 173 (71.78%) belonged to a family with income below USD 189.00 (which corresponds to minimum Brazilian wage), and 167 (69.29%) were dependent on government benefits (Family Grant). One-hundred and sixty-six (68.88%) women were from the Bororó village; of these, 73.86% were from the Guarani-Kaiowá ethnic group.

Fig 1

Sampling flowchart showing the number of samples collected and diagnosis methods performed.

An overall prevalence of TV infection using the primer set TVK3/7 was 27.8% (67/241) [95% CI: 22.2–33.9], whereas this rate dropped to 7.41% (17/241) [95% CI: 4.1–11.0] on direct microscopy (Table 1).

Table 1

Prevalence of T. vaginalis by PCR and direct microscopy and others cytologic finds in direct microscopy in indigenous women from Dourados/MS reserve.

	n	%	95% CI*
T. vaginalis (PCR)	67	27.80	22.20–33.90
T. vaginalis (DM)	17	7.41	4.10–11.00
G. vaginalis (DM)	21	8.71	5.50–13.00
C. albicans (DM)	8	3.31	1.40–6.40
Cytologic change	24	9.96	6.49–14.45
ASC-US (DM)	10	4.14	2.00–7.50
Suggestive HPV (DM)	14	5.81	3.21–9.55

*Clopper-Pearson test. PCR: Polymerase Chain Reaction. DM: Direct Microscopy, ASC-US: atypical squamous cells of unknown significance.

Direct microscopy findings

Direct microscopic analysis showed the presence of G. vaginalis and C. albicans in 21 (8.71%) [95% CI: 5.5–13.0] and 8 (3.31%) [95% CI: 1.4–6.4] women, respectively. In addition, human papillomavirus (HPV) infected lesions and atypical squamous cells of unknown significance (ASC-US) were observed in 14 (5.81%) [95% CI: 3.21–9.55]) and 10 (4.14%) [95% CI: 2–7.5]) women evaluated. The presence of concomitant infections was observed in 6 of 67 women, 3 (4.47%) with TV and G. vaginalis; 3 (4.47%) with TV and C. albicans, and 5 (7.45%) with TV and lesions suggestive of infection by HPV and ASC-US (Table 1).

Sociodemographic status and behavior characteristics

The highest proportions of TV-positive women belonged to age groups 18 to 29 years 35.08% [95% CI: 26.22–46.67], from Bororo village 33.13% [95% CI: 26.03–40.85], 34.78% [95% CI: 21.35–50.25] were unemployed, and 31.53% [95% CI: 24.76–39.37] depended on government benefits. A high prevalence of TV infection was seen in single women 39.29% [95% CI: 27.58–52.37], 34.21% [95% CI: 21.21–50.11] of them had multiple sex partners; 29.3% [95% CI: 23.31–35.88] did not use condoms, and 60% [95% CI: 14.66–94.73] had previous history of STIs. In addition, the two women who used illegal drugs were TV positive (Table 2).

Table 2

Prevalence of T. vaginalis in indigenous women in relation to sociodemographic and behavioral variables.

Variable		N	%	Positive	Negative	TV %	95% CI*
Socioeconomic characteristics
Age (years)
	18–29	81	33.60	29	52	35.80	26.22–46.67
	30–39	66	27.38	19	47	28.79	18.30–41.25
	40–49	50	20.74	14	36	28.00	16.23–42.49
	50–59	33	13.69	5	28	15.15	5.11–31.90
	≥60	11	4.56	1	10	9.09	0.23–41.28
Village
	Bororó	166	68.88	55	111	33.13	26.03–40.85
	Jaguapiru	75	31.12	12	63	16.00	8.55–26.28
Ethnicity
	Guarani (Kaiowá)	178	73.86	52	126	29.21	22.65–36.48
	Guarani (Nhandeva)	3	1.24	1	2	33.33	0.84–90.57
	Terena	40	16.60	9	31	22.50	10.84–38.45
	Outros	20	8.30	5	15	25.00	08.66–49.10
Education
	Up to 8 years	200	82.98	58	142	29.00	22.28–35.82
	Over 9 years	41	17.02	9	32	21.95	10.56–37.61
Employment status
	Employed	46	19.09	16	30	34.78	21.35–50.25
	Unemployed	195	80.91	51	144	26.15	20.14–32.91
Income (USD)/Family Unit
	< 283	173	71.78	53	120	30.63	23.86–38.08
	283–566	60	24.90	14	46	23.33	13.38–36.04
	567–1132	7	2.90	0	7	0	0–40.96
	>1133	1	0.41	0	1	0	0–97.50
Government Benefit
	Family Grant	167	69.29	53	114	31.73	24.76–39.37
	Retirement	21	8.71	5	16	22.31	08.22–47.17
	None	47	19.50	9	37	19.15	9.15–33.26
	Do not know	6	2.49	0	6	0	0–45.93
Behavioral characteristics
Marital status
	Single	56	38.50	22	34	39.29	27.58–52.37
	Married	185	61.50	45	140	24.32	18.71–30.99
History of cancer
	Yes	17	7.05	5	12	29.41	10.31–55.96
	No	224	92.95	62	162	27.68	21.93–34.03
Alcohol use
	Yes	42	17.43	12	30	29.57	15.72–44.58
	No	199	82.57	55	144	27.64	21.55–34.41
Tobacco use
	Yes	28	11.62	9	19	31.14	14.88–52.35
	No	213	88.38	58	155	27.23	21.37–33.73
Illicit drug use
	Yes	2	0.83	2	0	100.00	15.81–100.00
	No	239	99.17	65	174	27.20	21.66–33.31
Share objects (syringe and hygiene supplies)
	Yes	10	4.15	6	4	60.00	26.24–87.84
	No	231	95.85	61	170	26.41	20.84–32.59
Partners in the past 1year
	> one	203	84.23	54	149	26.60	22.10–33.07
	< two	38	16.73	13	25	34.21	21.21–50.11
Condom use in the last yearcondom use in the last year
All women
	Yes	26	10.79	4	22	15.38	4.36–34.87
	No	215	89.21	63	152	29.30	23.31–35.88
Married women
	Yes	16	8.64	1	15	6.28	0.15–30.23
	No	169	91.35	44	125	26.03	19.59–33.33
Single women
	Yes	11	19.64	4	7	36.36	10.92–69.20
	No	45	80.36	18	27	40.00	25.69–55.66
History of STIs
	Yes	5	2.07	3	2	60.00	14.66–94.73
	No	236	97.43	64	172	27.12	21.55–33.27
Urethral Discharge
	Yes	25	10.37	7	18	28.00	12.07–49.39
	No	216	89.63	60	156	27.70	21.92–34.26

*Clopper-Pearson exact.

Factors associated with TV infection

Additional analysis of the possible risk factors for TV infection showed that the indigenous women who live in the Bororó village have almost three times more risk of infection (p = 0.009), the risk is two times among single women (p = 0.043), and sharing objects increases the risk of infection by four times (p = 0.04). No statistically significant association was found with non-use of condoms (p = 0.261) and aged under 30 years (p = 0.1295). However, when combined variables were analyzed, the risk of TV infection increased four times (p = 0.005) in the group of single women aged below 30 years and three times (p = 0.000) in single women who do not use condoms (Table 3).

Table 3

Distribution of women based on dependence correlation with T. vaginalis infection.

	X2	p-value	OR	95% CI	p-value
Socioeconomic characteristics
Age < 30	3.89	0.030	1.63	0.91–2.93	0.129
Village Bororó	7.55	0.004	2.60	1.29–5.22	0.001
Schooling less than 8 years	0.84	0.237	1.45	0.65–3.23	0.467
Income less than 283.00USD	2.45	0.078	1.70	0.87–3.33	0.159
Government income dependent	4.197	0.027	2.18	0.99–4.76	0.069
Behavioral characteristics
Single women	4.79	0.023	2.01	1.07–3.79	0.043
Share objects	5.39	0.030	4.18	1.14–15.31	0.050
Do not use a condom	1.30	0.181	2.28	0.75–6.88	0.261
Singles under 30 years	9.31	0.004	4.08	1.56–10.64	0.005
Single and not using condom	13.56	0.000	3.11	1.67–5.78	0.000

Discussion

This is the first study that measures the extent of TV infection among women from an indigenous population in Brazil. Very few existing studies do not have enough data to describe the prevalence and associated factors for trichomoniasis and other STIs in this population [15-17]. Here, we observed a prevalence of 27.8% of the TV infection, which was dependent of laboratory diagnostic technique used [7]. Indeed, PCR (27.8%) and direct microscopy (7.41%) diagnosis showed a considerable difference in the prevalence of TV and might be due to factors, such as bacterial load and test sensitivity [18].

Direct microscopy is frequently used in clinical because it is inexpensive, fast, and non-labor-intensive compared with alternatives such as wet mount preparation, culture, and PCR [19]. In addition, direct microscopy is commonly used in basic health units in Brazil, for the diagnosis of cellular changes, and of pathogenic microorganisms, although with low sensitivity [20], especially in women who do not have symptoms [21]. However, a disadvantage of this convenience is the lower detection rate of direct microscopy relative to PCR as observed in our study (3.75 lower). Taking into account that most cases of TV are asymptomatic or have common symptoms among uninfected patients [19, 22], increasing even more the number of underdiagnosis cases of the disease [10], it is imperative to carry out population screening and to implement information measures on the disease as well as more specific and precise diagnostic methods [19, 22].

The results of this study are similar to the already published ones where the higher prevalence of TV is found in a highly vulnerable population: people deprived of their liberty, sex workers, and populations with low socioeconomic indexes [2, 4, 8, 9]. In this study, 71% of the women were observed to have a family income of less than 189.00 USD, which represents less than 20% of the average family income from the rest of Brazil [19, 22]. The high rates of unemployed women (80.91%), women dependent on government benefits (79.9%), or with low education (less than eight years) (83%), corroborate with other data that indicate the low socioeconomic level of this population [20, 23].

Several demographic and behavioral factors have been associated with TV infection. Our study did not show any association between level of education and family income, but an association was observed in women who depended on government benefits. Also, an association between living in the Bororó village (33.13%) and TV infection was observed, against 16% in the Jaguapiru village, showed that the risk of TV infection increased by 2.5 times in women living in the Bororó village. Data already described in the literature show that the Bororó has a lower socioeconomic index than Jaguapiru village [23], and our study found that only 13% of the women living in the Bororó village had economic activity, while this percentage rises to 32% for women living in the Jaguapiru village. Those findings might be related with the low socioeconomic index, but more studies are necessary to determine if any other factors can be involved.

We also observed that the use of condoms has a low association with TV infection in this population but is important to report that among women said they did not use condoms, 78% were married women. Considering the prevalence among the group of the married women who do not use condoms, the prevalence of TV was 26.03% and among married women who use condoms the prevalence was 6.25%. Besides that, for single women, the risk of TV infection increases by two times and a strong relationship between TV infection and single women who do not use condoms is observed. A prevalence rate of 35.80% was found in women aged below 30 years and TV infection; this number becomes higher (57.89%) when stratified women aged below 30 years and single.

Concomitant infection between TV and G. vaginalis and TV and C. albicans were found in 3 women, respectively. The concomitant infection is commonly found in cases where TV makes the vaginal environment susceptible to infection by C. albicans, and other microorganisms, such as G. vaginalis joins leading to the development of bacterial vaginosis (BV) [24-30]. The presence of atypical squamous cells of unknown significance was observed in 10 of the sample population, and HPV characteristic lesions were observed in 14 women, and among them, 5/24 of women who show lesion on the pap smear, were infected concomitantly with TV. Although HPV has not been diagnosed, the injuries indicates the presence of this viral infection [28, 31]. Some studies have investigated the relationship between BV and STIs, as well as ASC-US and other changes found in the pap smear [28, 32]. These data suggest that the overall prevalence of TV in the indigenous women evaluated may be much higher than the 27.8% found in this study.

There are a few limitations in our study. The participants were enrolled from Basic Health Units of the two indigenous reserves, which affects the generalizability of the results, because sampling is convenient, and thus possible biases may occur, for example, women who had a better chance of accessing the basic health unit, women who are more likely to seek a health service or also women who had some symptom. Sample distribution was unequal between the two reserves, and we must be careful about establishing comparisons. Another limitation is related to variables obtained through the questionnaire, once participants were reluctant to answer some questions, mainly those related to sexual behavior and the use of alcohol and illegal drugs, and thus introducing reporting biases.

In conclusion, a high prevalence of TV infection in indigenous women from two indigenous reserves in the Midwest region of Brazil was observed in this study. Certain groups such as (i) women dependent on government benefits, (ii) single women aged below 30 years who do not use condoms were most likely to have TV infection. In addition, TV infection is underdiagnosis in this population, therefore, improved STI surveillance, with the use of more accurate diagnostic methods, as well as proper management of the risk factors in different indigenous population groups, is vital for health promotion and prevention efforts.

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5 Aug 2020

PONE-D-20-17319

PREVALENCE OF SEXUALLY TRANSMITTED INFECTIONS IN INDIGENOUS BRAZILIAN WOMEN: WATCH OUT FOR Trichomonas vaginalis INFECTION

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Reviewer #1: This is a very good article, with relevant data from a population in which little is known. The data presented here have great relevance in the general context, as it is a gap in the literature and evidence knowledge that tends to generate actions in the field of public health. The article is well structured and meets the steps foreseen for a scientific article. All methodological steps were well described, identifying all the steps performed in this study.

However, there is a problem with the objectives proposed in the article. The purpose of the study does not correspond to what is developed in the body of the article. There is a focus throughout the article, from the introduction, analysis, and discussion, on the infection by trichomoniasis, but in the objectives, it informs to evaluate also other STIs, mainly viral hepatitis, HIV, and syphilis. I believe that an important decision needs to be made: better to structure and discuss the data in the light of the other sexually transmitted infections presented in the objective, or focus on the discussion in the analysis of the findings of Trichomonas vaginalis infection. The latter seems to be more viable and close to the one presented so far. Maintaining this analysis alone does not make the work of little relevance.

In addition to these indications, I present some specific considerations. Follow below:

1. Title - I didn't understand why part of the title is in capital letters and part is not.

ABSTRACT

1. “The prevalence of the 3.73% for T. pallidum and 0.41% for HIV were found in rapid tests.” Note that the prevalence of viral hepatitis was equal to 0. It is also an important fact.

2. Identify that it is a cross-sectional study and the period that the data collection occurred - "A cross-sectional study was 71 conducted between January and December 2018" was only mentioned in the article´s methodology.

3. "The prevalence of STIs, mainly TV, is among the highest in the world". About what? Among women? Compared to what other infections?

INTRODUCTION

1. “The main and most prevalent STIs described in the literature are caused by Chlamydia 42 trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis (TV), and Treponema pallidum.” This information is very important. What is the reference? Where is this described?

2. “Trichomoniasis is the most common STI caused by TV.” The information is incomplete. Where? Between who? What is the reference?

3. The focus of the introduction is on trichomoniasis. It scarcely reports on other STIs, especially viral hepatitis, HIV, and syphilis, as indicated in the study's objective.

METHODOLOGY

1. “Considering an expected prevalence of 15%” - Prevalence of what? Line 74

2. “Alere DetermineTM 118 Syphilis TP rapid test was used for the diagnosis of syphilis.” It is important to describe what this TR Identifies. Do you investigate antibodies? Do you identify if you have active (acute) syphilis or the presence of antibodies in the body (may indicate a serological scar)?

RESULT

1. “USD 283.00” - It´s important to contextualize this information for Brazilians. How was this variable categorized? What is the criterion used for this categorization? What does this mean in the Brazilian context in the period studied? Is it the minimum wage?

2. “human 156 papillomavirus (HPV) infected lesions” - was described in the results without commenting on the summary, methodology, or introduction. It is important to contextualize and present all the data that will be presented in the Results section, at least in the methodology.

3. Present the sociodemographic and behavioral variables in the methodology, describing in detail each variable and its categories.

TABLES

1. “Table 2. Distribution of indigenous women based on socioeconomic and behavioral 172 variables” - Is the prevalence of TV not described in this table? In the title, you should mention that too.

2. “Share objects” - What objects are shared?

3. “Condom use” - In which relationships? With a steady partner? With a casual partner? In what period in life? In the last 6 m? Partners in the past 1year “> one and

DISCUSSION

1. The discussion and analysis of associated factors are restricted to TV infection. The other STIs are not even mentioned. The work is actually about TV.

2. “This difference is might be due to factors, such as bacterial load and test sensitivity [12]. These factors contribute to an underdiagnosis of the disease; once direct microscopy is the only clinical diagnostic method applied [4]. ” - This finding is extremely relevant and should be better discussed. Including, to envision the impact of this in the planning in the actions in the health services.

3. “This data indicate that prevalence rates of infection increases due to socioeconomic factors in this population.” - Line 213-214. The difference in prevalence in these two regions can be explained by the difference in socioeconomic development, but from the data presented we cannot say that it is due to this. Other factors can determine this discrepancy, an example of the community viral load and STI prevalence.

4. “On the other hand, for single women, the risk increases by two times (p = 0.043) and, a strong relationship between TV infection and single women who do not use condoms (p = 0.000) is observed, increasing the risk of having TV infection (p = 0.000) by three times. ” - Line 216-219. This data should be better developed and explained. Condom use can be an important factor in the association between relationship and infection. Discuss this association better.

5. It´s important to discuss better that it is a sampling due to convenience and the bias caused by the study population comes from a health service.

6. In the important conclusion, reinforce the use of more sensitive methods for the diagnosis of VT, considering that there is a discrepancy when comparing the methods of analysis. This is an important finding in this study.

Reviewer #2: The manuscript is well written and the experimental design is adequate to the proposed objectives. The results of the study point to the high prevalence of STI, especially T. vaginalis, in the indigenous population of a region of Brazil and, although the sample size is small, it demonstrates the vulnerability of this specific population to STIs. However, the text needs some adjustments to be published.

1. In the abstract, the authors state in the methods that they will make rapid tests for hepatitis B and C but do not report any information in the results. In addition, the authors should draw some conclusions about the comparison of molecular and T. vaginalis microscopy tests.

2. In the methodology, it would be interesting if in the questionnaire the question about sexual partners included indigenous people and other people outside the community.

The authors must inform how the quality control of the DNA extraction of the samples was carried out. The DNA extracted from the culture of ATCC 30236 strain was used as a positive control of the reaction, but we need to make sure that the negative samples had viable DNA. An internal control of the reaction would be interesting, with the amplification of a human gene to guarantee the result.

The authors cite the collection of Vaginal samples (line 91) for PCR and Direct microscopic examination. They do not mention blood collection for the performance of rapid tests. Authors should add this information.

In line 116, the authors put Federal University of Espírito Santo, Vitória, Brazil after citing the rapid HIV test. Does this mean that the test was the test manufacturer?

At the end of the methodology, the authors do not mention which test was performed to detect anti-HCV.

The authors should mention in the methodology which procedures were adopted for the women in the study when the studied infections occurred

3. In results, on line 140, the authors claim that 73.86% were from the Guarani-Kaiowá ethnic group. And the rest of the group? Authors must inform ethnicities in the methodology, when mentioning Bororó and Jaguapiru village (line 79)

In line 149, hepatitis C is again cited. Authors should add hepatitis C to other topics if they want to continue with the information.

Table 1 mentions Cytologic change, however, this information is not included in any previous topic, neither in the abstract nor in the methodology. Authors should review this.

Table 1 is confused in the "n in TV PCR" column, as it concerns only T. vaginalis and in the table several other agents are mentioned. In my opinion, this description can be made in the text, without requiring a table representation. The percentage of 25.37% in T. vaginalis in Direct Microscopy was confused and has no reference in the text.

The authors do not mention the observation of C. albicans (line 155) in the abstract, nor do they mention in the methodology that they would also perform cytology in the collected cervical samples, to justify the finding of possible injury by HPV and ASC-US.

In table 2, the authors cite Ethnicity. This information must also be included in the methodology. In addition, in the table it would be interesting to detail "others" (which is written in Portuguese), because in the Guarani-Nhandeva ethnic group only 3 individuals are mentioned.

In table 2, the authors must adjust the decimal places of all percentage results.

**********

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Reviewer #1: No

Reviewer #2: No

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26 Aug 2020

Revision note

August 26th, 2020.

Dr. Joerg Heber

PLOS ONE - Editor-in-Chief

RE: PONE-D-20-17319R1

Dear Editor

Thank you very much for inviting us to submit a revised version of our manuscript entitled: “Prevalence of sexually transmitted infections in indigenous Brazilian women: watch out for Trichomonas vaginalis infection (PONE-D-20-17319)” for publication in Plos One. Indeed, the reviewers have raised a number of important concerns. We have now made a through revision of the manuscript taking into account these points, which helped in improving the quality of the manuscript. Please find bellow a point-by-point response to the reviewer’s and to your comments.

Reviewer #1:

This is a very good article, with relevant data from a population in which little is known. The data presented here have great relevance in the general context, as it is a gap in the literature and evidence knowledge that tends to generate actions in the field of public health. The article is well structured and meets the steps foreseen for a scientific article. All methodological steps were well described, identifying all the steps performed in this study.

However, there is a problem with the objectives proposed in the article. The purpose of the study does not correspond to what is developed in the body of the article. There is a focus throughout the article, from the introduction, analysis, and discussion, on the infection by trichomoniasis, but in the objectives, it informs to evaluate also other STIs, mainly viral hepatitis, HIV, and syphilis. I believe that an important decision needs to be made: better to structure and discuss the data in the light of the other sexually transmitted infections presented in the objective, or focus on the discussion in the analysis of the findings of Trichomonas vaginalis infection. The latter seems to be more viable and close to the one presented so far. Maintaining this analysis alone does not make the work of little relevance.

In addition to these indications, I present some specific considerations. Follow below:

TITLE

1. I didn't understand why part of the title is in capital letters and part is not.

R: We have corrected the existing error and adapted the title to a version of the work in which we specifically deal with TV.

“Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women” (P1/L1)

ABSTRACT

1. “The prevalence of the 3.73% for T. pallidum and 0.41% for HIV were found in rapid tests.” Note that the prevalence of viral hepatitis was equal to 0. It is also an important fact.

R: We received the comment about the possibility of maintaining the work only on TV, we accepted this suggestion and reformatted the document keeping only the data on T. vaginalis and microscopic findings.

2. Identify that it is a cross-sectional study and the period that the data collection occurred - "A cross-sectional study was 71 conducted between January and December 2018" was only mentioned in the article´s methodology.

R: We corrected the absence of the information.

“We conducted a cross-sectional study between January and December 2018,” (P2/L17)

3. "The prevalence of STIs, mainly TV, is among the highest in the world". About what? Among women? Compared to what other infections?

R: We added the information in order to supply the suggestion.

“The high prevalence TV found in this population is comparable to highly vulnerable populations, as prisoners, sex workers and women in regions with low socioeconomic levels, moreover, seems to be an underdiagnosis for this infection”. (P2/L31)

INTRODUCTION

1. “The main and most prevalent STIs described in the literature are caused by Chlamydia 42 trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis (TV), and Treponema pallidum.” This information is very important. What is the reference? Where is this described?

R: We corrected the absence of reference.

“Rowley J, Vander Hoorn S, Korenromp E, Low N, Unemo M, Abu-Raddad LJ, et al. Chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. Bull World Health Organ [Internet]. 2019 Aug 1 [cited 2020 Jan 24];97(8):548-562P.”

“WHO. Global health sector strategy on sexually transmitted infections 2016–2021. Towards ending STIs. Report. Geneva: 2016 June. Report No. WHO/RHR/16.09; 2016.”

2. “Trichomoniasis is the most common STI caused by TV.” The information is incomplete. Where? Between who? What is the reference?

R: There was an inconsistency in the writing that was corrected.

“Trichomoniasis, caused by TV, is the non-viral STI most common in the world [3, 4]” (P3/L43)

3. The focus of the introduction is on trichomoniasis. It scarcely reports on other STIs, especially viral hepatitis, HIV, and syphilis, as indicated in the study's objective.

R: We accepted this suggestion and reformatted the document, keeping only the data on TV and microscopic findings.

METHODOLOGY

1. “Considering an expected prevalence of 15%” - Prevalence of what? Line 74

R: We corrected the inconsistency in the text.

“Considering an average prevalence of T. vaginallis of 15% (4–27%)” (P4/L79)

2. “Alere DetermineTM 118 Syphilis TP rapid test was used for the diagnosis of syphilis.” It is important to describe what this TR Identifies. Do you investigate antibodies? Do you identify if you have active (acute) syphilis or the presence of antibodies in the body (may indicate a serological scar)?

R: We removed information about other STIs from the document and kept only information about TV and microscopic findings.

RESULT

1. “USD 283.00” - It´s important to contextualize this information for Brazilians. How was this variable categorized? What is the criterion used for this categorization? What does this mean in the Brazilian context in the period studied? Is it the minimum wage?

R: We consider the comment and add the information.

“Family with income below USD 283.00 (which corresponds to minimum Brazilian wage)” (P7/L148)

2. “human 156 papillomavirus (HPV) infected lesions” - was described in the results without commenting on the summary, methodology, or introduction. It is important to contextualize and present all the data that will be presented in the Results section, at least in the methodology.

R: We added information in the summary and introduction in order to remedy the deficiency in the text.

“Direct microcopy also found 21 (8.71%) and 8 (3,31%) women were infected with Gardnerella vaginalis and Candida albicans, respectively. In addition, 10 women presented atypical squamous cells of unknown significance and 14 lesions suggestive of HPV.” (P1/L27)

“The diagnosis of TV in the public health network is basically performed through direct microscopy of a sample obtained in the Pap smear test. Although the Pap smear has specificity for T. vaginalis, there are sensitivity limitations that prevent the use of this technique for the diagnosis of infection [3, 5]. The main focus of this technique is on the search for cellular changes such as atypical granular cells and squamous lesions, other findings such as Gardnerella vaginallis infection or Candida albicans, end up being in the background, which can also be related with problems in the female reproductive tract, especially the imbalance of bacterial flora, increasing the risk of infection by other STIs. [6, 7, 8]” (P2/L49)

3. Present the sociodemographic and behavioral variables in the methodology, describing in detail each variable and its categories.

R: Variables were added to the text.

“A questionnaire addressing demographic was given to the participants contemplating the following variables: village (Bororó and Jaguapirú), and ethnicities (Guarani-Kaiowa, Guarani Nhandeva, Terena, and others not predominant in the region); socioeconomic data (age, employment status, Income, government benefits, and years of schooling); clinical data: history of cancer, symptoms, and other STIs diagnoses; and behavioral factors: marital status, number of partners in the last year, alcohol use, tobacco use, illicit drug use, share objects (Syringes and personal hygiene supplies), condom use. Categorical variables were represented as “Yes” or “No”, and numerical variables were categorized later.” (P5/L90)

TABLES

1. “Table 2. Distribution of indigenous women based on socioeconomic and behavioral 172 variables” - Is the prevalence of TV not described in this table? In the title, you should mention that too.

R: We fixed the error in the table title.

“Prevalence of T. vaginalis in indigenous women in relation to sociodemographic and behavioral variables” (P9/L184)

2. “Share objects” - What objects are shared?

R: were added to the text.

“Syringes and personal hygiene supplies” (Table 2)

3. “Condom use” - In which relationships? With a steady partner? With a casual partner? In what period in life? In the last 6 m? Partners in the past 1year “> one and

R: We reformulated the paragraph in order to better describe the relationship between condom use and TV infection in this population, we also formatted table 1 to include the requested information.

DISCUSSION

1. The discussion and analysis of associated factors are restricted to TV infection. The other STIs are not even mentioned. The work is actually about TV.

R: We changed the text to keep only Infection by TV, since the information about other infections does not include enough numbers to confidently describe the risk factors.

2. “This difference is might be due to factors, such as bacterial load and test sensitivity [12]. These factors contribute to an underdiagnosis of the disease; once direct microscopy is the only clinical diagnostic method applied [4]. ” - This finding is extremely relevant and should be better discussed. Including, to envision the impact of this in the planning in the actions in the health services.

R: We discussed the result better.

“Direct microscopy is frequently used in clinical because it is inexpensive, fast, and non-labor-intensive compared with alternatives such as wet mount preparation, culture, and PCR [19]. In addition, direct microscopy is commonly used in basic health units in Brazil, for the diagnosis of cellular changes, and of pathogenic microorganisms, although with low sensitivity [20], especially in women who do not have symptoms [21]. However, a disadvantage of this convenience is the lower detection rate of direct microscopy relative to PCR as observed in our study (3.75 lower). Taking in to account that most cases of TV are asymptomatic or have common symptoms among uninfected patients [19,22], increasing even more the number of underdiagnosis cases of the disease [10], it is imperative to carry out population screening and to implement information measures on the disease as well as more specific and precise diagnostic methods [19,22].” (P12/L205)

3. “This data indicate that prevalence rates of infection increase due to socioeconomic factors in this population.” - Line 213-214. The difference in prevalence in these two regions can be explained by the difference in socioeconomic development, but from the data presented we cannot say that it is due to this. Other factors can determine this discrepancy, an example of the community viral load and STI prevalence.

R: We reformulated the statement, making it clear that this possibility can be explained by other factors not covered in the study.

“Those findings might be related with the low socioeconomic index, but more studies are necessary to determine if any other factors can be involved.” (P13/L234)

4. “On the other hand, for single women, the risk increases by two times (p = 0.043) and, a strong relationship between TV infection and single women who do not use condoms (p = 0.000) is observed, increasing the risk of having TV infection (p = 0.000) by three times. ” - Line 216-219. This data should be better developed and explained. Condom use can be an important factor in the association between relationship and infection. Discuss this association better.

R: We reformulated the paragraph in order to better describe the relationship between condom use and TV infection in this population, we also formatted table 1 to include the requested information.

“We also observed that the use of condoms has a low association with TV infection in this population (p = 0.261) but is important to report that among women said they did not use condoms, 78% were married women. Considering the prevalence among the group of the married women who do not use condoms, the prevalence of TV was 26.03%, among married women who use condoms the prevalence was 6.25% (Table 2). Besides that, for single women, the risk of TV infection increases by two times (p = 0.043), and a strong relationship between TV infection and single women who do not use condoms (p = 0.000) is observed. A prevalence rate of 35.80% was found in women aged below 30 years and TV infection (p = 0.03); this number becomes higher when stratified women aged below 30 years and single (p = 0.04) (Table 3).” (P13/L236)

5. It´s important to discuss better that it is a sampling due to convenience and the bias caused by the study population comes from a health service.

R: We insert the information in the text.

“because sampling is convenient, and thus possible biases may occur, for example, women who had a better chance of accessing the basic health unit, women who are more likely to seek a health service or also women who had some symptom.” (P14/L260)

6. In the important conclusion, reinforce the use of more sensitive methods for the diagnosis of VT, considering that there is a discrepancy when comparing the methods of analysis. This is an important finding in this study.

R: We insert the information in the text.

“TV infection is underdiagnosis in this population, therefore, improved STI surveillance, with the use of more accurate diagnostic methods,” (P14/L270)

Reviewer #2:

The manuscript is well written, and the experimental design is adequate to the proposed objectives. The results of the study point to the high prevalence of STI, especially T. vaginalis, in the indigenous population of a region of Brazil and, although the sample size is small, it demonstrates the vulnerability of this specific population to STIs. However, the text needs some adjustments to be published.

R: Dear reviewer, some of the issues raised are related to STIs other than TV, the other reviewer indicated the possibility of making the document more objective, removing this data and maintaining only TV. We chose to follow this suggestion. In this sense, the questions raised about the other STIs were removed from the document.

1. In the abstract, the authors state in the methods that they will make rapid tests for hepatitis B and C but do not report any information in the results. In addition, the authors should draw some conclusions about the comparison of molecular and T. vaginalis microscopy tests.

R: We inserted the information related to the comparison between the molecular test and microscopy. The information on other STIs were removed from the document, following suggestion from the reviewer 1.

“Therefore, a routine test program, as well as a review of the diagnostic method used, is encouraged for proper management.” (P2/L33)

2. In the methodology, it would be interesting if in the questionnaire the question about sexual partners included indigenous people and other people outside the community.

R: We accepted the suggestion rose by the reviewer, it really would be something of great value for the work, but unfortunately the issue was not addressed, as it could bring some constraints to the participants and they could deny the enrolment in the study.

The authors must inform how the quality control of the DNA extraction of the samples was carried out. The DNA extracted from the culture of ATCC 30236 strain was used as a positive control of the reaction, but we need to make sure that the negative samples had viable DNA. An internal control of the reaction would be interesting, with the amplification of a human gene to guarantee the result.

R: We carry out the quality control of the samples through spectrophotometry, agarose gel of the extracted samples and by PCR for β-globin gene. Unfortunately, the information’s were missed in the first version from the manuscript, but were added as suggested by the reviewer

“DNA was quantified using the UV-Vis Biodrop µLite Spectrophotometer (Biochrom, USA). DNA integrity was evaluated by agarose gel and by amplification of β-globin gene (268 base pairs) representing the internal control of the reaction.” (P5/L111)

The authors cite the collection of Vaginal samples (line 91) for PCR and Direct microscopic examination. They do not mention blood collection for the performance of rapid tests. Authors should add this information.

R: We follow the suggestion from the reviewer 1 and removed all the informations about other STIs from the text, thus keeping the work focused only on TV infection.

In line 116, the authors put Federal University of Espírito Santo, Vitória, Brazil after citing the rapid HIV test. Does this mean that the test was the test manufacturer?

R: Yes, it deals with the place where the test was produced, there was a small flaw in the writing. But we decided to remove information about HIV and other STIs from the manuscript.

At the end of the methodology, the authors do not mention which test was performed to detect anti-HCV.

R: In the same way as the previous questions, we remove this information, keeping only TV infection as the focus of the manuscript.

The authors should mention in the methodology which procedures were adopted for the women in the study when the studied infections occurred

R: Our study had a partnership with the basic health units and all the information obtained were summitted to the health unit of the units, where appropriate procedures were carried out. We also add this information in the text.

“The exams results were submitted to the Health Unit, where the necessary treatment measures could be taken for the patients in whom an infection was detected.” (P5/L102)

3. In results, on line 140, the authors claim that 73.86% were from the Guarani-Kaiowá ethnic group. And the rest of the group? Authors must inform ethnicities in the methodology, when mentioning Bororó and Jaguapiru village (line 79)

R: We include ethnicities in the methodology.

“Data and samples were obtained during their visit to the Basic Health Units of the two indigenous reserves evaluated: Bororó and Jaguapiru village, where Guarani (Kaiowá and Nhandeva) and Terena ethnicities predominate. (P4/L87)

In line 149, hepatitis C is again cited. Authors should add hepatitis C to other topics if they want to continue with the information.

R: We removed data about other STIs from the manuscript.

Table 1 mentions Cytologic change, however, this information is not included in any previous topic, neither in the abstract nor in the methodology. Authors should review this.

R: We added information in the summary and introduction in order to correct this issue..

“Direct microcopy also found 21 (8.71%) and 8 (3,31%) women were infected with Gardnerella vaginalis and Candida albicans, respectively. In addition, 10 women presented atypical squamous cells of unknown significance and 14 lesions suggestive of HPV.” (P1/L27)

“The diagnosis of TV in the public health network is basically performed through direct microscopy of a sample obtained in the Pap smear test. Although the Pap smear has specificity for T. vaginalis, there are sensitivity limitations that prevent the use of this technique for the diagnosis of infection [3, 5]. The main focus of this technique is on the search for cellular changes such as atypical granular cells and squamous lesions, other findings such as Gardnerella vaginallis infection or Candida albicans, end up being in the background, which can also be related with problems in the female reproductive tract, especially the imbalance of bacterial flora, increasing the risk of infection by other STIs. [6, 7, 8]” (P2/L49)

Table 1 is confused in the "n in TV PCR" column, as it concerns only T. vaginalis and in the table several other agents are mentioned. In my opinion, this description can be made in the text, without requiring a table representation. The percentage of 25.37% in T. vaginalis in Direct Microscopy was confused and has no reference in the text.

R: We removed the information that was causing confusion in table 1.

The authors do not mention the observation of C. albicans (line 155) in the abstract, nor do they mention in the methodology that they would also perform cytology in the collected cervical samples, to justify the finding of possible injury by HPV and ASC-US.

R: We added information in the summary and introduction to correct this issue in the manuscript.

“Direct microcopy also found 21 (8.71%) and 8 (3,31%) women were infected with Gardnerella vaginalis and Candida albicans, respectively. In addition, 10 women presented atypical squamous cells of unknown significance and 14 lesions suggestive of HPV.” (P1/L27)

“The diagnosis of TV in the public health network is basically performed through direct microscopy of a sample obtained in the Pap smear test. Although the Pap smear has specificity for T. vaginalis, there are sensitivity limitations that prevent the use of this technique for the diagnosis of infection [3, 5]. The main focus of this technique is on the search for cellular changes such as atypical granular cells and squamous lesions, other findings such as Gardnerella vaginallis infection or Candida albicans, end up being in the background, which can also be related with problems in the female reproductive tract, especially the imbalance of bacterial flora, increasing the risk of infection by other STIs. [6, 7, 8]” (P2/L49)

In table 2, the authors cite Ethnicity. This information must also be included in the methodology. In addition, in the table it would be interesting to detail "others" (which is written in Portuguese), because in the Guarani-Nhandeva ethnic group only 3 individuals are mentioned.

R: In the study design, the ethnic groups that are predominant were considered to be the Guarani and Terena ethnic groups, the Guarani ethnic group, in turn, is divided into two subgroups, determined Kaiowa and Nhandewa, during the questionnaire this subgroup was divided, but most of the patients declared themselves Guarani-Kaiowa. With regard to the determined group "others", these are groups that are considered to have a lower proportion within the population. This proportion was described in the methodology according to the request.

“A questionnaire addressing demographic was given to the participants contemplating the following variables: village (Bororó and Jaguapirú), and ethnicities (Guarani-Kaiowa, Guarani Nhandeva, Terena, and others not predominant in the region); socioeconomic data (age, employment status, income, government benefits, and years of schooling); clinical data: history of cancer, symptoms, and other STIs diagnoses; and behavioral factors: marital status, number of partners in the last year, alcohol use, tobacco use, illicit drug use, share objects (syringes and personal hygiene supplies), condom use. Categorical variables were represented as “yes” or “no”, and numerical variables were categorized later.” (P5/L90)

In table 2, the authors must adjust the decimal places of all percentage results.

R: It has been corrected and adjusted.

We hope that the manuscript can be now reconsidered for publication in Plos One. Please, let me know if any further change in the manuscript is required.

Yours Sincerely,

Silvana B. Marchioro (corresponding author)

DVM, PhD

Submitted filename: Response to reviewers.docx

Click here for additional data file.

15 Sep 2020

PONE-D-20-17319R1

Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women

PLOS ONE

Dear Dr. Marchioro,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Catherine E Oldenburg

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thank you for addressing the reviewers' comments. Reviewer 1 has two additional minor suggestions for the article.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The article presented here is better structured and discussed. It is indicated for publication, according to my assessment. The indications made in the first review were corrected satisfactorily, and I don´t have any more major requests.

I have only two indications to do. The title of figure 1 (Fig 1. A sampling flowchart showing the number of samples collected and diagnostic methods performed) is found in the middle of the article (line 152-153). This must appear immediately before the figure, and not alone in the middle of the text. The authors return the results data in the discussion, once again, the indication of the tables. I believe that this indication of the tables in the discussion is not necessary (lines 234 and 239), given that it was made in the results.

This is an article relevant to the field of public health and the data here exposed are well presented. I take this opportunity to thank you for the invitation and opportunity to review this work. Nothing more to say.

Kind regards,

F. S.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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17 Sep 2020

Revision Note

September 17th, 2020

Dr. Catherine E Oldenburg

Academic Editor

PLOS ONE

Dear Editor

Thank you very much for inviting us to submit a revised version of our manuscript entitled: “Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women (PONE-D-20-17319R1)” for publication in PLoS One. We reviewed the points raised by reviewer 1 in this second review. Please find bellow a point-by-point response to the reviewer’s and to your comments.

We hope that the manuscript can be now reconsidered for publication in PLos One. Please, let me know if any further change in the manuscript is required.

Yours Sincerely,

Silvana B. Marchioro (corresponding author)

DVM, PhD

Comments to the Author

Reviewer #1: The article presented here is better structured and discussed. It is indicated for publication, according to my assessment. The indications made in the first review were corrected satisfactorily, and I don´t have any more major requests.

I have only two indications to do. The title of figure 1 (Fig 1. A sampling flowchart showing the number of samples collected and diagnostic methods performed) is found in the middle of the article (line 152-153). This must appear immediately before the figure, and not alone in the middle of the text.

R: We appreciate your observation. The title of the figure was inserted in the text following the journal rules in the guide for authors. (Figure captions must be inserted in the text of the manuscript, immediately following the paragraph in which the figure is first cited (read order). Do not include captions as part of the figure files themselves or submit them in a separate document).

The authors return the results data in the discussion, once again, the indication of the tables. I believe that this indication of the tables in the discussion is not necessary (lines 234 and 239), given that it was made in the results.

R: We excluded the indication of the tables and the p-values from the discussion as suggested by the reviewer. Thanks for watching this mistake and we apologize for not fixing it in the first time.

This is an article relevant to the field of public health and the data here exposed are well presented. I take this opportunity to thank you for the invitation and opportunity to review this work. Nothing more to say.

Kind regards,

F. S.

Reviewer #2: (No Response)

Submitted filename: Responses to Revielwers.docx

Click here for additional data file.

24 Sep 2020

Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women

PONE-D-20-17319R2

Dear Dr. Marchioro,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Catherine E Oldenburg

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

7 Oct 2020

PONE-D-20-17319R2

Prevalence and factors associated with Trichomonas vaginalis infection in indigenous Brazilian women

Dear Dr. Marchioro:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Catherine E Oldenburg

Academic Editor

PLOS ONE