Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda.

BACKGROUND: Tuberculosis (TB) is the leading cause of death among people living with HIV (PLWH), and current evidence suggests that heavy alcohol users have an increased risk of developing TB disease compared to non-drinkers. Not known is whether the increased risk for TB disease among alcohol users may reflect higher rates of latent TB infection (LTBI) among this population. We assessed the latent TB infection prevalence based on tuberculin skin testing (TST) and examined association with current alcohol use among HIV-infected persons on antiretroviral therapy (ART) in south-western Uganda.
METHODS: We included PLWH at the Mbarara Regional Hospital HIV clinic, who were either current alcohol consumers (prior 3 months) or past year abstainers (2:1 enrolment ratio). Participants were recruited for a study of isoniazid preventive therapy for LTBI. TST was performed using 5 tuberculin units of purified protein derivative. The primary outcome was a positive TST reading (≥5mm induration), reflecting LTBI. We used logistic regression analyses to assess the cross-sectional association between self-reported current alcohol use and a positive TST.
RESULTS: Of the 295 of 312 (95%) who returned for TST reading, 63% were females and 63% were current alcohol drinkers. The TST positive prevalence was 27.5% (95% confidence interval [CI]: 22.6% - 32.9%). The odds of a positive TST for current alcohol users compared to abstainers was 0.76 (95% CI: 0.41, 1.41), controlling for gender, age, body mass index, history of smoking, and prior unhealthy alcohol use.
CONCLUSIONS: The prevalence of LTBI among PLWH on ART in south-western Uganda was moderate and LTBI poses a risk for future infectious TB. Although alcohol use is common, we did not detect an association between current drinking or prior unhealthy alcohol use and LTBI. Further studies to evaluate the association between LTBI and different levels of current drinking (heavy versus not) are needed.

Introduction

Globally, tuberculosis (TB) is the leading cause of mortality among people living with HIV (PLWH) [1]. In 2018, there were an estimated 0.9 million new cases of TB disease globally amongst PLWH [1] and an estimated quarter of the world’s population is infected with TB (latent TB infection, LTBI) [1-3]. Progression to active TB, either through primary progression or reactivation of LTBI is 20–30 times higher in PLWH than those without [2]. In sub-Saharan Africa (SSA), LTBI in the general population is estimated to range from 34–75% [4-6], with no difference found by HIV status in recent studies [5, 6]. However, TB preventive therapy is recommended for PLWH in high burden TB settings because of the high risk of progression to active disease [1, 2, 7]. In Uganda, the current TB incidence rate is estimated at 200 per 100,000 population. This is lower than that of South Africa, where the TB incidence rate is estimated at 520 per 100,000 but is higher than incidence reported in low TB burdened areas of less than 100 per 100,000 population [1].

Alcohol use is a leading risk factor for global disease burden and health loss [8], including TB [9], and heavy alcohol consumption is associated with a 2.5-fold increased risk of active TB disease compared to individuals who did not drink alcohol [9, 10]. This elevated TB disease risk may be attributed to a higher LTBI prevalence among people who are heavy alcohol consumers, possibly due to social factors such as frequenting bars that may increase exposure opportunity [11-13]. Alternately, excess TB disease burden may reflect a faster rate of progression from primary infection or LTBI to active disease, due to impaired immune function in heavy alcohol users [11, 14]. It is important to know if alcohol drinkers in TB- and TB/HIV-burdened areas have a higher prevalence of LTBI than non-drinkers, given that the recommendations for isoniazid preventive therapy (IPT) in low resource settings caution against IPT use in regular and heavy alcohol users [7, 15], although IPT reduces all-cause mortality among HIV-infected persons by 32–62% [16, 17] and reduces the incidence of TB reactivation beyond the benefit of using ART alone [18].

Uganda has a prevalence of HIV at 6.2% [19], and heavy alcohol consumption is estimated at 25% among PLWH in those settings [20-23]. While some literature cites no association between alcohol use and TB infection [6, 24], there is little detail specifically targeting PLWH on ART. Hence, we sought to: i) Estimate the prevalence of LTBI confirmed using the tuberculin skin test (TST) among PLWH on ART and ii) Evaluate the association between current alcohol use and TST positivity. We hypothesized that participants with self-reported current alcohol consumption have higher odds of having a positive TST.

Methods

Study setting and population

We collected data during the screening visit for the Alcohol Drinkers’ Exposure to Preventive Therapy for TB (ADEPTT) Study, which is part of the Uganda Russia Boston Alcohol Network for Alcohol Research Collaboration on HIV/AIDS (URBAN ARCH). The primary aim of the ADEPTT study is to estimate the rate of hepatotoxicity to isoniazid (6 months INH) treatment among adults (> = 18 years) who are HIV/LTBI co-infected and are current (prior 3 months) alcohol drinkers and to compare that rate to hepatotoxicity among non-drinkers (abstaining at least one year). We screened PLWH who participated in prior URBAN ARCH studies of alcohol use in the Immune Suppression Syndrome (ISS) clinic of Mbarara Regional Referral Hospital (MRRH) in south-western Uganda from 2010–2017 [25, 26].

Eligibility criteria was based on the main ADEPTT study and included: 1) age ≥ 18 years, 2) fluency in English or Runyakole (the local language), 3) HIV-infected on a non-nevirapine containing ART regimen for at least six months, 4) living within 2 hours travel time from the clinic with no plans to move, 5) no evidence of active TB infection based on WHO symptoms criteria, 6) no prior use of TB medicines for treatment and/or prevention of TB, 7) being either a self-reported current (prior 3 month) drinker or abstainer (at least one year since drinking), 8) having alanine aminotransferase [ALT] and aspartate aminotransferase [AST] ≤ 2x of the upper limit of normal (ULN), 9) women who were pregnant.

Study procedures and variables collected

The data for the analyses came from three sources: the ADEPTT screening process, prior URBAN ARCH research data, and the ISS clinic database. Data were linked via study and clinic identification numbers.

Tuberculin Skin Testing (TST)

The TST was conducted by placing 0.1 ml containing 5 tuberculin units of purified protein derivative (PPD) (Tubersol; Sanofi Pasteur Limited, Toronto, Ontario, Canada) on participants’ forearms using the Mantoux method. The indurations were read by trained clinical research assistants (CRAs) and cross-checked by another CRA using a TST tape measure between 48 and 72 hours after placement. TST may have reduced sensitivity in immune-compromised individuals such as those with HIV infection [27], nonetheless, in this study, an induration of 5mm or greater was considered a positive TST result (the cut off recommended for those with HIV) [28]. WHO recommends using either TST or interferon-gamma release assays (IGRA) [1] for latent TB detection.

Outcome variable

The outcome measure was a positive TST result during ADEPTT screening, as described above.

Independent variable

The primary predictor of interest was current alcohol consumption, defined as self-reported alcohol use within 3 months prior to the TST. This variable was obtained by self-report during the ADEPTT screening procedures.

Covariates

Gender (male, female) was collected during ADEPTT screening. Data from prior URBAN ARCH study visits included lifetime history of smoking (never versus ever) and “prior unhealthy alcohol use”. Prior unhealthy alcohol use was determined using the Alcohol Use Disorders Identification Test—Consumption (AUDIT-C), which was modified to ask participants to answer the AUDIT-C questions based on the period in their life during which they drank the most. Those meeting AUDIT-C cut-offs for this prior drinking period (> = 3 for women and > = 4 for men, [29]) were considered prior unhealthy drinkers. These measurements from prior URBAN ARCH study visits were collected 3–5 years before the screening visit for ADEPTT.

Date of birth, marital status, ART initiation date, body mass index (BMI) and most recent HIV viral load were linked from ISS clinic data. Age and duration of ART at TST placement were calculated from these data. Age was categorized as <35 or ≥35 years; BMI was categorized as low (<18.5), normal (18.5–24.9), overweight (25–29.9), and obese (≥30); viral load was categorized as ≤1000 and >1000 copies/ml.

Statistical methods

We conducted descriptive analyses of all variables, overall and by self-reported current alcohol use. We calculated frequencies and proportions for categorical variables; we calculated means and standard deviations (std dev) and medians with interquartile ranges (IQR) for continuous variables. We ran unadjusted logistic regression models and multivariable logistic regression models to assess the association between current alcohol use and a positive TST. The multivariable model controlled for age, gender, BMI, smoking history, and prior unhealthy alcohol use. These covariates were chosen for inclusion in the multivariable analysis a priori based on literature of the risk factors that may increase TB disease cases [1, 30]. We also conducted exploratory analyses to assess whether the association between alcohol use and TST positivity differed by gender, and present multivariable results stratified by gender. We also compared the distributions of gender, age, BMI and alcohol use of the persons that were included and those screened but excluded either because they did not meet the main study inclusion criteria or did not return for TST reading on time.

Ethical considerations

Written informed consent was sought from participants before screening. The study activities were approved by the Ethics review boards of Mbarara University of Science and Technology (#11/10-16), The Uganda National Council of Science and Technology (# HS 2183), University of California San Francisco (# 16–19093), and Boston University (# H-35809).

Results

Characteristics of the study participants

We approached 418 prior URBAN ARCH participants for ADEPTT Study screening; 106 persons were excluded for reasons that were not mutually exclusive, including: being on nevirapine (n = 31), having had previous active TB (n = 14), having previously taken TB medicines for LTBI (n = 25), having consumed alcohol in the past year but not the past 3 months (n = 8), having elevated baseline AST and/or ALT results (n = 18), living out of catchment area (n = 15), not on ART equal or more than 6 months (n = 1), pregnant (n = 2), not cleared of active TB (n = 2) and declining participation (n = 7). 312 participants received a TST. Seventeen participants did not return for PPD reading within 72 hours, leaving 295 for this analysis (Fig 1). A comparison between the 295 persons included and the 123 persons excluded from the analyses did not show any difference by alcohol consumption, age, gender, or BMI.

Fig 1

Eligibility flow.

Of the 295 who met inclusion criteria, 63% were female, median age was 38 years (IQR: 32 to 45), 56% were married, 92% had been on ART for at least one year, 97% were virally suppressed (≤1000 RNA copies), and median BMI was 23.3 (IQR: 20.8 to 27.5) (Table 1). Seventeen percent reported ever smoking in their lifetime. Approximately two-thirds (63%) were self-reported current alcohol drinkers, and a similar proportion (57%) were prior unhealthy alcohol users. Eighty-one percent (n = 87) of males were self-reported current drinkers, compared to 54% (n = 100) of the females (Table 1). Among those who reported prior unhealthy alcohol use, 79% (n = 127) self-reported current drinking during ADEPTT screening, compared to 45% (n = 55) of those without prior unhealthy alcohol use.

Table 1

Characteristics of eligible study participants in south-western Uganda, overall and by self-reported alcohol use at screening (n = 295).

	N (%)	Self-reported alcohol abstainers (at least 12 months) (n = 108)	Self–reported current (prior 3 months) alcohol users (n = 187)
TST result a
TST-positive (≥5 mm induration)	81 (27.5)	32 (39.5)	49 (60.5)
TST-negative (<5 mm induration)	214 (72.5)	76 (35.5)	138 (64.5)
Sex a
Male	108 (36.6)	21 (19.4)	87 (80.6)
Female	187 (63.4)	87 (46.5)	100 (53.5)
Age (median (IQR))b	38 (32–45)	40 (32.5–48)	37 (32–43)
<35	103 (34.9)	33 (32.0)	70 (68.0)
≥35	192 (65.1)	75 (39.1)	117 (60.9)
Smoking history (lifetime) c
Ever	50 (17.3)	15 (30.0)	35 (70.0)
Never	239 (82.7)	88 (36.8)	151 (63.2)
Body Mass Index (median (IQR))b	23.3 (20.8–27.5)	23.5 (20.8–27.5)	23.1 (20.8–27.6)
Low (<18.5)	24 (8.5)	9 (37.5)	15 (62.5)
Normal (18.5–24.9)	149 (52.8)	54 (36.2)	95 (63.8)
Overweight (25–29.9)	70 (24.8)	26 (37.1)	44 (62.9)
Obese (> = 30)	39 (13.8)	14 (35.9)	25 (64.1)
Marital status b
Not married	128 (43.8)	53 (41.4)	75 (58.6)
Married	164 (56.2)	54 (32.9)	110 (67.1)
Prior unhealthy alcohol use c
No	123 (43.3)	68 (55.3)	55 (44.7)
Yes	161 (56.7)	34 (21.1)	127 (78.9)
Duration of ART b
< 1 year	23 (7.8)	8 (34.8)	15 (65.2)
> = 1 year	272 (92.2)	100 (36.8)	172 (63.2)
HIV viral load (median (IQR)) b	75 (20–75)	75 (20–75)	75 (40–75)
< = 1000	287 (97.3)	104 (36.2)	183 (63.8)
>1000	8 (2.7)	4 (50.0)	4 (50.0)

a ADEPTT Study screening data;

b ISS Clinic data;

c Prior URBAN ARCH study visit data.

TST results

Of the 295 participants, 81 were TST positive (27.5%, 95% confidence interval [CI]: 22.6%, 32.9%). Among the current drinkers, 49 out of 187 (26%) were TST positive, while 32 out of 108 (30%) of abstainers were TST positive (Table 2). The unadjusted odds of a positive TST among current drinkers compared to abstainers was 0.84 (95% CI: 0.50, 1.43). The unadjusted odds of a positive TST among prior unhealthy alcohol users compared to participants with no prior unhealthy alcohol use was 1.08 (95% CI: 0.64, 1.82) (Table 2).

Table 2

Unadjusted and adjusted Odds Ratios (OR) and 95% confidence intervals (95% CI) for TST-positive results among study participants who completed TST screening (n = 295).

	TST-positive a (n = 81) N (%)	TST-negative a (n = 214) N (%)	Unadjusted OR (95% CI)	p-value	Adjusted OR (95% CI)	p-value
Alcohol use–self-report at screening a				0.53		0.39
Abstainer (≥12 months)	32 (29.6)	76 (70.4)	1.00		1.00
Current drinker (≤3 months)	49 (26.2)	138 (73.8)	0.84 (0.50, 1.43)		0.76 (0.41, 1.41)
Sex a				0.24		0.35
Male	34 (31.5)	74 (68.5)	1.00		1.00
Female	47 (25.1)	140 (74.9)	0.73 (0.43, 1.23)		0.73 (0.37, 1.42)
Age b				0.73		0.98
<35 years	27 (26.2)	76 (73.8)	1.00		1.00
≥35 years	54 (28.1)	138 (71.9)	1.10 (0.64, 1.89)		1.01 (0.56, 1.80)
BMI b				0.17		0.19
Low (<18.5)	11 (45.8)	13 (54.2)	1.00		1.00
Normal (18.5–24.9)	35 (23.5)	114 (76.5)	0.36 (0.15, 0.88)		0.38 (0.15, 0.94)
Overweight (25–29.9)	19 (27.1)	51 (72.9)	0.44 (0.17, 1.15)		0.50 (0.18, 1.40)
Obese (> = 30)	11 (28.2)	28 (71.8)	0.46 (0.16, 1.34)		0.57 (0.18, 1.82)
BMI (continuous) (median (IQR)) b	23.0 (20.2–27.5)	23.4 (21.0–27.6)	1.00 (0.95, 1.05)	0.96	-
Prior unhealthy alcohol use c				0.77		0.66
No	34 (27.6)	89 (72.4)	1.00		1.00
Yes	47 (29.2)	114 (70.8)	1.08 (0.64, 1.82)		1.14 (0.63, 2.07)
Smoking history c				0.30		0.80
Never	64 (26.8)	175 (73.2)	1.00		1.00
Ever	17 (34.0)	33 (66.0)	1.41 (0.73, 2.70)		1.10 (0.53, 2.31)

a ADEPTT Study screening data;

b ISS Clinic data;

c Prior URBAN ARCH study visit data.

In multivariable analysis, the adjusted odds ratio [aOR] of a positive TST for current alcohol users compared to abstainers was 0.76 (95% CI: 0.41, 1.41) after controlling for gender, age, BMI, prior unhealthy alcohol use and history of smoking (Table 2). There was also no evidence of an association between positive TST and prior unhealthy alcohol use (aOR 1.14, 95% CI: 0.63, 2.07). The relationship between current alcohol use and a positive TST did not appear to differ by gender (p-value for interaction = 0.86): aOR: 0.68 (95% CI: 0.22, 2.05) for males and aOR: 0.89 (95% CI: 0.41, 1.91) for females (stratified analyses, Table 3).

Table 3

Adjusted Odds Ratios (OR) and 95% confidence intervals (95% CI) for TST-positive results among study participants who completed TST screening (n = 295), stratified by sex.

	MALES a (n = 108)		FEMALES a (n = 187)
	Adjusted OR (95% CI)	p-value	Adjusted OR (95% CI)	p-value
Alcohol use–self-report at screening a		0.49		0.76
Abstainer (≥ 12 months)	1.00		1.00
Current drinker (≤3 months)	0.68 (0.22, 2.05)		0.89 (0.41, 1.91)
Age b		0.10		0.28
<35 years	1.00		1.00
≥35 years	0.43 (0.16, 1.18)		1.50 (0.72, 3.13)
BMI b		0.08		0.43
Low (<18.5)	1.00		1.00
Normal (18.5–24.9)	0.37 (0.11, 1.21)		0.32 (0.07, 1.42)
Overweight (25–29.9)	1.20 (0.27, 5.39)		0.31 (0.07, 1.48)
Obese (> = 30)	-		0.45 (0.09, 2.21)
Prior unhealthy alcohol use c		0.58		0.85
No	1.00		1.00
Yes	1.34 (0.48, 3.75)		1.07 (0.50, 2.29)
Smoking history c		0.65		0.93
Never	1.00		1.00
Ever	1.25 (0.47, 3.33)		0.95 (0.27, 3.32)

a ADEPTT Study screening data;

b ISS Clinic data;

c Prior URBAN ARCH study visit data

Discussion

We found that LTBI prevalence among HIV-infected persons on ART in south-western Uganda was 27.5% (95% CI: 22.6%, 32.9%). This was a little lower than the prevalence reported in other LTBI based studies in SSA that ranged from 34–75% [4-6]. Differences may be attributed to variations in TB burden in different communities and differing TB infection control practices. At the ISS clinic from which research participants were recruited for this study, there is routine clinical screening for TB symptoms, isolation of TB suspects to a designated space, and expedited referral for TB investigation and treatment procedures for confirmed TB cases.

We found no differences between the odds of a positive TST among HIV-infected current alcohol users compared to abstainers in crude analyses or after controlling for age, gender, BMI, smoking history, and prior unhealthy alcohol use. This study provides descriptive information and the first estimates of LTBI among PLWH by current alcohol use. While there is literature on the association between heavy alcohol use, alcohol use disorders, and TB disease [9, 10, 14, 31, 32], this study evaluates the association between alcohol use and LTBI. It is possible that the higher rates of active TB disease referenced in unhealthy alcohol users may more likely be primary TB than reactivation given a higher likely exposure in bars and congested social drinking venues [11, 13]. However, it has also been observed that unhealthy alcohol consumption may disrupt some immune pathways and render reactivation to active TB disease more likely [11, 14]. Our data, which showed only the prevalence of latent TB infection, was not sufficient to allow us to examine these pathways. Further research in this area is needed.

We additionally found no relationship between prior unhealthy alcohol use and TST positivity. This result contrasts the higher odds of LTBI in drinkers in China and India [33, 34], but is consistent with results found in South Africa [6, 24]. This may suggest that ‘any’ current and/or prior unhealthy alcohol use may be playing a lesser role in determining the body’s response to a tuberculin test among PLWH on ART. The findings may be influenced by the lack of information on the frequency, magnitude and timing of current alcohol use. Although there may be a possibility that TST negative participants had not yet mounted a persistent immune response to mycobacterium tuberculosis bacteria, this may not be the case in our study participants who had been on antiretroviral therapy (ART) for more than 6 months and more than 95% of them were virally suppressed. Our work highlights the need to further examine how different levels of current alcohol use impact the risk of latent TB infection and progression to disease among drinkers in multiple settings.

This study has several limitations. The sample size was relatively modest due to multiple exclusion criteria for the parent study that screened out various groups of participants such as those with elevated alanine and aspartate aminotransferase—ALT and AST) more than 2 times the upper limit of normal. This was for the participants’ safety as the INH to be given is potentially hepatotoxic. Since heavy alcohol drinkers have a higher risk of having elevated liver function tests, some might have been potentially eliminated at that point which may potentially affect generalizability. A comparative analysis between the participants included and those excluded from the analyses did not show any difference by alcohol consumption, age, gender, or BMI. In addition, self-reported alcohol consumption may be subject to recall bias. Our primary variable of current drinking was collected for study recruitment purposes, rather than to detect unhealthy drinking. Another limitation is that our additional variable to represent prior unhealthy drinking as well as some other variables of interest were collected 3–5 years prior. The median duration since last URBAN ARCH visit was 44.6 months (IQR: 36.2–53.9). Thus, the duration of time relative to TST assessment is inconsistent across the exposure variables and “lifetime” smoking and prior unhealthy alcohol use may be subject to misclassification due to the time lag. Although TST may not be a perfect test for LTBI, it is accepted by WHO [1] as one of the tests to detect latent TB. Both TST and interferon-gamma release assays (IGRA) perform reasonably well in high-TB burden settings and correlate well with proxy measures of exposure to mycobacterium tuberculosis among the HIV-infected persons and contacts exposed to smear positive index cases [35]. Additionally, the concern in literature about TST’s reduced sensitivity among the immunocompromised [27] was minimal in this study since the participants were on ART for more than six months and were clinically and virally stable. False negative results could also arise from the window period (2–8 weeks) before development of cell-mediated immunity estimated just after exposure to mycobacteria.

In summary, we found that LTBI by TST response among HIV-infected persons on ART in south-western Uganda was similar to global statistics though slightly lower than in prior SSA studies. Although prior unhealthy alcohol use existed in more than half the cohort, we did not detect an association between current alcohol use and LTBI. More research is needed to determine the effect of various magnitudes of alcohol drinking and LTBI as well as the mechanisms for increased TB disease among drinkers in the literature.

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10 Mar 2020

PONE-D-19-35988

Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda

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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: No

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

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. General comment: This manuscript generally adds to the body of knowledge on issue of LTBI. The authors need to explain the policy implications of their results given TB preventive therapy (TPT) is being recommended for all people with HIV. At a prevalence of 27.5% in the study population, do we seem to be recommending to 72.5% that do not need it. Or it is also possible that the 72.5% had not yet mounted a good immune response to reflect positivity?

2. Line 56: The authors should reference the latest global TB report (2019)

3. Line 64: Authors advised to cross check the latest incidence. It has come down lately

4. Line 166: Authors should indicate the IRB approval numbers

5. Line 173: The breakdown of the participants screened doesn’t total up to 106. Totals up to 118

6. Line 177: 418 minus total excluded= 300 not 312. Authors should crosscheck these figures in the section 172-181

7. Tables 2 and 3: The authors need to explain why they included all the factors in the multivariate analysis yet they were insignificant at bivariate? If these had biological plausibility, then they need to mention it and provide the appropriate references

8. Lines 226-237: Paragraph is too long. Could be split into 2 i.e. prevalence of LTBI and then the paragraph on odds of TST positivity given alcohol use history. The authors need to explain why there was no association between TST positivity among HIV infected current alcohol users and yet literature shows alcohol consumption is a big risk factor for TB. Is it possible that the TB among alcohol users is the primary TB instead of reactivation?

Reviewer #2: The introduction and abstract were generally clear and readable. A minor stylistic note is not to report results that are not findings of the study without reference, Since it is not practise to reference in abstracts perhaps it would be useful to say “there is evidence to suggest alcohol users have an increased risk of developing disease...” {leave it in intro line 72} There is a clear outline of the aim and synopsis of what is known and what is not known this is good. The purpose and hypothesis are also clearly defined. In the Methods section eligibility and sample are well described. The reliability and validity of using TST as a primary instrument are described in the discussion but should appear in the methods. It would be useful to have the complete definitions for “prior unhealthy” consumption. Systematic bias maybe introduced by only including TST screened for LTBI prevalence estimate. TST. There may be a potential systematic reason why these people did not present (too ill/heavy drinkers). The result tables are clear and readable. There is no stratification by alcohol use, the independent variable, it is an all or nothing measure. Therefore, the evidence is not sufficient for conclusions drawn in line 245 -247{This may suggest that alcohol use may be playing a lesser role in acquiring TB infection among PLWH on ART, compared to its role in TB disease progression}. These are merely measuring of association the lack of sample decreased the statistical value of these measures. For example, men report drinking (87%) but there are less men are in the study (37%). Drawing any links between alcohol use and TST response seem tenuous at best.

**********

6. 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.

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Reviewer #1: No

Reviewer #2: No

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27 Apr 2020

Re: Responses to Manuscript PONE-D-19-35988: “Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda”

Thank you all for the valuable time you spent reviewing our manuscript and the very thoughtful suggestions given to us. We also appreciate the opportunity to respond to the editor’s and reviewer’s recommendations. Below, please find a summary of the editor and reviewer’s comments and our responses to indicate how each point is addressed in the revised manuscript.

The Academic Editor gave the following communication:

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Response: We have made adjustments to the following sections of the manuscript: The acknowledgments and the disclosure of conflict of interest. The details are included in our response to number 3 under Journal requirements.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future.

Response: This is not applicable to our manuscript. This paper is based on a standard clinical screening procedure for the main study enrolling participants into a tuberculosis preventive therapy study using Isoniazid (INH). The clinical procedure conducted on each participant was a tuberculin skin test (TST) to detect latent tuberculosis infection (LTBI). For this clinical procedure, we used steps on a CDC manual chart for tuberculin testing (Reference #27 in the manuscript).

The Journal requirements to be addressed included:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have read and abided by the style requirements for PLOS ONE including those for file naming. We have made some adjustments to the paper including: changes on the title page, changing manuscript text to double spacing and limiting the manuscript sections and sub-sections to 3 clear heading levels.

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

Response: This paper is based on screening steps before enrollment into the main tuberculosis prevention study. The questionnaires for the 5 screening steps in English and the one step which was translated into Runyankole (step 1) will be submitted as Supporting Information.

3. Thank you for stating the following in the Competing Interests section:

"I have read the journal's policy and the authors of this manuscript have the following competing interests: [Dr Debbie Cheng serves on Data Safety and Monitoring Boards for Janssen. The remaining authors have no conflicts of interest to declare."

We note that one or more of the authors are employed by a commercial company: Janssen

3.1 Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Response: We have updated the financial disclosure statement in the revised manuscript to read: All authors have no conflicts of interest to declare. (line 316)

Although Dr Debbie Cheng serves on Data Safety and Monitoring Boards for Janssen (as a consultant, not employee), after further evaluation, because Dr Cheng’s presence on a Data Safety Monitoring Board on a Janssen study for Crohn’s Disease is not in any way related to the TB preventive study from which this paper is written and does not in any way interfere with any steps in the research work from which this manuscript is derived, nor do we believe it interfered with the objective presentation of the peer review, we have hence removed that detail from this paper.

The amended online Funding Statements may include: “The funders (NIAAA) provided support in the form of effort based salaries for the following authors [WM, RF, DC, NE, CN, JA, BL, KJ, JH (NIAAA) and NIAID to DC only, but did not have any role in the study design, data collection and analysis, decision on where to submit for publication, nor preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

3.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Response: We have updated the financial disclosure statement as mentioned in response to 3.1. We have also adjusted the acknowledgments section in the manuscript (lines 309-311) to read: This study was supported by Grants from National Institute on Alcoholism and Alcohol Abuse: U01 AA020776 and K24 AA022586 (PI: Hahn); U24AA020779 (PI: D Cheng) and NIH/NIAID P30AI042853 (support to D Cheng).

We apologize for the misquote in the previous paper. Debbie Cheng is not the PI of the NIH/NIAID P30AI042853 grant although she is supported by this grant whose PI is Dr S Cu-Uvin.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

Response: We have consulted with the Institutional Review Boards and Committees that approved the main study. We find that there are no legal restrictions on sharing a de-identified data set once all the partners have agreed. We have prepared an anonymized dataset of the 295 participants on whom detailed analysis was done.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Response: We have prepared an anonymized dataset of the 295 participants on whom detailed analysis was done. This will be submitted as a Supporting Information file.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Response: The corresponding author has an ORCID iD in the Editorial Manager (0000-0002-6694-2645). We thank you for sharing the link to the elaborate video for instructions given. This has been very helpful.

Responses specific to Reviewer #1’s comments:

1. General comment: This manuscript generally adds to the body of knowledge on issue of LTBI. The authors need to explain the policy implications of their results given TB preventive therapy (TPT) is being recommended for all people with HIV. At a prevalence of 27.5% in the study population, do we seem to be recommending to 72.5% that do not need it. Or it is also possible that the 72.5% had not yet mounted a good immune response to reflect positivity?

Response: As noted in the manuscript, the 27.5% prevalence is only somewhat lower than other estimates of LTBI prevalence in similar settings. Although there is a possibility that some of the 72.5% were actually latently infected with TB but had not yet mounted persistent immune response to mycobacterium tuberculosis bacteria, we think that this is likely to be minimal because our study participants had been on antiretroviral therapy (ART) for more that 6 months and more than 95% of them were virally suppressed. We have now added this to the discussion (lines 272-276).

Because our findings are consistent with others’ and because our primary goal was to examine LTBI among those who consume alcohol compared to those who do not, there are no significant policy implications. We do not dispute the global call to expand access to TB preventive therapy because TB is the world’s top infectious disease killer from a single disease agent (WHO Global Tuberculosis Report 2019).

2. Line 56: The authors should reference the latest global TB report (2019)

Response: We have referenced the World Health Organization Global TB Report (2019) as Reference 1. We thank you for the advise to add this reference to our paper.

3. Line 64: Authors advised to cross check the latest incidence. It has come down lately

Response: The authors have checked the latest incidence from the WHO Global TB report 2019 and replaced the old incidence rates quoted in old paper with the latest incidence. This appears in lines 66 to 68 of the revised manuscript.

4. Line 166: Authors should indicate the IRB approval numbers

Response: Thank you for this communication. We have indicated the IRB approval numbers for the main study (lines 179-181 in the revised manuscript).

5. Line 173: The breakdown of the participants screened doesn’t total up to 106. Totals up to 118

Response: The authors appreciate this feedback. We have revised this section of the results to add a statement indicating that the reasons for excluding the 106 participants were not mutually exclusive (line 187). Some participants had more than one reason for exclusion and although counted under each exclusion criteria, the aggregated exclusion total counted each individual once. The full description is given in Figure 1.

6. Line 177: 418 minus total excluded= 300 not 312. Authors should crosscheck these figures in the section 172-181

Response: We apologize for the previous lack of clarity in line 177 of the old paper. With the response given in 5 above, we hope it is clearer how we came up with 312 as the participants who received TST (418 -106 = 312).

7. Tables 2 and 3: The authors need to explain why they included all the factors in the multivariate analysis yet they were insignificant at bivariate? If these had biological plausibility, then they need to mention it and provide the appropriate references

Response: We included several variables in the multivariate analysis model apriori (even when they were non-significant in the bivariate analysis) as we were concerned about confounding, which is not defined based on p-values. All of these variables have biological plausibility, that is, are documented high risk factors for progression to active TB (Refs 1,2,15), thus they were included regardless of statistical significance.

8. Lines 226-237: Paragraph is too long. Could be split into 2 i.e. prevalence of LTBI and then the paragraph on odds of TST positivity given alcohol use history. The authors need to explain why there was no association between TST positivity among HIV infected current alcohol users and yet literature shows alcohol consumption is a big risk factor for TB. Is it possible that the TB among alcohol users is the primary TB instead of reactivation?

Response: We have split the previous long paragraph into 2 as advised. The first paragraph now occupies lines 244-251. The second paragraph has been expanded to include an explanation of the study findings compared to literature (lines 252-265).

It is possible that higher rates of active TB disease referenced in unhealthy alcohol users is more likely primary TB than reactivation given a higher likely exposure in bars and congested social drinking venues (Refs 11,13). However, it has also been observed that unhealthy alcohol consumption may disrupt some immune pathways and render reactivation to active TB disease more likely (Refs 11,14). Our data, which showed only the prevalence of latent TB infection, was not sufficient to allow us to examine these pathways. Further research in this area is needed.

Responses Specific to Reviewer #2’s comments:

The introduction and abstract were generally clear and readable. A minor stylistic note is not to report results that are not findings of the study without reference, Since it is not practise to reference in abstracts perhaps it would be useful to say “there is evidence to suggest alcohol users have an increased risk of developing disease...” {leave it in intro line 72}

Response: Thank you for this guidance. We have made the change advised in the abstract (line 31) but left it in the introduction, line 72 of revised manuscript).

There is a clear outline of the aim and synopsis of what is known and what is not known this is good. The purpose and hypothesis are also clearly defined. In the Methods section eligibility and sample are well described. The reliability and validity of using TST as a primary instrument are described in the discussion but should appear in the methods.

Response: We have moved the sentences describing the reliability and validity of using TST as a primary instrument from the discussion to the methods sections. These now appear in lines 131-136 within the methods section.

It would be useful to have the complete definitions for “prior unhealthy” consumption.

Response: We have expanded the definition for ”prior unhealthy” consumption in lines 147-151 of the methods section of the revised manuscript.

The study defined prior unhealthy consumption (based on prior URBAN ARCH data) using the Alcohol Use Disorders Identification Test- Consumption (AUDIT-C), and modified those questions to ask about the period of the participant’s life during which they drank the most. Then, we used the normal cutoffs (>=3 for women, >=4 for men) to classify people as “prior unhealthy” drinkers. This description is in the “covariates” section.

Systematic bias maybe introduced by only including TST screened for LTBI prevalence estimate. There may be a potential systematic reason why these people did not present (too ill/heavy drinkers).

Response: We note the valid concern raised. We may not rule out the systematic bias completely since the steps to determine eligibility were hinged towards the main TB preventive study. For instance, to be approached for screening, one had to be on ART for equal or more than 6 months. Given this pre-set eligibility, most participants were stable clinically, regardless of whether they were heavy drinkers or not. We screened participants who participated in prior URBAN ARCH studies of alcohol use which included all spectrum from mild to heavy alcohol users.

We also acknowledge the fact that the parent study eligibility criteria included having liver function tests (specifically alanine and aspartate aminotransferase - ALT and AST) less or equal to 2 times the upper limit of normal. This was for the participants’ safety as the INH to be given is potentially hepatotoxic. Since heavy alcohol drinkers have a higher risk of having elevated liver function tests, some might have been potentially eliminated at that point. We have added this to the study limitations lines 281-285.

The result tables are clear and readable. There is no stratification by alcohol use, the independent variable, it is an all or nothing measure. Therefore, the evidence is not sufficient for conclusions drawn in line 245 -247 {This may suggest that alcohol use may be playing a lesser role in acquiring TB infection among PLWH on ART, compared to its role in TB disease progression}. These are merely measuring of association the lack of sample decreased the statistical value of these measures. For example, men report drinking (87%) but there are less men are in the study (37%). Drawing any links between alcohol use and TST response seem tenuous at best.

Response: Thank you for this suggestion. In this study, we found no difference in LTBI prevalence in current or prior unhealthy alcohol users compared to abstainers. We have removed the bracketed statement above and replaced it with: this suggests that “any” current alcohol use and/or prior unhealthy alcohol use may be playing a lesser role in determining the body’s response to a tuberculin test among PLWH on ART (lines 268-270) . In another new study being done among HIV infected patients in care, our team of research partners will assess the prevalence and correlation of LTBI among current heavy alcohol drinkers.

6. 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.

Response: This has been noted. Thank you very much for sharing this information.

Thank you again for the consideration and review of our manuscript. We hope that with these revisions, the manuscript is now suitable for publication in PLOS ONE.

Submitted by;

Dr Winnie R Muyindike

Mbarara University of Science and Technology/Referral Hospital, Mbarara, Uganda

Email: wmuyindike@gmail.com

Phone: +256 772 521619

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

12 May 2020

PONE-D-19-35988R1

Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda

PLOS ONE

Dear Dr. Muyindike,

Thank you for submitting your manuscript to PLOS ONE. We provisionally accept the manuscript, but would like to give you the opportunity to address the final minor comments raised by the reviewer. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Nicky McCreesh

Academic Editor

PLOS ONE

[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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Line 31: Comments have been addressed, but I would still add “….current evidence…”

Line 131-136: Comments have been addressed, but phrasing is still in discussion form, maybe best to some of it in the discussion as a limitation of this work.

(line 131) “TST may not be a perfect test for LTBI but it is widely accepted as a reasonable proxy [28] and”

AND

(line 134) “Both tests perform reasonably well in high-TB burden settings and correlate well with proxy measures of exposure [28].” I would move back to discussion and expand.

I would also move the sentences around in the method section (line 134/5) “TST may have reduced sensitivity in immune-compromised individuals such as… “ before the sentence starting (line 130) “A TST…”

281-285 comments have been addressed, but I would leave out (line 286) “However, the exclusions do not introduce a systematic bias that we see.”

line 245 -247 comments were addressed.

**********

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.

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

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment 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 Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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.

7 Jun 2020

Responses specific to Reviewer #1’s comments:

Comment to question, “Have the authors made all data underlying the findings in their manuscript fully available? reviewer 1 said No:

Response: We have prepared an anonymized dataset for all the 418 participants screened prior to conducting the tuberculin skin test (TST). The dataset that is attached on the 2nd resubmission includes those included for detailed analysis (295) and those excluded from the detailed analysis (123) who didn’t meet the criteria for inclusion for detailed analysis. This will be submitted as a Supporting Information file.

Responses specific to Reviewer #2’s comments:

Line 31: Comments have been addressed, but I would still add “….current evidence…”

Response: We have added the word ‘current’ in the suggested position in line 31

Line 131-136: Comments have been addressed, but phrasing is still in discussion form, maybe best to some of it in the discussion as a limitation of this work. (line 131) “TST may not be a perfect test for LTBI but it is widely accepted as a reasonable proxy [28] and”

and (line 134) “Both tests perform reasonably well in high-TB burden settings and correlate well with proxy measures of exposure [28].” I would move back to discussion and expand.

Response: We have moved the suggested parts back in the limitations paragraph of the discussion and expanded. The previous line 131 and 134 now appear in the limitations under discussion, lines 292 to 299.

I would also move the sentences around in the method section (line 134/5) “TST may have reduced sensitivity in immune-compromised individuals such as… “ before the sentence starting (line 130) “A TST…”

Response: We have moved the suggested sentences around in the methods section. The previous lines 134/5 is now line 131. It has been inserted before the previous sentence which described the 5mm reading for a positive TST which is now line 132.

281-285 comments have been addressed, but I would leave out (line 286) “However, the exclusions do not introduce a systematic bias that we see.”

Response: We have deleted the line that previously read, “ However, the exclusions do not introduce a systematic bias that we see.”

Thank you again for the consideration to process our manuscript for publication. We hope that with these revisions, the manuscript is now suitable for publication in PLOS ONE.

Submitted by;

Winnie R Muyindike

Submitted filename: Response to Reviewers June 06 2020.doc

Click here for additional data file.

12 Jun 2020

Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda

PONE-D-19-35988R2

Dear Dr. Muyindike,

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.

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Reviewers' comments:

23 Jun 2020

PONE-D-19-35988R2

Tuberculin skin test positivity among HIV-infected alcohol drinkers on antiretrovirals in south-western Uganda

Dear Dr. Muyindike:

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.

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on behalf of

Dr. Nicky McCreesh

Academic Editor

PLOS ONE