Detection of Chikungunya virus in bodily fluids: The INOVACHIK cohort study.

BACKGROUND: Chikungunya is a widely distributed, re-emerging tropical disease caused by the chikungunya virus (CHIKV). Little is known about the duration for which CHIK RNA are detectable in bodily fluids, especially genital secretions, and current evidence is based on small series or case reports. An understanding of viral dynamics across different body compartments can inform diagnostic testing algorithms and public health prevention interventions.
METHODOLOGY: A prospective cohort study was conducted to assess the presence and duration of detectable levels of CHIKV RNA in blood, urine, saliva, semen, and vaginal secretions. Men and women (≥ 18 years) with a positive reverse transcriptase-polymerase chain reaction (RT-PCR) test for CHIKV in the acute phase (1-14 days) of the disease were included. After enrollment, clinical data and samples were collected every 15 days over the first 2 months, and a final collection was performed 3 months after recruitment. The Kaplan-Meier interval-censoring method and the parametric Weibull model were fitted to estimate the median time of viral persistence until the lack of CHIKV RNA detection among all body fluids. Punctual estimates of the median time of CHIKV RNA persistence for each fluid were estimated using a 95% confidence interval (CI).
RESULTS: From April to December 2019, 170 participants were screened. Of these, 152 (100 women) were enrolled in the study. The median and interquartile range (IQR) ages for men and women were 39.3 (IQR: 26.9, 50.7) and 43.5 (IQR: 33.8, 53.6) years, respectively. CHIKV RNA was detected in 80.3% (122/152) of serum samples, 23.0% (35/152) of urine samples, 30.3% (46/152) of saliva samples, 14.3% (6/42) of semen samples, and 20.2% (20/99) of vaginal secretion samples. The median time until the loss of CHIKV RNA detection was 19.6 days (95% CI, 17.5-21.7) in serum, 25.3 days (95% CI, 17.8-32.8) in urine, 23.1 days (95% CI, 17.9-28.4) in saliva, and 25.8 days (95% CI, 20.6-31.1) in vaginal secretion. The number of semen samples available was too small to make statistical estimates, but a last positive sample was obtained from a participant 56 days after the onset of symptoms.
CONCLUSIONS: CHIKV RNA could be detected in all bodily fluids studied, including genital secretions during the acute and convalescent phases and additional studies on viral infectivity in semen and vaginal secretions are warranted.

Introduction

Chikungunya is a widely distributed, re-emerging tropical disease caused by the chikungunya virus (CHIKV) and transmitted by Aedes aegypti mosquitoes [1,2]. Prior to 2013, CHIKV cases and some outbreaks were identified in several countries in Africa, Asia, Europe, and the Indian Ocean Islands. In 2013, the first local transmission of the CHIKV in the Americas was identified in the Caribbean countries and territories. The virus then spread throughout most of the Americas in 2014, especially in Brazil [1,2]. To date, three CHIKV genotypes are known: West African, Asian, and East Central South African (ECSA), of which the last two are prevalent in Brazil [3,4].

Clinically, the disease has three main phases: acute, post-acute, and chronic. In the acute phase, symptom onset ranges from 2–12 days following bites by an infected mosquito. This phase is associated with an abrupt onset of fever, headache, arthralgia, myalgia, fatigue, prostration, and rash. Severe joint pain is the most prevalent symptom, described in 90% of cases. The post-acute phase appears after 14 days of illness, following the febrile period. At this stage, joint pain is observed, which may last for up to 3 months. Finally, in the chronic phase, articular manifestations are seen, which are usually debilitating and can persist for many years [5,6].

Routinely, diagnosis is performed using serum or plasma samples [7]. However, the alternative use of urine and saliva samples for molecular diagnosis has been described in the acute phase of flavivirus infections, such as the West Nile virus [8], dengue virus (DENV) [9], and Zika virus (ZIKV) infections [10]. In addition, several studies have suggested a more extended detection and persistence of ZIKV in selected body fluids, such as saliva, urine, semen, sweat, and rectal samples [11-15].

Gardner et al. showed that oral fluid (saliva) of CHIKV-infected animals and humans might contain infective CHIKV in the acute phase of the disease. Human saliva samples were obtained from 13 CHIKV-positive patients who presented with hemorrhagic manifestations [16]. The prolonged detection of ZIKV RNA in semen has been described in some studies [13,17,18]. Although studies involving the isolation of CHIKV in saliva, urine, and semen samples are scarce, during an outbreak of chikungunya in French Polynesia, the virus was detected in saliva and urine samples in the acute phase of the disease [19]. Bandeira et al. reported CHIKV RNA in semen and urine samples 30 days after symptom onset, bringing new perspectives for alternative diagnostic forms and mechanisms of infection transmission [20], with implications for its prevention and control.

This study aimed to estimate the presence and duration of detectable levels of CHIKV RNA in bodily fluids, namely serum, saliva, urine, semen, and vaginal secretion in the acute and convalescent phases of the disease.

Methods

Ethics statement

INOVACHIK was a prospective cohort study conducted at the Acute Febrile Illness Laboratory, Oswaldo Cruz Foundation outpatient clinic in Rio de Janeiro, Brazil. The institutional review board reviewed and approved the study protocol (CAAE: 06779019.0.0000.5262). Written informed consent wasobtained before participation from all patients.

Study site and cases management

Patients admitted to the hospital or the intensive care unit were not targeted for enrollment to avoid bias towards patients with more severe disease. Thus, the patients enrolled in the study were screened at a general febrile illness outpatient clinic for more generalizable findings. Patients seen at this outpatient clinic are either referred by other health units in Rio de Janeiro or spontaneously seek care.

Men and women aged ≥18 years who had developed acute fever or arthralgia (with or without a rash) and no evident focus of bacterial infection within the previous 7 days were enrolled. A standard case report form was used to record information about the epidemiological and clinical features. We defined fever as an axillary temperature ≥ 37.5°C. Patients with symptoms reported for up to 7 days were included in the study. The first visit (with fluid collection) was performed on different days, depending on the patient’s arrival at our clinic.

Clinical data and biological samples were collected every 15 days for 2 months, with a final 3-month collection. The first samples for all fluids were collected in the first week of symptoms, and the second samples were collected within 14 days after the onset of symptoms (+/- 3 days as visit window). Data regarding clinical signs and symptoms was collected during the acute phase (1–14 days). All patients were tested over the study duration (3 months), despite undetectable RT-PCR results in all body fluids collected during a visit.

Laboratory tests

Serum, urine, saliva, semen, and vaginal secretion specimens were collected at enrollment, every 15 days for 2 months, and at the 3-months follow-up.

The samples were tested for CHIKV using real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Following the manufacturer’s instructions, RNA was extracted using the QIAmp Viral RNA Mini Kit. The general procedures for rRT-PCR for chikungunya, zika, and dengue have been described elsewhere [21,22,23]. The RT-PCR mix was prepared using the GoTaq Probe 1-Step RT-qPCR System and was run using the Applied Biosystems 7500 Real-Time PCR System. Cycle threshold (Ct) values lower than 38 and sigmoid curves were considered positive. In addition, serum was tested for anti-CHIKV-IgM according to the manufacturer’s protocol.

As a reference laboratory, all measures to avoid cross-contamination within the samples were adopted. There were different areas designated only for PCR: 1. In two large rooms, RNA extraction (manual or automated) was performed in cabinets with UV light or in a biological safety cabinet, with an adjacent room for adding samples in the PCR mix; 2. One “clean” room was used for mix preparation only; 3. One room with thermocyclers was used for the amplifications.

A non-template control (NTC) was used to check for the absence of sample cross-contamination, contamination of reagents, consumables, and environment. The NTC is a negative "sample" that can be water or negative plasma extracted simultaneously with the clinical samples and included in the amplification process. In all steps, tips with barriers were used, most consumables were disposable, and gloves were changed frequently. In addition, aseptic cleaning was frequently performed in all rooms.

Testing for ZIKV and DENV by rRT-PCR, was also performed for serum samples collected during the acute phase of the disease using a commercial kit (ZDC) from the Instituto de Tecnologia em Imunobiológicos Biomanguinhos. The ZDC kit was approved by the Agência Nacional de Vigilância Sanitária/ANVISA (registry #80142170032). Urine specimens collected during the acute phase were also tested for ZIKV using rRT-PCR. In cases where the ZDC Kit detected DENV, the protocol by Lanciotti et al. was used to identify DENV subtypes [24]. All study tests were performed at the National Reference Laboratory for Epidemiological Surveillance of Arbovirus in the Laboratory of Flavivirus at the Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.

Statistical analyses

The sociodemographic variables were described using frequencies and proportions for categorical variables and medians and ranges or interquartile ranges (IQRs) for continuous variables [25].

An exploratory analysis was performed by calculating summary statistical measures, and the violin plot was used to assess the distribution of the persistence time of the fluids. The Kaplan–Meier (K–M) curve technique was used to analyze time-to-event outcomes and estimate the probability of survival at various time intervals. Graphs were used to illustrate survival; in this case, the persistence time for each fluid over time [26]. The time until the lack of RNA detection in each bodily fluid was defined as the number of days between the onset of CHIKV symptoms and the first negative RT-PCR result. We assumed that CHIKV RNA in all specimens was detectable on the day of symptom onset. Patients who never tested positive during the study were excluded from the analysis, even if they had only one visit. Parametric Weibull regression models were used to estimate the time until the loss of CHIKV RNA detection in body fluids. Medians and 95% confidence intervals (CIs) were used to report the results. We estimated survival functions and 95% CIs for the Weibull model with median and 95th percentiles [27].

The Weibull curve was used as a smoother curve for the Kaplan–Meier estimator’s distribution. The Weibull curve showed the best choice as a smoothing curve to represent the Kaplan–Meier estimator’s distribution for all studied fluids. Statistical analysis was conducted using R, version 3.6 (R Core Team, 2020) and IBM SPSS Statistics 22.0.

Results

Participants characteristics

From April 10th to December 5th, 2019, A total of 170 participants were screened. Of these, 152 patients were enrolled in the study. The reasons for exclusion of the 18 potential participants were as follows: unspecified viral disease (n = 13), bacterial tonsillitis (n = 1), syphilis (n = 1), mononucleosis (n = 1), influenza virus (n = 1), and adverse cutaneous reaction (n = 1), as shown in Fig 1.

Fig 1

Flow diagram of INOVACHIK Cohort Study.

The median and interquartile range (IQR) age was 39.3 (IQR; 26.9, 50.7) for men and 43.5 years (IQR; 33.8, 53.6) for women. The majority of the study population was women (n = 100, 65.8%). Most patients were born in the state of Rio de Janeiro (82.6%). Table 1 shows the main sociodemographic characteristics of the study population.

Table 1

Sociodemographic characteristics of the study population, April—December 2019, Rio de Janeiro, Brazil.

Characteristics	n	%
Female	100	65.8%
Male median age (IQR)	39.3 (26.9–50.7)
Female median age (IQR)	43.5 (33.8–53.6)
Race
Black	24	15.8%
White	76	50.0%
Mixed race	51	33.6%
Yellow	1	0.7%
Education level
Elementary School	44	29.0%
High School	66	43.4%
College	42	27.6%
Marital Status
Single	70	46.1%
Married / Stable Union	66	43.4%
Divorced / Separated	13	8.6%
Widowed	3	2.0%

IQR: interquartile range.

Signs, symptoms, and comorbidities

Arthralgia (99.3%), fever (99.3%), prostration (94.7%), headache (86.8%), taste alteration (81.6%), chills (76.3%), myalgia (71.7%), and retroorbital pain (53.3%) were the most common symptoms reported. Complaints were associated with a rash in 84.9% of these patients. Joint swelling was also a common sign (61.2%), especially in the hands, ankles, and knees. Table 2 shows the signs and symptoms in the acute phase of the disease.

Table 2

Signs or Symptoms at Acute Phase of the Disease (1–14 days).

Sign/Symptoms	(n)	Percentage
Fever	151	99.3%
Arthralgia	151	99.3%
Prostration	144	94.7%
Headache	132	86.8%
Rash	129	84.9%
Taste alteration	124	81.6%
Chills	116	76.3%
Pruritis	115	75.7%
Anorexia	114	75.0%
Myalgia	109	71.7%
Backache	100	65.8%
Nausea	95	62.5%
Edema	93	61.2%
Eye pain	81	53.3%
Photophobia	70	46.1%
Sweating	50	32.9%
Diarrhea	47	30.9%
Abdominal Pain	46	30.3%
Lymphadenopathy	36	17.1%
Eye Congestion	30	19.7%
Dyspnea	28	18.4%
Vomiting	26	17.1%
Light Bleeding	26	17.1%
Odynophagy	25	16.4%
Runny nose	23	15.1%
Nasal congestion	21	13.8%
Otalgia	18	11.8%
Cough	16	10.5%
Hoarseness	14	9.2%
Dysuria	9	5.9%

The most frequent comorbidities observed were high blood pressure (19.7%), allergic rhinitis (19.1%), and arthrosis (9.9%). Coinfection with HIV was found in eight patients (5.3%), but their clinical presentations did not differ from those of the rest of the study population.

Zika and Dengue virus coinfection

Among the 152 enrolled participants, three (2.0%) had confirmed ZIKV infection, as assessed by rRT-PCR in the serum (n = 2) and urine (n = 1). A positive rRT-PCR result for DENV was found in four participants (2.6%), and all presented with DENV-2 infection.

Specific IgM antibodies against CHIKV

Specific IgM antibodies against CHIKV were detected in 146 participants (96.1%). Six participants had a single visit with negative IgM antibodies against CHIKV; therefore, it was not possible to document the occurrence of seroconversion. The lack of IgM class antibody production in these participants was probably because sample collection was performed 2 days after symptom onset in five participants and 3 days after symptom onset in another participant.

CHIKV detection in bodily fluids

Among the enrolled participants, 122 had detectable CHIKV RNA (80.3%) in serum in at least one specimen (Table 3), and eight (5.3%) had CHIKV RNA detection in more than one serum sample. CHIKV RNA was detected in urine samples only once in 35 (23.0%) of 152 participants (Table 3). CHIKV RNA was detected in urine samples from 30/100 (30.0%) female participants and only 5/52 (9.6%) male participants. Among the 152 enrolled participants, 46 (30.3%) had CHIKV RNA detected in at least one saliva specimen (Table 3), four (2.6%) of whom had positive results more than once. Of the 52 male participants, 42 provided at least one semen sample. Only six participants (14.3%) had detectable CHIKV RNA in semen, of which two had a second detection. All but one female participant provided at least one vaginal secretion specimen for RT-PCR analysis. CHIKV RNA was present in 20 participants (20.2%), with a single detection performed in 19 participants and twice in one participant. Of note, six participants had their first CHIKV RNA detection at or after the third study visit (≥1 month after symptom onset).

Table 3

Detection of CHIKV RNA in Body Fluids, According to Gender for 152 enrolled participants*.

Body Fluid	Total Patients	Positive Patients	Detection Percentage	Male n (%)	Female n (%)
Serum	152	122	80.3%	45 (86.5)	77 (77.0)
Urine	152	35	23.0%	5 (9.6)	30 (30.0)
Saliva	152	46	30.0%	14 (26.9)	32 (32.0)
Semen	42	6	14.3%	6 (14.3)	Not Applicable
Vaginal Secretions	99	20	20.2%	Not Applicable	20 (20.2)

* Data were derived from a combination of all study visits. The first samples (for all fluids) were collected within the first week after symptom onset. Samples were collected every 15 days for 2 months and at 3 months of follow-up. Each participant collected a maximum of six samples for each body fluid.

Of the 152 enrolled participants, 114 were included in the persistence analysis. The reasons for exclusion were as follows: only reactive anti-Chikungunya IgM during the whole study (n = 25) without a positive RT-PCR result, the first detection of CHIKV RNA in any bodily fluids occurred during or after the third study visit (n = 6), coinfection with DENV-2 (n = 4), and ZIKV (n = 3). A total of 34 (29.8%) participants were lost to follow-up for unknown reasons, and 80 (70.2%) completed the study for persistence analysis.

The median time for the loss of CHIKV RNA detection was 19.6 days (95% CI, 17.5–21.7) in the serum (Fig 2), 25.3 days (95% CI, 17.8–32.8) in urine (Fig 3), 23.1 days (95% CI, 17.9–28.4) in saliva (Fig 4) and 25.8 days (95% CI, 20.6–31.1) in vaginal secretions (Fig 5). The number of semen samples available was too small for statistical estimation. Nevertheless, the maximum detection of CHIKV RNA was observed 56 days after the onset of symptoms in a study participant.

Fig 2

Survival curves (Kaplan–Meier analysis) of CHIKV RNA persistence in Serum, with Weibull fit.

Fig 3

Survival curves (Kaplan–Meier analysis) of CHIKV RNA persistence in Urine, with Weibull fit.

Fig 4

Survival curves (Kaplan–Meier analysis) of CHIKV RNA persistence in Saliva, with Weibull fit.

Fig 5

Survival curves (Kaplan–Meier analysis) of CHIKV RNA persistence in Vaginal Secretion, with Weibull fit.

Table 4 shows the percentiles from the Weibull models and their 95% CIs until the loss of CHIKV RNA in selected body fluids. The 95th percentile of time was 39.7 days (95% CI, 35.7 to 43.7) in serum, 68.4 days (95% CI, 49.2 to 87.7) in saliva, 52.9 days (95% CI, 41.8 to 63.9) in urine, and 48.8 days (95% CI, 37.7 to 59.8) in vaginal secretions based on the Weibull model.

Table 4

Percentiles from the Weibull models until loss of CHIKV RNA in body fluids (in days).

Body Fluid	Percentile	0.95 LCL	0.95 UCL
	25th
Serum	12.83	10.98	14.68
Saliva	13.93	8.37	19.49
Urine	14.09	9.78	18.39
Vaginal Secretions	17.65	13.34	21.96
	Median
Serum	19.60	17.47	21.73
Saliva	25.31	17.83	32.80
Urine	23.14	17.91	28.37
Vaginal Secretions	25.84	20.61	31.07
	75th
Serum	27.38	24.79	29.96
Saliva	40.53	30.11	50.96
Urine	34.22	27.58	40.86
Vaginal Secretions	34.90	28.26	41.54
	95th
Serum	39.69	35.73	43.65
Saliva	68.42	49.18	87.66
Urine	52.87	41.83	63.91
Vaginal Secretions	48.75	37.71	59.78

LCL: Lower Confidence Limit, UCL: Upper Confidence Limit

Figs 6–9 shows RT-PCR Ct values for CHIKV detection for each fluid studied, allowing the understanding of the strength of the signal in the different body fluids over time. Samples with a Ct < 38 (dashed line) were considered positive. Each circle indicates a positive result. As expected, Ct values were lower for all body fluids mainly in serum samples in the acute phase suggesting higher viral loads.

Fig 6

CHIKV Cycle Threshold (Ct) by days after the onset of symptoms in Serum.

Fig 9

CHIKV Cycle Threshold (Ct) by days after the onset of symptoms in Vaginal Secretion.

Real-time reverse transcription PCR cycle threshold values for Chikungunya virus. Samples with Ct <38 (dashed line) were considered to be positive. Each circle indicates a positive result.

Discussion

This longitudinal study reported CHIKV detection by RT-PCR in several bodily fluids, including genital secretions, during the acute and convalescent phases of the disease (up to 3 months). We demonstrated that CHIKV RNA was detected more than 30 days in all the fluids studied. In addition, serum, urine, and saliva had detectable virus levels and persistence for more than 60 days, while urine had them for more than 90 days. To the best of our knowledge, this was the first cohort study to assess the persistence of CHIKV RNA in genital fluids (vaginal secretions and semen).

Females outnumbered male participants in the diagnosis of chikungunya. Similar results have been described in other studies [28-30]. A combination of fever, arthralgia, and prostration was the most prevalent presentation in our cohort, which is consistent with the results described by Anwar et al. [29].

Since 2014, the presence of co-circulating arboviruses (dengue, zika, and chikungunya) has increased the chance of coinfection. Epidemiological findings from a surveillance study for acute febrile illnesses including 948 participants, showed that 247 (26.1%) had evidence of an acute arboviral infection, of which 224 (23.6%) were single infections and 23 (2.4%) were coinfections [31]. Specifically, 13 (1.4%) patients tested positive for DENV/CHIKV coinfection and nine (0.9%) for CHIKV/flavivirus coinfection [28]. In another study, Dos Santos et al. reported five (9.6%) patients with coinfection with DENV-2 among 52 participants diagnosed with chikungunya [32]. Our cohort had similar results where coinfection of ZIKV and CHIKV was reported in three (2.0%) participants and 14 patients (9.2%) had reactive acute-phase anti-DENV IgM. DENV-2 was detected in only four participants (2.6%). We did not observe differences in symptom severity in patients with these coinfections.

We observed that the detection rate of CHIKV RNA was significantly higher in blood, saliva, and urine during the first week of symptom onset, which is consistent with other studies reporting viral presence during the acute phase of the disease [19,20]. In addition, saliva and urine did not increase the detection rate of CHIKV RNA in the acute phase of the disease, and, in concordance with Musso et al., blood was the sample of choice for chikungunya diagnosis [19]. CHIKV RNA persistence in the serum in our study was longer than expected. Most literature reports showed that CHIKV RNA in serum declines to undetectable levels within 1–2 weeks after symptom onset [33-35].

We also detected CHIKV RNA in urine 95 days after symptom onset. A similar study by Bandeira et al. reported the maximum viral persistence in urine after 30 days [20]. Interestingly, in our cohort, CHIKV RNA was detected in 30% of urine samples from female participants and in only 9.6% of male participants. We did not find reports evaluating RT-PCR RNA detection rates in urine samples by sex, but contamination by menstrual blood can be a reasonable explanation, although all the guidelines for urine collection were given to the study participants. Additionally, the collection was not performed during the menstrual period.

To the best of our knowledge, this is the first prospective study to monitor and detect CHIKV RNA in vaginal secretions. We detected CHIK RNA up to 46 days after the acute onset of symptoms in vaginal secretion samples. We did not perform statistical estimates for semen as the number of samples was small, but the maximum detection of CHIKV RNA in semen was 56 days after the onset of symptoms in a study participant.

Although we detected CHIKV in semen and vaginal secretions, it was impossible to assess its potential for sexual transmission as viral isolation was not attempted. In addition, this study did have an appropriate study design to establish sexual transmission due to the endemic nature of the infection, making it difficult to ascertain the actual route of transmission, sexual or vectorial, but additional studies on viral infectivity are warranted. Therefore, it was out of the scope of this study to assess the sexual transmission of CHIKV.

Chikungunya diagnosis in humans is mainly based on RNA detection in serum or plasma samples. However, we have demonstrated that saliva and urine could be considered as potential alternative samples for diagnosis in the acute and convalescent phases of the disease. Diagnostic algorithms using urine or saliva as alternative samples have the advantage of being quick, easy-to-perform, and being less invasive than blood collection. The demonstration of longer persistence of CHIKV in bodily fluids may help diagnosis in later stages of the disease.

This study has some limitations. 1) As the visits and sample collections were scheduled every 15 days, we may have underestimated the exact viral persistence time in the different body fluids; 2) the median duration of CHIKV in semen was evaluated in a small number of patients, because of difficulties in sample collection, mainly due to joint pain in the acute phase of the disease; and 3) the follow-up time was limited to 90 days, making it impossible to assess the maximum persistence of CHIKV in all bodily fluids.

Knowledge of chikungunya viral persistence, infectivity and epidemiology can inform recommendations for control, treatment, and prevention of the disease, and contribute to public health programs.

12 Nov 2021

Dear Dr Martins,

Thank you very much for submitting your manuscript "Detection of Chikungunya Virus in bodily fluids: The INOVACHIK Cohort Study." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

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

Methods

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

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

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

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

-Were correct statistical analysis used to support conclusions?

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

Reviewer #1: See below

Reviewer #2: The methods are generally well-described, but some points should be clarified:

A description of how the RNA was extracted (Trizol, RLT, AVL, etc), cDNA generation, and if any post-PCR analysis was performed is missing.

Line 111: what do the authors mean by "focus of infection"? Please clarify the specific criteria for the time post symptom onset and positive CHIKV PCR to the time of enrollment in the study?

Line 141-142: What is the rationale or experimental data for the assumption that all the fluids would be positive at symptom onset?.

Line 144-145: What is the rationale for censoring these individuals? If they are still positive at the end of the study, this would still indicate CHIKV RNA persistence, supporting the other data that CHIKV RNA persists long-term.

Table 1: Race/ethnicity data: are these the correct terminologies for different race and ethnicities?

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

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

Reviewer #1: See below

Reviewer #2: The results are clearly presented in the figures and tables. Some of the data are not shown, and the paper would benefit from the following added:

Line 166-175: Please report the signs and symptoms in a table.

Line 181-184: Please report the IgM ELISA titers in a figure.

Figure 2: The median and maximum time to a negative CHIKV RNA result is reported in the text, but it would be useful to have these numbers also represented in the figure or in a separate table.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

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

-Is public health relevance addressed?

Reviewer #1: See below

Reviewer #2: The introduction and conclusions/discussion could be reworked to highlight the public health significance of the work. For example, in the discussion, the authors could discuss how the urine or saliva test is less-invasive compared to blood tests for viral RNA. The downside is that the viral RNA is most consistently detected in the serum (80% positivity in this dataset). The authors could also expand upon the difference in detecting viral RNA compared to isolating infectious virus and any implications that this might have.

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Editorial and Data Presentation Modifications?

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

Reviewer #1: (No Response)

Reviewer #2: Lines 26-27: Needs a little more information in the abstract background section. A statement that introduces CHIKV is missing. A statement is needed about what was done and why the detection of CHIKV in body fluids may be important for public health.

Line 52: "...are warranted."

Line 52: Expand on the conclusion section- what are the implications for long-term detection of CHIKV RNA in body fluids?

Line 67: "...warranted."

Line 69: State where CHIKV is geographically distributed.

Line 73: CHIKV disease is generally described in three phases: acute, post-acute, and chronic. An expansion of the clinical symptoms of each phase is needed.

Line 83: There should be a more complete description of what has been shown for CHIKV detection from body fluids over each phase of infection. Also add the citation with Joy Gardner, et al. 2015 Plos One that showed infectious CHIKV recovery from patient saliva and compared it to animal models.

Line 83: Remove the word "other". Is anything known for other alphaviruses (RRV, ONNV, MAYV, VEEV, etc) in terms of detection in other body fluids such as saliva?

Line 183-184: In reference to the six participants that had a single visit with negative IgM antibodies- what was their time post symptom onset?

Line 271-276: Expand this paragraph to discussing CHIKV transmission as a whole. Please include a statement about how transmission occurs primarily through mosquitos but we do not know if transmission may occur by saliva or sexual contact (as with ZIKV for example).

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Summary and General Comments

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

Reviewer #1: In this manuscript, Martins et al. used RT-PCR to evaluate body fluids, collected at enrollment and out to three months from Brazilian patients with confirmed chikungunya virus (CHIKV) infection, for the presence of CHIKV RNA. CHIKV RNA was detected in all sample types (e.g., serum, urine, saliva, semen, vaginal secretions) at different frequencies and for different lengths of time since enrollment. A major strength of the study is the remarkable paucity of this type of virological data for CHIKV infection in humans. A major limitation of the study is the absence of synovial fluid, joint-associated tissues or other musculoskeletal tissues. Nevertheless, the study contributes important virological information. However, some of the presentation of the data is confusing and/or incomplete. Specific comments to improve the manuscript are outlined below.

1. The presentation of the data in Table 2 is confusing. Is this data derived from the initial sample collection at the time of enrollment? Or is it combined data from multiple collections? In some cases, the text says “in at least one specimen” or other similar statements. The presentation of these data needs to be clarified and improved. In addition , it also seems important to include information about the time of disease onset relative to collection of the initial samples.

2. Which collection times were used for the persistence analysis and which ones were used for the acute analysis (is this Table 2?). This should be clarified.

3. The details of RNA isolation, cDNA generation, and the RT-PCR assay are missing and should be included in the materials and methods section.

4. The authors state that any reaction with a Ct value lower than 38 and with sigmoid curves was considered positive. It would improve the study to include the Ct values detected to give readers an opportunity to understand the strength of the signal in the different body fluids over time.

5. Given the sensitivity of RT-PCR and the potential for false positives, the authors should report the methods and measures used to minimize contamination and other possible sources of false positive signals.

6. The clinical data presented from lines 166-175: Are these the signs and symptoms that were present at the time of study enrollment and first sample collection? This is not clear.

7. The authors indicate that clinical data were collected throughout the study, however, these are not reported. It would be useful to compare the clinical presentation to the decline in viral RNA signal in samples. Is there any relationship to the resolution and/or persistence of clinical symptoms and signs?

Reviewer #2: In this work, the authors describe a clinical study where they sampled body fluids from CHIKV patients (serum, saliva, urine, semen, and vaginal secretions) throughout the post-acute phase of disease. No previous group has examined viral RNA persistence in over time in all these body fluids, and no group has examined viral RNA in semen and vaginal secretions. The authors found that CHIKV RNA generally persists in body fluids for about 20-25 days. However, viral RNA detection in the serum was still the most reliable readout, with most (80%) of the cohort having CHIKV RNA positivity in the serum over the post-acute phase. The study would benefit from a discussion of the implications of these data for public health. It would also benefit from additional reasoning for why they were looking for viral RNA in body fluids other than serum, and more organization and flow in the introduction and conclusions section.

The following points should be addressed:

1. In the introduction, there should be a more detailed discussion of what we know about CHIKV RNA detection in patient body fluids (serum, urine, saliva, etc) during each infection phase- the acute, post-acute phase, and chronic phase. In addition, CHIKV detection in body fluids from CHIKV animal models could enhance the discussion and provide some comparison.

2. The long-term detection of vRNA shed in bodily fluids could reflect either infectious virus in the bodily fluids, or vRNA in the absence of infectious virus. The genome copy number would be useful because high copy numbers may be more suggestive that infectious virus is present. The authors should report CHIKV levels as genome copies (or Ct's if they don't have genome copies) relative to days after symptom onset. In addition, please describe the detection limit of the PCR assay.

3. In Figure 2- I'm not an expert on statistics, but the Weibull curve diverges from the KM curve in the serum, urine, and saliva data, but not as much in the vaginal secretion data. Why is this the case? Are both statistical analysis needed?

4. The conclusions section could use more discussion of the significance of CHIKV detection in body fluids. It would also benefit from a more organized structure. For example, there could be a paragraph devoted to CHIKV RNA detection in acute phase and a separate paragraph on long-term detection.

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Reviewer #1: No

Reviewer #2: No

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5 Jan 2022

Submitted filename: Response to Reviewers_04.01.2022.docx

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9 Feb 2022

Dear Dr Martins,

We are pleased to inform you that your manuscript 'Detection of Chikungunya virus in bodily fluids: The INOVACHIK Cohort Study.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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

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

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

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

Best regards,

William B Messer

Associate Editor

PLOS Neglected Tropical Diseases

Benjamin Althouse

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

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

Methods

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

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

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

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

-Were correct statistical analysis used to support conclusions?

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

Reviewer #1: Please see summary

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

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

Reviewer #1: Please see summary

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

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

-Is public health relevance addressed?

Reviewer #1: Please see summary

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Editorial and Data Presentation Modifications?

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

Reviewer #1: Please see summary

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Summary and General Comments

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

Reviewer #1: The authors have addressed the comments from the prior review and the manuscript is substantially improved. The inclusion of Ct values in new Fig 3, clarification of sample collection and symptom assessment, inclusion of assay details and methods to prevent contamination, and other modifications have greatly strengthened the report.

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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

17 Feb 2022

Dear Dr Martins,

We are delighted to inform you that your manuscript, "Detection of Chikungunya virus in bodily fluids: The INOVACHIK Cohort Study.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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

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

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases