Secondary prevention of silicosis and silico‐tuberculosis by periodic screening of silica dust exposed workers using serum club cell protein 16 as a proxy marker

Abstract Background and Objectives Silicosis is a neglected and widely prevalent occupational disease in India and several other countries such as China, South Africa, Brazil, etc. It is an irreversible, incurable, and progressive disease with high morbidity and mortality, which is mostly caused by occupational exposure to silica dusts. Silicosis is usually detected at an advanced stage, when effective intervention is not possible. But early detection appears to be a cost‐effective way to control it. There is a need for some suitable biomarker, which could detect silicosis at an early stage for further necessary intervention. This study aimed to estimate the lung damage in silicotic subjects and its relationship with serum CC16 as a proxy marker. The ultimate objective was to explore whether CC16 could be used as a screening tool for early detection of silicosis. Methodology Radiographs of 117 workers having radiological evidences of silicosis were evaluated in accordance with International Labour Organisation (ILO) Classification of chest radiographs and were categorized as mild, moderate, and severe lung damage using a lung damage scoring system, made for the purpose of this study. The concentration of CC16 in serum was determined by enzyme‐linked immunosorbent assay. Result It was observed that serum CC16 values were significantly decreased in relation to increasing lung damage. The mean ± standard deviation (SD) serum CC16 value in mild lung damage group was 8.4 ± 0.87 ng/mL as compared to 4.0 ± 2.10 ng/mL in moderate and 0.7 ± 0.21 ng/mL in high lung damage groups. On the other hand, CC16 value of control (healthy) population was found to be 16.3 ± 3.8 ng/mL. Conclusion Result of the study concluded that serum CC16 might be used as a periodic screening tool for early detection of silicosis and for it's secondary prevention. It may be viewed as a new approach toward control of silicosis, and an appropriate policy may be adopted.


| INTRODUCTION
Respiratory public health is currently facing several challenges such as increasing burden of chronic obstructive pulmonary diseases (COPDs) due to growing air pollution, disturbing occurrences of influenza epidemics, increasing multidrug-resistant tuberculosis (MDR-TB) etc. 1 On the top of it, the entire world is currently facing a highly distressing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With this backdrop, it is difficult to accept that a preventable occupational disease such as silicosis continues to threaten the health of so many people worldwide just due to lack of required initiatives for it's control. Silicosis is a widely prevalent occupational lung disease caused by inhalation of crystalline silica dust and is marked by inflammation with scarring in the form of nodular lesions on the lungs. 2 Common morbidity due to silicosis includes COPD, asthma, hypersensitivity pneumonitis, nephropathy, stomach cancer, etc. [3][4][5] India has a huge burden of silicosis with an estimated 8.5 million workers in the year 1999 at risk of silicosis as per data available on national health portal. 6 It may have at least doubled in 20 years, in view of increased population and industrialization. Many studies have been conducted among silica-exposed workers depicting the prevalence of silicosis that ranged between 12% and 50%. [7][8][9][10][11] Presently, silicosis is detected on the basis of opacities present over chest radiographs by using "ILO Classification of Radiographs for Pneumoconiosis," which often makes the diagnosis delayed as it is asymptomatic or mildly symptomatic in the initial stage of the disease and often ignored by the workers. Since silicosis is an incurable, irreversible, and progressive disease with high morbidity and premature mortality, early detection appears to be the only way for secondary prevention of it. Hence, there is a need for some suitable biomarker, which can detect silicosis among dust-exposed workers in its early stage, and thereby the workers could be identified and removed from further exposure to silica dust. In this regard, Indian Council of Medical Research-National Institute of Occupational Health (ICMR-NIOH), India, had conclusively established that serum club cell protein 16 (CC-16) could be used as a biomarker for early detection of silicosis. 12 However, the earlier study did not establish the relationship between various grades of lung damage and its corresponding serum CC16 value. Hence, an observational study was conducted to categorize the lung damage based on approximate quantification of silicotic damage using chest radiograph to mild, moderate, and high/severe and assessing its relationship with serum CC16 level. The ultimate objective of this study is to investigate whether serum CC16 could be used as a proxy marker/predictor and as a periodic screening tool among occupational silica dust-exposed workers for early detection of silicosis. Early indication of silicosis through periodic assessment of serum CC16 among workers with history of occupational silica dust exposure appears to be an useful method. This may be confirmed by the chest X-ray for secondary prevention of silicosis as well as for other purposes such as notification to the local authority and compensation to be paid to the victims as per law of the country. Hence, this study was initiated.

| METHODOLOGY
This was an occupational health clinic-based cross-sectional study.
The participants were workers of stone mines and stone quarries and living in the neighboring areas of their workplaces in Faridabad, Haryana and Jodhpur, Rajasthan. One hundred and forty-nine subjects with and without radiological evidence of silicosis were subjected for this study. Of them, 32 subjects were healthy controls with no history of occupational exposure to silica dust. The radiographs of both 117 workers with evidence of silicosis and 32 healthy controls were evaluated using International Labour Organisation (ILO) radiography guidelines for detection of silicosis.
Evaluation was performed for approximate quantification and categorization of lung damage. 13 Each radiograph was examined for quality of X-ray plate. Following this, acceptable X-ray plates were categorized in to mild, moderate, and severe silicotic lung damage.
For determination of lung damage, following four factors were con-   Amount of lung damage was calculated using size of opacity on chest X-ray. The size and shape of small opacities were scored as p = 1, q = 2, and r = 3. If more than one form (shape/size) of opacities was identified (mixed opacities), then the most common opacities were termed as primary opacity, while the next common opacities were secondary opacity. In case of mixed opacities, scoring was carried out as p/q = 1.5, q/r = 2.5, etc. Irregular small opacities are also categorized as s = 1, t = 2, u = 3, s/t = 1.5, etc.

| Size of large opacity (L)
Weightage of lung damage was given as per large opacity: A = 1, B = 2, and C = 3.

| Zones of lungs involved (Z)
Weightage is given according to number of zones involved. If one zone is involved, score given is 1 and if all six zones involved score given in 6.

| Categorization of approximate lung damage using lung damage score (LDS)
The total lung damage score (LDS) was obtained by multiplying small opacities (X) with profusion of opacities (Y) and number of affected lung zones (Z). To this, score of large opacity (if any) is added (L).
Lung Damage Score (LDS) = (X*Y * Z) + L. Using above scoring system, the extent of lung damage was categorized as: Severe/high when LDS = > 15, moderate when LDS = 7 to 15, and mild when LDS = 1 to 6.

| Blood sample collection
About 5 mL blood sample was collected from each study subjects for estimation of serum CC16 by the trained laboratory technicians from the eligible and willing study participants. Relevant demographic information was also collected from each subject using a pretested questionnaire. Study questionnaires along with blood samples were transported to the laboratory of ICMR-NIOH for further processing.
Concentration of CC16 in serum was determined by enzyme-linked immunosorbent assay (ELISA) method.

| Data management
Data were edited on the same day following collection and transported at ICMR-National Institute of Occupational Health. Data were entered and analyzed using Epi Info software, Windows-7.2 version.

| Institute ethics committee approval
The study was approved by the Institutional Ethics Committee (IEC) of ICMR-NIOH. A written informed consent was obtained from all the study participants before initiating this study.

| RESULTS
Mean age, duration of exposure, subjects with lung damage scores in various lung damage categories, and serum CC16 levels are shown in Table 1. Mean age of silicotic subjects varied between 46.2 and 50.5 year and that of healthy subjects was 37.5 year. All study subjects were males. Mean work duration for mild, moderate, and severe silicotic subjects was 28.6, 21, and 19.8 years, respectively. The result showed that out of total 117 silicotic participants, mild lung damage was observed in eight workers (6.8%), whereas moderate and high/severe lung damage were observed in 76 workers (65%) and 33 workers (28.2%), respectively. Mean LDS values for mild, moderate, and severe categories were 3.3 (n = 8), 9.9 (n = 76), and 19.9 (n = 33), respectively. These subjects had been working in sandstone mines and stone quarries. The radiographs of 32 healthy controls were within normal limits. Mean serum CC16 value of control population was 16.3 ± 3.8 ng/mL. It was observed that serum CC16 values were significantly decreased in relation to increasing lung damage (ie, mild, moderate, and high) among the study subjects. The mean serum CC16 value in mild lung damage category was 8.4 (SD ± 0.87) ng/mL as compared to 4.0 (SD ± 2.1) ng/mL in moderate and 0.7 (SD ± 0.21) ng/mL in high or severe lung damage categories ( Figure 1). It is interesting to note that duration of exposure is inversely associated with the amount of lung damage in this study. This may be attributed to the fact that varying quantity of dust with varying silica content may be inhaled by them over the years ( Table 1). Differences of mean of various categories of LDS were found to be statistically significant (moderate vs mild and high vs moderate) as indicated by P = <.01 (Table 1).
Similarly, differences of mean value of serum CC16 levels in various lung damage categories were found to be statistically significant (moderate vs mild and high vs moderate) indicated by P = <.01 (Table 1).

| Serum CC16-a possible biomarker for secondary prevention
The study observed that most silicotic subjects were detected when they had moderate lung damage (65%; n = 76) followed by severe lung damage (28.2%; n = 33). Only 6.8% (n = 8) of the studied workers were detected with mild lung damage ( Figure 1). This evidences that most silicotic subjects are detected when lung damage progresses to the extent of moderate to severe stage. The results also indicated an inverse relationship between the extent of lung damage and the serum CC16 value. Differences of mean serum CC16 values from healthy to mild lung damage, mild to moderate damage, and moderate to high damage are statistically significant suggesting serum CC16 might be used as a screening tool for early detection of silicosis cases among silica dust exposed workers. Earlier studies showed that smoking reduced serum CC16 to slight extent, and cessation of smoking elevates serum CC16 level. 12,14,15 None of the study participants was current smoker in this study. So, adjustment of serum CC16 value related to smoking habit was not required. It was observed that most silicosis patients quit their smoking habits following detection of silicosis on medical advice or when they develop distressing respiratory symptoms such as dry or productive cough with suspicion by the health care workers during their first consultation with them. Serum CC16 levels are inversely proportional to the amount of lung damage among silica dust-exposed workers as observed in this study ( Figure 1). So, if screening is performed in selected population with history of silica dust exposure, serum CC16 appears to be useful for early detection of silicosis as evidenced in this study as well as in earlier study. The present study was conducted among the workers with history of occupational exposure to silica dust. The dusty workplace exacerbates symptoms of bronchial asthma, chronic bronchitis, and COPD, and hence the persons suffering from such medical conditions cannot sustain for longer duration in dusty environment and eventually leave the job. It was also observed in our earlier study that serum CC16 value does not vary with age and/or gender. Hence, it could be used as a screening tool for early This study also indicates that the lung damage may be categorized to mild, moderate, and severe based on the declining level of serum CC16 value among silicotic subjects; mild lung damage = >6 to 9 ng/ mL, moderate lung damage = >3 to 6 ng/mL, and severe lung damage = 3 ng/mL or less. Hence, early silicosis may be considered if serum CC16 value remains between >6 and 9 ng/mL. Since smoking reduces CC16 value by 1 to 2 ng/mL, above range will include both smokers as well as nonsmokers for screening purpose from operational point of view under a public health programme. Some workers might have silica dust exposure history as well as serum CC16 value of just above 9 ng/mL. They may be in their very early pathological process of silicosis. But usually, these subjects are not detectable by chest X-ray, hence, unsuitable for confirmation. Considering all these, 9 ng/mL may be the cut-off value for detection of silicosis at an early stage.
In a country like India and similar other countries where burden of silicosis is high, periodic estimation of serum CC16 value using above-said method among dust exposed workers will be helpful in T A B L E 1 Age, duration of exposure, and lung damage category with score and serum CC16 reducing silicosis burden by its early detection and effective intervention. On the other hand, silicosis is intimately associated with silicotuberculosis due to reduced lung immunity. [16][17][18] High prevalence of tuberculosis among silica dust-exposed workers has been depicted by other research studies. 19,20 The findings of earlier study suggested that highest levels of CC16 in X-ray confirmed silicosis patients were around 9 ng/mL. This study has also showed that silicosis with mid lung damage had mean serum CC16 levels of 8.4 ± 0.87 ng/mL.
Hence, the study intends to highlight that early silicosis may be suspected when serum CC16 value is 9 ng/mL or slightly lower provided the subjects are nonsmokers or stopped smoking for at least 1 to 2 months before the test date. This is expected to ensure confir-

| Policy decision toward control of silicosis
Silicosis is a neglected public health disease with huge burden in India.
Most qualified physicians are not in a position to diagnose it properly due to lack of training during their medical practicing tenure or earlier Moreover, this will prevent not only silicosis by stopping/minimising further dust expose but also its associated co-morbidity, silico-tuberculosis. Silico-tuberculosis could detected at early stage by periodic screening of silicotic workers' sputum using CBNAAT/True-NAAT.
We understand that the same holds true for other similar countries with high burden of silicosis such as South Africa, China, Brazil, Thailand, Australia, some European countries, etc. They would also be benefitted initiating similar silicosis intervention programme in their countries using CC16 or similar marker for early detection as suggested by a recent study. 22 International organizations such as WHO, The Union, etc, should come forward coordinating and guiding member countries and taking this matter forward further in order to achieve the desired goal. The concept of secondary prevention of silicosis using an effective screening tool such as serum CC16 may be validated in above-said countries to establish its potential benefits both nationally as well as internationally.

| CONCLUSION
Silicosis is a neglected occupational disease and associated with high morbidity and premature mortality. The effective initiatives toward its prevention and control in India is practically non-existent.
Considering its huge burden, the country needs an urgent policy decision toward initiating national silicosis control programme for dual benefit of control of both silicosis as well as silico-tuberculosis.
Secondary prevention of silicosis and silico-tuberculosis by using a suitable and cost-effective biomarker such as CC16 may be viewed as a new approach of the proposed national silicosis control programme.

ACKNOWLEDGMENTS
The efforts of all laboratory staff of ICMR-NIOH as well as ESIC, Delhi, who provided laboratory support for this study are greatly appreciated with due recognition. Authors also gratefully acknowledge the contribution provided by the study participants.

FUNDING
Intramural funding from ICMR-National Institute of Occupational Health.

CONFLICT OF INTEREST
The provision of financial support does not in any way infer or imply endorsement of the research findings by either agency. The authors declare no conflict of interest relating to the material presented in this article. Its contents, including any opinions and/or conclusions expressed, are solely those of the authors.
Supervision of clinical work: Umesh Chandra Ojha.
The manuscript was prepared in consultation with all authors, and final draft to be published was approved by all authors.
Manuscript has been read and approved by all the authors, that the requirements for authorship as stated earlier in this document have been met, and that each author believes that the manuscript represents the sincere and honest work.

TRANSPARENCY STATEMENT
The lead author (Kamalesh Sarkar) affirms that the manuscript is an honest, accurate and transparent account of the study being reported and no important aspects of the study have been omitted. Also declares that any discrepancy from the study as planned (if relevant) have been explained.

ETHICS STATEMENT
The study was duly reviewed and approved by Institutional Ethics Committee (IEC) of National Institute of Occupational Health.