Indian J Med Res 151, April 2020, pp 319-325 Quick Response Code:

DOI: 10.4103/ijmr.IJMR_1799_18
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Club cell protein 16 as a biomarker for early detection of silicosis

Nibedita Naha1, Jaseer C.J. Muhamed1, Avinash Pagdhune1, Bidisa Sarkar2,# & Kamalesh Sarkar†
imK/icbKo55CAlRxwKTQm+VAr0Du6A== on 06/17/2024

1
Division of Biochemistry, †ICMR-National Institute of Occupational Health, Ahmedabad, Gujarat &
2
Department of General Medicine, KPC Medical College & Hospital, Kolkata, West Bengal, India

Received September 26, 2018

Background & objectives: Clinically silicosis is diagnosed by chest X-ray showing specific opacities along
with history of silica dust exposure. Diagnosis is invariably made at an advanced or end stage when it
is irreversible. Moreover, silicosis patients are susceptible to develop tuberculosis. Therefore, a suitable
biomarker for early detection of silicosis is needed. This study evaluated the suitability of club cell protein
(CC16) as a biomarker for early detection of silicosis.
Methods: This pilot study included 121 individuals from X-ray-confirmed/advanced silicosis, moderate
silica dust-exposed workers and healthy controls from western India. CC16 levels were quantified in
serum samples through ELISA. Sensitivity and specificity of CC16 values at different cut-off points were
calculated in both non-smokers and smokers.
Results: Serum CC16 level was significantly (P<0.01) decreased in X-ray confirmed advanced silicosis
patients (4.7±3.07 ng/ml) followed by moderately exposed workers (10.2±1.77 ng/ml) as compared to
healthy non-exposed individuals (16.7±3.81 ng/ml). Tobacco smoking also caused a significant decrease
of serum CC16 concentration in both healthy (10.2±1.12 ng/ml) and advanced silicosis workers
(2.6±2.28 ng/ml) compared to non-smokers. Sensitivity and specificity of CC16 values were also found to
be ≥83 per cent for screening all categories of individuals.
Interpretation & conclusions: Because of high sensitivity and specificity, serum CC16 could be used as
predictive biomarker for suspicion and early detection of silicosis, which would help in reducing/delaying
premature deaths caused by silicosis. It would also control silicotuberculosis additionally.

Key words Club cell protein - dust exposure - early detection biomarker - serum - silicosis

Silicosis is an irreversible occupational disease of agate, construction sites and non-metallic product
respiratory system caused by the reaction of lung tissue manufacturing units such as, refractory, ceramic, glass,
(parenchyma) to dust containing crystalline silica or mica, structural clay are prone to silicosis1. Although
silicon dioxide of respirable size (<10 µ in diameter). silicosis occurs mostly in the occupational exposure
People with varying length of exposure of 2-15 yr set-up, a sizable number of cases have also been reported
or more in the industries like mines, stone quarry, due to non-occupational exposure to silica dust2-4.

Present address: Department of Community Medicine, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha
#

© 2020 Indian Journal of Medical Research, published by Wolters Kluwer - Medknow for Director-General, Indian Council of Medical Research
319
 320 INDIAN J MED RES, APRIL 2020

Unfortunately, most silicosis cases remain undiagnosed Material & Methods

at an early stage due to asymptomatic nature of the initial
The study was conducted by the ICMR-National
stage of the disease, lack of a suitable biomarker for early
Institute of Occupational Health (ICMR-NIOH),
detection, poor health-seeking behaviour of the workers
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Ahmedabad, India, during the months of March to

and poor occupational healthcare delivery services at the
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May, 2018. Prior approval of the study was obtained

working areas, particularly in unorganized sectors. from the Institutional Ethics Committee. Written
Patients with silicosis are prone to develop informed consent was obtained from each participant
pulmonary tuberculosis (i.e., silicotuberculosis), before initiation of the study.
probably due to destruction of alveolar macrophages. A total of 121 persons were included in this
Differential diagnosis is difficult unless the physician pilot study by the convenient purposive samples
is aware of the occupational dust (silica) exposure (n=121) from three different categories, viz., no
history of the workers, which is very subjective
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exposure (healthy controls), moderate dust exposure

in nature. Moreover, superimposition of silicotic (ceramic factory workers) and X-ray confirmed
nodules and tuberculous infiltration in X-ray film and silicosis patients (advanced stage). Clinical
difficulty in isolation of Mycobacterium tuberculosis information was collected through health check-up and
from sputum of silicotuberculosis patients as silicotic personal interview using a semi-structured field-tested
fibrosis prevents its discharge into sputum, make the questionnaire. Of them, 25 workers were from the
situation more complicated5. Therefore, a suitable bio- sanitary ceramic production industry in Gujarat
marker is needed for early detection of silicosis, which with varying period (1-8 yr) of silica dust exposure
would not only prevent or control advanced silicosis (considered as moderately exposed to silica dust) and
cases but also silicotuberculosis, a neglected tropical 40 workers were X-ray confirmed silicosis patients
disease in the developing countries. (i.e., long duration exposure to silica dust) as per
A number of anti-inflammatory biomarkers for International Labour Organization (ILO) classification21
early detection of silicosis have been tested, but most of of chest X-ray, who worked in the agate industry
these were non-specific and hence found unsuitable for in Gujarat and sand stone mines in Rajasthan.
silicosis detection6,7. The club cell protein 16 (CC16) Furthermore, 65 age- and sex-matched healthy controls
is one of the potentially immunosuppressive or (volunteers coming from ICMR-NIOH) without
anti-inflammatory proteins secreted from non-ciliated any occupational history of silica dust exposure
epithelial cells of bronchoalveolar epithelium8,9. CC16 were included (9 refused to provide blood samples,
is proposed to be a peripheral marker of respiratory hence excluded). Of these healthy controls, 24 were
epithelial injury that protects respiratory tract against categorized in smoker subgroup (at least 5 cigarettes/day
oxidative stress-induced inflammation9-11 and passively for the last 2 months) because smoking might reduce
diffuses in bronchoalveolar-blood barrier to plasma12. serum CC16 value like other lung biomarkers22.
CC16 deficiency has been reported to be associated Those suffering from chronic lung or kidney disease,
with an increase susceptibility of lung to viral infections hypertension, diabetes, exsmokers and with regular
and oxidative stress in experimental rodent model13-15. intake of analgesics or anti-inflammatory drugs were
In human, a polymorphism of CC16 gene, localized to excluded (n=6) from this study.
a region linked to airway diseases has been discovered Sample collections and processing: Sample collection
in association with an increased risk of developing and processing were done as per the standardized
childhood asthma16,17 and integrity of lung epithelium18. method used in previous study23. For this purpose,
Though the exact physiological mechanism of CC16 venous blood (4 ml) was collected aseptically by
remains unknown, but evidence suggesting significant trained medical laboratory technicians using Vacutainer
reduction of CC16 in silica dust-exposed workers with needle and holder (Becton Dickinson, USA) and was
no change in respiratory symptoms, normal chest X-ray transported to the laboratory immediately. Serum was
and lung function tests19,20 indicates that CC16 could separated on the same day and stored at −25°C till
be an early asymptomatic detection tool for silicosis serum CC16 analysis was done.
and silica-exposed population at risk. Therefore, the
present study was carried out to assess the feasibility Club cell protein measurement: The concentration
to develop CC16 as a potential biomarker for early of CC16 in serum was determined by ELISA kit
detection of silicosis. specific to human as per manufacturer’s protocol
 NAHA et al: CC16: BIOMARKER FOR EARLY SILICOSIS 321

(BioVendor, USA). The absorbance was read at 450 nm silica dust exposure was 4.2±3.9 yr in the ceramic
in a standard microplate reader (BioTek, USA), which industry workers; and the same was 23.1±9.88 yr in the
was proportional to the concentration of CC16 protein X-ray confirmed advanced silicosis workers from the
present in the sample and the value was calculated agate and sand stone mines. Similarly, in the smoker
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from the standard curve. group, 24.3±12.7 yr was the mean duration of silica
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dust exposure for the advanced silicosis workers.

Statistical analysis: The normality of the data was
CC16 levels in healthy controls, moderate silica
checked by Kolmogorov-Smirnov test24. ANOVA dust-exposed workers and X-ray-confirmed advanced
(one-way classification) was applied to test the silicosis patients: In this present study, mean serum
differences in CC16 levels among exposure groups, for CC16 concentrations were 16.7±3.81, 10.2±1.77 and
smoking and non-smoking subgroups. Sensitivity and 4.7±3.07 ng/ml, respectively, in the healthy controls,
specificity analysis at a given cut-off value of serum moderately silica dust-exposed workers and X-ray
imK/icbKo55CAlRxwKTQm+VAr0Du6A== on 06/17/2024

CC16 was calculated considering the mean values confirmed advanced silicosis patients without any
with SDs for healthy non-smokers, moderate silica smoking habit (Fig. 1). In the three X-ray-confirmed
dust-exposed workers and X-ray confirmed silicosis silicosis workers, serum CC16 levels were found
workers (longer duration of silica exposed)25,26. to be below the detection limit of the ELISA kit
Similarly, cut-off value for healthy smoker and (i.e., 46 pg/ml). The results revealed significantly
advanced silicosis workers with smoking habit were (P<0.01) lower serum CC16 levels in the moderately
calculated. The CC16 value for moderate silica dust silica dust-exposed workers and X-ray confirmed
exposure individuals with smoking habit could not be silicosis workers compared to the healthy controls in
measured as no individual of this kind was available the non-smoker group. In smoker group also confirmed
for participation in this study. silicosis patients had significantly (P<0.01) lower
serum CC16 values compared to healthy controls
Results (Fig.1). Smoking had an additional lowering effect
Participants in the smokers group were on serum CC16 levels over the non-smokers in all
analysed separately to assess its additive effect categories of the individuals (Fig.1). However, no
over silica-induced lowering of CC16 values in the significant difference of serum CC16 levels was
non-smokers. In this study, about 30.6 per cent (37 of found in X-ray-confirmed silicosis workers of the two
121) participants were smokers, and of them, 35.1 per different occupational set ups such as, agate industry
cent (13 of 37) had silicosis. Of the 40 X-ray confirmed and sand stone mines.
silicosis workers, 30 (75%) had varying degree of
respiratory discomfort including shortness of breath/ Sensitivity and specificity of serum CC16 test: There
dyspnoea; 20 per cent of these silicosis patients (n=8) was a continuous fall in serum CC16 levels from
were suffering from silicotuberculosis. However, no 22.5 to 0.2 ng/ml in different categories of the study
participants in both the non-smoker and smoker groups.
such respiratory morbidity was found in the moderately
Hence, it was decided to have a cut-off value for each
exposed workers to silica dust.
category considering the mean values with SDs, to
Age and occupational silica dust exposure have the maximum possible sensitivity and specificity
history of various categories of both the non-smoker at a given cut-off value. In this study, best possible
and smoker groups are shown in the Table. In the cut-off values were obtained for non-smokers: 13.0 ng/
non-smoker group, the mean duration of moderate ml to separate out the healthy controls from moderately

Table. Age and duration of silica dust exposure of various categories of non‑smoking and smoking study participants
Parameters Non‑smokers (n=84) Smokers (n=37)
(mean±SD) Healthy Moderate silica X‑ray‑confirmed/ Healthy X‑ray‑confirmed/
controls dust‑exposed advanced controls advanced
(n=32) workers (n=25) silicosis (n=27) (n=24) silicosis (n=13)
Age (yr) 37.5±11.41 31.7±7.38 52.0±8.67**,†† 38.9±12.55 52.8±11.24δδ
Duration of exposure (yr) Nil 4.2±3.90 23.1±9.88†† Nil 24.3±12.71††
P<0.01 vs. respective healthy controls, P<0.01 vs. moderate silica dust‑exposed workers, P<0.01 vs. respective healthy controls
** †† δδ
 322 INDIAN J MED RES, APRIL 2020

A
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imK/icbKo55CAlRxwKTQm+VAr0Du6A== on 06/17/2024

Fig. 1. Mean±SD of serum club cell protein (CC16) levels with range in various categories of non-smoker (A) and smoker (B) groups.**P<0.01
vs. healthy controls, ††P<0.01 vs. silica dust-exposed workers, δδP<0.01 vs. healthy controls.

exposed silica dust and 7.0 ng/ml for separating out smokers, the sensitivity was 83 per cent (20/24), and
X-ray-confirmed/advanced silicosis from moderately for advanced silicosis smokers, it was 85 per cent
exposed asymptomatic individuals (Fig. 2A). With the (11/13) (Fig. 2B). However, specificity could not be
above cut-off value, sensitivity and specificity were calculated as true negative could not be found out due
calculated for healthy controls as well as advanced to non-availability of the smokers in moderate silica
silicosis cases. The sensitivity for the healthy controls dust-exposed workers.
was 88 per cent (28/32) and for the advanced silicosis
individuals was 92 per cent (22/24). The specificity Discussion
for healthy controls was 96 per cent (24/25) and for
advanced silicosis individuals, was 92 per cent (22/24) The present study showed a significant
(Fig. 2A). In the smoker group, healthy and advanced reduction in serum CC16 levels in individuals in
silicosis individuals had the cut-off values of 9.0 and three different occupational set ups in Western India
5.0 ng/ml, respectively (Fig. 2B). Hence, for healthy (Gujarat and Rajasthan), while comparing with the
 NAHA et al: CC16: BIOMARKER FOR EARLY SILICOSIS 323

A
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imK/icbKo55CAlRxwKTQm+VAr0Du6A== on 06/17/2024

Fig. 2. Sensitivity and specificity of serum club cell protein (CC16) test at given cut-off values in various categories of non-smoker (A) and
smoker (B) groups. Results represent the range and mean of serum CC16 levels with higher and lower side cut-offs.

non-exposed healthy controls. Previous studies have X-ray changes and confirmed silicosis cases, and
shown a decline of serum CC16 levels with silica dust their relationship with serum CC16 values15,27. CC16
exposure, but no study has conclusively evidenced concentrations in BAL fluid correlate with serum
on healthy, early silica-exposed cases without having levels, suggesting that serum could be used to infer
 324 INDIAN J MED RES, APRIL 2020

local CC16 lung biology28. Furthermore, alteration of smoking with X-ray confirmed silicosis reduced
serum CC16 reflects early toxic effects of silica on the the cut-off value, suggesting additional destruction
respiratory epithelium of the miners; thus, it ensures of lung epithelium in silicosis with smoking habit.
the efficiency of CC16 molecule as an early detection Shinkins et al25 opined that sensitivity and specificity
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marker by using it as a screening tool for suspicion/ along with cut-off values were useful for reporting
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prediction of silicosis during exposure to silica dust the transparent and accurate clinical diagnosis. If
particles in the workplaces8. periodic screening of silicosis with CC16 is initiated,
it would alert the dust-exposed workers regarding
Lowering of serum CC16 values by smoking in this
their lung health status and would help to delay the
study, indicated that tobacco smoking along with silica
advancement of the disease if preventive measures
exposure aggravated the disease progression more
are undertaken.
among the smokers. Celli and Owen28 corroborated the
possibility of CC16 as a modulator of cigarette smoke- Since silicosis is an irreversible progressive
imK/icbKo55CAlRxwKTQm+VAr0Du6A== on 06/17/2024

induced respiratory morbidity. Park et al22 revealed disease, early detection is beneficial. In absence of
a reduced serum CC16 level linked with accelerated a suitable biomarker for early detection of silicosis,
decline in lung function test after adjustments for age, the present findings suggest that the low serum
sex, race, smoking status, airway reactivity and body CC16 value below or equal to 7.0 ng/ml along with
mass index, etc. occupational exposure history of silica dust may be
an effective biomarker for early detection of silicosis.
High sensitivity and specificity are essential Early detection of silicosis will also help in reducing
components for any public health screening program26. silico-tuberculosis in the country.
As per Florkowski29, sensitivity and specificity are
critically dependent on clinical context and vary with Acknowledgment: Authors thank Servshri R. Kumar and P.
the cut-off values chosen for a particular diagnostic test. Upadhayay for CC16 ELISA analysis, and Servshri K. Pandit, J.
Patel, H. Makwana, K. Pathak, S. Dodia, J.R. Parikh and Dr S
Serum CC16 protein is not directly related to silicosis,
Yadav for assistance in field sample collection. Also, voluntary
rather it is indicative of the amount of healthy lung tissue participation of the individuals are acknowledged.
available; and in this study, observed values ranged
from 22.5 to 0.2 ng/ml from the healthy non-smokers Financial support & sponsorship: This study was funded
to moderate silica dust-exposed with moderate lung by an intramural research grant received from the ICMR-National
damage to X-ray-confirmed/advanced silicosis with Institute of Occupational Health, Ahmedabad.
significant lung damage. In the present study, cut-off
values of 13.0 and 7.0 ng/ml were considered for the Conflicts of Interest: None.
healthy non-smokers to separate out from the early
initiation of silicosis (asymptomatic stage) and from References
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For correspondence: Dr Kamalesh Sarkar, Director, ICMR-National Institute of Occupational Health, Meghaninagar,
Ahmedabad 380 016, Gujarat, India
e-mail: [email protected]