Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high-risk population, northeastern Thailand.

BACKGROUND: Cholangiocarcinoma (CCA) is a category of lethal hepatobiliary malignancies. Previous studies have found that Opisthorchis viverrini infection and diabetes mellitus (DM) are closely correlated with CCA. However, few studies have discussed the association of CCA with a combination of both O. viverrini infection and DM. This study aimed to assess the correlation of CCA with various combinations of O. viverrini infection and DM among a high-risk population in northeastern Thailand.
METHODOLOGY: This study included participants from 20 provinces in northeastern Thailand who had been screened for CCA in the Cholangiocarcinoma Screening and Care Program (CASCAP) between 2013 and 2019. Histories of O. viverrini infection and DM diagnosis were obtained using a health questionnaire. CCA screening used ultrasonography with a definitive diagnosis based on histopathology. Multilevel mixed-effects logistic regression was performed to quantify the association, which is presented as adjusted odds ratios (aOR) and their 95% confidence intervals (CI). PRINCIPAL
FINDINGS: Overall, 263,776 participants were included, of whom 32.4% were infected with O. viverrini, 8.2% were diagnosed with DM, and 2.9% had a history of both O. viverrini infection and DM. The overall rate of CCA was 0.36%. Of those infected with O. viverrini, 0.47% had CCA; among those with DM, 0.59% had CCA and among those infected with O. viverrini and had DM, 0.73% had CCA. Compared with participants who were not infected with O. viverrini and were non-DM, the aOR for those infected with O. viverrini and with DM was 2.36 (95% CI: 1.74-3.21; p-value <0.001).
CONCLUSIONS: The combination of O. viverrini infection and DM was highly associated with CCA, and these two conditions had a combined effect on this association that was greater than that of either alone. These findings suggest that CCA screening should have a strong focus on people with a combination of O. viverrini infection and DM.

Introduction

The liver fluke, Opisthorchis viverrini is a food-borne trematode which is a key public health problem with a well-documented distribution in Thailand, Lao PDR, Cambodia, Myanmar, and Vietnam [1,2]. One of the most serious consequences of this liver fluke infection is its association with subsequent cholangiocarcinoma (CCA). Globally the highest prevalence of O. viverrini infection, and the highest incidence rates of CCA, are found in Thailand, particularly northeastern Thailand [3-6], where O. viverrini infection prevalence was estimated at 17% in 2009 [2]. Another study in 2014 reported the prevalence of O. viverrini infection was about 23%, and it was more common in males and among people aged 40–49 years [7].

The main source of infection with O. viverrini is the consumption of undercooked or fermented fish (freshwater cyprinid fish) [2,8-10]. This habit is deeply embedded in the food culture of northeastern Thailand, as well as the broader lower Mekong region [11]. Re-infection with O. viverrini following curative treatment occurs in around 10% of cases due to this behavior [12]. Continued consumption of raw fish will lead to individuals experiencing cycles of O. viverrini infection, treatment, and re-infection, a serious problem in highly endemic areas and one that leads to increased risk of progression to development of CCA [3-6].

O. viverrini is not the only risk factor for CCA. Several other diseases have been linked with the condition, including diabetes mellitus (DM) [13,14]. Recent studies have reported that DM is a risk factor for both intrahepatic and extrahepatic subtypes of CCA [15]: patients with DM were 1.6 times more likely to be incident cases of CCA compared with those without DM [16]. In addition, a retrospective study has shown that, although not statistically significant, DM is associated with shorter survival of CCA patients [17]. The relationship between CCA and a combination of O. viverrini infection and DM has also been investigated in hamsters, revealing that infection with liver fluke during diabetes leads to more serious disease in the liver than is due to either condition alone [18]. However, there has been no previous investigation in humans of any relationship between CCA and a combination of O. viverrini infection and DM. This is particularly important in northeastern Thailand as the region, as well as having high O. viverrini infection rates, is also experiencing increasing prevalence of DM. Among rural females in the northeastern part of the country, one study found a prevalence of 8.5% [19]. In addition, the National Health Examination Survey reported an increasing prevalence of DM in the Thai population from 7.7% in 2004 to 9.9% in 2014 [20].

In northeastern Thailand a large-scale CCA screening project is underway run by the Cholangiocarcinoma Screening and Care Program (CASCAP). In this study, residents of northeastern Thailand provide information on both O. viverrini infection history and any previous DM diagnosis. They are then screened for CCA and associated hepatobiliary abnormalities. In this study we used CASCAP data to assess associations between the risk of CCA development and O. viverrini infection history among people with and without DM. We hypothesize that the group who were infected with O. viverrini and who also had a positive DM diagnosis will have a higher risk of CCA than other groups.

Materials and methods

Ethics statement

The research protocol was approved by Khon Kaen University Ethics Committee for Human Research, reference number HE631061. The data were provided from the Cholangiocarcinoma Screening and Care Program (CASCAP). The CASCAP data collection was conducted according to the principles of Good Clinical Practice, the Declaration of Helsinki, and national laws and regulations about clinical studies. It was approved by the Khon Kaen University Ethics Committee for Human Research under the reference number HE551404. All patients gave written informed consent for the study.

Study design

This cross-sectional study collected data from the Cholangiocarcinoma Screening and Care Program (CASCAP), northeastern Thailand. CASCAP is the first project for CCA screening in a high-risk population with a community-based bottom-up approach [21]. The CASCAP screening program aims to recruit all residents of northeastern Thailand. This is achieved using multiple methods and settings including tertiary-care hospitals, district-level hospitals and through mobile screening sessions at the sub-district level. This study includes all participants who were screened for CCA and enrolled in the CASCAP database up to the end of 2019 (n = 263,776). In addition to being screened for CCA, participants also filled out a questionnaire containing socio-demographic information, history of O. viverrini infection, diagnosis with DM, as well as other health and lifestyle information.

Study setting and population

Northeastern Thailand (or Isan) is Thailand’s largest region comprising 20 provinces. It is bordered by the Mekong River and Laos to the East and Cambodia to the Southeast. The population of northeastern Thailand is approximately 21 million (around one-third of the total population of Thailand).

Our study includes all people who participated in CASCAP from 20 provinces separated into two groups (screening and walk-in). The screening group were people attending health facilities for reasons unrelated to symptoms that could be associated with CCA, and who were invited to undergo routine ultrasonography (US) screening for CCA. The walk-in group comprised people who attended the hospital with symptoms indicating CCA and underwent histopathological diagnosis for CCA. Both screening and walk-in groups provided information regarding history of O. viverrini infection and DM diagnosis. The sample included in our study were those enrolled in the CASCAP database from 2013–2019 with a total of 263,776 subjects (Fig 1).

Fig 1

Data selection process.

Flowchart showing the number of participants at each stage of data selection, leading to the final analysis set.

Study outcome and independent variables

The primary outcome for this study was CCA which was defined as histologically positive diagnosis and categorized into two groups (no/yes). CCA was diagnosed through US, CT/MRI, and histopathological examination. Positive US results were defined as those indicating liver mass and/or bile duct dilatation, so-called suspected CCA. Those included in this group were referred to receive either CT or MRI scans. For the results of CT/MRI, there were only two categories—positive or negative for CCA. Biopsy confirmation for positive CCA was then needed. Participants who did not have suspicious US nor CT/MRI results were classified as negative for CCA. Each US examination was performed by either radiologists or well-trained general practitioners and was verified centrally by a radiologist at a tertiary hospital. The CT/MRI was mainly done by radiologists at tertiary hospitals. The data of histological findings were based on the standard protocol of the tertiary hospital at Khon Kaen University, Thailand.

The independent variables include the combinations of lifetime history of O. viverrini infection and diagnosis of DM. These were categorized into four groups, namely, uninfected with O. viverrini and being non-DM (OV- & DM-), uninfected with O. viverrini and having DM (OV- & DM+), infected with O. viverrini and being non-DM (OV+ & DM-), and infected with O. viverrini and having DM (OV+ & DM+). The co-variates include gender, age at enrollment, highest achieved educational level, occupation, history of smoking cigarettes defined as no/yes (current orever), drinking alcohol defined as no/yes (current or previous), history of eating raw fish defined as no/yes (current or previous), and history of praziquantel treatments (PZQ) defined as never/once/twice/three times or more. PZQ is used to treat O. viverrini infections. The level factors comprise individual, and province factors.

Statistical analysis

Categorical demographic characteristics were summarized using frequencies and percentages. Continuous data were summarized by their mean, standard deviation (SD), and minimum and maximum values. The rate of CCA was calculated based on a normal approximation to a binomial distribution. A multilevel mixed-effects logistic regression model was applied to consider the hierarchical structure of the subjects, where the individual (level 1) was nested within the provinces (level 2). The level of association between the combination of O. viverrini infection and DM diagnosis adjusted for all other co-variates (fixed effects) and CCA were presented as adjusted odds ratio (aOR) and their 95% confidence intervals (CI). This model was adjusted for the variation of individual-level, and province-level effects (random effects). The highest value of maximum likelihood was estimated to assess the fitness of the model. All test statistics were two-tailed and a p-value of less than 0.05 was considered statistically significant. All analyses were performed using a statistical package, STATA version 15 (Stata, College Station, Texas, USA).

Results

Descriptive summary

A total of 263,776 participants who underwent screening for CCA were enrolled in our study (Table 1). Participants were aged from 18 to 110 years with a mean age of 55.7 (SD = 9.3) years. Almost two-thirds of them were female (60.7%). The majority had only completed primary school or had not completed any formal education (76%) and worked as farmers (84.3%). Among study participants, overall prevalence of O. viverrini infection was 32.4%, and overall prevalence of DM was 8.2%. Among the participants, 62.3% (164,258) were in the OV- & DM- group, 5.4% (14,163) were OV- & DM+, 29.5% (77,831) were OV+ & DM-, and 2.8% (7,524) were in the OV+ & DM+ group. Fig 2 shows the gender distribution of each of these groups. Females outnumbered males in the OV+ and DM+ group. Fig 3 The highest percentage of participants who had OV+ and DM+ were in the >60 years category at 4.4% (3,483/78,625).

Table 1

Demographic characteristics of participants according to O. viverrini infection and diabetes mellitus status.

The data are presented as frequencies and percentages for the overall sample and separated by O. viverrini infection and diabetes mellitus status.

Characteristics	Total	Not infected with O. viverrini (OV-)		Infected with O. viverrini (OV+)
		DM-	DM+	DM-	DM+
	n = 263,776 (%)	n = 164,258 (%)	n = 14,163 (%)	n = 77,831 (%)	n = 7,524 (%)
Gender
Female	160,128 (60.7)	101,698 (61.9)	10,014 (70.7)	43,402 (55.8)	5,014 (66.6)
Male	103,641 (39.3)	62,556 (38.1)	4,149 (29.3)	34,426 (44.2)	2,510 (33.4)
Age groups (years)
< 50	72,549 (27.7)	50,257 (30.8)	1,834 (13.1)	19,639 (25.4)	819 (10.9)
50–60	110,774 (42.3)	68,333 (41.9)	6,019 (42.8)	33,261 (43.0)	3,161 (42.4)
> 60	78,625 (30.0)	44,528 (27.3)	6,201 (44.1)	24,413 (31.6)	3,483 (46.7)
Mean (SD)	55.7 (9.3)	54.9 (9.3)	59.2 (8.4)	56.2 (9.2)	59.7 (8.1)
Educational levels
Primary and lower	200,418 (76.0)	121,550 (74.0)	11,608 (81.9)	60,996 (78.4)	6,264 (83.2)
Secondary	51,554 (19.5)	34,664 (21.1)	1,994 (14.1)	13,919 (17.9)	977 (13.0)
Certificate and higher	11,804 (4.5)	8,044 (4.9)	561 (4.0)	2,916 (3.7)	283 (3.8)
Occupation
Unemployed	8,841 (3.4)	5,120 (3.1)	1,080 (7.6)	2,179 (2.8)	462 (6.2)
Farmer	222,444 (84.3)	137,590 (83.8)	11,280 (79.7)	67,341 (86.5)	6,233 (82.8)
Others	32,491 (12.3)	21,548 (13.1)	1,803 (12.7)	8,311 (10.7)	829 (11.0)
Smoking history
No	204,765 (77.6)	129,475 (78.8)	11,762 (83.1)	57,465 (73.8)	6,063 (80.6)
Yes, current or previous	59,011 (22.4)	34,783 (21.2)	2,401 (16.9)	20,366 (26.2)	1,461 (19.4)
Alcohol consumption
No	145,099 (55.0)	91,097 (55.5)	8,960 (63.3)	40,327 (51.8)	4,715 (62.7)
Yes, current or previous	118,677 (45.0)	73,161 (44.5)	5,203 (36.7)	37,504 (48.2)	2,809 (37.3)
History of raw fish eating
No	21,651 (8.2)	15,513 (9.4)	1,058 (7.5)	4,644 (6.0)	436 (5.8)
Yes, current or previous	242,125 (91.8)	148,745 (90.6)	13,105 (92.5)	73,187 (94.0)	7,088 (94.2)
Praziquantel treatments
Never	137,570 (52.1)	120,459 (73.3)	10,576 (74.7)	6,069 (7.8)	466 (6.2)
Once	96,982 (36.8)	30,361 (18.5)	2,388 (16.9)	58,654 (75.3)	5,579 (74.2)
Twice	18,887 (7.2)	7,622 (4.6)	642 (4.5)	9,544 (12.3)	1,079 (14.3)
Three times and more	10,337 (3.9)	5,816 (3.6)	557 (3.9)	3,564 (4.6)	400 (5.3)

Abbreviations: SD, Standard deviation; n, Number of participants; DM, Diabetes mellitus

Fig 2

Breakdown of study population according to O. viverrini infection and diabetes mellitus status.

Data show percentages of the study population falling into each of the four possible combinations of O. viverrini infection and diabetes mellitus status according to gender and overall.

Fig 3

Breakdown of study population according to O. viverrini infection and diabetes mellitus status.

Data show percentages of the study population falling into each of the four possible combinations of O. viverrini infection and diabetes mellitus status according to age groups.

Rate and association summary

Table 2 reveals the associations between frequency of O. viverrini infection, DM status and CCA diagnosis. From a total of 263,776 participants, 0.36% were diagnosed with CCA. The prevalence rate of CCA was 0.47% for those who had ever been infected with O. viverrini, 0.59% for those with DM. Compared to participants who were not infected with O. viverrini, the aOR for CCA among those infected with O. viverrini was 1.63 (95% CI: 1.37–1.92; p-value <0.001). Compared to participants who did not have DM, the AOR for CCA among those with DM was 1.50 (95% CI: 1.24–1.82; p-value <0.001). These effects controlled for co-variates comprising gender, age at enrollment, educational levels, occupation, smoking cigarettes, drinking alcohol, eating raw fish, and PZQ treatments (fixed effect), and the variation of province levels (random effects).

Table 2

Association of O. viverrini infection, diabetes mellitus diagnosis and other factors with cholangiocarcinoma using multilevel mixed-effects logistic regression.

The data are presented as number of participants, number and percentage having cholangiocarcinoma, crude odds ratios with their 95% confidence intervals and p-values from likelihood-ratio chi-square tests, and adjusted odds ratios and their 95% confidence intervals and p-values from likelihood-ratio chi-square tests for each factor.

Factors	Number	CCA		Crude analysis			Adjusted analysis
		Cases	%	cOR	95% CI	P-value	aOR	95% CI	P-value
Overall	263,776	944	0.36	NA	NA	NA	NA	NA	NA
O. viverrini infection						<0.001			<0.001
No	178,421	544	0.30	1			1
Yes	85,355	400	0.47	1.61	1.41–1.83		1.63	1.37–1.92
Diabetes mellitus						<0.001			<0.001
No	242,089	815	0.34	1			1
Yes	21,687	129	0.59	1.71	1.42–2.06		1.50	1.24–1.82
Gender						<0.001			0.039
Female	160,128	336	0.21	1			1
Male	103,641	608	0.59	2.77	2.42–3.17		0.81	0.67–0.99
Age groups (years)						<0.001			<0.001
< 50	72,549	87	0.12	1			1
50–60	110,774	335	0.30	2.42	1.91–3.06		2.14	1.69–2.72
> 60	78,625	518	0.66	5.25	4.18–6.60		4.68	3.69–5.93
Educational levels									<0.001
Primary and lower	200,418	734	0.37	1		<0.001	1
Secondary	51,554	109	0.21	0.55	0.45–0.67		0.66	0.54–0.82
Certificate and higher	11,804	101	0.86	2.30	1.86–2.83		1.67	1.30–2.15
Occupation									<0.001
Unemployed	8,841	44	0.50	1		<0.001	1
Farmer	222,444	685	0.31	0.64	0.47–0.87		0.82	0.60–1.12
Others	32,491	215	0.66	1.33	0.96–1.85		1.71	1.21–2.42
Smoking history						<0.001			<0.001
No	204,765	419	0.20	1			1
Yes, current or previous	59,011	525	0.89	4.32	3.80–4.92		2.69	2.23–3.25
Alcohol consumption						<0.001			<0.001
No	145,099	239	0.16	1			1
Yes, current or previous	118,677	705	0.59	3.70	3.20–4.29		2.35	1.97–2.81
History of raw fish eating						<0.001			<0.001
No	21,651	31	0.14	1			1
Yes, current or previous	242,125	913	0.38	2.86	1.99–4.09		2.01	1.40–2.90
Praziquantel treatment						<0.001			<0.001
Never	137,570	401	0.29	1			1
Once	96,982	257	0.26	0.87	0.74–1.02		0.58	0.48–0.71
Twice	18,887	153	0.81	2.43	2.02–2.94		1.51	1.21–1.87
Three times and more	10,337	133	1.29	3.70	3.03–4.53		2.27	1.83–2.83

Abbreviations: NA, Not applicable; cOR, Crude odds ratio; aOR, Adjusted odds ratio; 95% CI, 95% confidence interval of adjusted odds ratio

The combination of O. viverrini infection and DM, was associated with the highest rate of CCA, 0.73% (55/7,524). The highest CCA rate was 1.35% (34/2,510) found in males in the OV+ & DM+ group (Fig 4). Fig 5 shows the number of CCA cases according to the combination of O. viverrini infection and DM and separated by age.

Fig 4

Percentages of individuals with cholangiocarcinoma in relation to O. viverrini infection and diabetes mellitus status overall (A) and according to gender (B).

Data show the rate of cholangiocarcinoma as percentage for overall O. viverrini infection and diabetes mellitus groups, and combination of O. viverrini infection and diabetes mellitus separated by sex.

Fig 5

Numbers of cholangiocarcinoma cases by age and in relation to O. viverrini infection and diabetes mellitus status.

Data shows the number of cholangiocarcinoma cases according to combination of O. viverrini infection and diabetes mellitus according to age in year.

Multilevel mixed-effects logistic-regression model results for the association of combinations of O. viverrini infection and DM diagnosis with CCA are reported in Table 3 and Fig 6. Compared to participants in the OV- & DM- group, the crude analysis showed an OR of 2.5 (95% CI: 1.88–3.31; p-value <0.001) for CCA among those in the OV+ & DM+ group. In multivariable analysis, the aOR for CCA among those in the OV+ & DM+ group was 2.36 (95% CI: 1.74–3.21; p-value <0.001). These effects were controlled for co-variates comprising gender, age at enrollment, educational levels, occupation, smoking cigarettes, drinking alcohol, eating raw fish, PZQ treatments (fixed effects), and the variation of province level (random effects).

Table 3

Association of combinations of O. viverrini infection and diabetes mellitus with cholangiocarcinoma using multilevel mixed-effects logistic regression.

The data are presented as numbers of participants, numbers and percentages having cholangiocarcinoma, crude odds ratios and their 95% confidence interval and p-value from likelihood-ratio chi-square tests, and adjusted odds ratios and their 95% confidence interval and p-values from likelihood-ratio chi-square tests for various combinations of O. viverrini infection and diabetes mellitus.

Factors	Number	CCA		Crude analysis			Adjusted analysis
		Cases	%	cOR	95% CI	P-value	aOR	95% CI	P-value
Combination of O. viverrini infection and diabetes mellitus
OV- & DM-	164,258	470	0.29	1		<0.001	1		<0.001
OV- & DM+	14,163	74	0.52	1.81	1.41–2.31		1.56	1.21–2.00
OV+ & DM-	77,831	345	0.44	1.63	1.42–1.88		1.64	1.38–1.96
OV+ & DM+	7,524	55	0.73	2.50	1.88–3.31		2.36	1.74–3.21

Abbreviations: OV, Opisthorchis viverrini; DM, Diabetes mellitus; CCA, Cholangiocarcinoma; cOR, Crude odds ratio; aOR, Odds ratio adjusted for gender, age, educational levels, occupation, smoking cigarettes, drinking alcohol, eating raw fish, and praziquantel treatment; 95% CI, 95% confidence intervals

Fig 6

Adjusted odds ratios for the association of cholangiocarcinoma with O. viverrini infection and diabetes mellitus status.

Data show the magnitude of association of cholangiocarcinoma, comparing groups infected with O. viverrini (OV+), diabetes mellitus (DM+), and combinations of these (OV- & DM+, OV+ & DM- and OV+ & DM+). In each case, the comparison is against the group without diabetes mellitus or O. viverrini infection.

Discussion

We investigated the association of CCA with various combinations of DM and O. viverrini infection in Northeast Thailand. Our study revealed that CCA was highly associated with the combination of O. viverrini infection and DM and that this association was higher than that of CCA with either O. viverrini infection alone or DM alone. To the best of our knowledge, these findings represent the first epidemiological observation among a large population in Northeast Thailand concerning co-occurrence of DM and O. viverrini, and the association with CCA risk.

The proportion of our participants (32.4%) who had a history of O. viverrini infection was higher than noted in 2014, in a study which reported a prevalence of about 23% across a few provinces in northeastern Thailand [7]. This is likely because CASCAP is a large screening program for CCA that covers the entire region. We also found that 21,687 participants (8.2%) had DM, which is close to the previously reported prevalence in northeastern Thailand of about 8.5% [19]. The overall rate of CCA in our study was 0.36%. Of those infected with O. viverrini, 0.47% had CCA; among those with DM, 0.59% had CCA and among those in the OV+ & DM+ group, 0.73% had CCA. Given the nature of the study, where CCA case rates were calculated from among those who underwent screening, it is difficult to compare these results with other studies. However, these results are concerning given that northeastern Thailand is estimated to contribute 63% of CCA cases nationally, while containing only about one-third of the population of Thailand [22].

We found that participants who had been infected with O. viverrini had a 63% higher chance of having CCA than those who were not infected. Our study was conducted in an area endemic for O. viverrini infection, a major risk factor for development of CCA [23-26]. In line with other previous reports [13-15], our study also found that the chance of having CCA increased by 50% for people who had DM. Our study area, besides being a liver-fluke endemic area, also has an increasing incidence of DM [19,20]. Epidemiological studies are increasingly indicating DM as a risk factor for CCA [27-32]. DM plays a key role in influencing carcinogenesis and promoting progression of CCA [28].

Our findings showed CCA had the greatest association with the OV+ & DM+ group in both crude and multivariable analysis, and that the association was larger than for the O. viverrini infection group or the DM group alone. This is consistent with a previous study conducted in a hamster model [18]. Potential mechanisms to explain this increased CCA risk have been explored in some studies. For example, O. viverrini infection worsens diabetes-related damage to the liver and bile ducts in rodents, CCA patients’ tissues, and cultured human CCA cells [18,33]. As well, co-occurrence of opisthorchiasis and DM in hamsters and tissue cultures of human liver cells has been found to lead to more severe disease in the liver than is caused by either condition alone [18]. High glucose levels can stimulate the proliferation and metastatic ability of CCA cells derived from patients with chronic O. viverrini infections [33].

Other studies have assessed whether DM is a risk factor or protective factor in patients diagnosed with biliary tract diseases. One study in a non-endemic area for liver-fluke infection reported that DM may be a protective factor against CCA development in those with biliary tract diseases. However, DM was also associated with significantly increased risk of CCA in patients without these conditions [14]. This finding is consistent with another study in northeastern Thailand, which found that, before praziquantel (PQZ) treatment, O. viverrini infection had a protective effect against hyperglycemia and risk of metabolic disease. However, serum levels of HbA1c and HDL significantly increased during the six months following PQZ treatment [34]. In addition, a study among high-risk subjects for CCA who were diagnosed with periductal fibrosis (PDF) by ultrasonography found that DM had a protective effect against PDF [35]. The association between DM and O. viverrini-associated CCA needs further investigation both in molecular and epidemiological studies. There is still some possibility that the increased risks for CCA development that are associated with infection by O. viverrini coexisting with DM may be coincidental rather than causative.

In conclusion, our findings have revealed that O. viverrini infection and DM diagnosis are highly associated with CCA, with those positive for both O. viverrini infection and DM being at the highest risk. These findings suggest that CCA-screening programs, as well as health-education efforts, should concentrate not only on O. viverrini infection and DM separately but also should have a particular focus on those with a combination of these two risk factors.

Limitations of the study

A limitation of our study was that the data regarding history of O. viverrini infection and DM diagnosis were self-reported by participants, resulting in potential recall bias in the study results. This may particularly affect reporting of O. viverrini infections by older participants in whom infection may have occurred a long time prior to study participation. Detailed dietary data were not collected. All participants reported a history of previously consuming raw/fermented fish but information on frequency, interval and amount of consumption was not assessed. This information may have given more explanatory power to the differences in CCA observed between groups of study participants.

Recommendations

This study was conducted in northeastern Thailand and may not reflect the general population of the country or of SE Asia more generally. Further study is necessary in the region to test the generality of our results. Nevertheless, the methodology and results of our study can be used as a guideline in formulating clinical practice and future research priorities.

9 May 2021

Dear Dr. Suwannatrai,

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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: Feasible

Reviewer #2: = objective clearly stated

= study design is appropriate

= Hypothesis missing. proposed to clearly spell-out the hypothesis in the last paragraph of the instroduction

= sample size is fixed as CASCAP data was used

= statistical analysis should be reviewed by statistician

= yes, data originates from large-scale ongoing screening program. Ethics statement is not given in manuscript. Should be added.

please address the following important points:

= please provide for all variables used in this analysis a clear definition:

1. outcome: please state whether CCA was defined as histologically positive CCA or also other assessments.

2. O. viverrini infection: state clearly that this is a reported history of O. viverrini infection. Was the question restricted to a time period or any prior diagnosis.

3. Diabetes assessment was also reported by the patient. What exactly was asked. Ever diagnosed? Has the current treatment been included

4. smoking: please clarify if current smoking, ever smoking, smoking quantity etc.

5. drinking alcohol: please clarify what is considered drinking alcohol? is linked with quantity etc. or provide question.

6. eating raw fish: please specify what exactly was asked to participant (ever eaten raw fish, fish sauce, fermented fish etc.)

= please improve the statement of the statistical analysis in order to be:

When performing the adjusted analysis, did the authors adjust always for all the other variable. In Table 2 is it correct that for each aOR provided the 9 other variables were used for adjustment. Or, did the author conduct one multivariable analysis and report the adjusted results?

--------------------

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: Clear presentation

Reviewer #2: = results presented match the analysis plan

= result presentation can be improved:

1. In descriptive summary

- please add also overall prevalence of Opisthorchis infection, DM and CCA.

2. Table 1

- please add in all columns a column-percent in brackets as it is done in the "Total" column. This will add more numbers to the table but provides the reader an easier insight into the data which currently difficult because the reported number of observations in the subgroups are huge.

3. Table 2

This is the key results table of the manuscript. Hence it is worthy to add elements to provide easier insights in the data (landscape presentation might be necessary). Suggested to:

- add column percent in the "number" column

- insert a column next to "number" column (left of "%CCA" column) in which absolute number of CCA cases are reported

- insert two column in which 95% Ci and p-value of cOR are reported

- insert a column in which 95% Ci and p-value of aOR are reported

- please clarify the role of the reported p-value

4. Table 3

- please modify the table in analogy of table 2

- the content of this table is largely represented by Fig 6. Therefore, table 3 could be moved to a supplementary file

5. Fig 1

- can the exact dates of used registration period be given (1 Jan 2013 - 31 Dec 2019)?

6. Fig 2

- please use also colors.

- please add overall section with prevalence of O. viverrini infection and DM

7. Fig 3

- please use also colors

8. Fig 4

- please use also colors

- suggested to add overall section with CCA incidence in Ov alone and DM alone

9. Fig 5

- please use also colors

- suggestion: it might be clearer with the smoothed age-prevalence curves

10. Fig 6

- very nice! It could replace Table 3, see above

- please provide exact aOR above the rhomboid symbol

11. All tables and figures

- please provide in addition to the title a descriptive text with details in order to make the table/figure self-explanatory

--------------------

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: Somewhat weak

Reviewer #2: The authors report on a CCA risk increase when Ov infection and DM is present and was highest when both exposure were present. However, the discussion regarding the internal validity of the study is rather weak. The recall bias is mentioned in the limitations but could be addressed more clearly. Further other biais as seletion bias is likely to present. In Fig 1 clearly shows that almost 1 million people were excluded from the analysis. Could they have resulted in a selection biais. In addition, study participants consisted of "screening" and "walk-in" group patient groups. In how far could this fact have influenced the observed associations? Finally, existing evidence on the underlying biological mechanisms is given in the introduction. However, it would be great if this section in the introduction (lines 118-125) could be moved to the discussion and could be elaborated on in more detail. It is interesting that one cited article (Tsai et al, Int J Cancer, 2015; ref 14) reports on a negative association between DM and CCA.

Consequently, the conclusion of the article could be adapted to this discussion part and tuned down a bit. However, the public health message is nicely stated and more attention should be given to the multi-morbidity groups.

--------------------

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: = Please, carefully English edit the text. E.g. "had O. viverrini positive" or "had DM positive" is not correct English; line 101: drop "old", line 133-4: suggest to revise sentence; line 173: pathological or histological? line 163-4: Ov infection prevalences are higher in Laos than in NE Thailand; reported CCA incidence are worldwide highest in NE Thailand;

= Please check references and edit according to the journals recommendations. Italicize genus/species names.

= please check sentence on line 105: meaning seems inadequate. Most Ov re-infection are due to raw/fermented fish consumption

--------------------

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: General Comments

This is a large scale cross-sectional retrospective study on the effects of co-morbidity of Ov infection and DM on cholangiocarcinogenesis.

The story is smiple and the experimental design is straightforward. The results are clear-cut.

In contrast, the MS is lengthy, wordy with many repetitions. Introduction and Discussion can be shortened considerably to make MS much more impressive.

Specific Comments

Although this MS has a co-author of native-English using scientist, there are numerous typos and English usage problems, although those does not affect the scientific quality of this work.

1) Typically, for study areas, “Northeast of Thailand” in the title, but “Northeastern Thailand”, “Northeast Thailand”, “Thailand’s Northeast region” in other places. I believe the Thai government’s official expression is “the Northeast Thailand” and “northeastern Thailand”. Consistency is required.

2) Opishorchis viverrini is the name of parasite, whereas Diabetes mellitus is the name of disease. Whenever you are talking about co-morbidity, or combination of O. viverrini infection and DM, you should use “opisthorchiasis” or “O. viverrini infection” instead of “O. viverrini”

Discussion section is too much repeat of the results. What is important is how DM and Ov infection mutually affect to lead carcinogenesis. Please modify discussion to provide more about possible mechanisms of co-morbidity of opisthorchiasis and DM on CCA genesis.

Reviewer #2: Congratulations! This is a extremely nice and most valuable report. It is based on a unique data base, CASCAP. The conclusions on the study are of importance for Southeast Asia, particularly because of O. viverrini rates are high in many places and diabetes prevalence is in the increase.

--------------------

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Reviewer #1: No

Reviewer #2: No

Figure Files:

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Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

23 Jun 2021

Submitted filename: Response to reviewers_20210620.docx

Click here for additional data file.

6 Jul 2021

Dear Dr. Suwannatrai,

Thank you very much for submitting your manuscript "Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high risk population, northeastern Thailand" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations, especially the English usage.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Sutas Suttiprapa, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Banchob Sripa

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

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: (No Response)

Reviewer #2: please see general comments

--------------------

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: (No Response)

Reviewer #2: Authors have improved the presentation of the results. In the result tables of the risk analysis p-values are provided. Please specify from which statistics the p-values are (from the LR chi2 or z-statistics?). Please specify in the table legend. If the p-value is from z-statistics then it should be given on the same row as the OR and the 95% CI of the OR.

The Figures and Tables have been improved and are now self-explanatory. However, the explanatory text need to be English edited.

--------------------

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: (No Response)

Reviewer #2: good and have been improved

--------------------

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: It is still necessary that the text is English edited.

Please, check Table 3. The presentation is confusing. Explanations of the adjustments should be given in the legend. The p-value seems to be indicated in the wrong row. The crude and adjusted OR should be always corrected abbreviated (aOR, cOR) and the abbreviations consistently used (in this table and in the entire manuscript). Please also introduce the aOR and cOR in the analysis section of the methods.

--------------------

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: Comments to PNTD-D-21-00483R1

This revised version is acceptable after some corrections for English usages.

Especially attention is required for the usage of the abbreviations like OV and DM for the group names.

Consistent use of abbreviation is required.

l. 89: were >> are

l.94: people >> those

l.103: delete “and” after Thailand

l.107: delete “in Thailand”

l.110: by >> with

l.120: in >> of

l.119-120: change to “has shown that, although statistically not significant, DM was associated with shorter survival of CCA patients [17].

l.121: reported >> revealed

l.123: delete “the”

l.135: underway as the

l.148: requested from >> provided from

l.193: This group were >> Those included in this group were

l.196: negative results >> negative results group

l.199: data on >> data of

l.202: diagnosis with DM >> diagnosis of having DM

l.202: in to >> into

In the l.201-205 in the M&M section, the authors have defined 4 groups of participants using abbreviation of OV for O. viverrini infection and DM for diabetes mellitus.

In the Results section, the group names have been used inconsistently. Typical example can be seen in l.237-245, but inconsistency throughout in the Results and Discussion.

Consistent use of abbreviation is required.

Reviewer #2: This is a very interesting study. The authors have addressed all the points raised in my first assessment.

--------------------

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Reviewer #1: No

Reviewer #2: No

Figure Files:

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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

30 Jul 2021

Submitted filename: Response to Editor and reviewers.docx

Click here for additional data file.

17 Aug 2021

Dear Dr. Suwannatrai,

We are pleased to inform you that your manuscript 'Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high-risk population, northeastern Thailand' 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.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

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

Best regards,

Sutas Suttiprapa, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Banchob Sripa

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

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: (No Response)

Reviewer #2: OK

**********

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: (No Response)

Reviewer #2: OK

**********

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: (No Response)

Reviewer #2: OK

**********

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: OK

**********

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: (No Response)

Reviewer #2: The authors have addressed all comments / suggestions.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

3 Sep 2021

Dear Dr. Suwannatrai,

We are delighted to inform you that your manuscript, "Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high-risk population, northeastern Thailand," 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.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

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