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Introduction

Cystic fibrosis (CF) is a multi-system disease primarily involving the respiratory and gastrointestinal tracts. This autosomal recessive condition arises from loss of function of the CF transmembrane conductance regulator (CFTR) [1]. Major advances in respiratory therapies, nutritional optimisation and enzyme supplementation have transformed this disease and increased survival by decades [2, 3]. Medications that modulate CFTR show promise in improving quality of life and overall health [4, 5]. However, as individuals with CF are living longer, there is increasing incidence of comorbid conditions such as CF-related diabetes (CFRD) [6]. Present data suggest that 50% of adults will develop CFRD by the age of 50 years [7]. Individuals with CF have a high metabolic rate and need to consume sufficient energy to maintain their BMI [8, 9]. Pulmonary function is closely associated with nutritional status [10, 11]. Because of the central role of reduced insulin secretion capacity, CFRD could impact the ability to retain and utilise this energy, putting individuals in a catabolic state [8, 9], while the related hyperglycaemia could favour exacerbations further increasing the metabolic requirements [12, 13]. Early detection and treatment of CFRD could thus minimise its impact on individuals’ overall health and survival [8, 14].

Due to the insidious onset of CFRD and low reliability of simpler alternative screening methods [7, 15], an annual screening for glucose abnormalities is recommended from 10 years of age using a 2 h OGTT [16]. Glucose sampling is performed prior to and 2 h after ingestion of a standardised glucose load (1.75 g/kg, to a maximum of 75 g). A fasting glucose level ≥ 7.0 mmol/l and/or a 2 h OGTT glucose level ≥ 11.1 mmol/l are used to diagnose CFRD. These diagnostic criteria were derived from prospective trials of complications of hyperglycaemia in type 1 and type 2 diabetes and are based upon the risk of developing diabetic retinopathy [17, 18]. Long-term vascular complications are important outcomes but do not reflect the primary pulmonary and nutritional consequences of CFRD [17]. Moreover, the sensitivity and specificity of the standard OGTT are poor for CFRD and there is high variability in 2 h post-OGTT glucose in sequential OGTTs in a given individual [6, 17].

OGTT screening is associated with a high burden for patients and healthcare teams [19]. Improving the reliability of testing would improve its clinical predictive value and would provide more meaningful data. We explored whether a 2 h OGTT with 30 min sampling of blood glucose might improve the reliability and reproducibility of this test for the purpose of determining CFRD risk and the risk of accelerated clinical (pulmonary function and/or nutritional) decline. The primary aim of this paper was to investigate which blood glucose variables derived from an OGTT with 30 min sampling most strongly correlate with the risk of developing CFRD and key clinical measures in CF, such as BMI and pulmonary function. The secondary aim of this paper was to investigate whether these intermediary time points could provide additional data, so that OGTT frequency scheduling could be improved.

Methods

Design and setting

This cross-sectional and prospective analysis was performed using data collected from the Montreal CF Cohort (MCFC). This cohort was created in 2004 and is an ongoing systematic diabetes screening programme that includes over 300 adults with CF [6]. The included individuals undergo an OGTT, blood sampling and evaluation of pulmonary function (forced expiratory volume in 1 s [FEV1]) every 1–2 years [20]. Our analysis included data from 2004 until May 2019.

Inclusion and exclusion criteria for participation in the MCFC have been described previously [20]. In brief, the main exclusion criteria were prior diagnosis of CFRD, the use of CFRD modulator therapies, pregnancy, previous pulmonary transplantation, and recent pulmonary exacerbations or contraindicated medication use. In the case of pulmonary exacerbations or use of medication that could interfere with glucose metabolism [6], the inclusion procedure was modified as previously described [11].

Participants

Included in this study were adults (aged ≥18 years) with CF who did not have any missing plasma glucose measurements from their OGTTs and who had undergone at least two OGTTs. A consort diagram explaining the participants included in this study is shown in Fig. 1.

Fig. 1
figure 1

CONSORT diagram describing the inclusion of participants in this study. KM, Kaplan–Meier

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Some of the study participants were diagnosed with de novo CFRD by a single OGTT at initial screening. These individuals were not receiving treatment for CFRD at the time of the evaluation but were referred to an endocrinologist. They were included in the baseline analyses but not in the longitudinal assessment of diabetes risk.

The protocol was approved by the Research Ethics Committee of the Centre Hospitalier de l’Université de Montreal and of the Montreal Clinical Research Institute. Informed consent forms were reviewed and signed by all participants.

Clinical and biological data

All measures were taken on the same day as the diabetes screening OGTT. Age, sex and genotype were collected by chart review. Genotype was categorised into three groups based on CFTR expression and function as previously described [11]. Body weight (kg), standing height (cm), BMI (kg/m2) and FEV1 were measured as described previously [6]. For this study, FEV1 is expressed as a percentage of the predicted value. HbA1c was measured using fasting serum samples, as described previously [20].

OGTT

OGTT was realised in standardised conditions as previously described [6]. In brief, plasma glucose levels were measured every 30 min (fasting [G0], 30 min [G30], 60 min [G60], 90 min [G90] and 120 min [G120]). Plasma insulin levels were also measured every 30 min (I0, I30, I60, I90 and I120). Plasma glucose levels were measured immediately after the OGTT with a Glucose Analyzer (YSI 2900; YSI, USA). Insulin samples were frozen at −80°C and later measured in duplicate using human insulin RIA (Linco Research, USA).

The peak glucose level (Gmax) and peak insulin level (Imax) were evaluated and defined as the highest plasma glucose or insulin level recorded at any time point of the OGTT. The total AUC for glucose (GAUC) and insulin (IAUC) were calculated using GraphPad Prism (GraphPad Software, CA, USA). Using IAUC and GAUC, insulin secretion normalised for glucose was calculated by dividing IAUC over GAUC (AUCINS/GLU). AUCINS/GLU was also calculated for first-phase (early, 0–30 min) and second-phase insulin secretion (late, 30–120 min). Finally, the insulin sensitivity index (ISI) was estimated using the Stumvoll index [21]. If two or more insulin values were missing during an OGTT, the peak and AUC levels were excluded. No glucose data points were missing.

Participant categorisation

OGTT results were classified into different glucose tolerance groups based on OGTT G0 and G120 values [16]. Participants with a G0 ≤ 7.0 mmol/l (126.1 mg/dl) and a G120 ≤ 7.7 mmol/l (138.7 mg/dl) were placed in the normal glucose tolerance (NGT) group. Impaired glucose tolerance (IGT) is defined as having a G0 ≤ 7.0 mmol/l and a G120 > 7.7 mmol/l but <11.1 mmol/l (200.0 mg/dl). Finally, participants with de novo CFRD had a G0 > 7.0 mmol/l and/or a G120 ≥ 11.1 mmol/l.

Statistical analysis

Descriptive statistics were computed for all variables of interest. The mean ± SD are used to present data, as well as the range for clinical characteristics. All continuous data were evaluated for normality of distribution and parametric and non-parametric tests were used, as necessary. Spearman’s correlation was used to evaluate the relationship between nutrition, lung function and glucose levels. Spearman’s correlation was also used for observational prospective analyses. Bonferroni correction was used to minimise false positives. Significance was accepted when p ≤ 0.003. A one-way ANOVA test was also performed to compare baseline characteristics of three groups categorised based on Gmax. Statistical analysis was performed using SPSS software (version 25; IBM, USA). Significance was accepted when p ≤ 0.05.

To compare glucose tolerance groups and the number of participants with Gmax at each OGTT time point, a χ2 logistic regression was used. The adjusted residuals were also calculated and an adjusted residual ≥ ±2.0 implied that a group had significantly greater (+) or lower (−) frequency than expected.

Finally, to investigate the risk of developing CFRD, a Kaplan–Meier survival analysis comparing the Gmax groups was performed using GraphPad Prism (GraphPad Software, USA). The Mantel–Cox test was used to calculate a p value for the Kaplan–Meier analysis. A p value ≤0.05 implied significance.

Results

Characterisation of participants at baseline

Of the 304 adult CF patients in the MCFC, 11 were excluded from this study. Thus, a total of 293 adults with CF met the inclusion criteria and were included for cross-sectional analysis. Of the included participants, 131 were female sex (44.7%) and 162 (55.3%) were male. The mean age of participants was 25.5 ± 7.9 years old and 80.1% displayed pancreatic insufficiency. Almost half (48.8%) of the participants were homozygous for the ΔF508 mutation, while 40.3% were heterozygous and 9.2% were neither homozygous nor heterozygous. In terms of clinical status, the mean ± SD BMI was 21.7 ± 2.9 kg/m2 and the mean ± SD FEV1 was 73.0 ± 21.9%. The proportion of participants classified according to glucose tolerance was as follows: NGT, 56.3%; IGT, 29.0%; de novo CFRD, 14.7%.

The mean ± SD G0 was 5.45 ± 0.78 mmol/l and the G120 was 7.99 ± 3.28 mmol/l (electronic supplementary material [ESM] Table 1). The mean ± SD Gmax was 11.7 ± 3.12 mmol/l, with over half of the participants (61.8%) exhibiting Gmax at 60 min; 17.4% experienced Gmax at 30 min, 15.4% at 90 min and 5.5% at 120 min. The Gmax was higher than the G120 in 94.5% of the participants. Most participants with NGT experienced Gmax at 30 and 60 min, while most of the participants in the IGT and CFRD groups had a Gmax at 60 and 90 min (ESM Fig. 1). Of the 16 participants with a Gmax at 120 min, 11 were diagnosed with CFRD.

Relationship between baseline clinical measures and OGTT indices

Neither G0 nor G120 were significantly correlated with BMI or FEV1 (Table 1). Although there was no correlation between Gmax and BMI, Gmax was negatively associated with FEV1 (r = −0.18, p = 0.003). GAUC was inversely correlated with FEV1 and BMI but the associations were not statistically significant.

Table 1 Association between clinical status and OGTT glucose and insulin levels at baseline
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I0 was positively associated with BMI (r = 0.23, p < 0.0001), but not FEV1. I120 did not have significant correlations with BMI or FEV1. Both IAUC and AUCINS/GLU had a significant positive correlation with BMI (r = 0.22, p < 0.0001 and r = 0.20, p = 0.001, respectively) but not FEV1.

Relationship between prospective clinical measures and baseline OGTT indices

From the 293 participants included at baseline, only 185 CF patients (with a mean follow-up of 7.5 ± 4.2 years) were included in observational prospective analyses due to the exclusion of individuals who discontinued their participation in the MCFC or who were diagnosed with de novo CFRD between their initial and most recent visit. The same analyses comparing OGTT indices with nutritional and clinical status performed at baseline were repeated for the follow-up data. We found similar patterns as with the analyses done at baseline, although none of the associations reached statistical significance (ESM Table 2).

We calculated the rate of change ([follow-up – baseline]/ time in months) of BMI and FEV1, and investigated their relationship with baseline OGTT variables (ESM Table 3). None of the correlations were significant.

Relationship between Gmax category and risk of developing CFRD

We investigated the risk of developing CFRD in the 185 participants for whom prospective data were available. To do this, we subdivided participants, according to their Gmax at the initial screening OGTT, into the following Gmax groups: <8.0, 8.0–8.9, 9.0–9.9, 10.0–10.9, 11.0–11.9, 12.0–12.9, 13.0–13.9 and ≥14.0 mmol/l. No additional groups were created past Gmax < 8.0 mmol/l or ≥14.0 mmol/l (ie. Gmax = 7.0–7.9 or 14.0–14.9) as there would have been insufficient data. As the Gmax increased, CFRD risk over the course of 10–15 years increased as well (p = 0.0029) (ESM Fig. 2).

For simplicity, we did further analysis using fewer groups. Three distinct patterns of CFRD risk were identified in participants: low-risk group, Gmax < 8.0 mmol/l (n = 20); intermediate-risk group, Gmax 8.0–12 mmol/l (n = 116); and high-risk group, Gmax > 12.0 mmol/l (n = 49). We performed another Kaplan–Meier analysis according to the low-, intermediate- and high-risk groups (Fig. 2). Participants who had a Gmax < 8 mmol/l were at significantly lower risk of developing CFRD over the course of 10–15 years (p = 0.0077). In fact, none of the participants in this Gmax group developed CFRD. The risk of developing CFRD increased as the Gmax threshold increased. In the intermediate-risk group, 27 (23.3%) participants developed CFRD over the study period, while 15 (30.6%) in the high-risk group developed CFRD. Furthermore, of the participants in the intermediate-risk and high-risk groups, 107 displayed NGT at baseline and 21 of them (19.6%) developed CFRD.

Fig. 2
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Kaplan–Meier analysis for risk of developing CFRD according to Gmax category (in mmol/l) at initial screening OGTT of adult participants with CF

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We compared the clinical characteristics of the low-, intermediate- and high-risk groups (Table 2). The intermediate- and high-risk groups had significantly higher proportions with pancreatic insufficiency; 93.9% of the high-risk group and 73.9% of the intermediate-risk group were being treated with pancreatic enzymes (p < 0.0001). There were no significant differences in sex, age, BMI, body fat % or CFTR mutation class between the three groups. The low-risk group had a significantly greater FEV1. HbA1c was lowest in the low-risk group; both the low- and intermediate-risk groups had lower fasting and AUC plasma glucose levels compared with the high-risk group (p < 0.0001). Total insulin secretory capacity (AUCINS/GLU), first-phase AUCINS/GLU and second-phase AUCINS/GLU were greater in the low-risk group than the other groups and became worse as the risk level increased (p < 0.017, Table 2). The low-risk group also exhibited a higher ISI compared with the other groups (p < 0.0001). However, there was no difference in fasting or total (AUC) insulin secretion between groups. Participants with low CFRD risk also reached their Gmax earlier (p = 0.001) and all had NGT (p = 0.001). To further investigate the relationship between HbA1c and Gmax, we performed a non-linear regression at baseline and observed that HbA1c increased in accordance with Gmax for Gmax values ≥12 mmol/l (Fig. 3).

Table 2 Characteristics of adults with CF at baseline, according to Gmax
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Fig. 3
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Association of HbA1c (mmol/mol) with Gmax (mmol/l) in adult participants with CF (n = 293)

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Discussion

With a large prospective observational cohort, in adults with CF, we found that the maximum glucose attained during an OGTT screening test with 30 min sampling is strongly associated with the risk of developing CFRD over a 10 year period. None of the patients with a Gmax < 8 mmol/l developed diabetes. Conversely, those with a Gmax > 12 mmol/l had a very high risk of developing CFRD within 5 years. These variables could help to discriminate individuals with high vs low risk of developing CFRD and thus modulate the screening schedule.

Current clinical diagnostic testing for CFRD uses a two time point OGTT. A recent report, however, highlighted that Gmax during a five time point OGTT had stronger correlation with lung function and nutritional status than G120 in a retrospective analysis of 27 paediatric patients with CF [22]. In a larger adult cohort, we have further investigated these findings by analysing prospectively the relationship between Gmax and the risk of developing CFRD. We identified low-risk (Gmax < 8 mmol/l), intermediate-risk (Gmax 8–12 mmol/l) and high-risk (Gmax > 12 mmol/l) groups, with striking difference in CFRD risk.

Individuals with CF who had a Gmax < 8 mmol/l were more likely to have a heterozygous CFTR genotype, though not significant. They had significantly better pulmonary function and were more frequently pancreatic sufficient at baseline. Our data support a study by Brodsky et al. which found that 1 h glucose levels >7.8 mmol/l in individuals with CF were associated with poorer pulmonary function [23]. Furthermore, blood glucose levels ≥8.0 mmol/l are associated with the appearance of glucose in airway fluid, possibly favouring increased bacterial growth [12]. Our findings contrast those of a study by Hameed et al., which found that a Gmax ≥ 8.2 mmol/l predicted a decline in weight SD score over a 12 month period in paediatric patients with CF [24]. Although we found no difference in BMI between the Gmax groups, the message that individuals with a Gmax < 8 mmol/l are at lower risk of CFRD and clinical status deterioration remains. Participants in our study who had a Gmax < 8 mmol/l generally presented lower G0 and their peak insulin response was faster than the other groups. These findings show that individuals with CF who have Gmax < 8 mmol/l should be considered as being at low risk of CFRD and merit less frequent diabetes screening.

Although most clinical differences were observed between the low-risk group and the other groups, CFRD risk progressively increased between each group. While the low-risk group had no occurrences of CFRD over 10 years, there was a 25-fold increase in CFRD risk between the low- and intermediate-risk groups, as well as between the intermediate- and high-risk groups. There was a 50-fold increase in CFRD risk between the low- and high-risk groups. Furthermore, our results support a previous study, using data from the same cohort as ours, reporting that HbA1c values ≥5.5% (36.6 mmol/mol) were associated with higher CFRD risk [25]. The low-risk group had a mean HbA1c of 35.6 mmol/mol (5.41%), while the intermediate- and high-risk groups had a mean HbA1c > 37.4 mmol/mol (5.57%). HbA1c sensitivity in the detection of CFRD is poor compared with other types of diabetes and values can be normal even in individuals with overt CFRD [15]. Thus, Gmax might be a better variable for identifying CFRD risk. Clinically, the intermediate- and high-risk groups had a mean FEV1 that was 15% lower than the low-risk group. The prevalence of pancreatic insufficiency was remarkably higher in the intermediate- and high-risk groups, highlighting the importance of this easily accessible factor. Our findings support other studies that have already highlighted the importance of pancreatic insufficiency in overall disease severity and CFRD risk [26, 27].

Similar to work by Prentice et al., we ran regression analyses at baseline and prospectively to investigate the clinical relevance of OGTT variables [22]. We found that there was a stronger negative association between measures of FEV1 with Gmax than G120 at baseline. As previously reported, insulinaemia variables were more associated with BMI than blood glucose variables [28], with I0, Imax, AUCINS and AUCINS/GLU all being positively associated with BMI. Similar associations were found at follow-up. Prospectively, we found that FEV1 and BMI did not worsen at a faster rate in correlation with Gmax, G120 or any insulinaemia variables. In stark contrast to the landmark paper by Lanng et al. [29], we found that nutritional status and lung function deterioration does not seem to precede CFRD onset in our modern cohort.

The measurement of insulin during an OGTT is rare and the methods are not standardised. Thus, in this context, insulin data was used to further characterise individuals with CF based on their CFRD risk level rather than to investigate its predictive value. The low-risk group had more favourable efficiency of insulin secretion (AUCINS/GLU) for total, early- and late-phase secretion. The intermediate-risk group only had better early-phase AUCINS/GLU than the high-risk group. The high-risk group was half as efficient as the low-risk group for early-phase AUCINS/GLU, and only 60% as efficient for total and late-phase AUCINS/GLU. These findings demonstrate that individuals with CF who secrete less insulin in response to glucose and have lower beta cell function have a higher CFRD risk, a common theme found in other studies [30, 31]. We also found that low-risk individuals had more favourable ISI, which worsened as CFRD risk increased. We have previously observed that emerging insulin resistance, in the context of limited insulin secretion capacity over a long period of time, in ageing individuals with CF is an important factor contributing to the onset of CFRD [6, 32]. In addition, the more efficient early-phase AUCINS/GLU in low-risk individuals could explain why they more frequently hit their Gmax earlier during the OGTT. The insulin peak became more delayed as CFRD risk and glucose tolerance worsened, with most of the high-risk individuals attaining their Gmax at 60 min. As research regarding insulinaemia variables and CFRD risk is still novel, these results underline the importance of further investigation.

Finally, we must consider the practicality of our suggestion of a 2 h OGTT with 30 min sampling. The advantage is the potential to improve the sensitivity of this clinical test; the limitations are in cost and more intensive testing for patients and healthcare teams. Adherence to OGTT screening is already <50% at many centres [2, 25, 28, 33], mainly due to perceived burden from both sides [19]. We found that a five time point OGTT provided better assessment of CFRD risk but its applicability in a clinical setting needs to be established. We therefore wanted to understand which time points could create the most useful protocol for determining CFRD risk in a clinical setting. In this cohort, we found that the intermediate (30, 60 and 90 min) time points alongside Gmax analysis could provide better assessment of CFRD risk than the standard 0 and 2 h time points. Moreover, a subgroup of participants with NGT and a Gmax ≥ 8 mmol/l hit their Gmax at intermediate time points and had a higher CFRD risk. These findings support those of Dobson et al., who reported that 60 and 90 min OGTT values may be more sensitive than 2 h values in detecting glucose intolerance in CF patients [34]. Hameed et al. also found that increased glucose values at earlier (30 min) time points could identify glycaemic abnormalities and higher probabilities of insulin deficiency [35]. Furthermore, Coriati et al. found that a shortened 90 min OGTT with 30 min sampling provided excellent sensitivity and specificity for IGT and CFRD screening. Thus, the 2 h OGTT might be shortened to 90 min without compromising CFRD risk stratification or clinical care [36]. These findings suggest that intermediate time points could potentially provide better assessment of future CFRD development and clinical status decline, while allowing shortening of the screening test to provide some relief to patients and healthcare teams [28]. Validation studies need to be performed to balance burden vs increased blood samples during a potentially shorter OGTT with better assessment of CFRD risk, thus allowing targeted screening focusing on high-risk patients without systematic annual screening in all patients.

Our study had some limitations. First, this analysis is derived from a relatively homogeneous French-Canadian population implying limitations for external validity. Still, the data are based on a large and well-characterised cohort with long-term follow-up. Second, the MCFC consists mainly of younger and healthier individuals compared with other cohorts. However, data from individuals in the MCFC mirror those reported for North American populations of similar age [37]. Third, as with any observational analysis, causality cannot be established and a lower Gmax and lower prevalence of pancreatic insufficiency may be markers of lower disease severity and/or better therapeutic compliance.

Conclusions

In adults with CF, Gmax is strongly associated with the risk of developing CFRD. No participant with a Gmax < 8 mmol/l developed CFRD over 10 years. Furthermore, a higher Gmax is associated with a greater proportion of pancreatic insufficiency and poorer lung function and insulin secretory capacity. Our findings suggest that a CF-specific OGTT with intermediate time points would optimise the reliability of this test, leading to better CFRD risk stratification and OGTT scheduling. Further investigation into translating this into a practical clinical screening test is needed.