For the SARS outbreak in Hong Kong, it is possible to compare our results with previously published estimates.

Our estimate of the average effective reproduction number prior to the first global alert (R = 3.6, 95 percent CI: 3.1, 4.2) is more precise than the estimate obtained by assuming an exponential increase in the number of cases (R = 3.5, 95 percent CI: 1.5, 7.7) (8) and more precise than the estimated lower bound excluding "super-spreading events" (R > 2.7) (7).

Our estimate of the average effective reproduction number after the first global alert (R = 0.7, 95 percent CI: 0.7, 0.8) is much higher than the previously estimated lower bound excluding "super-spread events" (R > 0.14) (7).

This comparison illustrates that the estimation algorithm presented here allows more precise estimation of the effec- tive reproduction numbers for infectious diseases under more general assumptions than was previously possible.


The effectiveness of control measures against SARS can be estimated if it is assumed that the sudden decrease in the effective reproduction number for SARS around the time of the first global alert was indeed caused by the implementa- tion of control measures.

The decrease in the reproduction number from 3 to 0.7 then suggests that the control measures taken around this time prevented approximately 77 percent (100 × (3 - 0.7)/3 percent ≈ 77 percent) of all potential secondary infections.

This effectiveness suffices for eventu- ally stopping infections in situations where each case causes up to 4.3 (1/(1 - 0.77) ≈ 4.3) secondary infections.

Recalling that before implementation of control measures, each SARS case produced, on average, approximately three secondary infections, this effectiveness has been barely sufficient for the eventual interruption of the chain of human-to-human transmission of the SARS virus.


As the direct threat of a worldwide SARS epidemic has waned, the question arises as to how we can use the experi- ence with SARS to improve infection control against new infectious diseases.

Our analysis of the epidemic curves for SARS, as reported for Hong Kong, Vietnam, Singapore, and Canada, shows how crucial the rapid implementation of control measures has been in limiting the impact of the epidemics, both in terms of preventing more casualties and in terms of shortening the period during which stringent infection control measures were in place.

A first lesson from the several SARS epidemics is that a timely alert against a new infectious disease is most essential.

Our anal- ysis suggests that the control measures implemented prevented approximately three quarters of all potential secondary infections; this may be insufficient to stop another new infectious disease.

A second lesson, then, is that it is crucial to estimate the transmission potential of a new emerging disease as soon as possible and to establish whether additional, more stringent control measures are required.


Health officials faced a daunting task with the emergence of severe acute respiratory syndrome (SARS) last year: forecasting the trajectory of an emerging infectious disease and implementing effective control measures, even as the etiologic agent was still being identified.

Investigators initially had little to go on beyond crude epidemiologic data such as the timing of new cases (the epidemic curve).

With such limited data, it was difficult to disentangle two fundamental epidemiologic quantities: the time from one transmission of the infection to the next, known as the serial interval or generation time, and the average number of secondary cases resulting from each infection, known as the reproductive number.


A simple example illustrates the problem.

Compare two idealized diseases, A and B. Disease A has a short generation time of 4 days but has relatively low transmissibility, such that each primary infection generates two secondary infections.

Disease B has a longer generation time of 8 days but is more transmissible, such that each primary infection generates four secondary infections.

The initial epidemic curves for these two infections will be nearly superimposable, with approximately a doubling of new cases every 4 days.


(These calculations are approximate.

In fact, the generation time for any given transmission will be a random variable, with some distribution.

This distribution will affect the precise doubling time of the epidemic and will also affect the shape of the epidemic curve in the period before the epidemic enters an exponential growth phase [1–3].) Thus, investigators cannot separate generation time from reproductive number on the basis of the epidemic curves alone.

As a result, they are limited in their ability to predict the efficacy of potential interventions.

Despite nearly identical epidemic curves, an outbreak of disease A can be stemmed by an intervention that reduces transmission threefold, while disease B will continue to spread even with such control measures in place.


In the current issue of the Journal, Wallinga and Teunis [4] present a statistical escape from this analytical Catch-22.

Their approach is this: Once epidemiologists have traced the chains of transmission in a single isolated outbreak, they can use the transmission network to infer the distribution of generation times for that disease.

This distribution, which results from the biology of host-pathogen interaction, should be relatively constant in comparison with transmission rates, which will vary from location to location and over the course of an epidemic as the pool of susceptible persons declines and as control measures are implemented.

Therefore, investigators can use this generation time distribution as a grounding point from which to infer the reproductive number, R, from epidemic curves observed in other settings where extensive contact tracing may not have been achieved.

Most importantly, Wallinga and Teunis describe an elegant method for following the instantaneous reproductive number as it evolves over time in a single epidemic.

They provide a means of transforming the time series of cases, along with a “known” distribution for the generation time, into a time series of estimated values for the instantaneous reproductive number on each day.


This method allows nearly real-time tracking of the effect of control measures and other changes in an epidemic on the level of transmission ongoing in the population.

Estimates are not precisely real-time, because accurate estimates of the instantaneous reproductive number on a given date cannot be made until some time after that date.

Specifically, one can only estimate the number of persons a given case has infected when a sufficient amount of time has passed that all patients with secondary cases generated by that case (and by other, surrounding cases) have become infected, become symptomatic, and had their infections reported.

While this lag period (slightly more than the generation time, plus the incubation period, plus the reporting delay) could be decades long for diseases with extended latency periods such as human immunodeficiency virus disease, in the case of SARS the lag period is a matter of a few weeks, and the information provided is as fresh as one could obtain, given the available data.


Wallinga and Teunis’s elegantly simple approach is related to more complex applications of back-calculation that have been used to derive incidence of infection from data on disease prevalence or incidence in human immunodeficiency virus disease [5] and to derive annual average reproductive numbers from case data in the United Kingdom epidemic of bovine spongiform encephalopathy [6].

The present work is distinguished by its real-time applicability to a rapidly transmitted disease and its requirements for only the serial interval distribution and the epidemic curve as inputs.


In their report [4], Wallinga and Teunis demonstrate the power of this analytical approach by improving our understanding of the 2003 SARS outbreaks in Hong Kong, Vietnam, Singapore, and Canada.

The authors find that despite the very different epidemic curves observed in each location, pre-intervention reproductive numbers appear to have been similar.

Furthermore, they infer that control interventions appear to have reduced transmission rates approximately fourfold in each location—enough to stop the epidemics, but only barely.


Like any model, this one has some limitations, which the authors note.

First, the method makes the simplifying assumption of independence of transmission events—that the assignment of a source to any case A is independent of the assignment of the source to each other case B. This is, at best, an approximation.

For example, if different persons vary in their degree of infectiousness (e.g., because of differences in viral shedding) or in their duration of infectiousness, the numbers of secondary infections per individual will be overdispersed (even within the same short time period).

Put another way, if we know that case i was still infectious at a time when case j became infected, we know that case i is a likely candidate for having infected other secondary cases around the same time.

Second, the method assumes that interventions do not affect the generation time of the pathogen.

In reality, when interventions involve improved monitoring followed by either isolation or effective treatment, most transmissions will occur early, before detection.

As a result, the mean generation time will be reduced, and the algorithm presented here could lead to an overestimate of the instantaneous transmission rate for the period subsequent to intervention.


So will Wallinga and Teunis’s approach work despite these limitations? Computer simulations offer some limited evidence that it will, but further study is warranted to assess its performance under various departures from its assumptions.

If the method proves robust, it will be a very useful tool for policy-makers in the midst of an outbreak.

Rapid assessment of the reproductive number of an emerging infection in the absence of interventions is a crucial first step in understanding and controlling the disease.


Control of a disease in a population requires that the reproductive number be brought below 1 and kept there.

By assessing the initial reproductive number (before the implementation of control measures), health officials can estimate the magnitude of the control measures that will be necessary.

As the infection spreads in a particular population and as control measures are instituted, real-time tracking is needed to establish the impact of control.

The success of a control program for a disease like SARS, in which isolation of symptomatic patients and quarantine of contacts were the key control measures, can be measured in part by “process” indicators, such as the time from the onset of symptoms in a case to the isolation of that case to prevent transmission, or the fraction of incident cases that were identified by contact tracing prior to symptom onset.

However, real-time measurements of disease transmission, using methods like the one developed by Wallinga and Teunis, will provide “bottom-line” evidence of whether an epidemic is being brought under control.

During the 2003 SARS outbreak, health officials used a conservative definition—no new cases in a time span exceeding twice the longest known incubation period for the infection—to declare an epidemic fully controlled in a particular jurisdiction, but health officials responsible for daily control efforts need a more immediate and quantitative measure of the effects of control measures as they work toward the goal of eliminating the local epidemic.

Wallinga and Teunis’ method of tracking the instantaneous reproductive number provides just such a measure.


More broadly, new tools are needed to facilitate data entry, management, visualization, analysis, and forecasting in the early days of an outbreak.

For example, epidemiologists attempting to track a natural outbreak of a disease like SARS or pandemic influenza or the deliberate release of a biologic agent would benefit from the ability to manipulate a rapidly changing database with ease, to map spatial and temporal patterns in disease incidence according to the home, school, hospital, or workplace of cases, and to perform key analyses in ways that have been thought through and validated in advance.

As data become available during the course of an epidemic, public health officials should have at their disposal tools to allow the integration of these data into real-time predictions of the relative costs and benefits of potential control strategies.

Many such tools were developed in real time during the 2001 epizootic outbreak of foot-and-mouth disease in the United Kingdom [7], and since then, refined methods for “predictive” vaccination strategies have been proposed [8].

It would be encouraging to see similar effort being put toward planning for human disease outbreaks.


Spurred by the perceived threat of biological terrorism, current epidemiologic efforts to prepare for outbreaks have taken two major forms.

The first is the development of “syndromic surveillance” systems designed to detect the first anomalous cases and alert health officials that an outbreak is under way [9].

While they are well suited to this purpose, such systems are not designed to provide much additional information once the outbreak has been identified.

Other efforts have concentrated on modeling particular attack scenarios with different pathogens, particularly smallpox [10, 11] and anthrax [12, 13].

These models provide valuable a priori guidance about the range of possible responses to given scenarios, but they require, of necessity, untestable assumptions about the size and nature of the outbreak; moreover, to some degree, each of these models gives results whose application is limited to the pathogen under consideration or pathogens that closely resemble it.

As with syndromic surveillance, the usefulness of such models is greatest prior to or at the moment of an attack, but unless the models are designed to incorporate up-to-the-minute data on the state of the outbreak, they will provide limited real-time assistance once an outbreak is under way.


Given the indisputable creativity of Nature and the potential creativity of biologically sophisticated evildoers, we cannot expect to have an appropriate a priori model for every outbreak.

Therefore, development of tools for understanding and responding to a novel outbreak as it unfolds is a pressing task, and one that differs from those that have received the most epidemiologic attention thus far.

Transmission models, as well as the spatial-temporal analysis tools used in syndromic surveillance, may be readily adapted to meet the need for analysis of and response to an outbreak in progress, but the adaptation requires additional work.

The technique described by Wallinga and Teunis would be a valuable component of a suite of tools that should be made available to public health officials before the next important outbreak occurs.


We thank Drs.

Lipsitch and Bergstrom [1] for their thoughtful commentary on our article [2], which appears in this issue of the Journal. They discuss the method we developed in the context of real-time analysis of case notifications.

We would like to add a few more thoughts along these lines.


The contribution of the method toward real-time analysis of incoming notifications is, as Lipsitch and Bergstrom aptly summarized, to provide “a means of transforming the time series of cases… into a time series of estimated values for the instantaneous reproductive number on each day” [1].

The transformation is from calendar time to generation time and from number to relative increase in number.

The key variable involved in this transformation is the generation interval, which is the time interval between a primary case and a secondary case.

Literature on this key variable has been scant and has been blurred by the use of confusing terminology.

We cannot think of a better introduction to the concept of generation intervals than was provided by Professor Paul Fine in a recent article in this journal [3].

Fine illustrates the relatedness of the generation interval, epidemic curves, transmission chains, and reproduction numbers.

Our contribution is simply to specify these relations further.


In our article [2], we emphasized that under certain technical conditions, only a few simple computational steps are needed to transform the time series of severe acute respiratory syndrome cases into a time series of estimated reproduction numbers.

Here we would like to add that such a transformation could be carried out for any infectious disease that is transmitted from person to person.

Lipsitch and Bergstrom [1] correctly note that a short generation interval facilitates real-time interpretation of incoming case reports; for infections with a long generation interval (such as human immunodeficiency virus, herpesvirus, or mycobacterial infection), the transformation might be less useful.


At the moment, we are studying the possibility of extending the method to account for reporting delay, unobserved cases, and heterogeneous transmission.

However, the finding that transformation is in any case feasible, and in many cases computationally trivial, opens up perspectives for estimating reproduction numbers from time series of case notifications.


The reason for emphasizing the relevance of real-time estimation of reproduction numbers of emerging infectious diseases is that after syndromic surveillance systems have signaled the emergence of a new disease, we know little more about the novel infectious agent than that each case has been infected by another case and that successive generations of cases are separated by a typical generation interval.

This is not enough information with which to build a mathematical transmission model, but it is sufficient for real-time estimation of reproduction numbers, and hence for real-time estimation of the additional control effort required.


Lipsitch and Bergstrom assert that, when the next important outbreak arrives, such almost parameter-free estimation tools for reproduction numbers can be a valuable addition to syndromic surveillance systems and mathematical transmission models.

We could not agree more.


In recent years, a tremendous effort has been made to identify approaches for combatting metabolic disturbances and mito- chondrial dysfunction, such as those seen in ageing [1] and type 2 diabetes mellitus [2, 3] by specifically targeting the sirtuin (SIRT) enzyme family [4].

SIRTs are NAD + -dependent deacetylating enzymes that regulate cellular metabolism [5].

To date, seven mammalian SIRT enzymes (SIRT1-7) have been identified, each having its own characteristic tissue and subcellular compartment expression, enzyme activity and tar- gets.

We kindly refer readers to Houtkooper et al [6] for a comprehensive review on SIRTs.


Several SIRT-targeting strategies have been deployed, dem- onstrating the metabolic benefits of SIRT activation.

In mice, a SIRT1 gain-of-function mutation evoked a metabolic profile that protected against insulin-resistant diabetes by increasing hepatic insulin sensitivity, hepatic glucose tolerance and overall metabol- ic efficiency [7, 8].

Moreover, a proposed SIRT1 activator, SRT1720, increased mitochondrial respiration and improved in- sulin sensitivity [9], mimicking the signalling profile observed with caloric restriction [10] in high-fat-diet (HFD)-challenged mice.

Resveratrol, an AMP-activated protein kinase (AMPK)- activating polyphenol that activates SIRT1, improved skeletal muscle mitochondrial function in healthy obese men, in individ- uals with type 2 diabetes and in first-degree relatives of those Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00125-019-4831-3) contains a slideset of the figures for download, which is available to authorised users.

The concept of influencing NAD + bioavailability to activate the SIRTs was recently proposed for combatting metabolic distur- bances and mitochondrial dysfunction in humans [13, 14].

This is supported by reports that decreased NAD + bioavailability con- tributes to metabolic disturbances in ageing mice [15, 16] and humans [17, 18], and also in a rodent model of type 2 diabetes mellitus [16].

SIRTs are important consumers of NAD + and de- pend on this rate-limiting substrate to act as metabolic sensors, responding to the level of available NAD + .


Considering the limited scope of this review, we will not digress into detail of the NAD + metabolism and refer the read- er to more comprehensive reviews on this topic [5, 19-21].

Briefly, however, as NADH is the predominant electron donor to the electron transport chain, NADH/NAD + redox potential is an important indicator of the bioenergetic status of the cell and is tightly regulated [21].

The cytosolic and mitochondrial NADH/NAD + and NADPH/NADP + redox states are strongly connected.

These states depend on the formation of NAD + from NADH through cellular processes, such as the glycolytic enzyme activity, the citric acid cycle and the electron transport chain [20], thereby exemplifying the essentiality of NAD + and its redox potential within cellular metabolism.


The NAD + pool is maintained through a continuous process of biosynthe- sis and breakdown, stemming from the salvage and the Preiss- Handler pathways or from de novo biosynthesis at one end, and enzymatic consumption at the other [20] (Fig. 1).


When NAD + levels rise, SIRTs activate and deac(et)ylate or mono- ADP-ribosylate a variety of metabolic substrates, such as per- oxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and forkhead box protein O1 (FOXO1).

This elicits an array of metabolic adaptations, including mitochondrial biogenesis in skeletal muscle [19] and enhanced oxidative metabolism in skeletal muscle, brown adipose tissue and the liver [22, 23].

On a physiological level, this may lead to im- proved insulin sensitivity [24, 25], improved metabolic flexi- bility [26] and increased mitochondrial function [26, 27].


Exercise and caloric restriction share a common denomina- tor in that they affect AMPK activity, which can modulate NAD + bioavailability (Fig. 2).

To support this, AMPK acti- vation in C2C12 myotubes increases cellular NAD + levels and, in turn, activates SIRT1 and the subsequent PGC-1α- dependent upregulation of mitochondrial and lipid metabo- lism [28].

An increased demand for energy by the cell, such as during exercise, activates AMPK.

With this in mind, it was shown that exercise induces the expression of nicotin- amide phosphoribosyltransferase (NAMPT), the rate- limiting enzyme that converts nicotinamide into NAD + [29], thereby increasing NAD + bioavailability [30, 31].

The induc- tion of NAMPT expression through AMPK has been sug- gested to be a mechanistic adaptation to the metabolic stress derived from both exercise and caloric restriction [32-34].


Moreover, exercise in rats has been demonstrated to induce


Various research groups have pursued sustained SIRT activa- tion through an increase in endogenous NAD + bioavailability.

Preclinical research in ageing or HFD-challenged mice has shown that boosting NAD + levels by supplementation with NAD + precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside, attenuates age-related de- cline of muscle strength [1, 36], increases lifespan and healthspan [36].

In addition, oxidative metabolism and activa- tion of SIRT1 and SIRT3 are enhanced in HFD-fed mice sup- plemented with NAD + precursors [26].

In aged mice, NAD + precursor supplementation also restored arterial SIRT1 activ- ity, which was associated with improved vascular function and decreased aortic stiffness [37].

These findings demonstrate the feasibility of altering NAD + bioavailability and subsequent SIRT activation.


More specifically, in HFD-fed mice, exogenous adminis- tration of the NAD + precursor NMN was demonstrated to be a viable method of increasing endogenous NAD + bioavailabil- ity and inducing SIRT1 activity, thereby attenuating the effects of the HFD and improving glucose tolerance and hepatic in- sulin sensitivity [16].

Long-term administration of NMN was also found to mitigate the age-associated decline in energy metabolism, insulin sensitivity and lipid metabolism [36].

Similarly, supplementation of HFD-challenged mice with nic- otinamide riboside (another NAD + precursor), also improved hepatic insulin sensitivity [26].

Additionally, an improved glu- cose tolerance and lipid profile were observed in mouse models of age-induced type 2 diabetes upon NMN supple- mentation [16].


The NAD + precursors nicotinic acid and nicotinamide have also been used to supplement HFD-challenged mice, increas- ing hepatic NAD + levels and improving glucose tolerance.

In one study, nicotinamide proved to be a more potent booster of NAD + than nicotinic acid as it was also found to specifically alter the expression of SIRT1, SIRT2 and SIRT6 [38].

Lastly, Acipimox, a synthetic nicotinic acid analogue, has been shown to elevate NAD + in C2C12 myotubes [39].


Together, these preclinical data suggest that dietary supple- mentation of NAD + precursors can increase NAD + levels and beneficially affect metabolic health.


Preclinical research has explored compounds that can inhibit the NADases CD38 [40] and poly(ADP-ribose) polymerase-1 (PARP-1), reducing the enzymatic competition for their shared substrate, for example by reducing their NAD + -bind- ing capacity, and thus enhancing SIRT1 activity (Fig. 2).

Following this line of thought, a decrease in PARP-1 activity coincides with a rise in SIRT activity and NAD + levels in worms [41] and mice [27], with PARP-1 −/− mice displaying a leaner phenotype with higher energy expenditure compared with PARP-1 +/+ mice.

In line with this, in skeletal muscle, PARP-1 inhibitor-induced increases in SIRT1 activity were accompanied by improved mitochondrial function, enhanced energy expenditure and endurance performance [42].

In endo- thelial progenitor cells, PARP-1 inhibition also preserved cel- lular NAD + content [43].

Similarly, Cd38 knockout mice have elevated NAD + levels and are protected against HFD-induced


Regular exercise and caloric restriction are well known to im- prove metabolic health in humans [47].

Alongside improving insulin sensitivity, metabolic flexibility and mitochondrial func- tion, exercise also upregulates the expression of NAMPT in hu- man skeletal muscle [48] (Fig. 2).

Endurance-trained athletes have a twofold higher expression of NAMPT in skeletal muscle compared with baseline levels in sedentary obese, non-obese and type 2 diabetic individuals.

After completing a 3 week training intervention, the non-obese group displayed increased NAMPT expression over baseline.

NAMPT levels correlated positively with PGC-1α expression, mitochondrial content, maximal mito- chondrial ATP synthesis in skeletal muscle and overall maximal aerobic capacity [48].

Concordantly, increased skeletal muscle SIRT3 content and PGC-1α expression were reported in men who were sedentary obese at baseline after a 12 week aerobic exercise intervention [49].

In a 6 week one-leg endurance exer- cise intervention, NAMPT protein levels only increased in the trained leg as compared with the untrained leg [34], further supporting the paradigm of activating the NAD + /SIRT axis through exercise and NAMPT induction.


Continuing, during a caloric restriction-induced weight- loss intervention, NAMPT and subsequent SIRT1 expression were found to be increased in adipose tissue of healthy obese participants [50] when compared with healthy lean partici- pants.

The participants were studied prior to, and after 5 months and 12 months, of the intervention, with the inter- vention resulting in a loss of 17.1% of body weight in the obese group.

At baseline, gene expression of SIRT1, SIRT3, SIRT7 and NAMPT were significantly lower and PARP-1 ac- tivity significantly higher in the obese participants when com- pared with the lean group, indicating a state of low NAD + bioavailability in obese individuals.

With weight loss, SIRT1 expression increased, whereas PARP-1 activity declined in the subcutaneous adipose tissue of the obese group [50].


Evidence that a state of obesity or overnutrition indeed lowers NAD + levels also comes from studies of longer-term overfeeding using an HFD for 8 weeks in young, healthy men.

This result- ed in reduced NAD + levels and SIRT activity in skeletal mus- cle when compared with baseline [51].

This was further sup- ported by PGC-1α hyperacetylation in the same skeletal mus- cle biopsies.

Concurring with these findings, a study in young adult monozygotic twins (n = 26 obesity-discordant pairs and n = 14 obesity-concordant pairs) reported that obesity was as- sociated with lower NAD + /SIRT axis activation in subcutane- ous adipose tissue [14].

Together, these findings suggest that a state of energy abundance is prone to reduce the activity of the NAD + /SIRT axis and that inducing a state of energy demand may aid to restore NAD + levels.


From a human dietary perspective, Trp, nicotinic acid, nico- tinamide, and nicotinamide riboside are the predominant NAD + precursors currently used in intervention trials, with nicotinamide riboside being the latest addition to the array of dietary NAD + precursors (Fig. 1).

The efficacy and safety of treatment with each of these NAD + precursors are discussed in more detail below.


Nicotinamide Phase 0 and phase 1 trials have demonstrated tolerance and safety of nicotinamide in daily pharmacological doses up to 3.5 g [52-56] and single doses of up to 6 g [57-59].

However, at doses above this, nicotinamide can be- come hepatotoxic [60].


Nicotinic acid and Acipimox Nicotinic acid is the most effec- tive pharmacological drug available for elevating HDL- cholesterol and lowering total cholesterol, LDL-cholesterol and triacylglycerol levels, thereby reducing the overall cardio- vascular risk profile of the user [61].

However, nicotinic acid can elevate plasma glucose levels by inducing insulin resis- tance following a rebound increase in circulating NEFAs [62].

This poses a challenge when using nicotinic acid as (add-on to statin) therapy for dyslipidaemia in individuals with impaired glucose tolerance, impaired fasted glucose or type 2 diabetes, with the reduction in overall cardiovascular disease risk on one hand and compromised glycaemic control on the other.

The worsening of hyperglycaemia with nicotinic acid use would possibly require additional therapeutic fine tuning to be implemented on an individual level to maintain glycaemic control.

Alternatively, a reduction in the dose of nicotinic acid could improve glycaemic control, however, this may require acceptance of reciprocal compromise of the lipid profile or additional therapy to be initiated.


A large clinical trial evaluated the efficacy of nicotinic acid as a treatment for hypercholesterolaemia, with a daily dose of 1-3 g, for a duration of 96 weeks [63].

Overall, nicotinic acid was well tolerated.

However, flushing was reported as a major adverse event.

In contrast to nicotinamide, nicotinic acid is a vasoactive compound [64] and activates the G protein- coupled receptor, GPR109A, thereby inducing flushing [65].

In an attempt to reduce the occurrence of flushing and improve adherence, synthetic and extended- and sustained-release for- mulations of nicotinic acid were developed.

Acipimox is a synthetic nicotinic acid analogue and, thereby, an NAD + NADH NADH supplementation has also been used to boost NAD + levels in humans.


In a small study, 80 adults with chronic fatigue syndrome received daily doses of 20 mg of NADH combined with 200 mg of coenzyme Q 10 and were compared with placebo-treated individuals [68, 69].

The in- tervention improved reported fatigue [68] and increased max- imal heart rate after 8 weeks of treatment [69] but did not alter body weight or blood pressure.

Additionally, in peripheral blood mononuclear cells (PBMCs), the intervention signifi- cantly reduced NAD + levels and increased NADH levels, thus, significantly lowering the NAD + /NADH ratio over base- line.

Furthermore, ATP content and citrate synthase activity were significantly increased in PBMCs [68].

Unfortunately, it cannot be distinguished whether the observed results were solely attributed to NADH supplementation considering the co-administration of coenzyme Q 10 in this study.


In contrast to nicotinic acid, nicotin- amide riboside is not vasoactive and does not cause flushing [70], thereby overcoming one of the adverse effects of nico- tinic acid supplementation.

In a recently published placebo- controlled, double-blind, randomised, phase 1 crossover trial, a daily dose of 1000 mg of nicotinamide riboside for 6 weeks was demonstrated to be well tolerated and adverse events were no more frequent than in the placebo arm [71].

These findings are confirmatory of the preceding phase 1 trials [72-74].

Additionally, nicotinic acid adenine dinucleotide (NAAD) has been confirmed as a reliable and sensitive biomarker for assessing changes in NAD + levels following nicotinamide riboside supplementation [72].


Daily nicotinamide riboside supplementation of up to 2000 mg can effectively enhance blood NAD + levels, achieving higher steady-state concentrations over baseline [73].


Concordantly, a more recent study demonstrated that nicotin- amide riboside supplementation increased NAAD and NAD + levels by~60% in PBMCs.

In this study, the effect of 6 weeks of nicotinamide riboside supplementation vs placebo was tested in healthy middle-aged and older adults.

It was also found that 6 weeks of nicotinamide riboside supplementation tended to im- prove systolic blood pressure and pulse-wave velocity, both of which are markers of cardiovascular health [71].

However, no effect of nicotinamide riboside supplementation was found on physical performance outcomes, such as the 4 metre or 6 minute walk test, handgrip strength or maximum torque.

Moreover, met- abolic variables, such as V O 2max during a treadmill exhaustion test, respiratory exchange ratio, and insulin sensitivity assessed by an IVGTT, did not differ between the groups.

From these findings, it was concluded that long-term nicotinamide riboside supplementation is a viable strategy for enhancing NAD + in humans and potentially has cardiovascular benefits that require further exploration in larger trials.


Most recently, an RCT of daily treatment with 2000 mg of nicotinamide riboside for 12 weeks was reported, evaluating safety, insulin sensitivity and other metabolic variables in 40 healthy, obese, middle-aged men [75].

Overall, nicotinamide riboside was well tolerated and only four adverse events were reported: pruritus, excessive sweating, bloating and transient changes in stools.

Nicotinamide riboside supplementation in- creased NAD + metabolism, as was seen by an increase in uri- nary metabolites.

Using the hyperinsulinaemic-euglycaemic clamp technique, insulin sensitivity was found to be un- changed before and after supplementation and when compared with the placebo condition.

In addition, resting energy expen- diture and respiratory exchange ratio were not affected by nic- otinamide riboside supplementation.

Also, intrahepatic lipid content and body composition remained unchanged in the treatment group vs baseline and compared with the placebo group.

Finally, a significant but modest increase in serum tri- acylglycerol levels was detected after nicotinamide riboside supplementation when compared with baseline values.

The authors concluded that this study was underpowered and future studies should be larger and focus on other variables of meta- bolic health, such as intrahepatic lipid content, which showed significant changes in rodents [76, 77] treated with nicotin- amide riboside and approached significance in this study.


Tryptophan Another dietary NAD + precursor, Trp, is an es- sential amino acid and is metabolised into NAD + through de novo biosynthesis in the liver and kidneys [20].

This route is critical for maintaining the NAD + pool, even though the con- version ratio of Trp to NAD + is low in humans, averaging 60:1 [78].

Nonetheless, Trp is deemed capable of meeting the met- abolic demands of NAD + metabolism in nicotinic acid- and nicotinamide-deficient diets, and is well tolerated at high doses, between 30 and 50 mg/kg bodyweight, apart from drowsiness/sleepiness [79].

Recently, higher circulating Trp levels were identified as a predictive marker for the development of type 2 diabetes in a large prospective Chinese cohort [80].

However, to date, no dietary supplementation studies are available that directly as- sess whether boosting NAD + through Trp might be metabol- ically beneficial in humans.


The drawback of pharmacological strategies involving CD38 and PARP-1 inhibition is the original intended therapeutic use in malignancies [81, 82].

As such, no clinical trials with PARP-1 or CD38 inhibitors that focus on improving metabol- ic variables have been conducted in humans.

This, however, does not imply that this strategy must be abandoned altogeth- er, as a viable work-around to exploit the theoretical metabolic benefit of inhibition of NAD + consumers may present itself in due time, allowing us to assess their efficacy in clinical trials.


The current evidence base from preclinical research on NAD + is setting the stage for trials in humans by identifying the points at which intervening in the NAD + metabolism process seems to be clinically and physiologically relevant (see Summary text box).

Even though many results have not been replicated in humans at this point in time, phase 0 and phase 1 trials have proven the feasibility and safety of NAD + boosting in humans.

As most evidence that increased NAD + levels may be beneficial to human metabolism comes from indirect ob- servations, such as exercise and weight loss interventions, the assessment of efficacy in well-powered phase 2 and phase 3 trials is urgently awaited in order to draw clear conclusions.

Additionally, studies in metabolically disturbed individuals must be considered as these are more in line with the preclin- ical models used.

To date, generally healthy populations have been included in studies in this area, in which the range of improvement may be too small to detect significant changes.

The combination of strategies to increase NAD + , such as ex- ercise, caloric restriction, or CD38 and PARP-1 inhibitors, with NAD + precursor supplementation may also be consid- ered, to evaluate added efficacy of such approaches, as seen in mice [15] (see Recommendations text box).


Currently, a number of clinical trials (Table 1) are underway in which NAD + precursor supplementation is being used to improve (often disturbed) metabolic health variables.

The coming years will prove whether the promising results ob- served in preclinical studies can indeed find human translation.


To the Editor: Schrauwen and colleagues have recently reviewed the emerging field of NAD+ metabolism in Diabetologia [1].

Their manuscript includes a description of a clinical trial of dietary nicotinamide riboside supplementation effects on whole body insulin sensitivity in obese men, conducted by us, together with our colleagues at Aarhus and Copenhagen Universities, Denmark [2].

As posted on clinicaltrials.gov (NCT02303483), this placebo-controlled, randomised clinical trial used M value determined by the hyperinsulinaemic–euglycaemic clamp (HEC) technique, the gold-standard approach for directly quantifying insulin sensitivity, as the primary endpoint.


We report a negative outcome of the trial, and while this may be disappointing in terms of the possible future use of nicotinamide riboside supplementation for the prevention of type 2 diabetes, we believe it is important to publish such outcomes and to cite these correctly.

We were therefore disconcerted to find the following description of our manuscript in the review by Schrauwen and colleagues: ‘The authors concluded that this study was underpowered and future studies should be larger and focus on other variables of metabolic health’.

We find this to be a severe misrepresentation of a central conclusion of our work.


As outlined in our manuscript, power calculations were conducted prior to enrolment into our study to determine an appropriate sample size for detecting clinically meaningful differences in the primary endpoint and to try to evade type I and type II errors resulting from insufficient sample size.

We powered the study to detect a difference in the HEC-derived M value of 1.5 mg kg−1 min−1, which has previously been achieved with insulin-sensitising drugs or exercise [3, 4].

We used a conservative estimate of the SD of 1.6 mg kg−1 min−1 in the power calculation, based on in-house experience from previous clamp examinations [2].


The primary endpoint, the M value, was not affected by nicotinamide riboside supplementation (p for interaction = 0.68).

However, the actual SD for the difference between the baseline and the end-of-study M value within the placebo group was only 0.48 mg kg−1 min−1.

Therefore, in retrospect, the assumed SD was probably too conservative.

With an SD of 0.5 and n of 40, we would be able to detect a significant difference in the M value of 0.45 mg kg−1 min−1 with a power of 0.80.

Hence, we do not suspect that the lack of effects of nicotinamide riboside can be attributed to the study being underpowered (i.e. a type II error).


In addition to the M value, our study included several predefined secondary endpoints, and the combined outcomes of these were also negative.

In line with standard study design, the trial was powered to detect significant differences in the primary endpoint and, therefore, where the risk of a type II error was discussed in our paper, this was in terms of the secondary endpoints [2].


When citing a clinical trial, it is important to respect the distinction between endpoints.

When quoting the authors as concluding that the study was underpowered, unless otherwise stated, it will be assumed that this conclusion refers to the primary outcome of the study.

Dietary supplements, such as nicotinamide riboside, are already commercially available and are being promoted as facilitating healthy ageing in humans by an industry that is not subject to the same strict regulatory demands as the traditional pharmaceutical industry.

This stresses the need for scientific testing of safety and efficacy of dietary supplements in rigorously designed human clinical trials, and the outcomes of such trials should be reported correctly in subsequent review papers, irrespective of whether the outcome is positive or negative.


To the Editor: Jessen and colleagues [1] have commented on our recent review of NAD+ metabolism in Diabetologia [2].

In their letter, they comment on the discussion and citation of a clinical trial that they performed, in which dietary nicotinamide riboside was supplemented in obese men to assess the effects on whole body insulin sensitivity [3].


We concede to the argument made by Jessen and colleagues that it is important to make a clear distinction between endpoints when citing clinical trials, as this may otherwise lead to unwanted assumptions regarding the endpoint(s) that are discussed.

In our review, we did not have the intention to suggest that the study by Dollerup et al [3] was underpowered for its primary endpoint, insulin sensitivity, but unfortunately omitted to add ‘for these secondary endpoints’, for which we do apologise.


Nevertheless, the overall message and conclusion of our review does not change and we agree with Jessen and colleagues that more human studies are needed to test the efficacy of dietary supplements in rigorously designed human clinical trials.


Forty HCC patients with portal hypertension who were treated with TIPSS between 1995 and 2012 were included in the analysis.

Medical records and imaging studies were analysed.

The indication for TIPSS implantation, pro- cedure-related complications, treatment success and overall survival were assessed.


TIPSS implantation was performed in 23 patients (57.5%) due to treatment refractory ascites, in 14 patients (35.0%) due to recurrent variceal bleeding and in three patients (7.5%) due to ascites and variceal bleeding.

Primary tech- nical success was assessed in all patients.

After TIPSS implantation, no variceal bleeding reoccurred and ascites was controlled in 74.1%.

No severe procedure- related complications and no deterioration of liver function were observed.

Post-TIPSS hepatic encephalopathy occurred in 40.0% of all patients.

30-day, 90-day-, 1-year- and 5-year survival rates were 97.5%, 75.0%, 42.5% and 7.5%, respectively.

Median overall survival after TIPSS implantation was 180 days.


Transjugular intrahepatic portosystemic shunt implantation is an effective and safe treatment for portal hypertension in patients with HCC.


Portal hypertension in patients with liver cirrhosis can lead to the development of ascites and/or oesophageal varices that can cause life-threatening upper gastrointes- tinal bleeding.

The development of these complications is associated with increased mortality and morbidity.

1, 2 Furthermore, liver cirrhosis is a major risk factor for the development of hepatocellular carcinoma (HCC).

3 As HCC typically represents a late-stage complication of liver cirrhosis, therefore, most of these patients are also affected by symptomatic portal hypertension (SPH).

Importantly, portal hypertension in HCC patients may not only be due to liver cirrhosis but also due to portal vein thrombosis (PVT) that can occur during HCC pro- gression as a result of increased clotting or portal vein invasion.


Previous studies reported an incidence of portal hypertension of approximately 30% in patients with HCC, but as in these studies portal hypertension has been evaluated using clinical parameters instead of portal vein pressure measurement, a higher frequency of portal hypertension in these patients is estimated.

4, 5 Impor- tantly, portal hypertension directly affects treatment of HCC.

6 Indeed, according to the current guidelines of the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) liver cirrhosis with portal hypertension is considered as a relative contraindication for hepatic resection because of the high risk of post-operative liver failure.

7, 8 Furthermore, complications of portal hyper- tension in patients with HCC contribute to the poor prognosis.

9-11 Despite the high incidence and poor prog- nosis of portal hypertension in HCC patients, however, there are no established guidelines for the treatment of SPH in patients with HCC.


During the last 25 years the TIPSS has been estab- lished as an effective and minimally invasive procedure for the treatment of portal hypertension and its compli- cations.

12, 13 Refractory ascites, recurrent and also acute variceal bleeding, hepatic hydrothorax, hepatorenal syndrome and Budd-Chiari syndrome are the most com- mon and accepted indications for TIPSS implanta- tion.

14-24 Next to absolute contraindications for TIPSS insertion such as right heart failure, severe hepatic fail- ure, systemic infection and uncontrolled hepatic enceph- alopathy, hepatic malignancy is considered a relative contraindication.

12, 25 Of note, few studies with small sample size investigated the implantation of TIPSS in patients with hepatic malignancy including primary liver cancers as well as hepatic metastases from extrahepatic malignancies.

These studies showed a good efficacy of TIPSS implantation and no association with severe pro- cedure-related complications.

26-28 However, these studies are characterised by rather small patient cohorts and by hepatic tumours of different entities.

In addition, data to the OS are limited.

To address this important clinical issue, we analysed the OS, safety and efficacy of TIPSS implantation in HCC patients presenting with SPH due to advanced liver cirrhosis with or without PVT.


From November 1995 to December 2012, 40 patients with HCC and SPH received TIPSS implantation at our institution.

Patients with intra - or extrahepatic cholan- giocarcinoma or metastases from extrahepatic malignan- cies were excluded from this analysis.

Decision for TIPSS implantation in HCC patients was discussed individually for each patient regarding technical feasibility and hepatic expansion of HCC.

Due to these inclusion criteria, approximately 15% of HCC patients with severe SPH (three of 20 patients per year) received TIPSS implantation.


In 23 patients, TIPSS was performed after diagnosis of HCC has been established.

The median time from HCC diagnosis to TIPSS implantation was 6.0 (1-38) months in these patients.

Seventeen patients were admitted to our Liver Centre for evaluation of TIPSS implantation and HCC was suspected in the pre-interventional setting with color doppler ultrasound (CDUS).

We confirmed HCC diagnosis as cited below.

Although diagnosis of HCC was established, we performed TIPSS implantation as planned.


The diagnosis of HCC was based on histology or unequivocal radiological features in at least two con- trast-enhanced imaging techniques (CT, magnetic reso- nance imaging (MRI), ultrasound) according to the current EASL guidelines which have been applied retro- spectively.

7 Centrally located tumour masses were local- ised in segment VIII, V and IV according to Couinaud.

29 Diagnosis of liver cirrhosis was defined on ultrasound, CT or MRI, clinical findings and laboratory tests.

Clinically significant portal hypertension was defined as (i) presence of oesophageal varices or gastric varices, TIPSS implantation in patients with HCC 13652036, 2015, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.12994 by Mcgill University, Wiley Online Library on [18/05/2024].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (ii) thrombocytopenia with platelet count <100 9 10 9 /L associated with splenomegaly and (iii) presence of asci- tes.

The presence of oesophageal varices was assessed by endoscopy.

30 SPH was defined as presence of ascites or one or more episode of variceal bleeding.


According to Moore et al., treatment refractory ascites was defined as (i) nonresponse to sodium-restricted diet and diuretic treatment, (ii) intolerance of diuretic ther- apy and (iii) rapid recurrence of ascites after therapeutic paracentesis.

31 Response to TIPSS treatment was defined as follows: absence of clinically detectable ascites with or without diuretic therapy or ascites needing no further paracente- sis and/or no further variceal bleeding episodes after TIPSS implantation.

Development of large amount of ascites as well as variceal bleeding after TIPSS implanta- tion was defined as nonresponse.


All patients underwent follow-up examination.

During follow-up none of our HCC patients was transplanted.

Patients receiving TIPSS had routine physical examina- tion and laboratory testing before TIPSS and 1 and 3 months after TIPSS implantation and thereafter fol- low-up was performed in 3-month intervals.

The median follow-up time after TIPSS implantation was 211 days (3-2919 days).

On each admission, CDUS was per- formed to evaluate shunt function.

Shunt insufficiency was defined as (i) maximum flow velocity less than 60 cm/s or above 180 cm/s within the shunt or less than 30 cm/s in the portal vein, 13 (ii) recurrent or increasing ascites and (iii) recurrent variceal haemorrhage.

Shunt dysfunction resulted in TIPSS revision and during this intervention shunt dysfunction was confirmed by portog- raphy and an elevated portosystemic pressure gradient by more than 12 mmHg.


Diagnosis of hepatic encephalopathy (HE) was estab- lished in clinical examination and West-Haven criteria were applied to assess severity of HE.

32 HCC-specific treatment was determined according the Barcelona Clinic Liver Cancer (BCLC) classification.

7, 33 Treatment response was assessed using contrast-enhanced CT or MRI and the modified RECIST (mRECIST) criteria were applied.

34 Model of end-stage liver disease (MELD) score and Child-Pugh stage were determined according the medi- cal records.

Intrahepatic tumour expansion was classified according to the Milan criteria.


Before TIPSS implantation, CDUS was performed in all patients to evaluate if TIPSS procedure was technically possible and to assess portal and hepatic vein patency and vascular abnormalities.


The technique of TIPSS implantation has been described previously.

15, 36-38 In brief, a transjugular approach was used to advance a puncture needle into a hepatic vein.

Puncture of a por- tal vein was done using ultrasound guidance.

After suc- cessful puncture portography was performed, following dilatation of the parenchymal tract with introduction of the stent graft.


Before and after TIPSS implantation, portal vein pressure and central venous pressure were measured to calculate the pre- and post-shunt portosys- temic gradient (PSG).

Technical success was assessed by final portography.


In 16/40 patients (40%) covered stents (Viatorr; W. L. Gore & Associates, Inc., Flagstaff, AZ, USA) were used while in 24/40 patients (60%) bare metal stents (BMS; Palmaz stents; Cordis, Miami, FL, USA, Wallstent; Boston Scientific, Malborough, MA, USA, Luminexx; Bard Medical Division, Covington, GA, USA) were implanted.


The decision whether BMS or covered stents were implanted was an individual deci- sion of the TIPSS- performing operator and was not defined in the study protocol.


All patients provided written informed consent for TIPSS implantation.

This study was performed in accordance with the Declaration of Helsinki and it has been approved by the local ethics committee of the University Hospital of Freiburg (no. EK 37/12).


The present study was an observational study.

Patients were analysed retrospectively from the day of TIPSS implantation.

All patients were followed-up until death or last contact.

The primary endpoint was overall sur- vival calculated from the day of TIPSS implantation.

The cut-off point for survival data was 15th of June 2013.

Furthermore, primary endpoints were efficacy and safety of TIPSS implantation in HCC patients.

In patients receiving HCC therapy, treatment response was defined as the secondary endpoint.


Continuous variables are expressed as median with range (minimum-maximum), whereas categorial vari- ables are reported as frequencies and percentages.


For continuous variables differences were determined using Wilcoxon-Mann-Whitney and Kruskal-Wallis tests, whereas v 2 tests or Fisher's Exact tests were used for categorial variables.

Differences in laboratory parame- ters before, 1 and 3 months after TIPSS procedure were 128 Aliment Pharmacol Ther 2015; 41: 126-136 ª 2014 John Wiley & Sons Ltd D. Bettinger et al. 13652036, 2015, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.12994 by Mcgill University, Wiley Online Library on [18/05/2024].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License assessed using Friedman tests.

For sub-analyses of statistically significant Kruskal-Wallis or Friedman test, the Bonferroni correction was applied.

P values <0.05 were considered being significant.


OS was calculated using Kaplan-Meier analyses with death being recorded as event.

Differences in OS were calculated using log-rank tests.

Statistical analyses were performed with SPSS (Version 20.0; IBM, New York, NY, USA) and GraphPad Prism (Version 5; GraphPad Soft- ware, San Diego, CA, USA).


Table 1 summarises the demographical and clinical char- acteristics of the 40 enrolled patients at time of TIPSS implantation.

The leading aetiology of liver disease was chronic alcohol abuse (62.5%) followed by chronic hepa- titis C virus (HCV) infection (27.5%) and NAFLD (5.0%).


TIPSS implantation was performed due to ascites in 23 of 40 patients (57.5%).

In 14 of 40 patients (35.0%), TIPSS was indicated after oesophageal variceal haemor- rhage.

In four of these patients, an emergency TIPSS implantation for an acute bleeding episode was per- formed.

In all of these patients, HCC was known before emergency TIPSS implantation.

Three patients (7.5%) had treatment refractory ascites and experienced former variceal bleeding leading to TIPSS implantation.


In 13 of 40 patients (32.5%), portal hypertension was aggravated by PVT.

In 11/13 patients (84.6%) PVT was diagnosed concomitantly with HCC and tumour invasion was confirmed by computed tomography (CT) and therefore being classified as malignant PVT.

In two patients, PVT without tumour invasion of portal vein was diagnosed more than 6 months before diagnosis of HCC, so that these PVTs were described as nonneoplas- tic.

Among the patients with PVT 11/13 patients pre- sented with refractory ascites.


Sixteen patients (40.0%) were within the Milan criteria while 24/40 patients (60.0%) were outside.

Mean size of the largest HCC nodules each was 3.7 AE 1.8 cm.

In 15 of 40 patients (37.5%), HCC was located centrally.

Table 2 presents the tumour characteristics including location of HCC, the size of the largest HCC nodule and the extent of PVT in all 40 patients who were allocated to TIPSS implantation.


The shunt was successfully placed in all 40 patients.

After TIPSS implantation, portal vein pressure (PVP) decreased from median 26.5 (15-38) mmHg to 21 (10- 27) mmHg.

Portosytemic gradient (PSG) decreased from median 20.0 (13.0-33.0) mmHg to 8.0 (2-16) mmHg (P < 0.001).

After TIPSS implantation in patients with variceal haemorrhage no further bleeding episodes appeared.

Response to TIPSS was observed in 20 of 27 patients treated for ascites (74.1%, Figure S1; including the patients both with ascites and variceal bleeding).


During TIPSS implantation, no severe complications were observed, especially no tumour rupture or severe haemorrhage.

In four of 17 patients (23.5%) with oesophageal varices, TIPSS implantation was performed to control acute bleeding.

One of these patients was in a haemorrhagic shock and multi-organ failure and death occurred.

In another patient, acute renal failure developed which was rescued by conservative treatment.


Laboratory tests with regard to liver function revealed no significant impairment in liver function (Figure S2).

Indeed, INR and albumin remained stable (data not shown) whereas an increase in bilirubin levels was observed after TIPSS implantation (1.7 mg/dL vs. 2.4 mg/dL vs. 2.9 mg/dL, P = 0.018).

AST and ALT lev- els did not increase significantly.

Overall, MELD score did not change significantly after TIPSS implantation.


TIPSS insufficiency was suspected when clinical symp- toms of portal hypertension reappeared and/or when reduction in blood flow within TIPSS was diagnosed in CDUS.

Eight of 40 patients (20.0%) were diagnosed with shunt insufficiency during follow-up CDUS examination.

TIPSS thrombosis occurred in three of 40 patients (7.5%) within 30 days after TIPSS implantation.

All patients with shunt thrombosis had bare metal stents (BMS).

Only in one patient shunt dysfunction was due to tumour invasion of the TIPSS as confirmed by CT.


During follow-up, only one patient developed lung metastasis 7 years after diagnosis of HCC and 6 years after TIPSS implantation.

This patient had no invasion of portal vein and HCC was not located in central seg- ments excluding tumour cell seeding by TIPSS implanta- tion.


During the follow-up period, 36 of 40 (90.0%) patients with TIPSS implantation died.

Median OS after TIPSS implantation was 180 days (0-443.4).

30-day and 90-day survival rates were 97.5% and 75.0%, respectively.

One-year and 5-year survival rates were 42.5% and 7.5%, respectively (Figure 1).


In 34 of 36 patients (94.4%) the cause of death was progression of tumour disease.

Two patients died due to multi-organ failure after TIPSS implantation.


Patients with PVT had a median OS of 5.2 months (4.3-6.0), while OS in patients without PVT was 17 months (3.2-18.7).

This difference in survival did not reach statistical significance (P = 0.890, Figure 2).


In patients with PVT, there were no life-limiting technical complications.


After successful TIPSS implantation, 11 of 40 patients (27.5%) were treated with selective segmental or subseg- mental transarterial approaches.

These patients had preserved liver function and had sufficient portal vein flow.

Nine patients were treated with transarterial chemoembolisation (TACE), while two patients did not receive embolisation after transarterial chemoperfusion (TAC).

Treatment response was evaluated in eight of 11 patients according to the modified RECIST (mRECIST) criteria (Figure S3).

One patient showed complete remis- sion (CR), five patients had stable disease (SD) and in two patients follow-up imaging confirmed progressive disease (PD).

Overall, four patients had complications after transarterial treatment of HCC.

Two patients presented with HE and one patient each developed


Portal hypertension and HCC are common complica- tions in patients with advanced liver cirrhosis.

Portal hypertension may result in life-threatening complications such as acute variceal bleeding or hepatorenal syndrome and is therefore an important cause of morbidity and mortality in these patients.

As HCC also represents a late-stage complication of liver cirrhosis, several patients present with portal hypertension and HCC.

To date, however, no guidelines for the treatment of portal hyper- tension in HCC patients are established.


In patients without HCC TIPSS has been shown to be a minimally invasive method to treat portal hypertension and its associated complications.

15, 16, 19 However, pri- mary hepatic malignancy is considered a relative contra- indication for TIPSS implantation although relevant information does not exist to support this recommenda- tion.

To date, only few studies with small cohorts have focused on the efficacy and safety of TIPSS implantation in patients with hepatic malignancies including HCC and hepatic metastases (Table 3).

Therefore, we set out to analyse HCC patients with portal hypertension due to PVT and advanced liver cirrhosis receiving TIPSS implantation at our institution with regard to efficacy and safety of TIPSS insertion as well as OS.


First, we focused on feasibility and safety of TIPSS implantation.

Importantly, apart from SPH as a result of liver cirrhosis, we also included 13 patients (32.5%) with PVT.

These patients are of special interest as TIPSS implantation may be associated with higher rates of tech- nical failure and complications.

39, 40 In patients with liver cirrhosis and PVT, TIPSS implantation is feasible, effective and safe in selected patients.

41 However, TIPSS implantation in malignant PVT is discussed controver- sially.

In our cohort, all procedures were performed suc- cessfully and we observed no severe procedure-related complications such as tumour rupture or severe proce- dure-related complications neither in patients with PVT nor in our other patients.

These results show that severe procedure-related complications are rare in patients with HCC as long as these patients are strictly selected (pre-interventional CDUS for exclusion of portal and


One reason for the restriction to perform TIPSS pro- cedure in HCC patients is the risk of tumour seeding as TIPSS may traverse malignant tumour tissue, especially in centrally located tumours.

A hypothetical risk is the development of pulmonary metastases spreading via the portal venous system.

However, in our study, only one patient developed pulmonary metastasis 6 years after TIPSS implantation and, therefore, we did not associate this metastasis with TIPSS.

Wallace et al. examined nine patients with intra- and extrahepatic malignancies receiv- ing TIPSS due to portal hypertension.

They observed development of pulmonary metastases in two of nine patients 10 and 5 months after TIPSS implantation.

44 Liu et al. detected seven of 58 patients with metastases during follow-up after TIPSS implantation.

42 In our cohort, none of the patients with centrally located HCC developed metastases, but interpretation of these data is difficult as follow-up of our patients may have been too short (211 days) and 1- and 5-year survival rates were 42.5% and 7.5%, respectively.


Second, we evaluated efficacy of TIPSS in patients with HCC.

TIPSS was highly effective in both patients with ascites as well as bleeding and PSG decreased sig- nificantly.

Ascites has been shown to be an important negative prognostic factor in patients with HCC within the Milan criteria undergoing nontransplant therapies.

45 Therefore, it is important to control ascites in these patients and in this setting TIPSS implantation may con- tribute to improved survival.


In 20.0% of our patients shunt insufficiency occurred.

TIPSS thrombosis occurred in three patients (7.5%) within the first months after TIPSS implantation.

Wallace et al. reported a similar rate (8%) of shunt occlusion within the first 30 days.

27 More- over, Saxon et al. even observed a shunt occlusion rate of 17% within 30 days after TIPSS implantation in their cohort of 116 patients with liver cirrhosis, but without HCC.

46 Several mechanisms have been discussed that may explain this early shunt occlusion in patients with and without HCC.


Trauma of malignant and also of not-malignant liver tissue may stimulate coagulation leading to shunt occlusion and especially tumour patients may be at higher risk due to a procoagulant imbalance.

Furthermore, it is also possible that tumour may invade the shunt.

However, as no autopsies have been performed in our study cohort we cannot prove this hypothesis.

Interestingly, Saxon et al. provided evi- dence that bile duct damage during TIPSS implantation is a major cause for early shunt occlusion.

47 In summary, further prospective studies are warranted to determine the role of shunt occlusion in these patients and to eval- uate anticoagulation strategies as a preventive measure.


Third, we set out to determine the incidence of post-TIPSS HE.

In several studies, rates of HE range from 13 up to 47%.

In the cohort by Liu et al., HE occurred in 25.9% of the patients.

28 Wallace et al. even reported a rate of 44%.

27 We also observed a similar rate of post-TIPSS HE with 40.0%.

One of these patients developed severe grade IV HE and died of multiorgan failure.


Fourth, we analysed OS in our HCC patients receiv- ing TIPSS implantation.

Median OS was 180 days after TIPSS procedure.

Two patients died shortly after TIPSS procedure due to multi-organ failure.

One of these patients had TIPSS implantation to control acute vari- ceal haemorrhage, but bleeding could not be controlled sufficiently.

The other patient developed severe grade IV HE and also died of multi-organ failure.

Our median OS was significantly higher compared to the study by Liu et al. who reported a median OS of 77 days after TIPSS implantation.

28 Their 30-day survival rate was 49%.

Notably, in comparison to our study their patients had advanced tumour disease as all patients had PVT and in our cohort tumour size was significantly smaller, which contributes to our good survival.

Wallace et al. reported a 30-day survival rate of 84%.

27 Our data even showed a rate of 97.5%.

This high 30-day survival rate can be explained by strict selection for TIPSS implanta- tion in our patient cohort.

Furthermore, OS in patients with PVT was lower than in patients without PVT reflecting the more advanced tumour stage in these patients.

But, importantly, there were no technical com- plications after TIPSS implantation in patients with PVT leading to in-hospital death and in these patients prognosis was especially limited by progression of tumour disease.

Fifth, we analysed the efficacy and safety of HCC treatment after TIPSS implantation.

27.5% of patients with TIPSS were treated with transarterial therapies.

In nine patients, TACE was performed whereas the remain- ing two patients were treated with transarterial chemop- erfusion (TAC) without embolisation.

TACE takes the advantage of the hepatic dual blood supply resulting in embolisation of the tumour feeding hepatic arteries while portal venous flow to normal hepatocytes is preserved.

In a patent TIPSS, the hepatic portal venous perfusion is altered as portal venous flow is decompressed in the sys- temic circulation.

Therefore, TACE in TIPSS patients has been considered a relative contraindication as the result of hepatic ischaemia might be increased.

Although HCC derives his blood primarily from hepatic arteries, portal venous contribution is observed in some HCCs.

48, 49 This important change in the blood supply may aggra- vate tumour necrosis and therefore, TACE in TIPSS patients may be associated with increased hepatotoxicity.


In our cohort, no patients developed severe hepatotoxic- ity.

Severe complications were an episode of acute HE in two patients and the development of hydropic decom- pensation and spontaneous bacterial peritonitis, but these complications were manageable by conservative treat- ment.

These findings are in accordance with the morbid- ity and mortality rates in other studies.

50, 51 Moreover, we evaluated efficacy of transarterial treatment in our HCC patients with TIPSS.

Most of our patients had stable disease and only one patient had complete remission.

In accordance with our findings, Kuo et al. showed that the rate of complete remission after TACE is significantly higher in patients without TIPSS compared to TIPSS patients.

51 This may be due to arterioportal shunting which is common after TIPSS implantation.

52 This may have a negative effect on TACE efficacy, especially as it may divert a portion of the chemoembolic agent away from the tumour.

Overall, it is important to state that using selective transarterial approaches, TACE in TIPSS patients maybe safe and can lead to stable tumour disease.


In conclusion, we were able to show that TIPSS implantation in patients with HCC is an effective and safe procedure and selective HCC treatment after TIPSS implantation is possible and safe.

Considering the limita- tions of our study namely the retrospective design with a missing control group, the long observation period from 1995 to 2012, the small sample size and also the individ- ual decision for TIPSS implantation in HCC patients, further well-designed prospective studies comparing HCC patients with portal hypertension treated with TIPSS or standard medical treatment are needed to ana- lyse the effect of TIPSS implantation on OS in these patients cohort.


Guarantor of the article: Michael Schultheiß, MD.


Author contribution: Dominik Bettinger: study concept and design, analysis and interpretation of the data, drafting of the manuscript and statistical analysis.

Eva Kn€ uppel: acquisition of the data.

Wulf Euringer: performed the TIPSS implantation.

Hans Christian Span- genberg: study concept and design and study supervi- sion.

Martin R€ ossle: performed the TIPSS implantation and critical revision of the manuscript.

Robert Thimme: interpretation of the data, supervision of the study and critical revision of the manuscript.

Michael Schultheiß: supervision of the study, drafting and critical revision of the manuscript.

All authors approved the final version of the manuscript.


Hepatocellular carcinoma (HCC) complicating cirrhosis occurs more frequently as cirrhosis becomes advanced and therefore HCC often co-exists with significant liver dysfunction and portal hypertension.


In the widely accepted Barcelona-Clinic Liver Cancer (BCLC) staging system patients with very advanced liver disease (i.e. Child–Pugh stage C) have the worst outlook [1].


Indeed, in the early natural history studies in HCC, a significant proportion of deaths were due to complications of portal hypertension [2].


The presence of HCC, and in particular a central lesion, is considered a relative contra-indication to TIPSS though there is little evidence to indicate poor outcomes in this patient group [3].


To explore this issue further, Bettinger et al. report their experience of TIPSS in 40 patients with HCC and symptomatic portal hypertension [4].


This report, from a centre with extensive experience of TIPSS, highlights several issues with regard to indications, patient selection, and outcomes for patients with HCC.


First, patient selection was strict: only 15% of patients with HCC and symptomatic portal hypertension were selected for TIPSS.


The indications for TIPSS were frequently refractory ascites and varices though only 10% were for acute, uncontrolled variceal haemorrhage.


Although the broad criteria for selection were specified, factors considered to be absolute contraindications were not reported and perhaps surprisingly some patients with malignant portal venous involvement were treated with TIPSS.


Second, the clinical outcomes after TIPSS were acceptable, with technical success and complications similar to published data, despite most patients having bare metal TIPSS and the presence of portal vein thrombosis.


However, without stratification by BCLC stage it is difficult to determine how these patients have survived in comparison to their peers.


TIPSS may therefore have a role in the management of patients with HCC and symptomatic portal hypertension in selected cases and in experienced centres.


The use of TIPSS here appears to have been largely palliative, successfully providing control of portal hypertension and symptoms, and on occasion allowing the use of selective chemoembolization.


Any benefit in terms of survival, however, could only be inferred from further comparative studies.


With great interest, we read the editorial by Rowe and Tripathi and thank the authors for their comments concerning our paper published in the current issue of AP&T [1], [2].

Patients with end-stage liver disease are often affected by portal hypertension and hepatocellular carcinoma (HCC).

Symptomatic portal hypertension (SPH) is associated with impaired survival in HCC patients.

In our retrospective study investigating TIPSS implantation in HCC patients, we were able to show a good clinical control of SPH in HCC patients, especially in those with small HCC [2].


As the authors state correctly, our patients were highly selected and the decision to perform TIPSS implantation was made on an individual basis.

Due to the retrospective design of our study, it was not entirely possible to evaluate the absolute contraindications in patients with HCC and SPH who were not allocated to TIPSS implantation.

However, main contraindications included uncontrolled tumour growth and poor performance status of a given patient, but absolute and relative contraindications need to be clearly defined in further studies.

Indeed, another important issue to be investigated will be the role of TIPSS implantation in patients with malignant portal vein thrombosis in HCC patients.


Furthermore, Rowe and Tripathi point out that it will be important to stratify these patients according to the BCLC classification to determine overall survival.

We fully agree with this comment and indeed further studies are needed that include a control group of HCC patients with SPH, who are therefore treated with medical or endoscopic treatment.

In our opinion, it will be important to focus on patients with BCLC A suffering from SPH and are therefore not allocated to curative surgical resection.

TIPSS may resolve SPH and thus, these patients may be allocated to surgery.

Further prospective studies including a control group should focus on this important issue.


In summary, our retrospective observation study highlights several important points and clinical relevant aspects, which can be evaluated in further well-designed prospective studies.


The introduction of continuous glucose monitoring (CGM) represents one of the most important advancements within diabetes treatment and self-management over the last decades.

CGM provides easy access to current glucose levels, glucose trends, and the retrospective analysis of glucose excursions, thus facilitating easier and better diabetes management for both people living with diabetes (PLWD) and health care professionals.

As a result, PLWD using CGM technology show improvement in HbA1c, glucose variability, hypoglycemia prevalence, overall well-being, and treatment satisfaction and have less fear of hypoglycemia compared to SMBG [1-3].


One critical issue with this technology remains the quality of the accuracy of glucose measurements, even though vast improvements in this regard have become evident over the last years [4-7].

Nevertheless, most current CGM have already reached accuracy levels of SMBG and are, therefore, labeled for nonadjunctive use by regulators, meaning that CGM can be utilized for treatment decisions without the subsequent SMBG confirmation [8-11].

Furthermore, as CGM enables easy assessment of the time spent in target range (TIR), which has been shown to be a valid marker of glycemic control alongside HbA1c, there has been an evolution of individual treatment guidelines for CGM use with a focus on TIR [12].

However, further improvement in CGM accuracy, particularly in the hypoglycemic range, is crucial for the development of reliable diabetes technology, especially for automated insulin delivery systems, as well as for reducing the burden of diabetes management for PLWD.


Another key aspect of CGM technology that is crucial to adherence is comfort in both wearing and inserting the sensor.

Registry data show that wearing discomfort is the Biosensors 2022, 12, 106.

https://doi.org/10.3390/bios12020106 https://www.mdpi.com/journal/biosensors prevailing factor for CGM discontinuation [13].

Therefore, new developments in diabetes technology aim to reduce discomfort in diabetes management and, thus, facilitate better quality of life with diabetes.


As CGM technology is made available to a growing number of PLWD, the negative impact on the environment and natural resources increases due to the use of disposable products and accumulation of plastic and medical waste, including hazardous parts such as insertion needles.

This problem is increasingly being addressed not only by environmental organizations but also by users of diabetes technology and patient organizations.

There- fore, the Diabetes Technology Society started its Green Diabetes Initiative to promote the development of medical devices in terms of sustainability and reduce the environmental impact associated with advancements in diabetes technology [14].


In the present analysis, we aimed to assess the accuracy and usability of a novel CGM system that is the first to feature predominately reusable components with needle- free insertion.


In this monocentric, open-label, non-randomized, single-arm clinical study, eight in dividuals with type 1 diabetes were equipped with two sensors of the GlucoMen ® Da CGM system (Waveform Cascade, A. Menarini Diagnostics, Florence, Italy; Figure 1) t wear at home.

This novel CGM features a needle-free insertion system containing a single use, disposable sensor, the reusable transmitter to be recharged after 14 days of use (fo up to 5 years), the reusable sensor insertion tool (for up to 5 years), the transmitter charg ing unit with a USB port, and the GlucoMen ® Day App supported by Android and iO devices allowing for viewing glucose levels and trends, for calibrating the system, and fo setting alarms.


The sensors were simultaneously inserted into the subcutaneous adipose tissue o the opposite sides of the lower abdomen and worn for 14 days.

Additionally, participant were requested to calibrate the CGM devices once daily and perform 8-9 finger-prick glu cose tests per day (GlucoMen Day METER, A. Menarini Diagnostics, Florence, Italy).


The primary objective was to assess the device's accuracy compared to a laborator reference instrument (YSI 2300, Yellow Springs, OH, USA).

The secondary objectives in cluded comparing CGM to SMBG and a precision assessment by evaluating the agreemen between the two sensors worn in parallel.

Additionally, a usability assessment includin a 10-item questionnaire with an ordinal rating scale asking about insertion pain, adhesiv adherence, problems with calibration, reliability of readings, usability of and satisfactio with the mobile application, device wearability, and overall experience was performed.


On days 4 and 10 of the study, a 5-h meal and insulin challenge was performed at the research center.

Each participant consumed a standardized meal containing 100 g of carbohydrates and received an increased insulin bolus (the regular bolus insulin dose plus 20%) subcutaneously.

Venous plasma and capillary blood samples were collected simultaneously every 20 min.

Plasma glucose was measured on site with YSI, while SMBG was performed using the study-specific BG meter.


CGM accuracy was assessed by calculating MARD, MAD (mean absolute difference), Median ARD, Median AD, and CEG for both CGM/YSI and CGM/SMBG matched pairs.

Following a frequently used approach in glucose monitoring system accuracy evaluations, MARD (and median ARD), as a percentage error, was calculated for glucose above the 100 mg/dL threshold only (i.e., values in the 100- to 400-mg/dL range), while, for the lower glucose values (ranging from 40-99 mg/dL), it calculated the absolute error as MAD (and median AD) [15,16].

The lag time between CGM and blood glucose data was determined for each sensor and applied prior to calculating MARD and MAD.

The lag time was calculated as the shift in time that provided the best CGM/reference correlation, using the Poincarè method (time shift giving the best R 2 vs. references), and was adjusted for SMBG and YSI data and for YSI data individually for each sensor.

The average lag time was calculated as the weighted average (based on the respective N of data) of the lag time of all the sensors.

All statistical analyses were performed following the intention-to-treat principle.


Eight adult participants (3 females (37.5%), age 41.6 ± 13.3 years, BMI 28.0 ± 6.1 kg/m 2 , HbA1c 55.6 ± 12.2 mmol/mol, diabetes duration 13.9 ± 6.5 years) completed the study.

During the study period, on average, 94.4% of the theoretically possible data was collected and used for analysis after applying the exclusion criteria for data recording (estimated CGM signal).


Overall, the 450 CGM/YSI matched pairs available for analysis were generated from glucose data collected within the range of 40-400 mg/dL. This resulted in a MARD of 9.7 (±9.4)% and a MAD of 20.5 (±18.7) mg/dL, as summarized in Table 1.

By analyzing specific glucose ranges, it was observed that sensor accuracy as assessed by MARD was better in the 201-400-mg/dL range compared to the 100-200-mg/dL range (6.1% vs. 10.7%), while MAD was lower in the 40-70-mg/dL range compared to the 71-99-mg/dL range (19.5 vs. 20.9 mg/dL).

Furthermore, both MARD and MAD were lower on day 4 compared to day 10 (7.4% vs. 11.4% and 17.7 vs. 27.3 mg/dL).

CEG analysis showed that 84.9% of CGM/YSI data pairs was in the clinically acceptable zone A, while the combined percentage for zone A and B was 97.8.

CEG is displayed in Figure 2a.


During sensor use at the research center and at home, a total of 1957 CGM/SMBG matched data pairs were collected in the range of 40-400 mg/dL. The overall MARD was 13.1 (±12.8)% for values between 100-400 mg/dL, while the MAD for glucose values between 40-99 mg/dL was 16.6 (±16.8) mg/dL, as summarized in Table 1.

Similar to what was observed for YSI, the MARD and MAD were lower in the higher glucose range (13.4% for 100-200 mg/dL vs. 12.2% for 201-400 mg/dL and 20.2 mg/dL for 40-70 mg/dL vs. 15.2 mg/dL for 71-99 mg/dL), while the CEG analysis showed a combined percentage of 98.2 for zones A and B (Figure 2b).


Glucose values recorded by the two sensors worn in parallel were evaluated using mean ± SD and coefficient of variation (CV) for each participant.

An average SD of 11.1 mg/dL and an average CV of 9.7% were observed, demonstrating acceptable agreement between the two sensors.


The participants were requested to complete a questionnaire containing 10 questions with an ordinal five-scale answer rating.

When asked about pain perception at insertion, 50% stated that the procedure was painless and 25% claimed it to be less painful than finger pricking.

The remaining 25% found the insertion as painful as routinely used SMBG (12.5%) or more painful than finger pricking (12.5%).

There was also predominant satisfaction with sensor adhesive, wearability, calibration procedure, and user-friendliness of the dedicated mobile application.

The results of the usability assessment can be found in the supplementary appendix (Figure S1).


High CGM accuracy is crucial for good diabetes management, especially when CGM readings are used for insulin dosing both alone or in combination with open/closed-loop systems.

Accurate readings are also essential for precise calculations of time in range (TIR), a parameter that has become increasingly popular for treatment decisions and the overall assessment of glycemic control.

By modeling the 7-point glucose profiles collected in the DCCT (Diabetes Control and Complications Trial), it was shown that TIR was strongly associated with development of microalbuminuria and retinopathy progression, suggesting that it represents a valid surrogate indicator of microvascular complications [17].


So far, MARD has been commonly used to assess overall CGM accuracy, while MAD is used to assess accuracy in the low glycemic range.

Good CGM performance is generally assumed in systems with an overall MARD < 10%, although analytical performance cannot be fully assessed by a single parameter [18].

In the present analysis, the GlucoMen ® Day CGM achieved a MARD of 9.7% compared to YSI during meal/insulin challenges (with one daily calibration), while compared to SMBG, a MARD of 13.1% was obtained over the 14-day wear period.

The higher MARD calculated for SMBG may result from lower accuracy associated with glucose meters compared to laboratory reference.

This finding is often seen during assessment of CGM devices at home [19-21].

The results of the present analysis are comparable to those obtained for other CGM systems such as the real-time (rt) CGM G6 (Dexcom, San Diego, CA, USA) with a MARD of 9.9% or the intermittently scanned (is) CGM systems Freestyle Libre 1 and 2 (Abbott, Chicago, IL, USA) with respective MARDs of 12.0% and 9.2% in adults compared to YSI [22-24].


Furthermore, similar to earlier findings in CGM systems, there was an increase in MARD/MAD in the low glycemic range while accuracy was higher in hyperglycemic ranges [25-27].

Since this effect was more pronounced during the meal challenges in our analysis, it can be assumed that the assessed CGM system is more accurate following post- prandial glucose excursions.

Interestingly, MAD was slightly lower in the 40-70-mg/dL range compared to the 71-99-mg/dL range, suggesting an increased or at least stable accuracy in relevant hypoglycemia, whereas other CGM systems show an impaired ac- curacy when BG levels drop below 70 mg/dL [22,25,27].

Given the fact that most CGM systems are prescribed due to impaired hypoglycemia awareness, CGM accuracy in hypo- glycemia is essential for giving a timely warning and taking adequate countermeasures to avoid hypoglycemia.


In addition to MARD/MAD as measures of numerical accuracy, clinical accuracy, as expressed by CEG, should also be evaluated to support the sensor performance data and should be favored over Clarke Error Grid or Continuous Error Grid analysis [15].

In our study, the CEG analysis showed 98% of all data pairs in the clinically relevant zones A and B for both reference methods, thereby verifying the clinical accuracy of GlucoMen ® Day.

For comparison, the CEG analysis of Freestyle Libre 1 and 2 showed a respective 99.7% and 99.9% of matched pairs in zones A and B [23-25].


Advancements in diabetes technology have so far drastically decreased the burden for people with diabetes.

The reduction of painful procedures associated with diabetes management improved quality of life and facilitated good glycemic control [28-30].

The replacement of SMBG and introduction of CGM in pediatric patients with diabetes was a crucial advance in reducing finger pricking.

However, especially in young children, sensor insertions are reported to be one of several barriers of this technology regardless of the significant advances of CGM use for children and parents in diabetes management [31].

Further reduction of painful procedures may lead to increased adherence in CGM users, which is known to be age-dependent and especially poor in adolescents [32].


In the present analysis, the CGM system with needle-free insertion intended for ages 6 and above resulted in high user satisfaction in the majority of users claiming the insertion procedure to be painless or less painful than finger pricking.


An important innovative feature of GlucoMen ® Day CGM is reusability of most components, resulting in reduced environmental impact.

The wish for reducing the ecological footprint of diabetes technology is frequently expressed by users and patient organizations, especially in the face of the present climate crisis.

Common CGM insertion systems often include bulky, disposable applicators that contribute to a substantial increase in plastic and potentially hazardous medical waste accompanied by a significant amount of packaging waste.

This represents a rising problem worldwide and is fueled by the, per se, favorable, increasing availability of CGM.

Looking at data from the DPV (Diabetes Patienten Verlaufsdokumentation) and the T1D exchange registry, CGM use in type 1 diabetes increased from 6% in 2011 to 38% in 2018 [33].

In absolute numbers, CGM use is presumably even higher due to its prevalence in type 2 diabetes and is expected to grow due to increasing reimbursement by healthcare providers.

The growing ecological burden associated with diabetes technology is already acknowledged by the scientific community and is subject to ongoing discussion [14,34].


GlucoMen ® Day CGM includes a rechargeable transmitter and a reusable needle-free sensor insertion applicator, both of which can be used for up to 5 years.

Due to reduced insertion pain and reusable components, this and similar systems can help promote sustainable diabetes technology and stimulate other positive developments in this sector, such as biodegradable components.

Therefore, regulatory bodies should aim to establish rules for design and development of diabetes technology with reusable components that meet medical needs and are environmentally friendly.

Additionally, waste reduction should be accounted for in cost-effectiveness calculations and reimbursement strategies for such products.


The present analysis suggests that the GlucoMen ® Day CGM is a user- and environ- mentally friendly system that meets the current clinical requirements for state-of-the-art CGMs.

The reduced ecological impact of this needle-free system has to be emphasized and may support further advances in sustainable diabetes technology.


In their recent article entitled “Accuracy Assessment of the GlucoMen® Day CGM System in Individuals with Type 1 Diabetes: A Pilot Study” [1], Hochfellner and colleagues presented the results of an accuracy and usability evaluation of a novel continuous glucose monitoring (CGM) system.

While we appreciate the importance of assessing the accuracy of CGM systems, we would like to comment on certain aspects of the data analysis, presentation of results and conclusions.


In the section describing the data analysis (Section 2.2), it is stated that “the lag time between CGM and blood glucose data was determined for each sensor and applied prior to calculating MARD and MAD”, where MARD and MAD are common accuracy parameters representing mean absolute relative and mean absolute deviation between CGM and comparator measurements, respectively.

This correction of lag time before accuracy determination was common in microdialysis-based systems [2,3] or for prototype CGM systems [4].

However, in market-ready CGM systems intended to provide real-time glucose data such as the system examined in the article, the accuracy should be evaluated with respect to the CGM values displayed to the user.

The effect of the lag time correction on the reported accuracy parameters cannot be assessed because neither the average lag time mentioned in Section 2.2. nor MARD and MAD results before lag time correction are presented.

In general, however, a lag time correction leads to a reduction in MARD and MAD.


Regarding the MARD, it should be pointed out that for its calculation, only glucose levels ≥100 mg/dL were used (Table 1), whereas for glucose levels <100 mg/dL, MAD results were presented.

This is an important detail missing from the abstract and the discussion, where MARD results were reported.


As a consequence of choosing to correct for time lag and to use only glucose levels ≥100 mg/dL for MARD calculation, we would argue that the accuracy results reported in the article cannot be compared to the cited findings of CGM performance studies without lag time correction and MARD results determined across the full glucose range [5–7].

Another aspect of the reported study that impairs the comparability to previous results is the low sample size of eight subjects, which is not discussed.


Due to these differences in methodology between this and other studies, we therefore question the validity of the conclusion that the “analysis suggests the GlucoMen® Day CGM [ . . . ] meets the current clinical requirements for state-of-the-art CGMs”.


We thank Dr. Freckmann et al. [ 1] for the comment and for the detailed review of our data.

Referring to the first point about the correction of lag time, we agree that the chosen approach has some limitations in the interpretation of the results.

This aspect can be discussed together with the second point concerning mean absolute relative difference (MARD) data representation.

The main aim of this study was to conduct an evaluation of the GlucoMen® Day CGM system, which had recently been launched on the market by A. Menarini Diagnostics based on available data related to the CE registration study.

For this reason, we limited the number of enrolled participants to just eight, resulting in limited statistics, and the methods and metrics for analysis were chosen according to the same methods used in the system CE registration study [2].

Thus, actually, the results met the predefined accuracy criteria of the trial, but we can agree that they cannot be consistently compared with other research on CGM accuracy assessment.


In order to avoid any confusion, such results can be better clarified as follows: when comparing CGM to PG, the MARD for values ≥ 100 mg/dL was 9.7 [2.6–14.6]%.

The MARD for values ≥ 100 mg/dL for CGM vs. SMBG was 13.1 [3.5–18.6]%.

The mean absolute difference (MAD) for glucose values < 100 mg/dL was 20.5 ± 18.7 mg/dL (vs. PG) and 16.6 ± 16.8 mg/dL (vs. SMBG).

The preference of MAD for lower glycemic intervals has mainly a mathematical reason: the relative (percentage) bias between a measurement and its reference increases the lower the reference value.

Based on this principle, for example, ISO 15197:2015 for BGMs specifies to use an absolute bias for values below 100 mg/dL and % bias at values higher or equal to 100 mg/dL. Such considerations are also widely discussed in the literature [3].

Therefore, also given the small sample size of this study, MAD was considered to be more appropriate for lower glucose concentration ranges, while the chosen cut-off of 100 mg/dL was derived for comparison purposes with available data for this system and other CGM studies [2,4].


To conclude, the present analysis confirmed that the GlucoMen® Day CGM is a user- and environmentally friendly system with performance in line with previously reported accuracy criteria.

In future studies, the system will be more extensively evaluated based on current metrics to determine whether it meets the clinical requirements for state-of-the-art CGMs and user expectations.


The ecologic niche for S. aureus in humans is in the anterior nares, where S. aureus can be identified most consistently in humans [27].

One-quarter to one-third of healthy persons har- bor S. aureus in the nose at any time [27].

Although S. aureus can also be found on the skin or in the rectum or vagina, the nose appears to be the primary reservoir for replication and spread to other bodily sites.

This idea is supported by studies that have revealed that, if S. aureus nasal carriage is temporarily eliminated by use of an intranasal topical antibiotic (e.g., mu- pirocin), colonization often simultaneously disappears from other colonized body sites [28].

Nasal S. aureus isolates are often identical to strains that later cause clinical infection, as assessed by PFGE [29, 30].


The proof of principle demonstrating the importance of nasal colonization's role in the pathogenesis of S. aureus infection is that, when colonization is eradicated, the risk of clinical infec- tion is lowered [27].

This observation has spurred attempts to eradicate S. aureus from the nose to prevent infection among high-risk groups.

Unfortunately, nasal decolonization strategies have limitations.

Short-term eradication is generally successful, but patients are often later recolonized with the same strain [26, 28, 31].

This suggests that recolonization from sources exogenous to the nose can occur.

Another limitation is that recolonizing strains are sometimes resistant to the previously used intranasal topical antibiotic [32].


The relationship between colonization and infection has been a fundamental tenet in the pathogenesis of S. aureus.

However, there are very few data on the association between CA-MRSA infection and nasal MRSA colonization, and there are even fewer data from prospective studies.

Indeed, a common feature of outbreak and nonoutbreak (i.e., endemic) CA-MRSA infec- tions is the lack of identifiable endogenous sources of MRSA (i.e., asymptomatic carriage in the anterior nares).