As stated in Table 2.13 [1], we have indeed observed a few anesthesiologists who had very low scores in succession [3].

To detect such patterns quickly – as would be desired if there were impairment from whatever cause – we nightly perform Bernoulli Cumulative Sum (CUSUM) monitoring (the mathematics of this monitoring was described in our Anesthesia and Analgesia Statistical Grand Rounds article [3]).

Low score detection occurs within 50 ±14 (median ± quartile deviation) days [3].

Thus, contrary to the letter, low scores are detected (using Bernoulli CUSUM) far before biannual ongoing professional practice evaluation analyses are performed using mixed effects logistic regression [1, 4].


As stated in Appendix 3, there is significant positive correlation between mean scores and percentages of scores equal to the maximum (Kendall’s sb +0.36, P <0.001) [1].

This is evidence of concurrent validity and the opposite of Dr. Ramsay’s hypothetical scenario.


As stated in Table 2.2 [1], questions’ responses are appropriately highly correlated (Cronbach alpha 0.948 ± 0.001 standard error), which is shown also in Appendix 3 and the first paragraph of the results [5].

Individual question responses do not create many different and commonly observed scores (contrary to the letter) [5].


Thus, the scenario and examples considered in Dr. Ramsay’s letter are unrealistic.

In addition, the following addresses the implication that the comparisons made with our described analyses are inaccurate.


As shown in Figures 3-6 and Appendices 7 and 8, valid logistic regression’s comparison to the mixed-effects inference is each anesthesiologist’s mean score equally weighting each rater – not the mean pooled score, as used in the scenario [1, 6].

We previously showed that the two are different because of the inequality of the variabilities of scores among raters (P < 0.001) [6].


As shown in Figures 3 and 4 and Appendix 5, the mean scores cannot be reliably compared among anesthesiologists, once the anesthesiologists have received feedback and learned how to provide better supervision, unless adjustment is made for the leniency of raters [1].

Thus, the letter’s use of mean scores is unreliable in practice [1].

As shown in Appendix 2, models of mean scores adjusted for rater leniency violate the basic statistical assumptions of the inference (e.g., normal distributions) [1].


Consequently, and in noted contrast to our approach, even though use of mean scores may be desired, Dr. Ramsay’s letter does not suggest how they can be used reliably or with any validity.


There is a decades-old debate around the extent, mecha- nism and 'evolutionary scale' of sexuality among human protozoan pathogens (Tibayrenc & Ayala 1991, 2002, 2012, 2013, 2014a).

The debate stems from a wider dis- cussion around the frequency of genetic exchange in many microparasites (Tibayrenc & Ayala 1991; Maynard Smith et al. 1993; Heitman 2010).

At the heart of the issue lies the importance of sex for pathogen fitness (i.e. the generation of novel adaptive allelic repertoires-or escape from Muller's ratchet) weighed against recombina- tional load (i.e. the disruption of favourable interactions between alleles at different loci in clonal lineages) (Barton & Charlesworth 1998).

Even among protozoan parasite species where genetic exchange is now well characterized (e.g. Plasmodium falciparum, Trypanosoma brucei brucei), population genetic evidence for its occur- rence may vary between disease foci (e.g. Duffy et al. 2013).

The frequency of genetic exchange must be defined, in evolutionary terms, by the fitness advantage sex confers in a given environment or at a given period- icity of environmental change.

However, our ability to explore the adaptive significance of sex at biologically relevant scales-for example within hosts, vectors and disease outbreaks-is severely limited by our inability to detect recombination events.


Standard statistical popula- tion genetic tools may be too blunt to provide clarity where genetic exchange is relatively infrequent, 'covert' or non-Mendelian (De Meeus et al. 2006; Heitman 2010).

Similarly, where the participants in genetic exchange events are genetically similar, distinguishing parents from progeny is challenging.

In the light of the apparent lack of evidence for sex, some have suggested, therefore, that sex must be rare and/or inconsequential in most proto- zoan pathogen populations (Tibayrenc & Ayala 1991, 2002).

This clonal hypothesis has been subject to a spate of recent refinement and re-iteration (Tibayrenc & Ayala 2012, 2013, 2014a,b).

Broadly speaking, the model of pre- dominant clonal evolution (PCE) proposes that genetic recombination in many parasitic protozoa is 'too rare to break the prevalent pattern of clonality,' such that there is 'restrained recombination on a evolutionary scale' (Tibayrenc & Ayala 2012, 2014b).

Furthermore, the model defines inbreeding, selfing and homogamy as 'particular cases of PCE' (Tibayrenc & Ayala 2014b).


Here, we examine the value of an alternative perspective on sexu- ality in parasitic protozoa, with specific focus on many of the organisms at the centre of the debate, namely medically important trypanosomatids, Giardia and Toxo- plasma.

We attempt to reconcile PCE with the presence of recombination machinery in parasite genomes (El-Sayed et al. 2005; Ramesh et al. 2005), experimental proof of genetic exchange events in the laboratory (Gaunt et al. 2003; Poxleitner et al. 2008; Akopyants et al. 2009; Sadlova et al. 2011; Peacock et al. 2014) and an increasingly large body of evidence for widespread genetic exchange in the field (Cooper et al. 2007; Balmer et al. 2011; Ramirez et al. 2012; Duffy et al. 2013; Baptista Rde et al. 2014; Rogers et al. 2014).

Working from the assumption that genetic exchange may be common among protozoan parasites, © 2014 John Wiley & Sons Ltd Molecular Ecology (2014) 23, 4195-4202 we ask-why is sex so difficult to detect in natural populations?


Geographically and temporally stable genetic subdivisions in protozoan pathogen populations are defined variously as species, subspecies, assemblages and discrete typing units (DTUs).

Genetic divergence thresholds for species definitions are not clearly defined between genera.

Indeed, among human trypanosomatid parasites and Giardia, the levels of genetic divergence that 'species', 'subspecies,' 'DTU' and 'assemblage' describe are approximately equiva- lent (Jerlstrom-Hultqvist et al. 2010; Yeo et al. 2011; Boite et al. 2012).

Genetic subdivisions often have eco-epidemio- logical significance, and these subdivisions must also be taken into account when evaluating the frequency of genetic exchange-it makes little sense to address each par- asite species (or genus) as a whole.

Divergent, non-recom- bining genetic subgroups in a sample can inflate genetic linkage statistics and mask recombination events that occur between more closely related individuals (Maynard Smith et al. 1993).

Non-recombining subdivisions may simply har- bour further non-recombining groups-the so called Rus- sian doll hypothesis (Tibayrenc & Ayala 2014a).

However, much of the available evidence supports the former asser- tion.


Genomic comparisons across three genomes represen- tative of Giardia intestinalis assemblages A, B and E indicate minimal recombination (Xu et al. 2012).

Similarly, interas- semblage recombination is absent when several thousand Giardia isolates are compared using fewer markers (Takumi et al. 2012).

In contrast, good evidence of genetic exchange was detected among a series of epidemiologically linked Giardia assemblage AII cases from Peru (Cooper et al. 2007).

In Leishmania and Trypanosoma cruzi, interspecies (or inter-DTU) hybridization occurs only sporadically (e.g. Nolder et al. 2007; Roellig et al. 2013), although highly suc- cessful genotypes can result (Lewis et al. 2011).

However, those studies that evaluate intraspecies/intra-DTU diver- sity, as with Giardia, almost invariably find evidence for widespread genetic exchange (Rougeron et al. 2009, 2011; Ocana-Mayorga et al. 2011; Ramirez et al. 2012; Rogers et al. 2014).


The role T. brucei subspecies play as barriers to genetic exchange is less clear.

The boundary between T. b. brucei and T. b. rhodesiense is highly porous, with a single, exchangeable, virulence determinant (the human serum resistance-associated gene) often defining the latter (Balmer et al. 2011).

Overall, population genetic and geno- mic data now support substantial genetic exchange within and between T. b. brucei and T. b. rhodesiense (Balmer et al. 2011; Goodhead et al. 2013) but not, it seems, within Try- panosoma brucei gambiense I, where all available evidence points to clonality (Koffi et al. 2009).


Thus, there is evidence among many protozoan parasites that broad population genetic subdivisions-species, sub- species, assemblages or DTUs-act as partial reproductive barriers.

The relative frequency of genetic exchange between and within parasite population genetic subdivisions has led to considerations of what some term the 'evolutionary scale' of such events (Tibayrenc & Ayala 2012)-a concept we explore more fully in Table 1.


Generally, reliable estimates of recombination frequency are best derived from popula- tions of individuals that are un-subdivided in space and time.

Thus, even once species, subspecies, assemblages or DTUs are accounted for, population genetic analysis of spa- tio-temporally dispersed collections of individuals impedes the detection of genetic exchange.


In general, recent studies that define reproductive mode in diploid protozoan patho- gens have all derived estimates from minimally subdivided population samples (Koffi et al. 2009; Rougeron et al. 2009; Ocana-Mayorga et al. 2011), even down to the level of the same host (Llewellyn et al. 2011).

However, at very local scales-exactly where physical contact between parasites might facilitate hybridization-one inevitably encounters the problem of discriminatory power.


There are two ways to approach the problem of detecting genetic exchange where it is suspected but difficult to prove owing to the apparent similarity of circulating strains.

The first is simply to increase the discriminatory power of the molecular markers used.

The second is to tar- get hybrid zones-where recently diverged, genetically dis- tinguishable subpopulations come into contact.


Recent population genomic analysis of Leishmania infantum from a restricted locality in Turkey efficiently demonstrates the result where both approaches are applied simultaneously (Rogers et al. 2014).

Leishmania infantum isolates from ele- ven sand flies (Phlebotamus tobbi) and one human were sub- mitted to Illumina HiSeq genome sequencing.

Genomic patterns of polymorphism within and between clones were then evaluated.

Identifying L. infantum reference strain JPCM5 as similar to one putative parent, the authors were able to define 'parentage blocks' across the 12 study genomes.

The authors suggest the data are consistent with a population formed from a single hybridization event between two divergent L. infantum strains, followed by several rounds of interbreeding between these hybrids (Rogers et al. 2014).


It is crucial to highlight that an earlier study of six of the isolates using fewer genetic markers- 14 microsatellite loci-detected no specific evidence to suspect the strains were genetic hybrids (Gouzelou et al. 2012).

In fact, typical of tens, if not hundreds of T. cruzi and Leishmania populations described in the literature (e.g. Llewellyn et al. 2009; Rougeron et al. 2011, 2009)-the six strains Gouzelou et al., genotyped where moderately diverse with heterozygosity deficit over loci.

Genome-scale population data for parasitic protozoa are likely to force us to reconsider many former conclusions based on limited numbers of genetic markers.

Rogers et al. ( 2014) is a case in point.


It follows that signatures of hybridization are poten- tially detectable among multiple parasite populations given sufficient discriminatory power.

Thus, it is hard to accept that 'PCE' explains genetic diversity among parasitic proto- zoa (Tibayrenc & Ayala 2012) while we are still largely © 2014 John Wiley & Sons Ltd 4196 N E W S A N D V I E W S : O P I N I O N 1365294x, 2014, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.12872 by Mcgill University, Wiley Online Library on [18/05/2024].

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Perhaps we are


Rogers et al. ( 2014) may have specifically targeted vector- isolated L. infantum in the hope of detecting hybrids.

Experimental hybrids in Leishmania major and Leishmania donovani have both been derived from crosses in sand flies (Akopyants et al. 2009; Sadlova et al. 2011).

The recent identification of haploid gametes in T. brucei unequivocally identifies the Tsetse fly salivary gland as the site of genetic exchange (Peacock et al. 2014).

The definitive site of genetic exchange in T. cruzi is perhaps the mammalian host (hybrids emerged after passage of parents through Vero cells) (Gaunt et al. 2003), although further hybridization events in the laboratory have not been observed since.

In Giardia, genetic exchange (in-crossing) has been detected at the environmental cyst stage of the life cycle (Poxleitner et al. 2008).


However, the concept of a 'definitive host or vector' should not be a major preoccupation for population geneticists.

The allelic signature of sex in a recombining population should be detectable regardless of where the event takes place.

The same is technically true for the hap- loid life stages of Toxoplasma gondii.

The identity of the definitive host of T. gondii-the domestic cat-is well estab- lished, even though the population structure of most human T. gondii isolates worldwide appears highly clonal (Sibley et al. 2009).

As such, the role that sex in the T. gon- dii definitive host has in potentiating short-term clonal epi- demics has been overlooked until fairly recently (Wendte et al. 2010).


In Wendte et al. 's (2010) study, an outcrossing event generated several haploid progeny, detectable in non-human strains (e.g. chickens, water samples).

How- ever, among strains recovered locally from humans, only a single haploid variant was identified.

This human clone was atypical of the archetypal I, II and III non-recombining global human lineages, but strongly related to nearby non- human strains, suggesting a recent local emergence.


Thus, while definitive individual hosts or vectors are perhaps irrelevant, a failure to sample non-human transmission cycles may overlook recombinant clones, that is a focus on human sampling may simply detect a rapidly expanding clonal genotype.

An important parallel exists in T. brucei rhodesiense where serial human clonal outbreaks against a backdrop of recombination in the non-human populations have long been suspected (MacLeod et al. 2000; Duffy et al. 2013).


The molecular machinery for meiotic cell division is thought to have evolved early in eukaryotes (Cavalier- Smith 2002).

Sequence comparisons among metazoa, protists, fungi and yeasts indicate homology among many


More complete descriptions of genetic exchange mechanisms described experimentally in each species are included in the body of the article.

It is of note that different sexual or parasexual mechanisms are potentially present in the same species.

Thus, a mecha- nism of genetic exchange observed in the laboratory and its predicted impact may not correspond precisely to the patterns of genetic exchange observed in natural populations.

Cytological mechanisms of genetic exchange may there- fore be variable within and among protozoan pathogen species.


Many population genetic models, on the other hand, implicitly assume Mendelian sexuality.

At single loci, standard interpretations of Mendelian segregation make little sense in populations that might be at least par- tially clonal and/or undergo nonmeiotic or unorthodox meiotic sex.

In long-term clonal organisms, excess hetero- zygosity is better attributed to allelic sequence divergence (ASD) than outbreeding (Mark Welch & Meselson 2000).

In reality, however, excess heterozygosity is only rarely observed in parasitic protozoa (e.g. Koffi et al. 2009).

In such cases, recent hybridization between divergent parents followed by clonal expansion may be a more cogent expla- nation than ASD (e.g. Lewis et al. 2011).


Mendelian inter- pretations of heterozygous deficit are frequently made in Leishmania populations (Rougeron et al. 2009, 2011).

How- ever, loss of heterozygosity events (such as gene conver- sion and mitotic recombination) might be responsible for observed intragenomic variability.

Dispersed loss of hetero- zygosity is reported in T. cruzi (Llewellyn et al. 2009; Yeo et al. 2011); ploidy cycles involving the loss of entire haplo- types occur in Candida (Hickman et al. 2013); and internu- cleus transfer results in homokaryosity in Giardia (Yu et al. 2002).

Nonetheless, genome-scale analysis of natural Leish- mania hybrids does suggest some level of inbreeding (Rogers et al. 2014).


While Mendelian interpretations of single locus segrega- tion in parasitic protozoa can be misleading, measures of genetic linkage between loci should robustly detect non- Mendelian sexuality.

Pairwise linkage equilibrium between microsatellite loci has been detected in several trypanoso- matid populations (Ocana-Mayorga et al. 2011; Duffy et al. 2013), indicative of sex.


Unfortunately, excess homozygos- ity in parasite populations is most commonly accompanied by linkage disequilibrium (Llewellyn et al. 2009; Rougeron et al. 2009; Ramirez et al. 2012).

Indeed, high homozygosity reduces the impact of recombination, prolonging the maintenance of disequilibrium.

Thus, claims of 'inbreeding' based on individual loci cannot be corroborated by interlo- cus linkage equilibrium (Rougeron et al. 2009), even in highly genetically diverse populations (Llewellyn et al. 2009).

The insensitivity of interlocus measures of linkage to anything but fairly frequent genetic exchange events is likely to complicate matters (de Meeus & Balloux 2004).


The difficulty of obtaining 'hard evidence' for genetic exchange in protozoan pathogen populations using a lim- ited number of nuclear genetic markers has led us and oth- ers to explore diversity outside the nuclear genome (Ramirez et al. 2012; Baptista Rde et al. 2014).


In most try- panosomatids, plastid inheritance is generally uniparental after genetic exchange (Gaunt et al. 2003; Akopyants et al. 2009; Lewis et al. 2011; Rogers et al. 2014), although it may be transiently biparental in the immediate aftermath of the event (Gibson & Garside 1990).

Identity by descent means that plastid genome introgression events are easily detect- able against a background of frequent nuclear genetic exchange.

Where nuclear genetic exchange is asymmetric (unequal contributions from each parent), comparisons between un-phased nuclear and plastid phylogenies also show high levels of incongruity.

In T. cruzi at least, inter- DTU hybridization events can be highly asymmetric (e.g. Roellig et al. 2013), whereby nuclear transfer from one of the two parents involved in a cross is frequently undetect- able.

Similar observations can be made using higher-resolu- tion genetic markers within the DTU T. cruzi I in Colombia (Ramirez et al. 2012).

Parallel approaches reveal introgres- sion in T. b. brucei (Balmer et al. 2011), and our understand- ing of the frequency of genetic exchange in several Leishmania species could benefit, as it could for T. gondii, where apicoplast inheritance is uniparental during tachyzo- ite crosses in the definitive host (Ferguson et al. 2005).


Thus, plastid vs. nuclear genome comparisons are useful in detecting cryptic genetic exchange at fine scales where nuclear allele frequency-based indices lack power.

There are, however, two caveats.

First, mutation rate differentials between nuclear and plastid genomes could lead to appar- ent incongruence in the absence of introgression (Tibayrenc & Ayala 2013).

However, where two near-identical nuclear genotypes have radically different mitochondrial genomes, and donors and recipients can be clearly identified within the study population, rate variation is unlikely to play a confounding role (Ramirez et al. 2012).

Second, it is plausi- ble that plastid exchange can occur in the absence of nuclear genetic exchange.

For mitochondria especially, where mutation rate and thus Muller's ratchet is acceler- ated by an order of magnitude with respect to the nuclear genome, regular outcrossing is likely to be selectively advantageous (Haag-Liautard et al. 2008; Neiman & Taylor 2009).


As the clonal theory suggests (Tibayrenc & Ayala 2012, 2013), in some parasite populations, it is likely that we fail to detect genetic exchange events because they are genu- inely extremely rare.

Among many human isolates of T. gondii (Sibley et al. 2009) and T. b. gambiense I (Koffi et al. 2009), for example, sex appears absent.

However, among almost all other protozoan parasites populations discussed, evidence of a role for frequent, although not obligate, genetic exchange in defining parasite diversity is mounting.

Rogers et al. ( 2014) estimate the mean rate of meiotic events as one per 7.5 9 10 4 per mitotic cell divisions in L. infan- tum.

Given that approximately 10 times this number of Leishmania promastigotes can be found in a single sand-fly midgut, the figure suggests tens of crossing-over events per vector-a non-negligible rate population-wide.


Protozoan parasite populations, where excess homozy- gosity persists and inbreeding is inferred, are purported © 2014 John Wiley & Sons Ltd N E W S A N D V I E W S : O P I N I O N 4199 1365294x, 2014, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.12872 by Mcgill University, Wiley Online Library on [18/05/2024].

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While theoretically sound, the definition may be invalid when one considers the evidence from natural populations.


First, most diploid parasite pop- ulations that display excess homozygosity over loci are highly genetically diverse (Llewellyn et al. 2009; Rougeron et al. 2009, 2011; Ocana-Mayorga et al. 2011; Gouzelou et al. 2012), including at the level of individual hosts (Llewellyn et al. 2011).

Thus, any level of inbreeding in its classical sense (herein defined as genetic recombination within clo- sely related parasite populations, as distinct from selfing) should generate large numbers of heterozygotes-which is apparently not the case.


Only strict selfing-perhaps resembling ploidy cycles in C. albicans (Hickman et al. 2013)-could simultaneously explain high genetic diversity and excess homozygosity in diploid parasitic protozoa.


However, selfing is a poor means of escaping Muller's ratchet-extinction is a likely outcome in the absence of mitigating factors (e.g. Loewe & Cutter 2008).

It is thus difficult to envisage the advantage of strict selfing over clonality, unless sex represents an obligate portion of the parasite lifecycle and there is a shorter-term fitness benefit to avoid outcrossing, such as the maintenance of multilo- cus drug resistance genotypes in P. falciparum (Razakan- drainibe et al. 2005).


Even so, with reference to Table 1, the theoretical fitness benefits of clonality in the short term are likely to be balanced or outweighed by those of sex at the same scale.


The second reason why excess homozygos- ity observed in parasite populations is probably not a 'par- ticular case of clonality' we have already addressed and simply relates to discriminatory power.


Small sets of mar- ker loci are no substitute for genomes.


Population genomic findings from L. infantum (Rogers et al. 2014) could well be corroborated across many other populations of diploid parasites previously defined as homozygous.


In this case, signatures of moderate inbreeding derived for a small number of microsatellite loci can in fact obscure strong signatures of genomic hybridization and sex.


In conclusion, while we acknowledge the importance of clonal propagation to parasitic protozoa, we propose that sex is fundamental to organismal fitness across multiple evolutionary scales and occurs more frequently than previ- ously suspected.

Traditionally, inappropriate sampling across genetic subdivisions, poor discriminatory power of molecular markers, insufficient sampling from non-human reservoirs and inadequate population genetic models have all hindered our ability to detect genetic exchange events in natural populations of parasitic protozoa.

With the advent of population genomics, discriminatory power is no longer a limiting factor.

However, appropriate study design and sample collection remain vital considerations if we are to accurately describe population genomic struc- tures and the natural dynamics of genetic recombination in the genome-sequencing era.


The predominant clonal evolution (PCE) model of micropathogens proposed by us has been challenged by a recent paper in Molecular Ecology.

We review the main tenets of our model and show that the criticisms raised by the paper’s authors are based on papers that are either misunderstood or misquoted.

We argue that the PCE model and its recent developments (in particular the ‘Russian doll model’ dealing with micro-clonal evolution) are supported in most cases when adequate data are available [1].

Predominant clonal evolution is defined as strongly restricted recombination, not absence of recombination.

This definition is widely accepted, not only by us, but also by many authors working on parasitic protozoa, fungi, bacteria, and viruses [2].

This means that evidencing occasional bouts of recombination or hybridization does not falsify the PCE model.

More specifically, we propose that PCE is verified when the species considered has reached a ‘clonality threshold’ beyond which clonal evolution overcomes the effects of recombination in the long run [3].

The increase in the phylogenetic signal can be tested by classical linkage disequilibrium (LD) tests and phylogenetic analyses [4].

One of the most important consequences of PCE in a given species is the existence, due to restricted recombination, of discrete genetic subdivisions that are extremely stable in space and time [5].

We have proposed to call these entities: ‘near-clades’, to replace the many terms found in the literature [6].

A near-clade is a phylogenetic line that is clouded by occasional recombination/hybridization.

However, once the species has crossed the ‘clonality threshold’, the discreteness of the near-clades becomes clearer and clearer when more relevant data are considered [7].

The term ‘clade’ is not appropriate, as some recombination goes on among the near-clades in most, if not all, micropathogen species surveyed by us [8].

This feature of the model has considerable evolutionary and epidemiological consequences [9].

As a matter of fact, the near-clades, due to their stability and discreteness, constitute appropriate targets for molecular epidemiology (strain typing), and tend to exhibit different phenotypic characteristics, including with respect to epidemiology and clinical features [10].

The authors aim to invalidate this remarkable finding, by stating that ‘it makes little sense to address each parasite species (or genus) as a whole’, because these ‘genetic subdivisions act as reproductive barriers’ [11].

The authors omit to mention that evidencing the genetic subdivisions (that is to say: the near-clades) and showing that they act as reproductive barriers has been possible only through the PCE approach, and constitutes one of its main achievements [12].

This way of presenting the affair amounts to making believe that the main conclusion of the PCE approach was self-evident, which is quite untrue: it is the result of long-term research of more than 30 years, started at a time when pathogen population genetics was almost nonexistent [13].

This statement of the authors is inappropriate, because the job still has to be completed or perfected for many pathogen species, and only the PCE approach will allow to do so [14].

Strangely, the authors consider that the PCE approach at the level of the whole species makes little sense.

However, in the case of Toxoplasma gondii, they accept the hypothesis of PCE based on data that consider the whole species [15].


Within-near-clade PCE: the ‘Russian doll’ (RD) model This model aims at exploring the level of genetic exchange, not among the near-clades, but rather, within each of them [16].

As a matter of fact, many authors consider that there is more recombination within the near-clades than between them [17].

The RD model takes as null hypothesis that within the near-clades, recombination is potentially random (not just that occasional recombination can occur), because the expectations for random recombination are well known (linkage equilibrium) [18].

The working hypothesis is that the near-clades each exhibit a miniature picture of the whole species, with the two main features of the PCE model, namely (i) strong LD, or non-random association of genotypes occurring at different loci, and (ii) lesser near-clades [19].

The RD approach simply consists in testing the PCE model within each of the near-clades [20].

Ramírez & Llewellyn attempt to invalidate RD by favouring the null hypothesis [21].

However, the examples they advance are either weak or misleading [22].

Some of them are even counterexamples of what the authors try to demonstrate [23].

In the case of Giardia, the only case cited by the authors to falsify RD is not based on a population genetics approach, but rather on the sequencing of a few genes [24].

The conclusions of this article are based on observed discrepancies between the phylogenetic trees based on three loci for seven strains [25].

This is a possible indication that some genetic exchange may go on (although other explanations are possible), but says nothing about the frequency of recombination [26].

Moreover, with such a limited set of data, the risk of statistical type II error (impossibility to reject the null hypothesis of random recombination, not because it is proven, but because the test lacks power) is high [27].

Lastly, this study does not concern a whole Giardia ‘assemblage’ (= near-clade), but rather, the subassemblage A2 of the assemblage A [28].

The mere existence of this subassemblage, which shows that assemblage A is subdivided into discrete units, is in itself evidence supporting RD [29].

Two of the examples cited about Leishmania do not concern intra-near-clade recombination but rather, the whole species [30].

They have therefore nothing to do with the RD controversy [31].

Moreover, the ‘widespread genetic exchange’ that the examples are supposed to evidence is actually selfing/inbreeding, which is considered by us and many, if not most, authors working on pathogens population genetics as a particular case of PCE [32].

Lastly, the tests used to infer selfing/inbreeding in Leishmania are based on the hypothesis that these parasites are diploid, while convergent results rather favour that Leishmania exhibits widespread aneuploidy [33].

This bias has been underlined by us, as well as by Ramírez et al. [ 34].

Another study, repeatedly cited by Ramírez & Llewellyn, is actually an illustrative counterexample of what they try to demonstrate, and a talking illustration for RD [35].

By high-resolution sequencing, Rogers et al. evidence a monophyletic subdivision (near-clade) within Leishmania donovani/infantum [36].

They do find indication of hybridization within this near-clade [37].

However, what Ramírez & Llewellyn omit to mention is that Rogers et al. clearly state that ‘extensive LD between chromosomes is consistent with rare recombination and supports clonality as the primary reproductive mode’ and that there is ‘mainly clonal reproduction in the parasite population’ [38].

The data presented do not make it possible to know whether there are lesser near-clades within this monophyletic subdivision [39].

However, they do show that clonality is the dominant mode of reproduction within this near-clade, which supports a RD pattern [40].

The studies cited concerning Trypanosoma cruzi are not strong evidence against RD [41].

The first one is consistent with recombination in a small subpopulation of the TCI near-clade in Ecuador [42].

However, as we have shown, this ‘domestic’ population is poorly defined (33% of selvatic strains are included in it, while the ‘selvatic’ clade comprises 12% of domestic strains) [43].

The risk of type II error is high, due to very limited sampling (only 12 truly ‘domestic’ strains) [44].

Additionally, the ‘selvatic’ clade shows strong LD and clonality, which favours the RD hypothesis [45].

Observing a lack of congruence between mitochondrial and nuclear phylogenies indicates at best limited introgression, as it can be observed even between different biological species [46].

Another study has postulated ‘substantial recombination’ in Brazilian strains of the TCII near-clade [47].

However, this study relies on limited sampling and is subject to type II error [48].

There is a strong contradiction between the hypothesis of ‘substantial recombination’ (with lack of LD) and the evidence for clear structuration of these populations, even in sympatry, as shown by STRUCTURE analysis [49].

A recently published study deals with TCI T. cruzi populations isolated from selvatic triatomine bugs in Bolivia [50].

The data are compatible with frequent genetic exchange, however, as the authors assert their data are prone to type II error and should be corroborated by more extended sampling [51].

While focusing on these disputable examples consistent with the null hypothesis, Ramírez & Llewellyn omit to cite the many cases presented by us, which strongly support the RD model, including in T. cruzi, Leishmania, Toxoplasma, Giardia and Cryptococcus [52].

This is all the more astonishing, since some of the best examples of RD patterns were found in the authors’ own data [53].

As a matter of fact, convergent results show that, within T. cruzi, the near-clade TCI is subdivided into several smaller near-clades that are widespread and stable in space and time, including in sympatry [54].

These ‘mini-near-clades’ have been corroborated by several different molecular markers, including the mini-exon gene, the cytochrome B gene, multilocus PCR-RFLP and SSUrDNA [55].

Contrary to what the authors state, within Colombian TCI strains, there is LD [56].

As a matter of fact, the p values for the LD test and the Ia index of association (another LD test) are 4 x 10^-4 and 0.037, respectively [57]. As noted by Tomasini et al., this is evidence for LD, and not for the opposite, as wrongly concluded by the authors [58].

Similarly, within TCI selvatic strains, strong LD evidences ‘widespread clonality, infrequent recombination’ [59].

Lastly, strong evidence for a RD pattern within Argentinian TCI strains has been recently published [60].

We do not state that the RD pattern is presently verified in all pathogens. But we do argue that it has been strongly supported by many data, especially in T. cruzi, Leishmania infantum, Cryptococcus neoformans, Giardia and Toxoplasma gondii, in each case when genetic and population samplings are enough and adequate [61].


Conclusion We argue that the PCE hypothesis is not just an artefact, and that it is the most parsimonious model for many, if not most pathogens, including viruses, bacteria, parasitic protozoa and fungi [62].

Our conclusions are based on a large set of published data, dealing with bacteria (48 species), yeasts and fungi (nine species), parasitic protozoa (21 species) and viruses (11 species or categories) [63].

These data are easily accessible to any reader wanting to verify our proposals [64].

If looking for recombination in pathogens amounts to ‘searching for a needle in a haystack’, it obviously means that recombination is severely restricted; in other words, that the PCE model is supported [65].

When it is not, like in some populations of Plasmodium falciparum, or in Helicobacter pylori, one has no trouble finding clear indications for recombination without searching in a haystack [66].

Until now, the use of more resolutive markers has reinforced the PCE and RD hypotheses [67].

As an example, near-clades and RDs in Toxoplasma gondii are better evidenced by a set of highly resolutive markers than by more conventional tools such as multilocus sequence typing [68].

Similarly, whole-genome sequencing in Neisseria meningitidis uncovers the existence of deep phylogenies that were not apparent with multilocus sequence typing [69].

As a matter of fact, contrary to what Ramírez & Llewellyn state, lack of resolution should favour the null hypothesis (random recombination) rather than PCE, even when highly resolutive markers make it possible to find ‘the needle in the haystack’ [70].

Concerning the amount and frequency of recombination, we have called for discarding vague, subjective assertions, such as ‘widespread genetic exchange’, ‘more frequent than expected’, which amount to a conflict between the supporters of the half full and the half empty bottle [71].

We have proposed, rather, to use a clear-cut PCE criterion relying on the notion of a ‘clonality threshold’ [72].

If, within a given species, a growing phylogenetic signal is observed as more relevant data are obtained, it means that restricted recombination efficiently counters the effects of genetic exchange, and that the near-clades that subdivide the species will tend to diverge more and more [73].

This PCE-specific signal should be looked for, first at the level of the entire species [74].

Only once the near-clades have been clearly evidenced by this approach, and are fully recognized by most authors (which is still the case for a limited number of species only), RD patterns should be looked for within them, but only in a second round [75].


Tibayrenc and Ayala raised several interesting objections to an opinion piece we recently published in Molecular Ecology [1].

Our piece examined the value of an alternative perspective to their theory of predominant clonal evolution (PCE) on the prevalence and importance of genetic exchange in parasitic protozoa.

In particular, our aim was to establish whether population genetic signatures of clonality in parasites were representative of true biological/evolutionary processes or artefacts of inadequate tools and inappropriate or inadequate sampling.

We address Tibayrenc and Ayala’s criticisms and make a detailed response.

In doing so, we deny the consensus that Tibayrenc and Ayala claim around their views and dismiss much of the language which Tibayrenc and Ayala have introduced to this debate as either arbitrary or inaccurate.

We strongly reject accusations that we misunderstood and misquoted the work of others.

We do not think the PCE provides a useful framework for understanding existing parasite population structures.

Furthermore, on the eve of the population genomic era, we strongly urge Tibayrenc and Ayala to wait for the forthcoming wealth of high-resolution data before considering whether it is appropriate to refine or re-iterate their PCE hypothesis.


In our piece, we suggested that ‘Genetic divergence thresholds for species definitions are not clearly defined between genera’ pointing out that genetic divergences between major genetic subdivisions called variously ‘species’ (e.g. Leishmania), ‘subspecies,’ (e.g. Trypanosoma brucei) ‘DTU’ (e.g. Trypanosoma cruzi) and ‘assemblage’ (e.g. Giardia duodenalis) are approximately equivalent [2].

We suggested that lumping these subdivisions together in analyses made ‘little sense’ as it will artificially inflate genetic linkage statistics and mask recombination occurring within lineages [3].

Rather than accepting this potential flaw in experimental design, in their response Tibayrenc and Ayala (tangentially) appropriate the finding of reproductively isolated subgroups in pathogen populations as a major achievement of their PCE model [4].

This comment is not a criticism of our manuscript per se, but we do strongly disagree with it.


In our opinion, the discovery of major genetic subdivisions in parasitic protozoa is the result of the collective efforts of hundreds of researchers over the last 40 years and, yes – the self-evident result of (i) genotyping the same conserved molecular markers across studies; (ii) finding that intermediate genotypes between major subdivisions are exceedingly rare.

It is categorically not one of the ‘main achievements’ of the PCE.

In our review, we merely wished to point out that if genetic exchange occurs, it is more likely to happen within these subdivisions than between them.


Later in their critique, Tibayrenc and Ayala cite a population genomic study of Neisseria meningitidis as evidence of ‘deep phylogenies’ in pathogen populations [5], presumably to further bolster the PCE model.

Careful reading of this commendable study (and personal correspondence with its authors) reveals precisely the pattern of population diversity we highlighted: deep subdivisions with little or no exchange between them; intense lateral exchange of genetic information within them.

In still yet another recent review, Tibayrenc and Ayala conveniently dismiss this finding as ‘invisible sex’ [6], regardless of the considerable genetic diversity present among strains involved and the possible fitness benefits associated (virulence factors, antimicrobial resistance, etc) [see also Table 1 in [1]].

Importantly, Budroni et al. ’s findings absolutely contradict the expectations of Tibayrenc and Ayala’s much discussed and little supported ‘Russian doll’ hypothesis [7].


In the second section of their comment, Tibayrenc and Ayala go on further to defend their ‘Russian doll’ theory, suggesting that the data we present in support of ‘sex-within subdivisions’ is ‘weak’ and ‘misleading’ [4].

In fact, we presented the available data in a balanced fashion, including examples where evidence of genetic exchange was not uncovered [8], as well as where it was [9].

Crucially we focused on studies that examine diversity among large ‘minimally subdivided population samples’ in space and time – that is, where one could have reasonable confidence about the population genetic parameters derived from them.

It is hardly fair to call this ‘misleading’.

We did not cite the 2013 article they mention that contained an error in a P-value interpretation [10]; however, we accept that there was an error for which correction has been submitted.


Tibayrenc and Ayala suggest we furthermore ‘omit to cite the many cases presented by us [them] that support the Russian Doll model’ [4].

We invite the reader to review these ‘many cases,’ as we have performed – most are simply not convincing.

Almost all the studies they cite have sample sizes an order of magnitude lower than ours and those that we cite, as well as low numbers of conserved genetic markers used for genotyping.

In one case, Tibayrenc and Ayala totally misconstrue the study findings: Minning et al. 2011 found decoupling between copy number variation and DTU identity ‘strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.

’ [ 11] – the opposite meaning to that portrayed by Tibayrenc and Ayala [12].


The overall picture that emerges from existing data – including studies cited by ourselves and Tibayrenc and Ayala – is a spectrum of within-subdivision population structures, some sexual, some occasionally sexual and some clonal.

The Russian doll hypothesis is valid as a hypothetical model.

However, the statistical tools on which it is based are highly sensitive to anything but completely random associations between loci at a population level.

Thus, only truly panmictic populations – rare enough in sexual species let alone non-Mendelian or parasexual pathogens – will depart from the RD model as stated [4].

We believe that a model that ignores the shades of grey in population structure between different pathogens and pathogen populations is not fit for purpose.

It is a lovely metaphor, but it is an extremely poor summary of the data.


We stand accused of using ‘vague and subjective’ assertions in our opinion piece [4].

This is a shame, as in our review, we set out to try and define some of the increasingly impenetrable lexicon developed by Tibayrenc and Ayala to describe PCE [7, 12, 6].

Specifically, we attempted to define the meaning of the term ‘evolutionary scale’ as Tibayrenc and Ayala use it.

However, we did not explore the opacity the recently invented term ‘clonality threshold’ [13, 4] which, although pseudo-quantitative, is in fact equally as vague as a phrase we used – ‘widespread genetic exchange’ [1] which was the target of their comment in this context.

Even the word ‘predominant’ in ‘predominant clonal evolution’ is open to wide interpretation.

Another term used by Tibayrenc and Ayala that concerns us is Avises’s so-called congruence principle [4].

We can find no reference to this in the book they cite [14].

However, where the word ‘congruence’ is used in Avise 2004; it describes a means of intervalidating genetic distance measures because they give the same tree topology, not intervalidating topologies from different genetic markers because they support clonality as Tibayrenc and Ayala suggest [4].


We hope Tibayrenc and Ayala will join us in agreeing that there is no ‘one size fits all’ when it comes to making assertions about something as complex as pan-pathogen population genetic structure.

Vague language is appropriate to phenomena that are incompletely understood.

However, the use of language that implies precision where there currently is none is highly misleading.


A final concern mirrors one raised in a similar exchange earlier between Rougeron et al., and Tibayrenc and Ayala regarding Leishmania population genetics [15].

The energy and enthusiasm which Tibayrenc and Ayala have applied to their ‘clonal hypothesis’ over the years has given rise a large body of self-authored and co-authored work (including no less than eight reviews since 2012).

In their criticism of us, Tibayrenc and Ayala suggest that many of the instances of genetic exchange we cite are ‘considered by us [them] and many, if not most, authors working on pathogen population genetics as a particular case of PCE.

’ In line with Rougeron et al., we argue this consensus does not in fact exist.

There is a large (and rapidly growing) body of work around the PCE; however, it is almost all authored, co-authored or edited by Tibayrenc and Ayala.


In our review, we highlight the importance of population genomic data in providing definitive measures of population-level genetic exchange in protozoan pathogens [1].

We cited a recent study that demonstrated multiple genetic exchange events in L. infantum at a disease focus in Turkey [16].

By also citing an earlier study that used 14 microsatellite markers on the same strains but found no specific evidence of genetic exchange, we aimed to demonstrate the relatively improved power of genome-scale sequencing approaches [17].


In their comment, Tibayrenc and Ayala suggest we ‘omit to mention’ that there is a lot of clonal reproduction going on in this population.

Far from it, we clearly state the estimated frequency of meiotic events: approximately 1 per 7.5 x 10^4 mitotic events [16].

We would further highlight that the data produced by Rogers et al. 2014 were consistent with not just a single out-crossing event, but multiple later genetic exchange events among the progeny of this cross.


Population genomic studies to establish parasite mating systems should be subject to the usual caveats around study design that we discuss in our review: minimally spatio-temporally subdivided samples, no lumping together of subdivisions, etc [1].

However, if these caveats can be satisfied, the precision that accompanies the estimation of recombination rate, as well as the estimation of the likely exchange mechanism (via interpretation of the genomic distribution of break points, mosaics, aneuploidies, loss of heterozygosity, etc), will be unprecedented.


We are amazed to find that Tibayrenc and Ayala have chosen to re-iterate their views now, in advance of the majority of the data on which any clear understanding should be founded.


Evidence of genetic exchange in our view does not depend on demonstration of absolute panmixia.

Tibayrenc and Ayala insist that the predominant clonal evolution model is not incompatible with presence of recombination, or recombination of crucial impact (‘but on an evolutionary scale’) or frequent selfing. And yet, the impact of hybridisation and genetic exchange is overwhelmingly irrefutable, with multiple examples both from experimental and from natural populations, with profound and recent epidemiological impact, including the emergence of epidemic clones.

We therefore prefer an approach that is unencumbered by entrenched adherence and justification of the unnecessarily complicated and conceptually outdated ‘predominant clonal evolution’ hypothesis.


Knee osteoarthritis (OA) is a leading cause of disability, 1-4 with no approved agents proven to alter the disease course.

Joint replacement surgery remains the only definitive for end- stage disease, with knee OA accounting for 97% of all knee replacement (KR) surgeries, the numbers and costs of which are rising each year.

5 Thus, there is an urgent need for new effective treatment options for knee OA.


Bone plays an important role in OA pathophysiology and is considered an important treatment target.

Since mechanical factors play a central role in OA development, 6-8 and bone adapts readily to mechanical stresses, 9 it is essential to understand the long-term effects of modulating bone remodelling via antiresorptive agents on the course of OA.

Trials of antiresorptive agents have had conflicting results.


For bisphosphonates, trials have focused primarily on medium-term and short-term structural endpoints, such as joint-space width on radiograph and bone marrow lesions on MRI, with the majority not finding a protective effect.

10-1214 Observational studies have also focused primarily on structural changes, but their interpretation is limited by potential confounding and selection bias.

1617 Additionally, efficacy of bisphosphonates in an animal model of OA suggests that it varies based on stage of disease at which it is initiated.


While bisphosphonates may have beneficial articular cartilage effects, there are theoretical concerns about their long-term effects related to decreasing bone turnover.

Because of its high bone turnover state, OA bone may be relatively hypomineralised and weaker, and thus more easily deformed, 1920 but may also have increased stiffness overall due to increased subchondral bone thickness and volume.

21 Whether bisphosphonates could increase stiffness and interfere with bone adaptation to mechanical stresses, which could be detrimental in OA in the long term, requires clarification before further pursuit of this as an OA treatment.


The most clinically relevant long-term sequela of knee OA is end-stage disease leading to joint replacement surgery.

If bisphosphonates could reduce the risk of KR, it would suggest that these agents have a beneficial effect on the disease process, and in turn, can prevent or delay a costly surgical intervention.

On the other hand, if long-term bisphosphonate use has a negative impact on bone's mechanical properties, then this class of drugs should not be pursued for further development for OA management.

We therefore evaluated the relation of bisphosphonate use to KR surgery.


The Health Improvement Network (THIN) is a UK general practitioner (GP) electronic database that has anonymised health data on >12 million patients.

22 The information available in THIN is collected by GPs as part of routine patient care, which is deidentified and integrated into a central database for research purposes.

THIN contains information on demographic factors, referrals, hospitalisations, laboratory test results and prescriptions ordered by GPs, including the dose, strength and formulation of medication.

Diagnoses and test procedures are recorded with read codes.

Prescriptions written by GPs are recorded Ann Rheum Dis.

Author manuscript; available in PMC 2019 February 13.

automatically in the database as drug codes, with the use of a coded drug dictionary (Multilex).

Quality is checked regularly and the information from this database has been found to be representative of the UK population.


Eligible subjects of the current study consisted of women aged 50-89 years during 2003 to 2012 who were enrolled in THIN for at least 1 year without a diagnosis of knee OA prior to inclusion in the study sample.

They were also required to have at least one GP visit or prescription during the year prior to their knee OA diagnosis.


We excluded subjects who were on bisphosphonates prior to their knee OA diagnosis, on non-bisphosphonate antiresorptive agents or on bisphosphonates that are prescribed only for Paget's disease or metastatic disease in the UK (clodronate, tiludronate); those with a KR prior to a knee OA diagnosis; and those with rheumatoid arthritis.

We also excluded subjects deemed unlikely to be a candidate for KR surgery, including subjects with body mass index (BMI) >40 kg/m 2 , history of joint infection, high-risk cancer (pancreatic, oesophageal, gastric or metastatic cancer) or comorbidities with poor prognosis (end-stage renal disease on dialysis and severe pulmonary disease requiring supplemental oxygen).

In our main analyses, we also excluded individuals with incomplete covariate data for BMI, alcohol and smoking status, with sensitivity analyses conducted in which we used multiple imputation to enable inclusion of such subjects.


We conducted a sequential (ie, time-stratified) propensity score-matched cohort study to examine the relation of bisphosphonate initiation to the risk of KR among older women with incident knee OA.

We divided the study period (1 January 2003-31 December 2012) into ten 1-year cohort accrual blocks.

We propensity score-matched each bisphosphonate initiator with a non-initiator within each cohort accrual block using a greedy-matching algorithm.

23 The propensity score for initiating bisphosphonates was computed using logistic regression within each cohort accrual block.


For bisphosphonate initiators, the date of the first bisphosphonate prescription was considered the index date.

Non-initiators were assigned an index date randomly within the cohort accrual block.

The covariate assessment period for the propensity score modelling was prior to the index date.


Variables included in the propensity score model were: sociodemographic factors (age, sex, BMI, smoking status, alcohol use and socioeconomic status), knee OA characteristics (duration and analgesic use), potential indications for bisphosphonate (osteoporosis, osteopaenia and fractures (hip, wrist, vertebral, fragility and multiple fractures over time)), bone mineral density assessment, glucocorticoid use, falls and fall risk, comorbidities (see table 1), healthcare usage (GP visits and hospitalisations) and medications (see table 1).

Covariate balance was assessed with standardised mean differences, all of which were <0.1.


The first bisphosphonate prescription was identified using Multilex drug codes in THIN which have been used in prior studies.

2425 We focused on the bisphosphonates approved for use in osteoporosis in the UK, which include etidronate-calcitonin combination therapy, ibandronate, pamidronate, risedronate and zolendronate.


Trials of bisphosphonates in knee OA have not been able to examine the risk of KR surgery due to the longer duration of follow-up required to examine this clinically relevant endpoint.

Interestingly, though the clinical scenario and mechanisms are distinct from that of OA, bisphosphonates have been demonstrated to reduce the risk of revision surgery after joint replacement, potentially through antiresorptive effects minimising periprosthetic bone loss and osteolysis.

30 Nonetheless, while there are potentially beneficial effects of bisphosphonates and other bone-modulating agents on bone and cartilage, 38 study results in OA have been conflicting to date.


We previously demonstrated in a spinal OA trial that alendronate reduced spinal osteophyte and disc space narrowing progression, 39 and an observational study also noted less bone pathology on knee imaging by MRI (bone marrow lesions and subchondral bone attrition) and knee pain.

16 A trial of risedronate with 1-year follow-up suggested symptom benefits, and minimal non-significant structure benefits based on joint-space width on radiograph. 10 However, a subsequent larger trial with 2-year follow- up failed to demonstrate any symptom or structure (joint-space width) benefits, 11 and a small subset (<5%) of this trial that had MRIs demonstrated no difference in bone marrow lesions between treatment and placebo.

12 Analysis of prevalent bisphosphonate users in the Osteoarthritis Initiative (OAI) cohort demonstrated no significant difference in joint-space width versus non-users 17 ; this may be partly related to the prevalent drug user study design used and analytic approach.

40 More recently, a trial evaluating intravenous zoledronic acid demonstrated significant differences in bone marrow lesion volume and pain between treatment and placebo arms at 6 months, but not at the 12-month follow-up.

14 Taken together, these studies have raised questions about whether potential benefits are being missed by use of relatively insensitive radiographs.


Additionally, another concern is whether there are potential adverse longer term effects of bisphosphonates on OA pathology, such as may arise from inhibiting bone turnover, which may in turn lead to increased bone stiffness due to increased mineralisation and subsequent theoretical adverse biomechanical consequences.


Our findings indicate that bisphosphonates do not appear to have long-term adverse effects on the course of knee OA, and may in fact reduce the risk of KR surgery, suggesting that the clinical course may be less severe and/or have a slower rate of progression.

In the OAI, bisphosphonate use was associated with a slower trajectory of joint-space width loss and of MRI-based three-dimensional bone shape change, an imaging biomarker predictive of OA onset.

4142 Thus, there does not appear to be a reason for bisphosphonates to be avoided in those with knee OA, and in fact, their use may be beneficial, supporting the ongoing investigation of bone modulation as an OA therapeutic target.


Potential limitations of this study deserve comment.

Because THIN does not have bone density results, there is a concern for residual confounding by indication since individuals with lower bone mineral density are more likely to be on a bisphosphonate and are also more likely to have a lower BMI.

We attempted to address this by including factors in the propensity score that are likely reflective of bone mineral density and risk of osteoporosis.

The diagnostic code for osteoporosis and osteopaenia were not as frequently recorded as one would expect for individuals on bisphosphonates, likely reflecting the fact that GPs do not always record the condition for which bisphosphonates are being prescribed.

In a manual review of patient profiles, we noted that all patients on bisphosphonates without an Ann Rheum Dis. Author manuscript; available in PMC 2019 February 13.

osteoporosis code (including those who had an osteopaenia code) were universally on full- strength doses of a bisphosphonate, usually with ≥1 bone mineral density test and/or ≥1 fracture suspicious for an osteoporotic fracture.


Further, the code for osteoporosis and/or osteopaenia often occurred well after initial bisphosphonate prescription.

Additionally, existing published literature provides some reassurance that bone mineral density status is unlikely to importantly affect our study results.

Low systemic bone mineral density has either been associated with increased risk of knee OA progression or no increased risk.

43-46 Based on such data, one would expect bisphosphonate initiators, who presumably may have lower bone mineral density, to have potentially either no or increased risk of knee OA progression.

In contrast, we found that bisphosphonate initiators experienced a protective effect even while accounting for BMI.

Additionally, vitamin D, which is commonly taken as over-the-counter medication without prescription, particularly in multivitamins, is not reliably recorded within THIN in a standardised fashion, and was low but well balanced between the two groups in our study sample.

Nonetheless, their use is unlikely to importantly confound our results given the lack of association of vitamin D with knee OA in two recent rigorously conducted clinical trials.

4748 Though effects on KR cannot be determined from these trials, some studies have suggested that joint-space width may be predictive of KR surgery.


We also examined the relation of incident bisphosphonate use to hip fracture to determine whether we could replicate the findings from existing published data; such a replication would suggest that the propensity score model likely adequately captured most important confounders.

In this analysis, we found the HR to be similar to the effect noted for clinical fractures in the Fracture Intervention Trial.

49 Finally, we performed a sensitivity analysis to address the degree of unmeasured confounding that would have to be present to nullify our results.

For bone mineral density and/or vitamin D to result in a fivefold alteration in the risk of KR while also having a prevalence differential of 30% between the two groups is highly unlikely.

Thus, at minimum, it appears that bisphosphonates do not have a detrimental effect and may indeed have a potential beneficial effect.


Secular trends precluded robust active user comparator analyses, particularly as many women initiated oestrogens prior to knee OA diagnosis.

Further, these results are primarily of relevance to older women; whether there should be different biological effects in middle- aged women or in men is not clear, but cannot be extrapolated from this study.

Our approach to addressing this clinically important question also


Subjects were followed from the index date until KR, death, loss-to-follow-up (e.g., transfer out of GP practice), age >90 years, last date of data collection by GP or end of study (31 December 2013).

To examine the relation of incident bisphosphonate use to KR surgery, we conducted Cox proportional hazards regression using age as the time scale with an intention- to-treat approach, additionally adjusting for the covariates used in the propensity score model.


To explore the possibility of residual confounding, we performed additional sensitivity analyses.

In the first, we re-ran the above analyses without including BMI in the propensity score to determine indirectly how much of an influence BMI has on the effect estimate.

We also did not include alcohol or smoking so that our model was not limited to those with complete data.

We also performed multiple imputation by imputing missing BMI, alcohol and smoking data in five blocked datasets that were each used to create propensity score- matched datasets that were analysed separately with a summary effect estimate calculated.


Second, we conducted age-matched, BMI-matched and OA duration-matched analyses, in which bisphosphonate initiators were matched 1:4 with non-initiators, adjusting for all other potential confounders used in the propensity score model.

Third, this latter analysis was repeated using active comparator drugs as the referent group: (1) oestrogen initiators; (2) initiation of any other bone-modulating agent (eg, strontium, calcitonin, raloxifene, teriparatide and oestrogen).

Fourth, to account for potential selection bias due to death or loss to follow-up (eg, individuals transferring out of their GP practice or other issue), we applied inverse probability weighting.

3435 To account for competing risk of death, we implemented the Fine and Grey approach. 36 Finally, to assess the influence of unmeasured and residual confounding factors, such as bone mineral density, we performed a sensitivity analysis using an array approach, which is helpful for exploring the effect of residual confounding over a wide range of parameter values and effects, and to examine the extent of confounding necessary to fully explain the observed effect estimate.

37 All statistical analyses were performed using SAS V.9.3 (SAS Institute, Cary, North Carolina, USA).


From among 27 295 subjects who met our inclusion and exclusion criteria, we identified 2225 bisphosphonate initiators.

From these, 2006 bisphosphonate initiators were propensity- score matched to the same number of non-initiators.

Eighty-four per cent of the bisphosphonate initiators were prescribed alendronate; the remaining were risedronate (13%), ibandronate (2%) and etidronate-calcitonin combination therapy (1%).

The mean age of the cohort was 76, and the mean BMI was 27.3 kg/m 2 .

Overall, covariate data were well-balanced among the initiators and non-initiators (table 1).

Approximately 60% of the initiators and the non-initiators appeared to have an indication for bisphosphonate use prior Ann Rheum Dis.

Author manuscript; available in PMC 2019 February 13.

Author Manuscript to the index date; however, this proportion increased to 82% among the initiators, and remained fairly steady among the non-initiators (63%) over the follow-up period.


The mean follow-up time for subjects was approximately 3 years for both groups (table 2).

During the follow-up period, 208 KR surgeries were performed overall, with 138 in bisphosphonate initiators and 170 in the non-initiators.

The mortality rate was similar in both groups (39.7 and 38.2 per 1000 person-years in the initiators and non-initiators, respectively).

The crude incidence rate of KR was 22.0 per 1000 person-years among the bisphosphonate initiators and 29.1 per 1000 person-years among the comparator group, with a crude incidence rate ratio of 0.75 (95% CI 0.60 to 0.94) (table 2).

The HR for KR related to bisphosphonate initiation was 0.74 (95% CI 0.59 to 0.93), and results did not change materially when additionally adjusted for the potential confounders used in the propensity score (adjusted HR 0.76 (95% CI 0.60 to 0.95)).


Because approximately 15% of subjects had missing values for either BMI, smoking and/or alcohol, we performed a sensitivity analysis in which we created the propensity score without including these variables.

In this analysis, with 2379 initiators of bisphosphonates and the same number of propensity score-matched non-initiators, the adjusted HR was similar to the main result (0.79, 95% CI 0.64 to 0.98).

In analyses using multiple imputation, the adjusted HR was also similar to the main result (0.72, 95% CI 0.59 to 0.89).

We also conducted a sensitivity analysis in which we matched each bisphosphonate initiator to four non-initiators by age (within 2 years), BMI (±0.5 kg/m 2 ) and OA duration (within 2 years).

The adjusted HR from this matched cohort study was 0.35 (95% CI 0.26 to 0.47).

To further address confounding by indication, we compared bisphosphonate initiation to that of an active comparator drug (oestrogen and any bone-modulating agent, respectively), matched on age, BMI and OA duration, and obtained results similar to our main results (adjusted HR 0.75 (95% CI 0.30 to 1.87).

The smaller sample sizes and lower number of KR outcomes precluded precise estimation of these effects.


When we accounted for censoring due to loss to follow-up or death with inverse probability weighting, the results remained similar to the main results (HR 0.75, 95% CI: 0.59 to 0.94).

The competing risk of death analysis also provided similar results (HR 0.76, 95% CI 0.61 to 0.86).

Finally, in a sensitivity analysis to examine the potential effect of residual confounding on our results, we found that in order for an unmeasured confounder to nullify the observed HR, that confounder would need to be associated with a fivefold difference in risk of the outcome (KR) and have a 30% difference in prevalence among bisphosphonate initiators versus non-initiators.

Such a scenario would be highly unlikely (figure 1).


In this large cohort of older women with incident knee OA derived from the general population, we found that bisphosphonate initiation was associated with a ~25% lower risk of KR surgery than those who did not initiate bisphosphonates.

Our findings were consistent across several sensitivity analyses that were performed to address potential residual confounding, missing data and selection bias, suggesting that it is unlikely for unaddressed biases to result in bisphosphonate initiation to have a detrimental effect.


With great interest, we read the recent article by Neogi et al entitled, ‘Effect of bisphosphonates on knee replacement (KR) surgery’ [1].

In the study, the authors concluded that in this population-based cohort of older women with incident knee osteoarthritis (OA), those with incident bisphosphonate users had lower risk of KR than non-users of bisphosphonates, which was further supported by another large cohort study [2].

The strengths of this study include a propensity score-matched cohort design, Cox proportional hazards regression to control for potential confounders and sensitivity analyses focused on residual confounding.

Meanwhile, the authors acknowledged the limitations of their work.

We applaud and congratulate their important work for clinical practice.

However, several important points should be further discussed.


Generally, determinants of patient preferences for KR are OA severity, the level of knee pain, disability, and the weakness of quality of life, and more importantly, their willingness and access to KR.

In the current study, authors tended to agree that reduced risk of KR has resulted from weakened OA progression after bisphosphonates use.

However, recent high-level evidence indicated that bisphosphonates neither provide pain relief and function improvement nor radiographic progression in knee OA [3][4].

Therefore, we wonder that bisphosphonates may affect the willingness and access to KR of patients with OA, thus reducing the risk of KR.


Actually, many clinical and sociocultural factors, such as racial differences (African Americans and whites), social support and an educational intervention included a decision aid, have vital important influence on patient preferences for KR [5][6][7].

More importantly, a recent study revealed that patient preferences for KR were strongly associated with knee pain severity in patients with OA with health insurance, but their inverse relationship disappeared in patients without health insurance [8].

Also, another study suggested that despite worse baseline knee pain and function, black participants had much lower adjusted risk of having total KR (TKR) than white participants [9].

Thus, these indicated that sociocultural factors, but not OA severity, may have more significant influence on the willingness and access to KR.


However, Neogi and colleagues seemed to ignore these important sociocultural factors in Cox proportional hazards regression, propensity score model and sensitivity analyses.

We strongly recommend additional analyses based on aforementioned risk factors, which may further increase the robustness and credibility of the current study.


Additionally, the authors assessed the influence of bisphosphonates use on the risk of KR in patients with OA.

However, there were no any data involving the baseline characteristics of OA severity, the level of knee pain, disability and the weakness of quality of life between both cohorts.

Moreover, the influence of the dose, route, time point or measuring instrument of bisphosphonates administration on the risk of KR was unclear, so further subgroups should be warranted.

Meanwhile, the primary outcome (the risk of KR) may be involved in many similar terms, such as TKR, revision KR, hemi-KR or KR in both knees.

As with us, some readers may be confused about the notion.

Therefore, the primary outcome should be clearly defined.


To sum up, we respect the great work done by the authors, but the study should be interpreted with the aforementioned limitations and further analyses should be performed.


We thank Dr Li and colleagues [1] for their interest in our paper.

As outlined in our paper [2], we used data from a large general practitioner electronic health records database for our study.

These data are collected and recorded as part of routine clinical care with diagnostic codes.

As such, factors such as willingness to undergo and access to knee replacement (KR), knee pain severity, disability, quality of life and others are not available in this database, and we were thus unable to examine these as potential confounders.

We were, however, able to account for socioeconomic status (Townsend Deprivation Index), and performed sensitivity analyses demonstrating that substantial residual confounding is unlikely.


The assessment of bisphosphonate exposure was based on prescription data, and alendronate was the most common bisphosphonate prescribed (84%), as described in the paper [2].

The potential mechanisms by which bisphosphonates may confer the noted effects were beyond the scope of this paper.

Nonetheless, it is not clear that bisphosphonate initiation would alter one’s willingness to undergo KR or access to KR such that a protective effect would be noted in our study.


We included both total and unicompartmental primary knee arthroplasty since either surgery would indicate substantially symptomatic knee osteoarthritis, but did not include revision surgery since the reasons for that procedure are not for severe/end-stage osteoarthritis; this is the same approach used by the other studies we had cited on this matter.

As per standard time-to-event analyses, subjects were censored at the first KR, among the other censoring factors outlined in the paper.


Animal and human studies have demonstrated that water drinking may elicit a pressor response (May & Jordan, 2011).

Jordan, Shannon, Grogan, Biaggioni, and Robertson (1999) were the first to demonstrate a potent pressor response (increase of up to 33 mmHg in systolic blood pressure) after water intake in patients with severe orthostatic hypotension (i.e. patients with autonomic failure).

Thereafter, they observed that the magnitude of the pressor response was attenuated in healthy elderly subjects and completely abolished in young healthy subjects after water intake (Jordan et al., 1999, 2000; Schroeder et al., 2002; Scott, Greenwood, Gilbey, Stoker, & Mary, 2001).


The reason for the blunted pressor response after water drinking in healthy young c 2018 The Authors.

Experimental Physiology c 2018 The Physiological Society subjects is not fully understood, but previous authors suggested that the arterial baroreflex might play a role.

This idea is supported by animal experiments, in which mice with intact baroreflexes did not respond to water intake (McHugh et al., 2010).

In contrast, water elicited a substantial increase in arterial blood pressure after inter- ruption of the afferent baroreflex arc by sinoaortic denervation (McHugh et al., 2010).


In humans, water drinking increases efferent sympathetic outflow in the absence of a pressor response in young healthy subjects (Scott et al., 2001), suggesting that baroreflex function is preserved; however, to our knowledge, this has not been tested directly.


In contrast, in patients with autonomic failure and in hypertensive and healthy older adults, water intake induces sympathoexcitation, 1318 wileyonlinelibrary.com/journal/eph Experimental Physiology.

2018;103:1318-1325. 1319 which is accompanied by a significant pressor response suggestive of impaired baroreflex function (Callegaro et al., 2007; Cariga & Mathias, 2001; Jordan et al., 1999, 2000).


Interestingly, previous studies have demonstrated that the central mechanism of osmosensation and systemic osmoregulation involves neurons of the central baroreflex regulatory regions (i.e. periaqueductal grey; Bourque, 2008).

Indeed, stimulation of the ventrolateral periaqueductal grey enhanced sympathetic baroreflex sensitivity in humans (Sverrisdóttir et al., 2014).

However, the extent to which an alteration in arterial baroreflex function may contribute to arterial blood pressure responses to water intake in healthy humans remains unclear.


Therefore, the present study was undertaken to test the hypothesis that water drinking increases the arterial baroreflex control of muscle sympathetic nerve activity (MSNA) in young healthy subjects.


To test this hypothesis, MSNA, arterial blood pressure and heart rate were continuously recorded before, during and for 60 min after ingestion of water or saline, and spontaneous arterial baroreflex-MSNA gain was calculated.


We chose 60 min of data collection based on previous studies indicating that the peak MSNA response occurs ∼30 min after water ingestion in young healthy subjects (Scott et al., 2001).


The study was approved by the Fluminense Federal University ethics committee (CAAE: 53503816.0.0000.5243).

Written informed consent was obtained from all participants, and all experimental procedures conformed to the standards set by the Declaration of Helsinki, except for registration in a database.


Ten healthy and recreationally active male volunteers with a mean (±SD) age of 24 ± 2 years, body weight of 77 ± 8 kg and height of 177 ± 6 cm participated in this study.

Subjects had to be asymptomatic, non-smokers, normotensive and non-diabetic.

None of the subjects was receiving prescribed or over-the-counter medications, and all were confirmed to have no heart disease.

The subjects were recruited through posters placed on the university campus.

Subjects were requested to abstain from caffeinated beverages for 12 h and from strenuous physical activity and alcohol for a minimum of 24 h before any experimental sessions.

Subjects were a minimum 2 h postprandial upon arrival to the laboratory.


Subjects were studied in the supine position at an ambient room temperature of ∼24 • C. Arterial blood pressure and heart rate were measured on a continuous beat-to-beat basis using finger photoplethysmography (Finometer Pro; Finapres Medical Systems, Arnhem, The Netherlands) and a single-lead ECG (PowerLab 4/25T and Laboratory Chart Pro 7 software, AD Instruments, Castle Hill, NSW, Australia), respectively.

Brachial arterial blood pressure


• What is the central question of this study?

Water drinking increases muscle sympathetic nerve activity (MSNA), and it increases arterial blood pressure (ABP) in older populations but not in young healthy subjects.

Does an increase in gain of arterial baroreflex control of MSNA contribute to maintenance of ABP after water drinking in healthy young subjects?


The gain of arterial baroreflex control of MSNA was increased and remained elevated 60 min after water drinking (500 ml) but remained unchanged after saline intake.

An enhancement in gain of arterial baroreflex control of MSNA contributes to the maintenance of ABP after water drinking in young healthy subjects, probably via osmosensitive mechanisms.


of the dominant arm was measured with an automated digital sphygmomanometer (EM-759-E; Omron, Kyoto, Japan) to validate Finometer measurements of absolute blood pressure in real time during data collection.

Respiratory movements were monitored using a strain-gauge pneumograph placed in a stable position around the abdomen (MLT1132 respiratory belt; ADInstruments).


Multiunit recordings of postganglionic MSNA were obtained by inserting a unipolar tungsten microelectrode percutaneously through the intact skin and positioning it into muscle nerve fascicles of the peroneal nerve of the right leg at the fibular head.

A reference electrode was placed subcutaneously 2-3 cm distal.

The recording electrode was adjusted until a site was found where nerve activity displayed a pulse synchronous pattern of spontaneous bursts, had a signal-to-noise ratio of 3:1, was increased during an end-expiratory breath-hold or Valsalva manoeuvre and was unresponsive to an unexpected loud noise or skin stroking (Vallbo, Hagbarth, Torebjörk, & Wallin, 1979).

The raw signal was amplified (×100,000), filtered (bandwidth 700-2000 Hz), rectified and integrated (time constant 0.1 s) to obtain a mean voltage neurogram (Iowa Bioengineering, Iowa City, IA, USA).

Sympathetic bursts were identified using a fully automated program (Hamner & Taylor, 2001); burst identification was then corrected by inspection by a single blinded observer (L.C.V.).

The MSNA was quantified as burst incidence (in bursts per 100 heart beats) and burst frequency (in bursts per minute).

In one of the 10 participants, we were able to obtain a quality MSNA signal only during the saline protocol.


After instrumentation, participants rested for ∼15 min to permit stabilization of all cardiovascular variables.

Thereafter, a 10 min base- line was performed, in which the subject rested quietly while all neurocardiovascular variables were continuously recorded.

The sub- jects then ingested 500 ml of bottled water (n = 9, pH 7.5; [Na + ], 1.5 mmol l −1 ; [Ca 2+ ], 3.1 mmol l −1 ) in <5 min.


The MSNA, arterial blood 1469445x, 2018, 10, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/EP087095 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 1320 VIANNA ET AL.

F I G U R E 1 Original records illustrating muscle sympathetic nerve activity (MSNA, top panel) and arterial blood pressure (bottom panel) at baseline (a) and at 30 min after the ingestion of 500 ml of bottled water (b) pressure, heart rate and respiration were measured for 60 min after the completion of drinking.


In a subset of subjects (n = 6), a control visit was performed, in which 500 ml of saline was ingested and all experimental measures were performed in an identical manner to the water drinking visit.


The temperature of the water and saline was controlled at 22 • C.


The participants were advised to ingest 500 ml of water 2 h before the experiments to start the study protocol with a suitable hydration status (Teixeira, Ramos, Marins, & Ricardo, 2015; Vianna, Oliveira, Silva, Ricardo, & Araujo, 2008) and were asked to void their bladder immediately before the experiments (Fagius & Karhuvaara, 1989).


In addition, the participants' sensation of bladder filling was reported at the end of each experiment on a 0-10 scale, with zero representing a complete lack of this sensation and 10 representing an irresistible desire to void (Fagius & Karhuvaara, 1989).


Baseline MSNA, arterial blood pressure and heart rate were calculated as mean values over a 5 min period.

After consumption of water or saline, 5 min averages were calculated every 15 min.

The same segments were used to assess arterial baroreflex control of MSNA by analysing the relationship between spontaneously occurring fluctuations in diastolic blood pressure and MSNA, as previously described (Fisher et al., 2015; Kienbaum, Karlssonn, Sverrisdóttir, Elam, & Wallin, 2001; Prodel, Barbosa, Mansur, Nobrega, & Vianna, 2017; Sundlöf & Wallin, 1978; Vianna et al., 2012).

Briefly, the diastolic blood pressure for each cardiac cycle within a data collection period was grouped into 3 mmHg pressure bins.

The slope of the relationship between MSNA burst incidence and diastolic blood pressure was identified using linear regression analysis, with a minimum r value of −0.5 used as the criterion for accepting slopes (Kienbaum et al., 2001; Sundlöf & Wallin, 1978; Young, Deo, Chaudhary, Thyfault, & Fadel, 2010).

All data were weighted to account for the number of cardiac cycles within each pressure bin, thus removing bias attributable to bins containing a small number of cardiac cycles.


In addition, spontaneous cardiac baroreflex sensitivity (cBRS) was assessed using the sequence technique as previously described (Holwerda et al., 2016; Parati, Di Rienzo, & Mancia, 2000; Teixeira et al., 2018a).

The sequence technique is based on the identification of consecutive beats in which progressive increases (or decreases) in systolic blood pressure (input variable) are followed by a progressive lengthening (or shortening) in RR interval (output variable).

Briefly, sequences of three or more consecutive and concurrent beats with corresponding increases or decreases in systolic blood pressure and RR interval were identified as arterial baroreflex sequences (Cardio- Series v2.4, Brazil).

Sequences of at least three consecutive cardiac cycles were detected only when the variations in systolic blood pressure and RR interval were ≥1 mmHg and ≥1.0 ms, respectively.


A linear regression was applied to each individual sequence, and only those sequences in which r was >0.85 were accepted (Kardos et al., 2001; Zollei, Paprika, & Rudas, 2003).

The slopes of the systolic blood pressure and RR interval relationships for up and down sequences were then calculated and averaged for a measure of spontaneous cBRS.


Comparisons were made using univariate repeated-measures ANOVAs, and significant main effects were evaluated with LSD post hoc analyses when appropriate.

Statistical significance was set at P < 0.05, and values are presented as means ± SD.


Figure 1 shows an original recording of one representative sub- ject, illustrating the MSNA and arterial blood pressure responses at 1321 TA B L E 1 Muscle sympathetic nerve activity (MSNA) and cardiovascular responses to water and saline drinking baseline and at 30 min after water drinking.

Group summary data of all neurocardiovascular variables at rest and at each time point after water drinking are presented in Table 1.

After water drinking, MSNA burst frequency and burst incidence were increased within 15 min and remained elevated above baseline for the remainder of the protocol (P < 0.05), whereas mean blood pressure and heart rate remained unchanged (P > 0.05).

In comparison, after saline drinking, all neurocardiovascular variables remained unchanged from baseline throughout the protocol (all P > 0.05).

Figure 2 illustrates the changes (∆) from baseline in MSNA burst frequency (Figure 2a), MSNA burst incidence (Figure 2b) and mean blood pressure (Figure 2c) for the 60 min period after ingestion of either bottled water (grey bars) or saline (open bars).


The arterial baroreflex gain of MSNA burst incidence was significantly enhanced (i.e. more negative) within 15 min [∆−1.04 ± 1.05 (95% confidence interval −1.84, −0.23) bursts (100 heart beats) −1 mmHg −1 ] and remained elevated for the duration of the study, reaching a peak during the 30th minute [∆−2.07 ± 0.98 (95% confidence interval −2.82, −1.32) bursts (100 heart beats) −1 mmHg −1 ] after water drinking (Figure 3, grey bars).

In contrast, after saline drinking, the gain of arterial baroreflex bursts incidence was not different from baseline at any time point (Figure 3, open bars).


The spontaneous relationship between systolic blood pressure and RR interval remained unchanged after water intake (baseline, 22.2 ± 11.9 ms mmHg −1 ; 30 min, 23.6 ± 12.4 ms mmHg −1 ; and 60 min, 26.0 ± 12.8 ms mmHg −1 ; P > 0.05) and during the saline drinking (base- line, 21.6 ± 6.6 ms mmHg −1 ; 30 min, 22.8 ± 6.9 ms mmHg −1 ; and 60 min, 25.0 ± 8.0 ms mmHg −1 ; P > 0.05).

Similar results were found


The main new finding of the present study is that water drinking enhanced the gain of the arterial baroreflex control of MSNA in young healthy men.

Indeed, the slope of the relationship between diastolic blood pressure and MSNA was increased 15 min after ingestion of water and remained elevated throughout 60 min.

Furthermore, mean arterial blood pressure, heart rate and cBRS were unchanged after water intake.

In comparison, after ingestion of saline, all neuro- cardiovascular variables were unchanged.

Collectively, these findings support the concept that an enhancement in the gain of the arterial baroreflex control of MSNA contributes to the maintenance of arterial blood pressure after water drinking in young healthy subjects and that these responses are likely to be mediated by osmosensitive mechanisms.


The sympathoexcitatory effects of water drinking are well established in both animals (McHugh et al., 2010) and humans (Callegaro et al., 2007; Scott et al., 2001).

Previous reports have demonstrated that water-induced increases in sympathetic outflow were accompanied by significant elevations in arterial blood pressure in patients with autonomic failure and elderly subjects but not in young healthy subjects (Cariga & Mathias, 2001; Jordan et al., 1999, 2000).


The findings of the present study advance our understanding of how healthy young subjects are able to buffer the increases in arterial blood pressure after water drinking.

For the first time, we demonstrate that an enhancement in the gain of arterial baroreflex control of MSNA may counteract the effects of water-induced increases in sympathetic outflow to maintain arterial blood pressure.


In line with this, previous animal reports have shown that mice with intact baroreflexes did not respond to water intake, whereas water elicited a substantial increase in arterial blood pressure after interruption of the arterial baroreflex arc via sinoaortic denervation (McHugh et al., 2010).

In addition, it has been shown that patients with autonomic failure present impaired arterial baroreflex control compared with healthy counterparts (Biaggioni, 2017).

Taken together, these findings suggest that intact arterial baroreflex function is essential for the maintenance of blood pressure after water drinking.


The precise pathways mediating the improvement in arterial baroreflex-MSNA gain after water intake remain unclear, but some relevant points warrant discussion.


The pressor response to water drinking may involve osmosensitive transient receptor potential vanniloid 4 (Trpv4).

Water absorption induces a hyposmolar stimulus in the portal circulation, probably involving activation of Trpv4 (Lechner et al., 2011; McHugh et al., 2010), which triggers a sympathetic reflex (Jordan et al., 2000) and may increase arterial blood pressure.


Indeed, duodenal infusion of water [25 l (g body weight) −1 ] decreases portal osmolality in both wild-type and Trpv4 −/− mice (all had undergone sino- aortic denervation), but only wild-type mice demonstrated increases in arterial blood pressure (McHugh et al., 2010).


Collectively, these data suggest that the pressor response induced by water ingestion involves sympathetic activation and that the Trpv4 channel is an important mediator of the afferent input of the response (McHugh et al., 2010).


In addition to the well-described sympathoexitatory effects of water intake, the present finding illustrates the impact of water drinking on the arterial baroreflex.


The central mechanisms of osmosensation and systemic osmoregulation are not fully understood, but we can speculate that the hepatic portal osmoreceptors signal hyposmotic stimuli via the splenic nerve activating different brain regions, including the peri- aqueductal grey (Bourque, 2008).

Indeed, stimulation of the ventro- lateral periaqueductal grey has been shown to enhance sympathetic baroreflex sensitivity in humans (Sverrisdóttir et al., 2014); however, whether this osmoregulatory circuit plays a role in water intake- induced elevations in arterial baroreflex-MSNA gain requires further investigation.

The osmopressor response to water is apparent in patients with autonomic failure and elderly subjects but not in young healthy populations.

We reasoned that in young healthy subjects, the pressor response to water was not observed owing the fact that osmoreceptor- mediated increases in MSNA were counteracted by an enhancement in the gain of arterial baroreflex control of MSNA.

Indeed, we found that the arterial baroreflex-MSNA gain was increased 15 min after water intake and remained elevated for the remainder of the protocol.

Inter- estingly, we did not find any changes in cBRS after water ingestion.


The reason for these differential findings are unclear, but might be associated with greater changes in sympathetic outflow compared with vagal outflow after water drinking, the latter being more representative of spontaneous cBRS.

Nevertheless, there are several reports in the literature indicating differential changes in cardiac and MSNA baroreflex control after a variety of interventions (Young et al., 2010), and a lack of relationship between cardiac and sympathetic baroreflex sensitivity has been reported in healthy humans (Dutoit et al., 2010; Taylor et al., 2015).


In addition, it is important to note that the methods used to measure cardiac and sympathetic baroreflex assess different components of the arterial baroreflex system.

More specifically, baroreflex-MSNA gain does not take into account the end-organ response, whereas the cBRS gain includes the end-organ response (Parati et al., 2000).


In this sense, further investigation is warranted to clarify the mechanisms by which water drinking differentially affects sympathetic and cardiac baroreflex sensitivity.


In the present study, we used spontaneous baroreflex measures derived from MSNA burst incidence and not total MSNA.

The rationale for this approach was based on previous studies demonstrating that the gain of arterial baroreflex control of MSNA burst incidence was similar to sympathetic baroreflex sensitivity obtained using pharmacological manipulations of arterial blood pressure (Dutoit et al., 2010; van Schelven, Karemaker, Blankestijn, & Oey, 2008).


In contrast, spontaneous arterial baroreflex gains calculated from measurements using total MSNA have been suggested to be influenced more by non- baroreflex inputs (Dutoit et al., 2010; Kienbaum et al., 2001), which might limit the interpretation of such measures.


Indeed, several studies have reported weak relationships between burst area and diastolic blood pressure, whereas burst incidence consistently demonstrates strong relationships with diastolic blood pressure (Dutoit et al., 2010; Keller et al., 2004; Kienbaum et al., 2001; Ogoh, Fisher, Raven, & Fadel, 2007; Rudas et al., 1999).


It is important to note the potential limitations of the present study.

We included only men and, given existing sex differences in blood pressure regulation (Hart & Charkoudian, 2014; Joyner, Wallin, & Charkoudian, 2016; Kim et al., 2011; Teixeira et al., 2018b), our results may not be generalizable to women.

In addition, the arterial baroreflex control of MSNA was calculated based on a closed-loop measure using spontaneous fluctuations in MSNA and blood pressure only.


We recognize that open-loop assessments of arterial baroreflex gain using perturbational methods, such as the infusion of vaso- active drugs (i.e. modified Oxford), allow for a more comprehensive examination of arterial barorefex control of MSNA.

Nevertheless, the slope of the relationship between MSNA and diastolic blood pressure derived from the closed-loop spontaneous analysis method has been shown to be correlated with the slopes derived using the open-loop modified Oxford approach for assessing arterial baroreflex-MSNA gain (Dutoit et al., 2010).


Furthermore, important to our study design, we were interested in making arterial baroreflex measurements at multiple time points to characterize the response after water ingestion fully, and repeating the modified Oxford technique at baseline and then every 15 min for 60 min was not feasible, particularly as it is standard to perform at least two modified Oxford trials at each time point (Holwerda et al., 2016; Rudas et al., 1999).


In summary, the present study demonstrates, for the first time, that bottled water, but not saline intake, enhances the gain of arterial baroreflex control of MSNA in young healthy men.

These data support the concept that the lack of a pressor response after water ingestion in young healthy subjects may be attributed to water-induced increases in the gain of arterial baroreflex control of MSNA.


We read with great interest the recent study by Vianna et al. ( 2018), who should be congratulated for their novel and enlightening findings concerning the acute effect of water ingestion on arterial baroreflex function of healthy young individuals.

Aiming to provide some additional information for better understanding and extrapolation of these findings to broader populations and conditions, we would like to underscore a few methodological and technical issues, mainly regarding the estimation of baroreflex sensitivity (BRS).


One crucial parameter, especially when assessing haemodynamic and baroreflex parameters and their responses to various interventions, is the body position [1], [2].

Subjects in the sitting, supine or other positions may exert different blood pressure (BP) and heart rate values, even without any intervention.

Furthermore, BRS changes between different body positions are unclear, because the existing published data are controversial.

Thus, it would be relevant to know whether the findings of this study remain the same for subjects who ingest water in postural positions other than supine.

More specifically, the subjects in this study ingested 500 ml of bottled water probably in the sitting position, but all measurements were performed in the supine position.

It might be meaningful to explore the same BRS and BP changes after water ingestion in the sitting or standing positions, which might be more indicative to daily habits.