Capsular perforation can be characterized as threatened perforation, covered perforation, free perforation, or sub- trigonal perforation.

2,4 Threatened perforation is described as an area of diverging capsular fibers through which peripro- static fat might be visible.

A covered perforation is similar to a threatened perforation but differs in degree with fatty tissue freely visible at the site of perforation, however, firmly covering the perforated hole.

A free perforation is a perfo- ration in the capsule through which irrigation fluid can be seen to run in and out, with little to no periprostatic fat ob- served at the margins of the wound.

In a subtrigonal perfo- ration, a false passage is created during insertion of the resectoscope or catheter, penetrating through the prostate and prostatic capsule into the direction of the trigone.

This is likely to occur in large prostates especially with large median lobes.

Threatened and covered perforations do not change the postoperative course of the patient, whereas free or sub- trigonal perforations may lead to significant extravasation of irrigation fluid into the extraperitoneal space.

2 Bladder injury during morcellation is another potential perioperative complication of HoLEP, with injury rates re- ported in 0.5% to 18.2% of cases.

1,2 If unrecognized, large full-thickness injuries can allow extravasation of irrigation fluid into the extraperitoneal or intraperitoneal space.

The extravasation of irrigation fluid can mimic the presentation of a free capsular perforation with fluid extravasation.

Although it has not been reported, theoretically there is also the risk of bowel injury with a full thickness injury to the bladder.

If there is suspicion for bladder injury, the bladder should be examined.

Any injuries should either be repaired or managed conservatively with an indwelling catheter.

Bladder injuries can be avoided by achieving optimal hemostasis and bladder distention before starting morcellation.

Morcellation should take place away from the bladder mucosa.


As these cases demonstrate, a capsular perforation can mi- mic the presentation of a bladder injury during morcellation.

If there is any suspicion of capsular perforation or bladder injury, an abdominal examination should be performed in- traoperatively.

Peak airway pressures may be increased or there may be a mismatch in irrigant inflow and outflow.

With high suspicion of injury, both cystoscopic inspection of the bladder urothelium and additional imaging such as a cystogram or CT cystogram are indicated.

If recognized correctly, the patient can be spared the morbidity of an exploratory lapa- rotomy or laparoscopy and instead be treated conservatively with Lasix and bladder drainage through a urethral catheter.

Patients who are clinically asymptomatic with normal vital signs can be discharged, generally on POD 1.

Repeat imaging to ensure there is no bladder injury can be obtained on an outpatient basis.


A rare complication of HoLEP is prostatic capsular per- foration leading to large extravasation of irrigation fluid into the retroperitoneum, resulting in a distended abdomen.

This presentation may be similar to that of a bladder injury, how- ever, if there is low suspicion of a bladder injury, conservative measures can be taken for effective treatment.


No competing financial interests exist.


Holmium laser enucleation of the prostate (HoLEP) is a well-established treatment option for benign prostatic hyperplasia (BPH) that has been gaining popularity in the United States.

HoLEP is a minimally invasive procedure that involves enucleation of the prostate with subsequent morcellation and serves as an alternative to transurethral resection of the prostate (TURP), which is still considered the gold standard of treatment in many institutions and practices.


The literature demonstrates that HoLEP provides improvement of International Prostate Symptom Score, quality of life, flow (Qmax), and post-void residuals [1].

It has also shown to have decreased or at least similar complication rates, such as bleeding and need for catheterization, compared with a TURP [1][2].

In addition, HoLEP allows for the opportunity of increased removal of prostatic tissue.

These findings have made the procedure increasingly more popular and favorable.

However, HoLEP is well known for having a steeper learning curve and initial increased cost of materials compared with a standard TURP [3].


This recent report describes the inherent risks of morcellation during HoLEP procedures with multiple case reports [4].

The purpose of this editorial is to demonstrate the relative safety of morcellation during HoLEP.


In the first case, the authors report prolonged morcellation time and concern for bladder injury because of poor observation associated with bleeding at the bladder neck [4].

Although exact details are unclear from the report, it needs to be emphasized that a clear endoscopic view is the most important aspect for safe morcellation.

The first step of morcellation is to ensure the bladder is appropriately distended to ensure appropriate observation and to decrease risk of bladder injury.

To achieve this, there should be a well-trained OR staff to monitor irrigation as well as maintaining two open inflow ports.

This is always needed to assist with identification of the previously enucleated prostatic tissue and maintain bladder distention [5].


One of the benefits of Holmium lasers is the relative ease of achieving hemostasis to greatly reduce the amount of bleeding during a procedure and the laser has known tissue penetration of about 5 mm.

Maintaining hemostasis throughout the entirety of morcellation is critical.

Bleeding must be controlled meticulously to ensure a clear endoscopic view before morcellation [6].

Unfortunately, this crucial step appears to have been performed inadequately based on the description from the report.

It is also important to maintain a minimal distance between the endoscope and the morcellator to improve precision and accuracy of morcellation, which, again, will decrease risk of bladder injury [6].


As long as adequate hemostasis is achieved and a clear view of the adenoma and bladder is maintained, the incidence of injury should be minimal.

Guidelines for safe morcellation should always be adhered to.

If there is any question with regard to observation or excessive bleeding, morcellation should be halted immediately in favor of achieving better hemostasis and reidentification of crucial landmark.

Failure to follow set standards puts the patient at increased risk for injury.


HoLEP has proven to be a powerful technique in treating BPH.

Its advantages over a standard TURP procedure have been well described in the literature.

Proper safety standards exist to prevent bladder injury.

In particular, maintaining observation with constant irrigation, achieving adequate hemostasis, and maintaining a safe minimal distance between the camera and morcellator are all necessary to reduce risk of complication, and retrieval of tissue should be performed in a manner to minimize strain on the urethra, bladder neck, and bladder itself.

Although this report illustrates the inherent risks of HoLEP, specifically morcellation, the information provided suggests that standard safety techniques were not adequately followed.

When performed appropriately, morcellation is a relatively safe procedure after enucleation of the prostate.


We appreciate the review provided by Stutz et al. in response to our case report on the management of capsular perforation during holmium laser enucleation of the prostate (HoLEP).

There are several valid points made by the authors with regard to the safety, efficacy, and durability of HoLEP for patients with outlet obstruction secondary to prostatic enlargement.

The letter gave a concise overview of the proper safety standards to prevent bladder injury during morcellation for patients undergoing this operation [1].


The purpose of our case report was not to emphasize prevention and management of a bladder injury/perforation, which has been well documented previously [2][3].

Instead, it was to share that in our experience of >800 cases at our institution since 2009, these 2 cases were memorable as they demonstrate how prostatic capsular perforation can resemble the intraoperative signs of a bladder injury.

It should be reiterated that, in both cases, neither patient actually suffered a bladder injury from morcellation.

Yet, the physical examination findings of a distended abdomen, paired with changes in peak airway pressures or a mismatch in irrigant inflow and outflow resembled that of a bladder injury.

As the case report highlights, the proper management of this clinical situation is vastly different from that of exploring for a suspected bladder injury, as capsular perforation can be managed conservatively with Lasix and bladder decompression [4].


The steep learning curve for HoLEP has been well documented and experienced HoLEP practitioners will recognize that visualization during morcellation can be impacted by multiple factors, including hemostasis, degree of bladder distention, prostatic size, prior indwelling catheter, and the concomitant administration of anticoagulants.

If safe technique during morcellation is adhered to with low suspicion of bladder perforation, the etiology of the physical examination findings can be attributed to a capsular perforation.

Urologists performing HoLEP should be aware of all the potential complications of HoLEP, and the nuances in their presentation, work-up, and management.


Endotoxin is the biological activity that is potential within the Lipopolysaccharide (LPS) macromolecule which is a major outer membrane component of Gram-negative (GN) bacteria (Figure 1) [1-3].

Each E. coli bacterial cell has approximately 10 6 LPS molecules [3].

The biological activities of endotoxin in humans and other species are potent and broad ranging.

These activities are mediated mostly by the lipid-A residue within the molecule.

For these reasons endotoxin has long been identified not only as a potential marker of GN infection [4,5] but also as a mediator and hence a potential target for specific anti-endotoxin therapies [6-10].

Surprisingly however, these potentials have not been realized.

The conflicts among over 100 studies of endotoxin as a potential marker of GN infection are reviewed elsewhere [4].


The focus of this review are the disconnects within the literature bearing on the relationship between endotoxemia and sepsis on the one hand and in assessing the potential of anti-endotoxemia therapies including antibody therapy designed to neutralize the biological activity of endotoxin on the other.


There is an extensive literature relating to O-polysaccharide specific antibodies generated by vaccination which mediate protection through an antibacterial effect which is not considered here.


The term "endotoxin" has been attributed to Richard Pfeiffer.

In the 1890's he made the distinction between the toxic properties that were endogenous within the GN bacterial cell, which he termed "endotoxin" versus those released outside the cell, which were termed "exotoxin" [11].

The exact identity of the endotoxin molecule was unknown for several decades and the structure-activity relationship in the mediation of the biological activities of endotoxin has only recently become clear.

In the mid 1980's the lipid-A moiety of the lipopolysaccharide molecule of Escherichia coli was totally chemically synthesized in a form available for studies of the structure activity relationship of this molecule [12].

With these studies, it became apparent that the biological activities of endotoxin could be attributed to the lipid-A component of the lipopolysaccharide (endotoxin) molecule (Figure 2).

The study of lipid-A partial structures have further clarified this structure-activity relationship [13].

Other microbiological and biochemical studies have identified the mechanisms regulating the synthesis of lipid-A within Gram-negative bacteria and specific variations in the structure of lipid-A which may have relevance to the pathogenesis of GN infection [14,15].


In studying endotoxin and the effects of anti-endotoxin interventions, several pharmacological properties of endotoxin that are unusual for a toxin should be noted.

Firstly, there are numerous effects induced by administration of endotoxin to experimental animals and humans.

Which of these effects is the most relevant correlate of survival in sepsis is sometimes not clear [16].

Recent review articles list over 20 humoral, cellular, immunological and metabolic effects.

Second, unlike most other toxins, the biological effects of endotoxin are not a uniform gravimetric property of the molecule.

The quantity of endotoxin is usually interpreted as the amount of biological activity in comparison to a reference endotoxin preparation assayed in parallel.

For this reason the concentration of endotoxemia is confusingly and variably reported in weight units (picograms) and sometimes in units of endotoxin O-Specific chain Core region n Lipid-A Monosaccharide Ethanolamine Glucosamine Phosphate 2-keto-3-deoxyoctonic acid, KDO Long chain fatty acid 2592 activity (Endotoxin Units; EU/mL).

The equivalence is dependent on the choice of reference endotoxin but is often approximated as 1 EU/mL ≈ 100 pg/mL [4].


Endotoxin has several other interesting properties.

It has long been recognized that human plasma accentuates some of the effects of endotoxin [17-20].

Moreover, the toxicity of endotoxin is indirectly mediated and endotoxin itself is not cytotoxic, a phenomenon which is best exemplified in the C3H/HeJ mouse strain [21,22].

This inbred mouse strain is resistant to the effects of endotoxin and this non-responsiveness is a genetically determined trait.

Moreover, the susceptibility to endotoxin can be restored by the transfer of macrophage cells in bone marrow transplants from histo-compatible C3H/HeN strain mice which have normal endotoxin responsiveness.

This observation implicated the mediation of the effects of endotoxin by a genetically encoded host receptor.

This observation ultimately enabled the identification of the gene for the endotoxin recognition (lps), and in turn, the LPS receptor mechanisms.


Endotoxin has potent biological activities in humans [23-25].

In humans, biological responses are apparent at doses as small as 4 ng/kg [25] whereas in other species doses of 1 mcg/kg (rabbits [26], sheep [27]) or as high as 2 mg/kg (rats [28], pigs [29], and non-human primates [30-33]) are required for response.

The difference in endotoxin activity between species is a confounding factor in preclinical studies in animals towards defining the role of endotoxin as a mediator in the pathogenesis of GN infections and in the development of anti-endotoxin antibodies.

The curious extreme sensitivity of humans to the effects of endotoxin is problematic for the development of anti-endotoxin therapies in that replication in experimental animals requires either large doses of endotoxin or artificial interventions to sensitize the experimental animals to the effects of endotoxin.

One such intervention is the administration of galactosamine to mice to achieve levels of sensitivity at doses of endotoxin that are comparable to that seen in humans [28].


A further biological property of endotoxin that is most unusual for a toxin is tolerance [34-39].

Doses of endotoxin in experimental animals that would otherwise be lethal can be tolerated after pre-treatments by sub-lethal doses [34].

In the 1920's, bacterial preparations containing endotoxin were in therapeutic use to induce a fever as a therapy for cancers and syphilis in humans [37].

Humans, as with other species, will acquire tolerance on repeated dosing.

Endotoxin doses as much as 200-fold higher may be required to achieve a fever response equivalent to that achieved with the initial dose.


There was a series of fascinating experiments undertaken initially in rabbits and later in humans [38-40] to investigate the relevance of tolerance toward GN infections.

Studies were undertaken in the 1960's using prisoner volunteers who were rendered tolerant to endotoxin by repeated doses of endotoxin and who were subsequently deliberately infected with Salmonella typhi (i.e., typhoid) or Pasteurella tularensis (i.e., tularemia).

These studies demonstrated that tolerance to endotoxin could be induced by administration of Salmonella endotoxin even during the course of an experimental typhoid illness [38].

Moreover, "Despite unequivocal activation of the endotoxin tolerance mechanisms……, the febrile and toxic course of typhoid fever proceeded unabated" [38].

There is some evidence that anti-endotoxin antibodies contribute to endotoxin tolerance [40].


There is an historical footnote to these studies with Pasteurella tularensis.

The role of endotoxin in the pathogenesis of tularaemia has recently been investigated and found to be complex.

While the LPS 2593 of this organism is considered to be an important virulence factor, surprisingly the immuno-biological activity of this molecule has been found to be weak [41].


The lipopolysaccharide molecule from all clinically relevant GN bacteria studied consists of three components against which an antibody could be directed.

These are; a polysaccharide chain, a core oligosaccharide and a lipid component, lipid-A (Figure 2) [11,24].

The LPS molecule, and in particularly, the lipid-A component, has been described as an "information rich" molecule, with many possible sites for specific recognition by prokaryotic and eukaryotic proteins.

The structure of the polysaccharide chain of the LPS molecule is highly variable even within species of GN bacteria whereas the lipid-A molecule is broadly conserved across GN bacteria of different types (see below) [24].

The core regions binding sites of LPS being located in the cell wall may be less accessible to antibody binding due to their location in the LPS molecule (Figure 1).


The polysaccharide consists of a repeating saccharide unit in a chain configuration which is hydrophilic and antigenic.

Being the outer most part of the LPS molecule, this is presumably more readily accessible to antibody binding than are the core regions of the LPS molecule.

In Enterobacteriaceae (e.g., Escherichia coli), the polysaccharide is variable in length and in composition between bacterial strains, and confers the bacterial strain's O-antigen specificity.

Some bacterial genera have less complete LPS structures.

For example, the O-specific antigenic chain is not present in mucosal pathogens such as Neisseria, Bordetella and Haemophilus and the LPS in these genera have only a core oligosaccharide.

The core oligosaccharide can be divided into an inner core and outer core region and for the genus Chlamydia even the outer core region is not present and these genera have only an inner core [3,11].


In developing antibodies to lipoplysaccharide, the site of antibody binding may determine the anti-endotoxin activity.

Lipid-A contains the molecular components which critically determine the endotoxic activity of LPS [42,43].

The structure of E. coli lipid-A consists of a diglucosamine with two phosphates and six acyl (fatty acid) chains (hexaacyl LPS).

Two 3-hydroxymyristate (fatty acid) chains are attached directly to each of the two glucosamines with two secondary ("piggyback") chains also attached.

The fatty acids may be laurate (twelve carbon, C12), myristate (C14) with sometimes palmitate (C16) found as the secondary acyl chain, and the primary (glucosamine-linked) acyl chains typically having 12 carbons.

This hexaacyl LPS structure is optimal for recognition by the CD14-MD-2-TLR4 receptor.

The LPS molecule from Gram-negative bacteria other than E. coli may have more or fewer acyl chains, longer acyl chains, branched acyl chains, unsaturated acyl chains, only one phosphate, or other modifications.

The degree of recognition of the various hexaacyl and non- hexaacyl LPS structures by the CD14-MD-2-TLR4 receptor determines the mediation of endotoxic biological activity.


The structure of the lipid-A monomer also determines its molecular shape [44-46].

These shapes are variably conical or cylindrical depending on the ratios between the hydrophobic versus hydrophilic regions.

The most endotoxically active lipid-A monomer structure is conical whereas less active lipid-A is cylindrical.

The activity of endotoxin can be increased by sonication [47].

The biological significance of these supra-molecular structures, and the relative importance of LPS aggregates versus LPS monomers towards host recognition, remains unclear.

At concentrations above a critical micelle concentration, lipid-A monomers aggregate into supra-molecular structures determined by the molecular shape of the lipid-A. These supra-molecular structures

are sometimes large enough to be viewed under electron microscopy [46].


Lipopolysaccharide-binding protein (LBP) is a serum protein which binds to the lipid A component of bacterial endotoxin and facilitates its delivery to the CD14 antigen on the macrophage, where pro-inflammatory cytokines are released and a cascade of host mediators is initiated.

The neutrophil granular protein bactericidal/permeability-increasing protein (BPI) competes with LBP for endotoxin binding and functions as a molecular antagonist of LBP- endotoxin interactions [48,49].


The specific nature of this binding between these serum proteins and LPS has prompted attempts to develop synthetic LPS binding peptides for possible anti-endotoxin based treatments of sepsis.

Preclinical studies with these synthetic peptides demonstrate that they can combine excellent selectivity for LPS binding together with suppression of LPS-induced cytokine release in vitro and protection from lethal LPS induced septic shock in vivo [50-52].

Moreover, the mean concentration required to inhibit growth (mean inhibitory concentration, MIC) and the concentration required to achieve anti-LPS activity with these peptides are comparable [51,52].

Interestingly, the molecular interaction between these peptides and LPS which results in this neutralization of biological activity is evident as a bio-physical change in the LPS supra-molecular structure.

With peptide binding, the lipid-A part of LPS is converted from its "endotoxic" conformation, being the cubic aggregate structure, to an inactive multi-lamellar structure.

Peptides that bind to lipid-A have direct anti-bacterial activity as a consequence of the key structural role of lipid-A within the bacterial membrane structure.


Whereas remarkably diverse lipid-A structures are found in the LPS molecules produced by different Gram-negative bacteria, the requirements for maximal activation of animal cells are rather restricted [53-57].

The lipid-A structure that has been most extensively studied and the one that is optimally sensed by the CD14-MD-2-TLR4 receptor is that from E. coli.

This lipid A consists of a bis-phosphorylated di-glucosamine to which are attached six saturated fatty acyl chains with lengths of 12 or 14 (occasionally 16) carbons.

LPS molecules that have this structure are commonly referred to as "hexaacyl LPSs" in contra-distinction to other lipid-A structures which have four, five or seven (i.e., non-"hexaacyl LPSs") fatty acyl chains.


Figure 3 shows the interaction between hexaacyl lipid-A and the CD14-MD-2-TLR4 receptor [13].

The amphipathic lipid A molecule consists of negatively-charged (indicated by "Θ") phosphate groups and hydrophobic fatty acyl chains.

The MD-2 co-receptor protein consists of a "binding pocket" which is lined with hydrophobic amino acid residues and charged amino acids at the mouth of the MD-2 pocket; the former and latter accommodate the fatty acyl chains and the phosphate groups of lipid-A, respectively.


The next part of the figure shows lipid-A inside the MD-2 pocket and also in contact with TLR4, with which it forms a 1:1 complex.

Upon lipid-A binding, this TLR4-MD-2 complex dimerizes with a second complex.

The bound lipid-A engages the TLR4 molecule in the second complex (TLR4*) at two main dimerization interfaces; the first being between the 6th acyl chain of lipid A (which extends out of the pocket) with uncharged amino acids (cloud labeled "neutral") on TLR4*, the second being between the negatively-charged 1-phosphate (1-PO 4 ) on lipid-A with positively-charged amino acid residues (cloud labeled "+") on TLR4*. Hexaacyl lipid-A structures are common among Enterobacteriaceae such as E. coli whereas non-Enterobacteriaceae, such as Pseudomonas aeruginosa may have a non-hexaacyl lipid-A structure.


The structural differences in lipid-A among clinical GN bacterial isolates is a relatively recent finding.

The possible clinical relevance of these differences between the lipid-A structures of colonizing versus environmental GN bacteria remains to be determined.


Even were the perfect anti-endotoxin antibody were to be available tomorrow, we would still need to identify the patient population in which it should be used [58,59].

This is generally regarded as the patient population with sepsis.

What is sepsis [60]? Sepsis is a clinical syndrome for which defining objective diagnostic and prognostic tests are lacking and continue to evolve [61-64].

Forty years ago Lewis Thomas said that "It is our response that makes the disease" [63], a comment which succinctly describes sepsis as being the body's systemic response to suspected or proven infection rather than the infection per se.


In 1991, The American College of Chest Physicians (ACCP) and the Society of Critical Care Medicine (SCCM) convened a panel of 35 experts to derive a standard set of definitions to rapidly identify and triage those patients who might benefit from novel anti-endotoxin antibody therapies that were about to enter clinical trials [65].

The resulting consensus criteria were initially termed the Bone criteria and subsequently evolved into the Systemic Inflammatory Response Syndrome (SIRS) which consists of the presence of at least two of the following; ● White blood cell count (WCC) >12,000/mm, <4000/mm 3 or with >10% immature neutrophils.


The main advantage of SIRS and related criteria are their relative simplicity which led to widespread clinical application.

Sepsis, severe sepsis and septic shock are common in the ICU setting with each occurring in as many as 10% of ICU admissions [66-70].

Sepsis and septic shock are associated with substantial attributable mortality.

In the CUB-Rea database, the risk of death was 54% in those with versus 28% in matched controls without septic shock giving an estimated attributable risk of 26% [66].


However, the limitations in using these defining criteria of sepsis are that;


● They are too sensitive, as non-infection related conditions are included [61];


● The sepsis clinical syndrome is non-specific as to whether the causative organism is Gram-negative bacterial or other [64];


● Being a reflection of the host response, SIRS has an associated morbidity and mortality which is similar whether or not the SIRS is associated with a documented infection [67,68]; and ● As many as a third of episodes are found to occur outside of the ICU setting [68].

The limitations of the current methods for defining and testing for sepsis is best illustrated by a contrast with the field of cardiovascular medicine where in the last ten years, the availability of the serum cardiac troponin test, a rapidly available serum test of cardiac injury, has transformed the management of patients presenting with acute coronary syndromes [71].

A rapid test for sepsis with this sensitivity and specificity is not yet available.


A rapid test for endotoxemia would appear to be a logical method for identifying patients with the potential to benefit from anti-endotoxin therapies, and this approach has been used in some studies of anti-endotoxin antibodies.

This is to be further explored with newer endotoxemia assays such as the Spectral Endotoxin Activity Assay ® as used in the EUPHRATES trial.

However to date, the use of a rapid test for endotoxemia as a method for patient stratification has not proved to be practical due to issues relating to the available assays for endotoxemia [4].

Even within the endotoxemia positive sub-groups of patients within clinical trials, the results of studies of anti-endotoxemia therapies have not been as expected (see below).

Moreover the interpretation of endotoxemia assays whether qualitative or quantitative are not simple [4,72,73].

The levels of endotoxemia encountered in patients with sepsis vary over a range of several log-fold.

Among patients with meningococcemia this range is from 10 to 10,000 ng/L [74] and among patients with sepsis this range is from undetectable (<2 pg/mL) to 5 ng/mL [4].

Interestingly it has been reported that endotoxemia is detectable in runners at the conclusion of a marathon at levels comparable to those with sepsis [75].


In the development of anti-endotoxin antibodies as potential therapeutic agents, the following questions need to be considered in their clinical evaluation;


● What do bacteremia and endotoxemia separately contribute towards the attributable mortality of Gram negative sepsis?


● Is GN bacteremia a single entity?


● Can endotoxemia be considered as a single entity?


● Is the attributable mortality dependent on the underlying mortality risk?


● Is the onset of severe sepsis an indication that the detrimental pathophysiological process has passed the point of no return and hence anti-endotoxemia therapy at this point is futile?


● Is the disconnect bridgeable or is a more fundamental explanation required?


None of the above questions can be satisfactorily resolved in an animal model of sepsis.

For example, GN bacteremia is clearly not a single entity despite the fact that experimental models commonly have used a single bacterial species, typical E. coli, as the challenge inoculum.

Amongst various GN bacteremia types there is a well-defined ranking of mortality risk with the risk being lowest for E. coli and highest for GN bacteremias such as Pseudomonas aeruginosa.

Likewise, endotoxemia should not be regarded as a single entity given the recently described differences in the lipid-A structures among the different GN bacteremia types.

Also, the relationship between endotoxemia versus GN bacteremia both with respect to their mutual concordance and as predictors of outcome among patients with suspected sepsis is not simple [76-78].


Endotoxemia is not detectable for at least 20% and up to 50% of patients with GN bacteremia.

Moreover, the relevance of endotoxemia detection to prognosis is dependent on the co-detection of GN bacteremia, the GN bacteremia type and the underlying mortality risk in the study population [76,77].

This has been examined in a series of meta-analyses and L'Abbé plots using data derived from 100 studies of patients with sepsis in various settings.

The most recent analyses examined the relative strength of endotoxemia and GN bacteremia as independent predictors of increased mortality risk [77] and the confounding relationship with the GN bacteremia type [78].


The most recent of these analyses has attempted to identify the separate contributions toward the impact of endotoxemia versus GN bacteremia on the mortality risk among various studies of patients in various settings [77].

The mortality risk was analyzed from 35 studies for which individual patient data was available.

This data was stratified into four distinct groups; GN bacteremia and endotoxemia co-detected (patient group 1), GN bacteremia alone (group 2), endotoxemia alone (group 3) each versus patients with neither endotoxemia nor GN bacteremia detected (group 4).

The difference in mortality risk between group 4 versus respectively groups 1, 2 and 3 can be represented in a summary 2598 odds ratio (OR) or risk difference (RD).

In addition, the heterogeneity in risk across these 35 studies can be visualized in the "scatter" within Figure 4.

Also as group 4 is generally the largest group, the mortality risk for this group can be taken as representative of the underlying mortality risk for each study.


The co-detection of endotoxemia with GN bacteremia is most predictive of increased mortality risk (OR 6.9; 4.4-11.0; RD 19%; 11%-27%) versus the detection of neither (Figure 4a).

However, this was most apparent amongst a sub-group analysis of studies undertaken outside an ICU setting and is a finding which is associated with marked heterogeneity [4,77].

By contrast, the detection of either endotoxemia (Figure 4c) or GN bacteremia (Figure 4b) in isolation versus the detection of neither is associated with borderline elevation of risk (OR < 2.0; RD < 10%) and, moreover, is associated in The heterogeneity is most apparent in the contrast of mortality risk for patient groups one versus four (Figure 4a).

In this regard, there is an unequal distribution in GN bacterial isolates among the patients of groups 1 and 2.

E. coli bacteremia isolates are less common in group 1 versus group 2 whereas for Pseudomonas bacteremia isolates the converse applies.

Moreover, while the underlying patient risk was higher for studies in an ICU setting versus studies in other settings (Figure 4), as might be expected, the additional mortality associated the detection of either or both of endotoxemia or GN bacteremia was less apparent in the studies of patients at high versus low underlying risk [77].


A further analysis of the individual patient data from studies of patients derived from eight ICU studies and small numbers from an additional 6 non-ICU studies had been undertaken [78].

These patients were identified to have GN bacteremia with or without endotoxemia (i.e., groups one and two).

This analysis has unexpectedly revealed that the detection of endotoxemia in association with E. coli bacteremia has no prognostic value in any setting [78].

It is notable that this lack of prognostic value with E. coli bacteremia is at variance with the experience for GN bacteremia overall.

Note that E. coli bacteremia is often the most common GN bacteremia category in any clinical series.

This finding is also surprising given the extensive use of endotoxin derived from E. coli in experimental models.

This factor is yet to be explored as a possible basis for the disconnect between the pre-clinical and clinical experience with anti-endotoxin antibodies.


Some have questioned whether the onset of severe sepsis indicates that the detrimental pathophysiological process has passed the point of no return and hence anti-endotoxemia therapy at this point is futile.

The answer to this question can only be speculated.

However, there are parallels with antibiotic therapy for which there is now clear evidence that early effective antibiotic therapy is critical to optimal activity and survival of patients with septic shock [79] or conversely, late antibiotic therapy could be considered relatively ineffective.

Moreover, combination antibiotic therapy may improve survival [80].

The question then becomes what additional benefit from early effective combination antibiotic and anti-endotoxin therapies could be expected? Only an adequately designed clinical trial can answer this question.


However, no simple explanation has emerged to account for this "disconnect" and there are numerous paradoxical discrepant findings between the pre-clinical studies on the one hand and the randomized controlled clinical trials of the same agent on the other [88-93].

Hence the term "endotoxin-sepsis disconnect" could be extended to positive results from the early clinical studies with anti-endotoxin therapies which raised expectations for progress in the development of these therapies.

Failure to replicate findings with specific anti-endotoxin antibodies in later studies, also represents a paradoxical "disconnect" [91,92].

Anti-inflammatory agents that have been developed to block mediators (e.g., anti-TNF, anti-PAF) whose release is stimulated by endotoxin and hence are "down-stream" are not considered here although these also each exemplify a paradoxical disconnect.

Some additional literature is cited and discussed further in [93].


Indeed more broadly, no adjuvant approach to the treatment of sepsis has emerged from the extensive pre-clinical research.

This extensive literature dealing with the pre-clinical and clinical evaluations of candidate anti-sepsis agents is complex and difficult to summarize.

Some examples of "disconnect" are reviewed here to the extent that they have a bearing on the pre-clinical and clinical evaluation of anti-endotoxin antibodies for adjuvant therapy in Gram-negative sepsis.


Despite 40 years of research with at least six candidate anti-endotoxin antibody therapies each having shown promise in pre-clinical and early clinical trials, no anti-endotoxin antibody approach to the treatment of sepsis has emerged with proven clinical benefit.

In relation to anti-endotoxin therapies, the extent to which the mediation of survival benefit in experimental studies with the various anti-endotoxin therapies relate to the binding of endotoxin, the neutralization of endotoxin activity versus the clearance of either endotoxemia or even bacteremia are each contentious and unclear [91,94].

For example, a review article which reviewed the evidence for and against one extensively studied hypothesis,-that antibodies to the inner core raised by immunization with enterobacterial deep rough mutants confer broad spectrum protection during Gram-negative bacterial sepsis, cites over 200 references published up to 1997 [95].

The most paradoxical suggestion is that some of the anti-endotoxin monoclonal antibodies and recombinant proteins under study had been contaminated by endotoxin in the production process [96,97].

This endotoxin contamination from the source bacteria could account for the difficulties in replicating the protection in later pre-clinical studies.


A question of particular interest in this contentious area of research is the evidence pertaining to the relationship of survival benefit to reductions in levels of circulating endotoxemia.

Unresolved issues that could bridge the pre-clinical versus clinical disconnect is the possibility that anti-endotoxin 2601 therapies may be more effective in some patient sub-groups if only these could be identified [87].

In this regard the differences in lipid-A structure that have recently been discovered between E. coli versus other Gram negative bacteria together with the finding that endotoxemia has no impact on mortality when detected in association with E. coli in contrast to other types of GN bacteremia remain an important basis for re-appraisal of this old literature [4,78].


Some of the anti-endotoxin therapies including antibodies which have reached clinical studies are summarized in Tables 1-3.

The agents listed in these tables have anti-endotoxin activity which is thought to be mediated either through binding to lipid-A or through blocking the effects of endotoxin presumably at the level of the receptor.

However, even these pre-clinical studies of binding and mechanism of action are controversial and in some cases have been difficult to reproduce [98].

Not shown in these tables is a range of other biological agents designed to inhibit downstream immunological mediators whose release is triggered by endotoxin which have been evaluated in randomized clinical trials of the patient group with sepsis.

Recent commentaries [87,99] and a systematic review of 45 reviews of animal studies of therapeutic interventions for sepsis found evidence of over-extrapolation of the preclinical research findings and an under-appreciation of the deficiencies and risk of bias in study methods [100].

Moreover, an optimal animal model needs to be more than simply an "intoxication" model [135,139-141].

Any beneficial therapeutic effect would presumably need to be additional to that provided by supportive and other therapies such as antibiotics.

The desirable type of study design is one which more closely reflects the sepsis dynamic in the clinical setting but this is difficult to achieve.


Controlled models of septic shock have been developed in dogs and other animals by workers at the National Institutes of Health (NIH) in attempts to disentangle the separate contributions of clearance of endotoxemia versus bacteremia toward outcome.

These models have generated unexpected and paradoxical observations bearing on the relative importance of clearance of bacteremia versus endotoxemia in relation to the effects of experimental therapeutic interventions on outcome [142-154].


In the dog, implantation of an intra-peritoneal clot infected with various selected Gram-negative and Gram-positive challenge bacteria induces bacteremia together with cardiovascular changes characteristic of septic shock leading to lethality [142].

The outcomes of any therapeutic interventions can then be studied under controlled conditions simulating a clinical trial including randomized treatment assignment and investigator blinding [146].

This model enables the study of the relative effects of intervention with antibiotics, cardiovascular support and anti-endotoxin therapies on hemodynamic changes, survival time, quantitative levels of bacteremia as well as quantitative levels of endotoxemia.


A series of paradoxical observations have emerged from these studies which are of interest to the questions bearing on the relationship of the effect of anti-endotoxin agents to the clearance of bacteremia versus endotoxemia and toward improved outcome.

Amongst these studies were several that addressed variations in the type of bacterial challenge, the strain type, virulence factors and a range of therapeutic interventions [142-145] which are reviewed elsewhere [155].

Only those studies that are most relevant to the development of anti-endotoxin therapies including anti-endotoxin antibodies are reviewed here.


A strikingly paradoxical observation arose when the following putative anti-endotoxin interventions were studied; two anti-endotoxin therapies; a monoclonal anti-endotoxin antibody (HA-1A) [146], reconstituted human HDL lipoprotein [152], and plasma exchange [151].

These interventions were each separately studied versus control treatments in this model for the effect on survival and the associated levels of bacteremia and endotoxemia following the E. coli challenge.

The effects of these interventions on the levels of endotoxemia resulted in a lowering [152], or no effect [146], or were not measured [151].

Surprisingly, in each case, the therapies either failed to increase survival or even paradoxically resulted in worsened cardiovascular effects or shortened survival despite similar quantitative levels of bacteremia versus control treated animals.


Further studies also generated somewhat unexpected results bearing on bacteremia and endotoxemia in relation to the protective effect of various monoclonal antibodies (MAB) against an E. coli challenge in this canine and in other models.

Both an isotype matched core region specific MAB and an O-side chain type specific MAB conferred protection additional to that provided by antibiotic and supportive therapy in this canine model [143].

The mechanism of protection differed.

For the O-side chain specific MAB protection was mediated through enhanced clearance of the bacteremia and endotoxemia.

Whereas the core region specific MAB, while protection was mediated through decreased circulatory collapse, this occurred without any measurable reduction in the bacteremia or endotoxemia compared to control treated dogs.

This NIH group also investigated the protective effect of recombinant granulocyte colony stimulating factor (rG-CSF) versus control treatment and the relationship between clearance of bacteremia and endotoxemia and outcome in a canine pneumonia model induced by an E. coli intra-tracheal challenge [154].

The enhanced peripheral blood neutrophil counts following prophylactic administration of rG-CSF was associated with reduced levels of endotoxemia and improved survival in this canine model of E. coli pneumonia compared to control treatment.

The quantitative bacteremia counts were similar in the treatment and control groups.


The summation of these paradoxical observations made over the course of these studies reviewed here together with other studies that have been reviewed elsewhere [4,155] led this group of investigators to speculate as to whether endotoxemia has continued importance once the inflammatory response and the clinical manifestations have been initiated in that possibly… "tolerance to endotoxemia may develop within hours [51], and persistently high levels of endotoxin may not be central to the manifestations of the sepsis syndrome" [144].


Another research group has also examined a monoclonal anti-endotoxin antibody (HA-1A) as well as an alternate anti-endotoxin intervention, bactericidal/permeability increasing protein (BPI) which is an endogenously produced human endotoxin neutralizing protein, against a lethal E. coli at a high (10 11 cfu/kg inoculum) dose challenge.

The plasma endotoxin levels were significantly reduced compared to no treatment after the use of BPI but not with HA-1A.

However, whilst both treatments attenuated cytokine release, neither improved survival [156].


The impetus for the development of anti-endotoxin therapies had been supported by a range of clinical observations (Table 1) [157,158].

In the 1970's, several clinical studies suggested that the presence in acute phase patient serum of high levels of antibodies to core antigens of lipopolysaccharide as expressed in a mutant E. coli J5 strain correlated with improved outcome of patients with sepsis [101-103].

More recent observational studies have again demonstrated the correlation between the measurable levels of anti-endotoxin core antibodies in pre-operative serum with adverse post-operative events in patients undergoing cardiac surgery [107].


These early observations led to intervention studies using various preparations of anti-sera prepared from volunteer donors specifically immunized using the mutant E. coli J5 strain [104-107].

Anti-sera production was considered to be both an expensive and an impractical undertaking for clinical use outside of investigational studies.

Later studies used either immunoglobulin preparations produced commercially from selected blood donations screened on the basis of anti-sera titres or specific monoclonal antibodies.


The results of these intervention studies are contentious as there were a number of different study designs leading to difficulties in interpretation and even reproducibility [157,158].

Among these studies there were differences in anti-endotoxin antibody or agent, differences in study population and differences in study end point.

Various preparations of anti-sera or immunoglobulins were used, some containing both IgM and IgG, other preparations only IgG. Some studies used a therapeutic strategy whereas other studies chose to target high risk patient groups as a prophylactic strategy.

The end point of interest varied, in some studies being the occurrence of GN bacteremia, this was particularly the 2605 case for studies of a prophylactic approach (Table 1).

Whereas the end point in other studies was mortality, this was the case more so for studies of a treatment approach (Tables 2 and 3).


Some of these studies used normal (non-immunized) immunoglobulin preparations as the intervention for control group patients.

However, the choice of normal immunoglobulin preparations for control group patients is problematic as it is possible that even these may have anti-endotoxin efficacy in patients with sepsis [115,159,160] even with possible mortality benefit [159], although not seen in all studies [160-164].


A small number of studies listed in Tables 1-3 have used the detection of endotoxemia as either a criterion for study entry or as a quantitative outcome of intervention [113-115].

In this regard, the studies undertaken in patients with meningococcemia are of particular interest for three reasons.

Unlike other Gram negative infections, meningococcemia is clinically distinctive and rapid recognition and early treatment is feasible.

Second, endotoxemia occurs more commonly with meningococcaemia than with other Gram negative bacteremias [64].

Finally, there is evidence from Norwegian investigators implicating a relationship between quantitative levels of endotoxemia and outcome among patients with meningococcaemia [74].

However, three studies of anti-endotoxin therapies have failed to find any significant overall mortality benefit in this condition (Table 2).

Moreover, one study that examined the effect of endotoxemia detection on outcome using a logistic regression model found no significant interaction effect from the intervention [109] and another study examined levels of endotoxemia but did not report the impact on outcome [110].


There are several possible explanations for the disappointing results in these studies undertaken in patients with meningococcemia.

Adequacy of dosing and speed of administration of the intervention have been considered amongst a range of study design aspects.

For example, in the 1992 French study [108] a significant increment in the serum level of anti-J5 antibody was not apparent in the intervention group post infusion of anti-J5 antiserum.

However, the evidence implicating a relationship between quantitative levels of endotoxemia and outcome amongst patients with meningococcaemia has undergone a reappraisal.

Recent studies from the same group of Norwegian investigators has found a close correlation between levels of endotoxemia and bacterial DNA detected using quantitative PCR methods among patients with systemic meningococcal disease [165].

Hence the level of bacteremia is likely to be a confounder in the correlation between the level of endotoxemia and outcome in this condition.


A number of these anti-endotoxin treatment studies have examined the sub-group of patients with detectable endotoxemia for any different treatment response [115,166].

Notable amongst these was a sub-study from the HA-1A monoclonal antibody which concluded that HA-1A reduced mortality amongst the 27 patients with detectable endotoxemia but not amongst 55 patients without detectable endotoxemia [166].

However mostly these have been small sub-groups.

For other clinical trials, the results in the endotoxemia positive sub-groups of patients have also not been as expected [131].


One parameter not previously considered in the interpretation of these study results is the type of GN bacteremias seen.

Given that the impact of co-detection of endotoxemia with GN bacteremia on patient prognosis is unequal for different types of GN bacteremia, even amongst Enterobacteriaceae [78] it is plausible that a high prevalence of E. coli bacteremias among any of the studies in Tables 1 and 3 could account for the lack of therapeutic success.

However, this is speculative and the proportion of E. coli bacteremias among the overall total GN bacteremias for most studies is unknown.

In the development and clinical evaluation of anti-endotoxin antibodies, the recent published clinical experience with polymyxin is of interest [167-180].

Polymyxin B is a potential anti-endotoxin intervention as there is strong evidence for endotoxin binding and neutralization in pre-clinical studies.

Polymyxins are from a group of cyclic cationic polypeptide antibiotics and these have well characterized lipopolysaccharide binding, which is presumably mediated through binding to lipid-A, and which is associated with inhibition of endotoxin activity as measured in vitro and in whole animal studies.


It is notable that the endotoxin antagonist property of polymyxin-B is not simple as it is dependent in part on the bacterial origin of the LPS.

Amongst LPS preparations from eight different GN bacteria, Polymyxin was most inhibitory against the LPS of E. coli and least inhibitory against the LPS of N. meningitidis.

An additional paradoxical finding from this study was that while polymyxin at low concentrations was inhibitory to the effects of endotoxin, at concentrations greater than 50 mcg/mL polymyxin acted synergistically with endotoxin in inducing IL-1 secretion [167].


While toxicity limits the clinical use of polymyxin B as an antibiotic, polymyxin B can be bound to a solid phase such as a hemo-perfusion column [169].

This enables hemo-perfusion as a method for the removal of circulating endotoxemia through exposure to immobilized polymyxin B without the systemic toxicity.

This method and others [181] are being actively explored in clinical trials.


Cruz et al. [ 171] systematically reviewed the polymyxin-B hemo-perfusion literature published to 2006 and found 28 publications.

The results of these studies were heterogeneous.

Many were small, non-randomized and unblinded studies.

Also, there was possible duplicate publication amongst these studies.

All but two of the 28 publications had come from groups in Japan although since 2006 several studies from European centres have been published.


Whilst the Japanese studies suggest that levels of endotoxemia were reduced by polymyxin-B hemo-perfusion and that there was a reduction in mortality of nearly 50% [172], the experience among several small European studies has been inconsistent.

These European studies, which all had fewer than 70 patients, have tested for either reductions in endotoxemia or mortality using polymyxin-B based or other hemo-perfusion interventions [173-177].

These studies found either no mortality benefit with a reduction in endotoxemia [173,174], or a reduction in mortality with an unknown effect on endotoxemia [175], or neither finding [176,177].


The most promising result of all studies came from the EUPHAS study (Early Use of Polymyxin Hemoperfusion in Abdominal Septic shock) which studied post-surgical patients with severe sepsis or septic shock secondary to intra-abdominal infection.

This study was conducted in 10 Italian ICU's over a 3 year period (2004-2007) and was terminated after an interim analysis revealed that a statistically significant and seemingly impressive reduction in clinical severity indices and mortality had become apparent between the groups after the enrolment of 64 patients.

The reduction in 28 day mortality achieved in this study was as follows; 11 of 34 (33%) patients with polymyxin-B hemo-perfusion versus 16 of 30 (53%) patients with conventional therapy [175].


However, the interpretation of the EUPHAS study is unclear for several reasons [178-180].

One is that even with 64 patients, it is a small study.

Also, the number of bacteremias found in the two groups was unequal; 16 among the patients receiving polymyxin-B hemo-perfusion versus only 3 among the patients receiving conventional therapy, a highly significant and unexplained imbalance.

Hence whether the efficacy of the polymyxin is dependent on the clearance of endotoxemia or of bacteremia remains unclear.

A third criticism is that survival had been prolonged by a few days only.

In this 2607 regard the study results were statistically significant only according to an analysis based on the relative survival time (adjusted hazard ratio 0.36; 95% CI: 0.16-0.8) but not by an analysis of absolute survival (Fisher's exact test; p = 0.13) A final criticism of the EUPHAS study is that endotoxemia levels were not measured.

These study deficiencies are to be addressed in two follow up multi-center studies, the EUPHRATES (Evaluating Use of Polymyxin Hemo-perfusion in an RCT of Adults treated for Endotoxemia and Septic shock) which is to be undertaken in North American ICU's together with the EUPHAS2 which is to be undertaken in Italian centers.


Another European study of endotoxemia adsorption using hemo-perfusion randomized 143 septic patients with suspected Gram-negative sepsis to receive either standard therapy alone versus standard therapy plus an extracorporeal endotoxin absorber.

In this study [181], the endotoxin absorber was immobilised human serum albumin (iHSA), not polymxin.

There was a non-significant trend toward lower endotoxemia levels with the extracorporeal endotoxin absorber treatment but no difference was found in the primary end point overall (APACHEII score) or survival.


Hence despite proven in vitro efficacy, the clinical efficacy of polymyxin remains unclear in clinical studies.

Moreover, whether any clinical efficacy is contingent on reduction in detectable levels of endotoxemia versus on anti-bacterial effects for example also remains unclear.


Endotoxin is an attractive target against which to develop adjuvant therapies such as antibodies to aid in the management of sepsis.

However, there are multiple challenges toward the development of these therapies.

The first is the design of suitable experimental models with which to test the effects of anti-endotoxin antibodies.

The extreme sensitivity of humans to endotoxin versus laboratory animals and the relative anti-bacterial versus anti-endotoxin effects of candidate antibodies are major considerations here.


More broadly, the exact role of lipid-A and the importance of endotoxemia in the mediation of sepsis both remain to be clarified.

The disconnect has increased with the recent progress in understandings of the complexities of the lipid-A structure function relationship on the one hand and the heterogeneous relationship between endotoxemia detection and the outcome of sepsis on the other.

For these reasons, endotoxemia, as with GN bacteremia, should no longer be considered as a single entity.

Moreover, the pathophysiological relevance of endotoxemia is dependent on the presence and type of concomitant GN bacteremia and possibly also on the underlying patient risk.


However, an overarching challenge toward the further development of anti-endotoxin antibodies, as with other anti-endotoxin therapeutics, is the need to acknowledge the substantial and increasing disconnect between the pre-clinical versus clinical experience.


I have read with interest James C. Hurley’s very good review [1]. I totally agree that a reappraisal of the use of anti-endotoxin antibodies in Gram negative infections is warranted.


In a study [2] we showed the possible association between endotoxin (LPS) and morbidity and mortality in septic shock.

This was a study in healthy primates (vervet monkeys).


We found, when these anesthetized primates received an LD100 iv infusion of Echerichia coli (E. coli) over one hour, both E. coli and endotoxin concentration significantly increased during the E. coli infusion.

The anti-endotoxin (anti-LPS) on the other hand decreased significantly.


Interestingly, when the animals succumbed, their LPS concentration was still raised, but there were no viable E. coli.

There was also only a small amount of anti-LPS present.


Hence, endotoxin concentration rather than circulating E. coli bacteria may be an important pathogen responsible for the high mortality experienced during E. coli shock.


This is in agreement with Spink et al. [ 3] and now Hurley [1] who suggested that endotoxin which forms an integral part of the outer cellular membrane of gram negative bacteria (GNB) participates in the genesis of shock.


In our review [4] and some of the other papers we published in this field [5–9], we refer to successful preliminary studies using anti-lipopolysaccharide IgG (anti-LPS).

The anti-LPS both present prior to the insult or given after the insult, would seem to inactivate plasma endotoxins and combat Gram-negative bacteria in sepsis.

Thereby, as Hurley [1] suggests, may form part of a possible new form of therapy.


The question that needs to be addressed is: how best to accomplice this? What part of the endotoxin should be attacked, the O-specific chain or the smaller Lipid-A, or even, if possible, both?


I appreciate the thoughtful comments from Dr. Brock-Utne [1] on my recent review of the disconnect between animal studies and clinical experience with anti-endotoxemia therapies [2].

Dr. Brock-Utne, the author of this commentary, has been a co-author of several important contributions in the area of human endotoxemia.

Their reports that strenuous exercise causes endotoxemia in ultradistance triathlon competitors are of great interest [3,4].

They had also reported how anti-LPS antibodies change in response following the appearance of endotoxemia in these athletes.

Their reports are important in that their publications are rare examples of systematic trans-species investigations of endotoxemia.

Moreover, the levels of endotoxemia in human athletes they observed were not trivial with levels as high as 0.294 ng/mL. These levels were 15 times the detection limit of the assay and at levels comparable to those seen in the blood of patients in the intensive care unit with sepsis.

Their reports of endotoxemia in these athletes are thought provoking and at the very least may help to explain fatalities with heat stroke [5].


However, the specific question they raise in their comment [1] as to whether anti-endotoxin therapy is feasible for Gram-negative sepsis is not simple.

In my review article I point to several levels of disconnect between animal model studies and human sepsis which are problematic towards addressing the question that Dr Brock-Utne has posed.

I am uncertain of the relevance of the observations of Brucellosis and its treatment published in 1948 [6] to the management of sepsis in the 21st century [7].

Moreover, it remains unclear what relevance the infusion of Echerichia coli (E. coli) endotoxin (LPS) into laboratory animals has to the development of therapies for human sepsis.

There are multiple instances of disconnect between findings in such laboratory experiments with promising anti-endotoxin agents and results of subsequent clinical studies [2].

For example, while animal model studies commonly use (E. coli) endotoxin, it is possible that this may have been the wrong endotoxin to study.

We recently found that endotoxemia detected in association with Gram-negative sepsis with E. coli bacteremia does not have a significant association with mortality [8,9] although endotoxemia with non-E. coli bacteremia does.

To me the broader question remains enigmatic and the disconnect between animal model research and human sepsis has increased rather than decreased with time.


The trial is registered at the Netherlands trial registry, #3440, www.trialregister.nl.

The data that support the find- ings of this study are available from the corresponding author, upon reasonable request.


All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: Support from the Netherlands Organisation for Scientific Research (NWO), VIDI 91711319 and the Dutch Brain Foundation for the submitted work.

No author has finan- cial relationships with any organizations that might have an interest in the submitted work in the previous 3 years.

No other relationships or activities that could appear to have influenced the submitted work.


Recently, Pijpers et al. ( 2019) reported in Brain on the use of onabotulinumtoxinA (ONA, Botox(cid:2), Allergan) for medication overuse withdrawal in patients with chronic migraine.

In their double-blind randomized controlled trial they conclude that ONA does not offer benefits.

Medication overuse in chronic migraine is a well-recognized problem [3].

Drug withdrawal is an integral part of all strategies to overcome it [4].

Apart from other substance specific withdrawal symptoms, transient increase in migraine may result.


The authors are unaware of two main aspects of ONA therapy of chronic migraine with far reaching consequences.

First, dose effect curves for ONA are rudimentary for most of its applications, and virtually non-existent for chronic migraine therapy.

This means we do not know whether there is a threshold dose for its efficacy, a saturation dose and what the relationship in between may be.

After all, ONA forehead doses of 17.5 MU are 50% of the PREEMPT forehead dose.

Besides, these ONA doses produce robust motor effects and somewhat higher doses are even exerting antidepressive effects [6].

Second, it is entirely unclear from where ONA’s chronic migraine effect originates.

It may well be the forehead where the PCB injections were placed.


The enormous ‘placebo effect’ of this study also raises suspicion that the placebo group was actually treated.

As the authors clearly cannot exclude a therapeutic effect in the placebo group, they may then just have merely compared a regular ONA therapy with a low dose ONA therapy.

When the authors are concluding that ONA is not effective, the exact opposite may be true: ONA is not only effective in chronic migraine, it may also be used in medication overuse withdrawal.

Having developed medication overuse to start with may actually be considered a reason to switch from conventional medication to ONA with its excellent adverse effect profile both, on acute and on long-term application.

If this is the strategy, that switch may very well be initiated before conventional medications are withdrawn.


We thank Prof. Dressler for his interest in our study and his honesty to report that he is shareholder of Allergan, the manufacturer of the drug tested in our trial, and that he holds patents on botulinum toxin and botulinum toxin therapy [1].


His letter offers us the opportunity to correct some misconceptions with respect to the perceived efficacy of botulinum toxin A (BTA) in chronic migraine with medication overuse (CM + MO).

It also enables us to correct some apparent misunderstandings with respect to the study design and to present new additional evidence, further supporting the conclusion that BTA does not afford any additional clinical benefit over withdrawal in patients with CM + MO.


Professor Dressler suggests that BTA is an effective treatment for chronic migraine.

We would like to put this claim into perspective.


While statistically significant, the greater reduction in headache days with BTA versus placebo was clinically hardly relevant: 1.8 days from 19.9 days at baseline (510%) [2][3][4][5].


Moreover, unblinding due to BTA-induced removal of facial wrinkling might well have biased outcome.

Up to 90% of participants in similar trials had correctly guessed their treatment, mainly because of the cosmetic effect of the treatment [6][7].


In a recent Cochrane review, the efficacy of BTA was disputed because of the large and unexplained heterogeneity in the studies [8].


To avoid unblinding, we administered 17.5 units of BTA in the forehead of participants in the placebo group.


This is very low compared to the 155 units used in the PREEMPT studies [2][3].


We are not aware of any published evidence that such low doses may afford clinically relevant improvement.


In fact, 17.5 units is significantly lower than the lowest dose ever reported for headache [9][8], psychiatric disorders [7], peripheral neuropathy [10], or regular cosmetic purposes [11].


In animal studies, low doses were clearly less effective [12] or not effective at all [13][14] on putative therapeutic modes of action.


In conclusion, there is no evidence that seven injections with in total 17.5 units BTA in the forehead might have caused a clinically relevant reduction in headache days.


Moreover, if such low doses were indeed effective, why then should we continue to treat patients with 31 injections totaling a 9-fold higher dose?


In his letter, Professor Dressler seems also mistaken with the study design and patient number.


To clarify, all 179 participants were advised to stop all headache medication and were randomly allocated (1:1) to receive BTA (n = 90; 31 injections; in total 155 units) or placebo (n = 89; seven injections with a total of 17.5 units BTA in the forehead and 24 injections with saline elsewhere).


Importantly, blinding was well kept.


BTA did not afford any additional clinical benefit over withdrawal alone [15].


The effect of withdrawal in our study was similar to that in other studies, showing similar proportions of patients reverting from chronic to episodic headache [16][17] or with at least 50% reduction in headache days [18][16][17].


In a recent randomized, controlled, clinical trial, patients with mainly CM + MO had 6.7 fewer migraine days after withdrawal [19].


In a recent review, it was recommended to implement withdrawal in the care of patients with CM + MO [20].


In summary, withdrawal is now recognized as a highly cost-effective therapy [21] leading to substantial reduction of headache days in most patients [17][22][19][20][21].


First described by Sir Astley Cooper in 1845, 10 the precise anatomy of the IMF remains controversial in the literature.

Multiple authors cite that a discrete inframammary crease ligament is found originating from the fifth rib medially to the sixth interspace laterally and inserts into the deep dermis at the level of the IMF.

11-13 Recent anatomic dissections of the breast have identi- fied dermal attachments to the deep and superficial fascia that constitute the foundation of the IMF rather than the presence of a distinct inframammary crease ligament.

These dissections have shown that the superficial fascia splits into a subcutaneous layer anterior to the gland (Camper's fascia) and a superficial fascia layer deep to the gland (Scarpa's fascia).

Below this Scarpa's layer, a layer of deep fascia lies on the anterior surface of the pectoralis major and serratus anterior muscle.

Collagen fibers from the superficial and deep layers of the superficial fascia trav- erse the gland to insert into the dermis at the level of the IMF.

14,15 Even though the anatomy of the IMF may be disputed and these ligamentous structures or fascial confluences may not be fully appreciated clinically, the IMF should still be reinforced for these high-risk patients because of their potential for inferior implant malposition.

A 3-point suture would ideally mimic dermal attachments between the superficial Camper's fascia, Scarpa's fascia, and deep fascia on the anterior surface of the pectoralis.


For the purposes of this study, 3-point suture fixation was only applied to primary subpectoral breast-augmentation pa- tients with an inframammary approach.

Implants, as well as the IMF, were selected based on tissue-based measurements.

4 After pocket dissection and implant placement, 16 a 3-point fixation suture (3-0 vicryl) was placed at the midpoint of the incision between the superficial fascia along the lower aspect of the IMF incision, the superficial fascia along the upper aspect of the IMF incision, and the deep fascia at the IMF (Figure 1).


This fixation was repeated 1 cm lateral and medial to the midpoint, closing the breast pocket.

The implant was protected during all points of fixation.

The 3-point suture fixation created dermal fascial attachments to the underlying deeper fascia, reinforcing the IMF.

Of note, ap- propriately placed 3-point suture fixation will transmit a mild external indentation to the soft tissue that will dissipate over the subsequent 2 weeks.

The remainder of the incision was closed in layers.

A video demonstrating the IMF fixation suture at the IMF of the breast in a primary breast augmenta- tion is available as Supplemental Material.


We have applied this technique from 2010 through 2014 to approximately 600 high-risk primary-breast-augmentation patients in our practice.

All of these patients had at least 1 of the 6 aforementioned preoperative criteria; these pa- tients had increased stress on the IMF and/or iatrogenic weakness of the IMF.

No postoperative complications were reported, and implant malposition was less than 1% in this series.

All high-risk patients seemed to benefit equally from placement of a 3-point suture.

Future investi- gations are needed to determine which patient subtypes have the highest benefit from IMF fixation or if the presence of multiple criteria have a compounded risk of implant malposition.


Since implementing this protocol, no high-risk patients in the clinical practice have been excluded from this surgi- cal algorithm.

All patients have been seen approximately 1 week, 1 month, 3 months, 6 months, and 1 year postoper- atively.

Representative clinical cases are depicted in Figure 2 (constricted pole) and Figure 3 (narrow breast).


This article describes a single surgeon's experience with 3-point suture fixation as a preventive adjunct for implant malposition in primary breast augmentation.

This technique did not increase operative time, implant trauma, operative bleeding, or postoperative recovery.

Postoperatively, patients did not complain of increased pain or IMF distortion.

Long-term studies are needed; but anecdotally, implant mal- position has been less than 1% with this surgical adjunct in this high-risk population.

Prior to 2010, the incidence of implant malposition was 3% in this cohort.


Even though we have not seen any complications from this procedure in our series, this intervention is not 100% atraumatic.

Three-point suture fixation places increased suture material in the incision, possibly leading to a foreign body reaction.

Additionally, the suture causes a temporary fold deformity; however, this deformity subsides after a few weeks.

Furthermore, placement of a deep-fascial suture po- tentially increases the risk of hematoma or implant injury.

As a result of these possible adverse outcomes, we reserve the 3-point suture for high-risk patients.


This article focuses on technical refinements rather than a comparative series; as such, the specifics of patient demographics and/or clinical outcomes were not extrapo- lated.

A longer, larger investigation with detailed patient characteristics is needed to compare our series vs controls.

Despite these limitations, because this technical refine- ment is easily adaptable, sustainable, has minimal risk, and does not increase operative time, reporting the


No increased financial cost was associated with use of this technique.


By preemptively treating breast augmentation patients who are at high risk for IMF deformities, this IMF fixation suture can control the IMF and implant position.

This technical refinement for breast augmentation may help refine results and minimize future complications; however, future studies are needed for definitive conclusions.


Two recent studies describe suture techniques to control the inframammary fold (IMF) [1], [2].

Campbell et al. [ 1] use 3-0 Vicryl (Ethicon, Somerville, NJ) deep fascial sutures to reinforce the IMF, and report no complications in 600 patients and implant malposition in fewer than 1% of patients.

Similarly, Montemurro et al. [ 2] describe a “stable reset” of the IMF, using Quill barbed sutures (Surgical Specialties, Wyomissing, PA).

These authors [2] report that 1.15% of their 436 patients experienced bottoming out and 1.38% had a double bubble.

Campbell et al. [ 1] recognize the need to compare their results with controls.


In the legend to the authors’ Figure 3, Campbell et al. [ 1] state that they repositioned the IMF down to 7.2 cm, although its position next to a pigmented skin lesion appears unchanged.

Montemurro et al. [ 2] state that the existing IMF was lowered 1.5 cm in their clinical example.

This wording suggests that the degree of lowering was controlled and determined by the surgeons.


It is uncertain whether large sutures, such as 0 PDO Quill [2] that take longer to absorb, are advantageous.

Wounds are known to heal by a “one wound” concept [3], not respecting tissue layers.

Scar tissue seems to morph (cheese wire) around sutures.

At reoperation, permanent sutures are typically loose [4].

The strength of the bond depends on the scar tissue, not the suture [5].


To reduce bottoming out, the subpectoral pocket may be dissected slightly high on the chest, anticipating descent of the implant with time (Figure 1).

In many women, the incision may be located about 1 cm cephalad to the IMF, rather than exactly in the IMF as recommended by Montemurro et al. [ 2].

The dissection proceeds obliquely and superiorly to the inferior border of the pectoralis muscle, parallel to the fascial condensations.

It makes sense, whenever possible, to avoid dissection through the fascial condensations and horizontal ligaments [6] deep to the IMF by staying superior to them, rather than disrupting the IMF and then repairing it.

This safe dissection plane also minimizes the risk of the double bubble deformity.

The scar is still well hidden on the underside of the breast and is unlikely to be exposed when the patient wears a bikini top (an occasional problem if the scar is located too low).


Measurements reveal that the IMF drops after breast augmentation (Figure 1) [7].

Three months after surgery it has descended 0.71 cm on average (range, 0.06-1.55 cm) in patients treated with smooth, round, saline-filled implants inserted through an inframammary incision, and no extra reinforcement [7].

Textured implants are designed to adhere to local tissue and resist movement.

However, reports of bottoming out in some patients and photographs suggest that textured implants do settle [2].


In truth, the operator may have little control over the descent of the IMF.

The IMF descends gradually (Figure 1).

A recent cadaveric study introduces rib fixation using absorbable anchors [8].

In view of the dynamic nature of the IMF [7], a static repair may appear unnatural over time.

Reinforcement of the IMF potentially increases the risk of hematoma or implant damage [1], and causes more patient discomfort [2].


Comment is needed regarding inclusion rates.

Both studies claim 100% follow-up 6 months [2] or 1 year after surgery [1].

This is quite extraordinary.

Cosmetic surgery patients are notoriously unreliable in keeping long-term follow-up appointments, particularly for research purposes [9].

A 37% attrition rate at 1 year is typical [9].

Unfortunately, plastic surgeons are still not in the habit of measuring their results using a reference system and standardized photographs (Figure 1).

Only measurements can inform us as to the validity of our treatments, or lack thereof.


In a recent letter to the Aesthetic Surgery Journal, “Can We Really Control the Inframammary Fold (IMF) in Breast Augmentation?” Dr Swanson made claims regarding the inability to control the IMF during primary breast augmentation [1].

We were surprised by the comment and almost decided not to respond, however, we decided to offer the following.


Controlling IMF position and nipple: fold length is the single most important aspect of breast surgery and is often disregarded.

The surgeon, who pays no attention to this philosophy, is sure to have poor results; and the surgeon, who makes this a priority, will enhance results; however, controlling the IMF can never be 100% successful as many variables exist, the most unpredictable being – living tissue.


Our clinical practice has demonstrated an easily adaptable, low-risk, three-point IMF suture fixation technique to reinforce the IMF to decrease the risk of implant malposition in high-risk patients [2].

This technique places a three-point suture between the superficial fascia of the breast envelope, the superficial fascia of the IMF, and the deep fascia of the IMF, mimicking the ligamentous attachments discussed by Muntan and Matousek in independent anatomic studies [3][4].

Conversely, other authors believe the IMF to be purely a superficial fascial structure; and in this case, this 3-point suture is a non-anatomic reinforcement of the IMF.

Additionally, Montemurro et al suggested equivocal success with a low complication profile using barbed sutures to reinforce the IMF in primary breast augmentation [5].

Although our group has limited experience with barbed sutures in primary breast augmentation, the clinical findings of Montemurro et al support the anatomic studies and the success of suture fixation to control the IMF.


Our previous study demonstrated six indications to preemptively control the IMF:


(1) Surgical lowering of the IMF. (2) Utilization of implants 20% larger than recommended by the High-5 process.

(3) Nulliparous women with dense glandular breast tissue.

(4) Narrow breast width (BW < 11 cm).

(5) Shaped form stable implants.

(6) Constricted lower pole or tuberous breasts.


Because of skepticism regarding our lack of follow up and success of controlling the IMF with suture fixation, we have included further images to demonstrate our postoperative outcomes.

Figure 1 demonstrates preoperative and one-year postoperative images of a nulliparous women with dense glandular breast tissue after primary breast augmentation with three-point suture fixation.

Figure 2 demonstrates preoperative and one-year postoperative images of a patient with implants 20% larger than the High-5 system.


The surgeon has many techniques to control the IMF, and to dispute that is simply inaccurate; we recommend this algorithm to all surgical practices not only to enhance pre-surgical planning and patient education but also to optimize postoperative outcomes [6][7].


We aimed to identify all published data relating the presence of fQRS to cardiovascular end points in patients with AMI.

The electronic databases PubMed, MEDLINE, Embase, and Cochrane Li- brary were searched to find primary references and reviews.

Search terms included: fragmented QRS, QRS fragmentation, fQRS, coronary artery disease, CAD, myocardial infarction, MI, mortality, mor- bidity, survival, and prognosis.

These terms were combined with the search algorithm, for example, "fragmented QRS and myocardial infarction." The search was restricted to adults (>18 years of age) in English language peer-reviewed journals from 1960 to October 2015.


Abstracts of the articles published by the American College of Cardiology, the American Heart Association, the European Society of Cardiology, were also searched.

Reviews and reference lists of retrieved articles were hand searched for potentially relevant publication not previously identified in the database search.

The retrieved studies were examined to eliminate po- tential duplicates or overlapping data.

Our analysis is based on the guidelines of the Meta-analysis of Observational Studies in the Epidemiology Group.


Studies recruiting patients with STEMI and/or NSTEMI were included.

Diagnosis of an acute STEMI was made by the presence of new or presumed new ST-segment elevation at the J point in >2 contiguous leads of >0.2 mV in leads V 1 , V 2 , or V 3 , and >0.1 mV in other leads.

Marked ST depression, which was maximal in leads V 1 through V 3 , without ST segment elevation in other leads, was designated as posterior wall MI and included in the STEMI group.

23 Acute NSTEMI was defined by the detection of increases and/or decreases in cardiac biomarkers (troponin I), with >1 value above the 99th percentile of the upper reference limit, together with evidence of myocardial ischemia, which included typical symptoms of myocardial ischemia, electrocardiographic changes indicative of new ischemia (new ST-T changes) or the development of pathologic Q waves on ECG, or imaging evidence (nuclear imaging, echocardiography, or left ven- triculography) of a new loss of viable myocardium or new regional wall motion abnormality.

Studies recruiting patients with stable CAD or patients with unstable angina pectoris (normal cardiac biomarker levels obtained 6-8 hours after presentation) were excluded.

In addition, studies only including patients with ischemic or nonischemic cardiomyopathy, implantable cardioverter defibrillator (ICD), hypertrophic cardiomyopathy, congenital heart disease, Brugada syndrome, long QT syndrome, and Chagas' disease were excluded.


Only the studies that used 12-lead ECG during the index hospitalization of MI for the definition of fQRS were included.

The description of Das for definition of fQRS was searched for in the articles and other definitions such as "QRS distortion" were omitted.

1 Das has defined fQRS as; the presence of an additional R wave (R ) or notching in the nadir of the S wave, or the presence of 1R (fragmentation) in two contiguous leads, corresponding to a major coronary artery territory where the QRS duration <120 ms.

One study was excluded because the ECGs were obtained 2 months after AMI.

8 Persistent QRS fragmentation was defined as presence of QRS fragmentation throughout the hospital stay including the discharge ECG, or the last ECG performed in case of death.

Transient QRS fragmentation was defined as presence of fragmentation in at least one ECG but not in all ECGs recorded during the hospitalization or follow-up period.

Studies using methods other than 12-lead ECG such as vectorcardiography, magnetocardiography, and signal-averaged ECG were also excluded.


The primary outcome of interest was the occur- rence of a first fatal event during the study period.

Major adverse cardiovascular events (MACE) were defined as recurrent myocardial infarction, target vessel revascularization (percutaneous or surgical) or death from these events.


The risk of study bias was evaluated with Quality in Prognosis Studies (QUIPS) tool which includes six domains: participation, attrition, prognostic factor measurement, outcome measurement, study confounding, statistical analysis and reporting.

24 Publication bias was evaluated by generating a funnel plot of the logarithm of effect size against the standard error for each trial.


The significance between two groups was estimated by odds ratio (OR) and weighted mean difference (WMD) with a 2-tailed 95% confidence intervals (CI).

A fixed-effect model was used for homogenous studies, whereas a random-effect model was used for heterogeneous studies.

Statistic I 2 was used to describe the percentage of total across-studies variation due to study-to-study het- erogeneity.

Subgroup analyses were performed to explore and control potential confounders.

A two- sided P value <0.05 was considered statistically significant.

Statistical analysis was performed by using Review Manager 5.0 (The Cochrane Collaboration, Oxford, United Kingdom).


The study selection process is illustrated in Figure 1.

In total, 204 studies (excluding duplicates) were identified by our literature search.

After the exclusion of nonrelevant studies, case reports and reviews by title and abstracts, 41 studies including patients with acute MI were retrieved for further consideration.

In 27 of these studies, data on morbidity/mortality were not reported.

Three studies were excluded as morbidity/mortality data regarding subgroup analysis of STEMI/NSTEMI were not reported.

Finally, 10 studies were included in our systematic review.


The methodological quality of the included studies was generally good, without high risk of bias (Table 1).

In six studies with long-term follow- up, only Lorgis et al. reported data regarding loss to follow-up.

9-12, 16, 17 Six studies reported in-hospital adverse events, thus it may be assumed that no cases were lost to follow-up.

13-18 In addition, it was not clear how three studies accounted for potential confounders, raising the small possibility of result distortion.

13, 15, 18 The funnel plot did not suggest evidence of publication bias (Fig. 2).


The baseline characteristics of ten included studies are shown in Table 2.

The study size differed from 85 to 433 subjects and in total 2766 cases were included in this meta-analysis, 1064 of them were assigned in the fQRS (+) group (38.5%) and 1702 of them were assigned in the fQRS (-) group (61.5%).

Four studies were designed as prospective and others were retrospective trials.

9, 15-17 Male patients were predominantly enrolled in these studies.

The mean age of cases ranged from 54 to 71 years in fQRS (+) groups and 55 to 62 years in the fQRS (-) groups.

studies included patients with both STEMI and NSTEMI (n = 607).

11, 13 In total, 1626 patients (58.7%) had STEMI and 1440 patients had NSTEMI (41.3%).


All of the studies have defined QRS fragmen- tation using ECGs obtained during acute MI and mostly within 48 hours of hospitalization (Table 2).

The rate of QRS fragmentation ranged between 20% and 61% and was 38.5 % in total.

In the STEMI population, fQRS was detected in 568 of 1527 patients (37.2%) and in NSTEMI population, fQRS was detected in 327 of 939 patients (34.8%) which was not statistically different (P = 0.41).


All of the studies except for Sheng et al. reported left ventricular EF in study groups.

In six studies, left ventricular EF was lower in fQRS (+) (ranged between 35% and 45%) compared to fQRS (-) group (ranged between 43% and 50%).

9, 14-18 Whereas, three studies reported an insignificant difference of left ventricular EF between the groups.

10-12 Seven studies reporting left ventricular EF in mean ± standard deviation were analyzed with the random effects approach.

The overall effect showed that patients who had fQRS on admission ECG had a significantly lower LVEF than patients without fQRS (WMD −6.01, 95% CI [−9.08, −2.94], P < 0.00001) (Fig. 3)


In six studies the frequency of 3-vessel disease was reported. 10, 12, 14, 16-18 In four studies, the frequency of 3-vessel disease was higher in fQRS (+) group (ranged between 42% and 61%) compared to fQRS (-) group (ranged between 15% and 33%).

In total, the frequency of 3-vessel disease was 46.4% in fQRS (+) group and was 24.2% in fQRS (-) group (P < 0.01).Whereas, two studies re- ported an insignificant difference between groups regarding multivessel involvement.

12, 17 Most of the patients with STEMI were treated with primary percutaneous coronary intervention (77.6%).


Six studies reported in-hospital mortality 13-18 and six studies reported long-term adverse events.

9mortality rates but reported the outcome as MACE only.


Regarding in-hospital events, mortality was significantly higher in fQRS (+) group (99/733; 13.5%) compared to fQRS (-) group (47/1293; 3.6%) (OR 4.03; 95% CI, 1.81-8.94; P = 0.0006) (Fig. 4).

When only STEMI studies were included in the analysis, mortality was higher in fQRS (+) group (57/479; 11.9%) compared to fQRS (-) group (21/814; 2.6%) (OR 6.01; 95% CI, 2.37-15.23; P = 0.0002).

14-16, 18


Five studies reported mortality rates during a follow up period ranging between 6.6 and 28.2 months.

10-12, 16, 17 The mortality rate was higher in fQRS (+) group (89/473; 18.8%) compared to fQRS (-) group (54/1009; 5.3%) (OR 3.93; 95% CI, 1.92-8.05; P = 0.0002) (Fig. 5).

In addition the frequency of MACE was higher in fQRS (+) group (46.9%) compared to fQRS (-) group (14.6%) (OR 5.13; 95% CI, 2.77-9.51; P < 0.00001) (Fig. 6).

When only 3 NSTEMI studies are included in the analysis, mortality rate was higher in fQRS (+) group (17.7%) compared to fQRS (-) group (5.7%) (OR 3.42; 95% CI, 2.01-5.82; P < 0.00001).

10, 12, 17 When 2 studies which required persistence of fQRS(+) throughout the hospitalization period were excluded, 10, 14 presence of fQRS was still found to be correlated with higher risk of mortality in the remaining study population (OR 3.59; 95% CI, 1.99-6.47; P < 0.00001), in STEMI subgroup (OR 8.08; 95% CI, 2.65-24.65; P < 0.00001), 15, 16, 18 and in NSTEMI subgroup (OR 3.20; 95% CI, 1.46- 6.99; P < 0.00001).

12, 17


presence of fQRS is associated with higher risk of in-hospital and long-term mortality and adverse cardiovascular events in patients with AMI.


This is the first meta-analysis focused on STEMI and NSTEMI.

Two prior meta-analyses included patients with stable/unstable CAD, cardiomyopa- thy and ICD implantation.

19, 20 AMI is the most severe form of CAD which is associated with high risk of left ventricular failure, arrhyth- mias and mortality compared to stable patients.

Fragmentation of QRS has been shown to be correlated with myocardial scar, left ventricular dysfunction and arrhythmias in patients with various cardiovascular diseases.

2, 19 In fact, AMI is the initial event in most of patients that lead to heart failure, arrhythmias and ICD implantation.

Determination of QRS fragmentation during the acute phase of AMI (especially within 48 hours) is feasible and as we have shown in this meta- analysis, presence of fQRS may have clinical value in establishing patients with higher risk for short and long term adverse cardiac events.


In this analysis, we have found that presence of fQRS is correlated with short- and long-term adverse cardiac event regardless of the AMI type.

In addition, a higher portion of patients with fQRS had multivessel disease which may result in incomplete revascularization.

We have found that patients with fQRS (+) have lower LVEF compared to fQRS (-) patients.

This finding is concordant with previous reports which found lower LVEF, and larger left ventricular systolic and diastolic dimensions and volumes in patients with fQRS and CAD.

21, 25 As we included only AMI patients, we can assume that appearance of QRS fragmentation even in the early phase of AMI may indicate larger infarct size and worse LV systolic function.

This correlation may partially explain the worse prognosis in patients with fQRS (+).


The prognostic value of QRS fragmentation during the acute phase of ACS was first proposed by Das et al. 26 In that study, they have reported that fQRS had higher sensitivity for diagnosis of STEMI or NSTEMI compared to Q waves, T-wave inversion or ST-segment depression.

In addition, they have found higher long-term mortality rates in fQRS(+) group compared to fQRS (-) group.

This study was not included in this meta-analysis because the mortality and morbidity rates were not reported in the AMI subgroup.

In another study, Pietrasik et al. reported incidence of QRS fragmentation as 53% in AMI patients after 2 months.

8 They found that presence of QRS fragmentation and/or Q waves was not associated with long term adverse events.

Interestingly, they found that patients with resolved Q waves and persistent fQRS had the highest risk for long-term adverse events.


An important confounding factor in evaluation of QRS fragmentation in CAD patients is the timing of the ECG and persistence of QRS fragmentation throughout the study period.

The studies are heterogeneous and persistence of ECG findings were not reported in all studies.

In this meta- analysis, five studies reported data regarding dura- tion of QRS fragmentation 10, 11, 13, 14, 18 In subgroup analysis, we have found that documentation of QRS fragmentation within 48 hours of hospitalization is adequate and is correlated with worse outcome in STEMI and NSTEMI patients.

The data regarding the rate of persistence of QRS fragmentation after AMI is controversial, thus, to our opinion use of the ECGs obtained within 48 hours of hospitalization is adequate for risk stratification of patients.


The exact mechanism of fQRS on ECG has not been fully elucidated in the literature but most of the studies have concluded the main causative mechanism is cardiac fibrosis and scar- ring.

Presence of QRS fragmentation is accepted as a sensitive marker of myocardial scar after AMI.

2 Myocardial damage causes heterogeneity of my- ocardial segments and a conduction delay around the infarction zone or scar accounts for the reason for fragmentation in QRS.

Even if arrhythmic complications were not reported in detail in this analysis, sudden cardiac death is a major cause of death in patients with CAD and MI.

Thus, presence of fQRS may be correlated with arrhythmic compli- cations and SCD that leads to higher mortality risk.


Several potential limitations of present meta- analysis should be taken into account.

First, there is publication bias on considering only published studies.

In addition, language was restricted to English.

About half of the studies were retrospec- tive which warrants more large scale randomized controlled trials.

The duration of follow-up was not same for all studies.

In addition, it is hard to evalu- ate the additive prognostic value of QRS fragmen- tation besides reduced LVEF in patients with AMI.


Presence of fQRS on admission ECG was a predictor of mortality, MACE, deterioration of LV function and presence of multivessel disease in patients with STEMI and NSTEMI.

Further evaluation of clinical use of QRS fragmentation in patients with AMI are needed to establish the risk of arrhythmic complications and mortality in patients with fQRS.


Dear Editor,


We read with great interest the article by G€ung€or et al. [ 1] in the recent issue of your journal.

In this meta-analysis, the authors showed that presence of fQRS was a predictor of mortality, major adverse cardiovascular events (MACE), deterioration of left ventricle function, and presence of multivessel disease in patients with ST-segment elevation myocardial infarction (STEMI) and non-STEMI (NSTEMI).

We would like to add some comments and contributions to this article.


Development of fQRS is a dynamic process and appears approximately 24–48 hours after the onset of symptoms or after diagnosis and following MI necrosis [2].

Therefore, in the majority of studies, the 48th hour were investigated for the definition of fQRS.

Whereas the importance of the presence of on admission fQRS was investigated in two of the studies included in this meta-analysis [3], [4], in others the importance of the presence of fQRS in the 48th hour electrocardiography (ECG) was investigated.

The authors proposed that fQRS within 48 hours of hospitalization is adequate and correlated with worse outcome in the discussion section.

However, in the conclusion section, they indicated that fQRS on admission ECG was a predictor of mortality, MACE, deterioration of LV function, and presence of a multivessel disease in patients with MI.

Stating as fQRS on admission ECG in the conclusion section of the meta-analysis can cause confusion and misperception on the part of the reader, because the fQRS on admission ECG can disappear within the 48 hour follow-up.

For this reason, we think that it will be more correct to say fQRS developing within 48 hours.