- Email: [email protected]
New genetics of inheritable jaundice and cholestatic liver disease
COMMENTARY
patients were assigned to one of three treatment groups: stent implantation plus placebo, stent implantation plus abciximab, or balloon angioplasty plus abciximab. The primary outcome measure was the rate of death, myocardial infarction, or urgent repeat revascularisation at 30 days. The analysis is presented as two individual paired comparisons, with outcome in the stent+placebo group being compared with that in each of the abciximabtreatment arms. The principal conclusions rest on the stent+placebo vs stent+ abciximab analyses. They provide an appropriate and timely assessment of the impact of abciximab in coronary stenting. The inclusion of the balloon+abciximab group provides additional data, but the absence of a balloon+ placebo group makes it hard to define possible treatment interactions between the stent+placebo and balloon+abciximab groups. For the comparison between stent groups, abciximab therapy was associated with a significant reduction in the primary outcome event rate (5·3% vs 10·8%, p<0·001). The rates of death or large myocardial infarction are reported as a pre-specified secondary outcome measure and were also reduced in the abciximab group (3·0% vs 7·8%, p<0·001). Much of this advantage was related to a striking reduction in the incidence of non-Q-wave myocardial infarction in the immediate post-procedural period. Since definition of these events required at least a five-fold rise in cardiac enzymes, they are likely to be of prognostic significance. The rates of adverse cardiac events were substantial in all groups and higher than those reported for other recent stent trials. This difference probably reflects the more general patient population recruited in EPISTENT (multivessel and vein-graft disease, the various presentations of the acute coronary syndrome, and diffuse and long-segment disease). The routine analysis of cardiac enzymes may have detected some events that might have otherwise gone unrecognised though the vast majority of myocardial infarctions involved overt presentation with pain or electrographic changes. Abciximab therapy was not associated with increased bleeding or vascular complications, and it now seems clear that these adverse effects can be avoided with appropriate concomitant heparin therapy. In their conclusions the EPISTENT investigators declare that, “a new standard of care for the prevention of major adverse ischaemic outcomes with percutaneous coronary revascularisation procedures has emerged”. The prospect of another substantial increment in the procedural costs of coronary intervention must now be acknowledged and its implications addressed by both purchasers and providers of healthcare. We must await the medium-term results and cost-effectiveness analysis from this study. The impact of abciximab on repeat revascularisation will be important. Future clinical studies, perhaps with other IIb/IIIa antagonists and with longer therapy will be essential in the assessment of this potentially important advance.
R H Stables Royal Brompton Hospital,London SW3 6NP, UK 1
2
3
82
Fischman DL, Leon MB, Balm DS, et al. A randomised comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994; 331: 496–501. Serruys PW, de Jaegere P, Klemeneij F, et al. A comparison of balloonexpandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med 1994; 331: 489–95. Schomig A, Neumann FJ, Kastrati A, et al. A randomised comparison
4
5
6
of antiplatelet and anticoagulant therapy after the placement of coronary artery disease. N Engl J Med 1996; 334: 1084–89. The EPIC investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty, the EPIC Investigation. N Engl J Med 1994; 330: 956–61. The EPILOG Investigators. Platelet glycoprotein IIb/IIIA receptor blockade and low-dose heparin during percutaneous coronary revascularization. N Engl J Med 1997; 336: 1689–96. The CAPTURE Investigators. Randomised placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina. Lancet 1997; 349: 435 (correction Lancet 1997; 350: 744).
New genetics of inheritable jaundice and cholestatic liver disease Inheritable disorders characterised by jaundice or cholestasis were described over 50 years ago. In some, such as Crigler-Najjar syndromes I and II, the enzymatic defect in glucuronyl transferase (UDP[B]-GT) was quickly defined. However, in the cholestatic disorders, the molecular defect could not be defined because of lack of knowledge on how bile acids and other organic anions, such as bilirubin glucuronides, are secreted by hepatocytes. The discovery of bile-canalicular ATPdependent transporters for bile acids (sister P glycoprotein, SPGP), other organic anions (MRP2), and phospholipids (MDR3) has enabled the molecular characterisation of several inheritable hyperbilirubinaemias, cholestatic or otherwise. The Crigler-Najjar syndromes I and II are autosomally recessively inherited non-haemolytic unconjugated hyperbilirubinaemias. Type I carries a high risk of kernicterus and, through an unknown mechanism, the hyperbilirubinaemia in type II disease responds to phenobarbitone. Many mutations in UDP(B)GT have been described in different families with either type I or II disease and are responsible for producing the disease.1 Gilbert’s disease, the commonest inheritable unconjugated hyperbilirubinaemia, occurs in about 8% of the population, but the molecular defect, which is an extra TA sequence in the promoter for UDP(B)GT, occurs in 17%.2 Expression of the clinical syndrome, which has a benign course, requires the superimposition of haemolysis, infection, prematurity, or other acquired event. The Dubin-Johnson syndrome is a benign recessively inherited disorder characterised by conjugated hyperbilirubinaemia and defective biliary secretion of non-bileacid organic anions, including bilirubin glucuronides. A point mutation, which results in absence of function of MRP2, an ATP-dependent canicular transporter for these ligands, has been demonstrated.3 Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomally recessively inherited diseases in children. All are characterised by cholestasis associated with raised concentrations of plasma bile acids, defective bile acid secretion, progressive liver damage, and growth failure. On the basis of molecular genetic studies, PFIC has been divided into several categories. The availability of DNA marker sequences (ESTs) generated by the Human Genome Project has enabled the identification of large and frequently inbred families with individual disorders. The findings of the molecular genetic studies overlapped temporally with the discovery of specific ATPdependent transporters in the bile canaliculus. PFIC I (Byler disease) was described in an Old Order Amish pedigree descended from Jacob Byler. The defective gene was mapped to Chr18q21 by the screening of the genome for chromosomal sequences shared by patients
THE LANCET • Vol 352 • July 11, 1998
COMMENTARY
from the original Byler kindred. Probability calculations indicate that such sharing is unlikely to occur by chance. A mutated gene, designated FIC1, was identified and it codes for a P-type membrane ATPase.4 How this defect results in cholestasis and abnormal bile- acid transport is not known. Normal P-ATPases couple hydrolysis of ATP to translocating phosphatidylserine and phosphatidylethanolamine in the plasma membrane of many different cells. Perhaps alterations in hepatic membrane lipids affect the function of ATP-dependent transporters for bile acids and other ligands. Unexpectedly, mutations in FIC1 were also detected in patients with benign recurrent intrahepatic cholestasis (BRIC). This recessively inherited disorder usually begins in childhood and presents with episodic cholestasis of variable severity and duration. During remission, all clinical, laboratory, and morphological findings are normal and the disease is not progressive. Mutated FIC1 was identified by searching for chromosomal sequences shared by only three distantly related patients.3 Although BRIC and PFIC I share some clinical features, the former is progressive whereas the latter is intermittent. How the different phenotypes result from the same genetic defect remains obscure. A second form of PFIC resembles Byler disease clinically but occurs in non-Byler families, mainly in the Middle East and Europe. Homozygosity mapping and genome search in unrelated pedigrees associated the disorder with Chr2q24, from which the gene for SPGP was cloned.5 Remarkably, at almost the same time, SPGP was shown to be a bile-canalicular ATP-dependent bile-acid transporter.6 There are many different SPGP point mutations in patients with PFIC of this type. The genetic studies also indicate that SPGP is a major, if not exclusive, bile-canalicular ATP-dependent bile-acid transporter. A third PFIC group involves mutations in the class III multidrug resistance (MDR) P-glycoproteins (mdr2 in mice and MD3 in man), which mediate ATP-dependent translocation of phosphatidylcholine across the canalicular membrane. Mice with a disrupted mdr2 gene lack phospholipids in bile and develop progressive liver disease that resembles PFIC in man. Two different point mutations in MDR3 resulting in a non-functioning protein have been described in two children with PFIC associated with raised serum γ-glutamyl transpeptidase concentrations.7 In addition, virtually absent MDR3 mRNA has been described in Navajo Indian children with PFIC and neuropathy.8 In mdr2 knockout mice, bile acids are secreted normally and, in the absence of phospholipids, do not form micelles and retain detergent action that results in necrosis and fibrosis of small bileducts. Raised serum ␥-glutamyl transpeptidase concentrations are believed to result from small-bileduct damage and distinguish this PFIC group from PFICI and BRIC. The Alagille syndrome is an autosomal dominant disorder characterised by paucity of small bileducts, intrahepatic cholestasis, and abnormalities of the eye, heart, and vertebrae. Identification of rare patients with cytogenetic deletions results in mapping of the gene to Chr20p12, from which JAG1 was identified. JAG1 encodes for an unidentified ligand that binds to the notch receptor, which is critical for determination of cell fates in early development. Analysis of patients with Alagille syndrome who do not have deletions revealed several mutations in JAG1, each of which abolishes expression of the altered allele.9 Alagille syndrome is the first disease to be described
THE LANCET • Vol 352 • July 11, 1998
at the molecular level that affects development of the hepatobiliary system. These exciting advances illustrate how the Human Genome Project has facilitated the building of the bridge between clinical genetics, physiology, and human disease.The elucidation of the molecular defects permits specific diagnosis, genetic screening for detection of carriers, and understanding of the physiology of hepatocellular secretion. In addition, the door is slowly opening for the treatment of some of the more serious disorders by gene or cell therapy. To date, liver transplantation has been the only effective treatment.
Irwin M Arias Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA 1 2
3
4
5
6
7
8
9
Jansen PCM, Bosma PJ, Roy-Chowdhury J.The molecular biology of bilirubin metabolism. Prog Liver Dis 1995: 13: 125–50. Bosma PJ, Chowdhury JR, Bakker C et al. The genetic basis of te reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med 1995; 333: 1171–75. Paulusma CC, Kool M, Bosma PJ, et al. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 1997; 25: 1539–42. Bull LN, van Eijk, Pawlikowska L, et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998; 18: 219–24. Strautnieks SS, Kagalwalla AF,Tanner MS, et al. Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2p24. Am J Hum Gen 1997; 61: 630–33. Gerloff T, Stleger B, Hagenbuch B, et al.The sister of P glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998; 27: 10046–50. De Vree J, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 1998; 25: 282–87. Zhang X, Arias IM. Deficient MDR3 expression in liver from patients with Navajo neuropathy: a human homologue of md2-/mice. Hepatology 1997; 26: 69A. Oda T, Elkahloun AG, Pike BL, et al. Mutations in human jagged 1 gene are responsible for Alagille syndrome. Nat Genet 1997; 16: 235–42.
What techniques for diagnosis of ventilator-associated pneumonia? The diagnosis of ventilator-associated pneumonia (VAP), a nosocomial infection with substantial morbidity and an attributable mortality varying between zero and 30% in different populations,1,2 remains a challenge. In most instances, the diagnosis is based on non-invasive investigations, non-specific clinical criteria (fever, leucocytosis, new radiographic infiltrate), and isolation of microorganisms by non-quantitative analysis of endotracheal aspirates. This approach leads consistently to overestimation of the incidence of VAP because of inclusion of cases of purulent tracheobronchitis and tracheobronchial colonisation or non-infectious processes mimicking VAP—for example, pulmonary oedema, contusion, atelectasis, pleural effusions, or adult respiratory distress syndrome.3 Therefore, invasive bronchoscopic techniques such as bronchoalveolar lavage (BAL) or the collection of specimens with a protected specimen brush (PSB) have been proposed for quantifying lower-respiratory-tract organisms above a threshold concentration. Unfortunately, these procedures are timeconsuming, require rigorous adherence to bronchoscopic and microbiological techniques, and can impair blood-gas exchange, delay therapy, or lead to a sepsis-like syndrome. Moreover, they are not universally available. Their use in everyday practice remains controversial. Thus there is still no gold-standard for the diagnosis of VAP.4 83
patients were assigned to one of three treatment groups: stent implantation plus placebo, stent implantation plus abciximab, or balloon angioplasty plus abciximab. The primary outcome measure was the rate of death, myocardial infarction, or urgent repeat revascularisation at 30 days. The analysis is presented as two individual paired comparisons, with outcome in the stent+placebo group being compared with that in each of the abciximabtreatment arms. The principal conclusions rest on the stent+placebo vs stent+ abciximab analyses. They provide an appropriate and timely assessment of the impact of abciximab in coronary stenting. The inclusion of the balloon+abciximab group provides additional data, but the absence of a balloon+ placebo group makes it hard to define possible treatment interactions between the stent+placebo and balloon+abciximab groups. For the comparison between stent groups, abciximab therapy was associated with a significant reduction in the primary outcome event rate (5·3% vs 10·8%, p<0·001). The rates of death or large myocardial infarction are reported as a pre-specified secondary outcome measure and were also reduced in the abciximab group (3·0% vs 7·8%, p<0·001). Much of this advantage was related to a striking reduction in the incidence of non-Q-wave myocardial infarction in the immediate post-procedural period. Since definition of these events required at least a five-fold rise in cardiac enzymes, they are likely to be of prognostic significance. The rates of adverse cardiac events were substantial in all groups and higher than those reported for other recent stent trials. This difference probably reflects the more general patient population recruited in EPISTENT (multivessel and vein-graft disease, the various presentations of the acute coronary syndrome, and diffuse and long-segment disease). The routine analysis of cardiac enzymes may have detected some events that might have otherwise gone unrecognised though the vast majority of myocardial infarctions involved overt presentation with pain or electrographic changes. Abciximab therapy was not associated with increased bleeding or vascular complications, and it now seems clear that these adverse effects can be avoided with appropriate concomitant heparin therapy. In their conclusions the EPISTENT investigators declare that, “a new standard of care for the prevention of major adverse ischaemic outcomes with percutaneous coronary revascularisation procedures has emerged”. The prospect of another substantial increment in the procedural costs of coronary intervention must now be acknowledged and its implications addressed by both purchasers and providers of healthcare. We must await the medium-term results and cost-effectiveness analysis from this study. The impact of abciximab on repeat revascularisation will be important. Future clinical studies, perhaps with other IIb/IIIa antagonists and with longer therapy will be essential in the assessment of this potentially important advance.
R H Stables Royal Brompton Hospital,London SW3 6NP, UK 1
2
3
82
Fischman DL, Leon MB, Balm DS, et al. A randomised comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994; 331: 496–501. Serruys PW, de Jaegere P, Klemeneij F, et al. A comparison of balloonexpandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med 1994; 331: 489–95. Schomig A, Neumann FJ, Kastrati A, et al. A randomised comparison
4
5
6
of antiplatelet and anticoagulant therapy after the placement of coronary artery disease. N Engl J Med 1996; 334: 1084–89. The EPIC investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty, the EPIC Investigation. N Engl J Med 1994; 330: 956–61. The EPILOG Investigators. Platelet glycoprotein IIb/IIIA receptor blockade and low-dose heparin during percutaneous coronary revascularization. N Engl J Med 1997; 336: 1689–96. The CAPTURE Investigators. Randomised placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina. Lancet 1997; 349: 435 (correction Lancet 1997; 350: 744).
New genetics of inheritable jaundice and cholestatic liver disease Inheritable disorders characterised by jaundice or cholestasis were described over 50 years ago. In some, such as Crigler-Najjar syndromes I and II, the enzymatic defect in glucuronyl transferase (UDP[B]-GT) was quickly defined. However, in the cholestatic disorders, the molecular defect could not be defined because of lack of knowledge on how bile acids and other organic anions, such as bilirubin glucuronides, are secreted by hepatocytes. The discovery of bile-canalicular ATPdependent transporters for bile acids (sister P glycoprotein, SPGP), other organic anions (MRP2), and phospholipids (MDR3) has enabled the molecular characterisation of several inheritable hyperbilirubinaemias, cholestatic or otherwise. The Crigler-Najjar syndromes I and II are autosomally recessively inherited non-haemolytic unconjugated hyperbilirubinaemias. Type I carries a high risk of kernicterus and, through an unknown mechanism, the hyperbilirubinaemia in type II disease responds to phenobarbitone. Many mutations in UDP(B)GT have been described in different families with either type I or II disease and are responsible for producing the disease.1 Gilbert’s disease, the commonest inheritable unconjugated hyperbilirubinaemia, occurs in about 8% of the population, but the molecular defect, which is an extra TA sequence in the promoter for UDP(B)GT, occurs in 17%.2 Expression of the clinical syndrome, which has a benign course, requires the superimposition of haemolysis, infection, prematurity, or other acquired event. The Dubin-Johnson syndrome is a benign recessively inherited disorder characterised by conjugated hyperbilirubinaemia and defective biliary secretion of non-bileacid organic anions, including bilirubin glucuronides. A point mutation, which results in absence of function of MRP2, an ATP-dependent canicular transporter for these ligands, has been demonstrated.3 Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomally recessively inherited diseases in children. All are characterised by cholestasis associated with raised concentrations of plasma bile acids, defective bile acid secretion, progressive liver damage, and growth failure. On the basis of molecular genetic studies, PFIC has been divided into several categories. The availability of DNA marker sequences (ESTs) generated by the Human Genome Project has enabled the identification of large and frequently inbred families with individual disorders. The findings of the molecular genetic studies overlapped temporally with the discovery of specific ATPdependent transporters in the bile canaliculus. PFIC I (Byler disease) was described in an Old Order Amish pedigree descended from Jacob Byler. The defective gene was mapped to Chr18q21 by the screening of the genome for chromosomal sequences shared by patients
THE LANCET • Vol 352 • July 11, 1998
COMMENTARY
from the original Byler kindred. Probability calculations indicate that such sharing is unlikely to occur by chance. A mutated gene, designated FIC1, was identified and it codes for a P-type membrane ATPase.4 How this defect results in cholestasis and abnormal bile- acid transport is not known. Normal P-ATPases couple hydrolysis of ATP to translocating phosphatidylserine and phosphatidylethanolamine in the plasma membrane of many different cells. Perhaps alterations in hepatic membrane lipids affect the function of ATP-dependent transporters for bile acids and other ligands. Unexpectedly, mutations in FIC1 were also detected in patients with benign recurrent intrahepatic cholestasis (BRIC). This recessively inherited disorder usually begins in childhood and presents with episodic cholestasis of variable severity and duration. During remission, all clinical, laboratory, and morphological findings are normal and the disease is not progressive. Mutated FIC1 was identified by searching for chromosomal sequences shared by only three distantly related patients.3 Although BRIC and PFIC I share some clinical features, the former is progressive whereas the latter is intermittent. How the different phenotypes result from the same genetic defect remains obscure. A second form of PFIC resembles Byler disease clinically but occurs in non-Byler families, mainly in the Middle East and Europe. Homozygosity mapping and genome search in unrelated pedigrees associated the disorder with Chr2q24, from which the gene for SPGP was cloned.5 Remarkably, at almost the same time, SPGP was shown to be a bile-canalicular ATP-dependent bile-acid transporter.6 There are many different SPGP point mutations in patients with PFIC of this type. The genetic studies also indicate that SPGP is a major, if not exclusive, bile-canalicular ATP-dependent bile-acid transporter. A third PFIC group involves mutations in the class III multidrug resistance (MDR) P-glycoproteins (mdr2 in mice and MD3 in man), which mediate ATP-dependent translocation of phosphatidylcholine across the canalicular membrane. Mice with a disrupted mdr2 gene lack phospholipids in bile and develop progressive liver disease that resembles PFIC in man. Two different point mutations in MDR3 resulting in a non-functioning protein have been described in two children with PFIC associated with raised serum γ-glutamyl transpeptidase concentrations.7 In addition, virtually absent MDR3 mRNA has been described in Navajo Indian children with PFIC and neuropathy.8 In mdr2 knockout mice, bile acids are secreted normally and, in the absence of phospholipids, do not form micelles and retain detergent action that results in necrosis and fibrosis of small bileducts. Raised serum ␥-glutamyl transpeptidase concentrations are believed to result from small-bileduct damage and distinguish this PFIC group from PFICI and BRIC. The Alagille syndrome is an autosomal dominant disorder characterised by paucity of small bileducts, intrahepatic cholestasis, and abnormalities of the eye, heart, and vertebrae. Identification of rare patients with cytogenetic deletions results in mapping of the gene to Chr20p12, from which JAG1 was identified. JAG1 encodes for an unidentified ligand that binds to the notch receptor, which is critical for determination of cell fates in early development. Analysis of patients with Alagille syndrome who do not have deletions revealed several mutations in JAG1, each of which abolishes expression of the altered allele.9 Alagille syndrome is the first disease to be described
THE LANCET • Vol 352 • July 11, 1998
at the molecular level that affects development of the hepatobiliary system. These exciting advances illustrate how the Human Genome Project has facilitated the building of the bridge between clinical genetics, physiology, and human disease.The elucidation of the molecular defects permits specific diagnosis, genetic screening for detection of carriers, and understanding of the physiology of hepatocellular secretion. In addition, the door is slowly opening for the treatment of some of the more serious disorders by gene or cell therapy. To date, liver transplantation has been the only effective treatment.
Irwin M Arias Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA 1 2
3
4
5
6
7
8
9
Jansen PCM, Bosma PJ, Roy-Chowdhury J.The molecular biology of bilirubin metabolism. Prog Liver Dis 1995: 13: 125–50. Bosma PJ, Chowdhury JR, Bakker C et al. The genetic basis of te reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med 1995; 333: 1171–75. Paulusma CC, Kool M, Bosma PJ, et al. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 1997; 25: 1539–42. Bull LN, van Eijk, Pawlikowska L, et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998; 18: 219–24. Strautnieks SS, Kagalwalla AF,Tanner MS, et al. Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2p24. Am J Hum Gen 1997; 61: 630–33. Gerloff T, Stleger B, Hagenbuch B, et al.The sister of P glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998; 27: 10046–50. De Vree J, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 1998; 25: 282–87. Zhang X, Arias IM. Deficient MDR3 expression in liver from patients with Navajo neuropathy: a human homologue of md2-/mice. Hepatology 1997; 26: 69A. Oda T, Elkahloun AG, Pike BL, et al. Mutations in human jagged 1 gene are responsible for Alagille syndrome. Nat Genet 1997; 16: 235–42.
What techniques for diagnosis of ventilator-associated pneumonia? The diagnosis of ventilator-associated pneumonia (VAP), a nosocomial infection with substantial morbidity and an attributable mortality varying between zero and 30% in different populations,1,2 remains a challenge. In most instances, the diagnosis is based on non-invasive investigations, non-specific clinical criteria (fever, leucocytosis, new radiographic infiltrate), and isolation of microorganisms by non-quantitative analysis of endotracheal aspirates. This approach leads consistently to overestimation of the incidence of VAP because of inclusion of cases of purulent tracheobronchitis and tracheobronchial colonisation or non-infectious processes mimicking VAP—for example, pulmonary oedema, contusion, atelectasis, pleural effusions, or adult respiratory distress syndrome.3 Therefore, invasive bronchoscopic techniques such as bronchoalveolar lavage (BAL) or the collection of specimens with a protected specimen brush (PSB) have been proposed for quantifying lower-respiratory-tract organisms above a threshold concentration. Unfortunately, these procedures are timeconsuming, require rigorous adherence to bronchoscopic and microbiological techniques, and can impair blood-gas exchange, delay therapy, or lead to a sepsis-like syndrome. Moreover, they are not universally available. Their use in everyday practice remains controversial. Thus there is still no gold-standard for the diagnosis of VAP.4 83