221
Medicine
Chaisson et al
FIGURE 2. Confirmation of anti-MDA-5 antibodies in the
sera of Cases 1 and 2 using an immunoprecipitation assay.
35
S-methionine-labeled MDA-5, generated by in vitro
transcription/translation, was immunoprecipitated using serum
from Cases 1 and 2 (lanes 2 and 3) or 2 different normal controls
(lanes 4 and 5). Input 35S-methionine-labeled MDA-5 (not
subjected to immunoprecipitation) is shown in lane 1. These data
demonstrate that Cases 1 and 2 have anti-MDA-5 antibodies.
ventilation failed. He was transitioned to comfort care and died
shortly after ventilator support was withdrawn. Autopsy revealed
bilateral diffuse alveolar damage and acute interstitial pneumonitis. There was also focal intraalveolar hemorrhage, edema, and
acute inflammation in the left upper lobe. No pulmonary emboli
were identified. Paratracheal lymphadenopathy was noted with
the largest lymph node measuring 4.5 cm in greatest dimension,
benign on histologic examination. No other significant pathologic findings were seen on autopsy.
Postmortem, immunoprecipitation was performed on the
patient’s serum to evaluate the presence of MDA-5 antibody,
given the constellation of clinical findings and the absence of
another cause of rapidly progressive lung disease. Immunoprecipitations performed in our research laboratory using in vitro
transcription/translated 35S-methionine-labeled MDA-5 confirmed
the presence of MDA-5 antibodies in serum from this patient
(Figure 2, Case 1). Cause of death was ultimately determined to
be the result of rapidly progressive ILD, likely secondary to
MDA-5-associated dermato-pulmonary syndrome.
Case 2
A 60-year-old healthy white woman began noticing increasing fatigue, followed 2 weeks later by acute onset of night
&
Volume 91, Number 4, July 2012
sweats, chills, nonproductive cough, and dyspnea. She was treated
with 2 courses of antibiotics over 3 weeks, with improvement in
the dyspnea and chills, but continued fatigue. Two months later,
she noticed skin changes including periorbital edema, and erythema on her forehead, nose, chin, elbows, and axillae, and she
developed symmetric inflammatory arthritis involving her hands,
wrists, shoulders, knees, and ankles. She was started on a steroid taper by her primary care physician and referred for further
evaluation.
Complete blood count and electrolyte panel were unremarkable. ESR was 19 mm/h. TSH, CK, and aldolase were also
unremarkable. Serologies, including ANA, ANCA, RF, antiphospholipid antibody, antihistidyl tRNA synthetase (Jo-1) antibody,
cryofibrinogen, and cryoglobulins, were negative. Mammogram,
Pap smear, and colonoscopy were normal. Steroids were reinitiated at this point with improvement in the arthralgia, but persistence of the rash.
Two months later, the patient was admitted to the hospital
with worsening dyspnea and chest tightness. Chest CT showed
multifocal areas of ground-glass infiltrates. A third course of
antibiotics was prescribed with improvement in her dyspnea. A
prednisone taper starting at 40 mg daily was prescribed given the
persistent rash, arthralgia, myalgia, and fatigue. Only the arthralgia
improved with prednisone, and pulmonary symptoms worsened.
Repeat CT of the chest showed improved bilateral ground-glass
infiltrates compared to the initial scan, but worsening peripheral,
patchy airspace opacities. She also had developed new papules
of the palms and painful fissures at the lateral aspects of the
interphalangeal joints of the hands. Painful ulcers developed on
her wrists, forearms, arms and buttocks. She continued to have
active arthritis. She was started on methotrexate. Six months after
symptom onset, she had lost 60 lb, and her exercise tolerance
was limited to walking a few steps in her house.
She was hospitalized and transferred to our institution
for progressive dyspnea. A third chest CT scan showed multilobar consolidation (Figure 3A). Pulmonary function tests demonstrated a restrictive pattern with severely impaired gas transfer
(forced vital capacity EFVC^ 2.34 L, 69% predicted; forced expiratory volume in the first second EFEV1^ 1.87 L, 70% predicted; total lung capacity ETLC^ 3.79 L, 69.6% predicted;
diffusing capacity of lung for carbon monoxide EDLCO^ 8.75,
41.3% predicted). On examination the patient was afebrile,
normotensive, breathing 18 breaths/min, and was saturating
95% on room air. Her pulse was 98 beats/min. She had diffuse
nonscarring alopecia, upper eyelid edema, livedoid rash on her
FIGURE 3. A, Thoracic CT images with intravenous contrast enhancement showing scattered peripheral interstitial thickening throughout
both lungs and multiple subcentimeter ground-glass pulmonary nodules throughout all lobes of both lungs. B, Cutaneous ulceration
of the dorsal third metacarpophalangeal joint of the left hand.
222
www.md-journal.com
* 2012 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Medicine
&
Volume 91, Number 4, July 2012
chin and nasolabial folds, and V-neck erythema. Erythema was
present over the dorsum of the second to fifth metacarpophalangeal joints bilaterally with an ulceration on the dorsum
of the third left metacarpophalangeal joint (Figure 3B). She had
exquisitely painful ulcers at the tips and lateral borders of
the fingers, as well as a livedoid palmar rash. Palmar papules
were found overlying the second metacarpophalangeal joint as
well as thenar and hypothenar eminences. She had wrist and
elbow ulcers and bilateral painful ulcers on her buttocks. She had
no clinical evidence of weakness. An electromyogram was not
performed.
White blood cell count was 4270. Electrolyte panel was
unremarkable. Sedimentation rate was elevated at 45 mm/h.
Complement C3, complement C4, serum protein electrophoresis
and urine protein to creatinine ratio were normal. Repeat serologies, including a myositis panel (Quest Diagnostics assay), were
unremarkable with exception of ANA of 1:80 in speckled pattern and a positive anti-Ro antibody (ELISA assay). Ferritin was
elevated at 713 ng/mL. Skin biopsies from multiple sites (chest,
ulcers, palms, buttock ulcer) were nonspecific. A skin biopsy
from the chest rash showed rare dying keratinocytes, diffuse
pigment-laden macrophages, and a mild, superficial perivascular and interstitial infiltrate. Wedge biopsies of the lung (lingula,
left superior lower lobe, left inferior lower lobe) showed interstitial fibrosis with honeycombing.
She was treated with prednisone taper starting at 60 mg
daily and azathioprine 75 mg daily, escalated to 150 mg daily.
After 5 months on therapy her energy level improved and she
returned to activities in her home. Pulmonary function tests
showed preserved lung function (FVC 2.29 L, 69% predicted;
FEV1 1.9 L, 71% predicted; TLC 3.55 L, 65% predicted; DLCO
10.75, 44% predicted). The lesions on her fingers and the ulcerative lesions of her wrists and elbows persisted despite therapy, and were complicated by wound infection necessitating
surgical debridement and intravenous antibiotic therapy. No new
skin lesions appeared.
The patient gave written informed consent for further
research antibody testing given her constellation of symptoms
and progressive skin and lung disease. She was found to have antiMDA-5 antibody by immunoprecipitation assay (see Figure 2,
Case 2).
METHODS
We reviewed the MEDLINE database (National Library
of Medicine, Bethesda, MD) for publications between 1966
and July 6, 2011, using the following search terms: ‘‘amyopathic
dermatomyositis,’’ ‘‘clinically amyopathic dermatomyositis,’’ ‘‘dermatomyositis,’’ ‘‘interstitial lung disease,’’ ‘‘MDA-5,’’ ‘‘melanoma
differentiation-associated protein 5,’’ ‘‘anti-CADM-140,’’ and
‘‘anti-CADM-140 antibody.’’ We reviewed only articles written in English, and limited our search to humans. Additionally,
articles were excluded if they did not contain discussion of
clinical features of MDA-5 antibody, and/or only reviewed prior
studies. One study was excluded for insufficient data.36 Abstract and full text reviews were performed independently by
3 authors (NFC, JP, A-MO). Data extraction was performed by
the same 3 authors. Where possible, we attempted to identify
publications that may have used the same patients in duplicate
studies. Immunoprecipitations to confirm the presence of MDA-5
antibodies were performed as described2 using 35S-methioninelabeled MDA-5 that was generated by coupled in vitro transcription/translation from full-length cDNA encoding human
MDA-5 (Origene, Rockville, MD). One KL of serum was used
in each immunoprecipitation, and the immunoprecipitates were
* 2012 Lippincott Williams & Wilkins
MDA-5 Autoantibody and Dermato-Pulmonary Syndrome
visualized by fluorography after electrophoresis on 10% SDSpolyacrylamide gels.
RESULTS
We identified 11 studies that met our inclusion criteria
(Table 1). Most studies were case series or cross-sectional analyses. One case report was included. No clinical trials or prospective analyses were identified. Two pairs of studies likely
included data from similar patient cohorts. All studies but 1
evaluated Japanese or Korean patients; the other study involved
American patients. In all studies but 1, the presence of MDA-5
antibodies was identified only in patients with DM or CADM.
In the remaining study, a patient with systemic sclerosis was
MDA-5 antibody positive.15 Among MDA-5 antibody-positive
patients, 8 studies included data regarding presence of myositis.8,11,13,15,23,27,28,32 Accounting for studies that included overlapping patient cohorts, 80% of MDA-5-positive individuals
were clinically amyopathic (CK G300 and clinically insignificant muscle weakness).
It is notable that MDA-5 is a cytoplasmic protein, whose
expression is interferon (IFN)-regulated. To our knowledge there
are no systematic analyses of whether patients with MDA-5
autoantibodies also express other autoantibodies. The first patient described here was ANA negative, and the second patient
had low ANA titer and a positive anti-Ro antibody.
There was a significant prevalence of ILD (60%Y100%)
among MDA-5 antibody-positive individuals, although criteria
(radiographic changes or restriction on pulmonary function test)
for defining ILD varied by study (see Table 1). A significant
portion of patients (22%Y100%) was classified as having acute
or subacute ILD, characterized by evidence of progression of
ILD within 1 or 3 months, respectively. This was significantly
higher than MDA-negative patients (4%Y20%).8,23,32 One study
evaluated differences in chest CT scan findings between MDA-5
antibody-positive and -negative patients with DM-associated
ILD.32 Patients with MDA-5 antibody tended to have regions
of ground-glass attenuation in random distribution or focally in
the lower lobes of the lung, whereas MDA-5 antibody-negative
patients had predominately reticular changes in the lower lobes.
Significant skin findings in patients with MDA-5 antibodies
included skin ulceration, arthritis, mechanic’s hands, palmar papules, heliotrope rash, Gottron papules, periungual erythema,
and periungual telangiectasias (Table 2). Evidence from a systematic study of patients referred to a dermatology clinic7 indicates that the first 4 clinical findings are significantly more
prevalent in MDA-5 antibody-positive individuals than in MDA5 antibody-negative DM patients.
Six studies reported data on treatment, although most did
not include specific dosing or mention treatment algorithms
employed (Table 3). In all but 1 of the 6 studies,15 prednisone
or prednisolone was used. Other immunosuppressants included
cyclophosphamide, cyclosporine, azathioprine, IVIG, and, in 1
study, methotrexate. Only 1 study reported individual treatment
data.13 In that study, all MDA-5 antibody-positive patients received prednisolone initially, but no specific preference for
treatment could be identified beyond this.
Mortality data were provided in 8 studies.8,10,11,13,15,18,23,32
Accounting for studies that included overlapping patient cohorts,
overall mortality was 38% for MDA-5 antibody-positive patients, over follow-up periods ranging from 6 months to 14 years.
Two studies reported mortality data at 6 months8,23 that were
much higher in MDA-5 antibody-positive patients compared with
MDA-5 antibody-negative patients (57% Erange, 46%Y75%^ vs.
9% Erange, 8%Y9%^). One study that followed patients an
www.md-journal.com
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
223
224
www.md-journal.com
Nested
case-control
Multicenter
cross-sectional
Case series
Case series
Case series
Cross-sectional
27
13
23
11
15
8
Japanese
Japanese
Japanese
Japanese
Japanese
Japanese
Korean
Japanese
American
Japanese
Japanese
DM, CTD
DM
DM, CTD,
SSc with ILD
DM
DM and PM
MDA-5
DM
DM with ILD
DM, CTD,
IPF, healthy
controls
DM, CTD,
IPF, healthy
controls
DM, CTD
Population
Studied
30†
192
65
135
376†
255†
49
2
77
25
294†
30†
13
65
82
376†
42†
38
2
77
25
67†
8† (1/7)
13 (3/10)
14 (3/11)
21 (4/17)
43† (9/34)
8† (2/6)
9 (4/5)
2 (0/2)
10 (3/7)
12 (4/8)
27†‡ (NA)
100 (NA)
NA
NA
100 (50)
100 (92)
100 (100)
100 (77)
100 (84)
100 (53)
95 (91)
100 (75)
45.8 T 16
47.5 T 24.7
51.5 T 8.8
53.5 T 9.4
NA
44.5 T 12.7
53 T NA
57.3 T 9.4
43.6 T 14.6
47 T 15
60.5 T 10.9
75
46
63
14
44
NA
33
50
NA
58
NA
100
92
100*
95
93
88
66
100
60
100*
NA
Total Total Patients No. Patients
Age at
% With
%
Patients
With DM
With MDA-5 Onset (yr) CADM or DM
With
Studied
or CADM
(M/F)
(Mean T SD) (CADM Only) Deaths (%) ILD
100
54
71
79
NA
50
66
100
22
42
NA
% With
ILD and
A/SIP
20
4
NA
NA
NA
NA
NA
NA
5
8
NA
% With
ILD and
A/SIP
MDA-5-Negative
Patients
Abbreviations: A/SIP = acute or subacute interstitial pneumonitis, CTD = connective tissue disease, IPF = idiopathic pulmonary fibrosis, NA = not available; PM = polymyositis, SD = standard deviation,
SSc = systemic sclerosis/scleroderma.
*All patients had ILD as part of the inclusion criteria.
†Indicates possible duplication of patients between studies.
‡Number of patients with positive result by immunoprecipitation.
Cross-sectional
Case report
Case series
Cross-sectional
Cross-sectional
Study Type
18
10
7
32
28
Reference
Population
Demographic
Anti-MDA-5-Positive Patients
TABLE 1. Demographic Characteristics for Both Anti-MDA-5 Antibody-Positive and -Negative Patients, Previous Reports
Chaisson et al
Medicine
&
Volume 91, Number 4, July 2012
* 2012 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Medicine
&
Volume 91, Number 4, July 2012
MDA-5 Autoantibody and Dermato-Pulmonary Syndrome
TABLE 2. Skin Features and Biopsy Results Among Patients Who Were Anti-MDA-5 Positive by Immunoprecipitation
Findings
Gottron Periungual
Periungual
Skin
Palmar
Compatible
Heliotrope Papules Erythema Telanciectasias Ulceration Arthritis Papules Mechanic’s Fever With DM on
Reference Rash (%)
(%)
(%)
(%)
(%)
(%)
(%)
Hands (%) (%) Skin Biopsy (%)
10
7
27
13
23
15
8
100
70
50
56
15
68
38
100
70
75
86
92
68
25
NA
NA
NA
70
NA
NA
63
NA
90
NA
NA
NA
NA
NA
50
80
NA
30
NA
NA
NA
NA
70
50
42
69
NA
NA
NA
60
NA
NA
NA
NA
63
NA
60
NA
NA
NA
NA
NA
NA
NA
NA
74
69
NA
NA
0
NA
100
NA
NA
NA
NA
Abbreviations: See previous table.
average of 588 days reported 7 of 12 patients with MDA-5 antibody died compared to none in the MDA-5 antibody-negative
cohort. In all 8 studies, the majority of deaths in MDA-5 antibodypositive patients were attributable to progressive ILD.
DISCUSSION
DM encompasses a wide spectrum of disease, almost universally involving the skin, and frequently affecting muscle and
lung. The disease phentoypes are complex, with the combination of tissues involved in different individuals varying considerably.30 The disease associated with MDA-5 autoantibodies is
characterized by striking ILD and a distinct cutaneous phenotype (especially ulcers and vasculopathy), but minimal to absent
muscle inflammation and damage. This constellation of damage is suggestive of a common vascular target. The cutaneous and
pulmonary involvement in the absence of myositis leads us to posit
that this unusual dermato-pulmonary syndrome has distinct underlying causes and mechanisms. Although recent studies have
suggested that muscle disease can occur in MDA-5 antibodypositive patients, it is unusual to have significant muscle weakness or elevation of muscle enzymes in this setting.29,32
Detection of commercially available autoantibodies is also
rare in patients with anti-MDA-5. In the 2010 study by Gono
et al,11 only 21% of patients with MDA-5 antibodies had a
positive ANA. However, the presence of cytoplasmic staining by
immunofluorescence was noted in both of our patients and is
TABLE 3. Referenced Therapies Used in Patients With
Anti-MDA-5 Antibodies*
Reference
10
32
13
11
15
8
Therapy
Pulsed MP, MP, CsA, Tac, IVIG, AZA
MP, CsA, Cyc
Pulsed MP, MP, CsA, Cyc, IVIG
MP, Cyc, CI
Pulsed MP, CsA, Cyc, AZA, MTX
MP, immunosuppressants not otherwise defined
Abbreviations: AZA = azathioprine, CI = calcineurin inhibitor not
otherwise specified, CsA = cyclosporin A, Cyc = cyclophosphamide,
IVIG = intravenous immunoglobulin, MP = methylprednisolone, MTX =
methotrexate, Tac = tacrolimus.
*Specific algorithms were not addressed in any study, and individual
data were provided in only 1 study (reference 13).
* 2012 Lippincott Williams & Wilkins
characteristic in patients with MDA-5 antibody.27 In a patient
who presents with ILD, palmar papules, painful skin ulcers and
absent myopathy, absent nuclear staining and presence of cytoplasmic immunofluorescence pattern should raise suspicion for
MDA-5 antibodies.
Cutaneous Manifestations of MDA-5
The hallmark cutaneous manifestations of the DM spectrum include violaceous papules or plaques located on the dorsal aspect of the metacarpophalangeal or interphalangeal joints,
called Gottron papules; erythematous or violaceous macules
in the same distribution, called Gottron macules; and heliotrope
rash, which is a periorbital violaceous erythema. These findings,
which are present in over 70% of individuals with DM,20 occur with similar prevalence in both MDA-5 antibody-positive
and -negative patients. MDA-5 specific skin manifestations include 1) painful, erythematous papules, macules, or both on the
palmar surfaces of the metacarpophalangeal and interphalangeal joints that can have a central, ivory coloration, ulceration,
and sometimes manifest as 2 distinct papules on either side of
the joint; 2) cutaneous ulceration, found in 80% of these patients
compared to G20% of anti-MDA-5-negative DM patients. Ulcers can be found on the elbows and knees, lateral nailfolds, and
within Gottron papules. Patients with MDA-5 antibodies also have
a higher prevalence of tender gums and/or oral erosions compared with MDA-5 antibody-negative patients.7,13 Our 2 patients
both had palmar papules, and Case 2 had painful ulceration of
the hands, wrists, elbows, and buttocks. In neither case could a
clinical diagnosis of classic DM be made with certainty. Additionally, skin biopsies in both patients were nondiagnostic.
MDA-5-Associated Interstitial Lung Disease
The spectrum of ILD in patients with DM can range from
the asymptomatic individual with incidental radiographic or pulmonary function abnormalities, to rapidly progressive and fatal
disease. The reported prevalence of ILD among all patients with
an idiopathic inflammatory myopathy varies widely.5,21 In all
studies of MDA-5-associated disease we identified, the lung was
frequently involved and was often severely affected, highlighting the lung as a central focus in this syndrome. Studies indicate
that ILD progresses more rapidly in those patients with MDA-5
antibodies, with approximately 40% of MDA-5 antibody-positive
patients manifesting rapidly progressive ILD. In the largest group
of MDA-5 antibody-positive patients reported to our knowledge
to date,13 93% had ILD, using results from chest radiography,
chest CT, and pulmonary function tests. Patients were classified
www.md-journal.com
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
225
Medicine
Chaisson et al
as having rapidly progressive ILD if they had worsening dyspnea, hypoxemia, and progression of interstitial changes on radiography within 1 month of onset of symptoms. In general,
MDA-5 antibody-positive patients who have coexistent muscle
disease have a lower mortality than those without muscle disease.11
Both of our patients initially described pulmonary symptoms close
to the time of onset of skin manifestations, with pulmonary
symptoms preceding skin disease in 1 patient, and following
skin disease in the other.
We found only 1 study that looked at specific radiographic
findings in patients with MDA-5 antibodies.32 The findings of
ground-glass attenuation in MDA-5 antibody-positive patients
versus reticular patterns in MDA-5 antibody-negative patients
remains very nonspecific and is unlikely to change management
without further verification of underlying disease pathology by
the clinician. Additionally, these data need to be validated in a
large, blinded cohort before firm conclusions can be made. Pathologic data in patients with MDA-5 antibodies have not been well
described to date either. In 1 of our patients, wedge biopsy revealed usual interstitial pneumonia pattern, while in the other
a diffuse alveolar damage pattern was present. Findings of diffuse alveolar damage are uncommon in patients with CADM and
DM, and in these cases, patients generally have poor response
to therapy and dismal prognosis.22,25
MDA-5 Autoantibodies and Disease Pathogenesis
MDA-5 belongs to the family of retinoic acid-inducible
gene I (RIG-I)-like receptors (RLRs), a family of RNA helicases
that functions as cytoplasmic sensors of pathogen-associated
molecular patterns within viral RNA. To date, 3 RLR members
have been identified: RIG-I, MDA-5, and LGP2 (laboratory of
genetics and physiology 2). While all are broadly expressed in
most tissues, levels are typically low in resting cells but are greatly
increased with IFN exposure and after virus infection.17,34,35
The RLRs drive type 1 IFN production and antiviral gene expression, which mediates the intracellular immune response to
control virus infection. Recent studies suggest that RLRs may
also activate other inflammatory signaling pathways, including
the inflammasome.26 Different RLRs appear to detect infection
preferentially with different viruses. Interestingly, MDA-5 detects
several RNA viruses, including picornaviruses (like polio, coxsackie, and rhinovirus), flaviviruses (Dengue and West Nile), as
well as vaccinia (DNA virus). The striking vascular character of
the skin phenotype in MDA-5 antibody-positive patients and
the rapidly progressive lung disease suggest that blood vessels
are the primary target of the immune response, potentially initially targeted during virus infection, and driven later by type I
IFN pathways. Defining the mechanisms underlying the initiation and propagation of the anti-MDA-5 immune response is a
high priority. Elucidating the sites of MDA-5 expression in the
target tissue (and especially blood vessels at these sites) will be
important in this regard.
It has been noted that resistance to type 1 diabetes in humans is associated with polymorphisms that impair the function
of the mda5 gene, suggesting that the relative strength of induction of type I IFNs may be important in establishing and
propagating autoimmunity.3,24 Since uncontrolled viral infection on the one hand (inadequate viral sensing by MDA-5) or
heightened type I IFN-induced inflammation on the other (strong
MDA-5 function) both have the capacity to render an individual susceptible to tissue damage, it will be of interest to define
whether mda5 polymorphisms are present in these patients,
which either augment or impair MDA-5 expression and function, and thereby regulate downstream type I IFN effects. If augmented MDA-5 signaling and type I IFN activity play a role in
226
www.md-journal.com
&
Volume 91, Number 4, July 2012
the amplified ILD and vascular phenotype seen in this dermatopulmonary syndrome, this might have therapeutic implications
for newly available agents that inhibit type I IFN signaling. Alternatively, if these pathways are decreased in MDA-5 antibodypositive patients, the possibility that increased virus-induced
damage might play a role in initiating lung disease in these
patients could prompt a different set of approaches.
Response to Therapy
Effective therapeutic modalities to treat the MDA-5-associated
dermato-pulmonary syndrome have not yet been identified, and
no data exist comparing the effectiveness of 1 therapy over another. This is likely because this entity has only recently been
described, and it is difficult to diagnose given the current lack of
a widely available MDA-5 antibody assay. While no firm guidelines can be drawn from the literature regarding treatment of
MDA-5 antibody-positive individuals, many experts continue to
treat patients similarly to those who have DM and are MDA-5
antibody negative, usually beginning with corticosteroid therapy. Limited reports in the literature of patients with DM and
rapidly progressive ILD suggest cyclophosphamide and cyclosporine early in the disease may be beneficial.14,33 Cyclosporine
has also been used to treat ulcerative lesions in a patient with
CADM and intractable skin ulceration,31 but these observations
were made before the recognition of the MDA-5-associated entity. The poor outcomes associated with MDA-5 antibodies
strongly suggest that the optimal therapy has not yet been defined. It is possible that rapid, aggressive institution of immunosuppression might avoid irreversible lung injury if diagnosis
is prompt. On the other hand, standard approaches to DM and
associated conditions may not be appropriate for this condition;
instead, more specific therapy targeting type I IFNs, or potentially antiviral therapy if a pathogen is defined, may be more appropriate. In the current study, 1 patient died (Case 1) despite a
trial of IVIG, pulse dose prednisone, and cyclophosphamide.
The other patient (Case 2) received high-dose steroids and was
maintained on prednisone and azathioprine since her diagnosis,
but continued to have significant disease activity.
Outcomes
Data from previous studies show that the presence of
MDA-5 antibody is associated with increased mortality in patients
with DM and CADM compared to those who are MDA-5 negative.8,23,32 Although many studies did not describe the cause
of death, those that did suggested that the majority of individuals succumbed to ILD refractory to immunosuppressive therapy. The presence of MDA-5 antibodies clearly has prognostic
value for those who present with this characteristic dermatopulmonary syndrome. Although the numbers remain small, approximately 40% of MDA-5 antibody-positive patients died,
mostly within the first year. This was very different from MDA-5
antibody-negative patients, where 1-year mortality was 0%, and
overall mortality was G10%. In 2010, Gono et al9 reported
findings in individuals with CADM and ILD suggesting that
increased ferritin levels correlated with faster progression of
ILD. Presence of MDA-5 antibody was not measured in that
study, but studies of individuals with MDA-5 antibodies support the observation that the higher ferritin levels might be associated with increased ILD progression.10,11,32
Conclusion
In the current report, we describe 2 patients with a progressive dermato-pulmonary syndrome that defied diagnosis using
clinically available modalities. Unusual skin features included
* 2012 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Medicine
&
Volume 91, Number 4, July 2012
violaceous papules on the palmar surfaces of the metacarpophalangeal and interphalangeal joints, and painful ulcerative skin
lesions. Inflammatory muscle disease was absent. Standard diagnostic studies, including autoantibodies, skin biopsy, and VATS
lung biopsy, failed to reveal the underlying process. Antibodies
to MDA-5 were present, strongly suggesting that autoimmunity
to MDA-5 plays a role in generating this specific phenotype. In
these settings, when extensive evaluation is otherwise nondiagnostic, clinical suspicion for MDA-5 antibodies should be high,
and further testing should be aggressively pursued.
REFERENCES
1. Anonymous. Ventilation with lower tidal volumes as compared with
traditional tidal volumes for acute lung injury and the acute respiratory
distress syndrome. The Acute Respiratory Distress Syndrome Network.
N Engl J Med. 2000;18:1301Y1308.
2. Casciola-Rosen LA, Pluta AF, Plotz PH, Cox AE, Morris S, Wigley FM,
Petri M, Gelber AC, Rosen A. The DNA mismatch repair enzyme
PMS1 is a myositis-specific autoantigen. Arthritis Rheum.
2001;44:389Y396.
3. Downes K, Pekalski M, Angus KL, Hardy M, Nutland S, Smyth DJ,
Walker NM, Wallace C, Todd JA. Reduced expression of IFIH1 is
protective for type 1 diabetes. PLoS One. 2010;5:e12646.
4. Euwer RL, Sontheimer RD. Amyopathic dermatomyositis
(dermatomyositis sine myositis). Presentation of six new cases and
review of the literature. J Am Acad Dermatol. 1991;24:959Y966.
5. Fathi M, Dastmalchi M, Rasmussen E, Lundberg IE, Tornling G.
Interstitial lung disease, a common manifestation of newly diagnosed
polymyositis and dermatomyositis. Ann Rheum Dis.
2004;56:297Y301.
6. Fathi M, Lundberg IE, Tornling G. Pulmonary complications
of polymyositis and dermatomyositis. Semin Respir Crit Care Med.
2007;28:451Y458.
7. Fiorentino D, Chung L, Zwerner J, Rosen A, Casciola-Rosen L.
The mucocutaneous and systemic phenotype of dermatomyositis
patients with antibodies to MDA5 (CADM-140): a retrospective study.
J Am Acad Dermatol. 2011;65:25Y34.
8. Fujikawa K, Kawakami A, Kaji K, Fujimoto M, Kawashiri S,
Iwamoto N, Aramaki T, Ichinose K, Tamai M, Kamachi M, Nakamura H,
Ida H, Origuchi T, Ishimoto H, Mukae H, Kuwana M, Kohno S,
Takehara K, Sato S, Eguchi K. Association of distinct clinical subsets
with myositis-specific autoantibodies towards anti-155/140-kDa
polypeptides, anti-140-kDa polypeptides, and anti-aminoacyl tRNA
synthetases in Japanese patients with dermatomyositis: a single-centre,
cross-sectional study. Scand J Rheumatol. 2009;38:263Y267.
9. Gono T, Kawaguchi Y, Hara M, Masuda I, Katsumata Y, Shinozaki M,
Ota Y, Ozeki E, Yamanaka H. Increased ferritin predicts development
and severity of acute interstitial lung disease as a complication of
dermatomyositis. Rheumatology (Oxford). 2010;49:1354Y1360.
10. Gono T, Kawaguchi Y, Ozeki E, Ota Y, Satoh T, Kuwana M, Hara M,
Yamanaka H. Serum ferritin correlates with activity of anti-MDA5
antibody-associated acute interstitial lung disease as a complication of
dermatomyositis. Mod Rheumatol. 2011;21:223Y227.
11. Gono T, Kawaguchi Y, Satoh T, Kuwana M, Katsumata Y, Takagi K,
Masuda I, Tochimoto A, Baba S, Okamoto Y, Ota Y, Yamanaka H.
Clinical manifestation and prognostic factor in anti-melanoma
differentiation-associated gene 5 antibody-associated interstitial lung
disease as a complication of dermatomyositis. Rheumatology (Oxford).
2010;49:1713Y1719.
12. Gunawardena H, Betteridge ZE, McHugh NJ. Myositis-specific
autoantibodies: their clinical and pathogenic significance in disease
expression. Rheumatology (Oxford). 2009;48:607Y612.
* 2012 Lippincott Williams & Wilkins
MDA-5 Autoantibody and Dermato-Pulmonary Syndrome
13. Hamaguchi Y, Kuwana M, Hoshino K, Hasegawa M, Kaji K,
Matsushita T, Komura K, Nakamura M, Kodera M, Suga N, Higashi A,
Ogusu K, Tsutsui K, Furusaki A, Tanabe H, Sasaoka S, Muro Y,
Yoshikawa M, Ishiguro N, Ayano M, Muroi E, Fujikawa K,
Umeda Y, Kawase M, Mabuchi E, Asano Y, Sodemoto K, Seishima M,
Yamada H, Sato S, Takehara K, Fujimoto M. Clinical correlations
with dermatomyositis-specific autoantibodies in adult Japanese
patients with dermatomyositis: a multicenter cross-sectional study.
Arch Dermatol. 2011;147:391Y398.
14. Hirakata M, Nagai S. Interstitial lung disease in polymyositis and
dermatomyositis. Curr Opin Rheumatol. 2000;12:501Y508.
15. Hoshino K, Muro Y, Sugiura K, Tomita Y, Nakashima R, Mimori T.
Anti-MDA5 and anti-TIF1-gamma antibodies have clinical significance
for patients with dermatomyositis. Rheumatology (Oxford). 2010;49:
1726Y1733.
16. Kameda H, Nagasawa H, Ogawa H, Sekiguchi N, Takei H, Tokuhira M,
Amano K, Takeuchi T. Combination therapy with corticosteroids,
cyclosporin A, and intravenous pulse cyclophosphamide for acute/
subacute interstitial pneumonia in patients with dermatomyositis.
J Rheumatol. 2005;32:1719Y1726.
17. Kang DC, Gopalkrishnan RV, Lin L, Randolph A, Valerie K,
Pestka S, Fisher PB. Expression analysis and genomic characterization
of human melanoma differentiation associated gene-5, mda-5: a novel
type I interferon-responsive apoptosis-inducing gene. Oncogene.
2004;23:1789Y1800.
18. Kang EH, Nakashima R, Mimori T, Kim J, Lee YJ, Lee EB,
Song YW. Myositis autoantibodies in Korean patients with inflammatory
myositis: anti-140-kDa polypeptide antibody is primarily associated
with rapidly progressive interstitial lung disease independent of
clinically amyopathic dermatomyositis. BMC Musculoskelet Disord.
2010;11:223.
19. Kawachi Y, Maruyama H, Furuta J, Fujisawa Y, Nakamura Y, Takahashi T,
Otsuka F. Cutaneous deep necrosis with dermatomyositis: correlation
with interstitial pneumonia. Eur J Dermatol. 2007;17:345Y346.
20. Kovacs SO, Kovacs SC. Dermatomyositis. J Am Acad Dermatol.
1998;39:899Y922.
21. Labirua A, Lundberg IE. Interstitial lung disease and idiopathic
inflammatory myopathies: progress and pitfalls. Curr Opin Rheumatol.
2010;22:633Y638.
22. Lee CS, Chen TL, Tzen CY, Lin FJ, Peng MJ, Wu CL, Chen PJ.
Idiopathic inflammatory myopathy with diffuse alveolar damage.
Clin Rheumatol. 2002;21:391Y396.
23. Nakashima R, Imura Y, Kobayashi S, Yukawa N, Yoshifuji H,
Nojima T, Kawabata D, Ohmura K, Usui T, Fujii T, Okawa K, Mimori T.
The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific
autoantigen identified by the anti-CADM-140 antibody. Rheumatology
(Oxford). 2010;49:433Y440.
24. Nejentsev S, Walker N, Riches D, Egholm M, Todd JA. Rare variants
of IFIH1, a gene implicated in antiviral responses, protect against
type 1 diabetes. Science. 2009;324:387Y389.
25. Parambil JG, Myers JL, Ryu JH. Diffuse alveolar damage:
uncommon manifestation of pulmonary involvement in patients with
connective tissue diseases. Chest. 2006;130:553Y558.
26. Poeck H, Bscheider M, Gross O, Finger K, Roth S, Rebsamen M,
Hannesschlager N, Schlee M, Rothenfusser S, Barchet W, Kato H,
Akira S, Inoue S, Endres S, Peschel C, Hartmann G, Hornung V,
Ruland J. Recognition of RNA virus by RIG-I results in activation of
CARD9 and inflammasome signaling for interleukin 1 beta production.
Nat Immunol. 2010;11:63Y69.
27. Sato S, Hirakata M, Kuwana M, Suwa A, Inada S, Mimori T,
Nishikawa T, Oddis CV, Ikeda Y. Autoantibodies to a 140-kd polypeptide,
CADM-140, in Japanese patients with clinically amyopathic
dermatomyositis. Arthritis Rheum. 2005;52:1571Y1576.
www.md-journal.com
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
227
Medicine
Chaisson et al
28. Sato S, Hoshino K, Satoh T, Fujita T, Kawakami Y, Fujita T, Kuwana M.
RNA helicase encoded by melanoma differentiation-associated gene 5
is a major autoantigen in patients with clinically amyopathic
dermatomyositis: association with rapidly progressive interstitial lung
disease. Arthritis Rheum. 2009;60:2193Y2200.
29. Sato S, Kuwana M. Clinically amyopathic dermatomyositis.
Curr Opin Rheumatol. 2010;22:639Y643.
30. Shamim EA, Rider LG, Pandey JP, O’Hanlon TP, Jara LJ,
Samayoa EA, Burgos-Vargas R, Vazquez-Mellado J, Alcocer-Varela J,
Salazar-Paramo M, Kutzbach AG, Malley JD, Targoff IN,
Garcia-De la Torre I, Miller FW. Differences in idiopathic inflammatory
myopathy phenotypes and genotypes between Mesoamerican Mestizos
and North American Caucasians: ethnogeographic influences in the
genetics and clinical expression of myositis. Arthritis Rheum.
2002;46:1885Y1893.
31. Shimojima Y, Ishii W, Kato T, Hoshi K, Matsuda M, Hashimoto T,
Tanaka Y, Ikeda S. Intractable skin necrosis and interstitial pneumonia
in amyopathic dermatomyositis, successfully treated with cyclosporin
A. Intern Med. 2003;42:1253Y1258.
32. Tanizawa K, Handa T, Nakashima R, Kubo T, Hosono Y, Watanabe K,
Aihara K, Oga T, Chin K, Nagai S, Mimori T, Mishima M.
228
www.md-journal.com
&
Volume 91, Number 4, July 2012
HRCT features of interstitial lung disease in dermatomyositis with
anti-CADM-140 antibody. Respir Med. 2011;105:1380Y1387.
33. Yamasaki Y, Yamada H, Yamasaki M, Ohkubo M, Azuma K,
Matsuoka S, Kurihara Y, Osada H, Satoh M, Ozaki S. Intravenous
cyclophosphamide therapy for progressive interstitial pneumonia
in patients with polymyositis/dermatomyositis. Rheumatology (Oxford).
2007;46:124Y130.
34. Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, Miyagishi M,
Taira K, Foy E, Loo YM, Gale M Jr, Akira S, Yonehara S, Kato A,
Fujita T. Shared and unique functions of the DExD/H-box helicases RIG-I,
MDA5, and LGP2 in antiviral innate immunity. J Immunol. 2005;175:
2851Y2858.
35. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T,
Miyagishi M, Taira K, Akira S, Fujita T. The RNA helicase RIG-I
has an essential function in double-stranded RNA-induced innate
antiviral responses. Nat Immunol. 2004;5:730Y737.
36. Zahn S, Barchet W, Rehkamper C, Hornung T, Bieber T, Tuting T,
Wenzel J. Enhanced skin expression of melanoma
differentiation-associated gene 5 (MDA5) in dermatomyositis and
related autoimmune diseases. J Am Acad Dermatol. 2011;64:988Y989.
* 2012 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
ˇ˛A b s t r a c t
E u r o p e a n J o u r n a l o f I n t e r n a l M e d i c i n e 1 8 ( 2 0 0 7 ) 3 1 4 3 2 0
O r i g i n a l a r t i c l e
w w w . e l s e v i e r . c o m / l o c a t e / e j i m
A n t i - a n d p r o - o x i d a n t f a c t o r s a n d e n d o t h e l i a l d y s f u n c t i o n i n c h r o n i c c i g a r e t t e s m o k e r s w i t h c o r o n a r y h e a r t d i s e a s e
E . R o c c h i a , N , F . B u r s i c , P . V e n t u r a b , A . R o n z o n i a , C . G o z z i a , G . C a s a l g r a n d i a , L . M a r r i a , R . R o s s i a , M . G . M o d e n a a
a D e p t . M e d i c i n e e S p e c i a l i t a M e d i c h e , C h a i r o f T e r a p i a M e d i c a , U n i v e r s i t a d i M o d e n a e R e g g i o E m i l i a , I t a l y b D e p t . M e d i c i n e e S p e c i a l i t a M e d i c h e , C h a i r o f M e d i c i n a I I , U n i v e r s i t a d i M o d e n a e R e g g i o E m i l i a , I t a l y
c D e p t . E m e r g e n z a - U r g e n z a , C h a i r o f C a r d i o l o g i a , U n i v e r s i t a d i M o d e n a e R e g g i o E m i l i a , I t a l y
R e c e i v e d 2 2 A p r i l 2 0 0 6 ; r e c e i v e d i n r e v i s e d f o r m 1 8 S e p t e m b e r 2 0 0 6 ; a c c e p t e d 1 2 O c t o b e r 2 0 0 6
B a c k g r o u n d : E n d o t h e l i a l d y s f u n c t i o n i n c i g a r e t t e s m o k e r s h a s b e e n a s c r i b e d t o i n c r e a s e d o x i d a t i v e d a m a g e . T h e a i m s o f t h e p r e s e n t s t u d y w e r e t o c o m p a r e t h e e n d o t h e l i a l f u n c t i o n o f n o r m o t e n s i v e s m o k e r s w i t h t h a t o f n o n - s m o k e r s a n d t o e x a m i n e i t s r e l a t i o n t o s o m e p a r a m e t e r s r e p r e s e n t a t i v e o f o x i d a t i v e d a m a g e a n d o f a n t i o x i d a n t c a p a c i t y .
M e t h o d s : W e i n v e s t i g a t e d 3 2 c h r o n i c s m o k e r s ( 1 5 3 0 c i g a r e t t e s d a i l y ) a f f e c t e d b y c o r o n a r y h e a r t d i s e a s e , r a n g i n g f r o m a c u t e m y o c a r d i a l i n f a r c t i o n t o i n s t a b l e a n g i n a p e c t o r i s , a n d 2 8 m a t c h e d n o n - s m o k e r s w i t h o u t a n y d e f i n i t e r i s k f a c t o r s . A l l s u b j e c t s u n d e r w e n t a s s e s s m e n t o f n i t r i c o x i d e ( N O ) - d e p e n d e n t e n d o t h e l i a l f u n c t i o n , m e a s u r e d a s b r a c h i a l a r t e r y v a s o d i l a t a t i o n i n r e s p o n s e t o r e a c t i v e i s c h e m i a , u s i n g a s t a n d a r d i z e d e c h o g r a p h i c m e t h o d . P l a s m a a n d u r i n a r y l e v e l s o f N O w e r e a l s o m e a s u r e d i n a l l s u b j e c t s , a s w e r e u r i n a r y 1 5 - i s o p r o s t a n e F 2 t , p l a s m a s e r u m l i p i d s , h o m o c y s t e i n e ( H c y ) , a s c o r b i c a c i d , r e t i n o l , t o c o p h e r o l , a n d a l p h a - a n d b e t a - c a r o t e n e ( b y h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ) .
R e s u l t s : S m o k e r s s h o w e d a s i g n i f i c a n t l y l o w e r N O - m e d i a t e d v a s o d i l a t a t i o n r e s p o n s e ( 3 . 5 0 % v s . 6 . 1 8 % , p b 0 . 0 0 1 ) a n d h i g h e r l e v e l s o f u r i n a r y N O m e t a b o l i t e s a n d 1 5 - i s o p r o s t a n e F 2 t . T h e y a l s o h a d h i g h e r l e v e l s o f H c y ( p b 0 . 0 0 1 ) ; t h e s e v a l u e s w e r e s i g n i f i c a n t l y a n d i n v e r s e l y r e l a t e d t o N O s e r u m l e v e l s ( r = "0 . 5 1 2 , p b 0 . 0 0 1 ) . M o r e o v e r , s m o k e r s h a d a s i g n i f i c a n t a n d c o r r e s p o n d i n g r e d u c t i o n i n c i r c u l a t i n g l e v e l s o f a s c o r b i c a c i d , t o c o p h e r o l , a n d a l p h a - a n d b e t a - c a r o t e n e .
C o n c l u s i o n s : T h e p r e s e n t s t u d y s h o w s a c l e a r r e l a t i o n b e t w e e n e n d o t h e l i a l d y s f u n c t i o n ( N O p r o d u c t i o n i m p a i r m e n t ) a n d c i g a r e t t e s m o k i n g , e s p e c i a l l y i n t h e p r e s e n c e o f h i g h l e v e l s o f L D L - c h o l e s t e r o l . I t a l s o d e f i n e s s o m e m a r k e r s o f b o t h o x i d a t i v e d a m a g e a n d a n t i o x i d a n t p r o t e c t i v e c a p a c i t y i n t h i s c o n d i t i o n . T h e m o n i t o r i n g o f t h e s e f a c t o r s m a y b e a d v i s a b l e i n o r d e r t o a s s e s s t h e a m o u n t o f e n d o t h e l i a l d a m a g e . © 2 0 0 7 E u r o p e a n F e d e r a t i o n o f I n t e r n a l M e d i c i n e . P u b l i s h e d b y E l s e v i e r B . V . A l l r i g h t s r e s e r v e d .
K e y w o r d s : A n t i o x i d a n t s ; E n d o t h e l i a l d y s f u n c t i o n ; C i g a r e t t e s m o k i n g ; H o m o c y s t e i n e ; I s o p r o s t a n e ; N i t r i c o x i d e
1 . I n t r o d u c t i o n
C i g a r e t t e s m o k i n g r e p r e s e n t s a n i n d e p e n d e n t r i s k f a c t o r f o r c a r d i o v a s c u l a r d i s e a s e [ 1 ] . H o w e v e r , t h e r e l a t i v e r o l e o f n i c o t i n e [ 2 5 ] a n d o f o t h e r f a c t o r s ( h y p e r t e n s i o n , h y p e r c h o - l e s t e r o l e m i a ) [ 6 , 7 ] i n i n d u c i n g v a s c u l a r d a m a g e , a n d p a r t i c -
N C o r r e s p o n d i n g a u t h o r . U n i v e r s i t y o f M o d e n a a n d R e g g i o E m i l i a , D e p t . M e d i c i n e e S p e c i a l i t a M e d i c h e , C h a i r o f T e r a p i a M e d i c a , V i a d e l P o z z o 7 1 , P o l i c l i n i c o d i M o d e n a , I t a l y . T e l . : + 3 9 0 5 9 4 2 2 2 1 5 1 ; f a x : + 3 9 0 5 9 4 2 2 4 3 6 3 .
E - m a i l a d d r e s s : r o c c h i . e m i l i o @ u n i m o r e . i t ( E . R o c c h i ) .
u l a r l y e n d o t h e l i a l d y s f u n c t i o n , i s n o t w e l l - d e f i n e d . T h e l a t t e r , w h i c h i s w i d e l y c o n s i d e r e d t o b e a n i m p o r t a n t h a l l m a r k o f a t h e r o s c l e r o s i s , i s o f t e n t h e f i n a l r e s u l t o f d i f f e r e n t c o m p l e x m e c h a n i s m s , i n v o l v i n g t h e p r o d u c t i o n o f r e a c t i v e o x y g e n s p e c i e s ( f r e e r a d i c a l s a n d p e r o x y l r a d i c a l s ) [ 8 ] a n d l e a d i n g t o r e d u c e d n i t r i c o x i d e ( N O ) e n d o t h e l i a l a v a i l a b i l i t y .
I n t h e v a s c u l a t u r e , e n d o t h e l i a l N O ( E D N O ) p l a y s a k e y r o l e i n m a i n t a i n i n g t h e b l o o d f l o w b y m o d u l a t i n g p h y s i o - l o g i c a l v a s o d i l a t a t i o n [ 9 1 2 ] . A n o t h e r i m p o r t a n t p h y s i o l o g - i c a l r e a c t i o n o f N O i s t h e f o r m a t i o n o f t h i o n i t r i t e s o r S - n i t r o s o t h i o l s ( R S N O s ) [ 1 3 ] . T h e s e a d d u c t s f o r m i n t h e
0 9 5 3 - 6 2 0 5 / $ - s e e f r o n t m a t t e r © 2 0 0 7 E u r o p e a n F e d e r a t i o n o f I n t e r n a l M e d i c i n e . P u b l i s h e d b y E l s e v i e r B . V . A l l r i g h t s r e s e r v e d . d o i : 1 0 . 1 0 1 6 / j . e j i m . 2 0 0 6 . 1 0 . 0 0 6
G r o u p s
N o n - s m o k e r s ( n = 2 8 )
S m o k e r s ( n = 3 2 )
P o u l t r y
1 . 9 2 ± 0 . 1 5 1 . 6 6 ± 0 . 1 1
M e a t
1 . 6 4 ± 0 . 1 5 1 . 7 5 ± 0 . 1 6
L e a f y v e g e t a b l e s
2 . 3 1 ± 0 . 1 6 2 . 1 7 ± 0 . 1 3
T o m a t o e s a n d c a r r o t s
2 . 0 8 ± 0 . 1 6 2 . 0 3 ± 0 . 1 4
N u t s
0 . 9 5 ± 0 . 1 5 1 . 0 5 ± 0 . 1 4
M i l k
2 . 2 4 ± 0 . 2 3 2 . 0 0 ± 0 . 2 0
D a i r y p r o d u c t s
2 . 3 1 ± 0 . 1 2 2 . 5 5 ± 0 . 1 4
E . R o c c h i e t a l . / E u r o p e a n J o u r n a l o f I n t e r n a l M e d i c i n e 1 8 ( 2 0 0 7 ) 3 1 4 3 2 0
3 1 5
T a b l e 1
D i e t a r y r e p o r t i n t h e g r o u p s s t u d i e d , b a s e d o n a n e m p i r i c a l s c o r i n g s y s t e m f o r t h e d i f f e r e n t c l a s s e s
o f n u t r i e n t s
F r u i t
2 . 5 4 ± 0 . 1 6 2 . 5 2 ± 0 . 1 6
V a l u e s f r o m 0 t o 3 , c o r r e s p o n d i n g , r e s p e c t i v e l y , t o a n a b s e n c e o f t h e c l a s s i n t h e w e e k l y d i e t t o a n n u t r i t i o n a l b o a r d .
M e a n ± S . E .
o p t i m a l d i e t a r y i n t a k e , w e r e a s s i g n e d a c c o r d i n g t o a l o c a l
p r e s e n c e o f a t h i o l c o m p o u n d a n d o x y g e n a n d h a v e , i n t u r n , a n i m p o r t a n t r o l e i n r e d u c i n g v a s c u l a r t o n e a n d p l a t e l e t a g g r e g a t i o n [ 1 4 , 1 5 ] . A m o n g t h e d i f f e r e n t k i n d s o f R S N O s , t h e t r a n s f o r m a t i o n o f h o m o c y s t e i n e ( H c y ) , a w e l l - k n o w n i n d e p e n d e n t r i s k f a c t o r f o r a t h e r o t h r o m b o s i s , i n t o S - n i t r o s o - h o m o c y s t e i n e s e e m s r e l e v a n t . T h i s s u b s t a n c e , u n l i k e n a t i v e H c y , d o e s n o t s u p p o r t h y d r o g e n p e r o x i d e g e n e r a - t i o n a n d , h e n c e , c e l l t o x i c i t y , b u t s h o w s , o n t h e c o n t r a r y , w e l l - d e f i n e d a n d i m p o r t a n t v a s o d i l a t i n g a n d i n h i b i t i n g p r o p e r t i e s o f p l a t e l e t a g g r e g a t i o n a n d a d h e s i o n o f l e u k o - c y t e s t o v e s s e l w a l l s [ 1 6 , 1 7 ] . I n t h e p r e s e n c e o f a n e x c e s s o f r e a c t i v e o x y g e n s p e c i e s , t h e r e i s a r e d u c t i o n i n t h e a v a i l a b i l i t y o f e n d o t h e l i a l N O , d u e t o a g r e a t e r f o r m a t i o n o f p e r o x y n i t r i t e s , w h i c h i n d u c e s m a n y d i f f e r e n t d a m a g i n g e f f e c t s t o l i p i d , p r o t e i n , a n d n u c l e i c a c i d m e t a b o l i s m . T h e d e c r e a s e i n N O a v a i l a b i l i t y i n d u c e s d i f f e r e n t p r o - a t h e r o g e n i c e v e n t s , s u c h a s h y p e r - e x p r e s s i o n o f v a s c u l a r c e l l a d h e s i o n m o l e c u l e s ( V - C A M ; I - C A M ) , a n i n c r e a s e i n H c y i n n a t i v e ( a n d h e n c e t o x i c ) f o r m , a n d a r e d u c t i o n i n v a s o d i l a t o r r e s p o n s e [ 1 7 1 9 ] .
F o r t h e s e r e a s o n s , a n a s s e s s m e n t o f p r o - a n d a n t i o x i d a n t f a c t o r s t h a t a r e a b l e t o i n f l u e n c e e n d o t h e l i a l f u n c t i o n , a n d p a r t i c u l a r l y N O a v a i l a b i l i t y , s e e m s r e l e v a n t . I n t h e p r e s e n t s t u d y o f p a t i e n t s a f f e c t e d b y c o r o n a r y h e a r t d i s e a s e ( C H D ) a n d s t r a t i f i e d b y t h e p r e s e n c e / a b s e n c e o f s m o k i n g h a b i t , w e c o m p a r e d t h e f l o w - m e d i a t e d d i l a t a t i o n ( F M D ) i n r e s p o n s e t o r e a c t i v e i s c h e m i a a n d t h e c o r r e s p o n d i n g b i o c h e m i c a l m a r - k e r s o f b o t h o x i d a t i v e d a m a g e a n d a n t i o x i d a n t s t a t u s , i n o r d e r t o e s t a b l i s h w h i c h o f t h e s e m a r k e r s m a y b e m o r e u s e f u l f o r c l i n i c a l , d i a g n o s t i c u s e a n d c o n t r o l i n t e r v e n t i o n s [ 2 0 ] .
2 . M a t e r i a l s a n d m e t h o d s
2 . 1 . P a t i e n t s
T h e s t u d y w a s c o n d u c t e d i n a c c o r d a n c e w i t h t h e p r i n - c i p l e s o f t h e D e c l a r a t i o n o f H e l s i n k i . A l l p a r t i c i p a n t s g a v e t h e i r w r i t t e n i n f o r m e d c o n s e n t b e f o r e t h e s t u d y .
S i x t y s u b j e c t s ( a l l C a u c a s i a n ) w e r e r e c r u i t e d f o r t h e s t u d y . T h i r t y - t w o ( a g e d 5 0 ± 9 y e a r s , m e a n ± S D , 1 3 f e m a l e s ) w e r e c h r o n i c c i g a r e t t e s m o k e r s a n d 2 8 ( a g e d 5 2 ± 8 y e a r s , 1 5 f e m a l e s ) w e r e n o n - s m o k e r s . A l l o f t h e s u b j e c t s h a d p r e - v i o u s l y b e e n a d m i t t e d t o t h e c a r d i o l o g y u n i t o f o u r i n s t i t u - t i o n a n d d i a g n o s e d a s h a v i n g c o r o n a r y h e a r t d i s e a s e ( C H D ) , r a n g i n g f r o m a c u t e m y o c a r d i a l i n f a r c t i o n ( A M I ) t o i n s t a b l e a n g i n a p e c t o r i s ( I A P ) . S m o k i n g h a b i t ( 1 5 3 0 c i g a r e t t e s d a i l y ) w a s t h e s i n g l e o r l a r g e l y p r e d o m i n a n t r i s k f a c t o r i n t h e f i r s t g r o u p ; n o o t h e r d e f i n i t e r i s k f a c t o r s w e r e p r e s e n t i n t h e s e c o n d g r o u p . N o o t h e r d i s e a s e ( d i a b e t e s i n p a r t i c u l a r ) w a s o b s e r v e d i n e i t h e r g r o u p a n d a l l s u b j e c t s h a d s t o p p e d u s i n g d r u g s ( d i u r e t i c s , n i t r a t e s , A C E i n h i b i t o r s , w h e n p r e s e n t , i n s o m e c a s e s ) i n t h e l a s t 4 8 h p r i o r t o t h e s t u d y . T h e d i e t a r y h a b i t s o f t h e s u b j e c t s a r e p r e s e n t e d i n T a b l e 1 .
2 . 2 . A s s e s s m e n t o f b r a c h i a l a r t e r i a l r e a c t i v i t y
A p r e v i o u s l y v a l i d a t e d u l t r a s o u n d s t u d y o f b r a c h i a l a r t e r y r e a c t i v i t y w a s p e r f o r m e d i n a l l p a t i e n t s u p o n e n t e r i n g t h e s t u d y a n d 6 m o n t h s l a t e r u s i n g a n A c u s o n 1 2 8 X P / 1 0 m a i n f r a m e ( A c u s o n , M o u n t a i n V i e w , C a l i f o r n i a , U S A ) w i t h a 7 . 0 M H z l i n e a r - a r r a y t r a n s d u c e r . I m a g e s w e r e s t o r e d o n a s u p e r V H S v i d e o t a p e r e c o r d e r f o r f u r t h e r a n a l y s i s . T h e t e c h n i q u e f o r a s s e s s i n g b r a c h i a l a r t e r y F M D h a s a l r e a d y b e e n d e s c r i b e d i n d e t a i l e l s e w h e r e [ 2 1 , 2 2 ] . T h e m e t h o d o l o g y h a s a n i n t e r - o b s e r v e r v a r i a b i l i t y i n d i a m e t e r m e a s u r e m e n t s o f 0 . 4 5 ± 0 . 2 5 % , y i e l d i n g a c o e f f i c i e n t o f v a r i a t i o n o f 1 . 3 4 % a n d a c o e f f i c i e n t o f r e p e a t a b i l i t y o f 0 . 8 % . T h e s t u d y w a s p e r f o r m e d e a r l y i n t h e m o r n i n g a n d t h e s m o k e r s w e r e a d - v i s e d n o t t o s m o k e .
2 . 3 . C h e m i c a l s
A l l c h e m i c a l s , o f H P L C g r a d e , w e r e p u r c h a s e d f r o m F a r m i t a l i a ( M i l a n , I t a l y ) . A s c o r b i c a c i d , r e t i n o l , D - a l p h a - t o c o p h e r o l , a l p h a - c a r o t e n e , b e t a - c a r o t e n e , t o c o p h e r o l a c e - t a t e , r e t i n o l a c e t a t e ( t h e l a s t t w o a s i n t e r n a l s t a n d a r d s ) ,
T a b l e 2
M a i n l a b o r a t o r y a n a l y t e s i n e a c h g r o u p i n v e s t i g a t e d
G r o u p s A g e ( y e a r s ) N o n - s m o k e r s 5 2 ± 1 . 5
( n = 2 8 )
S m o k e r s 5 0 ± 1 . 7
( n = 3 2 ) M e a n ± S . E .
R B C ◊ 1 0 6 4 . 5 7 ± 0 . 1 0
4 . 4 9 ± 0 . 0 9
H b g / d l 1 3 . 3 2 ± 0 . 2 9
1 3 . 2 9 ± 0 . 3 4
L D L m g / d l 1 4 3 . 9 ± 1 0 . 6
1 7 4 . 7 ± 6 . 9
H D L m g / d l 5 0 . 1 ± 3 . 4
4 8 . 9 ± 3 . 2 5
T G L m g / d l 1 3 5 . 6 ± 2 3 . 4
1 3 4 . 8 ± 1 4 . 3
T S L m g / d l 5 1 9 ± 3 5
5 7 2 ± 2 3
G l u c o s e m g / d l 1 1 0 . 6 ± 5 . 8
1 0 9 . 3 ± 6 . 1
C r e a t i n i n e m g / d l 1 . 0 ± 0 . 0 4
0 . 8 5 ± 0 . 0 3
A l b u m i n g / d l 4 . 0 5 ± 0 . 1 0
4 . 0 3 ± 0 . 0 7
3 1 6 E . R o c c h i e t a l . / E u r o p e a n J o u r n a l o f I n t e r n a l M e d i c i n e 1 8 ( 2 0 0 7 ) 3 1 4 3 2 0
T a b l e 3
P l a s m a a s c o r b i c a c i d , t o c o p h e r o l ( v i t a m i n E ) t o t o t a l s e r u m l i p i d s r a t i o ( E / T S L ) , r e t i n o l , a l p h a - a n d b e t a - c a r o t e n e i n t h e g r o u p s i n v e s t i g a t e d
G r o u p s
N o n - s m o k e r s ( n = 2 8 )
A g e y e a r s
E G T A , g l u t h a t i o n e , o r t h o - p h e n y l e n d i a m i n e , n i t r a t e r e d u c - t a s e , a n d N A D H w e r e o b t a i n e d f r o m S i g m a ( M i l a n , I t a l y ) . A s c o r b a t e o x y d a s e w a s o b t a i n e d f r o m R o c h e ( M a n n h e i m , W e s t G e r m a n y ) . C a d m i u m g r a n u l e s w e r e o b t a i n e d f r o m A l d r i c h ( M i l a n , I t a l y ) . E c h i n e n o n e ( a s a n i n t e r n a l s t a n d a r d f o r c a r o t e n o i d s ) w a s a g e n e r o u s g i f t f r o m H o f f m a n n - L a R o c h e ( B a s e l , S w i t z e r l a n d ) .
2 . 4 . S a m p l e p r e p a r a t i o n
A l l s u b j e c t s , a f t e r a n o v e r n i g h t f a s t i n g , u n d e r w e n t b l o o d w i t h d r a w a l a n d u r i n e c o l l e c t i o n . T h e b l o o d s a m p l e s w e r e a n a l y z e d f o r l i p o s o l u b l e v i t a m i n s ( r e t i n o l , t o c o p h e r o l ) , a l p h a - a n d b e t a - c a r o t e n e , a s c o r b i c a c i d , n i t r i c o x i d e , H c y , a n d o t h e r r o u t i n e a n a l y t e s .
2 . 5 . D e t e r m i n a t i o n o f v i t a m i n s a n d c a r o t e n o i d s
A s c o r b i c a c i d i n p l a s m a w a s a n a l y z e d f o l l o w i n g t h e m e t h o d o f S p e e k e t a l . [ 2 3 ] . T h e a s c o r b i c a c i d c o n c e n t r a - t i o n w a s c a l c u l a t e d f r o m p e a k h e i g h t s w i t h t h e s t a n d a r d w o r k i n g s o l u t i o n a s a r e f e r e n c e . P l a s m a v i t a m i n s a n d c a r o t e n o i d s , a s w e l l a s t h e r a t i o b e t w e e n v i t a m i n E a n d t h e s u m o f t o t a l s e r u m l i p i d s , u s u a l l y e x p r e s s e d i n m g / d l ( E / T S L ) , w e r e a s s e s s e d f o l l o w i n g a p r e v i o u s l y p u b l i s h e d m e t h o d [ 2 4 ] .
2 . 6 . D e t e r m i n a t i o n o f n i t r i c o x i d e
N O w a s d e t e r m i n e d u s i n g G r e e n ' s m e t h o d , w h i c h r e l i e s o n t h e m e a s u r e m e n t o f n i t r i t e s a n d n i t r a t e s s i n c e t h e y a r e o b v i o u s l y t h e m o s t s t a b l e m e t a b o l i t e s [ 2 5 ] .
2 . 7 . D e t e r m i n a t i o n o f u r i n e 1 5 - i s o p r o s t a n e F 2 t
T h e d e t e r m i n a t i o n o f 1 5 - i s o p r o s t a n e F 2 t ( a l s o k n o w n a s 8 - i s o - p r o s t a g l a n d i n F 2 ± o r 8 - e p i - p r o s t a g l a n d i n F 2 ± ) i n u r i n e w a s m a d e a c c o r d i n g t o a c o m m e r c i a l l y a v a i l a b l e e n z y m e - l i n k e d i m m u n o a s s a y ( E L I S A ; M E D . D I A , S . G e r m a n o V e r c e l l e s e , V L , I t a l y ) . B r i e f l y , u r i n e s a m p l e s a r e m i x e d w i t h a n e n h a n c i n g r e a g e n t t h a t e s s e n t i a l l y e l i m i n a t e s i n t e r f e r e n c e s w i t h 1 5 - i s o p r o s t a n e F 2 t , c o n j u g a t e d t o h o r s e - r a d i s h p e r o x y d a s e ( H R P ) f o r b i n d i n g t o a p o l y c l o n a l a n t i b o d y s p e c i f i c f o r 1 5 - i s o p r o s t a n e F 2 t c o a t e d o n t h e m i c r o p l a t e s . T h e H R P a c t i v i t y r e s u l t s i n c o l o r d e v e l o p m e n t w h e n s u b s t r a t e i s a d d e d , w i t h t h e i n t e n s i t y o f t h e c o l o r p r o p o r t i o n a l t o t h e a m o u n t o f 1 5 - i s o p r o s t a n e F 2 t i n t h e s a m p l e s o r s t a n d a r d s [ 2 6 3 0 ] .
5 2 ± 1 . 5 5 0 ± 1 . 7
A s c o r b i c a c i d m m o l / L 4 1 . 2 ± 3 . 1
E / T S L r a t i o 2 . 5 1 ± 0 . 2 5
1 . 9 2 N N N ± 0 . 1 1
R e t i n o l m m o l / L
3 . 0 8 ± 0 . 1 9 2 . 7 6 ± 0 . 1 0
A l p h a - c a r o t e n e n m o l / L 3 4 . 0 ± 6 . 8
2 2 . 3 N N ± 2 . 9
B e t a - c a r o t e n e n m o l / L 1 7 3 ± 2 9 . 1
1 0 5 N N N ± 1 1 . 8
S m o k e r s ( n = 3 2 )
M e a n ± S . E . S t a t i s t i c a l a n a l y s i s ( A N O V A ) : N N p b 0 . 0 1 , N N N p b 0 . 0 0 1 .
3 6 . 5 N N ± 2 . 6
2 . 8 . D e t e r m i n a t i o n o f h o m o c y s t e i n e
B r i e f l y , b o u n d H c y i s r e d u c e d b y d i t h i o t h r e i t o l t o f r e e H c y , w h i c h i s t h e n e n z y m a t i c a l l y c o n v e r t e d t o S - a d e n o s y l - L - h o m o c y s t e i n e ( S A H ) b y S A H - h y d r o l a s e . S a m p l e a n d t r a c e r ( S A H f l u o r e s c e i n ) c o m p e t e f o r a m o u s e m o n o c l o n a l a n t i - S A H a n t i b o d y , a n d t h e i n t e n s i t y o f p o l a r i z e d l i g h t i s m e a - s u r e d . T h e c o n c e n t r a t i o n o f H c y i n p l a s m a i s i n v e r s e l y r e - l a t e d t o t h e f l u o r e s c e n c e p o l a r i z a t i o n [ 3 1 ] . H c y w a s m e a s u r e d b o t h a t b a s e l i n e ( f a s t i n g ) a n d a f t e r a n o r a l m e t h i o n i n e l o a d ( P M L H c y ; 6 h a f t e r 1 0 0 m g / k g m e t h i o n i n e ) .
2 . 9 . L a b o r a t o r y a n d s t a t i s t i c a l a n a l y s i s
O t h e r b i o c h e m i c a l i n d i c e s w e r e d e t e r m i n e d b y s t a n d a r d l a b o r a t o r y m e t h o d s . R e s u l t s a r e e x p r e s s e d a s m e a n ± S . E . M . B o t h i n t e r - g r o u p c o m p a r i s o n s a n d c o m p a r i s o n s b e t w e e n s m o k e r s a n d n o n - s m o k e r s w e r e p e r f o r m e d b y o n e - w a y A N O V A a n d l i n e a r c o e f f i c i e n t c o r r e l a t i o n ( r ) , e m p l o y i n g a n I B M P C c o m p u t e r ( I B M C o r p . A r m o n k , N e w Y o r k , U S A ) .
3 . R e s u l t s
T h e r e s u l t s a r e p r e s e n t e d i n T a b l e s 1 3 a n d F i g s . 1 3 . N o s i g n i f i c a n t d i f f e r e n c e i n d i e t a r y h a b i t s w a s f o u n d b e t w e e n t h e s m o k i n g a n d n o n - s m o k i n g g r o u p s ( T a b l e 1 ) . A c o m p l e t e o v e r v i e w o f t h e r o u t i n e a n a l y s e s p e r f o r m e d i s p r e s e n t e d i n T a b l e 2 . T h e b i o c h e m i c a l r e s u l t s o f t h e s u b j e c t s s t u d i e d , w h o w e r e s t r a t i f i e d a c c o r d i n g t o s m o k i n g h a b i t , o v e r l a p p e d q u i t e a b i t i n b o t h g r o u p s .
W i t h r e g a r d t o t h e p a r a m e t e r s e x p r e s s i n g t h e b i o l o g i c a l a n t i o x i d a n t p o t e n t i a l , w e o b s e r v e d t h a t p l a s m a a s c o r b i c a c i d , E / T S L r a t i o , a n d a l p h a - a n d b e t a - c a r o t e n e w e r e s i g n i f i c a n t l y l o w e r i n t h e s m o k e r s ( T a b l e 3 ) . M o r e o v e r , t h e F M D b y
F i g . 1 . P e r c e n t a g e o f b r a c h i a l a r t e r y f l o w - m e d i a t e d d i l a t a t i o n a f t e r u p p e r a r m o c c l u s i o n i n t h e n o n - s m o k i n g a n d s m o k i n g g r o u p s ( a n a l y s i s o f v a r i a n c e b e t w e e n t h e t w o g r o u p s ) .
v a s c u l a r r e a c t i v i t y w a s a l m o s t h a l v e d i n t h e s m o k i n g g r o u p ( F i g . 1 ) , a n d t h e d i f f e r e n c e b e t w e e n g r o u p s w a s h i g h l y s i g n i f i c a n t ( p b 0 . 0 0 1 ) . I n o r d e r t o c o n f i r m t h e h y p o t h e s i s o f a n E D N O - d e p e n d e n t i m p a i r e d v a s c u l a r r e s p o n s e , w e m e a s u r e d N O m e t a b o l i t e s i n b l o o d a n d u r i n e , o b t a i n i n g h i g h e r v a l u e s o f N O i n p l a s m a a n d l o w e r v a l u e s i n u r i n e o f n o n - s m o k e r s w i t h r e s p e c t t o s m o k e r s , a l t h o u g h w i t h o u t a c l e a r s i g n i f i c a n t d i f f e r e n c e ( F i g . 2 ) . M o r e o v e r , t h e i n c r e a s e i n u r i n a r y N O i n t h e s m o k i n g g r o u p s e e m e d q u a n t i t a t i v e l y r e l a t e d t o t h e n u m b e r o f c i g a r e t t e s s m o k e d d a i l y . T h e i s o p r o s t a n e l e v e l s w e r e a l s o h i g h e r i n t h e u r i n e o f s m o k e r s ( p b 0 . 0 0 1 ) , s u p p o r t i n g t h e i d e a o f a p o s s i b l e e f f e c t o f f r e e o x y g e n r a d i c a l s i n i n d u c i n g a r a c h i d o n i c a c i d m e t a b o l i t e s , s u c h a s i s o p r o s t a n e . F i n a l l y , H c y , w h i c h w a s n o t s i g n i f i - c a n t l y d i f f e r e n t b e t w e e n t h e t w o g r o u p s a t b a s e l i n e , s h o w e d a h i g h l y s i g n i f i c a n t i n c r e a s e ( p b 0 . 0 0 1 ) a f t e r t h e m e t h i o n i n e l o a d ( P M L H c y ) i n t h e s m o k i n g g r o u p ( F i g . 2 ) . W h e n p l a s m a P M L H c y w a s p l o t t e d a g a i n s t p l a s m a N O l e v e l s , a h i g h l y
F i g . 2 . A ) U r i n a r y a n d s e r u m n i t r i c o x i d e ( N O ) ; B ) u r i n a r y i s o p r o s t a n e ; a n d C ) p l a s m a h o m o c y s t e i n e ( b o t h b a s e v a l u e : H c y a n d p o s t - m e t h i o n i n e l o a d ; P M L H c y ) i n t h e n o n - s m o k i n g a n d s m o k i n g g r o u p s ( a n a l y s i s o f v a r i a n c e b e t w e e n t h e t w o g r o u p s ) .
F i g . 3 . D i f f e r e n t l i n e a r r e g r e s s i o n i n d i c e s b e t w e e n p l a s m a h o m o c y s t e i n e p o s t - m e t h i o n i n e l o a d ( P M L H c y ) a n d n i t r i c o x i d e ( N O ) , t o t a l s e r u m l i p i d s ( T S L ) , a n d v i t a m i n E t o t o t a l s e r u m l i p i d r a t i o ( E / T S L ) , r e s p e c t i v e l y .
s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n w a s o b s e r v e d ( F i g . 3 ) . S i m i l a r l y , a s i g n i f i c a n t d i r e c t c o r r e l a t i o n b e t w e e n P M L H c y a n d T S L a n d a t r e n d t o w a r d s a n i n v e r s e c o r r e l a t i o n b e t w e e n P M L H c y a n d E / T S L w e r e o b s e r v e d ( F i g . 3 ) . F i n a l l y , i n o r d e r t o e v a l u a t e t h e r e l a t i v e r o l e o f L D L - c h o l e s t e r o l l e v e l s w i t h r e s p e c t t o s m o k i n g h a b i t , w e s t r a t i f i e d b o t h t h e s m o k e r s a n d n o n - s m o k e r s a c c o r d i n g t o n o r m a l o r h i g h L D L - c h o l e s t e r o l s e r u m l e v e l s , o b t a i n i n g a s i g n i f i c a n t d i f f e r e n c e o n l y i n p l a s m a N O , w h i c h w a s r e d u c e d i n p r o p o r t i o n t o t h e L D L i n c r e a s e , b u t n o t i n F M D ( a s p e r c e n t a g e ) o r o t h e r s i g n i f i c a n t p a r a m e t e r s , w i t h i n b o t h t h e s m o k i n g a n d n o n - s m o k i n g g r o u p s .
4 . D i s c u s s i o n
W e c o m p a r e d t w o g r o u p s t h a t w e r e s i g n i f i c a n t l y h o m o g e n e o u s w i t h r e g a r d t o t h e t y p e o f i l l n e s s a n d l i f e
E . R o c c h i e t a l . / E u r o p e a n J o u r n a l o f I n t e r n a l M e d i c i n e 1 8 ( 2 0 0 7 ) 3 1 4 3 2 0 3 1 7
3 1 8 E . R o c c h i e t a l . / E u r o p e a n J o u r n a l o f I n t e r n a l M e d i c i n e 1 8 ( 2 0 0 7 ) 3 1 4 3 2 0
a n d a l i m e n t a r y h a b i t s , w i t h s m o k i n g c o n s i d e r e d a s t h e d i s c r i m i n a t i n g p a r a m e t e r . F M D , b y v a s c u l a r r e a c t i v i t y , w a s s i g n i f i c a n t l y r e d u c e d i n s m o k e r s . S i m i l a r o b s e r v a t i o n s w e r e m a d e w i t h r e g a r d t o N O , l e v e l s o f w h i c h w e r e h i g h e r i n n o n - s m o k e r s t h a n i n s m o k e r s . O n t h e o t h e r h a n d , b o t h t h e d e c r e a s e i n N O i n p l a s m a a n d t h e i n c r e a s e i n p l a s m a n i t r i t e s , t h a t w a s o b s e r v e d i n t h e s m o k e r s , s e e m t o s u g g e s t t h a t p l a s m a N O i s m o r e q u i c k l y m e t a b o l i z e d a s a r e s u l t o f h i g h e r o x i d a t i v e d a m a g e ( g r e a t e r p r o d u c t i o n o f r e a c t i v e o x y g e n s p e c i e s c a p a b l e o f i n a c t i v a t i n g N O ) i n t h i s l a s t g r o u p , a n d t h i s s e e m s q u a n t i t a t i v e l y r e l a t e d t o t h e n u m b e r o f c i g a r e t t e s s m o k e d d a i l y .
W e c h o s e t o a s s e s s i s o p r o s t a n e ( 1 5 - i s o p r o s t a n e F 2 t , a l s o k n o w n a s 8 - i s o - p r o s t a g l a n d i n F 2 ±) i n o r d e r t o b e t t e r e v a l u a t e t h e a m o u n t o f o x i d a t i v e d a m a g e , a s i t i s t h e f i n a l a n d s t a b l e p r o d u c t f o r m e d a s a r e s u l t o f t h e a c t i v a t i o n o f a n a l t e r n a t i v e p a t h w a y ( i . e . , d i f f e r e n t f r o m t h e o n e l e a d i n g t o t h r o m b o x a n e a n d p r o s t a g l a n d i n p r o d u c t i o n ) o f a r a c h i d o n i c a c i d m e t a b o - l i s m i n t h e p r e s e n c e o f r e a c t i v e o x y g e n s p e c i e s [ 2 6 ] . U r i n a r y 1 5 - i s o p r o s t a n e F 2 t w a s s i g n i f i c a n t l y i n c r e a s e d ( F i g . 3 ) i n s m o k e r s . A r e c e n t s t u d y , a d d r e s s i n g t h i s t o p i c i n d e e d , c o n - f i r m e d i t s r o l e a s a n i n d e p e n d e n t r i s k m a r k e r o f C H D [ 3 2 ] . W i t h r e g a r d t o H c y , w h i c h i s a l s o c o n s i d e r e d a n i n d e p e n d e n t r i s k f a c t o r o f v a s c u l a r d a m a g e , w e d i d n o t o b s e r v e a n y s i g n i f i c a n t d i f f e r e n c e i n t h e b a s e l i n e v a l u e s o f t h i s p a r a m e t e r b e t w e e n t h e t w o g r o u p s ; n e v e r t h e l e s s , P M L H c y v a l u e s w e r e s i g n i f i c a n t l y h i g h e r i n s m o k e r s , s u g g e s t i n g t h e e x i s t e n c e o f a s i g n i f i c a n t a l t e r a t i o n i n H c y m e t a b o l i s m i n t h e s e s u b j e c t s ( F i g . 3 ) . T h i s o b s e r v a t i o n i s i n a g r e e m e n t w i t h t h e d a t a c o n c e r n i n g a l i n k a g e b e t w e e n s m o k i n g a n d d e r a n g e m e n t o f H c y m e t a b o l i s m [ 3 3 , 3 4 ] a n d t h a t n o t o n l y m o d e r a t e l y h i g h b a s e l e v e l s o f H c y , b u t a l s o h i g h l e v e l s o f P M L H c y a l o n e , r e p r e s e n t a n i n d e p e n d e n t r i s k f a c t o r f o r v a s c u l a r d a m a g e [ 3 5 ] . I t i s l i k e l y t h a t i n s m o k e r s , w h e r e N O a v a i l a b i l i t y i s r e d u c e d , t h i s r e s u l t s i n a f a i l u r e o f N O - i n d u c e d d e t o x i f i c a - t i o n o f H c y a n d o f o t h e r H c y m e t a b o l i t e s w i t h t o x i c ( i . e . , v a s o c o n s t r i c t i v e ) e f f e c t s [ 3 6 , 3 7 ] . T h i s c o n s i d e r a t i o n i s s u p p o r t e d b y t h e i n v e r s e a n d s i g n i f i c a n t c o r r e l a t i o n w e o b s e r v e d b e t w e e n P M L H c y a n d N O ( F i g . 3 ) . N O a v a i l a b i l i t y p r o t e c t s f r o m e n d o t h e l i a l d a m a g e i n d u c e d b y H c y , a s N O s u p p l i e s a m e t a b o l i c p a t h w a y ( t h r o u g h t h e f o r m a t i o n o f S - n i t r o s o - h o m o c y s t e i n e ) t h a t i s a b l e t o p r o t e c t a g a i n s t H c y - i n d u c e d e n d o t h e l i a l o x i d a t i v e d a m a g e . T h i s p a t h w a y m a y b e c o m e e x h a u s t e d r a t h e r q u i c k l y i n t h e p r e s e n c e o f e x - c e s s i v e l y h i g h l e v e l s o f H c y o r e x c e s s i v e l y l o w l e v e l s o f N O , o r b o t h [ 1 6 , 3 8 ] .
T h e p a r a m e t e r s o f o x i d a t i v e d a m a g e ( p a r t i c u l a r l y u r i n a r y i s o p r o s t a n e a n d P M L H c y ) s h o w e d s i g n i f i c a n t a n d c o r r e s p o n d i n g r e s u l t s . T h u s , t h e p r e s e n t s t u d y c o n f i r m s w h a t i s k n o w n f r o m t h e l i t e r a t u r e , b u t w h a t a b o u t t h e a n t i o x i d a n t c a p a c i t y ? I n o r d e r t o e v a l u a t e t h i s i s s u e , w h i c h h a s n o t b e e n e x t e n s i v e l y e x p l o r e d i n c o n j u n c t i o n w i t h t h e d a t a p r e s e n t e d , w e a s s e s s e d t h e m o s t r e p r e s e n t a t i v e p a r a m e t e r s o f a n t i o x i d a n t a c t i v i t y , e x p r e s s i n g t h e a n t i o x i d a n t p o w e r o f b o t h t h e i n t r a - a n d e x t r a c e l l u l a r l i p i d p h a s e ( c a r o t e n o i d s a n d l i p o s o l u b l e v i t a m i n s ) a n d o f t h e i n t e r s t i t i a l
a q u e o u s p h a s e ( v i t a m i n C ) . V i t a m i n A l e v e l s s e e m e d b a r e l y r e d u c e d i n s m o k e r s , a s e x p e c t e d , f o r i t i s k n o w n t h a t p l a s m a c i r c u l a t i n g v i t a m i n A , w h i c h i s b o u n d t o r e t i n o l - b i n d i n g p r o t e i n ( R B P ) , i s p r e s e n t i n s m a l l a m o u n t s . I n c o n t r a s t , t h e v i t a m i n A s t o r e d i n l i v e r , a s p a l m i t i c e s t e r ( a b o u t 8 0 % o f t h e t o t a l a m o u n t o f v i t a m i n A i n t h e b o d y ) , i n c r e a s e s [ 2 4 ] . C i r c u l a t i n g v i t a m i n A i s p r o b a b l y m o r e i n d i c a t i v e o f l i v e r p r o t e i n s y n t h e s i s c a p a c i t y ( a s R B P ) t h a n o f v i t a m i n A b o d y c o n t e n t [ 3 9 ] . T h i s i s d i f f e r e n t f o r v i t a m i n A p r e c u r s o r s ( a l p h a - a n d b e t a - c a r o t e n e ) , a s t h e s e c o m p o u n d s a r e n o t s t o r e d i n t i s s u e s . T h e i r p l a s m a l e v e l s a r e g o o d i n d i c a t o r s o f t h e i r r e a l b o d y a v a i l a b i l i t y . O u r d a t a s h o w a s i g n i f i c a n t d e c r e a s e i n v i t a m i n A p r e c u r s o r s i n t h e s m o k i n g g r o u p , e s p e c i a l l y w i t h r e g a r d t o b e t a - c a r o t e n e ( T a b l e 3 ) . A l s o , v i t a m i n C l e v e l s w e r e r e d u c e d i n s m o k e r s . I t i s w e l l - k n o w n t h a t t h i s c o m p o u n d n o t o n l y h a s i m p o r t a n t , d i r e c t a n t i o x i d a n t a c t i v i t y i n t h e i n t e r s t i t i a l a q u e o u s d i s t r i c t [ 4 0 ] b u t i t a l s o e x e r t s a n i m p o r t a n t p r o t e c t i v e e f f e c t o n v i t a m i n E , p r o b a b l y t h e m o s t i m p o r t a n t a n t i o x i d a n t i n t h e l i p i d p h a s e [ 2 4 , 3 3 , 4 1 , 4 2 ] . V i t a m i n E ( t o c o p h e r o l ) l e v e l s w e r e s i m i l a r i n t h e t w o g r o u p s ; y e t , t h e a n t i o x i d a n t e f f e c t o f t o c o p h e r o l i s d i r e c t l y r e l a t e d t o l i p o p r o t e i n s , b y w h i c h i t i s c a r r i e d , p r o t e c t i n g t h e m f r o m o x i d a t i o n [ 4 3 ] . F o r t h i s r e a s o n , t h e c i r c u l a t i n g l e v e l s o f v i t a m i n E s h o u l d b e c o n s i d e r e d a s r e f e r r i n g t o c i r c u l a t i n g l i p i d l e v e l s a n d e x p r e s s e d a s E / T S L r a t i o . A s s h o w n i n T a b l e 3 , t h e E / T S L r a t i o w a s l o w e r i n s m o k e r s t h a n i n n o n - s m o k e r s [ 4 4 , 4 5 ] . T h e o b s e r v a t i o n o f a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n P M L H c y l e v e l s a n d t o t a l l i p i d s ( F i g . 3 ) s u p p o r t s t h e r e s u l t s o f t h e p r e s e n t s t u d y , c o n c e r n i n g a s i g n i f i c a n t l i n k a g e b e t w e e n d i f f e r e n t w e l l - k n o w n c a r d i o v a s c u l a r r i s k f a c t o r s ( H c y , L D L - c h o l e s t e r o l ) . N O c o n s u m p t i o n a n d , h e n c e , e n d o t h e l i a l d y s f u n c t i o n r e p r e - s e n t , i n o u r o p i n i o n , t h e f i n a l c o m m o n m e c h a n i s m o f d a m a g e o f a l l t h e s e f a c t o r s , t h r o u g h f r e e r a d i c a l g e n e r a t i o n [ 3 6 , 4 6 ] .
O n t h e o t h e r h a n d , o u r f i n d i n g s a l s o s u g g e s t a r o l e f o r a n t i o x i d a n t c o m p o u n d s i n t h e p r e v e n t i o n o f d a m a g e i t s e l f , p a r t i c u l a r l y f r o m t h e i n v e r s e t r e n d o f v i t a m i n E ( e x p r e s s e d a s E / T S L r a t i o ) w i t h r e s p e c t t o H c y l e v e l s ( F i g . 3 ) . O b v i o u s l y , f u r t h e r c a s e s c o u l d b e t t e r h i g h l i g h t s u c h a f i n d i n g . H o w e v e r , t h e p r e s e n t s t u d y d o e s , i n f a c t , c o n f i r m t h e p r o t e c t i v e e f f e c t s o f b o t h v i t a m i n C a n d v i t a m i n E a g a i n s t t h e o x i d a t i v e d a m a g e p r o m o t e d b y H c y [ 4 7 , 4 8 ] . A s a p r a c t i c a l c o n s e - q u e n c e , a l l o f t h e s e v i t a m i n f a c t o r s n e e d t o b e e v a l u a t e d i n c o n j u n c t i o n w i t h t h e m a r k e r s o f o x i d a t i v e d a m a g e .
I n s u m m a r y , t h e p r e s e n t s t u d y , w h i l e c o n f i r m i n g t h a t t h e e n d o t h e l i a l d y s f u n c t i o n , i n d u c e d b y c h r o n i c e x p o s u r e t o c i g - a r e t t e s m o k i n g , i s c l e a r l y d u e t o o x i d a t i v e d a m a g e , o u t l i n e s a s w e l l t h e i n v e s t i g a t i o n s t h a t a r e o f c l i n i c a l v a l u e . T h i s d a m a g e m a y , i n f a c t , b e a s s e s s e d b o t h d i r e c t l y b y s p e c i f i c b i o c h e m i - c a l m a r k e r s ( p a r t i c u l a r l y u r i n a r y N O a n d i s o p r o s t a n e ) a n d i n d i r e c t l y b y H c y l e v e l s a f t e r a m e t h i o n i n e l o a d . I t i s a l s o a s s o c i a t e d w i t h a r e d u c t i o n i n a n t i o x i d a n t c a p a c i t y ( b o t h h y d r o - a n d l i p o s o l u b l e f a c t o r s ) . B o t h o f t h e s e p a r a m e t e r s s h o u l d b e m e a s u r e d i n a n a c c u r a t e c l i n i c a l e v a l u a t i o n i n o r d e r t o d e f i n e t h e l e v e l o f d a m a g e a n d p o s s i b l e s t r a t e g i e s f o r i t s p r e v e n t i o n .
5 . L e a r n i n g p o i n t s
" T h e p r e v i o u s l y v a l i d a t e d u l t r a s o u n d s t u d y o f b r a c h i a l a r t e r y r e a c t i v i t y t o i s c h e m i a a p p e a r s t o b e a s u i t a b l e m e t h o d f o r s t u d y i n g e n d o t h e l i a l n i t r i c o x i d e ( N O ) a v a i l a b i l i t y .
" E n d o t h e l i a l d y s f u n c t i o n ( N O p r o d u c t i o n i m p a i r m e n t ) h a s b e e n c o n f i r m e d i n c i g a r e t t e - s m o k i n g p a t i e n t s a n d i n t h e p r e s e n c e o f a n i n c r e a s e i n L D L - c h o l e s t e r o l .
" U r i n a r y i s o p r o s t a n e a n d u r i n a r y l e v e l s o f h o m o c y s t e i n e p o s t - m e t h i o n i n e l o a d ( P M L H c y ) a r e p r o p o s e d a s u s e f u l m a r k e r s o f o x i d a t i v e d a m a g e i n c l i n i c a l s t u d i e s .
" B o t h h y d r o - a n d l i p o s o l u b l e a n t i o x i d a n t s a r e s h o w n t o b e d e p l e t e d i n c i g a r e t t e - s m o k i n g p a t i e n t s w i t h c o r o n a r y h e a r t d i s e a s e . A m o n g t h e m , t h e r a t i o b e t w e e n t o c o p h e r o l ( v i t a m i n E ) a n d t h e s u m o f t o t a l c i r c u l a t i n g l i p i d s ( E / T S L ) m a y b e c h o s e n a s a s e n s i t i v e m a r k e r o f e n d o t h e l i a l o x i d a t i v e d a m a g e i n o r d e r t o d e f i n e p r e v e n t i v e s t r a t e g i e s i n c l i n i c a l p r a c t i c e .
A c k n o w l e d g e m e n t
T h i s w o r k w a s m a d e p o s s i b l e b y f i n a n c i a l s u p p o r t f r o m t h e I t a l i a n M i n i s t r y o f U n i v e r s i t y a n d S c i e n t i f i c a n d T e c h n o l o g i c a l R e s e a r c h ( M U R S T ) , G r a n t n o . 6 0 R O C C 0 2 , t o t h e U n i v e r s i t y o f M o d e n a a n d R e g g i o E m i l i a .
ˇ˛A n t i b i o t i c r e s i s t a n c e
G e o f f S c o t t
I n a n e n v i r o n m e n t c o n t a i n i n g v a s t n u m b e r s o f m i c r o - o r g a n i s m s s a t u r a t e d w i t h o r r e p e a t e d l y e x p o s e d t o a n t i b i o t i c s , D a r w i n i a n t h e o r y p r e d i c t s t h e i n e v i t a b l e s e l e c t i o n o f r e s i s t a n t o r g a n i s m s . F o l - l o w i n g t h e i n t r o d u c t i o n o f a n e w a n t i b i o t i c , r e s i s t a n t s t r a i n s m a y b e r e p o r t e d w i t h i n a s l i t t l e a s 1 y e a r , a n d a f t e r a l a t e n t i n t e r v a l o f s o m e y e a r s , r e s i s t a n c e i n c r e a s e s d r a m a t i c a l l y a n d t h e v a l u e o f t h e a n t i b i o t i c f o r e m p i r i c a l t h e r a p y i s r e d u c e d .
S t a p h y l o c o c c u s a u r e u s i s n o w a l m o s t u n i v e r s a l l y r e s i s t a n t t o p e n i c i l l i n a p h e n o m e n o n f i r s t o b s e r v e d i n h o s p i t a l o u t b r e a k s o f s u r g i c a l s e p s i s i n t h e l a t e 1 9 4 0 s . T h i s o r g a n i s m h a s s h o w n a r e m a r k a b l e f a c i l i t y t o b e c o m e r e s i s t a n t t o e v e r y a n t i b i o t i c i n t r o - d u c e d , e v e n v a n c o m y c i n . R e d u c e d s e n s i t i v i t y t o v a n c o m y c i n ( v a n c o m y c i n - i n t e r m e d i a t e S . a u r e u s ) i s a s s o c i a t e d w i t h a t h i c k p e p t i d o g l y c a n c e l l w a l l . T h e c o m p l e x g e n e c a s s e t t e f o r v a n c o m y c i n r e s i s t a n c e i n E n t e r o c o c c u s s p p . ( v a n A ) c a n e a s i l y b e t r a n s f e r r e d t o S . a u r e u s i n v i t r o a n d h a s n o w b e e n d e t e c t e d i n m e t h i c i l l i n - r e s i s t a n t S . a u r e u s ( M R S A ) , m a k i n g t h e o r g a n i s m f u l l y r e s i s t a n t t o g l y c o p e p t i d e s . I t i s o n l y a m a t t e r o f t i m e b e f o r e s t r a i n s a r e s e e n t h a t a r e s e n s i t i v e t o o n l y a s m a l l n u m b e r o f n o v e l a n t i b i o t i c s i n d e v e l o p m e n t a n d p e r h a p s s o m e o f t h e o l d e r a n t i b i o t i c s ( e . g . t e t r a c y c l i n e , c h l o r a m p h e n i c o l ) . I n c o n t r a s t , s o m e o r g a n i s m s h a v e n o t y e t a c q u i r e d r e s i s t a n c e d e s p i t e h u g e p r e s s u r e s ; t h e s e i n c l u d e S t r e p t o c o c c u s p y o g e n e s , G r a m - p o s i t i v e a n a e r o b e s s u c h a s C l o s t r i - d i u m s p p . a n d P e p t o s t r e p t o c o c c u s s p p . a n d N e i s s e r i a m e n i n g i t i d i s ( t o p e n i c i l l i n ) .
A l m o s t a l l a n a e r o b e s w e r e c o n s i d e r e d t o b e s e n s i t i v e t o m e t r o - n i d a z o l e u n t i l r e c e n t r e p o r t s f r o m S p a i n , F r a n c e a n d t h e U S A s u g g e s t e d t h a t r e s i s t a n t B a c t e r o i d e s f r a g i l i s w i l l s o o n b e c o m e a s e r i o u s p r o b l e m .
E c o l o g y
R e s i s t a n c e i s d r i v e n b y a n t i b i o t i c u s e i n h u m a n a n d a g r i c u l t u r a l / v e t e r i n a r y p r a c t i c e . R e s i s t a n c e s e l e c t i o n o c c u r s b y s p o n t a n e o u s m u t a t i o n s o c c u r i n g a t a r a t e o f 1 0 9 1 0 6 d r i v e n b y t h e p r e s e n c e o f a n t i m i c r o b i a l s . R e s i s t a n c e e l e m e n t s a p p e a r i n s a p r o p h y t i c b a c t e r i a a s a r e s u l t o f a n t i b i o t i c u s e f o r g r o w t h p r o m o t i o n a n d p r o t e c t i n g c r o p s . W h e n t h e s e a r e e a t e n , r e s i s t a n c e m a y b e t r a n s f e r r e d t o h u m a n b a c t e r i a t h a t a r e o c c a s i o n a l l y p a t h o g e n i c . T h e l i k e l i h o o d o f f i n d i n g r e s i s t a n t o r g a n i s m s i n t h e g u t i s r e l a t e d t o t h e t o n n a g e o f a n t i b i o t i c u s e i n t h e c o u n t r y i n w h i c h t h e i n d i v i d u a l r e s i d e s ,
G e o f f S c o t t i s C o n s u l t a n t i n C l i n i c a l M i c r o b i o l o g y a t U n i v e r s i t y C o l l e g e L o n d o n H o s p i t a l s , L o n d o n , U K . H e q u a l i f i e d f r o m t h e R o y a l F r e e H o s p i t a l , L o n d o n , a n d t r a i n e d a t N o r t h w i c k P a r k H o s p i t a l , L o n d o n a n d U n i v e r s i t y C o l l e g e H o s p i t a l . H i s i n t e r e s t s i n c l u d e c o n t r o l o f t u b e r c u l o s i s a n d r e s i s t a n t i n f e c t i o n s , a n d c h a n g i n g h a b i t s .
a n d d e p e n d s o n t h e e a s e w i t h w h i c h a r e s i s t a n c e m e c h a n i s m c a n a r i s e . N o v e l r e s i s t a n c e i s u s u a l l y d e t e c t e d i n d i f f e r e n t p l a c e s i n t h e w o r l d a t a b o u t t h e s a m e t i m e , i m p l y i n g t h a t a n t i b i o t i c p r e s s u r e s a r e s i m i l a r w o r l d w i d e a n d t h a t b a c t e r i a h a v e l i m i t e d m e a n s o f d e a l i n g w i t h t h e p r o b l e m o f s u r v i v a l . F o l l o w i n g t h e a p p e a r a n c e o f o n e m u t a n t r e s i s t a n t p r o g e n y , r a p i d l y r e p l i c a t i n g b a c t e r i a c a n r e c o l o n i z e c a r r i e r s i t e s i n l e s s t h a n 2 4 h o u r s . A r e s i s t a n t m u t a n t o f M y c o b a c t e r i u m t u b e r c u l o s i s , w h i c h r e p l i c a t e s o n c e e v e r y 2 4 h o u r s , c a n r e c o l o n i z e d i s e a s e d l u n g w i t h i n 2 w e e k s .
O n c e r e s i s t a n c e h a s b e e n s e l e c t e d , o r g a n i s m s t r a n s f e r r e d f r o m p e r s o n t o p e r s o n c o n t i n u e t o b e r e s i s t a n t . W h e n t h e a n t i b i o t i c p r e s s u r e i s r e m o v e d , n o v e l c o l o n i z i n g a n d i n f e c t i n g f l o r a t e n d t o r e v e r t t o t h e s e n s i t i v e p h e n o t y p e . S o m e b a c t e r i a c o n t a i n i n g l a r g e r e s i s t a n c e p l a s m i d s a r e c o n s i d e r e d u n f i t a n d s e e m t o d i s a p p e a r f r o m t h e c l i n i c a l e n v i r o n m e n t m o r e e a s i l y t h a n o t h e r s .
R e s i s t a n c e a n d c h o i c e o f a n t i b i o t i c s
T h e t e r m a n t i b i o t i c r e s i s t a n c e i m p l i e s t h a t a p a r t i c u l a r a n t i b i o t i c i s i n e f f e c t i v e i n a c l i n i c a l i n f e c t i o n . T h i s m a y b e b e c a u s e t h e o r g a n i s m i s i n h e r e n t l y r e s i s t a n t t o t h e a n t i b i o t i c o r b e c a u s e i t i s i n a c c e s - s i b l e . I n v i t r o , r e s i s t a n c e i s d e f i n e d b y m e a s u r i n g t h e m i n i m u m i n h i b i t o r y c o n c e n t r a t i o n ( M I C ) a n d t h e m i n i m u m b a c t e r i c i d a l c o n c e n t r a t i o n ( M B C ) o f t h e a n t i b i o t i c a g a i n s t a n o r g a n i s m , u n d e r i d e a l l a b o r a t o r y c o n d i t i o n s , u s i n g a p p r o p r i a t e c o n t r o l s t o d e f i n e t h e c u t - o f f p o i n t s b e t w e e n r e s i s t a n t , i n t e r m e d i a t e a n d s e n s i t i v e . M e t h o d s o f t e s t i n g i n t h e l a b o r a t o r y a r e p r o b l e m a t i c , h o w e v e r , a n d a n y r e s u l t t h a t d o e s n o t c o r r e l a t e w i t h c l i n i c a l e x p e r i e n c e s h o u l d b e c h a l l e n g e d .
L o w M I C a n d h i g h M B C i m p l y t h a t a n a n t i b i o t i c i s b a c t e r i s t a t i c ; t h a t i s , i t i n h i b i t s t h e g r o w t h o f a n o r g a n i s m b u t i s u n a b l e t o k i l l i t . E v e n w i t h b a c t e r i c i d a l a n t i b i o t i c s , k i l l i n g i n v i v o n o r m a l l y r e q u i r e s a n i n t a c t i m m u n e s y s t e m . T h u s , t h o u g h b a c t e r i s t a t i c a n t i b i o t i c s a r e i n e f f e c t i v e i n n e u t r o p e n i c s e p s i s , b a c t e r i c i d a l a n t i b i o t i c s m a y s u p p r e s s i n f e c t i o n o n l y f o r i t t o r e c r u d e s c e w h e n t h e a n t i b i o t i c i s r e m o v e d . T h i s o b s e r v a t i o n g o v e r n s s t r a t e g i e s f o r t h e m a n a g e m e n t o f s u c h i n f e c t i o n s .
P a r a d o x i c a l l y , e v e n w h e n a n o r g a n i s m i s d e c l a r e d r e s i s t a n t t o a n a n t i b i o t i c b y i n v i t r o t e s t s , i t m a y a p p e a r t o b e e f f e c t i v e i n c l i n i c a l u s e ; t h i s m a y b e b e c a u s e M I C / M B C c a n b e e x c e e d e d i n v i v o b y g i v i n g a s u f f i c i e n t l y l a r g e d o s e . T h i s i s o f t e n s e e n i n l o w e r u r i n a r y t r a c t i n f e c t i o n a n d i n t h e t r e a t m e n t o f , f o r e x a m p l e , n o n - m e n i n g e a l p e n i c i l l i n - r e s i s t a n t p n e u m o c o c c a l i n f e c t i o n w i t h h i g h - d o s e p e n i c i l l i n .
E m p i r i c a l t r e a t m e n t : m a n a g e m e n t o f a n i n f e c t i o n b e f o r e b a c t e r i - o l o g y r e s u l t s a r e a v a i l a b l e d e p e n d s o n m a k i n g t h e c o r r e c t d i a g n o s i s a n d a s s e s s i n g t h e a n t i b i o t i c s e n s i t i v i t i e s o f t h e s u s p e c t e d o r g a n - i s m o r o r g a n i s m s f r o m l o c a l e p i d e m i o l o g i c a l k n o w l e d g e . O n c e c u l t u r e r e s u l t s a r e k n o w n , i t i s u s u a l l y a n o t h e r d a y b e f o r e i n v i t r o a n t i b i o t i c s e n s i t i v i t i e s a r e a v a i l a b l e . T h i s i s a c r i t i c a l p e r i o d i n t h e m a n a g e m e n t o f a n i n f e c t e d p a t i e n t a n d e x p l a i n s w h y , i n s e v e r e i n f e c t i o n s , b r o a d - s p e c t r u m a n t i b i o t i c s a r e o f t e n c h o s e n i n i t i a l l y w h e n t h e d e f i n i t i v e d i a g n o s i s i s u n c e r t a i n " a n d w h y w i t h h o l d i n g t r e a t m e n t m a y b e l i f e - t h r e a t e n i n g . T h i s p e r i o d i s g e n e r a l l y m o r e p r o t r a c t e d w h e n a n o r g a n i s m s u c h a s M . t u b e r c u l o s i s i s s l o w t o g r o w i n v i t r o . D e l a y i n t h e t r e a t m e n t o f t u b e r c u l o u s m e n i n g i t i s m a y l e a d t o p e r m a n e n t n e u r o l o g i c a l d a m a g e . W h e t h e r t h e c o r r e c t a n t i b i o t i c s w e r e c h o s e n i n i t i a l l y i s k n o w n o n l y w e e k s l a t e r .
A N T I B I O T I C S
M E D I C I N E 3 3 : 3
4 7 © 2 0 0 5 T h e M e d i c i n e P u b l i s h i n g C o m p a n y L t d
M e c h a n i s m s o f r e s i s t a n c e
R e s i s t a n c e m a y b e i n h e r e n t ( e . g . v a n c o m y c i n a g a i n s t G r a m - n e g a t i v e o r g a n i s m s , n i t r o f u r a n t o i n a g a i n s t P r o t e u s s p p . ) o r a c q u i r e d v i a g e n e t i c e l e m e n t s e n c o d i n g t h r e e f u n d a m e n t a l m e c h a n i s m s :
" p r o d u c t i o n o f i n a c t i v a t i n g e n z y m e s
" c h a n g e i n t h e t a r g e t s i t e
" e x c l u s i o n o f t h e a n t i b i o t i c f r o m t h e t a r g e t s i t e .
T h e l a t t e r m a y o c c u r b y r e s t r i c t i o n o f a c c e s s t h r o u g h p o r i n s ( o n l y i n G r a m - n e g a t i v e s ) o r b y a c t i v e e x c r e t i o n .
A n t i b i o t i c - i n a c t i v a t i n g e n z y m e s m a y b e p r o d u c e d i n v a s t e x c e s s , s u r r o u n d i n g t h e o r g a n i s m ( e . g . ≤- l a c t a m a s e f r o m S . a u r e u s ) , o r i n l i m i t e d a m o u n t s i n t h e p e r i p l a s m i c s p a c e o f G r a m - n e g a t i v e s . T h e e f f e c t i n v i v o i s s i m i l a r , b u t t h e l a t t e r o r g a n i s m s m a y a p p e a r s e n s i - t i v e i n v i t r o . C l a s s i c a l l y , t h i s i s s e e n i n E n t e r o b a c t e r s p p . r e s i s t a n t t o e x t e n d e d - s p e c t r u m ≤- l a c t a m a n t i b i o t i c s s u c h a s p i p e r a c i l l i n a n d c e f o t a x i m e ; e x p o s u r e o f t h e o r g a n i s m t o i n d u c e r s s u c h a s p e n i c i l l i n , c l a v u l a n a t e , c e r t a i n c e p h a l o s p o r i n s a n d i m i p e n e m m a y s w i t c h o n t h e p r o d u c t i o n o f c h r o m o s o m a l ( A m p C ) ≤- l a c t a m a s e s . T h e c l a s s i c a l T E M ( E s c h e r i c h i a c o l i ) a n d S H V ( K l e b s i e l l a s p p . ) ≤- l a c t a m a s e s ( w h i c h i n a c t i v a t e a m p i c i l l i n ) h a v e s h o w n a r e m a r k a b l e a b i l i t y t o m u t a t e t o e x t e n d e d - s p e c t r u m f o r m s ( w h i c h i n a c t i v a t e c e f o x i t i n a n d c e f o t e t a n ) a n d t o i n h i b i t o r r e s i s t a n c e ( n o t i n h i b i t e d b y c l a v u l a n a t e o r t a z o b a c t a m ) . C T X - M c e f o t a x i m a s e a r o s e b y e s c a p e f r o m t h e c h r o m o s o m e o f K l u y v e r a s p p . a n d i s n o w w i d e s p r e a d i n E n t e r o b a c t e r i a c e a e i n c l u d i n g E . c o l i .
O t h e r c l a s s i c a l i n a c t i v a t i n g e n z y m e s i n c l u d e c h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e a n d a m i n o g l y c o s i d e - m o d i f y i n g e n z y m e s .
T a r g e t s i t e c h a n g e m a y b e a s t r u c t u r a l a l t e r a t i o n p r e v e n t i n g b i n d i n g o f a n a n t i b i o t i c , o r a m e c h a n i s m w h e r e b y t h e m e t a b o l i c p a t h w a y t h a t i s n o r m a l l y i n h i b i t e d i s b y p a s s e d b y a n a l t e r n a t i v e o n e . T h i s i s s e e n i n s u l p h o n a m i d e r e s i s t a n c e a n d i n M R S A , w h i c h h a s a c q u i r e d a n o v e l p e n i c i l l i n - b i n d i n g p r o t e i n f r o m S t a p h y l o c o c c u s s c u i r i . I m p o r t a n t t a r g e t s i t e c h a n g e s i n c l u d e t o p o i s o m e r a s e s I I a n d I V ( q u i n o l o n e s ) , ≤ s u b u n i t o f D N A - d e p e n d e n t R N A p o l y m e r a s e ( r i f a m y c i n s ) a n d m e t h y l a t i o n o f 2 3 S t a r g e t ( 1 4 / 1 5 - m e m b e r e d m a c r o l i d e s ) .
E x c l u s i o n o f a n t i b i o t i c : p o r i n s a r e p r o t e i n s t r u c t u r e s e m b e d d e d i n t h e o u t e r b i l i p i d m e m b r a n e o f G r a m - n e g a t i v e o r g a n i s m s . T h e y c o n t r o l w h a t p a s s e s i n t o a n d o u t o f t h e c e l l o n t h e b a s i s o f m o l e c - u l a r s i z e a n d c h a r g e . M u t a t i o n s i n t h e g e n e t i c e l e m e n t s e n c o d i n g p o r i n s ( p e r m e a b i l i t y m u t a t i o n s ) m a y e x c l u d e o n e a n t i b i o t i c o r , m o r e c o m m o n l y , m u l t i p l e a n t i b i o t i c s . P s e u d o m o n a s a e r u g i n o s a h a s t w o o u t e r l i p i d m e m b r a n e s , p r o d u c e s ≤- l a c t a m a s e s c o n s t i t u t i v e l y , a n d t h e r e f o r e t e n d s t o b e m o r e r e s i s t a n t t h a n c o l i f o r m s .
A l t e r n a t i v e l y , o r g a n i s m s m a y a c t i v e l y e x c r e t e a n t i b i o t i c s , n o t a b l y t e t r a c y c l i n e s , m a c r o l i d e s a n d q u i n o l o n e s . E f f l u x p u m p s m a y b e u p - r e g u l a t e d . S . a u r e u s r e s i s t a n t t o e r y t h r o m y c i n b y a c t i v e e x c r e t i o n r e m a i n s s e n s i t i v e t o c l i n d a m y c i n , b u t t h e m o r e c o m m o n t a r g e t s i t e m u t a t i o n a f f e c t s s u s c e p t i b i l i t y t o b o t h a n t i b i o t i c s .
A c q u i s i t i o n o f r e s i s t a n c e
D i r e c t m u t a t i o n o f c h r o m o s o m a l g e n e s m a y l e a d t o r e s i s t a n c e , a n d n o n - f a t a l m u t a t i o n s a r e p a s s e d t o a l l p r o g e n y . A l t e r n a t i v e l y , s m a l l , m o b i l e , c i r c u l a r p i e c e s o f D N A t e r m e d p l a s m i d s m a y b e
p a s s e d f r o m o n e b a c t e r i u m t o a n o t h e r ( e v e n t o b a c t e r i a o f d i f f e r e n t s p e c i e s ) b y v a r i o u s m e c h a n i s m s s u c h a s d i r e c t t r a n s f e r b y t y p e I I p i l i ( F i g u r e 1 ) a n d p h a g e t r a n s f e r . P l a s m i d s r e p r o d u c e e a c h t i m e t h e o r g a n i s m d i v i d e s a n d c a n p r o b a b l y b e l o s t a s e a s i l y a s g a i n e d , g i v e n t h e c o r r e c t e n v i r o n m e n t .
T r a n s p o s o n s a r e l a r g e g e n e t i c e l e m e n t s o f t e n c o n t a i n i n g m u l - t i p l e g e n e s n e c e s s a r y t o c o n f e r p h e n o t y p i c r e s i s t a n c e . T h e y m a y e n c o d e p h e r o m o n e s , w h i c h a t t r a c t b a c t e r i a t o e a c h o t h e r a n d c a n b e t r a n s f e r r e d o n p l a s m i d s , g e n e r a l l y b e i n g i n c o r p o r a t e d i n t o t h e c h r o m o s o m a l D N A o f t h e n e w h o s t . F r e e D N A f r o m d e a d b a c t e r i a a n d e v e n m a m m a l i a n c e l l s c a n b e i n c o r p o r a t e d i n t o b a c t e r i a l c h r o m o s o m e s . A r e m a r k a b l e m e c h a n i s m o f r e s i s t a n c e i s s e e n i n p e n i c i l l i n - r e s i s t a n t S t r e p t o c o c c u s p n e u m o n i a e , w h i c h h a v e i n c o r - p o r a t e d a m o s a i c o f g e n e s i n t o t h e c h r o m o s o m e f r o m o t h e r r e s i s - t a n t ±- h a e m o l y t i c s t r e p t o c o c c i .
P r o b l e m a t i c r e s i s t a n t b a c t e r i a
G r a m - p o s i t i v e o r g a n i s m s
M R S A h a s r e p l a c e d m e t h i c i l l i n - s e n s i t i v e S . a u r e u s a s t h e m o r e c o m m o n c a u s e o f h o s p i t a l - a s s o c i a t e d s e p s i s i n m o s t p a r t s o f t h e w o r l d . T h e t a r g e t s o f t h e ≤- l a c t a m a n t i b i o t i c s a r e p e n i c i l l i n - b i n d i n g p r o t e i n s , c a r b o x y p e p t i d a s e s a n d t r a n s p e p t i d a s e s , w h i c h c a t a l y s e b r i d g i n g o f p e n t a p e p t i d e s u b u n i t s o f p e p t i d o g l y c a n . A l l s t r a i n s o f M R S A a l r e a d y p r o d u c e p e n i c i l l i n a s e , b u t t h e y n o w h a v e a n e w p e n i c i l l i n - b i n d i n g p r o t e i n ( P B P 2 ' ) t h a t i s n o t i n h i b i t e d b y m e t h i - c i l l i n a n d i t s c o n g e n e r s o x a c i l l i n a n d f l u c l o x a c i l l i n . T h e y a r e r e s i s t - a n t t o a l l ≤- l a c t a m a n t i b i o t i c s . T h e m e c A g e n e e n c o d e s P B P 2 ' , b u t r e q u i r e s e x p r e s s i o n o f s e v e r a l a n c i l l a r y g e n e s w i t h i n a t r a n s p o s o n f o r f u l l e x p r e s s i o n . T h u s , s o m e s t r a i n s w i t h m e c A m a y a p p e a r t o b e s e n s i t i v e t o m e t h i c i l l i n i n v i t r o . I n a d d i t i o n , a p l a s m i d e n c o d e s r e s i s t a n c e t o a v a r i a b l e n u m b e r o f o t h e r a n t i b i o t i c s .
S t r a i n s w i t h a t e n d e n c y t o s p r e a d e a s i l y a n d t o p r e d o m i n a t e i n h o s p i t a l s a r e t e r m e d e p i d e m i c M R S A ( E M R S A ) . T h e c u r r e n t e p i d e m i c o f E M R S A 1 5 o r E M R S A 1 6 i n t h e U K s t a r t e d i n 1 9 9 4 . I n t h e l a b o r a t o r y , t h e s e a r e d e t e c t e d b y t h e i r a n t i b i o g r a m , b u t o t h e r t y p i n g m e t h o d s ( e . g . p u l s e d - f i e l d g e l e l e c t r o p h o r e s i s o f c h r o m o - s o m a l D N A ) a r e n e e d e d t o s h o w t h a t t w o s t r a i n s a r e i n d i s t i n g u i s h - a b l e , t h u s i m p l y i n g t h a t c r o s s - i n f e c t i o n h a s o c c u r r e d . W h e n a f e w p a t i e n t s i n o n e o r t w o w a r d s a c q u i r e a n o v e l s t r a i n , s u c h a n o u t b r e a k c a n e a s i l y b e t r a c k e d a n d t h e n c o n t r o l l e d . W h e n E M R S A s t r a i n s b e c o m e e n d e m i c ( F i g u r e 2 ) , m o r e g e n e r a l m e a s u r e s a r e n e e d e d t o r e d u c e t h e r i s k t o p a t i e n t s a d m i t t e d t o t h e h o s p i t a l . M R S A m a y b e c o m e e n d e m i c i n n u r s i n g h o m e s , c r e a t i n g a p o o l f o r n o v e l i n t r o d u c t i o n i n t o h o s p i t a l ; t r a n s f e r o f p a t i e n t s f r o m w a r d t o w a r d a n d c o l o n i z a t i o n o f s t a f f l e a d s t o c o n t i n u e d e x p o s u r e o f p a t i e n t s t o n e w s t r a i n s a n d h e l p s m a i n t a i n e n d e m i c i t y .
I n f e c t i o n w i t h M R S A i s n o t u n t r e a t a b l e . S t r a i n s a r e o f t e n ( t h o u g h n o t p r e d i c t a b l y ) s u s c e p t i b l e t o g e n t a m i c i n o r o t h e r a m i n o g l y c o s i d e s , r i f a m p i c i n , c o - t r i m o x a z o l e , c h l o r a m p h e n i c o l o r c i p r o f l o x a c i n , a n d s o m e t i m e s t o t e t r a c y c l i n e s , m a c r o l i d e s , f u s i d i c a c i d a n d p s e u d o m o n i c a c i d . ( N o t e t h a t r i f a m p i c i n a n d f u s i d i c a c i d s h o u l d n e v e r b e u s e d a l o n e b e c a u s e r e s i s t a n t m u t a n t s a r e s e l e c t e d v e r y r a p i d l y . ) M R S A i s a l m o s t a l w a y s s u s c e p t i b l e t o g l y c o p e p t i d e s , t h o u g h s t r a i n s w i t h r e d u c e d s e n s i t i v i t y t o v a n c o m y c i n h a v e b e e n o c c a s i o n a l l y d e s c r i b e d i n p a t i e n t s w i t h c h r o n i c c o l o n i z a t i o n o r i n f e c t i o n w h o h a v e b e e n t r e a t e d f o r s e v e r a l w e e k s . 1 S o m e r a r e s t r a i n s o f S . a u r e u s a r e d e p e n d e n t o n v a n c o m y c i n t o a l l o w t h e m t o g r o w .
M E D I C I N E 3 3 : 3 4 8
© 2 0 0 5 T h e M e d i c i n e P u b l i s h i n g C o m p a n y L t d
A N T I B I O T I C S
P e n i c i l l i n - r e s i s t a n t S . p n e u m o n i a e s t r a i n s h a v e s h o w n a g r a d u a l p h a s e - s h i f t i n c r e a s e i n M I C t o p e n i c i l l i n o v e r s e v e r a l y e a r s . M I C i s a b o u t 0 . 0 0 1 m g / l i t r e i n s e n s i t i v e s t r a i n s a n d 1 m g / l i t r e i n r e s i s t a n t s t r a i n s . T h i s i s a c h i e v e d b y c h a n g e s i n p e n i c i l l i n - b i n d i n g p r o t e i n s w i t h l o w e r a f f i n i t i e s f o r ≤- l a c t a m s . I n t h e U K , s u c h s t r a i n s c u r r e n t l y r e p r e s e n t a b o u t 4 % o f t h o s e c a u s i n g i n v a s i v e i n f e c t i o n , b u t i n s o m e a r e a s ( e . g . S p a i n ) t h e r a t e i s a s h i g h a s 5 0 % . T h e s e s t r a i n s a r e u s u a l l y r e s i s t a n t t o m a n y o t h e r u s e f u l a n t i b i o t i c s , i n c l u d i n g c e p h a l o s p o r i n s , c h l o r a m p h e n i c o l a n d e r y t h r o m y c i n .
T r e a t m e n t w i t h h i g h - d o s e p e n i c i l l i n i s e f f e c t i v e i n p n e u m o n i a b u t n o t i n m e n i n g i t i s c a u s e d b y r e s i s t a n t s t r a i n s , s o m e o f w h i c h a r e s e n s i t i v e t o s e c o n d - g e n e r a t i o n a n d t h i r d - g e n e r a t i o n c e p h a l o - s p o r i n s . T h e m o r t a l i t y f r o m s e v e r e p n e u m o c o c c a l p n e u m o n i a r e m a i n s r e l a t i v e l y c o n s t a n t r e g a r d l e s s o f w h e t h e r t h e s t r a i n i s r e s i s t a n t t o p e n i c i l l i n , b u t m e n i n g i t i s c a u s e d b y a r e s i s t a n t s t r a i n i s m o r e l i k e l y t o b e f a t a l .
S p l e n e c t o m i z e d p a t i e n t s a r e a d v i s e d t o t a k e l i f e - l o n g l o w - d o s e o r a l p e n i c i l l i n p r o p h y l a x i s a g a i n s t t h e r a r e p o s s i b i l i t y o f o v e r - w h e l m i n g p n e u m o c o c c a l s e p s i s . I n t h e f u t u r e , s u c h p r o p h y l a x i s m a y b e c o m e l e s s e f f e c t i v e , b u t i t i s d i f f i c u l t t o i d e n t i f y a n a l t e r n a - t i v e s i m p l e a n d s a f e r e g i m e n .
G l y c o p e p t i d e - r e s i s t a n t e n t e r o c o c c i : e n t e r o c o c c i h a v e b e c o m e i m p o r t a n t c a u s e s o f n o s o c o m i a l p o s t o p e r a t i v e i n f e c t i o n i n t h e U S A . T h e y a r e l i k e l y t o c a u s e s e p s i s a n d p n e u m o n i a o n l y i n s e v e r e l y i l l p a t i e n t s i n t h e U K , a n d o c c a s i o n a l l y s t r a i n s a r e r e s i s t a n t t o v a n c o - m y c i n w i t h ( v a n A ) o r w i t h o u t ( v a n B ) t e i c o p l a n i n . T h e s e g e n e s r e q u i r e t h e a c t i o n o f c o m p l e x a c c e s s o r y g e n e s f o r f u l l e x p r e s s i o n .
v a n A e n c o d e s a s t r u c t u r a l c h a n g e i n t h e t e r m i n a l a m i n o a c i d o f t h e p e n t a p e p t i d e c h a i n o f p e p t i d o g l y c a n , f r o m D - a l a t o D - l a c . T h i s s u b s t i t u t i o n p r e v e n t s b i n d i n g o f v a n c o m y c i n , e n a b l i n g c o n s t r u c t i o n o f t h e d i p e p t i d e b r i d g e s i n p e p t i d o g l y c a n t o c o n t i n u e .
T y p i n g i n d i c a t e s w i d e h e t e r o g e n e i t y o f s t r a i n s , a n d t h a t c a r r i e r s u s u a l l y h a r b o u r m o r e t h a n o n e s t r a i n . T h i s i m p l i e s t h a t t h e t r a n s - p o s o n e n c o d i n g v a n A a n d t h e a c c e s s o r y g e n e s i s p r o m i s c u o u s a n d e a s i l y a b l e t o e n t e r t h e h o s t s e n t e r o c o c c i . T h e s e o r g a n i s m s a r e o f l o w v i r u l e n c e . I n f e c t i o n s m a y r e s p o n d t o s i m p l e a n t i b i o t i c s s u c h a s a m o x i c i l l i n i f t h e y a p p e a r s e n s i t i v e ( E n t e r o c o c c u s f a e c a l i s ) , b u t a r e g e n e r a l l y r e s i s t a n t t o a l l b u t a f e w n e w a n t i b i o t i c s . C o l o n i z a t i o n w i t h e n t e r o c o c c i i s d r i v e n b y c e p h a l o s p o r i n s a n d f l u o r o q u i n o l o n e s , t o w h i c h e n t e r o c o c c i a r e c o n s t i t u t i v e l y r e s i s t a n t .
G r a m - n e g a t i v e o r g a n i s m s
I n c o n t r a s t t o r e s i s t a n t G r a m - p o s i t i v e s , a g a i n s t w h i c h o l d a n t i - b i o t i c s o r a n t i b i o t i c s i n d e v e l o p m e n t a r e o c c a s i o n a l l y a c t i v e , s o m e G r a m - n e g a t i v e o r g a n i s m s ( p a r t i c u l a r l y n o n - g l u c o s e f e r m e n t e r s s u c h a s P s e u d o m o n a s , S t e n o t r o p h o m o n a s a n d A c i n e t o b a c t e r s p p . ) , e s p e c i a l l y i n I C U s , a r e r e s i s t a n t t o e v e r y u s e f u l a n t i b i o t i c a n d t h e r e a r e n o n e w a g e n t s i n d e v e l o p m e n t .
G l u c o s e f e r m e n t e r s : i n t e r m s o f r e s i s t a n c e a n d e n d e m i c / e p i d e m i c p r o b l e m s i n i l l , h o s p i t a l i z e d p a t i e n t s , K l e b s i e l l a , E n t e r o b a c t e r a n d S e r r a t i a s p p . a r e m o r e t r o u b l e s o m e t h a n E . c o l i . T h e y p r o d u c e S H V - t y p e ≤- l a c t a m a s e s c o n s t i t u t i v e l y , a n d m u t a t i o n s l e a d t o r e s i s t - a n c e t o a l l ≤- l a c t a m s o t h e r t h a n t h e c a r b a p e n e m s ( o r o c c a s i o n a l l y a z t r e o n a m ) . T h e y o f t e n l o s e s u s c e p t i b i l i t y t o q u i n o l o n e s a n d
A N T I B I O T I C S
a b
c d
1 D N A c a n b e t r a n s f e r r e d
a o n d i r e c t c o n t a c t , b , c v i a t y p e I I p i l i o r
d b y p h a g e s ( b a c t e r i a l v i r u s e s ) . ( b a n d c b y c o u r t e s y o f Z e n e c a . )
M E D I C I N E 3 3 : 3
4 9 © 2 0 0 5 T h e M e d i c i n e P u b l i s h i n g C o m p a n y L t d
a m i n o g l y c o s i d e s a n d b e c o m e e s s e n t i a l l y u n t r e a t a b l e . S o m e s t r a i n s o f S a l m o n e l l a e n t e r i c a ( p a r t i c u l a r l y t y p h i m u r i u m ) , a n d t h e e n t e r i c s a l m o n e l l a e ( e . g . S . t y p h i ) h a v e a c q u i r e d s t a b l e b r o a d r e s i s t a n c e t o a l l u s e f u l a n t i b i o t i c s . T h e s e a r e n o w p a n d e m i c .
N o n - g l u c o s e f e r m e n t e r s : P . a e r u g i n o s a h a s b e e n r e p l a c e d a s a t r o u b l e s o m e c a u s e o f n o s o c o m i a l i n f e c t i o n b y o t h e r e n v i r o n - m e n t a l a n d s k i n b a c t e r i a s u c h a s A c i n e t o b a c t e r b a u m a n i i v a r . c a l c o a c e t i c u s . S o m e s t r a i n s a r e r e s i s t a n t t o a l l a v a i l a b l e a n t i b i o t i c s , o t h e r s a r e s e n s i t i v e o n l y t o c a r b a p e n e m s a n d s o m e a m i n o g l y c o - s i d e s ( e . g . a m i k a c i n ) . I n r e s p o n s e t o l o n g - t e r m a n t i b i o t i c u s e o v e r m a n y y e a r s , B u r k h o l d e r i a c e p a c i a t e n d s t o r e p l a c e S . a u r e u s a n d P . a e r u g i n o s a a s c o l o n i z i n g f l o r a i n p a t i e n t s w i t h c y s t i c f i b r o s i s . I t m a y a l s o s h o w r e s i s t a n c e t o m a n y a n t i b i o t i c s a n d c a n c a u s e t r o u b l e s o m e c r o s s - i n f e c t i o n .
N e i s s e r i a s p p . : N . g o n o r r h o e a e i s i n t e r e s t i n g i n t h a t d i f f e r e n t s t r a i n s m a y e x h i b i t t h e t h r e e m e c h a n i s m s o f r e s i s t a n c e t o p e n i c i l - l i n ( r e d u c e d p e r m e a b i l i t y , c h a n g e s i n p e n i c i l l i n - b i n d i n g p r o t e i n s a n d ≤- l a c t a m a s e p r o d u c t i o n ) . E a c h c o n f e r s a s t e p w i s e i n c r e a s e i n M I C , a n d t h e r e i s n o c l e a r c u t - o f f b e t w e e n s e n s i t i v e a n d r e s i s t a n t s t r a i n s . M a n y s t r a i n s h a v e b e c o m e r e s i s t a n t t o o t h e r o r a l d r u g s s u c h a s c i p r o f l o x a c i n , t h o u g h t h e s e a r e c u r r e n t l y r a r e i n t h e U K .
N . m e n i n g i t i d i s h a s b e e n s l o w t o a c q u i r e r e s i s t a n c e t o p e n i c i l l i n , t h o u g h a f e w s t r a i n s i s o l a t e d r e c e n t l y i n S p a i n h a v e s l i g h t l y h i g h e r M I C s t h a n p r e d i c t e d . T h e v a l u e o f s u l p h o n a m i d e s w a s l a r g e l y l o s t b y t h e 1 9 7 0 s , a n d t h o u g h c h l o r a m p h e n i c o l r e m a i n s u s e f u l , s e c o n d - g e n e r a t i o n c e p h a l o s p o r i n s ( c e f o t a x i m e o r c e f t r i a x o n e ) s e e m t o g i v e t h e b e s t r e s u l t s i n c l i n i c a l t r e a t m e n t .
M . t u b e r c u l o s i s : r e s i s t a n c e t o f i r s t - l i n e a n t i t u b e r c u l o s i s a g e n t s h a s b e e n a m a j o r p r o b l e m i n t h e r e - e m e r g e n c e o f t u b e r c u l o s i s . I n s o m e c o u n t r i e s ( e . g . t h e s t a t e s o f t h e f o r m e r U S S R ) , m u l t i - d r u g - r e s i s t a n t s t r a i n s ( r e s i s t a n t a t l e a s t t o r i f a m p i c i n a n d i s o n i a z i d ) a r e e x t r e m e l y c o m m o n , a n d m o r e s o i n p a t i e n t s w h o h a v e b e e n t r e a t e d p r e v i o u s l y . I n t h e U K , l o n e r e s i s t a n c e t o i s o n i a z i d o c c u r s i n 5 % ( e x c e p t i n L o n d o n , w h e r e t h e r e i s c u r r e n t l y a n e p i d e m i c o f
o n e c l o n e r e s i s t a n t t o i s o n i a z i d t h a t s t a r t e d i n 1 9 9 5 a n d h a s l e d t o a r e s i s t a n c e r a t e o f 1 5 % i n a f f e c t e d a r e a s ) , a n d t o r i f a m p i c i n , e t h a m b u t o l a n d p y r a z i n a m i d e i n 1 % o r f e w e r . M u l t i - d r u g - r e s i s t a n t s t r a i n s a c c o u n t f o r 1 . 2 % . S t r a i n s r e s i s t a n t t o a l l a r e f o u n d t h r o u g h - o u t t h e c o u n t r y , b u t a r e m o r e c o m m o n l y s e e n i n L o n d o n .
M o s t p a t i e n t s c a n b e t r e a t e d s a t i s f a c t o r i l y w i t h a s t a n d a r d 6 - m o n t h r e g i m e n , b u t t h e r e s u l t s o f r o u t i n e a n t i b i o t i c s e n s i t i v i t y t e s t s a r e u n k n o w n f o r 5 "1 6 w e e k s a f t e r s e n d i n g s p e c i m e n s t o t h e l a b o r a t o r y , a n d p a t i e n t s w i t h r e s i s t a n t s t r a i n s n e e d m o r e t o x i c s e c o n d - l i n e d r u g s a n d p r o l o n g e d c o u r s e s o f t r e a t m e n t . R a p i d l i q u i d c u l t u r e a n d d e t e c t i o n o f g e n e s e n c o d i n g r e s i s t a n c e m a y i m p r o v e t h e s p e e d o f l a b o r a t o r y d i a g n o s i s .
S t r a t e g i e s f o r r e d u c i n g t h e i m p a c t o f r e s i s t a n c e
A n t i b i o t i c r e s i s t a n c e i s d r i v e n b y a n t i b i o t i c u s e . W h e n a n t i b i o t i c s a r e s u p e r s e d e d a n d t h e r e f o r e u s e d l e s s , s t r a i n s r e s i s t a n t t o t h e s e t e n d t o d i s a p p e a r .
I n t h e c o m m u n i t y : i n t h e U K , m o r e t h a n 8 0 % o f h u m a n u s e o f a n t i b i o t i c s o c c u r s i n t h e c o m m u n i t y , m o s t l y f o r r e s p i r a t o r y t r a c t i n f e c t i o n s . T h e S t a n d i n g M e d i c a l A d v i s o r y C o m m i t t e e , i n i t s r e p o r t T h e p a t h o f l e a s t r e s i s t a n c e , m a d e r e c o m m e n d a t i o n s t o r e d u c e i n a p p r o p r i a t e p r e s c r i b i n g . 2
" N o a n t i b i o t i c s s h o u l d b e g i v e n f o r s i m p l e c o u g h s a n d c o l d s .
" A n t i b i o t i c s s h o u l d n o t b e r o u t i n e l y p r e s c r i b e d f o r s o r e t h r o a t s , u n l e s s t h e r e i s e v i d e n c e o f s t r e p t o c o c c a l i n f e c t i o n .
" A n t i b i o t i c s a r e n o t r o u t i n e l y r e q u i r e d f o r a c u t e o t i t i s m e d i a a n d s i n u s i t i s - l i k e s y m p t o m s ; i f g i v e n , c o u r s e s c a n b e l i m i t e d t o 3 d a y s .
I n a d d i t i o n , 3 d a y s t r e a t m e n t s h o u l d s u f f i c e i n o t h e r w i s e h e a l t h y w o m e n w i t h u n c o m p l i c a t e d c y s t i t i s .
T h i s s t r a t e g y h a s b e e n u s e f u l i n r e d u c i n g p r e s c r i b i n g b y G P s , b u t a n x i e t y h a s r e s u l t e d f r o m a n e c d o t a l r e p o r t s o f a n i n c r e a s e i n b a c t e r i a l r e s p i r a t o r y i n f e c t i o n s .
I n h o s p i t a l , a n t i b i o t i c u s e c a n b e r e d u c e d b y s e v e r a l m e a n s .
N e w p a t i e n t s w i t h m e t h i c i l l i n - r e s i s t a n t S t a p h y l o c o c c u s a u r e u s a t U n i v e r s i t y C o l l e g e L o n d o n H o s p i t a l s , 1 9 9 1 1 9 9 9
4 5 0 4 0 0 3 5 0 3 0 0 2 5 0 2 0 0 1 5 0 1 0 0
5 0 0
I n f e c t i o n s C a r r i e r s
A N T I B I O T I C S
1 9 9 1
1 9 9 2 1 9 9 3 1 9 9 4
1 9 9 5
1 9 9 6
1 9 9 7
1 9 9 8 1 9 9 9
Y e a r
M E D I C I N E 3 3 : 3
5 0
© 2 0 0 5 T h e M e d i c i n e P u b l i s h i n g C o m p a n y L t d
2
A n n u a l i n c i d e n c e
A N T I B I O T I C S
S i t e s o f a c t i o n o f a n t i b i o t i c s a n d s o m e r e s i s t a n c e m e c h a n i s m s
G l y c o p e p t i d e
G l y c o p e p t i d e t o o l a r g e
A m i n o g l y c o s i d e s ≤- l a c t a m s
M u t a t i o n i n p o r i n
P o r i n
A m i n o g l y c o s i d e s M a c r o l i d e s C h l o r a m p h e n i c o l
R N A
G r a m - G r a m - p o s i t i v e n e g a t i v e
P e p t i d o g l y c a n
S u p e r c o i l e d D N A T o p o i s o m e r a s e s
Q u i n o l o n e s
F o l a t e s y n t h e s i s
S u l p h o n a m i d e s T r i m e t h o p r i m
S u b s t r a t e c h a n g e
M u t a t i o n
≤- l a c t a m s
P e r i p l a s m i c s p a c e ( ≤- l a c t a m a s e a c t i v i t y )
D N A
≤- l a c t a m a s e s
C h l o r a m p h e n i c o l a c t e y l t r a n s f e r a s e A m i n o g l y c o s i d e - m o d i f y i n g e n z y m e s
I n a c t i v a t i n g e n z y m e s
A c t i v e e x c r e t i o n
M a c r o l i d e s Q u i n o l o n e s T e t r a c y c l i n e s
" R o u t i n e u s e o f a n t i b i o t i c s f o r s u r g i c a l p r o p h y l a x i s s h o u l d b e r e d u c e d t o a m i n i m u m .
" A n t i b i o t i c s s h o u l d n o t b e s t a r t e d i m m e d i a t e l y i n a l l s u s p e c t e d i n f e c t i o n s . C e r t a i n p a t i e n t s ( e . g . t h o s e w i t h f e b r i l e n e u t r o p e n i a o r e v i d e n c e o f s e p t i c a e m i a ) r e q u i r e u r g e n t a n t i b i o t i c t h e r a p y , b u t i n m a n y o t h e r c a s e s ( e . g . m i l d p y r e x i a p o s t o p e r a t i v e l y ) , i t i s s a f e a n d u l t i m a t e l y p r e f e r a b l e t o w i t h h o l d a n t i b i o t i c s u n t i l c u l t u r e r e s u l t s a r e k n o w n o r t h e r e i s c l e a r e v i d e n c e o f b a c t e r i a l i n f e c t i o n .
" A n a l t e r n a t i v e i s t o d i s c o n t i n u e a n t i b i o t i c s a s s o o n a s i n f o r - m a t i o n i s a v a i l a b l e s u g g e s t i n g t h a t t h e p r o b l e m i s n o t b a c t e r i a l o r h a s r e s o l v e d i t s e l f . T h i s i s t e r m e d a n a n t i b i o t i c - s t o p p o l i c y , a n d t h e a i m i s t o e n c o u r a g e d o c t o r s t o a c t i v e l y r e v i e w t h e n e e d f o r a n t i b i o t i c s a f t e r , s a y , t h e s e c o n d d a y , g i v e n i n f o r m a t i o n o n c u l t u r e s a n d s u r r o g a t e m a r k e r s t h a t h a s b y t h e n b e c o m e a v a i l a b l e .
" C e r t a i n a n t i b i o t i c s c a n b e w i t h h e l d f r o m t h e h o s p i t a l f o r m u l a r y . T h i s i s t h e m a i n b e n e f i t o f a n a g r e e d a n t i b i o t i c p o l i c y . T h e c h o i c e o f r e s t r i c t e d a n t i b i o t i c s m a y b e d e c i d e d o n t h e b a s i s o f c o s t r a t h e r t h a n l i k e l y s e l e c t i o n o f r e s i s t a n c e . ( T h e s e a n t i b i o t i c s m u s t b e u s e d o c c a s i o n a l l y , h o w e v e r , w h e n r e s i s t a n c e t o a l l o t h e r a v a i l a b l e d r u g s h a s b e e n s e l e c t e d . )
" I n t h e o r y , a n t i b i o t i c s c a n b e r o t a t e d s u c h t h a t , f o r e x a m p l e , p r e d o m i n a n t l y p e n i c i l l i n s a r e u s e d a t s o m e t i m e s , a n d c e p h a l o - s p o r i n s o r q u i n o l o n e s a t o t h e r s . T h e r e i s l i t t l e c l e a r s c i e n t i f i c e v i d e n c e t h a t t h i s h a s a n y e f f e c t , a n d m a j o r , c o m p l i c a t e d s t u d i e s w o u l d b e n e e d e d t o d e t e r m i n e t h e e f f e c t o f c h a n g e i n u s e o n b o t h n o r m a l a n d i n f e c t i n g f l o r a . H o w e v e r , i t h a s b e e n s h o w n t h a t , i n a s e t t i n g o f h e a v y c e p h a l o s p o r i n u s e , d i s c o n t i n u a t i o n o f u s e o f t h i s c l a s s o f d r u g s l e a d s t o a r e d u c t i o n i n t h e r i s k o f c o l o n i z a t i o n w i t h g l y c o p e p t i d e - r e s i s t a n t e n t e r o c o c c i a n d C l o s t r i d i u m d i f f i c i l e - a s s o c i a t e d d i a r r h o e a .
O t h e r s t r a t e g i e s : d e v e l o p m e n t o f r e s i s t a n c e i n h u m a n p a t h o g e n s m i g h t b e d e l a y e d i f a n t i b i o t i c s w e r e n o t u s e d s o w i d e l y i n a n i m a l h u s b a n d r y , p a r t i c u l a r l y f o r g r o w t h p r o m o t i o n .
T h e f u t u r e
R e s i s t a n c e t o a n t i b i o t i c s i s o n e o f t h e g r e a t e s t t h r e a t s t o t h e s u c c e s s o f m o d e r n m e d i c i n e . I t h a s r e c e n t l y b e c o m e m o r e s e r i o u s b e c a u s e w e c a n n o l o n g e r b e s u r e t h a t a n y a n t i b i o t i c c h o s e n e m p i r i c a l l y w i l l w o r k , a n d b e c a u s e o f t h e e m e r g e n c e o f t o t a l l y r e s i s t a n t b a c - t e r i a . H o w t o r e d u c e r e s i s t a n c e w i t h o u t s i m p l y d i s c o n t i n u i n g u s e o f a l l a n t i b i o t i c s i s a d i l e m m a . W e d o n o t k n o w t o w h a t d e g r e e a n t i b i o t i c u s e m u s t b e r e d u c e d t o d e c r e a s e t h e s e l e c t i v e p r e s s u r e a n d a l l o w r e v e r s i o n t o s e n s i t i v i t y , n o r d o w e k n o w h o w t o p r o t e c t t h e f e w r e m a i n i n g d r u g s t h a t c a n b e u s e d t o t r e a t r e s i s t a n t i n f e c - t i o n s . D o c t o r s a n d v e t s s h o u l d o v e r c o m e t h e v i e w t h a t t h e y h a v e a n i n a l i e n a b l e r i g h t t o p r e s c r i b e e m p i r i c a l a n t i b i o t i c s , a n d s h o u l d s e t t a r g e t s f o r r e d u c t i o n o f t h e i r p e r s o n a l p r e s c r i b i n g .
ˇ˛A n t i b i o t i c s f o r a c u t e o t i t i s m e d i a : a m e t a - a n a l y s i s w i t h i n d i v i d u a l p a t i e n t d a t a
M a r o e s k a M R o v e r s , P a u l G l a s z i o u , C e e s L A p p e l m a n , P e t e r B u r k e , D a v i d P M c C o r m i c k , R o g e r A D a m o i s e a u x , I s a b e l l e G a b o u r y , P a u l L i t t l e , A r n o W H o e s
S u m m a r y
B a c k g r o u n d I n d i v i d u a l t r i a l s t o t e s t e f f e c t i v e n e s s o f a n t i b i o t i c s i n c h i l d r e n w i t h a c u t e o t i t i s m e d i a h a v e b e e n t o o s m a l l f o r v a l i d s u b g r o u p a n a l y s e s . W e a i m e d t o i d e n t i f y s u b g r o u p s o f c h i l d r e n w h o w o u l d a n d w o u l d n o t b e n e f i t m o r e t h a n o t h e r s f r o m t r e a t m e n t w i t h a n t i b i o t i c s .
M e t h o d s W e d i d a m e t a - a n a l y s i s o f d a t a f r o m s i x r a n d o m i s e d t r i a l s o f t h e e f f e c t s o f a n t i b i o t i c s i n c h i l d r e n w i t h a c u t e o t i t i s m e d i a . I n d i v i d u a l p a t i e n t d a t a f r o m 1 6 4 3 c h i l d r e n a g e d f r o m 6 m o n t h s t o 1 2 y e a r s w e r e v a l i d a t e d a n d r e - a n a l y s e d . W e d e f i n e d t h e p r i m a r y o u t c o m e a s a n e x t e n d e d c o u r s e o f a c u t e o t i t i s m e d i a , c o n s i s t i n g o f p a i n , f e v e r , o r b o t h a t 3 7 d a y s .
F i n d i n g s S i g n i f i c a n t e f f e c t m o d i f i c a t i o n s w e r e n o t e d f o r o t o r r h o e a , a n d f o r a g e a n d b i l a t e r a l a c u t e o t i t i s m e d i a . I n c h i l d r e n y o u n g e r t h a n 2 y e a r s o f a g e w i t h b i l a t e r a l a c u t e o t i t i s m e d i a , 5 5 % o f c o n t r o l s a n d 3 0 % o n a n t i b i o t i c s s t i l l h a d p a i n , f e v e r , o r b o t h a t 3 7 d a y s , w i t h a r a t e d i f f e r e n c e b e t w e e n t h e s e g r o u p s o f "2 5 % ( 9 5 % C I "3 6 % t o "1 4 % ) , r e s u l t i n g i n a n u m b e r - n e e d e d - t o - t r e a t ( N N T ) o f f o u r c h i l d r e n . W e i d e n t i f i e d n o s i g n i f i c a n t d i f f e r e n c e s f o r a g e a l o n e . I n c h i l d r e n w i t h o t o r r h o e a t h e r a t e d i f f e r e n c e a n d N N T , r e s p e c t i v e l y , w e r e "3 6 % ( "5 3 % t o "1 9 % ) a n d t h r e e , w h e r e a s i n c h i l d r e n w i t h o u t o t o r r h o e a t h e e q u i v a l e n t v a l u e s w e r e "1 4 % ( "2 3 % t o "5 % ) a n d e i g h t .
I n t e r p r e t a t i o n A n t i b i o t i c s s e e m t o b e m o s t b e n e f i c i a l i n c h i l d r e n y o u n g e r t h a n 2 y e a r s o f a g e w i t h b i l a t e r a l a c u t e o t i t i s m e d i a , a n d i n c h i l d r e n w i t h b o t h a c u t e o t i t i s m e d i a a n d o t o r r h o e a . F o r m o s t o t h e r c h i l d r e n w i t h m i l d d i s e a s e a n o b s e r v a t i o n a l p o l i c y s e e m s j u s t i f i e d .
L a n c e t 2 0 0 6 ; 3 6 8 : 1 4 2 9 3 5 S e e C o m m e n t p a g e 1 3 9 7
J u l i u s C e n t r e f o r H e a l t h S c i e n c e s a n d P r i m a r y C a r e , U n i v e r s i t y M e d i c a l C e n t r e U t r e c h t , t h e N e t h e r l a n d s ( M M R o v e r s P h D ,
C L A p p e l m a n M D ,
R A D a m o i s e a u x M D ,
P r o f A W H o e s M D ) ; D e p a r t m e n t s o f P a e d i a t r i c s a n d O t o l a r y n g l o g y , W i l h e l m i n a C h i l d r e n s H o s p i t a l , U n i v e r s i t y M e d i c a l C e n t r e U t r e c h t , t h e N e t h e r l a n d s
( M M R o v e r s ) ; U n i v e r s i t y o f O x f o r d , D e p a r t m e n t o f P r i m a r y H e a l t h C a r e , I n s t i t u t e o f H e a l t h S c i e n c e s , O x f o r d , U K
( P r o f P G l a s z i o u M D ,
P B u r k e F R C G P ) ; D e p a r t m e n t o f P e d i a t r i c s , U n i v e r s i t y o f T e x a s M e d i c a l B r a n c h G a l v e s t o n , T e x a s , U S A
( P r o f D P M c C o r m i c k M D ) ; C h a l m e r s R e s e a r c h G r o u p , C h i l d r e n s H o s p i t a l o f E a s t e r n O n t a r i o R e s e a r c h I n s t i t u t e , O t t a w a , O n t a r i o , C a n a d a
( I G a b o u r y ) ; P r i m a r y M e d i c a l C a r e , C o m m u n i t y C l i n i c a l S c i e n c e s D i v i s i o n , U n i v e r s i t y o f S o u t h a m p t o n , A l d e r m o o r H e a l t h C e n t r e , S o u t h a m p t o n , U K ( P r o f P L i t t l e F R C G P )
C o r r e s p o n d e n c e t o :
D r M a r o e s k a M R o v e r s
J u l i u s C e n t r e f o r H e a l t h S c i e n c e s a n d P r i m a r y C a r e , S t r a t e n u m 7 ∑ 1 0 9 , P O B o x 8 5 0 6 0 , 3 5 0 8 A B U t r e c h t , t h e N e t h e r l a n d s M . R o v e r s @ u m c u t r e c h t . n l
I n t r o d u c t i o n
A c u t e o t i t i s m e d i a i s o n e o f t h e m o s t c o m m o n c h i l d h o o d i n f e c t i o n s , t h e l e a d i n g c a u s e o f d o c t o r s c o n s u l t a t i o n s , a n d t h e m o s t f r e q u e n t r e a s o n f o r c h i l d r e n t o t a k e a n t i b i o t i c s . 1 E v i d e n c e f r o m s y s t e m a t i c r e v i e w s , h o w e v e r , s u g g e s t s t h a t a n t i b i o t i c s p r o v i d e o n l y m a r g i n a l b e n e f i t . 2 , 3 F u r t h e r m o r e , p r e s c r i b i n g a n t i b i o t i c s i s k n o w n t o e n c o u r a g e c l i n i c v i s i t s f o r s u b s e q u e n t e p i s o d e s , i n t e n s i f y p r e s s u r e o n c l i n i c i a n s t o p r e s c r i b e , i n c r e a s e a n t i b i o t i c u s e , a n d p r o m o t e a n t i b i o t i c r e s i s t a n c e . 4 6
G u i d e l i n e s t h e r e f o r e r e c o m m e n d s e l e c t i v e u s e o f a n t i - b i o t i c s f o r a c u t e o t i t i s m e d i a , e s p e c i a l l y i n c h i l d r e n a g e d 2 y e a r s o r o l d e r . I n c h i l d r e n y o u n g e r t h a n 2 y e a r s , n o c o n s e n s u s h a s b e e n r e a c h e d . S o m e g u i d e l i n e s r e c o m m e n d a n t i b i o t i c s f o r a l l t h e s e c h i l d r e n , 7 , 8 w h e r e a s o t h e r s a d v i s e a n t i b i o t i c s o n l y f o r c h i l d r e n u n d e r 2 y e a r s i f t h e y a r e s e v e r e l y a f f e c t e d o r h a v e p e r s i s t e n t s i g n s o f d i s e a s e o r r e l a t e d c o m o r b i d i t y . 9 , 1 0
R e l i a b l e i d e n t i f i c a t i o n o f s u b g r o u p s o f c h i l d r e n w h o d o , a n d d o n o t , b e n e f i t f r o m t r e a t m e n t w i t h a n t i b i o t i c s h a s n o t b e e n s t r a i g h t f o r w a r d , b e c a u s e i n d i v i d u a l t r i a l s h a v e b e e n t o o s m a l l f o r v a l i d a n d r e l i a b l e s u b g r o u p a n a l y s e s . A m e t a - a n a l y s i s o f t h e i n d i v i d u a l d a t a f r o m o r i g i n a l t r i a l s e n a b l e s t h e o p p o r t u n i t y t o i d e n t i f y s u b g r o u p s t h a t a r e m o s t l i k e l y t o b e n e f i t . W e t h e r e f o r e a i m e d t o i d e n t i f y s u b g r o u p s t h a t m i g h t b e n e f i t m o s t f r o m s u c h t r e a t m e n t .
M e t h o d s
S e l e c t i o n o f t r i a l s
W e d i d a s y s t e m a t i c s e a r c h o f t h e C o c h r a n e l i b r a r y , P u b M e d d a t a b a s e , E M B A S E , a n d t h e p r o c e e d i n g s o f t h e
i n t e r n a t i o n a l s y m p o s i a o n r e c e n t a d v a n c e s i n o t i t i s m e d i a . W e s e l e c t e d t r i a l s t h a t ( 1 ) u s e d r a n d o m a l l o c a t i o n o f c h i l d r e n , ( 2 ) i n c l u d e d c h i l d r e n a g e d 0 1 2 y e a r s w i t h a c u t e o t i t i s m e d i a , ( 3 ) c o m p a r e d a n t i b i o t i c s w i t h p l a c e b o o r n o t r e a t m e n t , a n d ( 4 ) h a d p a i n a n d f e v e r a s a n o u t c o m e . A l l t r i a l s w e r e a s s e s s e d f o r f o u r m a j o r q u a l i t y c r i t e r i a : p r o p e r r a n d o m i s a t i o n m e t h o d s ; d e g r e e o f f o l l o w - u p ; a n d b l i n d i n g o f t h e o u t c o m e a s s e s s o r , p a t i e n t , a n d c a r e g i v e r . A l l t r i a l s o b t a i n e d i n f o r m e d c o n s e n t a n d e t h i c s a p p r o v a l .
T h e p r i m a r y i n v e s t i g a t o r s o f a l l s e l e c t e d t r i a l s w e r e a s k e d f o r t h e r a w d a t a o f t h e i r t r i a l s . T h e d a t a t h u s o b t a i n e d w e r e t h o r o u g h l y c h e c k e d f o r c o n s i s t e n c y , p l a u s i b i l i t y , i n t e g r i t y o f r a n d o m i s a t i o n , a n d f o l l o w - u p . A f e w i s s u e s w e r e q u e r i e d w i t h t h e r e s p o n s i b l e t r i a l i n v e s t i g a t o r o r s t a t i s t i c i a n , a n d a l l w e r e r e s o l v e d .
O u t c o m e v a r i a b l e s
T h e p r i m a r y o u t c o m e w a s a n e x t e n d e d c o u r s e o f a c u t e o t i t i s m e d i a , w h i c h w a s d e f i n e d a s p a i n , f e v e r , o r b o t h a t 3 7 d a y s . W e u s e d t h i s c o m p o s i t e e n d p o i n t s i n c e b o t h f a c t o r s a r e r e l e v a n t f r o m c l i n i c a l a n d p a t i e n t s ( o r p a r e n t a l ) p e r s p e c t i v e s . F e v e r w a s d e f i n e d a s t e m p e r a t u r e o f 3 8 ∞ C o r h i g h e r , a n d p a i n w a s a s s e s s e d b y p a r e n t s a n d r e c o r d e d i n d i a r y f o r m ( a s e i t h e r y e s o r n o ) . B o t h o u t c o m e m e a s u r e s w e r e d i c h o t o m i s e d , s i n c e s e v e r a l t r i a l s m e a s u r e d t h e m i n t h i s w a y . F e v e r a n d p a i n w e r e a l s o s t u d i e d s e p a r a t e l y ( a s s e c o n d a r y o u t c o m e s ) . A d d i t i o n a l l y , t h e a d v e r s e e f f e c t s o f a n t i b i o t i c t r e a t m e n t m e n t i o n e d i n e v e r y t r i a l w e r e a n a l y s e d .
I n d e p e n d e n t p r e d i c t o r s o f a n e x t e n d e d c o u r s e o f d i s e a s e h a d b e e n e s t a b l i s h e d i n a n e a r l i e r s t u d y w i t h i n
w w w . t h e l a n c e t . c o m V o l 3 6 8 O c t o b e r 2 1 , 2 0 0 6
1 4 2 9
A r t i c l e s
A r t i c l e s
t h e s a m e s e t t i n g ( u n p u b l i s h e d d a t a ) . 1 1 W e u s e d t h e s e i n d e p e n d e n t b a s e l i n e p r e d i c t o r s i e , a g e ( <