PREVENTION AND CONTROL OF INFECTION
Nosocomial infections
and infection control
Aodhan S Breathnach
Abstract
Hospital patients are susceptible to infection because of underlying dis-
eases and medical interventions such as surgery, intubation or antibiotic
use, and also their exposure to microorganisms from other patients, the
hospital environment or hospital staff. An average of 5e10% of in-
patients have a nosocomial infection, with highest rates in surgical and
intensive care units. Most of these infections fall into one of five cate-
gories: line-associated infections and bacteraemia, surgical wound infec-
tion, nosocomial pneumonia, catheter-associated urinary tract infection,
and gastrointestinal infection, including Clostridium difficile and norovi-
rus. Many nosocomial infections are due to antibiotic-resistant organisms
such as meticillin-resistant Staphylococcus aureus and multi-resistant
Gram-negative organisms, which are selected by the antibiotic-rich hospi-
tal environment, but less resistant community pathogens may also cause
hospital infection. Measures to prevent nosocomial infection are varied,
and include aseptic handling of wounds, hand washing before and after
every patient contact, and restrained antibiotic use. Recent public anxiety
about nosocomial infection has led to increased political interest, with
mandatory surveillance and publication of infection rates, financial incen-
tives to reduce infections, and development of ‘care bundles’ e packages
designed to ensure compliance with several different control measures.
Keywords catheter-related infections; cross-infection; hospital infection;
meticillin-resistant Staphylococcus aureus (MRSA); microbial drug resis-
tance; nosocomial infection; surgical wound infection
Introduction
Nosocomial infections are those acquired in or associated with
hospitals. They are also known as hospital-acquired or
healthcare-associated infections. The usual definition of a
hospital-acquired infection is one that was not present or incu-
bating when the patient was admitted to a hospital or healthcare
facility; where there is doubt, a cut-off period of 48 hours after
admission is used. The terms hospital-acquired and healthcare-
associated are often used interchangeably, but ‘healthcare-asso-
ciated’ also has the wider meaning of any infection acquired as a
result of healthcare in any setting. The related term ‘iatrogenic’
refers to infection or illness specifically associated with medical
devices, procedures or therapies.
Any community infection can also occur in hospital, but there
are many factors in the hospital environment that lead to a
particular spectrum of infective problems. Nosocomial infections
are common, and may be serious or fatal. Some are unavoidable,
n S Breathnach MD FRCPath is a Medical Microbiologist at St
Aodha
George’s Hospital, London, UK. Competing interests: none declared.
MEDICINE 41:11
What’s new?
C Multi-resistant Gram-negative organisms such as Escherichia
coli, Klebsiella and Pseudomonas, are emerging as a significant
problem, with fears of significant numbers of untreatable in-
fections in the near future
C Antibiotic resistance is being increasingly seen as a global
problem, with factors such as over-the-counter antibiotics in
many countries, use of antibiotics in livestock, travel and health
tourism all contributing to selection and spread of resistant
organisms
C Antibiotic stewardship is increasingly emphasized, in order to
limit any avoidable selection of such resistant organisms. Many
hospitals employ a multidisciplinary approach to antibiotic
stewardship, with microbiologists, pharmacists and infection
control nurses all contributing
C Improved viral diagnostics means that the role of viral respi-
ratory and gastrointestinal infection in hospital is increasingly
recognized
but doctors’ duty of care to their patients extends to fundamental
matters such as basic hygiene, which may prevent patients
becoming infected, and avoidance of unnecessary antibiotics, to
discourage development of resistance.
Background
Despite the many recent advances in medical science, hospital-
acquired infections still cause considerable illness and some
mortality. It has been estimated that 5e10% of in-patients in
British and Irish hospitals have a nosocomial infection; the
prevalence is highest in surgical wards and intensive care units
(ICUs), and lowest in medical units.1 Apart from the significance
of harm resulting from medical or nursing care, nosocomial in-
fections cause a financial burden to hospitals, patients and so-
ciety. In a study carried out in the 1990s, the cost in the UK, in
terms of increased hospital stay and treatment, was estimated at
up to £1000 million/year.2 This figure does not include costs of
litigation and compensation, nor does it include costs to the
patient resulting from their infection.
Since 2000, nosocomial infections and antibiotic resistance
have become the subjects of considerable media, public and
political interest. Meticillin-resistant Staphylococcus aureus
(MRSA) bacteraemia was chosen as the subject of the first na-
tional reduction programme in England and Wales in 2004,
because it was relatively easy to measure (using straightforward
laboratory diagnosis) and was believed to be a rough marker of
general infection control standards. This was followed in 2007 by
a national 30% reduction target for Clostridium difficile in-
fections, with strict financial penalties for hospitals that did not
meet their target. There was considerable success in reducing
infection rates, though opinion is divided on whether an
organism-specific approach leads to more generalized improve-
ments in infection control, or, conversely, to skewing of re-
sources and attention, and therefore neglect of other nosocomial
649 Ó 2013 Elsevier Ltd. All rights reserved.
 PREVENTION AND CONTROL OF INFECTION
infections.3,4 In the USA, the Centers for Medicare and Medicaid
Services has reduced payments to hospitals for certain compli-
cations judged to be reasonably preventable e including several
nosocomial infections.5
Predisposing factors in nosocomial infection
It is helpful to consider the causes of nosocomial infection under
three headings: patient factors, medical/surgical interventions,
and the hospital microbial environment, as follows.
Underlying patient factors
Many patients are intrinsically vulnerable to infection: they are at
the extremes of age, debilitated, or have an underlying immuno-
deficiency (e.g. HIV). Neurological illness may lead to aspiration
pneumonia; and immobility and dehydration may encourage
urinary tract infection (UTI). Skin disease and bedsores allow
organisms to enter subcutaneous tissue. In addition, and impor-
tantly, the normal flora of all patients includes potential pathogens
such as Escherichia coli in the gut and S. aureus in the nose.
Medical and surgical interventions
Surgical incisions and intravascular devices provide means of
entry for pathogens. Urinary catheterization often causes UTIs.
Prosthetic joints and heart valves provide a protected niche for
bacterial growth, and immunosuppressive therapy allows even
low-virulence organisms to assume a dangerously pathogenic role.
Less obvious ways in which medical therapy can facilitate
infection include anaesthesia and ventilation (which predispose
to nosocomial pneumonia) and antibiotics (which alter normal
flora, reducing resistance to colonization by hospital organisms).
Inadequately disinfected endoscopes can transmit pathogens
such as Mycobacterium tuberculosis and Salmonella, as well as
hepatitis viruses.6
Hospital microbial environment
Patients may become infected with new organisms, from other
patients, staff or the environment. Transient hand carriage by
medical or nursing staff is thought to be the main route of spread,
but occasionally other routes are involved (e.g. airborne route for
respiratory pathogens). Overcrowding, understaffing and poor
hygiene, particularly hand washing, increase the risk of cross-
infection.
Antibiotic use in hospital has selected resistant organisms that
readily colonize and infect patients (Table 1). Many (e.g.
coagulase-negative staphylococci, enterococci) are of relatively
low virulence, but can cause severe illness in compromised pa-
tients; some (e.g. MRSA) can be as virulent as their more sen-
sitive counterparts. Multi-resistant Gram-negative organisms
have increased in relative importance. Such organisms include
members of the Enterobacteriaceae such as E. coli and Klebsiella,
in addition to Pseudomonas aeruginosa. These bacteria often
carry multiple resistance elements, including extended-spectrum
beta-lactamases or carbapenemases that confer resistance to
broad-spectrum cephalosporins and/or carbapenems such as
meropenem; they may also be resistant to aminoglycosides such
as gentamicin, and to ciprofloxacin. As a consequence, treatment
of these infections can be extremely difficult, and there may be
significant associated mortality. Global epidemics of such resis-
tant strains of bacteria are increasingly recognized, and in some
MEDICINE 41:11
areas these organisms are not confined to hospitals but are also
found in healthy individuals and in the environment. Factors
contributing to spread include antibiotic over-use in both the
hospital and the community, veterinary use of antibiotics
allowing resistant organisms to enter the food chain, availability
of over-the-counter antibiotics in some countries, and increased
travel, migration and health tourism.7 Antibiotic resistance is
further discussed in the article on pages 642-648 of this issue.
Common syndromes and problems in nosocomial infection
Most nosocomial infections fall into a few common categories.
Specialist units (e.g. burns, transplant, neurosurgery) see
different infections, in addition to the problems described below.
Infections in transplant patients are discussed in the article on
pages 000e000 of this issue.
Line-associated infections and bacteraemia
In most settings, one-quarter to one-third of cases of nosocomial
bacteraemia arise from an intravenous device, a similar propor-
tion are from an unknown source, and the remainder have
various sources, including the urinary tract, the gut and
ventilator-associated pneumonia.8
Intravenous devices provide both a break in the skin, allowing
entry of organisms, and a protected site for bacterial growth
shielded by a biofilm of platelets, fibrin and bacterial slime. The
risk is greater with increasing age of the line, central and multi-
lumen lines, and poor insertion technique or line care, which can
lead to infection of the insertion site or hub. Pre-existing skin
disease and colonization with pathogens such as MRSA also in-
crease the risk of infection. The heavy normal flora of the groin
means that femoral lines generally become infected, despite
careful insertion technique.
Almost 50% of nosocomial bacteraemia isolates are staphy-
lococci (either coagulase-negative or S. aureus, including MRSA)
and these are most likely to be line-associated. The remainder are
mainly Gram-negative (E. coli, Klebsiella, Enterobacter, Proteus
and Pseudomonas), with small numbers of enterococci and
Candida (not bacteria, but generally included in reviews of
‘bacteraemia’). Gram-negatives may be line-associated, but are
more likely to have a urinary source, or to arise from the gut in
patients with intra-abdominal pathology or neutropenia.
Presentation and diagnosis: line infections present with septi-
caemia or obvious infection of the exit site or tunnel, or are si-
lent. Other sequelae (which may manifest only after line
removal) include endocarditis and disseminated abscesses (e.g.
spinal, ophthalmic). Blood cultures and culture of the line tip are
the usual means of diagnosis.
Management: line infections (except some with coagulase-
negative staphylococci) are almost never eradicated by antibi-
otics unless the line is also removed. Infected lines are sometimes
left in situ when intravenous access is difficult, but this increases
the risk of septicaemic complications.
Nosocomial chest infections
Pneumonia is responsible for about 25% of all nosocomial in-
fections, but the diagnosis is often uncertain; in severely ill pa-
tients, there may be other explanations for fever, hypoxia and
650 Ó 2013 Elsevier Ltd. All rights reserved.
 PREVENTION AND CONTROL OF INFECTION

Typical hospital organisms

Usual location Risk factors for acquisition Typical infections Comments

Meticillin-resistant Nasal or cutaneous Antibiotic use, skin lesions, Skin/wound, orthopaedic, Occasional case reports
Staphylococcus carriage overcrowding, poor infection intravenous devices, of developing resistance
aureus control bacteraemia, endocarditis, to glycopeptides, which
respiratory tract, prosthetic are the agents of choice
devices for invasive infections
Enterococci and Gut, hospital Antibiotics e particularly Low virulence; typically, Vancomycin-resistant
vancomycin- environment cephalosporins, vulnerable ICU patients enterococci are more
resistant enterococci glycopeptides or renal patients, in whom correctly, but rarely,
use of glycopeptide antibiotics termed ‘glycopeptide-
is common resistant enterococci’; heat
and disinfectant tolerant,
so survive well in hospital
environment
Clostridium Gut, hospital Antibiotics (especially Antibiotic-associated Large outbreaks with fatal
difficile environment, cephalosporins, quinolones), diarrhoea/pseudomembranous cases reported; predisposed
elderly care wards poor hygiene colitis by loss of natural bowel
flora, hence several
suggested ‘alternative’
remedies based on the
principle of replacing this
flora (e.g. live yoghurt,
donor faeces)
Multi-resistant Gut, hospital Antibiotics, poor hygiene, Intra-abdominal, respiratory and Various species (e.g.
Gram-negatives environment, movement of patients between bloodstream infections in Acinetobacter, Klebsiella,
high-dependency hospitals and between vulnerable patients Enterobacter,
units countries Stenotrophomonas);
many are indolent
opportunists, but some
(e.g. Klebsiella,
Pseudomonas aeruginosa)
can cause aggressive,
virulent infections

NB: most nosocomial infections are caused by the patient’s own flora or by cross-infection with more sensitive organisms not listed above.

Table 1

pulmonary infiltrates on radiography (e.g. pulmonary oedema,
shock lung, segmental collapse). Furthermore, the respiratory
tract of hospitalized patients is often colonized with various or-
ganisms, and it is difficult to distinguish colonization from
genuine pneumonia.
Most studies implicate a variety of Gram-negative organisms
as the predominant causative agents, with doubts about the
significance of culture results as described above. S. aureus,
particularly MRSA, is the only commonly implicated Gram-
positive organism; pneumococci are seldom isolated, but may
be under-diagnosed. Thus, empirical treatment guidelines for
nosocomial pneumonia assume Gram-negatives to be the likely
cause and generally recommend agents with activity against
Gram-negative organisms.9
Hospital-acquired tuberculosis (TB) and Legionnaire’s dis-
ease: these are rare, but important because of their severity and
because of the possibility of outbreaks. Any sputum-smear-
positive TB patient who is not adequately isolated may spread
the organism to other patients; unrecognized multi-drug-resistant
TB poses the greatest risk, because these patients are likely to
remain infectious for longer. All contacts are at risk, but the
greatest risk of progression to disease is in HIV-positive and other
severely immunosuppressed contacts.10
Legionella infection is acquired from the environment; trans-
mission between patients does not occur. The organism normally
inhabits water, preferably at 20e40 C; sources in hospitals
include water-cooled ventilation systems (formerly the cause of
large outbreaks), shower heads and tap water. Patients most at
risk include the elderly, those with chronic respiratory disease,
and the immunosuppressed, particularly recent transplant re-
cipients. The infection presents as severe pneumonia; the or-
ganism grows poorly, so diagnosis usually depends on urinary
antigen detection.

MEDICINE 41:11 651 Ó 2013 Elsevier Ltd. All rights reserved.

 PREVENTION AND CONTROL OF INFECTION
Surgical site infections
Despite asepsis and antibiotic prophylaxis, surgical site infections
remain common. It is believed that infection usually arises from
the patient’s own skin flora, inoculated into the wound during
surgery; hair follicles and sweat glands cannot be completely
sterilized during skin preparation, so no surgical field is
completely sterile. Other possible sources include theatre staff
(who inevitably shed skin flakes and respiratory droplets),
perforated gut, and contaminated traumatic wounds.
The incidence of infection varies between hospitals and types
of surgery. In a recent large English survey, the lowest incidences
(2e3.5%) were associated with orthopaedic procedures and the
highest (10e15%) with amputations and abdominal surgery.
Severe underlying illness and lengthy operations increase the
risk; more than 35% of severely ill patients undergoing pro-
longed large bowel surgery develop infection. Staphylococci
(mostly S. aureus, including MRSA) cause almost 50% of in-
fections; most of the remainder are caused by Gram-negatives,
including E. coli and Pseudomonas.
Most infections are superficial and easily treatable. Deeper
infections are rarer, and may be catastrophic, particularly when
surgery involves bone, brain, a prosthesis, a vascular graft or a
transplant. Group A streptococcus (Streptococcus pyogenes) can
cause rapidly spreading, often fatal necrotic infections (necro-
tizing fasciitis).
Urinary tract infections
Urinary catheters are responsible for most nosocomial UTI: each
day, bacteriuria develops in 5% of catheterized patients; the risk
is greater with unhygienic insertion technique, greater age, de-
bility and dehydration. It is important to distinguish asymptom-
atic bacteriuria from symptomatic infection: it is reasonable to
treat symptomatic infection with antibiotics, but unless the
catheter is removed, the urinary tract will not be sterilized and
may become colonized with antibiotic-resistant organisms. For
this reason, investigation of urinary samples from asymptomatic
chronically catheterized patients is not recommended.11
Antibiotic-associated diarrhoea and C. difficile infection
C. difficile is the most common cause of hospital-acquired diarrhoea.
This important infection is further discussed on pages 654-657 of this
issue see also Table 1.
Nosocomial viral infections
Improved molecular diagnostic techniques mean that many more
nosocomial viral infections are now recognized that would pre-
viously have gone unreported. Such infections include viral
respiratory tract infections, including influenza, and norovirus
gastroenteritis. Norovirus in particular can cause rapidly
spreading hospital outbreaks affecting patients, staff and visitors.
Cross-infection with blood-borne viruses such as hepatitis B
or C, or HIV, is of great concern, but is fortunately rare because
of well-established control measures, including sterilization of
surgical instruments before use, and single use or sterilization of
needles and other pieces of equipment that come into contact
with sterile tissues and blood. Correct handling of blood samples
and disposal of used needles and other contaminated sharp
equipment is also important. In the past, attempts were made to
identify likely virus carriers and take greater precautions with
MEDICINE 41:11
Measures to control and prevent hospital-acquired
infection (current UK practice or recommendations)
Hand washing/cleaning (before and after patient contact): is said to
be the single most critical measure in preventing spread of organisms
such as meticillin-resistant Staphylococcus aureus (MRSA), yet
achieving compliance is challenging. Hand sanitizers using alcohol
gel or other disinfectant are a convenient alternative to washing,
though they do not kill bacterial spores (such as those of Clostridium
difficile), and may not inactivate norovirus.
Isolation and barrier nursing: of infectious patients (e.g. with
tuberculosis, MRSA or active diarrhoea).
Protective isolation: some immunocompromised patients are isolated
to protect them from infection, rather than to protect their contacts.
Aseptic approach: to insertion and handling of intravenous devices
and urinary catheters reduces infection rates.
Sterilization of surgical instruments and aseptic technique: reduces
entry of organisms into patients’ wounds.
Decontamination of endoscopes: endoscopes cannot withstand
autoclaving and often have complex channels; they are therefore a
major risk for transmission of infection.
Antibiotic stewardship: active efforts to ensure restrained antibiotic
use and adherence to antibiotic guidance, reduces selection of
resistant organisms, particularly C. difficile.
Surgical perioperative antibiotic prophylaxis: helps to prevent surgical
wound infection, particularly when there is a significant local flora at the
operative site (e.g. abdominal surgery), or when the consequences of
infection could be catastrophic (e.g. vascular graft surgery).
Correct disposal of contaminated sharps: (principally needles, glassware
and cannulae) into yellow ‘sharps bins’; do not re-sheath needles.
Vaccination of hospital staff: clinical staff should be either immune
to or vaccinated against conditions such as hepatitis B, rubella,
tuberculosis, influenza and varicella; this is to protect both staff
and vulnerable patients.
Screening of patients for carriage of MRSA or other resistant
organisms: this allows potentially infectious patients to be quickly
recognized and isolated.
Surveillance of nosocomial infection: this allows the scale of problems
to be measured, and comparison between hospitals, wards, etc. to be
made, and the effect of any interventions to be assessed.
Screening and partial exclusion of staff: staff who are known to be
infectious with hepatitis B/C or human immunodeficiency virus are
excluded from certain activities (e.g. surgery) through which they
might infect their patients.
Engineering and design considerations: many doctors are unaware of
the complexity of hospital design. Simple factors such as the availability
of sinks for hand washing and overcrowding of patients can have a
significant effect on the ability of staff to prevent cross-infection. Single
rooms must be ventilated appropriately e positive pressure for
protective isolation, neutral or preferably negative pressure for airborne
infections. Ideally there should be enough single rooms to isolate all
infectious patients. Filtered air must be supplied to theatres and some
isolation rooms, and the direction of airflow in theatres should be
controlled, from ‘clean’ to ‘dirty’ (i.e. lay-up area to theatre to anaesthetic
room). Hospital water supplies, and cooling towers that form part of the
air-conditioning system, must be kept free from Legionella.
Table 2
652 Ó 2013 Elsevier Ltd. All rights reserved.
 PREVENTION AND CONTROL OF INFECTION
them, but it was realized that carriers were often unrecognized.
Accordingly, the concept of ‘universal precautions’ has evolved,
based on the fact that any patient or sample might contain blood-
borne viruses and should be handled with this risk in mind.
Contaminated sharps waste should be disposed of in specific
yellow, rigid (needle-proof) containers, which are then inciner-
ated. Re-sheathing of needles after use was formerly a major
cause of sharps injuries and is now strongly discouraged.
Controlling and preventing nosocomial infections
The methods of controlling and preventing hospital infections are
as varied and complex as the infections themselves, and range
from engineering aspects of hospital design and maintenance, to
political decisions concerning vaccination policies, to personal
hygiene by staff and patients.12 The major interventions are sum-
marized in Table 2, and some have already been discussed above.
Some interventions such as asepsis in wound care and ster-
ilization of surgical instruments are long established. Other
measures such as universal mandatory hand hygiene or antibi-
otic restrictions are more recent and may be more challenging to
introduce, as they may conflict with long-established custom and
practice.
In recent years, infection control has been seen in the wider
context of improving patient safety, and there has been recog-
nition that prevention of nosocomial infections may depend on
several complementary interventions and control measures. This
has led to the introduction of ‘care bundles’ for several nosoco-
mial infections: combinations of practices that collectively reduce
the risk of infection, along with checklists or audit tools to ensure
compliance.13
Conclusion
Nosocomial infections remain common. While some are prob-
ably unavoidable, recent experience has shown that improved
attention and care can lead to significant improvements in
infection rates. Such improvements need a commitment at every
level of healthcare, from politicians to hospital executives to
ward staff. A Wilson J. Infection control in clinical practice. 3rd edn. Bailliere Tindall,
REFERENCES
1 Smyth ET, McIlvenny G, Enstone JE, et al. Four country healthcare
associated infection prevalence survey 2006: overview of the results.
J Hosp Infect 2008; 69: 230e48.
2 Plowman R, Graves N, Griffin MA, et al. The rate and cost of hospital
acquired infections occurring in patients admitted to selected spe-
cialties of a district general hospital in England and the national
burden imposed. J Hosp Infect 2001; 47: 198e209.
MEDICINE 41:11
3 Millar M, Coast J, Ashcroft R. Are methicillin-resistant Staphylococcus
aureus bloodstream infection targets fair to those with other types of
healthcare-associated infection or cost-effective? J Hosp Infect 2008;
69: 1e5.
4 Duerden B. Opinion on Millar, et al. ‘Are meticillin-resistant Staphy-
lococcus aureus bloodstream infection targets fair to those with
other types of healthcare-associated infection or cost-effective?’.
J Hosp Infect 2008; 69: 6e7.
5 Rosenthal MB. Nonpayment for performance? Medicare’s new reim-
bursement rule. N Engl J Med 2007; 357: 1573e5.
6 Spach DH, Silverstein FE, Stamm WE. Transmission of infection by
gastrointestinal endoscopy and bronchoscopy. Ann Intern Med 1993;
118: 117e28.
7 Sandora TJ, Goldmann DA. Preventing lethal hospital outbreaks of
antibiotic-resistant bacteria. N Engl J Med 2012; 367: 2168e70.
8 Jerassy Z, Yinnon AM, Mazouz-Cohen S, et al. Prospective hospital
wide studies of 505 patients with nosocomial bacteraemia in 1997
and 2002. J Hosp Infect 2006; 62: 230e6.
9 Masterton RG, Galloway A, French G, et al. Guidelines for the
management of hospital-acquired pneumonia in the UK: report of
the working party on hospital-acquired pneumonia of the British
Society for Antimicrobial Chemotherapy. J Antimicrob Chemother
2008; 62: 5e34.
10 Breathnach AS, de Ruiter A, Holdsworth GM, et al. An outbreak of
multi-drug-resistant tuberculosis in a London teaching hospital.
J Hosp Infect 1998; 39: 111e7.
11 Warren JW. Catheter-associated urinary tract infections. Int J Anti-
microb Agents 2001; 17: 299e303.
12 Ayliffe GA, Fraise A, Geddes AM, Mitchell K. Control of hospital
infection. 4th edn. London: Arnold, 2000.
13 Aboelela SW, Stone PW, Larson EL. Effectiveness of bundled
behavioural interventions to control healthcare-associated in-
fections: a systematic review of the literature. J Hosp Infect 2007;
66: 101e8.
FURTHER READING
Ayliffe G, Fraise A. Ayliffe’s control of healthcare-associated infection: a
practical handbook. 5th edn. Hodder Arnold, 2009.
2006.
WEBSITES
http://www.cdc.gov/hai/. Centers for Disease Control Hospital Infection
Program; US government web site dealing with all aspects of hospital
infections.
www.hpa.org.uk. UK Health Protection Agency; includes a section on
healthcare-associated infection. http://www.hpa.org.uk and http://
www.gov.uk/phe.
www.his.org.uk. UK Healthcare Infection Society.
www.hps.scot.nhs.uk/haiic/index.aspx. Scottish infection control website.
653 Ó 2013 Elsevier Ltd. All rights reserved.