Viral Infections in the Immunocompetent Host

1
Viral Infections in the Immunocompetent Host

• Corey Casper, M.D., M.P.H.
• Division of Infectious Disease, Department of
  Medicine
• The University of Washington
• Vaccine and Infectious Disease Institute,
• Fred Hutchinson Cancer Research Center

2
Outline

• Classification of Viruses
• Classical vs. Other Schemes
• Diagnosis of Viral Infections
• Common Viral Infections for the Infectious
  Disease Consultant

3
Classification of Viruses
4
Classification of Viruses

• Classic Taxonomy
• Nucleic Acid Structure
• DNA vs. RNA
• Single vs. Double Stranded
• Envelope
• Presence or absence
• Organization of genome
• Example Paramyxoviruses
• Mode of transcription
• Example Retroviruses
• Functional Taxonomy
• Group viruses by primary organ system involved in
  the pathology of disease
• Example Respiratory Viruses
• Group viruses with similar treatments
• Example Herpesviruses

5
Your Mother Knows Best?

• Which of the following viruses would you be most
  likely to acquire from touching a toilet seat?
  True MedCon Call!
• HIV
• Calicivirus
• Herpes Simplex Virus-2
• Parainfluenza

6
Viral Structure
7
Player or Bystander?

• A 63 y.o. man presents from an outside hospital
  with fever and headache for 2 weeks. Multiple
  blood, urine, CSF, and sputum cultures have been
  negative. Chest X-ray, full body CT and
  peripheral smear are all unremarkable. You are
  consulted by the medical team to assess whether
  the patients symptoms could be attributable to
  infection with CMV. Which of the following
  studies would support that diagnosis?
• 1,000 copies of CMV DNA by PCR from the
  peripheral blood
• Positive CMV IgM
• Positive urine CMV shell-vial culture
• None of the above

8
Diagnosis of Viral Infections - Direct
9
Diagnosis of Viral Infections - Indirect
10
Diagnostic Virology Culture

• Clinical specimen collected and either sent
  directly to lab or placed in viral culture medium
• Specimens then grown on number of different cell
  lines depending on type of virus suspected
• Diagnosis either by looking for CPE, or adding
  fluorescently-tagged antibodies to viral antigens
• Shell vial culture Diagnosis of CMV or BK
• Advantages Specific, sensitivity testing?
• Disadvantages Slow, not as sensitive as
  molecular diagnostics, not possible for all
  viruses

11
Diagnostic Virology DFA
Fluorescent label
Antibody to Viral Protein
Clinical Specimen
12
Diagnostic Virology EIA
13
Diagnostic Virology PCR

• Advantages
• Rapid
• Sensitive
• Quantitative
• Disadvantages
• Too sensitive?
• Specificity
• Costly

Source http//www.nupedia.com/newsystem/upload_fi
le/678/pcr.png
14
Case 1 HPI

• 18 y.o. woman from Sitka, Alaska who presents
  with fevers and abdominal pain for 2 weeks
• Initially presented to ED in AK 2 weeks PTA with
  dysuria and mild abdominal pain
• Treated with TMP-SMX without improvement
• Re-presented 3 days later with severe abdominal
  pain, headache and temperature to 102F. Had
  diffuse vesicular rash
• Admitted to hospital where she had the following
  labs/studies
• Normal CBC, SMA-7, negative UA, negative CXR and
  KUB
• AST 110, ALT 124, nml INR, GGT, Amylase, Alk Phos
• Hospital Course
• Subsequent multiple blood and urine cultures
  negative
• CT of chest, abdomen and pelvis negative
• Exploratory laparotomy found lesions on the liver
  as on the following slide
• Persistent fevers and abdominal pain despite
  Cefotetan, Doxycycline and Metronidazole
• Transferred to UWMC

15
Case 1 Hepatic Lesions
16
Case 1 Physical Exam on Transfer to UWMC

• T 38.9, HR 110, RR 22, BP 118/72
• Abd Diffuse TTP, no rebound or guarding
• Skin Adjacent rash
• GU Nml genitalia

17
The Herpesvirus Family

• HHV-1 Herpes Simplex 1 (HSV-1)
• Clinical Oral Herpes
• HHV-2 Herpes Simplex 2 (HSV-2)
• Clinical Genital Herpes
• HHV-3 Varicella Zoster Virus (VZV)
• Clinical Chickenpox, Zoster
• HHV-4 Epstein Barr Virus (EBV)
• Clinical Mono, lymphoma
• HHV-5 Cytomegalovirus (CMV)
• Clinical Retinitis, Pneumonitis, etc
• HHV-6/7 Roseolavirus
• Clinical Exanthem subitum
• HHV-8 Kaposis Sarcoma-Associated Herpesvirus
  (KSHV)
• Clinical KS, multicentric Castlemans disease,
  primary effusion lymphoma

18
Herpesvirus Family Characteristics

• Large, Enveloped DNA-viruses
• Envelope
• Transmission via mucosal surfaces
• Fomite acquisition is uncommon
• Large
• Smart!
• Evolved many complex mechanisms for immune
  evasion and pathogenesis
• DNA
• Use similar cellular machinery to human DNA, so
  therapy must find novel areas of difference (in
  contrast to HIV)
• Ubiquitous
• Except for HSV-2 and HHV-8, all infect more than
  50 of most populations worldwide
• Latency allows for life-long infection
• Intermittent reactivation and lifelong shedding
  can make understanding clinical symptoms and
  diagnostic tests challenging
• Long term infection with some herpesvirus can
  lead to cancer

19
Herpesvirus Therapy DNA Synthesis Inhibitors

• Aciclovir and ganciclovir require viral TK to
  make dGMP, then cellular kinases make dGTP which
  terminates DNA synthesis
• Cidofovir and foscarnet do not require TK
• Ribavirin depletes intracelluar GTP

Source Naesens and de Clercq Herpes 2001
20
Case 2

• 34 y.o. nurse presents with 3 weeks of coughing,
  post-tussive emesis, sinus congestion and malaise
  in January

21
Respiratory Viruses Clinical

• Heterogeneous group of viruses
• DNA and RNA, enveloped and naked
• Similar clinical presentations
• Seasonality is important

22
What goes around comes around

• http//depts.washington.edu/rspvirus/respiratory.h
  tm

23
Viruses in Health Care Workers, 2007-2008
24
Respiratory Viruses Pearls
25
Influenza Virus Strains

• Type A - moderate to severe illness - all age
  groups - humans and other animals
• - Subtypes of type A determined by hemagglutinin
  and neuraminidase
• Type B - milder epidemics - humans only -
  primarily affects children
• Type C - rarely reported in humans - no
  epidemics

26
Influenza Virus

• Neuraminidase
• Antigenic Determinant
• Confer virulence
• Allow viral mobility through
• Respiratory tract

• Hemagglutinin
• Binds virus to cell
• Confers target specificity

RNA

• M2 protein
• only on type A
• Allows H ions to enter virus to lower
• pH for viral uncoating

27
Influenza Antigenic Changes

• Hemagglutinin and neuraminidase antigens change
  with time
• Changes occur as a result of point mutations in
  the virus gene (antigenic drift), or due to
  exchange of a gene segment with another subtype
  of influenza virus (antigenic shift)
• Impact of antigenic changes depend on extent of
  change (more change usually means larger impact)

28
Making a Global Influenza Pandemic
OR
29
Influenza Clinical Features

• Incubation period 2 days (range 1-4 days)
• Severity of illness depends on prior experience
  with related variants
• Abrupt onset of fever, myalgia, sore throat,
  nonproductive cough, headache

30
Influenza Complications

• Pneumonia
• primary influenza
• secondary bacterial
• Reye syndrome
• Myocarditis
• Death 0.5-1 per 1,000 cases

31
Influenza Treatment Prophylaxis

• M2 Inhibitors
• Amantadine and rimantidine
• Effective against Influenza A
• 2007, gt97 of influenza was resistant
• SWINE FLU IS RESISTANT
• Inhibit viral replication
• Single mutation confers resistance, occurs with
  every 1,000-10,000 replications
• Neuraminidase inhibitors
• Oseltamivir (oral pill) and Zanamivir (inhaled)
• Effective against Influenza A and B
• 98 of H1N1 strains (except SWINE FLU) were
  resistant in 2008-9!
• Combination therapy?
• Oseltamavir, rimantidine and ribavirin have been
  shown to have combined efficacy

32
Respiratory Viruses Adenovirus

• Common cause of URI and keratoconjuntivitis. Has
  been occasionally associated with pneumonia in
  community outbreaks, diarrhea in children, and
  hepatitis.
• May cause cystitis or nephritis in transplant
  patients
• Treatment Supportive. IV cidofovir may be
  effective in the immunocompromised

33
Respiratory Viruses Parainfluenza

• Four subtypes
• PIV3 seen most commonly in severe infections
• In children, leading cause of croup. Can be a
  cause of severe lower respiratory tract illness
  in some children or transplant patients
• Treatment is supportive, but aerosolized
  ribavirin may be used in life-threatening cases

34
Respiratory Viruses Metapneumovirus

• Recently identified from retrospective series of
  unidentified respiratory illnesses.
• Serologic studies suggest most are infected by 5
  years of age, peak 6-12 months
• Mild URI in most, with rare progression to severe
  LRTI
• Wheezing is a common initial presentation
• Treatment is supportive

35
Respiratory Viruses Coronaviruses

• Large family of viruses with multiple animal
  hosts
• Generally cause non-specific symptoms such as
  fevers, myalgias, fatigue. May progresses to
  non-productive cough and dyspnea.
• Diagnosis is by PCR, and treatment is supportive
• SARS
• Newly identified virus associated with severe
  LRTI in Asia in 2003. Thought to be transmitted
  by contact with small mammals (civets) in Asia,
  spread between humans through respiratory
  droplets and feces
• Development of respiratory failure occurs in
  minority of cases, but may be more common in
  Asian persons

36
PCR for Respiratory Virus Detection

• Problem of inadequate specimens for immunoblot or
  DFA
• PCR is more sensitive and perhaps equally as
  specific
• Molecular Virology Lab now offers multiplex PCR
  for detection of 12 viruses

Kuypers, et al 2006
37
Gastrointestinal Viruses

• Most common viruses to cause gastrointestinal
  illnesses are Norovirus, calicivirus, rotavirus,
  astrovirus, and adenovirus
• Present with diarrhea, fever and/or abdominal
  pain. Children more often affected, although
  incidence high in institutional or closed
  settings (i.e. cruise ships)
• Transmission via fecal-oral route
• Diagnosis
• PCR of stool, or plasma PCR if disseminated
  disease suspected (adenovirus)

38
Case 3

• 21 year old UW student presents with fever to 39,
  headache, stiff neck and photophobia shortly
  after returning for Fall Quarter
• Student health service concerned about risk of
  meningitis epidemic

39
Case 3 Continued

• Physical examination revealed the following

40
Enteroviruses

• Large group of viruses including the subgroups
  poliovirus, echovirus, and coxsackieviruses
• Worldwide pathogens with most infections in
  summer and fall
• Chronic meningoencephalitis among persons with
  agammaglobulinemia
• Diagnosis
• PCR of stool, oropharynx or CSF

41
Enteroviruses Coxsackievirus

• Common causes of aseptic meningitis
• Heterogeneous and non-distinct exanthems (skin
  rashes). Exception Hand-Foot-Mouth
  (Coxsackievirus A16) with oral vesicles and
  papules/vesicles on palms and soles.
• Complications
• Group A
• herpangina (dysphagia with lesions on soft
  palate)
• Group B
• Myopericarditis

42
Case 4

• 62 y.o. man taken to HMC from cruise ship docked
  at Pier 66 with fevers, altered mental status,
  and weakness in the left leg

43
FLAVIVIRUSES

• Heterogeneous group of zoonotic / arthropod
  transmitted viruses
• West Nile Virus
• Dengue
• Yellow Fever
• Japanese Encephalitis
• St. Louis Encephalitis
• Tick-Borne Encephalitis
• Diagnosis
• Serology
• IgM during acute illness or IgG in convalescence
• Serum should be collected 8-10 days after illness
  onset.
• Follow up with a convalescent serum specimen
  obtained at least 2 weeks after the first
  specimen.
• CSF should be collected within 8 days of illness
  onset. IgM may appear in CSF earlier than in
  serum.
• IgM does not cross the blood brain barrier its
  presence in CSF indicates neuroinvasive disease.
• IgM antibody can persist for more than
• Non-specific (but this may be a good thing!)
• PCR
• Less sensitive, but useful in immunocompromised
  hosts

44
Flaviviruses West Nile Virus

• Rapidly emerging virus across U.S. since 1999
• WA one of the few states without any documented
  infections
• Transmitted from reservoirs in birds to human via
  mosquitoes
• Majority of infections are without symptoms or
  only with fever and malaise, but most severe
  complication is neurological (encephalitis and
  muscle weakness)

45
West Nile Distribution, 2008
46
Flaviviruses Dengue

• Tropical virus transmitted by Aedes aegypti (day
  biting) mosquito
• Illness characterized by high fever, headache
  (often retro-orbital), myalgias/arthralgias and
  rash
• Hemorrhagic fever or shock may occur shortly
  after resolution of fever. May be more common in
  persons previously exposed.

47
Flaviviruses Yellow Fever

• Endemic to sub-Saharan Africa and South America
• Transmitted by mosquito bites
• Symptoms range from constitutional to severe.
  Symptomatic patients likely to experience
  headache, altered mental status, icterus, and
  many have diffuse hemorrhage
• Preventable by vaccine, which may cause
  vaccine-induced encephalitis among young infants
  or the elderly

48
Other Flaviviruses

• Japanese Encephalitis
• High fevers and altered mental status
• Endemic to regions in Asia where mosquitoes
  interact with pigs and birds
• St. Louis Encephalitis
• Fevers and altered mental status, especially
  among the elderly.
• Seen in North, Central and South America as well
  as the Caribbean.
• Tick-Borne Encephalitis
• Infection via Ixodes species ticks
• Europe and Asia
• History persons with outdoor exposure.
• Presents with fever, but may progress to altered
  mental status and paralysis.

49
Case 5

• 28 year old latina sheep-sheerer from Oregon
  presents to UWMC with increasing lesion on hand

50
Poxviruses Orthopox

• Monkeypox recently spread by prairie dogs
• Cowpox cause milkers nodules on hands of dairy
  workers
• ORF nodule on hands, arms or face after exposure
  to ruminants
• Smallpox
• Diagnosis
• Electron Microscopy

51
Poxviruses Smallpox

• Smallpox is the only infectious disease
  eradicated with vaccination, now threatening to
  return in the setting of bioterrorism
• Infection via respiratory droplets or contact
  with infected lesions. Acquisition is largely
  asymptomatic for first 7-10 days, followed by a
  non-specific prodrome consisting of fevers and
  malaise.
• Patient becomes infectious upon development of
  rash. Typically, rash is maculopapular, starts in
  the oropoharynx/head/neck/upper extremities, and
  moves caudally. Lesions are usually in the same
  stage (i.e. vesicular, pustular, crusted), which
  differentiates the lesion from varicella.
• Diagnosis is by PCR or electron microscopy of
  vesicular fluid
• Treatment is supportive, although cidofovir may
  be effective if given early after infection.
  Vaccination within 4 days of exposure may
  mitigate course of infection

52
Poxviruses Parapox

• Molluscum contagiousum
• Umbilicated firm cutaneous
• May be more persistent in immunocompromised
  adults
• Typically is treated with curettage or
  cryotherapy.

53
Case 6

• 26 year old medical student wanders on to general
  medical ward with conjunctival hemorrhages,
  fever, and confusion

54
HEMORRHAGIC VIRUSES

• Filoviridae
• Ebola
• Marburg
• Bunyaviridae
• Hantavirus
• Rift Valley Fever
• Crimean-Congo Hemorrhagic Fever
• Arenaviridae
• Lassa virus
• Diagnosis
• Serology from CDC or PCR

55
Filoviridae

• Ebola and Marburg
• Acquired through contact with non-human primates
  in Africa
• Fevers and myalgias are followed by maculopapular
  rash, after which between 10 and 50 will develop
  disseminated intravascular coagulation

56
Bunyaviridae

• Rift Valley Fever
• Transmitted by Aedes mosquitos in sub-Saharan
  Africa
• Three clinical syndromes
• Non-specific febrile illness (90)
• Macular Retinitis / Vasculitis (10)
• Fulminant disease hepatic failure / hemorrhage
• Crimean-Congo Hemorrhagic Fever
• Transmitted by ticks in Southwest Asia, Middle
  East and Africa
• Hemorrhagic fever / DIC in 20-50
• Hantavirus
• Transmitted by wild rodents
• Two types
• Asian strains fever and renal failure
• North American strains fever and pulmonary edema

57
Arenaviruses

• Transmitted to humans via contact with rodents
• Endemic to Africa and South America
• Lassa fever
• Severe systemic illness with shock
• 20 mortality
• Lymphocytic Choriormeningitis Virus (LCMV)
• Aseptic meningitis with low mortality

58
Case 7

• You are called in the middle of the night because
  siblings child has high fevers and an unusual
  rash

59
PARVOVIRUS

• Smallest DNA virus
• Epidemiology
• Widespread infection
• 50 of adolescents and nearly all elderly persons
  have serum antibodies to Parvovirus B19
• Spread among close contacts by respiratory
  droplets or blood
• Clinical
• Cause of erhythema infectiousum (slapped cheek
  or 5th disease), arthritis, red cell aplasia or
  aplastic crisis, and hemophagocytic syndrome
• Fetal infection may lead to hydrops fetalis or
  miscarriage
• 10 fetal loss in 1st trimester pregnancies
• Risk of hydrops greatest in 3rd trimester
• Immunocompromised patients may have chronic
  low-level viremia which is not associated with
  disease
• Consider IVIG in non-immune, exposed pregnant
  women
• Diagnosis
• Plasma PCR
• Low reticulocyte count in presence of anemia
  could be an early diagnostic clue

60
Case 8

• 7 year old boy presents with fever and rash after
  visiting Hunan Province of China
• Also traveled to Hong Kong and San Francisco
• 11 other children had fever and similar rash

61
Measles (Rubeola)

• Virology
• Extremely infectious paramyxovirus spread through
  contact with respiratory droplets
• Nearly 2 week incubation period followed by
• Symptoms
• Constitutional symptoms
• Classic cough, coryza and Kopliks spots
  (small, bluish granules on erythematous buccal
  mucosa)
• Erythematous maculopapular rash spread
  cranio-caudally and may desquamate and involve
  palms / soles.
• Complications
• Pneumonia with secondary bacterial superinfection
• Encephalitis (may be chronic in subacute
  sclerosing panencephalitis)
• Diagnosis
• Serology

62
Measles Kopliks Spots
63
Mumps

• Paramyxovirus acquired through nasopharyngeal
  contact with respiratory droplets or fomites
• Extended (2-4 week) incubation period
• Clinical illness heralded by otalgia and parotid
  hypertrophy and sialadenitis, and may be followed
  by meningitis, encephalitis or orchitis
• Diagnosis
• Serology

64
Rubella (German Measles)

• Benign viral infection characterized by fever and
  maculopapular non-confluent craniocaudal rash.
• May occasionally be complicated by arthralgia
• Congenital infection
• May lead to fetal death and congenital
  abnormalities, including hearing loss, heart
  disease, cognitive delay

65

66
HSV-1 Epidemiology

• Prevalence
• Worldwide, 90 of people seropositive for HSV-1
  by age 40
• In US, approximately 50 and declining, but
  closer to 90 in groups with low SES
• Transmission
• Via saliva
• VesiclesgtUlcersgtAsymptomatic
• Culture positivity 80, 33 and lt25 respectively

67
HSV-1 Primary Infection

• Asymptomatic
• 47 of people with positive HSV-1 serology do not
  recall history of oral / genital ulcers
• Oral or Genital Ulcers
• Fever / pharyngitis in first 12-24 hours (oral)
• Vesicles by median of 7 days
• Resolved by 14 days
• Skin Infections
• Herpetic Whitlow
• Ocular Disease
• Leading cause of blindness worldwide is Herpes
  Keratitis!
• Encephalitis
• Neonates or immunocompromised
• Pneumonitis
• Neonates or immunocompromised
• Hepatitis
• Fulminant and fatal in 80

68
HSV-1 Recurrences

• Oral / Genital Ulcer Disease
• Recurrence rates vary greatly by individual
• 85 with prodrome 24h prior to lesion, then
  lesion x 8d
• HSV-1 recurs infrequently at genital sites
  (average once per year)
• Encephalitis
• Recurrences after primary encephalitis not
  uncommon (in contrast to HSV-2)
• First episode of encephalitis may result from
  reactivation of HSV-1 from oral primary in
  trigeminal ganglion
• Pneumonitis
• First episode of pneumonitis may result from
  aspirating reactivated oral HSV-1 during
  intubation or AMS

69
HSV-1 Diagnosis

• Serologic
• Detect IgG antibodies to HSV-1 and 2 gG (envelope
  glycoprotein)
• Develop within 7-21 days
• May be delayed by use of antivirals
• Some antibody assays have difficulty
  differentiating between HSV-1 and 2
• IgM testing is unreliable
• Virologic
• Looking for the virus
• Tzanck
• Insensitive and not specific
• DFA
• Rapid, specific and pretty sensitive for persons
  with active lesions
• Growing the virus
• Culture
• Sensitive, specific, time consuming (3-7 days)
• Amplifying viral DNA
• PCR
• Sensitive, specific, rapid
• Prone to contamination

70
VZV Natural History



Source Gilden DH, et. al. N Engl J Med 2000
342 635-645
71
Varicella Zoster Virus Clinical Syndromes

• Varicella (Primary Infection)
• 90-95 of persons by the age of 18 have had
  chickenpox
• Zoster (Recurrent)
• Common 30-50 cases per 1000 person years
• 20-30 of patients with HIV will develop Zoster
• More likely to be multi-dermatomal
• Associated with Immune Reconstitution
• 8 in one study
• Domingo P, Am J Med 2001 110605-9
• Chronic encephalitis
• Seen rarely outside immunocompromised persons
• Subacute headache, fever, altered mental status
• Acute Retinal Necrosis
• Weeks to months after varicella or zoster
• Likely due to hematogenous spread, so initial
  lesion at site distant to eye does not rule out
  ARN
• 75-85 chance of detachment leading to blindness,
  with little benefit from antivirals
• May prevent spread to contralateral eye

Source http//www.dermis.net/bilder/CD18/img0054.
jpg
72
EBV Primary Infection

• Asymptomatic
• Common in kids under 2
• May have negative Monospot
• Infectious Mono
• Fever, malaise, pharyngitis, lymphadenopathy,
  atypical lymphocytosis, splenomegaly without
  jaundice or hepatomegaly
• Hemophagocytic syndrome

73
EBV Malignancies after Chronic Infection

• Mechanism
• Persistent infection / activation of B cells
  coupled with viral immune evasion and control of
  cell cycle
• Burkitts
• Most common malignancy in childhood in Africa,
  along malaria belt
• HIV Associated Lymphomas
• Primary CNS
• NHL
• Nasopharyngeal Carcinoma
• Post-Transplant Lymphoproliferative Disorder
• Associated with degree of immunosuppression after
  SOT
• Lung (up to 9)gtHeartgtKidneygtLiver (1-2)
• Risk Factors Lymphocyte depletion (OKT3 or ATG),
  D/R-

74
EBV Diagnostic Tools and Cautions

• Serologic
• Heterophile Test (MonoSpot)
• Antibodies to sheep erythrocytes
• Develop in up to 70 of patients and may persist
  for gt 1 year
• Antibodies to EBV Proteins
• Viral Capsid Antigen (VCA)
• IgM develop immediately and rapidly fall, but
  laboratory test is difficult and prone to
  inaccuracy
• IgG develop rapidly and persist (not useful for
  diagnosis)
• Epstein Barr Nuclear Antigen (EBNA)
• Appears at the end of course of IM and persists
  for life
• Allows for viral latency
• Early Antigen (EA)
• Develop within 2-4 weeks and disappear
• Virologic (PCR)
• PCR for EBV from blood should be interpreted with
  caution
• May be found in blood from asymptomatic
  individuals
• Possibly due to B-cell stimulation
• Quantity does NOT predict development of
  malignancy
• Helpful from CSF to predict CNS lymphoma

75
HHV-8

• Diseases
• Kaposi Sarcoma
• Primary Effusion Lymphoma
• Multicentric Castleman Disease
• Prostate Cancer?
• Multiple Myeloma NO
• Pulmonary Hypertension - NO
• Prevalence
• General Population
• Random Blood Donors US 5, Italy 20-30, Middle
  East 20-30, Africa 20-100, South America 3-70,
  Asia 5-30
• High Risk Groups
• MSM 20-30 HIV-negative in US, 30-50 HIV-pos
• Recent study suggests that women in the United
  States may also have high rates of infection (16)

76
HHV-8 Pictures
77
(No Transcript)