TORCH:

RELIABLE SCREENING for EARLY

DIAGNOSIS
Loly RD Siagian

SIMPOSIUM ILMIAH PDS PATKLIN CAB.BALIKPAPAN

BALIKPAPAN, 2 APRIL 2017
SUBJECT
•* Overview
•* TORCH INFECTION
•* LABORATORY DIAGNOSTIC
•* SCREENING FOR EARLY DAGNOSTIC
TORCH is an acronym for a group of most common
infections which may lead to congenital diseases

They can be classed into 5 different groups:

T= Toxoplasma gondii (Toxoplasmosis)
O*
R= Rubella, also commonly known as ‘German measles’
C= Cytomegalovirus (CMV)
H= Herpes simplex virus (HSV)
* The O stands for a group of other serious infections, which includes Troponema pallidum
(Syphilis), HIV, Hepatitis B, Parvovirus B19, Epstein-Barr virus and many more

TORCH group assays detect the presence of antibodies to the above

listed pathogens to help characterize the state of infection.
Transmission of TORCH group infections from the mother to baby:

• Infections in the first trimester are usually the most dangerous

• The risk for permanent birth defects is higher at first trimester

Stegmann et al. (2002 Aug) Curr Womens Health Rep. 2(4), 253-8.
Karen et al. (2004) Clinical Infectious Diseases 38, 1035–7.
Menezes et al. (2009 May) BMC Pregnancy Childbirth. 7, 9 Suppl 1:S4.
 Aim of the TORCH testing
• To screen and determine the immune status of the mother
• To prevent mother-to-child transmissions
• To treat in time and prevent severe birth defects

The following 3 groups are targeted:

Patient group Purpose of the diagnosis

Mother To find out if TORCH infection occurred before or during pregnancy (guide to risk analysis)

Foetus To confirm or exclude the presence of a foetal infection

Infant To identify the pre-natal and/or post-natal exposure to the infection

Enders et al. (2012 Nov) J Clin Virol.

Murat et al. (2012 Nov) in Vaccine Immunol. 19(11), 1838-43.
Calimeri et al. (2012 Mar) Int J Gynaecol Obstet. 116(3), 211-3.
Qin et al. (2011 Aug) Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 25(4), 292-4.
Stegmann et al. (2002 Aug) Curr Womens Health Rep. 2(4), 253-8.
 The TORCH world is short of a common ground

• Guidelines – not always there and big geographical differences still exist

• Prevalence - varies by region and country but also within the same country

• Medical practice – treatment and diagnostic – are not similar everywhere

Assessing congenital risk requires multiple data points
Several tests and result interpretation are needed

The main questions when trying to determine congenital risk:

Questions? Solution
1. Has the mother been exposed to any of the TORCH pathogens before or Use IgG assays
during the pregnancy?
Or
1. What is the immune status of the mother? Could the fetus be at risk?
2. Is it an acute infection? Use IgM assays*
3. When did the infection occur or is it really an acute infection? Use IgG avidity assays

*IgM antibodies may persist many months and up to several years. In these cases it is critically important to perform the IgG avidity assay to
confirm if the infection is really acute (or primary).
Toxoplasmosis
Caused by Toxoplasma gondii, a unicellular protozoan parasite

Parasite lifecycle
Introduction

Toxoplasma gondii infections The mother may be infected by:

• Over 6 billion people have been

infected worldwide
• Cats play an important role in
transmission but also unwashed raw Handling or ingesting

vegetables undercooked meat

Improper handling of cat
litter
• Usually asymptomatic or has mild
symptoms
• No vaccine available A fetus may contract toxoplasmosis
through the placental connection
with its infected mother

Klaren et al. (2002) Ocul Immunol Inflamm. 10, 1–26.

Cook et al. (2000 Jul) BMJ. 15, 321(7254), 142-7.
Montoya et al. (2008 Aug) Clin Infect Dis 15, 47(4), 554-66.
Clinical features

Potential outcome of Toxoplasma gondii infections

• Early maternal infection may result with death
70
of the fetus and spontaneous abortion

Development of clinical signs (%)

60 61%
• Late maternal infection is less severe and fetal
damage is less likely 50

40

30
25%
Early and late consequences of
20
infection:
9%
• Death in-utero 10

• Hydro/microcephalus 0
• Eye defects Week 13 Week 26 Week 36
• Hearing loss
• Mental retardation etc.
Toxoplasma infection
Typical Immune Response

Low avidity IgG High avidity IgG

Primary infection Past infection
Relative antibody concentration

IgM
IgG
IgG avidity

Reactivation
or reinfection

Infection Days after infection Weeks after infection Months after infection Years after infection

Montoya and Liesenfeld 2004.

Diagnosis of Toxoplasma gondii

Antibody Screening Pregnancy Newborns

class test
IgM Yes. + (IgM antibodies may persist for Yes
May be used in prolonged time)
neonatal - (IgM rules out infection in pregnant
screening women during the previous two
trimesters)
IgG Yes + (identification of women at risk and Yes
those protected) (maternal antibodies may
- (women at risk of acquiring infection) persist until 12 months of
age)
IgG avidity No + (high avidity results rule out infection in No
recent 3–4 months)
Diagnosis of Toxoplasma gondii

START

Perform IgG and IgM test

IgG Negative IgG Positive IgG Negative IgG Positive

IgM Negative IgM Negative IgM Positive IgM Positive

Patient susceptible to infection. Patient immune to Toxoplasma gondii Early stage infection or non-specific Serological sign of acute Toxoplasma
Follow-up tests in 3-4 weeks and infection IgM. gondii infection or non-specific
continue with monitoring during the Follow-up test required after 2-3 stimulation of the immunological
pregnancy weeks system

Confirm positive finding

IgG, IgM serology in second sample,
avidity and PCR from amniotic fluid

Negative Positive
IgM negative, High avidity, PCR negative Further action may be required
Acute infection can not be confirmed Where acute Toxoplasma gondii infection confirmed, discuss pregnancy outcomes
with patient

Postnatal follow-up and care

STOP

Remington J.S. et al. Infectious Diseases of the Fetus and Newborn Infants, 5th ed., Philadelphia: W.B. Saunders, pp. 205-346.
Meek B. et al. (2001). Diagn Microbiol Infect Dis 41, 131-7.
German measles (Rubella)
Caused by a Rubella virus which is a member of the Togaviridae family

• Highly contagious; only minimal level of contact is

enough for transmission
• During pregnancy, it can lead to severe
complications to the fetus
• Roughly half of infected mothers show no
symptoms
• Vaccine-preventable
• Vaccination program available in many
countries (130 countries, covering 41% of
newborns)*, but infection still a threat in others
Best et al. (2007 Jun) Semin Fetal Neonatal Med. 12(3), 182-92.
De Santis et al. (2006 May) Reprod Toxicol. 21(4), 390-8.
Roush et al. (2007 Nov) JAMA. 14, 298(18), 2155-63.
Airborne transmission of Rubella virus * WHO , 2010
Rubella infection
Typical Immune Response

*Anti-rubella IgG and IgM antibodies appear almost at the same time when the rash appears
Thomas 2009.
Clinical features of Rubella infection
• Causing Congenital Rubella Syndrome (CRS),
fetal death and abortion
• CRS risk is higher during the first 12 weeks of
the pregnancy
The most frequent presentations of the CRS:
• Cataract
• Cardiac malformations
• Microcephaly
Eye anomalies may include cataracts, glaucoma, strabismus,
• Hearing loss nystagmus, microphthalmia, and iris dysplasia.

Best et al. (2007 Jun) Semin Fetal Neonatal Med. 12(3), 182-92.
De Santis et al. (2006 May) Reprod Toxicol. 21(4), 390-8.
Edlich et al. (2005) J Long Term Eff Med Implants. 15(3), 319-28.
Diagnosis of Rubella infections

START

Perform IgG and IgM test

IgG Negative IgG Positive IgG Negative IgG Positive

IgM Negative IgM Negative IgM Positive IgM Positive

Patient susceptible to infection. Patient immune to Rubella infection Early stage infection. Repeat test Recent acute Rubella infection,
Repeat tests in 2-3 weeks and required after 2-3 weeks reinfection, or non-specific
monitoring or immunization if stimulation of the immunological
required system

Confirm positive finding

IgG, IgM serology, avidity and PCR in
amniotic fluid

Negative Positive
IgM negative, High avidity, PCR negative Where acute Rubella infection confirmed, discuss pregnancy outcomes with
Acute infection can not be confirmed patient

STOP Postnatal follow-up and care

Mendelson at al. (2006) Reprod Toxicol. 21(4):350-82.

Manual for the Surveillance of Vaccine-Preventable Diseases: Available at: www.cdc.gov/vaccines/pubs/surv-manual/index.html.
Cytomegalovirus (CMV)
CMV is a member of Herpesviridae family

CMV infections
• The most common vertically transmitted viral
infection
• Common cause of mental handicap
• Transmitted person to person via contact of any
type of body fluid: urine, saliva, blood, tears, breast
milk, semen, feces and/or cervical secretions
• Affected children usually have young mothers of
low social status
• No vaccine available

Nigro G. (2009 Feb) J Matern Fetal Neonatal Med. 22(2), 169-74. CMV is a common cause of disabilities
Johnson et al. (2012 Jun) Clin Obstet Gynecol. 55(2), 521-30.
Mustakangas et al. (2000 Jun) Int J Epidemiol. 29(3), 587-91.
CMV infection
Typical Immune Response

Low avidity IgG High avidity IgG

Primary infection Past infection
Relative antibody concentration

Ig M
Ig G
Ig G avidity

Reactivation
or reinfection

Infection Days after infection Weeks after infection Months after infection Years after infection

Revello and Gerna 2004; Munro et al 2005.

Clinical features of CMV infections
Consequences of transmission of CMV from mother to
fetus can be very severe:
• Miscarriage and still-birth
• 90% are symptomless at birth but 10% will develop
symptoms
• The majority of the symptomatic cases (>80%) will lead to CMV infection in the cornea

permanent complications
• Most common symptoms at birth are:
• Mental impairment
• Hearing loss
• Visual impairment
Hearing loss

Foulon et al. (2008 Dec) Pediatrics. 122(6), e1123-7.

Enders et al. (2011 Nov) J Clin Virol. 52(3), 244-6. Epub (2011 Aug) 5.
Yinon et al. (2010 Apr) J Obstet Gynaecol Can. 32(4), 348-54.
Clinical features of CMV infections
Primary infection in early stage of pregnancy increases risk of severe symptoms of
congenital CMV

70
However…Maternal CMV infection
60 in early stages of pregnancy leads
Number of fetuses/babies

50 to higher risk of severe CMV

symptoms in the fetus
40

30

20 Symptomatic congenital infection

10 Symptomless congenital infection

0 No congenital infection
Pre-conception Trimester I Trimester II Trimester III

Revello and Gerna. J Clin Virol 2004.

Diagnosis of CMV infections

• The initial diagnosis is based on the detection of IgG and IgM

• IgM alone is not always reliable
• The age of an infection can be estimated by IgG-avidity tests.
Diagnosis of CMV infections

START

Perform CMV IgG and IgM test

IgG Negative IgG Positive IgG Negative IgG Positive

IgM Negative IgM Negative IgM Positive IgM Positive

Patient susceptible to infection. Patient is immune to CMV infection Early stage infection. Recent CMV infection, reinfection,
Repeat tests in and continue with Confirm the finding. reactivation, or non-specific
monitoring during the pregnancy Repeat test required after 2-3 weeks stimulation of the immunological
system

Confirm positive finding

IgG, IgM serology, IgG-avidity and PCR
in amniotic fluid and/or blood
samples

Negative Positive
IgM negative, High avidity, PCR negative Further action may be required
Acute infection can not be confirmed Where acute CMV infection confirmed, discuss pregnancy outcomes with the
patient

Postnatal follow-up and care

STOP

Munro, S.C., Hall, B., Whybin, L.R. et al. (2005). J Clin Microbiol 43(9), 4713-8.
Lazzarotto, T., Gabrielli, L., Lanari, M. et al. (2004). Hum Immunol 65, 410-415.
Guerra, B., Simonazzi, G., Banfi, A. et al. (2007). Am J Obstet Gynecol 196, 221-223.
Duff, P. (2007). A thoughtful algorithm for the accurate diagnosis of primary CMV infection in pregnancy. Am J Obstet Gynecol 196, 196-197.
Herpes simplex viruses 1 and 2

• Major congenital risk of transmission: during vaginal delivery

• Infection is particularly dangerous in
premature infants.
• Multiple organ infections (49%)
• 80 - 85% mortality
• Localized to Central Nervous System (CNS),
skin, eyes, oral cavity (50%)
• up to 40% mortality

Infants who survive the HSV encephalitis develop seizures,

mental retardation, and visual deficits.
Brown, Z. (2004 Aug) Herpes. 11 Suppl 3, 175A-186A.
Franca et al. (2004) Spec Care Dentist 24(5), 250-253.
Stamos et al. (1994) ediatr Clin North Am 41: 1017.33.
Diagnosis of HSV infections
Herpes simplex serology in pregnant women

• HSV type 1 & 2 specific IgG

• IgM testing is not reliable
• Cross-reaction with other Herpesviridea group viruses
• IgM serology cannot distinguish acute infection from the recurrent disease
• HSV-IgM antibodies may persist many months or years
IgM tests do not allow proper differentiation between primary and persistent infection

Production of IgG and IgM start at similar time

Titer

Primary HSV infection

Time after infection

Cowan et al. (2000 Apr) J Antimicrob Chemother.45 Suppl T3, 9-13.

Song et al. (2004 Apr) Sex Transm Infect. 80(2), 113-7.
Page et al. (2003 Aug) Sex Transm Infect. 79(4), 276-9.
Diagnosis of HSV infections
Perform HSV 1 & HSV type specific IgG test
HSV 1 and HSV-2
Negative
Check the immune status of the
partner
Partner is HSV Negative
If woman and her partner is in high
risk group, educate them with the
potential risks of HSV-2 infections and
protection
START
Pregnant women is HSV-1 IgG Positive HSV-2 IgG Positive
• Educate
Partner is HSV-2 Positive
• Antiviral treatment (supression of
the virus)
• Perform PCR from genital swap
samples
Perform PCR from the genital swap • Avoid vaginal delivery
sample of the woman
Educate the women and her partner:
• To avoid genital contact
• Use of condoms
• Antiviral treatment (supression of
the virus)
Adapted from Brown et al. (2005) Obstet Gynecol .106(4):845-56.
 Overview Toxo Rubella CMV HSV Syphilis

Why do we need to know HSV 2 serological-status?

• To control the spread of the infection

• To determine partner sero-status - majority of partners do not know they are infected
• To guide high risk groups to protect themselves
• Prevent the transmission

Smith et al. (2002) The Journal of Infectious Diseases 186(Suppl 1), S3–28.
Guidelines for the Management of Herpes Simplex Virus in Pregnancy. (2008) JUNE JOGC JUIN No 28.
Gardella et al. (2007) Clevland Clinic Journal of Medicine Vol 74 • No 3.
Diagnosis of HSV infections
When do we need to know HSV 2 serological-status?

In symptomatic patients: As a supplementary test when

lesions are negative by PCR and virus culture
In asymptomatic high risk group patients:
• Previous STD infection
• Partner is HSV-2 positive
• Patient has symptoms
In pregnancy
• To screen the expectant mother
• To evaluate the risk of aquiring the HSV-2 infection
• To determine the status of partner and provide counselling
Typical Herpes lesion

Smith et al. (2002) The Journal of Infectious Diseases 186(Suppl 1), S3–28.
Guidelines for the Management of Herpes Simplex Virus in Pregnancy. (2008) JUNE JOGC JUIN No 28.
Gardella et al. (2007) Clevland Clinic Journal of Medicine Vol 74 • No 3.
Syphilis

•WHO estimates 270,000 cases of congenital Syphilis

annualy
•Transmission possible at any stage of disease
•Infected new-born babies are asymptomatic at birth
•No vaccine available but treatable with antibiotics

Caused by gram negative bacterium “Treponema pallidum ssp. Pallidum”

Mindel, A. (1989) Primary and secondary syphilis, 20 years' experience. 2. Clinical features in Genitourin Med. (1989 Jan) 65(1), 1-3.
Clinical features of Syphilis
Syphilis during pregnancy
• Pregnant women are generally asymptomatic
• Nearly half of infected children die before or shortly after birth

Consequences of
congenital Syphilis

• Intrauterin growth • Blindness

restriction • Deafness
Congenital Syphilis
• Miscarriage • Facial deformity
• Stillbirth • Neurological problems
• Premature birth
Saloojee, H. et al. (2004) Bull World Health Organ 82 no.6, p. 424-430.
Mindel, A. (1989) Primary and secondary syphilis, 20 years' experience. 2. Clinical features in Genitourin Med. (1989 Jan) 65(1), 1-3.
Diagnosis of Syphilis

• Most pregnant mothers are symptomless and can only be identified

through serological screening
• In high risk-group pregnant women, repeated serologic testing at 3rd
trimester and at delivery is needed.
• Treponemal tests measure antibodies to Treponema pallidum or its
components and are very specific
• Non-treponemal tests are non-specific and measure antibodies to
cardiolipin-cholestrol-lecithin complex
Patterns of serologic reactivity to Treponema pallidum
FTA-ABs
treponemal
100
TPHA

% of patients who test positive

80

60

IgM untreated
40
VDRL/RPR
non-treponemal
20
treated

2 4 6 8 10 12 2 10 20
Time
of infection
weeks Time post infection years

primary secondary
lesion lesion

clinical
stages primary secondary latent tertiary
of syphilis (asymptomatic)

Peeling. Bulletin of the World Heatlh Organization 2004.

Summary
Toxo Rubella CMV Syphilis HSV

Causative agent
Parasite Virus Virus Bacterium Virus

Vaccination
Not available Available Not available Not available Not available

Source of major Primary infection. Primary infection. Primary infection. Active infection. No Primary infection.
congenital risk Immunity after Immunity after Reinfection and immunity to a new Reinfection and
infection. infection or vaccination reactivation possible infection. reactivation possible
Timing of major Throughout pregnancy,
congenital risk First trimester First trimester First trimester At birth
at birth
Treatment during No specific treatment
pregnancy No specific treatment Yes
Yes available Yes
available

Tests available by Elecsys Toxo IgM Elecsys Rubella IgM Elecsys CMV IgM Treponemal: HSV1 IgG
Roche Elecsys Toxo IgG Elecsys Rubella IgG Elecsys CMV IgG Elecsys Syphilis (Total) HSV2 IgG
(quant) (quant) TPLA2
Elecsys Toxo IgG avidity Elecsys CMV IgG avidity HSV 1 and 2 DNA Test
Non-treponemal:
CMV DNA quant RPR2
The safe and sure approach to Toxo screening

Flexibility to address every screening protocol (Elecsys® Toxo IgG)

Elecsys® Toxo IgM is first line of Elecsys® Toxo IgG is first line of Elecsys® Toxo IgM and Elecsys® Toxo
screening screening IgG are performed simultaneously

High sensitivity required – false- High sensitivity required – false- High sensitivity(IgM) andhigh specificity
negative results minimized! negative results minimized! required(IgG) –falseresultsminimized!

Toxo IgG result interpretation Toxo IgG result interpretation Toxo IgG result interpretation
Non-reactive: < 1 IU/mL Non-reactive: < 1 IU/mL Non-reactive: < 1 IU/mL
Indeterminate: ≥ 1 - < 3 IU/mL Indeterminate: ≥ 1 - < 3 IU/mL Indeterminate: ≥ 1 - < 30 IU/mL
Reactive: ≥ 3 IU/mL Reactive: ≥ 3 IU/mL Reactive: ≥ 30 IU/mL

25 March 2016 page 39 © 2014 Roche.

The safe and sure approach to Toxo screening
1. For marketswhere IgMandIgGare performedsimultaneously
Ultra-sensitive Elecsys® Toxo IgM assay
optimized to detect all potential acute
infections
Does not leave any suspicious case out
Minimizing the risk of missing an
acute infection
+
Elecsys® Toxo IgG test has been proven to Elecsys® Toxo IgG Avidity acts as a
ensure 100%specificity when used with the confirmatory test to exclude an early T. gondii
positive cut-off level of 30 IU/mL infection
All past infections are clearly identified Risk is clearly assessed
1. Leslé F et al. (2011 Oct) Eur J Clin Microbiol Infect Dis. 30(10), 1207-12.; 2. Murat JB et al. (2012 Nov) Clin Vaccine Immunol. 19(11), 1838-43.;
3. Van Helden J. (2009) Clin Lab. 55(7-8), 267-73.; 4. Jost C. et al. (2011 Nov) Clin Vaccine Immunol. 18(11), 1908-12.;
5. Prusa AR et al. (2010 Dec) Diagn Microbiol Infect Dis. 68(4), 352-7.; 6. S. Köhler et al. (2010) Eur J Clin Microbiol Infect Dis. 29, 359–363
The safe and sure approach to Toxo screening
2. For markets where IgM or IgG is performed first
Elecsys® Toxo IgG when used with the 3
IU/mL positive cut-off identifies all cases that
require further testing
All potential past infections are clearly
identified
Minimizing the risk of missing an
acute infection
+
Ultra-sensitive Elecsys® Toxo IgM assay Elecsys® Toxo IgG Avidity acts as a
optimized to detect all potential acute confirmatory test to exclude an early T. gondii
infections infection
Does not leave any suspicious cases out Risk is clearly assessed
1. Leslé F et al. (2011 Oct) Eur J Clin Microbiol Infect Dis. 30(10), 1207-12.; 2. Murat JB et al. (2012 Nov) Clin Vaccine Immunol. 19(11), 1838-43.;
3. Van Helden J. (2009) Clin Lab. 55(7-8), 267-73.; 4. Jost C. et al. (2011 Nov) Clin Vaccine Immunol. 18(11), 1908-12.;
5. Prusa AR et al. (2010 Dec) Diagn Microbiol Infect Dis. 68(4), 352-7.; 6. S. Köhler et al. (2010) Eur J Clin Microbiol Infect Dis. 29, 359–363
Clear discrimination between an acute and a past Rubella infection
IgM test very sensitive to early acute
infections
Early detection of an acute infection
Superior sensitivity
where it matters most
+
IgM test less sensitive to persistent IgM IgG test ultra sensitive to remote infections
Avoids false alarms Avoids unnecessary follow-up
Enders M. et al. (2013 Mar) Clin Vaccine Immunol. 20(3), 420-6.
Bollhagen R et al. (2006) 12th International Congress on Infectious Diseases, Portugal.
Grangeot-Keros L et al. (2009) ECCMID conference, Helsinki, Finland.
High precision diagnosis of acute CMV infections

The anti-interference
CMV assay
+
Detects only the IgM associated with an acute Anti-interference recombinant protein Elecsys® CMV IgG avidity helps to
infection by using a novel blocking agent to prevents cross reactivity with other herpes discriminate between acute and remote CMV
persistent IgM viruses infections

Focus on acute infections where the

Reacts only to CMV seroconversions Acts as a confirmatory test
congenital risk lies

1. Grangeot-Keros L. et al. (2010) ECCMID conference, Vienna, Austria.; 2. Revello MG et al. (2012 Dec) Eur J Clin Microbiol Infect Dis. 31(12), 3331-9.;
3. Baalawi F et al. (2010) Pathology. 42(6), 578-80.; 4. Grangeot-Keros L. et al. (2010 September) 3rd International Conference on Congenital Cytomegalovirus Infection, Paris, France.; 5. Revello M G
et al. (2009) ECCMID conference, Helsinki, Finland.
The new generation of HSV testing for informed pregnancy management

The new generation

of HSV testing
+
The right menu The anti-interference approach

HSV1 IgG + HSV2 IgG Anti-interference recombinant

The relevant tests to protein prevents cross reactivity
Superior specificity
assess congenial risk with other herpes viruses

No IgM test Anti-interference recombinant

Follows guidelines and protein prevents cross reactivity
Prevents cross reaction
KOL advocacy with other herpes viruses

1. Song et al. (2004 Apr) Sex Transm Infect. 80(2), 113-7.; 2. Strick et al. (2004 Jul) Expert Rev Mol Diagn. 4(4), 443-53.; 3. Fleming et al. (1997 Oct) N Engl J Med. 16, 337(16), 1105-11.; 4. Xu et al. (2006)
JAMA. 296(8), 964-973.; 5. Money D et al. (2008 Jun) Guidelines for the Management of Herpes Simplex Virus in Pregnancy. J Obstet Gynaecol Can. 30(6), 514-26.; 6. Brown ZA. (2000 May-Jun) Rev Med
Virol. 10(3), 141-4.; 7. Morrow R et al. (2006) Performance of a novel test for IgM and IgG antibodies in subjects with culture-documented genital herpes simplex virus-1 or -2 infection. Clin Microbiol Infect.
12, 463–9.
TERIMA KASIH