Pemeriksaan Bakteriologis

untuk Diagnosis TB

Basti Andriyoko
Laboratorium Patologi Klinik RSUP Dr. Hasan Sadikin
Bandung
 Topik pembahasan

Standar diagnosis TB : ISTC

Pemeriksaan Mikroskopis TB

Pemeriksaan Kultur TB

Pemeriksaan Molekuler pada TB

Alur diagnosis TB di Indonesia

 “Rules of the Lab”

• No lab test is perfect

• Do not order a lab test if you are not ready to deal with the
result
• Treat the patient, not the lab test
• For TB—There is a lot we still need to learn about
DST and molecular detection of drug resistance
 Discordance
 International Standards for
Tuberculosis Care, 3rd edition, 2014
“Early and accurate diagnosis is critical to
tuberculosis care and control”

Standards for Diagnosis: 1-6  3-6 Lab diagnosis

Standards for Treatment: 7-13  10,11 Lab follow up
Standards for Addressing HIV Infection and
other Co-morbid Conditions: 14-17
Standards for Public Health and Prevention:18-21
ISTC Standard 1
To ensure early diagnosis, providers must be aware of individual and
group risk factors for tuberculosis and perform prompt clinical
evaluations and appropriate diagnostic testing for persons with
symptoms and findings consistent with tuberculosis.

Penjelasan Standar 1:
Rapid molecular tests that increase both the speed and the sensitivity
for identifying Mycobacterium tuberculosis are increasingly available
and, in some situations as described in Standards 3, 5, and 6, are the
recommended initial diagnostic test.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 3
All patients, including children, who are suspected of having pulmonary
tuberculosis and are capable of producing sputum should have at least
two sputum specimens submitted for smear microscopy or a single
sputum specimen for Xpert® MTB/RIF* testing in a quality-assured
laboratory. Patients at risk for drug resistance, who have HIV risks, or
who are seriously ill, should have Xpert MTB/RIF performed as the
initial diagnostic test. Blood-based serologic tests and interferon-
gamma release assays should not be used for diagnosis of active
tuberculosis.
*As of this writing, Xpert®MTB/RIF (Cepheid Corp. Sunnyvale,
California, USA) is the only rapid molecular test approved by WHO for
initial use in diagnosing tuberculosis, thus, it is specifically referred to
by its trade name throughout this document.
International Standards for Tuberculosis Care, 3rd edition, 2014
ISTC

Penjelasan standar 3

• To establish a diagnosis of tuberculosis every effort must be

made to identify the causative agent of the disease. A
microbiological diagnosis can only be confirmed by culturing
M.tuberculosis complex or identifying specific nucleic acid
sequences in a specimen from any site of disease.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 4
• For all patients, including children, suspected of having
extrapulmonary tuberculosis, appropriate specimens from
the suspected sites of involvement should be obtained for
microbiological and histological examination. An Xpert
MTB/RIF test on cerebrospinal fluid is recommended as the
preferred initial microbiological test in persons suspected of
having tuberculous meningitis because of the need for a
rapid diagnosis.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC

Penjelasan standar 4

• it is recommended that Xpert MTB/RIF may be used as a

replacement test for conventional microscopy, culture,
and/or histopathology for testing of gastric lavage fluid and
specific non-respiratory specimens.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 5
• In patients suspected of having pulmonary tuberculosis
whose sputum smears are negative, Xpert MTB/RIF and/or
sputum cultures should be performed. Among patients with
sputum that is negative by smear and Xpert MTB/RIF who
have clinical evidence strongly suggestive of tuberculosis,
antituberculosis treatment should be initiated after
collection of specimens for culture examination

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 6.
• For all children suspected of having intrathoracic (i.e., pulmonary,
pleural, and mediastinal or hilar lymph node) tuberculosis,
bacteriological confirmation should be sought through examination of
respiratory secretions (expectorated sputum, induced sputum,
gastric lavage) for smear microscopy, an Xpert MTB/RIF test, and/or
culture.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 10
• Response to treatment in patients with pulmonary tuberculosis
(including those with tuberculosis diagnosed by a rapid molecular
test) should be monitored by follow-up sputum smear microscopy at
the time of completion of the initial phase of treatment (two
months).
• If the sputum smear is positive at completion of the initial phase,
sputum microscopy should be performed again at 3 months and, if
positive, rapid molecular drug sensitivity testing (line probe assays or
Xpert MTB/RIF) should be performed.
• In patients with extrapulmonary tuberculosis and in children, the
response to treatment is best assessed clinically.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC

Penjelasan standar 10

• Molecular tests, including Xpert MTB/RIF, are not suitable for

patient monitoring because these tests detect residual DNA
from non-viable bacilli. However, Xpert MTB/RIF is useful for
detecting rifampicin resistance in patients who remain
sputum smear positive after 3 or more months of treatment.

International Standards for Tuberculosis Care, 3rd edition, 2014

ISTC Standard 11
• Drug susceptibility testing should be performed at the start of therapy
for all patients at a risk of drug resistance.
• Patients who remain sputum smear-positive at completion of 3
months of treatment, patients in whom treatment has failed, and
patients who have been lost to follow up or relapsed following one or
more courses of treatment should always be assessed for drug
resistance. For patients in whom drug resistance is considered to be
likely, an Xpert MTB/RIF should be the initial diagnostic test. If
rifampicin resistance is detected, culture and testing for susceptibility
to isoniazid, fluoroquinolones, and second-line injectable drugs
should be performed promptly.

International Standards for Tuberculosis Care, 3rd edition, 2014

 WHO-approved microbiologic tests for tuberculosis

Test
• Sputum smear Microscopy
• Nucleic acid amplification tests
(NAATs) [other than Xpert MTB/RIF]
• Xpert MTB/RIF
• Automated liquid cultures and rapid
MPT64-based species identification
tests

International Standards for Tuberculosis Care, 3rd edition, 2014

Limit deteksi kuman dalam sputum berdasarkan metode
pemeriksaan

MGIT LJ FM ZN

•Limit deteksi pada MGIT: 10-100 kuman/ml sputum

•Limit deteksi pada ZN: 10000 kuman /ml sputum
•Limit deteksi pada Xpert MTB/RIF: 100-1000 kuman /ml sputum

 Lebih sensitif dibandingkan ZN, lebih sederhana pengerjaannya

Pemeriksaan Mikroskopis
Pewarnaan Ziehl Neelsen
 Mikroskopis, Pewarnaan BTA
Deteksi : 5.000-10.000 BTA/ml
Kekurangan
• Kurang sensitif : 20-80% pada TB paru
• Tidak spesifik MTBC
Keuntungan
• Murah, cepat
Kegunaan: Diagnostik dan follow up pengobatan
 WHO-approved microbiologic tests for tuberculosis
Test Site Major Findings/results of Systematic
Reviews
Diagnosis of Active Tuberculosis
Sputum smear Pulmonary Same-day sputum smear microscopy is as
microscopy accurate as standard smear microscopy.
Compared with the standard approach of
examination of two smears with light
microscopy over 2 days, examination of
two smears taken on the same day had
much the same sensitivity (64% for
standard microscopy vs 63% for same-day
microscopy) and specificity (98% vs 98%)
Pembacaan Mikroskopis Pewarnaan ZN
Pemeriksaan Kultur TB
 Kultur

Deteksi: 10-100 BTA/ml

• Padat : Ogawa, Lowenstein jensen
• Cair : MGIT, MODS
Konfirmasi diagosis TB
Positifitas kultur pada TB Paru: 85-90%.
 WHO-approved microbiologic tests for tuberculosis
Test Site Major Findings/results of Systematic
Reviews
Diagnosis of Active Tuberculosis
Automated Pulmonary TB • Automated liquid cultures are more sensitive
liquid and than solid cultures; time to detection is more
cultures and extrapulmonary rapid than solid cultures.
rapid TB • MPT64-based rapid immunochromatographic
MPT64-based tests (ICT) for species identification has high
species sensitivity and specificity.
identification
tests
 Kultur Padat

• Keuntungan: Pertumbuhan koloni dapat terlihat langsung,

dapat dilanjutkan dengan DST
• Kerugian: Inkubasi lama, 6-8 minggu, kontaminasi + 17%
 Kultur Cair
MGIT (Mycobacteria growth indicator tube)
• Keuntungan: Otomatis, Lebih cepat dari kultur
padat (7 hari pada smear positif), high
throughput capacity, DST.
• Kerugian: Mahal, biaya maintenance tinggi,
kontaminasi + 10%
• Sensitivitas: 81,5%
• Spesifisitas: 99,6%
 Kultur cair
MODS (Microscopic Observation Drug
Susceptibility)
• Keuntungan: Lebih cepat dari MGIT, murah,
dapat langsung dengan DST.
• Kerugian: risiko biosafety lebih tinggi,
memerlukan mikroskop inverted.
• Sensitivitas: 86%
• Spesifisitas: 70%
 Positif Palsu Kultur

• Kontaminasi silang:
 dari spesimen pasien lain
 Isolat kontrol
 peralatan kultur
 aerosol selama proses kultur
 Reagen terkontaminasi
• Masalah spesimen: salah melabel, tertukar,
clerical error.
Pemeriksaan Molekular /
Nucleic acid amplification test (NAAT)
 Kegunaan NAAT pada penyakit TB
• Mendeteksi keberadaan M.tuberculosis
 target spesifik DNA M.tuberculosis
• Drug susceptibility testing (DST)
 melihat apakah terdapat mutasi

Keuntungan NAAT
• Lebih sensitif dan spesifik dibandingkan mikroskopik
• Lebih cepat dibandingkan kultur
 Limitasi NAAT untuk Diagnosis TB

• Variasi gen yang menjadi target

IS6110, S1081, rpoB  sensitifitas berbeda
• Tidak dapat membedakan kuman hidup atau mati
• Tidak dapat digunakan untuk follow up terapi
 Limitasi NAAT untuk DST

• Gen target yang harus diperiksa banyak Obat Target gen

Rifampisin rpoB
• Tidak semua mutasi berhubungan dengan INH inhA & katG
phenotypic resistance → Silent muta ons : tidak fluoroquinolon gyrA
ada perubahan protein Kanamisin rrs & eis

• Kegunaan klinis: Apakah akan mempengaruhi Amikasin Rrs

penanganan pasien? Apakah mempercayai hasil Pyrazinamid pncA

NAAT, atau tetap menunggu hasil DST Etambutol embB

konvensional?
 WHO-approved microbiologic tests for tuberculosis
Test Site Major Findings/results of Systematic
Reviews
Diagnosis of Active Tuberculosis
Xpert MTB/RIF Pulmonary TB Xpert MTB/RIF used as an initial diagnostic test for
and detection of M. tuberculosis and rifampicin is
extrapulmonary sensitive and specific.
TB and RIF When used as an initial test replacing smear
resistance microscopy Xpert MTB/RIF achieved a pooled
sensitivity of 88% and pooled specificity of 98%.
The pooled sensitivity was 98% for smear-positive,
culture-positive cases and 68% for smear-negative
cases; the pooled sensitivity was 80% in people
living with HIV
Xpert MTB/RIF
• Rapid molecular test = Tes Cepat Molekuler (TCM)
• Catridge based nucleic acid amplification tests

Catridge
Alat GeneXpert
 Xpert MTB/Rif
- Mendeteksi M.tuberculosis complex, sekaligus melihat kepekaan
terhadap rifampisin
- Prinsip: Real Time PCR
- Target deteksi: rpoB Mtb wild type 81 bp

Bagian dari gen rpoB (81 bp)

HOT SPOT
>99% resistensi thd Rif akibat mutasi di hot spot
 Persiapan Sampel

4. Transfer 2ml campuran ke

2.Kocok 10-20 kali, lalu dalam catridge
diamkan 10 menit

3.Kocok lagi10-20 kali, lalu

1.Tambahkan reagen ke
diamkan 5 menit 5. Masukkan catridge ke
sputum dengan
perbandingan 2:1 dalam alat, mulai tes

Lihat hasil pemeriksaan

 Interpretasi Hasil
Xpert® MTB/RIF
MTB DETECTED;
RIF Resistance DETECTED
MTB DETECTED;
RIF Resistance NOT DETECTED
MTB DETECTED;
RIF Resistance INDETERMINATE
MTB Not Detected
Interpretation
MTB terdeteksi, Rifampisin
resisten
MTB terdeteksi, Rifampicin
resisten tidak terdeteksi
(sensitif)
MTB terdeteksi, Rifampicin
resisten belum dapat ditentukan
MTB tidak terdeteksi
Pengembangan Pemeriksaan TCM Xpert MTB/RIF
untuk spesimen non sputum  TB ekstra paru
Disesuaikan dengan kemampuan setiap fasilitas kesehatan
(tenaga dan sarana) :
• Kemampuan untuk pengambilan spesimen
• Kemampuan untuk mengolah spesimen untuk
pemeriksaan (Laboratorium)
Contoh:
Biosafety cabinet diperlukan pada pengolahan beberapa
spesimen.
Komunikasi klinisi dan Laboratorium
 Jenis spesimen ekstra paru yang telah
diujikan menggunakan Xpert MTB/Rif
• Jaringan • LCS
• FNAB • Urin
• Cairan pleura • Cairan tubuh lain (pericardium,
• Billas lambung peritoneal, synovial)
• Pus • Feses

Sensitivitas: 25 – 97%
Spesifisitas: 89 – 100%
Scott LE, et al. Diagnostic Accuracy of Xpert MTB/RIF for Extrapulmonary Tuberculosis Specimens: Establishing a
Laboratory Testing Algorithm for South Africa. JCM. 52:6. 1818-1823. 2014
 Line Probe Assay (LPA)
• Waktu :
• Identifikasi MTBC dan DST (1st line & 2nd line)
• Hanya untuk sampel sputum dengan smear positif atau kultur
positif
• Sensitivitas:
Smear positif: 85.7–94.6%
Smear negatif: 33.3–65.4%
• DST: Rif res: sensitivitas (97%), spesifisitas (99%) INH res:
sensitivitas (90%), spesifisitas, 99%).
LPA
Denkinger C. The TB diagnostic pipeline. 2017
Alur diagnosis TB dan TB Resistan Obat di Indonesia
PMK No.67 tahun 2016 Tentang Penanggulangan Tuberkolosis
PMK No.67 tahun 2016 Tentang Penanggulangan Tuberkolosis
Distribusi TCM GeneXpert Jawa Barat

• 53 alat
• Seluruh Kabupaten/Kota (kecuali: Kab.Garut, Kab.Sukabumi)
 Distribusi TCM GeneXpert Jawa Barat
Bandung Cimahi
1 RSHS 12 RS Mitra Anugerah Lestari
2 Labkes Jabar 13 PKM Cimahi Selatan
3 BBKPM Bandung
Tasikmalaya
4 RS Paru dr HA Rotinsulu
14 RS SMC Kab Tasikmalaya
5 RSUD Kota Bandung
15 PKM Ciawi Tasikmalaya
6 RS Al Islam Bandung
16 RSUD dr Soekardjo Kota Tasikmalaya
Kabupaten Bandung 17 PKM Karangnunggal Kab. Tasikmalaya
7 RSUD Lembang 18 Puskesmas Manonjaya
8 RSUD Soreang 19 PKM Cikatomas Tasikmalaya
9 RSUD Cililin
10 Labkesda Kab Bandung Barat
11 RSUD Al Ihsan
 Ciamis Kuningan
20 RSUD Ciamis 30 UPTD Labkesda Kab Kuningan
21 PKM Panumbangan Ciamis 31 RSUD Linggajati Kuningan
22 Puskesmas Banjarsari Kab Ciamis 32 RSUD 45 Kuningan
Cirebon Karawang
23 BKPM Prov Jabar Cirebon 33 Puskesmas Cikampek Kab Karawang
24 RSUD Gunung Jati Kota Cirebon 34 RSUD Karawang
25 RSUD Arjawinangun Kab Cirebon Bekasi
26 RSUD Waled Kab Cirebon 35 RSUD Kabupaten Bekasi
27 RS Paru Prov Jabar Sidawangi 36 RSUD dr Chasbullah Abdulmadjid
Cianjur Kota Bekasi
28 RSUD Sayang Kab Cianjur 37 RS Sentra Medika Cikarang
29 Puskesmas Ciranjang Kab Cianjur
 Bogor/Kab.Bogor
Depok
38 RSUD Kota Bogor
44 UPT PKM Kec Pancoran Mas Depok
39 RSUD Leuwiliang Bogor
45 RS Sentra Medika Cimanggis
40 RSUD Cibinong Kab Bogor
41 RSPG Cisarua Bogor
42 RSUD Ciawi Kab Bogor
43 RSUD Cileungsi
46 Sukabumi RSUD Syamsudin SH Kota Sukabumi
47 Purwakarta RSUD Bayu Asih Purwakarta
48 Pangandaran UPT Labkesda Pangandaran
49 Subang Puskesmas Pamanukan Subang
50 Majalengka PKM Talaga Kab. Majalengka
51 Indramayu RSUD Kab Indramayu
52 Banjar RSUD Kota Banjar
53 Sumedang RSUD Sumedang
54 Garut BBKPM Garut
 Kesimpulan

• Seluruh diagnosis TB harus melewati tahap

pemeriksaan laboratorium untuk mencari bukti
bakteriologis  alur diagnosis TB
• NAAT  TCM Xpert MTB/RIF, dapat digunakan
untuk menegakkan diagnosis TB dan memeriksa
resistensi terhadap rifampisin
• Pasien dengan riwayat pengobatan, harus
dilakukan pemeriksaan TCM Xpert MTB/RIF
sebelum terapi.
TERIMA KASIH