National Tb Standard Operating Procedures Manual

298 Pages·2017·1.96 MB·English

NATIONAL TUBERCULOSIS AND LEPROSY CONTROL PROGRAMME (NTBLCP)

FEDERAL MINISTRY OF HEALTH
DEPARTMENT OF PUBLIC HEALTH
NATIONAL TUBERCULOSIS AND LEPROSY CONTROL PROGRAMME

National Standard Operating Procedures
for Tuberculosis Laboratory Diagnosis

JANUARY 2017
2nd EDITION

National Standard Operating Procedures for Tuberculosis laboratory version 2 January, 2017
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NATIONAL TUBERCULOSIS AND LEPROSY CONTROL PROGRAMME (NTBLCP)

National Standard Operating Procedures for Tuberculosis laboratory version 2 January, 2017
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NATIONAL TUBERCULOSIS AND LEPROSY CONTROL PROGRAMME (NTBLCP)

National Standard Operating Procedures for Tuberculosis laboratory version 2 January, 2017
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NATIONAL TUBERCULOSIS AND LEPROSY CONTROL PROGRAMME (NTBLCP)

LIST OF CONTRIBUTORS (FIRST EDITION, 2012)

Obasanya, J. Ojika, G., Elom, E., Egbom,C., Sarkinfada,F., Okoro,L., Nwofor,A., Panwal,M.,
Bassey Eyo-Nsa,I., Ezati,N., Iwakun,M., Tubi,A., Ochei,K., Ameh,J., Ahmadu,I., Onazi,T.,
Makpu,S., Obeten,S., Umar A., Emenyonu,N., Allanana,J., Akintunde,E., Adebiyi,E.,
Omakwele,G., Nwokoye,N., & Baboolal,S.

LIST OF REVIEWERS

Ngige, E, Lawanson, A., Elom E.U., Panwal M, Tubi A., Ahmadu I., Nwokoye N., Ochei K. Udah
D., Akpakpan R .Nwofor, A.

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CONTENTS
FOREWORD .................................................................................................................................................. i
ACKNOWLEDGEMENTS ............................................................................................................................. ii
TABLE OF CONTENTS ... ………………………………………………………………………………iii
LIST OF ABBREVIATIONS.......................................................................................................................... iv
INTRODUCTION ........................................................................................................................................... 1
DIAGNOSTIC ALGORITHMS .........................................................................................................................
DIAGNOSTIC ALGORITHM OF PTB SUSPECT ......................................................................................... 2
DIAGNOSTIC ALGORITHM – SMEAR NEGATIVE PTB SUSPECT ........................................................... 3
DIAGNOSTIC ALGORITHM - MICROSCOPY CENTERS/PERIPHERAL LABORATORIES ...................... 4

DIAGNOSTIC ALGORITHM – ZONAL/STATE LABORATORIES ............................................................... 5
DIAGNOSTIC ALGORITHM – NATIONAL REFERENCE LABORATORIES ............................................... 6
SECTION A :TECHNICAL STANDARD OPERATING PROCEDURES ..................................................... 7
7.1 SOP ON SPECIMEN COLLECTION, STORAGE AND TRANSPORT ............................................. 12
7.2 SOP ON PREPARATION OF SMEARS, STAINING AND MICROSCOPY (ZN AND FM) .............. 19
7.3 SOP ON PROCESSING OF CLINICAL SPECIMENS: SPUTUM ..................................................... 29
7.4 SOP ON PROCESSING OF CLINICAL SPECIMENS OTHER THAN SPUTUM ............................. 36
7.5 SOP ON CULTURING OF MYCOBACTERA USING LJ MEDIUM ................................................... 47
7.6 SOP ON CULTURE OF MYCOBACTERA USING BACTEC MGIT 960 TB SYSTEM ..................... 54
7.7 SOP ON IDENTIFICATION OF MTBC USING RAPID TESTS (CAPILLA, SD BIOLINE, TBc) ....... 61
7.8 SOP ON IDENTIFICATION OF MTBC USING BIOCHEMICAL TESTS ........................................... 68
7.9 SOP ON IDENTIFICATION OF MTBC AND DRUG RESISTANCE TO INH, RIF AND SECOND-LINE
DRUGS USING HAIN LINE PROBE ASSAY (MTBDRplus and MTBDRsl) ............................................... 92
7.10 IDENTIFICATION AND DST USING NITRATE REDUCTION ASSAY ......................................... 121

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7.11 SOP ON IDENTIFICATION OF MTBC AND DRUG RESISTANCE TO RIF USING GENEXPERT TB
SYSTEM..... 127
7.12 SOP ON DRUG SUSCEPTIBILITY TESTING (SIRE, PZA) USING BACTEC MGIT 960 TB
SYSTEM.......... ......................................................................................................................................... 135
7.13 SOP ON DRUG SUSCEPTIBILITY TESTING (FIRST-LINE DRUGS) USING PROPORTION
METHOD......... .......................................................................................................................................... 144
7.14 SOP ON IDENTIFICATION OF MTBC AND NONTUBERCULOSIS MYCOBACTERIA (NTMS)
USING HAIN LINE PROBE ASSAY (MTBC, CM/AS) .............................................................................. 152
7.15 SOP ON DRUG SUSCEPTIBILITY TESTING (SECOND-line DRUGS) USING MGIT 960 TB
SYSTEM.......... ......................................................................................................................................... 177
7.16 SOP ON DRUG SUSCEPTIBILITY TESTING (SECOND-LINE DRUGS) USING AGAR
PROPORTION METHOD ......................................................................................................................... 187
APPENDICES ........................................................................................................................................... 202
8.1 PREPARATION OF 1% CARBOL FUCHSIN SOLUTION .............................................................. 202
8.2 PREPARATION OF 3% ACID ALCOHOL SOLUTION ................................................................... 211
8.3 PREPARATION OF 0.1% METHYLENE BLUE SOLUTION ........................................................... 213
8.4 PREPARATION OF AUROMINE O SOLUTION ............................................................................. 215
8.5 PREPARATION OF 1% ACID ALCOHOL SOLUTION ................................................................... 217
8.6 PREPARATION OF 0.1% POTASSIUM PERMANGANATE SOLUTION ...................................... 218
8.7 PREPARATION OF SODIUMHYDROXIDE-SODIUM CITRATE .................................................... 219
8.8 PREPARATION OF PHOSPHATE BUFFER .................................................................................. 222
8.9 PREPARATION OF LOWEINSTEIN JENSEN SLANTS ................................................................. 224
8.10 PREPARATION OF ANTIBIOTICS FOR SECOND-LINE DRUGS USING MGIT 960 ................. 229
8.10 PREPARATION OF ANTIBIOTICS FOR SECOND-LINE DST USING AGAR PROPORTION
METHOD......... .......................................................................................................................................... 233
8.11 PREPARATION OF AGAR PLATES WITH ANTIBIOTICS ........................................................... 236

INDEX................................................................................................................................................234-248
SECTION B: NON TECHNICAL STANDARD OPERATING PROCEDURES ...................................249-291

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LIST OF ABBREVIATIONS
AFB – Acid fast bacilli ACP – Adenosine cytosine phosphate
AG – Aminoglycosides AP – Alkaline phosphatase
AS – Atypical species ATCC – American typed culture collection
BAL – Bronchio-alveola lavage BAP – Blood agar plate
BCG – Bovis Bacille Calmette-Guerin BD – Becton Dickenson
BSC – Biological safety cabinet BSL2 – Biosafety level 2
BSL3 – Biosafety level 3 CC – Conjugate control
CFU – Colony forming units CM – Common mycobacteria
CSF – Cerebrospinal fluid CP – Cyclic peptides
DH O – Distilled water DNA – Deoxy-ribonucleic acid
2
DOTS – Directly observed treatment, short DST – Drug susceptibility test
course
EMB – Ethambutol FDA – Food and Drug Administration
FLQ - Fluoroquinolones FM – Fluorescent microscopy
GU – Growth units HEPA – High-efficiency particulate air
HPF – High power field ID – Identification
INH – Isonazid KNO – Potassium nitrate
3
LED—Light emitting diode
LJ – Lowenstein Jensen LPA – Line probe assay
MAC—mycobacterium avium complex

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M. – Mycobacterium MGIT – Mycobacteria growth indicator tube
MgCl – Magnesium chloride MTBC – Mycobacterium tuberculosis
2
complex
MGW—Molecular grade water
MOP – Method of proportion MSDS – Material safety data sheet
NaCl – Sodium chloride NALC – n-acetyl-l-cysteine
NaNO – Sodium nitrate NaOH – Sodium hydroxide
3
NTM – Nontuberculous mycobacteria OADC – Oleic acid dextrose catalase
PAS – Para-amino salicylic acid PBS – Phosphate buffered saline
PNB – P-nitro benzoate PCR – Polymerase chain reaction
PNM – Primer nucleotide mix PPE – Personal protective equipment
PCC – Probe check control PZA – Pyrazinamide
QA – Quality assurance QC – Quality control
RIF – Rifampicin RNA – Ribonucleic acid
SDW – Sterile distilled water SIRE – Streptomycin, isoniazid, rifampicin,
ethambutol
SOP – Standard operating procedure SPC – Sample processing control
TB – Tuberculosis TCH – Thiophene-2-carboxylic acid
hydrazide
UC – Universal control ZN – Zeihl-Neelsen

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INTRODUCTION
Tuberculosis still presents a major threat to the health of the Nigerian population. It is one of the
top 10 leading causes of hospital admissions and one of the leading causes of morbidity and
mortality in adults. Its recent relationship to HIV has introduced serious difficulties in case finding,
treatment and follow-up due to the dramatic increase in the number of cases.
There has been rapid expansion of the network of laboratories performing smear microscopy
since the inception of the NTBLCP in 1993. With the support of WHO, CDC/ASM, Global Fund,
USAID, ILEP, Agbami Co-venturers (Agbami Partners) and other stakeholders, the Culture/DST,
LPA and Xpert MTB/RIF services are also been provided in most TB laboratories in the country.
To standardize the procedures performed in these laboratories for the diagnosis of tuberculosis
cases including the resistant strains, there is need to harmonize the SOPs to inform and guide all
laboratory personnel on all the steps involved with minimal errors.

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