1.

Procedure

4.1 Principle Specimen quality from the moment of collection to the arrival of specimens at the laboratory where they will be cultured is the responsibility of the setting in which specimens are collected, that is, either the peripheral laboratory where patients were given sputum containers or the clinics where sampling/biopsy is performed. Since the laboratory is usually the only place where there is quality control of specimens received, laboratories at all levels must monitor quality indicators, e.g. the proportion of saliva sputum specimens, frequent late arrival of specimens, and report problems so that corrective action may be taken wherever necessary. Specimens sent to the laboratory should be of adequate volume, as specified below, accurately labelled for identification, and accompanied by a written laboratory request form according to WHO recommendations. Specimens should be sent to the laboratory as soon as possible after collection, in leakproof containers surrounded by absorbent material in a shock-resistant outer package that is properly labelled according to the national and/or international regulations for infectious material 4.3 Equipment and materials Wide-mouthed, unbreakable, leakproof, screw-capped containers. Containers should have a volume capacity of 50 ml and made of translucent material in order to observe specimen volume and quality without opening the container. 4.5 Detailed instructions for the procedure 4.5.1 Sample collection Sputum The large majority of specimens received for diagnosis are sputum samples.

If good specimens are to be obtained, patients must be instructed in how to produce sputum. Specimens should be collected in a separate, ventilated room or preferably outdoors. Keeping both hands on hips, cough forcibly and collect sputum in the mouth; spit the sputum carefully into a wide-mouthed, unbreakable, leakproof container and close the lid tightly. Ideally, a sputum specimen should be 35ml in volume, although smaller quantities are acceptable if the quality is satisfactory. If specimens are to be cultured using a centrifugation method (see SOP Specimen processing for culture), sputa should preferably collected directly into 50-ml centrifuge tubes to avoid the need for their transfer from one container to another. Label each specimen with the unique identification number from the laboratory request form. Note: Specimens are sometimes sent in formalin or bleach! It may therefore be advisable to remind the physician of collection conditions, the day before surgery.

Collect two or three specimens from each patient according to the policy.

4.5.2 Transport conditions

Sputa should be transported to the laboratory as soon as possible. If a delay of a few days cannot be avoided, keep specimens cool (refrigerated but not frozen) Up to a week in cold conditions will not significantly affect the positivity rate of smear microscopy; however, the additional growth of contaminants will result in an increased contamination rate on culture media. If the delay exceeds 3 days, an equal volume of cetyl pyridinium chloride (CPC; solution of 1% CPC in 2% sodium chloride) should therefore be added to sputum (see SOP preparation of reagents for culture). Sputum containing CPC can be kept for up to 7 days but must be kept at room temperature (>20 C since CPC crystallizes at lower temperatures). The addition of CPC must be indicated on the accompanying documents (see form below) because CPC has to be removed before culturing. Transport packaging The basic packaging system for local surface transport of all specimens consists of three layers (Annex 1):

Primary receptacle the specimen container packaged with enough absorbent material to absorb all fluid in case of breakage. Secondary packaging a second durable, watertight, leakproof packaging to enclose and protect the primary receptacle(s). Several cushioned primary receptacles may be placed in one secondary packaging, but sufficient additional absorbent material must be used to absorb all fluid in case of breakage.

For cold transportation conditions, ice or dry ice shall be placed outside the secondary receptacle. Wet ice shall be placed in a leakproof container;

Outer packaging secondary packagings are placed in outer shipping packagings with suitable cushioning material. Outer packagings protect their contents from external influences, such as physical damage, during transit.

For surface transport there is no maximum quantity per package. For air transport, no primary receptacle shall exceed 1 l for liquids or the outer packaging mass limit for solids. The volume shipped per package shall not exceed 4 l or 4 kg.

4.7 Quality control Before specimens can be accepted in the laboratory, the accompanying request forms must be checked carefully for identity (sample and request form labelled with the same number). Specimens that cannot be identified exactly will be not processed. Specimens should be examined on receipt of the sample, to ensure that they correspond in type, quantity, quality and volume to the appropriate criteria. Any deviations must be documented and noted on the final report since they may affect the results. The transport conditions and duration must be checked. Delays in transportation and/or exposure of specimens to extremes of temperature without protective measures must be documented and noted in the report.

2.

Related documents

Collins C, Grange J, Yates M. Organization and practice in tuberculosis bacteriology. London, Butterworths, 1985.

Guidance on regulations for the transport of infectious substances 20072008. Geneva, World Health Organization, 2007 (WHO/CDS/EPR?2007.2) available at http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2007_2/en/ind ex.html). Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA, United States Department of Health and Human Services, Centers for Disease Control, 1985. Laboratory services in tuberculosis control. Part II: Microscopy. Geneva, World Health Organization, 1998 (WHO/TB/98.258). Lumb R, Bastian I. Laboratory diagnosis of tuberculosis by sputum microscopy. Adelaide, Institute of Medical and Veterinary Science, 2005. Revised TB recording and reporting forms and registers version 2006. Geneva, World Health Organization, 2006 (WHO/HTM/TB.2006.373, available at http://www.who.int/tb/dots/r_and_r_forms/en/). Smithwick RW. Laboratory manual for acid-fast microscopy. Atlanta, GA, Center for Disease Control,1976.

Annex 1. Example of a triple packaging system

Annex 2. Request and reporting form for TB culture and Drug Susceptibility Test (DST)
Patient identification (ID): TB register number:____________ Previous TB register number:____________ MDR register number:_____________ Surname and first name of patient:________________________________ Sex:____ Ward / Department: __________________ *HIV-status: Pos / Neg / Unknown Age (yrs):_____

Address: _________________________________ _________________________________ new (never treated before for 1 month) relapse failure return after default chronic excretor MDR contact uncertain

TB Disease type and treatment history Site: pulmonary History: extrapulmonary (specify):_______________ Previous treatment: Cat.1 Cat.2 Cat.4 (second-line drugs) Other _________________ Origin of request: Region ID:_______________ ID:________________ Date specimen was collected: Local laboratory: ____/____/20____
st nd rd

District ID:_______________

Local laboratory

Specimen ID number:_______________ direct smear concentrated smear

smear result: 1 ____ 2 ____ 3 ____ specimen microscopy technique used: hot Ziehl-Neelsen cold staining fluorescence Specimen:

Request for testing at the reference laboratory: Reason: diagnosis follow-up at . months during treatment follow-up at . months after treatment specify):__________________ Requested tests: microscopy(type _______ )

sputum sputum in preservative, type other

culture

DST (first / second line)

Person requesting examination: Name:_________________________ Position:________________

Reference laboratory results: Date received in the Reference Laboratory _____/______/20_____ Reference Laboratory specimen ID:__________ Microscopic examination: previously reported on date _____/______/20_____ ID # Neg 1-9 1+ 2+ 3+ hot Ziehl-Neelsen direct smear cold staining concentrated smear fluorescence

Culture result: previously reported on date _____/______/20_____ ID # Contamin ated Neg Non-TB mycobacteria (species) 1-9 colonies actual count 10 100 col 1+

will follow Mycobacterium tuberculosis complex >100 - 200 col 2+ >200 col 3+

Results of M. tuberculosis drug susceptibility testing: phenotypic method used ______________________________________________ genetic method used _________________________________________________ ID # ____________ INH g/ml result Date: _____/______/20_____

will follow

Legend: S = susceptible; R = resistant; C = contaminated; ND = not done

Rifampicin

Ethambutol

Streptomycin

Pyrazinamide

Ofloxacin

Kanamycin

Signature:________________