D:\wwwroot\tripdatabasePlus_live\control\reindex\html\249724 TECHNICAL GUIDE Sputum Examination for Tuberculosis by Direct Microscopy in Low Income Countries Fifth edition 2000 International Union Against Tuberculosis and Lung Disease 68 boulevard Saint Michel, 75006 Paris, France WRITING COMMITTEE Mohammed Akhtar Gisela Bretzel Fadila Boulahbal David Dawson Lanfranco Fattorini Knut Feldmann Thomas Frieden Marta Havelková Isabel N de Kantor Sang Jae Kim Robert Küchler Frantz Lamothe Adalbert Laszlo Nuria Martin Casabona A Colin McDougall Håkan Miörner Graziella Orefici C N Paramasivan S R Pattyn Ana Reniero Hans L Rieder John Ridderhof Sabine Rüsch-Gerdes Salman H Siddiqi Sergio Spinaci Richard Urbanczik Véronique Vincent Karin Weyer On a draft document prepared by Adalbert Laszlo, for the International Union Against Tuberculosis and Lung Disease Graphic design: Edik Balaian PREFACE A technical guide for sputum smear microscopy, based on one initiated in 1969 by Dr J Holm, the then Director of the International Union against Tuberculosis, was first published in 1978 by the IUAT as the Technical Guide for Sputum Examination for Tuberculosis by Direct Microscopy . The guide was included in the third and fourth editions of the IUATLD's Tuberculosis Guide for Low Income Countries . It was designed to be a simple reference standard for the collection, storage and transport of sputum specimens and for the examination of sputum smears by direct microscopy. It was meant to address the needs of health care workers in low income, high prevalence countries which represent the bulk of the global tuberculosis caseload. More than twenty years have elapsed since its first publication, and the guide has remained unchanged throughout that time. Today, tuberculosis is one of the main causes of death from a single infectious agent among adults in low income countries, where it remains a major public health problem. The basic tool for TB diagnostic services, i.e., sputum smear microscopy, has not changed in its technical details in spite of major advances in modern diagnostic tech- nologies. However, the context in which it is applied, the National Tuberculosis Programme, has been refined to a considerable extent in the last two decades. The field use of the guide over the years has revealed omissions and inac- curacies that needed to be addressed. Furthermore, biosafety and quality assur- ance aspects of sputum smear microscopy were not sufficiently well covered in the previous edition. It was therefore felt that the IUATLD Technical Guide needed revision so it could better reflect its public health essence and keep up to date with modern TB control strategies. This document was carefully revised by members of the Bacteriology and Immunology Section of the IUATLD, by directors of the WHO/IUATLD Supranational TB Reference Laboratory Network and by other dis- tinguished professionals in the field of tuberculosis control. DR ADALBERT LASZLO Ottawa 2000 TABLE OF CONTENTS 1. SPUTUM MICROSCOPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Aims of tuberculosis laboratory diagnostic and follow-up services . . . . . . . . . . . 1 1.2 "Spot", "morning" and "spot" sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.3 "Morning" specimens for follow-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.4 Sputum containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.5 Collection of sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.6 Transportation of sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2. THE SPUTUM SMEAR MICROSCOPY LABORATORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1 Role of the laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 Physical environment of the laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3. THE STAINING METHOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.1 Preparation of Ziehl-Neelsen reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.1.1 Ziehl's carbol fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1.2 Aqueous phenol solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1.3 Decolourising agent solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1.4 Methylene blue counterstaining solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2 Smear preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2.1 Smearing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2.2 Fixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.2.3 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.2.4 Decolourising. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.2.5 Counterstaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2.6 Quality of smearing and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4. MICROSCOPIC EXAMINATION OF SPUTUM SMEARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.1 The microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.2 Operating the microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.3 Microscopic examination of smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.4 Grading of sputum smear microscopy readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.5 Preservation of smears for quality assurance testing . . . . . . . . . . . . . . . . . . . . . . . . . . 15 5. RECORDING AND REPORTING OF SMEAR MICROSCOPY RESULTS . . . . . . . . . . . . . . 15 5.1 The laboratory register . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 6. QUALITY ASSURANCE OF SPUTUM SMEAR MICROSCOPY . . . . . . . . . . . . . . . . . . . . . . . 17 6.1 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 6.2 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 7. DISINFECTION, STERILISATION AND DISPOSAL OF CONTAMINATED MATERIALS 18 8. BIOSAFETY IN THE TB MICROSCOPY LABORATORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 8.1 General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 8.2 Specific aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 9. MATERIALS MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 SUGGESTED READING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Annex 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 · Prevention of false positive results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 · Consequences of false positive results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 · Prevention of false negative results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 · Consequences of false negative results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Annex 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 · Care of the microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Annex 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 · Trouble-shooting guide for microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 1. SPUTUM MICROSCOPY 1.1 Aims of tuberculosis the health centre; "MORNING" specimens laboratory diagnostic consist of all sputum produced within one or and follow-up services two hours after rising. In low income and high tuberculosis preva- 1.3 "Morning specimens" for follow-up lence countries, sputum smear microscopy is, and is likely to remain for the foreseeable There are two phases in the treatment of future, the only cost-effective tool for diag- tuberculosis: the intensive phase , usually 2 nosing patients with infectious tuberculosis to 3 months, and the continuation phase, and to monitor their progress in treatment. which is 4 to 10 months, depending on the Sputum smear microscopy is a simple, inex- type of treatment. Regardless of the treat- pensive, appropriate technology method ment regimen, one "MORNING" sputum which is relatively easy to perform and to specimen is collected for follow-up at the end read. It yields timely results with a very high of the intensive phase of treatment to deter- sensitivity of detection of tubercle bacilli mine whether the patient can proceed to the transmitters, and provides most of the essen- continuation phase if the smear is negative tial laboratory-epidemiological indicators or, if the smear is positive, continue the inten- needed for the evaluation of the National sive phase. Another sputum specimen must Tuberculosis Programme (NTP). be taken during the continuation phase to check patient evolution and to detect possible treatment failure, and another upon comple- The aims of TB laboratory diagnostic ser- tion of chemotherapy to verify cure. Sputum vices within the framework of an NTP are: specimens at the end of treatment are often ­ diagnosis of cases difficult to obtain, as many patients have ­ monitoring of tuberculosis treatment stopped expectorating. The exact schedule of follow-up sputum examinations varies according to the drug regimen, and should be set out in the NTP Manual . 1.2 "Spot", "morning" and "spot" The patient is said to have completed sputum specimens for diagnosis treatment even if sputum specimens are not Under NTP conditions, the IUATLD recom- examined during and at the end of treatment. mends collecting three sputum samples "on The cure rate is the proportion of initially spu- the SPOT ­ early MORNING ­ on the SPOT", tum smear-positive patients who are declared preferably within two days, from each per- cured based on negative sputum smear son presenting at health centres, out-patient results on at least two occasions, including clinics, etc., with respiratory symptoms of one at the end of treatment. The objective of more than 3 weeks' duration. These samples the NTP is to achieve at least 85% cure rate are to be examined by smear microscopy in among new sputum smear positive TB cases the nearest laboratory. Under these condi- registered. tions, a case of sputum smear positive tuber- culosis is usually defined as a person pre- The IUATLD recommends : senting with respiratory symptoms with at ­ The examination of three sputum spec- least two positive sputum smear microscopy imens ­ "SPOT" + "MORNING" + examinations. "SPOT" ­ for the diagnosis of tubercu- This approach, also known as passive losis cases. case finding , detects about 80% of TB sus- ­ The examination of single "MORNING" pects ultimately positive on sputum smear sputum specimens on three occasions examination with the first specimen, an addi- for follow-up of treatment: one at the tional 15% with the second and a final end of the intensive phase , one during 5% with the third. "SPOT" specimens are the continuation phase, and one at the obtained when the TB suspects present at end of treatment . 1 A C B Figure 1 1.4 Sputum containers The health care worker should reassure persons suspected of having tuberculosis by The use of two kinds of sputum containers is explaining the reasons for the examination, recommended. One, available from UNICEF and give instructions on how to cough so that (Figure 1 A), is a rigid wide-mouthed screw- the expectoration is produced from as deep capped container made of unbreakable trans- down in the chest as possible. As an adjunct, parent plastic, readily disposable by burning, written instructions can be handed out if the which is used for most routine diagnostic person is literate. work. Its screw cap can be hermetically sealed to prevent desiccation of the sample The health care worker should make sure and leakage. that the specimen is of sufficient volume (3 to 5 ml) and that it contains solid or purulent The other, a screw-capped heavy glass material, the presence of which increases the container, such as the Universal bottle (Figure sensitivity of detection, and not just saliva. 2 A), is used for specimen transportation in However, if only saliva is obtained or, as fre- custom-made boxes that can be made of quently happens in "spot" sputum, volumes metal, wood or styrofoam. A wooden box is of less than 3 ml are produced, the specimen a sensible compromise in terms of sturdiness should nevertheless be processed, as it is and weight (Figure 2 B, C). The Universal sometimes likely to yield positive results. A bottle is reusable after disinfection by auto- sputum specimen can be classified by macro- claving for 30 minutes at 121oC and careful scopic examination as "salivary" when it cleaning. If an autoclave is unavailable, a consists mainly of saliva, "mucous" when it domestic pressure cooker is recommended. is mainly mucus, "purulent" when it appears yellow as pus, "muco-purulent" when there are visible yellowish particles in the mucus 1.5 Collection of sputum specimens and "bloody" when it contains blood. The presence of blood should always be noted The risk of infection for health care workers is because it is indicative of severe disease and highest when TB suspects cough; sputum could interfere with the reading of the smear. specimens should therefore be collected in the open air and as far away as possible from The health care worker should provide a other people. Failing this, a separate, well- sputum container with the health centre code ventilated room should be used. and the TB suspect or patient's identification 2 centre, and fixed smears can be sent to the nearest laboratory. This procedure is dis- couraged, however, because fixed smears are often of poor technical quality, having B been prepared by untrained personnel, and tend to decompose quickly in warm and humid climates. The health care worker should provide the TB suspect with a new, pre-labelled spu- tum container, explain how it should be used in the morning to collect the "MORNING" specimen and demonstrate how it should be securely closed before it is brought back to the health centre. 1.6 Transportation of sputum specimens A In countries lacking laboratory facilities that rely on specimen collecting units, transportation of specimens is required. Transportation is also required when opera- tional research projects of interest to the NTP are undertaken, such as a survey of TB drug resistance, etc. If culturing of specimens is required, the specimens should reach the lab- oratory within 3-4 days and should be refrig- erated while waiting for shipment. The most rapid and cost-effective means of trans- C portation should be selected. Contaminating flora do not affect the acid-fastness of mycobacteria but may liquefy the sputum, making smear preparation difficult and read- ing of slides unreliable. A list identifying the sputum specimens Figure 2 contained in the transport box and a com- must be written on the side of the container, pleted Request for Sputum Examination never on its lid. (Figure 1 C). The health care Forms (Figure 3) for each specimen must worker must ask the person being examined accompany the shipment. Before the ship- to bring it close to the mouth and expectorate ment from the health centre, the health care into it (Figure 1 B). This specimen is called a worker must verify for each transport box "SPOT" specimen. that: ­ the total number of sputum containers in If expectoration is not produced, the spu- the box corresponds to that on the accom- tum container must be considered as used panying list and that on the Request for and must be properly disposed of. Sputum Sputum Examination Forms; containers must be closed securely and if they are to be sent to a nearby laboratory, ­ the identification number on each sputum they should be placed in the appropriate container corresponds to that on the accom- transport box. Collected specimens should panying list and that on the Request for be kept in a cool place, batched and trans- Sputum Examination Forms; ported without much delay, i.e., at least twice ­ the accompanying Request for Sputum weekly, and processed as soon as possible. Examination Forms contain the requested Sputum can also be processed in the health information for each of the TB suspects. 3 REQUEST FOR SPUTUM EXAMINATION Figure 3. Request for sputum examination form 4 When this verification is completed, the first, 2 for second, 3 for third (Figure 12), health care worker: will be assigned to each specimen by labo- ­ dates the accompanying list; ratory staff. Results of the examination ­ puts the list and the Request for Sputum will be entered on the bottom half of the Examination Forms in an envelope which Request for Sputum Examination Form. The will be attached to the outside of the trans- Laboratory Serial Number begins with 1 on port box. 1 January each year and increases by one A laboratory code, a serial number and with each patient until 31 December of the a specimen sequence identifier, i.e., 1 for same year. 2. THE SPUTUM SMEAR MICROSCOPY LABORATORY 2.1 Role of the laboratory ­ perform all sputum smear microscopy requested in their catchment area, usually a In developing countries, most of the bacteri- district (50,000-100,000 inhabitants); ological diagnosis of tuberculosis is carried ­ act as referral center for specimen collect- out in peripheral or local laboratories, whose ing units; major responsibility is to provide diagnostic ­ co-ordinate with Regional (intermediate) microscopy for the NTP based on sputum Laboratories the referral of specimens smear examination by Ziehl-Neelsen ( ZN ) requiring culture and drug susceptibility test- staining. These laboratories, located in health ing; centres, health posts, hospitals, etc., usually have qualified technical personnel capable of ­ receive specimens during the opening performing ­ among other duties ­ sputum hours of the health centre; smear microscopy. They should be able to ­ send information to the Regional Labo- carry out the following functions: ratory; Figure 4 5 ­ comply with national quality assurance guidelines; ­ order, manage and store laboratory sup- plies. 2.2 Physical environment of the laboratory The detailed arrangement of the microscopy laboratory varies greatly depending on local Figure 5 conditions. It is difficult to generalise about the design of such laboratories since TB diag- ­ a sink (D) with running water for washing nostic services have, over time, been inte- hands; grated into existing general laboratory diag- ­ a bench area (E) for microscopy reading nostic services in many countries. Ideally, the directly below a window (Figures 4 and 8); TB microscopy laboratory should include the following distinct sections (Figure 4), adapted ­ a bench area or a table (F) for the labora- from Collins et al:1 tory register books and slide storage space (Figures 4 and 9); ­ a bench space or a table (A) for incoming specimen (Figures 4 and 5); ­ one well-lit work bench (B) for smear preparation (Figures 4 and 6); ­ a staining sink (C) with running water (Figures 4 and 7); Figure 6 1. Slide-holder for the preparation of smears 5. Alcohol lamp / Bunsen burner 2. Slide dryer 6. Forceps 3. Sputum container placed as close as possible to the 7. Metal waste receptacle with lid to receive infectious slide-holder on the right material 4. Wooden applicators 8. Box of engraved slides for the smears 6 Figure 9 bench: this plate should be about 80 cm wide, Figure 7 with borders 5 cm high. The front edge must be bent down at an angle of 90o to meet the edge of the table, thus facilitating manipula- tions (Figure 6). These must be conducted strictly over the surface plate, which should be decontaminated every day after use by soaking with a TB germicide (e.g., 5% phe- nol, 0.1% solution of sodium hypochlorite* [NaClO], also known as common household bleach, Chlorox, Javex, etc.). NOTE : If the technician is left-handed, it may be more convenient to arrange all (or Figure 8 most) items in Figure 6 in exactly the oppo- site position on the table (i.e., in a mirror ­ a locker (G) for the technicians' clothing image). (Figure 4). If the work bench is made of porous * Household bleach contains 5% of NaClO (50 g/litre); a 0.1% solution containing 1 g of NaOCl/litre is pre- material, a non-porous surface plate, such as pared by diluting 20 ml of household bleach in 1 litre formica, a marble slab, or a galvanised metal of water. This solution is used as an all-purpose dis- or aluminum sheet, should cover the work infectant for "clean conditions".2 3. THE STAINING METHOD 3.1 Preparation of Ziehl-Neelsen quently used option. The advantages of this reagents option, i.e., better standardisation and qual- ity assurance, outweigh the disadvantages of The method of choice for sputum smear long term storage. Cold staining procedures microscopy is the Ziehl-Neelsen (ZN) stain- such as the Kinyoun and Tan Thiam Hok ing technique because it is the only one that methods are not recommended, as evidence provides consistently good results without shows that they have difficulty detecting acid- the need for special equipment. Preparing the fast bacilli (AFB) in paucibacillary samples necessary reagents requires a weighing scale and the staining fades rapidly. Fluorescence that is not always available in a peripheral microscopy, which is recommended when laboratory, and preparing the reagents in the the daily workload exceeds 50 specimens, National Reference Laboratory or in the near- has no place in most peripheral laboratories est intermediate laboratory is therefore a fre- of low-income countries. 7 3.1.1 Ziehl's carbol fuchsin accompanying Request for Sputum Exam- ination Form. The use of new slides is rec- 3% fuchsin alcoholic stock solution (solution A) ommended; however, because they are often greasy, they tend to cling together and must Basic fuchsin*. . . . . . . . . . . . 3 g be cleaned with alcohol and then carefully 95% alcohol . . . . . . . . . . . . . up to 100 ml air-dried. When alcohol is not available the Place the required amount of fuchsin in a slides may be held over a flame to remove volumetric flask or measuring cylinder and, oils. Under weather conditions prevalent in adding enough ethanol or methylated spirit most low income countries, the use of slides to obtain a total volume of 100 ml, shake well in tropical packaging (each slide separated until completely dissolved. Small quantities from the next by a strip of impermeable of this solution should be filtered prior to paper) is recommended. Laboratory code, staining. serial number and sequence identifier can be engraved with a diamond marker on the 3.1.2 Aqueous phenol solution smear side and at one end of the slide. When diamond markers are not available, a dis- (solution B) carded round-tipped dental drill inserted in Phenol§ cristals . . . . . . . . . . . 5 g the tapered end of a discarded plastic pen Distilled water, if possible . . up to 90 ml can be used.3 An ordinary lead pencil can be Before adding water, liquefy the phenol used if frosted-end slides are available. crystals in a flask by gentle heating. To prepare the 0.3% Ziehl's carbol fuchsin 3.2.1 Smearing working solution, mix 10 ml of Solution A with 90 ml of Solution B. ­ Verify that the numbers on slides and con- tainers match. ­ Take sputum container corresponding to 3.1.3 Decolourising agent solutions the number on the slide. ­ Acid-alcohol solution ­ Open container carefully to avoid aerosol Alcohol 95% . . . . . . . . . . . . . . . . . . 970 ml production. Concentrated (35%) ­ Break a wood or bamboo stick applicator hydrochloric acid** . . . . . . . . . . . . 30 ml (Figure 10), select yellow, purulent particle of sputum with jagged end of the broken wood Or, when alcohol is unavailable: or bamboo stick applicator. Use the broken ­ 25% aqueous sulfuric acid solution Distilled water if possible . . . . . . . 300 ml Concentrated sulfuric acid . . . . . 100 ml * Pararosaniline chloride, Minimum dye content 88% Pour 300 ml of water into a 1 litre (C H NCl) Sigma P1528 or equivalent. 19 18 Erlenmeyer flask. Slowly add 100 ml of con- Staining powders are seldom pure, so a corrected weight should be used to ensure proper staining. The centrated sulfuric acid, allowing it to flow percentage of available dye content is frequently listed along the side of the flask. The mixture will on the original container label. The corrected weight heat up. Never pour water into concentrated is determined by dividing the desired amount of dye sulfuric acid ­ explosive spills may occur. by the decimal equivalent of the available dye. So, if the desired amount of dye is 3 g and the per cent avail- able dye is 75%, the actual amount of dye to be 3.1.4 Methylene blue counterstaining weighted is 3/0.75 = 4 g of impure dye. If the avail- solution 0.3% able dye content is 88% or more, there is no need to Methylene blue chloride . . 0.3 g correct the weight. Ethanol 95% (C H OH) ­ United States Pharmacopeia 2 5 Distilled water, if possible . . up to100 ml XVIII, 20, 1067 (1970). Can be of industrial grade. § Phenol approx. 99% (C H O) ­ Sigma P 3653 or 6 6 equivalent. 3.2 Smear preparation ** Concentrated hydrochloric acid (HCl) ­ can be of industrial grade. Concentrated sulfuric acid (H SO ) ­ can be of indus- Sputum containers are arranged in sequential 2 4 trial grade. order. Laboratory serial numbers must Methylthionine chloride, minimum dye content 82% match the corresponding information on the (C H CIN S) ­ Sigma M 9140 or equivalent. 16 18 3 8 Applicators are only used once. Discard by placing them in a waste receptacle containing 5% aqueous phenol solution or a 0.5% solution of sodium hypochlorite,* then autoclave or incinerate. CAUTION : Vapours are very toxic. 3.2.2 Fixing Fix dried smears by holding them with for- ceps and passing them smear side up over Figure 10 the flame 5 times for about 4 seconds (Figure 13). Do not heat-fix moist slides, and do not overheat . end of the two pieces of the applicator to break up larger particles. ­ Spread the sputum evenly over the central area of the slide using a continuous rotational movement (Figure 11); the recommended Figure 13 3.2.3 Staining Figure 11 ­ Place fixed slides on the staining rack in size of the smear is about 20 mm by 10 mm serial order, smeared side up. Slides should (Figure 12). be separated by a 1 cm gap, and should never touch one another. ­ Cover slides individually with filtered 0.3% Ziehl's carbol fuchsin working solution (Figure 14). Placing a strip of absorbent paper such as filter or even newspaper will hold the staining solution and prevent deposits of fuchsin crystals on the smear. ­ Heat slides from underneath with the flame of a Bunsen burner, an alcohol lamp or an alcohol soaked cotton swab until vapour starts to rise. Staining solution should never Figure 12 be allowed to boil. Do not allow the stain to dry (Figure 15). ­ Place slides on dryer with smeared surface upwards, and air dry for about 30 minutes. * Sodium hypochlorite is a strong oxidizing agent which is corrosive to metal. A 0.5% solution contain- ­ Re-cap sputum container, which should not ing 5 g of NaClO/litre, prepared by diluting 100 ml of be discarded before results are read and household bleach in 1 litre of water, is recommended recorded. for dealing with "dirty conditions".2 9 ­ Rinse slides gently with water to remove excess carbolfuchsin (Figure 16). ­ Drain off excess rinsing water from slides (Figure 17). Sputum smears appear red in colour. Figure 14 Figure 17 3.2.4 Decolourising ­ Cover slides with 25% sulfuric acid or acid-alcohol solution and allow to stand for 3 minutes, after which the red colour should have almost completely disappeared (Figure 18). If needed, repeat sequence until the Figure 15 red colour disappears, but do not over- decolourise. ­ Keep slides covered with hot, steaming car- bolfuchsin for 5 minutes by re-flaming as needed. Figure 18 ­ Gently wash away the sulfuric acid or acid alcohol and the excess stain with water (Figure 19). Drain off excess rinsing water Figure 16 from slides (Figure 20). 10 ­ Rinse slides individually with water (Figure 22). Figure 19 Figure 22 ­ Drain water off the slides, which are then allowed to air dry (Figure 23). Figure 20 3.2.5 Counterstaining ­ Cover slides individually with 0.3% meth- ylene blue counterstaining solution and allow to stand for 1 minute (Figure 21). Figure 23 The Ziehl-Neelsen staining procedure requires : ­ Staining for 5 minutes ­ Decolourising for 3 minutes ­ Counterstaining for 1 minute Figure 21 11 3.2.6 Quality of smearing and staining when held underneath the slide, the smear is too thick. ­ A properly stained smear should show a light blue colour due to methylene blue. If ­ Example of a good smear (Figure 24). dark blue, i.e., a newspaper cannot be read ­ Examples of bad smears (Figure 25) Figure 24 Figure 25 4. MICROSCOPIC EXAMINATION OF SPUTUM SMEARS 4.1 The microscope kept clean by wiping with lens paper on a daily basis. A binocular microscope with two objectives ­ a regular 40 x magnification objective and an oil immersion 100 x magnification objec- 4.2 Operating the microscope tive ­ and eyepieces of moderate magnifica- tion (8x or 10x) is required for the examina- ­ A drop of immersion oil is placed on a dry tion of smears (Figure 26). stained slide to increase the resolving power of the objective. To prevent cross contami- Microscopes equipped with the light-col- nation by AFB , the immersion applicator lecting mirror option are strongly recom- should not touch the slide. Cedarwood mended, as they are useful in the event of immersion oil should never be used, as it power failures and necessary in laboratories forms a thick paste upon drying that could that lack electricity. The mirror has one plane damage the lenses of the microscope. surface for artificial light and another con- "Makeshift" use of other oils, such as linseed, cave surface for natural light. An illuminator palm, olive, liquid paraffin, etc., is completely is built into the base of the microscope; a unsatisfactory. Some immersion oils can dis- halogen bulb provides good illumination. solve fuchsin stain,4 a circumstance that Halogen lamps have higher luminosity and might accelerate the fading of the ZN stain. longer life than tungsten lamps. Synthetic hydrocarbons and advanced poly- When not in use, microscopes should be mers with a refractive index of 1.5, non- kept in their case protected from dust, heat drying and non-hardening, with no solvent and humidity. Fungus growth is a constant capability, are recommended.* threat to the microscope's optical system: it can be inhibited by fitting the storage case * Type A or B immersion oil (R. P. Cargille Labs, Inc. with a 20-40 watt lamp, which is kept lit dur- Cedar Grove, NJ. Catalogue No. 16484, or VWR brand ing the storage of the microscope. The objec- Immersion Oil, Resolve, Catalogue No. 48218, or tive, eyepiece, condenser and light source are equivalent). 12 Figure 26 1) eye piece; 2) Diopter ring; 3) objective; 4) stage; 5) condenser; 6) diaphragm lever; 7) coarse focus knob; 8) fine focus knob; 9) light source. 13 ­ With the condenser raised to the upper- most position, the stained slide is placed on the stage and the light source is adjusted for optimal light by looking through the eyepiece and the regular 40 x objective. ­ An area containing more leukocytes (pus cells) than epithelial cells (more frequent in saliva) is selected before placing the drop of immersion oil. ­ By slowly changing to the immersion objective, a thin film of oil will form between the slide and the lens. The fine adjustment knob is used to focus the field; the lens should not be allowed to touch the slide. For more on the use and operation of the microscope, see reference 5. Figure 27 4.3 Microscopic examination of smears tically so that a second length can be read from right to left. There are about 100 immer- ­ Acid-fast bacilli appear bright red or pink sion fields in the 2 cm long axis of a smear. against the blue counterstained background. They vary greatly in shape, from short, coc- coid to elongated filaments; they can be uni- 4.4 Grading of sputum smear formly or unevenly stained, and can even microscopy results appear granular. They occur singly or in vari- able sized clumps, and typically appear as The information on the number of bacilli long, slender curved rods. found is very important because it relates to ­ The microscopic examination must be sys- the degree of infectivity of the patient as well tematic and standardised. It can start at the as to the severity of the disease. For this rea- left end of the smear. The reading begins at son, the report of the results of sputum smear the periphery of the field and ends at the cen- microscopy must be not only qualitative but tre (Figure 27). When the field is read, the also semi-quantitative. The IUATLD recom- slide is moved longitudinally to examine mends the following grading of results of adjacent fields. The slide can be moved ver- smear microscopy (Table 1). Table 1 IUATLD-recommended grading of sputum smear microscopy results AFB counts Recording/reporting No AFB in at least 100 fields 0/negative 1 to 9 AFB in 100 fields* Actual AFB counts 10 to 99 AFB in 100 fields + 1 to 10 AFB per fields in at least 50 fields ++ > 10 AFB per field in at least 20 fields +++ * A finding of 1 to 3 bacilli in 100 fields does not correlate well with culture positivity. The interpretation of the significance of this result should be left to the NTP and not to the microscopist. It is recommended that a new smear be prepared from the same sputum specimen and be re-examined. The reporting of actual AFB counts is recommended to allow a competent authority to determine whether the number fits the TB case definition of the NTP. In practice most microscopists read a few fields and confirm the finding by a quick visual scan of the remaining fields. 14 The microscopist should initial the smear the immersion oil from the smear by blotting result as well as other result entries in the with lens paper is discouraged because the laboratory register. smear might be scraped off the slide and The microscopist should take at least 5 the oil will never be thoroughly removed. minutes to read 100 fields, and should never Cleaning the slides of immersion oil by dip- be expected to process and read more than ping them in xylene (xylol)* and drying them 25 ZN-stained sputum specimens per day before storing them in slide boxes until the when working full time. No more than 10 to next supervision is recommended. Positive 12 specimens should be processed at one and negative slides should be kept in sepa- time. However, this situation seldom occurs rate slide boxes. Filled slide boxes should be even in the peripheral laboratories of high stored closed and as far removed from heat incidence countries. When TB sputum smear and humidity as possible until they are sam- microscopy is fully integrated in to the gen- pled for re-reading. Slides should not be dried eral primary health care services, the real and stored under direct UV light. The sam- challenge is to reach a workload high enough pling and re-reading of slides should be done to maintain testing proficiency. as soon as possible, because long term stor- age under tropical climatic conditions will cause fading of the ZN staining. 4.5 The preservation of smears for quality assurance testing * Xylene, mixed ACS Reagent Sigma X 2377 or equiv- Before storage of the slides, immersion oil alent. A safer, less toxic, less flammable xylene sub- must be washed from the smears. Cleaning stitute is available.6 5. RECORDING AND REPORTING OF SMEAR MICROSCOPY RESULTS A positive sputum smear is like a document suspects to be evaluated, which in turn upon which the diagnosis of pulmonary allows laboratory supply requirements to be tuberculosis is based. Results must be planned based on the number of reported recorded and copies of these records must smear positive cases. be kept in the laboratory. If possible, positive ­ The laboratory code, serial and sequence readings should be confirmed by a second number on the slide must be the same as that reader. Examined slides should be kept in the in the results section of the Request for laboratory for the period of time prescribed Sputum Examination Form . The upper por- by the NTP for the purposes of supervision tion of the Form must be accurately com- and proficiency testing (see chapter 6). pleted. The results of the smear examination should be recorded according to the IUATLD grading scale (Table 1). The report form is 5.1 The laboratory register then dated and signed by the laboratory's responsible officer. The IUATLD laboratory register (Figure 28) has two essential and useful features: it ­ All information from the laboratory form distinguishes between diagnostic sputum should be entered in full in the appropriate smear examination and treatment follow-up spaces of the Laboratory Register. All the microscopy, and allots a single line to each information requested in the laboratory reg- tuberculosis suspect examined, and not to ister must be entered, i.e., a blank space is each sputum specimen examined. This per- not a negative result but a missing record. mits the rate of smear positive cases among Positive results are entered in red ink. 15 Figure 28. Laboratory register for sputum smear microscopy 16 ­ Completed Request for Sputum Examin- ­ Upon completion of the examination of ation Forms must be sent back to the treat- each batch of submitted specimens, the date ment centre or the treating physician within of examination is recorded on the dispatch two working days. In case of a referral from list which is returned along with the trans- another health unit, the patient should port box to the originating health centre receive a copy of the completed form and the as soon as possible. Transport boxes are original must be sent to the treatment centre. cleaned with a cloth wet with a TB germicide Results should never be given to the patient (5% phenol or 0.1% sodium hypochorite) and only. If the patient fails to take the results to also returned to the health centre. Caution : the treatment centre, he or she may not both of these solutions are extremely corro- receive treatment. sive ­ protective gloves should be used. 6. QUALITY ASSURANCE OF SPUTUM SMEAR MICROSCOPY 6.1 Definitions analysed with the aim of looking for ways to permanently remove obstacles to success. Quality assurance of sputum microscopy is Data collection, data analysis, identification an indispensable part of an effective TB of problems and creative problem solving are Control Programme. It encompasses the key components of this process. It involves whole process of sputum collection, smear continued monitoring and identification of preparation, smear staining, microscopy, defects, followed by remedial action to pre- recording and reporting. vent recurrence of problems. The purpose of quality assurance pro- grammes is the improvement of the effi- ciency and reliability of smear microscopy 6.2 Procedures services. A quality assurance programme has Internal quality control of staining is manda- three main components: tory. New lots of staining solutions need to be ­ Quality control : Quality control is a process tested. This usually involves the staining of of effective and systematic internal monitor- known, unstained, positive and negative ing which aims to detect the frequency of smears. The inclusion of known, unstained errors against established limits of accept- smears each time staining is carried out in able test performance. Although it is not usu- the laboratory is also strongly recommended. ally feasible to determine error frequencies The re-reading of positive smears by another accurately, it is nevertheless a mechanism by technologist is highly desirable; in practice which tuberculosis laboratories can at least however, very few peripheral laboratories validate the competency of their diagnostic employ two TB microscopists. Direct obser- services. vation by an experienced observer of labo- ­ Proficiency testing : Also known as External ratory technicians performing their routine Quality Assessment, this is a programme tasks at all stages is an essential aspect of designed to allow participant laboratories to quality assurance. assess their capabilities by comparing their There are four principal methods of pro- results with those obtained with the same ficiency testing of smear microscopy: specimens in other laboratories of the net- ­ Sending smears from the Reference work, e.g., Regional and National Reference Laboratory to the peripheral laboratory for Laboratories. checking reading and reporting. ­ Quality improvement : Quality improve- ­ Monitoring the quality of sputum smear ment is a process by which the components microscopy in all its stages during super- of smear microscopy diagnostic services are visory visits in the field. 17 ­ Sending smears from the peripheral In the present context, quality improve- laboratory to the Reference Laboratory for ment consists of correcting deficiencies in re-reading. smear microscopy performance and reading by taking appropriate remedial action. It is ­ Sampling smears of registered patients the responsibility of the higher level labora- found in the District Tuberculosis Register tories of the network, i.e., Regional and All four methods have distinct advan- Central Reference Laboratories, to retrain tages and disadvantages; it is therefore technologists who demonstrate less than advisable to implement them according to optimal performance. For a more detailed dis- the needs and the circumstances of each cussion of quality assurance programmes in NTP . TB microbiology, see references 7 and 8. 7. DISINFECTION, STERILISATION AND DISPOSAL OF CONTAMINATED MATERIALS After the smears are examined, the lids of all 0.5% sodium hypochlorite solution and are used sputum containers are removed. Used fully submerged. Thereafter, these materials containers, lids and applicators are placed in can be disposed of by autoclaving. If an auto- a waste receptacle containing 5% phenol or clave is not available, all materials should be burned in an incinerator, an open pit or an empty oil drum (Figure 29). NB: If large num- bers of plastic containers are being burnt, the fumes produced are toxic. In the event that both burnable materials and glass sputum bottles are used, the latter should be discarded into a separate container so they may be boiled and washed for re-use. Figure 29 18 Other items such as slide holders, the dryer Slides with negative smears are boiled and the work surface should be soaked in 5% for half an hour in soap or detergent solution phenol or 0.5% sodium hypochlorite solution. (dishwashing liquid), washed under running After the slides have been quality con- water, wiped with cotton or cloth, air dried, trolled, the positive slides should be broken examined to confirm absence of scratches, and disposed of like other "sharps". Negative cleaned with an alcohol soaked cotton swab slides can either be disposed of, or if neces- and stored for re-use. sary, washed clean and re-used for non-TB TB slides, whether negative or positive, work (e.g., malaria, haematology). should never be re-used for TB work. 8. BIOSAFETY IN THE TB MICROSCOPY LABORATORY 8.1 General aspects until torn. This improper use affords a sense of false security and carelessness that often Laboratory workers are responsible for their impacts negatively on the biosafety condi- own safety and that of their co-workers. tions of the laboratory ­ contaminated gloves Transmission of Mycobacterium tuberculo- are used to handle or to operate laboratory sis results essentially from micro-aerosols, equipment that would otherwise never i.e., tubercle bacilli contained in droplet become contaminated. As the use of gloves nuclei, 1 to 5 microns in diameter, which are is impractical in most settings where this sufficiently small to reach lung alveoli, yet guide will be used, soaking hands in 70% sufficient large to adhere to the lining of the alcohol followed by washing with a detergent lung alveoli. solution, rinsing with water and drying with paper, is highly recommended. Infection control in the laboratory must aim at reducing the production of aerosols. Wearing conventional surgical masks Good ventilation is necessary for the protec- does not significantly reduce the risk of infec- tion of the laboratory staff from airborne tion by aerosol inhalation. The emphasis infectious droplet nuclei. An easy way to again is to be placed on the reduction of ensure ventilation and directional airflow is aerosols produced during laboratory proce- by judiciously locating windows and doors dures by adopting and strictly enforcing so that airborne particles are blown away Good Laboratory Practices.8 from the laboratory worker (see Figure 4). Eating, drinking and smoking are not per- Where electricity is available, extractor fans mitted in the laboratory. can be used to remove air from the labora- tory. Each time the technologists enter or leave 8.2 Specific aspects the laboratory they must wash their hands. Staff should wear protective clothing such as Laboratory procedures differ considerably in laboratory coats while exercising their duties, their potential to create aerosols: returning them to the lockers before leaving ­ Specimen collection the laboratory. Access to the laboratory Sputum from tuberculosis suspects is often should be restricted to laboratory staff only. collected in the laboratory for sputum collec- The wearing of disposable gloves for tion. This practice exposes laboratory work- smearing and staining is desirable; however, ers to a high risk of contagion by aerosols because they are meant to be discarded after and should not be allowed under any cir- each laboratory manipulation, their use rep- cumstance. As mentioned in Chapter 1, pre- resents a major expense for peripheral labs. cautions to lower this risk can be taken by Disposable gloves are for single use only, but instructing the tuberculosis suspects to cover in many laboratories they tend to be re-used their mouths while coughing and by having 19 them produce the specimen outdoors, where Expensive and sophisticated equipment is no the aerosols will be diluted and sterilised by substitute for good microbiology laboratory the UV light of direct sunlight. practice. Moreover, commercial type bio- safety cabinets (BSCs) require expert and ­ Smear preparation extensive yearly maintenance, an expense While opening sputum containers and the that is seldom considered at the time the smearing of slides may produce aerosols, equipment is purchased. Commercial type these manipulations entail less risk of trans- BSCs which are not properly maintained give mission than the unprotected coughing of a a false sense of protection, and the same smear positive patient. There is little evidence applies to the home made variety. Twenty that preparing sputum smears is correlated years of field experience in low income coun- with an increased risk of tuberculosis infec- tries have demonstrated the impracticality of tion. However, absence of evidence is not evi- the design proposed in the first edition of this dence of absence, and laboratory workers Guide. Therefore, BSCs are not mandatory in must be careful and remain vigilant at all peripheral laboratories that perform smear times. microscopy only. 9. MATERIALS MANAGEMENT To ensure the continuous flow of laboratory solutions are needed for each slide. It is fur- supplies, programmes must budget rationally ther assumed that 2 drops or 1/10 ml of oil are for requirements. The only quantifiable basis used for each slide. for planning is the number of patients The calculation is performed as follows: recorded and reported. The number and per- ­ the total number of smear positive patients centage of smear positive patients can be (new patients and retreatment cases) determined from the Laboratory Register. recorded on the previous two Quarterly Assuming that the smear positivity rate is Reports on Case-finding is entered under the 15%, that each tuberculosis suspect requires column headed "No. of patients"; three sputum examinations and that each ­ the requirements for the next half year (A) case of smear positive tuberculosis has three are calculated by multiplying the number of follow-up examinations, the number of patients by a predetermined factor, based on microscope slides and sputum containers the assumption that 10 suspects of TB need needed for each sputum smear positive case to be examined for each smear positive case; detected is (1 / 0.15) x 3 + 3 = 23 . ­ the reserve stock requirements (B) are Laboratory material requirements are rel- equal to twice the amount requirements for 6 atively small and for this reason are ordered months (A x 2); every 6 months rather than every 3 months ­ the amount of materials inventoried (C) in and the reserve requirement is estimated at the district store; one year's supply (Figure 30). ­ the total order (D) is the sum of the amount The amounts of basic fuchsin, methylene required for the next semester (A) plus the blue, ethanol and phenol are calculated from amount required for "reserve" stock (B) the IUATLD recommended method for ZN minus the inventoried amount (C) at the time staining, assuming that 5 ml of each of the the order form is completed. 20 0.1 0.1 0.7 9.7 1.7 1.1 30.5 41.6 14.4 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Per cent of cost 3.94 FF 13.12 17.06 4.006600 5.454554 0.013294 0.015748 0.095157 1.278225 0.217298 1.893364 0.148388 0.000029 0.000040 0.000748 0.000025 0.000019 0.000829 identified 2.19 0.66 2.84 US$ Cost per case (FOB) 0.667767 0.909092 0.002216 0.002625 0.015859 0.213038 0.036216 0.315561 0.024731 0.000005 0.000007 0.000125 0.000004 0.000003 0.000138 3 5 7 2 1 1 742 742 Total order 4,945 I=F+G­H 49,450 49,450 61,813 12,363 49,450 24,725 1 2 3 1 1 0 H 293 293 1,955 4,888 9,775 In stock 19,550 19,550 24,438 19,550 3 5 6 2 1 1 690 690 ment 4,600 Reserve require- G=2xF 46,000 46,000 57,500 11,500 46,000 23,000 2 2 3 1 1 0 345 345 ment 2,300 5,750 Running require- F=DxE 23,000 23,000 28,750 23,000 11,500 No E 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 of cases 23 23 case xAxC Require- ment per identified 0.34500 0.34500 2.30000 5.75000 0.00153 0.00230 0.00322 0.00092 0.00069 0.00046 D=(1/Bx3+3) 28.75000 23.00000 11.50000 C Annual depre- ciation 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.10 0.10 0.15 0.10 Figure 30. Taken from Reference 7 (Table V.6) B 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 Fraction of cases among suspects 1 1 A ment Require- per slide 0.01500 0.01500 0.10000 1.25000 0.25000 1.00000 0.50000 0.00007 0.00010 0.00140 0.00040 0.00020 0.00020 Unit 1 1 1 g 1 g 1 mL 1 mL 1 g 1 mL 1 mL 1 box 1 pack 1 1 1 reel 1 CALCULATING THE REQUIREMENTS FOR PERIPHERAL LABORATORIES AND COST OF MATERIAL PER IDENTIFIED CASE. Item Slides Sputum containers Methylene blue Basic fuchsin Immersion oil Sulfuric acid Phenol Xylene Methanol Filter paper sheets Lens tissue Slide storage box Wire loop holder Ni-Cr wire Microscope Total cost FOB Insurance, freight, storage, and distribution (30%) Total cost, including product, insurance, freight, storage, and distribution 21 References 3. De Kantor I N, Kim S J, Frieden T, Laszlo A, Luelmo F, Norval P Y, Rieder H L, 1. Collins C H, Grange J M, Yates M D. Valenzuela P, Weyer K. Laboratory ser- Organization and practice in tuberculosis vices in tuberculosis control. WHO Global bacteriology. London: Butterworths, 1985. Tuberculosis Programme. WHO/TB/ 2. Laboratory Biosafety Manual. 2nd ed. 98.258. Geneva: WHO, 1998. Geneva: WHO, 1993: pp 60-61. 4. Manual of norms and technical proce- 3. McDougall A C. An inexpensive slide dures for tuberculosis bacteriology. Part 1 marker made from a dental bur and a Smear microscopy. Technical note 26. plastic pen. Lep Rev 1992; 63: 79-80. Washington, DC: Pan American Health 4. Smithwick R C. Laboratory manual for Organization, 1984. acid-fast microscopy. 2nd ed. US 5. Manual for Laboratory Technicians. Department of Health, Education, and Revised National Tuberculosis Control Welfare, Public Health Service. Atlanta, Programme (RNTCP). Nirman Bhavan, GA: Centers for Disease Control, New Delhi, India: Central TB Division, Bacteriology Division, 1976. Directorate General of Health Services, 5. The Microscope. A Practical Guide. WHO Ministry of Health and Family Welfare, Project: ICP TUB 001. New Delhi, India: 1997. WHO Regional Office for South-East Asia, 6. Module for Laboratory Technicians. 1999. Nirman Bhavan, New Delhi, India: Central 6. McDougall A C. The use of xylene (xylol) TB Division, Directorate General of in medical laboratories. Lep Rev 1989; 60: Health Services, Ministry of Health and 67. Family Welfare, 1997. 7. Woods G L, Ridderhof J C. Quality assur- 7. Rieder H L, Chonde T M, Myking H, ance in the mycobacteriology labora- Urbanczik R, Laszlo A, Kim S J, Van Deun tory. In: Clinics in Laboratory Medicine. A, Trébucq A. The Public Health Service Vol 16, Number 3. Philadelphia, PA: W B National Tuberculosis Reference Labor- Saunders, 1996. atory and the National Laboratory 8. Kumari S, Bathia R, Heuck C C. Quality Network. Minimum requirements, role assurance in bacteriology and immunol- and operation in a low income country. ogy. WHO Regional Publication, South- Paris: IUATLD, 1998. East Asia Series No 28. New Delhi, India: 8. Fujiki A. TB microscopy. Tokyo, Japan: WHO Regional Office for South-East Asia, The Research Institute of Tuberculosis, 1998. Japan Anti-Tuberculosis Association, Japan International Cooperation Agency, Hachioji International Training Centre, Suggested reading 1998. 1. Bacteriology of tuberculosis. The speci- 9. Tuberculosis control: a manual of meth- men. Microscopy examination. Technical ods and procedures for integrated pro- note no. 26. Washington, DC: Pan grams. Scientific Publication no. 498. American Health Organization, 1984. Washington, DC: Pan American Health 2. Minamikawa M. Laboratory Manual for Organization, 1986. the National Tuberculosis Programme of 10. Enarson D A, Rieder H L, Arnadottir T, Nepal. National Tuberculosis Centre. Trébucq A. Management of tuberculosis: JICA/HMG National TB Control Project a guide for low income countries. 5th ed. (II). March 1998. Paris: IUATLD, 2000. 22 ANNEX 1 PREVENTION OF FALSE-POSITIVE SPUTUM RESULTS · Use new slides · Use a new applicator stick for each sample · Use filtered carbolfuchsin · Keep slides separate from one another while staining · Do not use staining jars · Do not allow carbolfuchsin to dry on the slide · Do not allow oil immersion applicator to touch the smear · Do not allow oil immersion lens to touch the smear · Label sputum containers, slides and laboratory forms completely and accurately · Cross check the number on the Request for Sputum Examination Form and sputum container before recording · Record and report results accurately CONSEQUENCES OF FALSE POSITIVITY · Unnecessary treatment - wastage of drugs · Loss of confidence in the NTP PREVENTION OF FALSE-NEGATIVE SPUTUM RESULTS · Make sure sample contains sputum, not just saliva · Make sure there is at least 3 ml of sputum · Select thick, mucopurulent particles for smearing · Smears should not be too thick nor too thin · Stain smears for 5 minutes · Decolourise smears for 3 minutes · Counterstain for 1 minute · Read all 100 fields before declaring the slide to be negative · Known positive control smears should show well stained AFB · Label sputum containers, slides and laboratory forms carefully · Cross check the number on the Request for Sputum Examination Form and on the sputum container before recording · Record and report results accurately CONSEQUENCES OF FALSE-NEGATIVE RESULTS · Patient remains untreated, resulting in suffering, spread of TB and death · Intensive phase treatment may not be extended, leading to inadequate treatment · Loss of confidence in the NTP 23 ANNEX 2 CARE OF THE MICROSCOPE The microscope is the centrepiece of the TB diagnostic services of the NTP. Proper handling and maintenance of the microscope by laboratory staff is essential to pro- long its useful life. The following points should be observed: · When not in use the microscope should be stored in a dry, dust and vibration free environment · Avoid exposing the microscope to direct sunlight and moisture and humidity · Use silica gel in the microscope storage box; restore by heating when silica gel becomes pink · Clean the microscope with lens paper before and after use · Wipe the surface of immersion lens with a piece of clean cotton before and after use. Do not use alcohol for cleaning lenses · Oil immersion lens should never touch the smear · Use fine focusing knob only while using the oil immersion lens 24 ANNEX 3 TROUBLE-SHOOTING GUIDE FOR MICROSCOPY PROBLEM POSSIBLE CAUSES SOLUTION Condenser too low Raise condenser Field is dim Diaphragm closed Open diaphragm Eye piece dirty Clean eyepiece Dark shadows in the field Eyepiece or objective A new eyepiece may be that move with eye piece contaminated with fungus needed when it is rotated Surface of eyepiece A new eyepiece may be scratched needed Smear portion of slide Turn slide over upside down Air bubble in the oil Move immersion oil The image is not clear objective from side to side Poor quality oil Change oil Lens dirty Clean lens Oil on the lens Clean lens Low power image Dust on upper surface of lens Clean lens not clear Broken lens A new lens is needed 25 COMPOGRAVURE IMPRESSION, BROCHAGE I M P R I M E R I E C H I R A T 42540 ST-JUST-LA-PENDUE JUIN 2000 DÉPÔT LÉGAL 2000 N° 9482 IMPRIMÉ EN FRANCE