Role of laboratory services in tb control

3. Dr. Ashraf El Adawy
Consultant Chest Physician
TB TEAM EXPERT – WHO
Masoura -Egypt

4. International Standards 2-6, 10, 11
Microbiologic Diagnosis of Tuberculosis

5. Significance of microbiologic testing for public health goals and patient care:
WHO global target of 70% case detection of new smear-positive cases
Rapid and accurate case detection coupled with effective treatment is essential to reduce the incidence of TB
Failure to perform a proper diagnostic evaluation before initiating treatment potentially:
1.Exposes the patient to the risks of unnecessary or wrong treatment
2. May delay accurate diagnosis and proper treatment

6. 6
Diagnosis of TB

7. 7

8. 1.Detection of infectious cases
2.Monitoring of treatment progress
3.Documentation of cure

9. Robert Koch was the first
to see Mycobacterium tuberculosis with his staining technique in Mach 24,1882.

10. Sputum Smear Microscopy
Sputum smear microscopy is the most important test for the diagnosis of pulmonary TB in many areas of the world
Direct smears (unconcentrated specimen) are most common
Assessment of laboratory quality is essential

11. Sputum Microscopy: Direct Smears
Direct smears of unconcentrated sputum:
–Fast, simple, inexpensive, widely applicable
–Extremely specific for M. tuberculosis in high-incidence areas
–Ziehl-Neelsen staining (carbol fuchsin type) most common

12. Sputum Smear Microscopy
Carbolfuchsin-based stains
Utilize a regular light microscope
Must be read at a higher magnification
Two types: Ziehl-Neelsen and Kinyoun. Both use carbolfuchsin/phenol as the primary dye
Smear is then decolorized with acid (HCI) alcohol and counter-stained with methylene blue

13. Ziehl-Neelsen (ZN) Stain

14. Direct smear microscopy
The direct smear microscopy of sputum is a reliable and simple technique for detecting Mycobacteria in order to diagnose pulmonary TB.
The method consists of microscopic examination of a specimen of sputum that has been spread on a slide, and stained by the Ziehl-Neelsen method.

15. Staining Characteristics
Mycobacteria are called Acid-Fast Bacilli (AFB) due to their microscopic appearance after decolorizing.
Organisms appear red on a blue background

16. 16
AFB smear
AFB (shown in red) are tubercle bacilli

17. The aims of TB laboratory diagnostic services within the framework of an NTP are:
1.Diagnosis of cases
2.Monitoring of tuberculosis treatment
17

18. Is used to diagnose tuberculosis in persons with suspected pulmonary disease and to identify sources of infection among persons with cough attending health facilities for any reason.
Sputum microscopy is also used to monitor the progress of infectious patients during treatment, including confirmation of cure.
18

19. M. tuberculosis is a gram-positive or frequently colourless bacterium
The detection of acid-fast bacilli in stained preparations examined under the microscope constitutes the first evidence of the presence of mycobacteria in a clinical sample.
The acid-fast characteristics of the microorganism are attributable to the high lipid content of the bacterial wall
19

21. The recommended approach comprises the classic Ziehl-Neelsen technique,which reveals M. tuberculosis as small, red-coloured curved rods (bacilli) over a bluish background
This technique is simple, very economical, and reproducible in any setting.
Visualisation is carried out under x1000 magnification in immersion oil and should last for at least 10 to 15 minutes.
21

22. Direct smear microscopy based on the Ziehl-Neelsen technique. M. tuberculosis appears as small, dark-red rods over a bluish background.
22

23. is an easy and quick procedure.
A minimum of three samples must be examined. (Morning samples are more likely to contain bacilli because secretions build up over night.)
Microscopic Direct Smear Examination

24. Routine sputum collection
-Patient provides, under supervision, an “on-the-spot” sample when he presents to the health facility
-
- Patient gets a sputum container to take home for an early- morning sample the following morning
Day 1 Sample 1
-Patient brings an early morning sample
- Patient provides another “on the spot” sample under supervision
Day 2 Sample 2
Sample 3
NB. If a patient is unable to produce a sputum sample, a nurse may help him to give a good cough and bring up some sputum. This must be done in a well-ventilated area, preferably in the open air.

25. Under NTP conditions, the IUATLD recommends collecting three sputum samples for the diagnosis of tuberculosis cases.
“on the SPOT – early MORNING – on the SPOT”, preferably within two days.
25

26. 26
Three sputum smears are optimal
8193100050100FirstSecondThird Cumulative Positivity

27. Mase SR, Int J tuberc Lung Dis 2007
Average yield of single early morning specimen: 86.4% Average yield of single spot specimen: 73.9%
Specimen Number
Incremental Yield of smear specimens
(of all smear-positive)
Incremental Sensitivity of smear specimens
(compared with culture)
1
85.8%
53.8%
2
11.9%
11.1%
3
2.4%
3.1%
Total
100%
68.0%
Performance of Sputum Microscopy

28. Collect specimens in a laboratory-approved, leak- proof container
Label all containers (name and date collected)
Collect specimens prior to initiation of therapy
Infection Control: Consider the safety of other patients and healthcare workers
–Collect sputum in well-ventilated area, preferably outdoors
Specimen Collection

29. Minimize contamination of specimens by:
–Instructing the patient to rinse mouth with water before collection
–Transport the specimen to the lab as soon as feasible after collection
Keep specimens refrigerated if possible
Specimen Collection

30. 30
Specimen Collection
For pulmonary specimens can be collected by:
1.Sputum sample
2.Induced sputum sample
3.Bronchoscopy
4.Gastric washing

31. 31
Easiest and least expensive method is to have patient cough into sterile container
HCWs should coach and instruct patient
Should have at least 3 sputum specimens examined
–Collected in 2 days intervals
–At least one early morning specimen

32. Importance of sample collection and processing
The basic recommendations for the handling of samples can be summarised as follows:
1.Whenever possible, sampling should be carried out before starting chemotherapy.
2.2. Sampling is to be carried out in open areas or well-ventilated rooms, and away from other people.
32

33. Importance of sample collection and processing
3. Sputum and urine samples are to be shipped in clean, wide-lipped glass or plastic containers with airtight screw-on covers.
4. The sample container should be labelled with the patient’s initials before shipment to the laboratory.
5. If the sample is obtained by direct methods (e.g., puncture of abscesses, cerebrospinal fluid, biopsy), an aseptic technique is required, placing the collected material in a sterile container.
33

34. 6. In direct sputum smear microscopy, one sample will yield on average 85% of positive readings, while two samples will yield 95% (i.e., the second sample raises the possibility of positivity by 10%), and three samples will afford 100%.
This is why the collection (and shipment) of three samples per patient is advised.
 Sending of more than three samples would add no further benefit.
34

35. 7. Since the most viable sputum sample is that collected in the morning, it is advisable, in the case of hospitalised patients, to obtain the three samples early in morning on 3 consecutive days
However, this procedure is problematic when sampling is performed at the peripheral level, since the patient would be required to present to the health care centre on 4 consecutive days: the first day being the consultation and the next 3 days to deliver the samples.
35

36. Thus, under control programme conditions, the first sample should be obtained at the time of consultation, while providing the patient with a second container for collection of the second sample early the next morning.
When the patient returns on the next day to deliver this second sample, the final third sample is collected.
36

37. In this way, the patient is only required to report to the health care centre twice, and three samples are obtained, with one sample being an early morning sample.
A similar procedure can be carried out in the case of urine sampling.
37

38. 8. The collected samples are to be sent immediately to the laboratory. If this is not possible, the samples should be kept in a refrigerator.
If only a smear microscopy evaluation is requested, there is no problem storing the sample for 7 to 10 days before shipment.
38

39. The problem only occurs when cultures are required. In which case, sample shipment should not be delayed for more than 4 to 5 days , storage in a refrigerator will be required.
If this measure is not adopted, the sensitivity of the culture decreases considerably, since the bacteria tend to die and will be unable to grow in culture.
39

40. 9. while the diagnostic yield of saliva is very low, it is not negligible, and many studies have shown that it can contribute a percentage of positivity readings that, although low, should not be ignored
Sputum stained with blood should also not be rejected, since they yield some diagnostic information.
40

41. The health care worker should make sure that the specimen is of sufficient volume (3 to 5 ml) and that it contains solid or purulent material, the presence of which increases the sensitivity of detection, and not just saliva.
However, if only saliva is obtained or, as frequently happens in “spot” sputum, volumes of less than 3 ml are produced, the specimen should nevertheless be processed, as it is sometimes likely to yield positive results.
41

42. 10. If the patient fails to expectorate, sputum can be induced with physiological saline aerosol, with the procedur performed in open areas or well- ventilated rooms.
In children who do not expectorate, gastric lavage can be performed on 3 consecutive days. The microbiological study of these samples has been shown to be useful
42

43. Specimen Collection Container: Specifications
50 ml capacity
Translucent or clear material
Single-use combustible material
Screw-capped with a water-tight seal
Easily-labeled walls

44. Specimen Collection
Three (3) specimens optimal for identifying infectious cases of tuberculosis
Make collection convenient and efficient for both patient and laboratory worker

45. Timing of Specimen Collection
Spot–Morning–Spot
 WHO/IUATLD Recommendation
1.Spot initial visit to the clinic
2.Early morning first sputum in the morning
3.Spot second visit to the clinic

46. Collection Considerations
Yield decreases rapidly after three specimens
Morning specimens on average better
–Collect three morning specimens from hospitalised patient

47. Follow-up Specimens for Monitoring Treatment
Collected during and at end of treatment
Early morning specimen

48. Specimen Collection: Safety
The patient is a greater danger to staff than the specimen!
Instruct patient to cover the mouth when coughing
Never collect sputum in the laboratory!
–Collect OUTSIDE
–Collect away from other people
Do not stand near patient during specimen collection

49. Advantages of Open Air Collection
Rapidly dilutes aerosols
UV light rapidly inactivates the bacilli

50. Patient Education: Collection
Best specimen comes from the lung
Saliva or nasal secretions are unsatisfactory
Remove dentures and rinse mouth with water
Need for three sputum samples for optimal diagnosis

51. Labeling Specimen Container

52. Specimen Receipt at Laboratory
Check specimens for quality:
–Volume (at least 3–5 ml)
–Describe sputum consistency (mucoid, purulent, bloody, or watery)
Register the specimen and allocate a laboratory serial number

53. Specimen Quality
Purulent
Mucoid

54. Specimen Quality
Saliva or Induced sputum (?)
Blood stained

55. Obtaining adequate good quality specimens is critical to ensure accurate and reliable AFB microscopy results
55

56. When reporting the results of the microscopic examination,the microbiologist should provide the clinician with an estimation of the number of acid-fast bacilli detected.
The number of bacilli observed is preferably scored by means of the following cross-system:
1.(...) Absence of acid-fast bacilli/100 microscopic fields
2.(+) 1-9 acid-fast bacilli/100 fields. Report numerically
3.(++) 10-99 acid-fast bacilli/100 fields
4.(+++) 1-10 acid-fast bacilli per field (observation of only 50 fields required)
5.(++++) > 10 acid-fast bacilli per field (observation of only 20 fields required)
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57. WHO and IUATLD Positive and Negative Report
oNegative Report: Negative for AFB where no organisms observed in 100 oil immersion fields
oPositive Report: Positive for acid-fast bacilli; provide AFB quantification

58. WHO/IUATLD Quantification scale Ziehl Neelsen
Number of AFB
Number of fields* examined
What to report
No AFB in 100 fields
100 fields
No Acid Fast Bacilli detected
1–9 AFB in 100 fields
100 fields
Record exact figure
(1 to 9 AFB per 100 fields)
10– 99 AFB in 100 fields
100 fields
1 +
1– 10 AFB in each field
50 fields
2 +
More than 10 AFB in each field
20 fields
3 +
* Oil immersion fields

59. Result record
Number of AFB in 100 fields
Negative
No AFB observed
Scanty
(record exact number observed)
1-9 AFB
+
10-99 AFB
++
100-999 AFB (or 1-10 per field)
+++
1000 or more
(or more than 10 AFB per field)
Examine each sample through the microscope. Systemically examine 100 fields for acid-fast bacilli (AFB). If AFB are present, count them and grade the quantity according to the scale below. If any AFB are present the result is positive.

60. Quantitation scale by WHO

61. Grading of microscopy Ziehl Neelsen staining smears
Examination
Result
Grading
No. of fields to be examined
More than 10 AFB per oil immersion fields
Positive
3 +
20
1-10 AFB per oil immersion fields
Positive
2 +
50
10-99 AFB per 100 oil immersion fields
Positive
1 +
100
1-9 AFB per 100 oil immersion fields
Scanty
Record exact number seen
100
No AFB per 100 oil immersion fields
Negative
0
100

62. Sputum smear microscopy for tubercle bacilli is positive when there are at least 10,000 organisms present per 1 ml of sputum.
Sputum smear microscopy requires 10,000 to 1,00,000 organisms/ml and acid fast bacilli (AFB) could be any pathogenic or saprophytic mycobacteria.
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63. “How reliable is smear microscopy?”
The amount of sputum on a slide for smear preparation is about 0.01 ml.
This is spread over an area of 200mm2 (10 x20mm).
Since the area of an oil-immersion field seen in the microscope is about 0.02mm2, 10 000 such fields would need to be screened
63

64. Thus, if a sputum specimen contains about 5000 bacilli per ml, the entire smear (if prepared as described) will contain about 50 bacilli.
 If these 50 bacilli were evenly distributed over the 10 000 fields of the smear, there would be one bacillus in 200 fields.
If 100 fields were examined the chance of finding this bacillus would be 50%.
64

65. Furthermore, to find one acid-fast bacillus in every 10 fields (or 10 in 100 fields) would require 1000 such bacilli to be present in the smear (10 000 fields) or 100 000 (105) per ml of sputum .
To find one acid-fast bacillus per field on the average would require 106 bacilli per ml of sputum Thus, a specimen that is consistently found to be positive would have to contain at least 100 000 AFB per ml.
65

66. These estimates are based on the assumption that the bacilli are evenly dispersed throughout the specimen, i.e. that each portion of material taken from the specimen will contain the same number of AFB spread evenly over the entire smear.
66

67. 67

68. it is known that bacilli are not evenly dispersed in a specimen, but are frequently found in clumps. Thus, when several samples are taken from a sputum specimen, the number of bacilli will vary from one sample to another.
68

69. Below a certain concentration of bacilli in a sputum specimen, the probability that AFB will be transferred from the specimen to the smear and found by microscopy approaches zero.
Although it has been estimated that, with optimal laboratory conditions, a positive smear can be obtained with only 100–1000 organisms per ml a more practical estimate is about 10 000 organisms per ml.
69

70. Smear microscopy is the technique of choice for the diagnosis of TB in all settings :
1) simplicity and reproducibility in any setting
2) speed
3) low cost
4) high specificity
5) the ability to delimit contagiousness
70

71. The major limitation of smear microscopy is its relatively low sensitivity , indeed, the great majority of cases are detected at fairly advanced stages of the disease.
The non-visualisation of acid-fast bacilli in a clinical sample does not rule out the diagnosis of TB, since the lowest detectable concentration of bacilli is 10,000/ml of sample.
 Thus, the technique only serves to detect very advanced and contagious cases of TB..
71

72. For instance, if a sputum sample only contains 5000 acid-fast bacilli/ml—still a high figure— and 0.01 ml is extended on the slide, the latter will contain only 50 bacteria, i.e., a single bacterium per 200 microscopic fields
If the technician examines 100 fields, the probability of seeing a bacterium is only 50%.
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73. 73

74. The sensitivity of smear microscopy is relatively limited. This implies that a negative result does not exclude the disease, since many false negative results may occur.
74

75. This possibility of yielding false-negative results (sensitivity) can be influenced by three important factors.
The first pertains to the stage of the disease. In this sense, sensitivity is high (80-90%) in a patient with TB who has a cavitary pattern on chest radiograph, but decreases in those who present with only TB infiltrates (50-80%), decreasing particularly in patients with nodular forms or masses (under 50%).
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76. The bacterial populations calculated for each of the different types of TB lesions.
76

77. The concentration of bacilli in the sputum is determined largely by the type of tuberculous lesion from which the bacilli originate.
Thus, a cavity about 2 cm in diameter (opening into a bronchus) may contain some 100 million tubercle bacilli, whereas a non-cavitated nodular lesion of the same size may contain only 100–1000 bacilli .
77

78. The second factor pertains to sample quality and performance of the technique.
it is essential to obtain the best samples possible, and many studies have shown that the highest sensitivities are achieved with purulent sputum, followed by mucopurulent and mucous samples, and saliva.
78

79. The last factor involves the time spent by the technician or microbiologist in examining the sample under the microscope.
It is known that 100 fields of the slide correspond to 1% of the smear, 200 fields to 2%, and 300 fields to 3%.
79

80. The examination of 300 fields, which is the number needed to ensure a negative result with a high degree of certainty, would take 15 to 20 minutes.
Persons studying the samples, however, often spend less time in examination.
Therefore, not spending enough time to examine the sample can lead to a false-negative result.
80

81. In turn, falsenegative results imply that contagious cases remain unidentified and are left untreated in the community.
81

82. False positive results of sputum smear microscopy
A false positive result means that the sputum smear result is positive even though the patient dose not really have sputum smear-positive PTB.
●This may arise because of the following:
1.Red stain retained by scratches on the slide;
2.Contamination of the slide.
3.Various particles that are acid-fast (e.g. food particle, dye precipitates, other micro- organisms).

83. The acid-fast staining characteristics with smear microscopy are common to all species belonging to the genus Mycobacterium, as well as to some fungal species.
As a result, the rest of the environmental mycobacteria appear the same under the microscope.
83

84. Some fungi, Nocardia species, or even food particles, dirt, or scratches on the slide can mislead the inexperienced observer. This may slightly reduce the specificity of the technique.
84

85. 85 False positive results
1. Food particles
2. Precipitated stains
3. Saprophytic AFB
4. Spores of B.subtlis
5. Fibres and pollen
6. Scratches on slide
7. Contamination through carry over of AFB from one smear to another

86. 86 Consequences of false positive results
● Patients are started on treatment unnecessarily
● Treatment is continued longer than necessary, in follow-up examinations
● Medications will be wasted
● Patients lose confidence in the programme

87. Causes of false negative results of sputum smear microscopy
Example
Type of problem
Patients provides inadequate sample
Sputum stored too long before smear microscopy.
Sputum collection
Faulty smear preparation and staining
Sputum processing
Inadequate time spent examining slide
Inadequate attention during examination
Sputum smear examination
Incorrect labeling of sample mistakes in documentation.
Administrative errors

88. 88 False negative results
 Inadequate sputum collection
avoid – saliva, nasal discharge
collect – bronchial sputum from depth of chest
 Inadequate storage of sputum / stained smears
exposure to direct sunlight
radiation ( UV light )
excessive heat / humidity
 Not taking mucopurulent portion of sputum
 Inadequate smear preparation
 Inadequate smear examination
 Administrative & recording errors

89. 89
Consequences of false negative smear results
Patients with TB will not be treated, resulting in
suffering, spread of TB and death
Intensive phase of treatment will not be extended for the required duration, resulting in inadequate treatment
Patient may lose confidence in the programme

90. AFB smear-positive patients are usually sick and seek treatment.
AFB smear-positive patients are much more likely to die if untreated.
Untreated, an AFB smear-positive patient may infect 10–15 persons/year.
Pulmonary Positive Patients

91. Advantages of AFB Smear Microscopy
Microscopy is a simple convenient test
 Requires minimal infrastructure and equipment
 Highly accurate, inexpensive and fast
 Accessible to the majority of patients
 Prioritizes infectious cases

92. 92 Advantages of sputum microscopy
1.More reliable than x-ray for the diagnosis of infectious TB
2.Simple to perform
3.Easy to read
4.Minimal infrastructure required
5.Inexpensive
6.Quick
7.Only tool to monitor and declare patients as “cured’’

93. Limitations of Microscopy
Can not distinguish between dead or live bacteria
High bacterial load >3000–5000 AFB /mL is required for detection
Can not do species identification
Can not perform DST

94. 94
Induced Sputum Collection
Induced sputum collection should be used if patient cannot cough up sputum on their own
Patient inhales saline mist, causing deep coughing
Specimen often clear and watery, should be labeled “induced specimen”

95. 95 Bronchoscopy
Bronchoscopy may be used:
1.If patient cannot cough up enough sputum
2.If an induced sputum cannot be obtained

96. 96 Gastric Washing
Usually only used if sample cannot be obtained from other procedures
Often used with children
Tube is inserted through nose and into stomach to obtain gastric secretions that may contain sputum

97. Chest radiography is useful for differential diagnosis of pulmonary disease among patients with negative sputum smears.
97

98. Compared radiographic severity and extent of culture-positive disease with microscopy results in concentrated sputa
98

99. 99

100. The IUATLD recommends:
The examination of three sputum specimens
“SPOT” + “MORNING” +“SPOT” – for the diagnosis of tuberculosis cases.
– The examination of single “MORNING” sputum specimens on three occasions for follow-up of treatment:
─one at the end of the intensive phase, one during the continuation phase, and one at the end of treatment.
100

101. A case of sputum smear positive tuberculosis is usually defined as a person presenting with respiratory symptoms with at least two positive sputum smear microscopy examinations.
This approach, also known as passive case finding, detects about 80% of TB suspects ultimately positive on sputum smear examination with the first specimen, an additional 15% with the second and a final 5% with the third.
101

102. Morning specimens” for follow-up
Regardless of the treatment regimen, one “MORNING” sputum specimen is collected for follow-up at the end of the intensive phase of treatment to determine whether the patient can proceed to the continuation phase if the smear is negative OR,
102

103. Morning specimens” for follow-up
if the smear is positive, continue the intensive phase.
Another sputum specimen must be taken during the continuation phase to check patient evolution and to detect possible treatment failure
Another sputum specimen must be taken upon completion of chemotherapy to verify cure.
103

104. The cure rate is the proportion of initially sputum smear-positive patients who are declared cured based on negative sputum smear results on at least two occasions, including one at the end of treatment.
 The objective of the NTP is to achieve at least 85% cure rate among new sputum smear positive TB cases
104

105. The patient is said to have completed treatment even if sputum specimens are not examined during and at the end of treatment.
105

106. Microscopic examination of sputum smears during and at the end of treatment
Sputum smear microscopy has a fundamental role in monitoring the response to treatment of infectious cases of pulmonary tuberculosis.
Smear examination should be performed at the end of the initial phase of treatment; if smears are still positive, the intensive phase should be extended for an additional month.
Smears should be examined during and at the end of the continuation phase to confirm cure.
106

107. ISTC Standard 10: Sputum Microscopy
 Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months).
If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed.

108. Sputum monitoring, new patients
108
Recommendation (Strong): if specimen obtained at end of intensive phase is smear +, repeat at end of third month. If still positive, obtain culture and DST
Failure: + bacteriology at 5th month or later, or MDR detected any time

109. Sputum monitoring, previously treated patients on first line drugs
109
Recommendation (Strong): if specimen obtained at end of intensive phase is sm +, obtain culture, DST

110. With short-course treatment regimens of high efficacy, smears can be positive at 2–3 months because of dead bacilli in patients with negative cultures.
Thus, treatment failure based on positive smear examination is not considered until the fifth month
 Negative smears during and at the end of treatment are required to declare a patient cured of tuberculosis.
110

111. Culture is not a priority test for systematic detection of cases.
Persons who are positive only on culture are less infectious than those who are also positive to microscopy.
Furthermore, culture is more expensive and complex than microscopy, and there is a relatively long delay until the result is available.
111

112. The last 15 to 20 years have seen a great increase in research on the diagnosis of tuberculosis (TB), with the introduction of numerous new techniques.
However, practically none of these techniques are indicated for the routine diagnosis of TB in countries with low- or middle-income levels.
112

113. Despite the advantages afforded by some of these novel techniques, they have not been able to replace smear microscopy and culture in their respective indications.
Further, most of these techniques are very expensive and complicated to perform.
Smear microscopy remains the cornerstone for case finding in low income countries
113

114. Radiometric Technology (BACTEC)
Radiometric method to detect early growth of mycobacteria in culture
BACTEC system, which employs a superscript 14 C-labeled substrate medium that is almost specific for mycobacteria.
Since its introduction, the BACTEC method has provided more rapid growth (average, 9 days), specific identification of M. tuberculosis (5 days), and rapid drug susceptibility testing (6 days).
Although radiometric technology cannot replace completely the classic myco bacteriologic methods, and may underestimate drug resistance, this is a valuable new tool.

115. Culture
Culture of mycobacteria is a much more sensitive test than smear examination and has been estimated to detect 10-100 viable mycobacteria per ml of sample and in case of active disease they are found to be 80% sensitive and 99% specific.
115

116. Culture of sputum is more sensitive than smear examination, but it takes 4 to 8 weeks before the result is known.
It also requires well-equipped laboratories with skilled staff.
Culture allows the study of anti-TB drug resistance. Culture

117. Diagnosis of Active TB
Sputum smear showing acid-fast bacilli (AFB) of Mycobacterium tuberculosis
Mycobacterial culture grown on egg based media called Lowenstein-Jensen (LJ) medium, generally grown on a solid slope as shown here

118. Although sputum microscopy is the first bacteriologic diagnostic test of choice, both culture and drug susceptibility testing (DST) can offer significant advantages in the diagnosis and management of TB.
Culture and Drug Susceptibility Testing

119. Cultures
To confirm the diagnosis even in smear negative.
To detect drug susceptibility and resistance.
To detect the bacilli in any specimen in extra- pulmonary tuberculosis.
Culture revealing this organism’s colonial morphology.

120. Löwenstein-Jensen solid culture medium showing the growth of colonies (rough, breadcrumb appearance) of M. tuberculosis.
120

121. Culture is not a priority test for systematic detection of cases.
Persons who are positive only on culture are less infectious than those who are also positive to microscopy.
Furthermore, culture is more expensive and complex than microscopy, and there is a relatively long delay until the result is available.
121

122. Traditional culture has always been made in solid medium, using coagulated egg (e.g., Lِ wenstein- Jensen, Coletsos) or agar (Middlebrook 7H10 and 7H11) as a base.
These should be the only media indicated for routine use in countries with low- or middle-income levels, with preference going to Lِ wenstein-Jensen medium
122

123. Role of mycobacterial culture in the diagnosis of tuberculosis
The probability of finding acid-fast bacilli (AFB) in sputum specimens by smear microscopy is directly related to the concentration of bacilli in the sputum
At concentrations below 1000 organisms per ml, the chance of observing bacilli in a smear becomes less than 10%.
In comparison, mycobacterial culture can detect far lower numbers of AFB, the detection limit being around 100 organisms per ml.
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124. Smear microscopy cannot reliably differentiate between the various pathogenic and non- pathogenic mycobacteria, which are all acidfast and morphologically alike.
 It therefore seems that, for the diagnosis of tuberculosis,both the sensitivity and the specificity of culture methods are far better than those of smear microscopy.
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125. Culture: Advantages
1.Higher sensitivity than smear microscopy (culture can make diagnosis despite fewer bacilli in specimen)
2.If TB suspected and sputum smears are negative, culture may provide diagnosis
3.Allows for identification of mycobacterial species
4.Allows for drug susceptibility testing

126. Culture: Disadvantages
1.Cost
2.Technical complexity
3.May take weeks to get results
4.Requires ongoing quality assurance
Therefore, more likely to be found in major referral centers.
Avoid delaying appropriate TB treatment in suspicious cases while awaiting results.

127. Limitations of Culture
1.Greater need for infrastructure, qualified staff, equipment, and additional safety measures
2.Increased time: weeks for result
3.More sensitive to technical deficiencies
4.Expensive

128. Culture: Solid Media
Solid media have the advantage that organisms (colonies) can be seen on the surface of the medium
Types most commonly used are:
Lowenstein-Jensen: egg-based
Middlebrook 7H 10 or 7H11: agar -based
Ogawa

129. MGIT Incubator
Culture: Liquid Media
 More sophisticated equipment
 Faster detection of growth
 Higher sensitivity than solid
media
 Can also be used for drug-susceptibility
testing
 Two examples:
BACTEC
MGIT
MGIT
BACTEC

130. Culture methods using solid media is the reference standard. It is less technologically intensive and media can be made locally.
The use of either liquid technique has advantages over solid media, can reduce time to culture results to 2-4 weeks, compared with 4-8 weeks for solid media.
Decisions to provide culture facilities for diagnosing TB and the methods to be used, clearly depend on financial resources, trained personnel, and the ready availability of reagents and equipment service.
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131. ISTC TB Training Modules 2009
Drug susceptibility testing should be performed at the start of therapy for all previously treated patients
Patients who remain sputum smear-positive at completion of 3 months of treatment and patients who have failed, defaulted from, or relapsed following one or more courses of treatment should always be assessed for drug resistance
Standard 11: Drug Susceptibility

132. What is the probability of obtaining a negative culture from a sputum specimen found positive by smear microscopy?
A negative culture result with a specimen containing tubercle bacilli may be due to various causes.
In patients receiving treatment, the organisms may have lost their ability to grow on culture media and be practically dead.
Patients being treated with a rifampicin-containing regimen often become culture-negative by about the third week of treatment, although they may still be sputum smear-positive: bacilli are dead or non- viable.
132

133. Sometimes the culture becomes negative before smear microscopy because the treatment provided to the patient makes the bacilli non-viable.
The mycobacteria continue to be eliminated by the host and continue to exhibit acid-fast staining characteristics.
This situation gives rise to falsepositive results owing to the existence of “non-viable bacilli”.
 Despite these results, such patients have very little potential to infect, and their course is favourable.
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134. What is the probability of obtaining a negative culture from a sputum specimen found positive by smear microscopy?
In patients who have not had treatment, sputum specimens may have been exposed to sunlight or heat, stored too long, dried out, or contaminated.
Excessive decontamination procedures before inoculation, over-heating during centrifugation, inadequate culture media, and deficient incubation may also result in a negative culture.
In a few instances, positive smears may be caused by non-tuberculous mycobacteria.
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135. Mycobacterial culture
Mycobacterial culture is the only means of ensuring a definite diagnosis of TB and the only acceptable method available for assessing patient follow-up and confirming cure.
For this reason, in countries with sufficient economic resources, all clinical samples suspected of containing mycobacteria should be grown in adequate culture media. However, there are limitations that reduce the use of culture in low and middle-income countries.
135

136. Culture offers several advantages that define it as the gold standard for the diagnosis and follow-up of TB cases
1.Cultures are much more sensitive than smear microscopy, and are able to detect as few as 10 bacteria per millilitre of sample.
2.Isolation in pure culture is necessary to correctly identify the isolated strains, since other mycobacteria appear identical to M. tuberculosis by smear microscopy.
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137. However, the logistical problems posed by culture limit its use, particularly in poorer countries. The main inconveniences of culture can be summarised as follows:
1. The main limitation of conventional culture is related to the slow divisional capacity of M. tuberculosis. This causes the time elapsed from sample receipt to reporting of the result to be no less than 4 to 6 weeks in conventional solid media, This is too long a wait for establishing a firm diagnosis.
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138. 2. The cost of culture is far greater than that of smear microscopy, and specific media are needed, with subsequent storage in an oven. Moreover, more specific training of personnel is required to perform cultures.
In view of the above considerations, it is not possible to use culture at the most peripheral levels of health care, unlike with smear microscopy.
Thus, when TB is clinically suspected and smear microscopy proves positive in this setting, treatment should be started and the patient registered as a TB case.
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139. in poor countries, where the main challenge continues to be access to smear microscopy evaluation for all symptomatic respiratory cases, culture is only indicated in special situations.
Priority in these poorer countries must be given to smear microscopy and treatment.
Culture would be reserved for cases of suspected resistance
139

140. Culture (not smear microscopy) is the only acceptable method for following up on a TB patient and for ensuring that cure has been effective.
However, under TB control programme conditions, the logistical problems posed by cultur examination make it necessary to resort to smear microscopy for patient follow-up, despite the fact that microscopy may not indicate the true course of the disease (e.g. smear-positive with negative culture results).
140

141. Culture would almost never be included in the diagnostic algorithm of patients initially presenting with negative smear microscopy results.
141

142. Culture and drug-sensitivity testing should be obtained, when feasible, for smear- negative TB and treatment failure.

143. Laboratory is the key Component in TB Control
NO LABS
NO DIAGNOSIS
NO TREATMENT
NO DOTS
NO TB CONTROL

144. 144
Conducted when patient is first found to have positive culture for TB
Determines which drugs kill tubercle bacilli
Tubercle bacilli killed by a particular drug are susceptible to that drug
Tubercle bacilli that grow in presence of a particular drug are resistant to that drug
Drug Susceptibility Testing

145. 145
Drug Susceptibility Testing
should be done if:
–Patient has positive culture after 3 months of treatment; or
–Patient does not get better
Drug susceptibility testing on solid media

146. Types of Drug-Resistant TB
Mono-resistant
Resistant to any one TB treatment drug
Poly-resistant
Resistant to at least any two TB drugs (but not both isoniazid and rifampin)
Multidrug- resistant
(MDR TB)
Resistant to at least isoniazid and rifampin, the two best first-line TB treatment drugs
Extensively drug-resistant
(XDR TB)
Resistant to isoniazid and rifampin, PLUS resistant to any fluoroquinolone AND at least 1 of the 3 injectable second-line drugs (e.g., amikacin, kanamycin, or capreomycin)

148. Bacteriologic Examination

149. Standard 1 (suspect case)
●All persons with unexplained cough lasting two–three weeks or more should be evaluated for tuberculosis.

150. Standard 2: Sputum Microscopy
All patients suspected of having pulmonary TB who can produce sputum should have at least two , and preferably three, sputum specimens obtained for microscopic examination.
When possible, at least one early morning specimen should be obtained.

151. Standard 3: Extrapulmonary Specimens
 For all patients suspected of having extrapulmonary TB, appropriate specimens from the suspected sites of involvement should be obtained for microscopy, culture and histopathological examination.

152. Pulmonary, 70%
Extrapulmonary, 21%
Both, 9%
Pleural, 18%
Lymphatic, 42%
Bone/joint, 11%
Genitourinary, 5%
Meningeal, 6%
Other, 12%
TB Cases by Form of Disease, United States, CDC, 2005
Peritoneal, 6%
Clinical Presentation: Extrapulmonary
Incidence/site may vary  TB can involve any organ
More common in HIV/TB (co-infection)

153. Standard 4: Evaluation of Abnormal CXR
All persons with chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination.

154. Evaluation of Abnormal CXR
Study from India:
2229 outpatients evaluated by CXR/culture
Of 227 cases deemed TB by CXR alone
–36% had negative sputum cultures for TB
Of 162 culture-positive cases of TB
–20% would have been missed based on CXR alone CXR alone is not enough!

155. Over-reliance on CXR without the use of sputum microscopy is a common practice in some areas
Data from a study done in a high-incidence country demonstrates just how misleading reliance on the CXR alone can be
Overall, radiographic examination for the evaluation of TB is most useful when applied as part of a systematic approach -- particularly, in the evaluation of persons whose symptoms and/or findings suggest TB, but who have negative sputum smears
155
Chest X-ray alone cannot confirm TB disease

156. Sputum Smear-Negative Patient
Criteria for diagnosis:
At least 3 negative sputum smears
Chest X-ray consistent with TB
Lack of response to broad-spectrum (non fluoroquinolone) antibiotic
Cultures must be attempted

157. Standard 5: Smear-negative Diagnosis
The diagnosis of sputum smear-negative PTB should be based on the following criteria:
At least three negative sputum smears (including at least one early morning specimen)
Chest radiography findings consistent with TB
Lack of response to a trial of broad-spectrum anti- microbial agents (avoid use of fluoroquinolone)
For such patients, if facilities for culture are available, sputum cultures should be obtained.
In persons with known or suspected HIV infection, the diagnostic evaluation should be expedited.

158. Standard 6
In all children suspected of having intrathoracic and extrapulmonary TB, specimens (sputum, extrapulmonary tissue) should be obtained for microscopy, culture, and histopathological (tissue) examination.
TB diagnosis should be based on chest radiography, history of TB exposure, positive TB test, and suggestive clinical findings if bacteriologic studies are negative.
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159. ISTC Standard 10: Sputum Microscopy
 Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months).
If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed.

160. ISTC TB Training Modules 2009
DST Drug susceptibility testing should be performed at the start of therapy for all previously treated patients
Patients who remain sputum smear-positive at completion of 3 months of treatment and patients who have failed, defaulted from, or relapsed following one or more courses of treatment should always be assessed for drug resistance
Standard 11: Drug Susceptibility

161. 161
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