DIAGNOSIS OF = TUBERCULOSIS

Newer=20 Diagnostic Techniques in Laboratory Diagnosis of Childhood=20 Tuberculosis

Sanjiv Nanda, Ashish Marwah, = Department=20 of Pediatrics,Postgraduate Institute of Medical Sciences,=20 Rohtak

A=20 Complete diagnostic work up of tuberculosis includes diagnosis of both = the=20 latent tubercular infection (LTBI) and active disease. Complete = diagnostic work=20 up includes:-

Clinical=20 features

Skin=20 testing

Radiological=20 diagnosis

Laboratory=20 examination

The=20 newer diagnostic techniques in the laboratory diagnosis of childhood=20 tuberculosis are as follows:-

I) culture

=20 =93Culture is the gold standard for definitive diagnosis of=20 tuberculosis=94.

Its=20 advantages are that it is more sensitive than microscopy, being able to = detect=20 as few as 10-100 bacteria/ml of digested, concentrated material. Growth = of the=20 organisms is necessary for precise species identification and drug = sensitivity=20 testing.

Various=20 methods are

1. =20 Conventional egg based solid medium such as Lowenstein Jenson=20 (LJ).

2. =20 Agar bases ones, i.e. middle brook 7H10 or = 7H11.

3. =20 Liquid media such as Kirschner=92s or middle brook 7H9/7H12 = broth. Its=20 modifications are: - BACTEC 460 TB system, MGIT 960 system and MB/Bac T=20 system.

Lowenstein=20 Jenson Medium

=20 Lowenstein Jenson is a selective and enriched egg based solid = medium. The=20 major constraint of this media is slow growth, taking about 8-12 weeks = as=20 incubation period. The drug susceptibility takes another 4=20 weeks.

Radiometric=20 Bactec 460 TB Method

The=20 bactec 460 TB system is a radiometric wherein 14C labeled palmitic acid = in 7H12=20 medium is used. Here, when the 14C labeled substrate present in the = medium is=20 metabolized, 14CO₂ is produced and measured by BACTEC system=20 instrument and reported as =93Growth Index (GI)=94=20 value.

Its=20 main advantages are:-

1. =20 Culture positive yield of bactec 12B is better than LJ (Table=20 2)

2. =20 Early detection of positive mycobacteria in cultures (Table=20 1)

3. =20 Differentiates typical and atypical mycobacteria within 5-6=20 days.

4. =20 Rapid and more standardized availability of drug resistance = report in 6=20 yo 7 days.

=20 Its main disadvantages are high cost and more medical = technologist=92s=20 time.

TABLE=20 1

Typical=20 Mean Time (Days) For Detection Of Mycobacterium = growth

Conventional=20 culture methods

BACTEC

Others

Smear=20 positive TB

20-25=20 days

9-13=20 days

9-20=20 days

Smear=20 negative TB

21-33=20 days

15-22=20 days

18-36=20 days

Non-tuberculous=20 mycobacterial infections

22-40=20 days

13-17=20 days

12-25=20 days

TABLE=20 2

Typical=20 Recovery Rates In Mycobacterium Infections Using Conventional Culture, = The=20 BACTEC 460 Tb & Other Liquid Based Culture Detection=20 Systems

Conventional=20 culture methods

BACTEC

460=20 TB

Others

Methods

Tuberculous=20 mycobacteria

79-95%

90-100%

77-97%

Non-tuberculous=20 mycobacteria

33-60%

76-85%

58-80%

Latest=20 Culture Methods

a)=20 BACTEC systems

This=20 assay system designed by Becton Dickinson, USA based on generation of=20 radioactive CO₂ from palmitic acid is well known and is = described=20 above.

b)=20 MGIT (mycobacterium growth indicator tube)

It=20 is a non radiometric automated system for growth and detection of = mycobaceria=20 with a capacity to incubate and continuously monitor 960 mycobacteria = growth=20 indicator tubes every 60 minutes for increase in=20 fluorescence.

c)=20 MB/ BACT:-

This=20 system (organon techaika) is adapted from strategy of colorimetric = detection of=20 CO₂. It is a continuous monitoring computerized database = management=20 system useful for drug susceptibility testing.

d)=20 Septichek

It=20 is a biphasic medium system consisting of an enriched selective growth = and a=20 slide with non selective middle brook agar on one side and two sections = on other=20 side =96 one with NAP and egg containing agar, second with chocolate=20 agar.

e)=20 Fast plaque for TB (reporter phages)

Mycobacterial=20 specific phages and reporter genes like luciferase have been = successfully used=20 for detection of growth and for assessing drug susceptibility testing.=20 Indication of viability is either emission of light from organism = because of=20 luciferase enzyme or production of a plaque as an indicator strain of=20 mycobacteria.

Advantages

-=20 Results in 48 hours.

-=20 Only detects live bacteria

II)=20 Advanced Skin Testing

A)=20 Quantiferon and ELISPOT assays

a)=20 In 2001, a new test, quantiferon TB or QFT, that measures the release of = IFN-g=20 in whole blood in response to stimulation by PPD was approved by US-FDA. = It is=20 an invitro test that measures a component of cell mediated immune=20 reactivity.

It=20 is based on quantification of IFN-g=20 released from sensitized lymphocytes in whole blood incubated overnight = with PPD=20 from M tuberculosis and control antigens. In this method whole blood = specimen of=20 donors in stimulated with ESAT-6 and CEP-10 antigen and then cultured. = Plasma=20 concentration of IFN-g=20 discharged is determined by quantiferon =96 CMI.

The=20 optimum cut off level determined by Harada et al was 0.35m/ml=20 for both ESAT-6 and CEP-10. This gave the test a sensitivity of 89% and=20 specificity of 98.1% in diagnosing TB infection.

Advantages=20 of QFT over Mantoux test:-=20

1. =20 Less affected by previous BCG vaccination

2. =20 Comparable efficacy with mantoux test in detecting TB=20 infection.

3. =20 Discriminated responses due to non tuberculous=20 mycobacteria.

A=20 sensitive enzyme linked immuno spot (ELISPOT) assay to detect T cells = specific=20 for mycobacterium tuberculosis antigen that are absent from M. bovis, = BCG and=20 other environmental mycobacteria has been developed recently. Also, = Elispot=20 correlates significantly more closely with M. tuberculosis exposure than = mantoux=20 test and is not affected by previous BCG = vaccination.

The=20 preliminary data that have come up show that ELISPOT offers a more = accurate=20 approach to diagnose children with latent tubercular infection than TST = (Mantoux=20 test).

B)=20 MPB-64 Patch Test

It=20 is a skin patch for diagnosing active tuberculosis. It provides an = approach to=20 distinguish active tuberculosis from PPD positive healthy controls. The = patch=20 when applied to skin delivers a soluble TB protein (MPB-64) directly to = skin.=20 Results are read after 72 hours.

The=20 test has a sensitivity of 88-98% and specificity = 100%.

C)=20 serology

Despite=20 dozens of studies published over the past several decades serology has = found=20 little place in routine diagnosis of TB in = children.

Problems=20 faced by serological testing in TB diagnosis

1. =20 Children tend to have low antibody titers making distinction from = natural=20 exposure antibodies more difficult.

2. =20 Antigenic cross reactions shared by M. tuberculosis with other=20 mycobacteria (e.g. NTM) in environment.

3. =20 Other reasons are background titers due to presence of anti M.=20 tuberculosis antibodies in persons with latent M. tuberculosis = infection, prior=20 active TB, BCG vaccination.

Several=20 recent studies, however, have used ELISA to detect antibodies to various = purified or complex antigens of M. tuberculosis in children. In most of = these=20 studies, IgG holds the great promise in diagnosing active disease in = children as=20 well as adults. However, sensitivity (29~75%) and specificity (50~92%) = are low=20 and not useful for confirmation of childhood Tb.

D)=20 Molecular Techniques (Nucleic Acid Amplification Technologies)=20

During=20 the last decade, several nucleic acid amplification technologies (NAAT) = have=20 been developed for direct detection and identification of mycobacterium=20 tuberculosis in clinical specimens. These methods have been able to = potentially=20 reduce the diagnostic time from weeks to days and are acquiring a = greater=20 relevance in field of laboratory TB diagnosis in modern=20 era.

a)=20 Polymerase Chain Reaction (PCR)

PCR=20 is the most commonly used technique of nucleic acid amplification used = for the=20 diagnosis of tuberculosis. It allows sequences of DNA present in only a = few=20 copies of mycobateria to be amplified in vitro such that the amount of = amplified=20 DNA can be visualized and identified. A number of target genes of = mycobacterial=20 DNA have been evaluated for diagnosis by PCR and various other genotypic = methods. The most common target gene used in PCR is IS6110. PCR (Roche = amplicor)=20 is approved by US-FDA for diagnosis of pulmonary tuberculosis on smear = positive=20 as well as smear negative specimens. These tests have not yet been = approved for=20 non pulmonary specimens since there is a dearth of reproducible data on = non=20 pulmonary specimens, possibly because of non specific inhibition of = polymerase=20 enzyme and to paucibacillary nature of these specimens. Modification of = PCR=20 using mRNA instead of DNA as target for amplification has also been = developed.=20 If appropriate sequence specific for M. tuberculosis are selected, = 10-100=20 organisms can be readily identified. PCR protocols for detection of = Indian=20 strains of microorganisms are available. The sensitivity of PCR for = detecting=20 respiratory TB is equivalent to 90% with a specificity of=20 95-99%.

b)=20 Real time PCR

The=20 basic principle is similar to that of PCR but in real time PCR system = the=20 detection of amplified product is based on detection and quantitation of = a=20 fluorescent reporter. Real time PCR is still a research tool and at = present is=20 not US-FDA approved for clinical diagnosis of=20 tuberculosis.

c)=20 Transcription mediated amplification (TMA) and nucleic acid sequence = based=20 amplification (NASBA) [Gen-Probe, USA]

TMA=20 also called mycobacterium tuberculosis direct (MTD) tests and NASBA are = based on=20 the isothermal amplification of rRNA. In this technique chemical, rather = than=20 biological amplification is used to produce nucleic acid, so that within = few=20 hours these tests distinguish between M. tuberculosis and non tubercular = mycobacteria. MTD is US-FDA approved for use in testing respiratory = samples=20 regardless of smear results.

The=20 sensitivity and specificity of this test is claimed to be 78-86% and = 90-100%=20 respectively.

d)=20 The Ligase Chain Reaction (LCx, Abbott = laboratories)

LCR=20 is a variant of PCR in which a pair of ologonucleolides is made to bind = to one=20 of the DNA target strands, so that they are adjacent to each other. A = second=20 pair of oligonucleolides is designed to hybridize to the same regions on = the=20 complementary DNA. The action of DNA polymerase and ligase in the = presence of=20 nucleotides results in the gap between adjacent primers being filled = with the=20 appropriate nucleotides and ligation of the primers. The LCR is = currently not=20 US-FDA approved and also no protocols developed for Indian strains are=20 available.

e)=20 Standard Displacement Amplification=20 (SDA)

It=20 is an isothermal invitro nucleic acid amplification technique, which is = based on=20 the ability of HincII to nick the unmodified strand of = hemiphosphorothiote form=20 of its recognition site and ability of exonuclease deficient klenow to = extend=20 3=92end at nick and displace the downstream DNA strand. Exponential = amplification=20 results from coupling sense and antisense reaction. Though additional = studies=20 are needed to evaluate this system, sensitivity and specificity of 87 to = 100%=20 and 99 to 100% respectively have been reported in the initial = studies.=20

It=20 uses PCR followed by reverse hybridization to identify the most = clinically=20 significant mycobacteria. The system proved reliable in routine = evaluations with=20 100% sensitivity from cultures and greater than 99 percent specificity. = It has=20 an additional benefit of identifying rifampicin=20 resistance.

Advantages=20 of Using NAAT for diagnosis of pulmonary = tuberculosis

1. =20 NAAT is as fast as and far more sensitive than smear microscopy = (results=20 in 24 hours) and by far faster than cultures.

2. =20 In smear positive TB suspects, NAAT closely matches sensitivity = and=20 specificity of cultures.

3. =20 NAAT has also been used for rapid diagnosis of MDR tuberculosis = directly=20 from specimen.

Role=20 of NAAT In Extrapulmonary Tuberculosis

There=20 is clearly a role of NAA assays in diagnosis of extrapulmonary=20 tuberculosis.

a)=20 Neurotuberculosis

PCR=20 has been extensively tried for the detection of M. tuberculosis specific = sequences in CSF as well as biopsy specimens of TBM and tuberculosis of = brain.=20 Sensitivity and specificity using different target sequences in=20 neurotuberculosis is as under:-

SPECIMEN

SENSITIVITY=20 (%)

SPECIFICITY=20 (%)

PCR=20 IS 6110

CSF

PCR=20 =96 TRC =96 4

CSF

PCR=20 =96 MPB 64

CSF

100

b)=20 Pleural effusion

Diagnosis=20 of pleural involvement in tuberculosis is made by appropriate pleural = biopsy and=20 microbiological examination of pleural fluid. It is time consuming and = direct=20 detection of M. tuberculosis is rare. PCR can help in confirming = diagnosis in=20 60% of specimens that are negative by mycobacterial culture. PCR has = also found=20 utility in diagnosis of skin, ocular, bone, genital and lymph node=20 tuberculosis.

Problems=20 Concerning Nucleic Acid Amplification Techniques:-

1.=20 False positive and false negative results: -=20 There has been a genuine concern of false positive results due to = contamination=20 occurring in clinics and laboratories. The problems of false positivity = can be=20 substantially reduced by proper laboratory design, strict discipline = about=20 collection and processing of the specimens, handling of reagents and use = of=20 certain blocking agents.

False=20 negative results are usually due to paucibacillary nature of disease, = uneven AFB=20 distribution and presence of inhibitors such as nucleases. False = negatives can=20 be avoided by using special capture resins and by testing multiple = samples as=20 inhibition is not found in every sample. Also use of internal controls = or spiked=20 controls is useful.

2.=20 Cost Factors: - In=20 a developing country like India cost factor for patient as well as = monumental=20 infrastructure facilities required for setting up of a high class = laboratory=20 assumes importance. However, it is heartening to note that prices of = primers and=20 reagents cost have reduced considerably in last 4-5 years and = considering the=20 advantages of rapidity, sensitivity and specificity cost should not be a = deterrent in adopting such techniques.

Drug=20 Resistance

Another=20 important aspect of laboratory diagnosis of TB is drug susceptibility = testing.=20 It is specially relevant in current scenario in India for M. = tuberculosis=20 infections because secondary drug resistance of 30 percent has been = reported in=20 different series. The spread of multidrug resistant tuberculosis = (MDR-TB) has=20 increased worldwide. MDR mycobacterium tuberculosis strains are those = resistant=20 to atleast rifampcin and isoniazed.

Various=20 Methods For Measuring Drug Resistance In M. Tuberculosis=20 Are:-

1.=20 Culture Method

i)=20 The absolute concentration method (e.g. LJ method):- Drug is = incorporated into=20 solid agar or LJ medium as two fold dilutions or used as a broth = dilution=20 medium. Resistance is defined as the lowest concentration of drug that = exhibits=20 growth (<20 colonies). Variations in inoculin size is major source = of error=20 and therefore should be standardized.

ii)=20 The resistance ratio method:- This is refinement of absolute = concentration=20 method that controls for variations in the MIC of test isolate divided = by MIC of=20 a standard susceptible strain such as H37Rv or by recently isolated = susceptible=20 wild type strains. If the ratio is two or less, or eight or more, the = isolate is=20 considered to be fully sensitive or highly resistant=20 respectively.

iii)=20 The proportion method (e.g Bactec 460 method):- In this method, the = strain is=20 classified as susceptible below a critical proportion of resistant = bacteria and=20 as resistant above it. This proportion for isoniazid and rifampcin is = one=20 percent. In practical terms, the proportion of drug resistant mutants = comes from=20 the ratio of number of colonies growing in drug free=20 medium.

Newer=20 Molecular Methods for Drug Resistance

Most=20 genotypic assays involve three main steps:-

a)=20 DNA isolation and purification

b)=20 Amplification of the nucleic acid

c)=20 Detection of mutation.

Various=20 genotypic methods for detection of drug resistance are direct DNA = sequences,=20 restriction fragment length polymorphism, PCR single strand conformation = polymorphism, dot spot, RNA-RNA mismatch heteroduplex mobility. Target = genes for=20 several antimicrobial drugs especially M. tuberculosis have been = identified and=20 rapid methods for identification of these mutants have been developed = e.g. many=20 studies have shown that Rifampcin resistance in 95-98 percent cases is = caused by=20 mutants in rpoB gene encoding the RNA polymerase b=20 subunit. Molecular typing ideally should give more accurate insight to = the drug=20 resistance pattern but reports show a poor correlation between clinical = picture=20 and molecular drug resistance pattern.

Conclusion

Considering=20 various methodologies, drug sensitivity by radiometric bactec and other = rapid=20 methods is recommended. Though testing by these methods is expensive = (Rs. 500=20 per drug) it still remains most accurate means of drug resistance = pattern, gives=20 quick result within 4-10 days and is currently recommended by WHO as the = method=20 of choice for testing MTB drug resistance. Molecular methods are = promising new=20 tools but need more standardization and = evaluation.

References

Singh=20 UB, Bhanu NV, Seth V. Diagnosis of mycobacteria in the laboratory. In: = Seth V, Kabra SK, eds. Essentials=20 of tuberculosis in children, 2^nd Ed, Jaypee brothers = medical=20 publishers (P) Ltd, New Delhi; 2003. p. 217-235.=20
Lodha=20 R, Kabra SK. Newer diagnostic modalities tuberculosis. Ind J Pediatr = 2004; 71:=20 221-7.=20
Brodie=20 D, Schlvger NW. The diagnosis of tuberculosis. Clinics in chest = medicine.=20 2005; 26: 247-71.=20
Katoch=20 VM. Newer diagnostic techniques for tuberculosis. Ind J Med Res 2004; = 120:=20 418-28.=20
Khan=20 EA, Starke JR. Diagnosis of tuberculosis in children increased needs = for=20 better methods. Emerging Infectious Diseases 1995; 1:=20 115-23.=20
Ganguly=20 NK. What is new in the diagnosis of tuberculosis? ICMR Bulletin 2002; = 32:=20 110-22.