Evaluation of the LiPA MYCOBACTERIA Assay for Identification of Mycobacterial Species from BACTEC 12B Bottles

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Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Evaluation of the LiPA MYCOBACTERIA Assay for Identification

of Mycobacterial Species from BACTEC 12B Bottles

NANCIMAE MILLER,* SUSANNA INFANTE,

AND

TIM CLEARY

Department of Pathology, Jackson Memorial Medical Center, University of Miami, Miami, Florida

Received 22 November 1999/Returned for modification 22 January 2000/Accepted 28 February 2000

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial

isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization

of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes

arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system.

This system identifies

Mycobacterium

species and differentiates

M. tuberculosis

complex,

M. avium-M.

intracel-lulare

complex, and the following mycobacterial species:

M. avium

,

M. intracellulare

,

M. kansasii

,

M. chelonae

group,

M. gordonae

,

M. xenopi

, and

M. scrofulaceum

. The mycobacteria were identified in the laboratory by a

series of tests, including the Roche AMPLICOR

Mycobacterium tuberculosis

(MTB) test, the Gen-Probe

AC-CUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat

shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients.

There was complete agreement between LiPA and the laboratory identification tests for 26

M. tuberculosis

complex, 9

M. avium

, 3

M. intracellulare

complex, 3

M. kansasii

, 4

M. gordonae

, and 5

M. chelonae

group (all were

M. abscessus

) isolates. Three patient samples were LiPA positive for

M. avium-M. intracellulare

complex, and all

were identified as

M. intracellulare

by the PCR-RFLP analysis. Seven additional mycobacterial species were

LiPA positive for

Mycobacterium

spp. (six were

M. fortuitum

, and one was

M. szulgai

). The LiPA

MYCOBAC-TERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR

amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

Although more than 70 mycobacterial species have been

described, relatively few of them are strictly pathogenic for

man or animals (19). While

Mycobacterium tuberculosis

com-plex strains are still responsible for the majority of

Mycobac-terium

infections worldwide, opportunistic infections due to

mycobacteria other than tuberculosis (MOTT) have been on

the increase, mainly as a consequence of the AIDS epidemic

(8, 21, 23). Among the mycobacterial species often implicated

in MOTT infections are

M. avium-M. intracellulare

complex,

M. chelonae,

M. abscessus,

M. kansasii, and

M. xenopi

(8, 19, 33).

M. gordonae

does not usually cause human infection but is

often encountered as a contaminant in clinical samples, and

discrimination from pathogenic species is a relevant diagnostic

issue (4).

The use of liquid cultures in the clinical laboratory improves

the ability to detect the growth of mycobacteria (14, 17, 26).

The radiometric method (BACTEC; Becton Dickinson, Sparks,

Md.) is a fast and sensitive liquid culture system (20). When a

BACTEC bottle is detected as positive, confirmation of the

presence of acid-fast bacilli is done by acid-fast staining and

the broth is plated on solid media.

M. tuberculosis

complex can

be identified rapidly by a variety of nucleic acid amplification

procedures that are commercially available (9, 30, 32). Rapid

identification of MOTT isolates growing on solid media can be

done by techniques such as thin-layer chromatography (11),

gas-liquid chromatography (30), high-performance liquid

chro-matography (10), and analysis with DNA probes (18). Recently

developed molecular methods, such as DNA probe tests (25)

and PCR-restriction fragment length polymorphism (RFLP)

analysis (28, 29), offer identification of this complex group of

organisms from a positive liquid culture medium prior to

de-tection of growth on solid media (3, 7). DNA probes

(ACCU-PROBE; Gen-Probe, Inc., San Diego, Calif.) can be used for

the rapid identification of

M. tuberculosis,

M. avium

and

M. intracellulare,

M. gordonae, and

M. kansasii

from solid culture

and directly from liquid culture systems (2, 3, 6, 7, 16, 26).

Unfortunately, the DNA probes are available for a limited

number of species, and without colonial morphology to guide

probe selection, testing with multiple probes may be necessary.

An algorithm based on growth rate in the BACTEC 12B bottle

and a fluorochrome smear quantitation to guide DNA probe

selection has been reported (16). PCR-RFLP analysis is a

reliable method for identification of MOTT, encompassing

identification of the entire range of organisms normally

iso-lated in a clinical laboratory (28, 29). The LiPA

MYCOBAC-TERIA test offers identification of a limited number of

com-mon mycobacterial species by PCR amplification of the 16 to

23S rRNA spacer region of

Mycobacterium

species followed by

hybridization of the biotinylated amplified DNA product with

14 specific oligonucleotide probes. The specific probes are

immobilized as parallel lines on membrane strips. The

objec-tive of this study was to evaluate the LiPA MYCOBACTERIA

(Innogenetics NV, Ghent, Belgium) assay for identification

and differentiation of specific mycobacterial species from

pos-itive BACTEC 12B liquid cultures. The assay was evaluated for

specificity, ease of use, and interpretation of results in a

rou-tine clinical laboratory.

MATERIALS AND METHODS

Culture and identification.Respiratory specimens submitted for culture were decontaminated with an equal volume of 5%N-acetyl cysteine-NaOH and con-centrated by centrifugation (24). The sediment was used to prepare two smears and to inoculate a selective 7H11 agar plate and a BACTEC 12B bottle (Becton Dickinson). BACTEC 12B bottles were incubated at 37°C in 5% CO2 and

monitored for growth for 6 weeks with a BACTEC 460 instrument according to the manufacturer’s instructions. When the growth index of a bottle reachedⱖ50,

* Corresponding author. Mailing address: Department of Pathology,

University of Miami, P.O. Box 016960, Miami, FL 33101. Phone: (305)

585-6258. Fax: (305) 585-0008. E-mail: nmiller@med.miami.edu.

1915

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a smear was prepared to confirm the presence of acid-fast organisms and the liquid medium was subcultured onto a blood agar and a 7H11 agar plate. Isolates of mycobacteria growing on solid media were identified by DNA probes (AC-CUPROBE; Gen-Probe, Inc.) forM. avium,M. intracellulare,M. gordonae, and

M. kansasiior by conventional biochemical tests performed according to

stan-dard protocols (13, 19).

AMPLICORM. tuberculosisPCR test.Respiratory specimens submitted for culture that were acid-fast organism smear positive were processed for PCR directly from the decontaminated concentrated specimen according to the in-structions of the package insert for the AMPLICORM. tuberculosistest (Roche Diagnostics, Indianapolis, Ind.), as previously described (5). In addition, PCR testing was also performed from positive BACTEC 12B bottles using 0.5 ml of the culture fluid concentrated by centrifugation at 16,000⫻gfor 15 min in a 1.5-ml screw-cap microcentrifuge tube. The pellet was resuspended in 100␮l of lysis buffer and processed in the same way as direct clinical specimens for the remainder of the procedure. All manipulations of positive smear specimens and BACTEC 12B bottles were performed in a biological safety hood. PCR ampli-fication and detection were performed according to the manufacturer’s guide-lines.

LiPA MYCOBACTERIA assay.Specimens were prepared for PCR amplifica-tion by removal of 0.2 ml from BACTEC 12B bottles. The pellet was resus-pended in 20␮l of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and placed in a 95°C heat block for 30 min, followed by centrifugation at 16,000⫻g

for 10 s. The tubes were placed in a⫺20°C freezer for 30 min. Upon thawing, samples were vortexed and centrifuged at 16,000⫻gfor 10 s. A reaction mix of 40␮l was prepared from the supplied amplification mixture containing de-oxynucleoside triphosphates, biotinylated primers, and thermostable DNA poly-merase, to which 10␮l of the processed specimen was added. The PCR was done in a Perkin-Elmer 9600 thermocycler with the following amplification profile: 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s for 40 cycles. The presence of the amplified product was verified by electrophoresis of 10␮l of the amplified product in a 2.0% agarose gel followed by staining with ethidium bromide. The expected size of the amplicon was a single band with a length of 400 to 550 bp. For hybridization, a 10-␮l sample of the amplified product was denatured in the hybridization trough, followed by addition of the hybridization solution provided in the assay kit and the membrane strip. The hybridization solution was pre-warmed to 62°C. The tray of strips was placed in a 62°C shaking water bath (80 rpm) with a lid and incubated for 30 min (model Gemini II incubator; Robbins Scientific, Sunnyvale, Calif.). After hybridization, two stringent washes were done at 62°C. The remainder of the procedure was done at room temperature using a rotary shaker at 80 rpm. Each strip was washed twice for 1 min using 2.0 ml of rinse solution, followed by addition of alkaline phosphatase-streptavidin conjugate solution for 30 min. Each strip was washed twice with rinse solution and once with 2.0 ml of the substrate buffer prior to incubation with the substrate

(5-bromo-4-chloro-3-indoylphosphate and nitroblue tetrazolium) solution for 30 min while being shaken. The color development was stopped by washing the strips twice in 2.0 ml of distilled water with shaking for 3 min. After hybridization and detection, each strip was aligned along the reading chart for interpretation using a green line at the top of the strip for reference.

PCR-RFLP analysis.The PCR-RFLP analysis to identify theMycobacterium

species was done by PCR amplification of a 439-bp segment of the mycobacterial 65-kDa heat shock protein gene (27, 28). Specimens from positive BACTEC 12B bottles were processed for PCR using the same method as that used for the LiPA assay. The PCR was done in a Perkin-Elmer 9600 thermocycler with the follow-ing amplification profile: 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s for 35 cycles.BstEII andHaeII (Sigma, St. Louis, Mo.) enzyme digestions of the am-plification product were performed, and restriction fragments were separated by agarose gel electrophoresis of a 3.0% gel composed of 2.0% high-resolution agarose (Sigma) and 1.0% routine-use agarose (Sigma). The molecular size standard (MspI-digested pUC18; Sigma) was placed in the gel every four lanes to reduce migration-related errors in interpretation of fragment sizes. Photographs were taken of the gels after ethidium bromide staining and were analyzed visually to determine the number and the sizes of the fragments present. Isolates were identified using a published PCR-RFLP analysis algorithm (28, 29). A website (www.hospvd.ch/prasite) was also used for pattern analysis and species identifi-cation.

RESULTS

LiPA PCR amplification of the 16 to 23S rRNA.

Amplified

product was detected in 57 of 60 patient samples after the first

amplification. The three negative samples were positive when

they were diluted 1:2 or 1:10 prior to amplification. The

am-plified product yielded a clear band in the range of 400 to 500

bp.

LiPA MYCOBACTERIA assay identification.

Figure 1

shows a representative sampling of results of the assay. Each

line number which was positive on the LiPA

MYCOBACTE-RIA strip was noted and used to determine the

Mycobacterium

species by using the probe alignment guide included in the kit

shown on the left side in Fig. 1. The conjugate control line and

Mycobacterium

species positive control line must be positive

for a valid result. The LiPA assay identified 60 mycobacterium

isolates from 59 patients (Table 1). One patient sample

pro-FIG. 1. Representative examples of results of the LiPA MYCOBACTERIA assay. The positions of the conjugate (conj.) control,Mycobacteriumgenus-positive control, and the 13 specific probes are shown on the left. The marker line at the top of the strip is used for orientation of the strip for analysis. Lanes: 1,M. fortuitum;

2,M. avium-M. intracellularecomplex; 3,M. gordonae; 4,M. chelonae; 5,M. kansasii; 6,M. avium; 7,M. tuberculosis; 8, conjugate control withoutMycobacteriumDNA

present. MYC,Mycobacteriumcomplex; MTB,M. tuberculosiscomplex; MKA,M. kansasii; MXE,M. xenopi; MGO,M. gordonae; MAIS,M. avium-M. intracellulare

complex; MAV,M. avium; MIN,M. intracellulare; MSC,M. scrofulaceum; MCH,M. chelonae.

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TABLE 1. Line probe assay results and identification of mycobacterial species by PCR and RFLP analysis of 60 patient samples

a

Sample Smearb _GIc _{Gel result}d _{LIPA result} _{LIPA ID} _{Other testing}e _RFLP

1 Neg 999 Clear 400 MYC, MTB M. tuberculosis MTB PCR MTB

2 1⫹ 999 Clear 400 MYC, MTB M. tuberculosis MTB PCR MTB

3 Neg 999 Smear 480–550 MYC Mycobacteriumspecies MFO biochem ID MFO

7 4⫹ 533 Clear 400, weak 850 MYC, MTB M. tuberculosis MTB PCR MTB

9 Neg 783 Clear 400, weak 850 MYC Mycobacteriumspecies MSZ biochem ID MSZ

10 Neg 999 Clear 400 MYC, MGO M. gordonae MGO probe MGO

11 Neg 71 Clear 400, weak 850 MYC, MGO M. gordonae MGO probe MGO

12 Neg 303 Clear 400, weak

clear 500 MYC, MKA-1 M. kansasii MKA probe MKA

13 2⫹ 999 Clear 400 MYC, MKA-1 M. kansasii MKA probe MKA

14 Neg 999 Clear 450 MYC Mycobacteriumspecies MFO biochem ID MFO

15 Neg 205 Clear 400 MYC, MAIS MAIS complex MIN probe MIN

16 Neg 61 Clear 400 MYC, MAIS, MIN M. intracellulare MIN probe MIN

19 2⫹ 999 Smear 450–500 MYC Mycobacteriumspecies MFO biochem ID MFO

21 Neg 999 Clear 400, clear 280 MYC, MTB M. tuberculosis MTB PCR MTB

22 Neg 999 Smear 400–550 MYC, MTB M. tuberculosis MTB PCR MTB

23 Neg 999 Smear 400–550 MYC, MCH-1, MCH-2 M. chelonaegroup III MCH group and MFO

biochem ID MAB and MFO

24 Neg 999 Smear 400–550 MYC, MCH-1, MCH-2 M. chelonaegroup III MCH group biochem ID MAB

26 Neg 999 450, smear 480–520 MYC Mycobacteriumspecies MFO biochem ID MFO

27 Neg 999 Clear 400 MYC, MAIS, MAV M. avium MAV probe MAV

28 Neg 999 Smear 400–450 MYC, MAIS, MIN, MCH-1,

MCH-2 M. intracellulareM. chelonaeIIIand MIN probe and MCHgroup biochem ID MIN and MAB

31 Neg 999 Clear 400 MYC, MCH-1, MCH-2 M. chelonaegroup III MCH group biochem ID MAB

34 Neg 999 Clear 400 MYC, MAIS, MIN M. intracellulare MIN probe MIN

35 Neg 999 Clear 400 MYC, MCH-1, MCH-2 M. chelonaegroup III MCH group biochem ID MAB

39 3⫹ 999 Clear 400 MYC, MAIS, MAV M. avium MAV probe MAV

43 Neg 999 Clear 400, smear

450–520 MYC Mycobacteriumspecies MFO biochem ID MFO

44 Neg 999 Clear 460 MYC Mycobacteriumspecies MFO biochem ID MFO

46 1⫹ 999 Clear 400 MYC, MAIS, MAV M. avium MAV probe MAV

49 Neg Neg Clear 380, 550,

smear 400⫹ Conjugate control only Nonmycobacterial No mycobacteria isolated

51 1⫹ 999 Clear 400 MYC, MAIS MAIS complex MIN probe MIN

53 Neg 999 Clear 400 MYC, MKA-1 M. kansasii MKA probe MKA

57 2⫹ 548 Smear 450–500 MYC, MAIS MAIS complex MAIS probe MIN

59 3⫹ 66 Weak 400 MYC, MTB M. tuberculosis MTB PCR MTB

60 3⫹ 23 Weak 450 MYC, MTB M. tuberculosis MTB PCR MTB

a_MYC,_{Mycobacterium}_{species; MTB,}_{M. tuberculosis}_{; MGO,}_{M. gordonae}_{; MAIS,}_{M. avium-M. intracellulare}_{complex; MIN,}_{M. intracellulare}_{; MAV,}_{M. avium}_;

MKA-1,M. kansasiigroup I; MCH-1,M. chelonaegroups I, II, III, and IV; MCH-2,M. chelonaegroup III; MFO,M. fortuitum; MSZ,M. szulgai; MAB,M. abscessus; ID, identification; biochem, biochemical testing.

b_{Smear, concentrated acid-fast organism smear negative (Neg, negative; 1}_⫹_{to 4}_⫹_{, positive).} c_{GI, growth index of BACTEC 12B bottle.}

d_{Appearance and size(s) of band(s) (in base pairs).} e_{Other testing, method of identification.}

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duced amplified product of the correct size but was negative in

the LiPA assay.

There was complete agreement between the LiPA

MYCO-BACTERIA assay and the laboratory identification tests for 26

M. tuberculosis, 9

M. avium, 3

M. intracellulare, 3

M. kansasii, 4

M. gordonae, and 5

M. chelonae

group (all were

M. abscessus)

isolates from positive BACTEC 12B bottles (Table 2). In one

patient sample, bands were present for both

M. intracellulare

and

M. chelonae. This patient has a history of cultures positive

for both organisms, and the PCR-RFLP yielded the same

iden-tification. There were six isolates of

M. fortuitum

and one

isolate of

M. szulgai; all were positive for the

Mycobacterium

species probe, which identified them as mycobacterial species.

The assay is not capable of species identification for

M. fortui-tum

and

M. szulgai.

The nine

M. avium

isolates were reactive with the

M. avium-M. intracellulare

complex probe (M. avium,

M. intracellulare,

M. scrofulaceum,

M. malmoense, and

M. haemophilum) and

the

M. avium

probe (M. avium,

M. paratuberculosis, and

M. sil-vaticum). The three

M. intracellulare

isolates were reactive with

the

M. avium-M. intracellulare

probe and the

M. intracellulare

probe. The four

M. gordonae

isolates were reactive with the

M. gordonae

probe. The three

M. kansasii

isolates were all

reactive with the

M. kansasii

group I probe (MKA-1) and

negative with the MKA-2 (group II) and MKA-3 (groups III,

IV, and V and

M. gastri) probes.

M. kansasii

isolates are

di-vided into five groups based on data derived from 16 to 23S

rRNA spacer nucleotide sequences (1). The MKA-1 probe

reacts with

M. kansasii

type I, the most frequent

M. kansasii

isolate from human sources worldwide (1).

M. kansasii

group

II, which is detected by the MKA-2 probe, is isolated from both

humans and the environment and is characterized by negative

hybridization in the ACCUPROBE assay.

M. kansasii

groups

III, IV, and V have rarely been isolated from humans but have

been found in environmental samples (1). The five

M. chelonae

solates were reactive with the MCH-1 probe (M. chelonae

groups

I, II, III, and IV) and the MCH-2 probe (M. chelonae

group

III).

M. chelonae

isolates are divided into four genotypical

clusters based on 16 to 23S rRNA nucleotide sequences (22).

The MCH-1 probe reacts with all four clusters, and the MCH-2

probe reacts with cluster III isolates, which encompass

M. che-lonae

and

M. abscessus. The PCR-RFLP analysis identified the

five

M. chelonae

isolates as

M. abscessus. One patient isolate

that was identified as an

M. chelonae

group isolate by the LiPA

assay and as

M. abscessus

by PCR-RFLP analysis had been

identified as

M. fortuitum

by conventional methods. Closer

inspection found the culture to contain two organisms,

M. for-tuitum

and

M. abscessus. We were unable to determine if both

species were detected in the LiPA assay since a specific probe

for

M. fortuitum

is not available and the reactivity with the

Mycobacterium

species probe may have been due to either or

both organisms. The presence of both organisms was

con-firmed by PCR-RFLP analysis of two different colonies from

the 7H11 agar plate.

DISCUSSION

The differentiation of species of

Mycobacterium

has

tradi-tionally been done by evaluation of growth characteristics and

biochemical testing. Rapid methodologies such as those using

DNA probes are limited by the number of available

commer-cial probes. In our laboratory, 50% of the acid-fast isolates

recovered from specimens are not MOTT; therefore, a

com-prehensive rapid detection method capable of identifying

mul-tiple species of mycobacteria in a single test would have a

significant impact. This paper describes the evaluation of a

practical method for the identification of mycobacterial DNA

amplified by PCR from acid-fast-bacillus-positive BACTEC

12B bottles. The LiPA line probe assay employs a reverse

hybridization reaction of biotin-labeled amplified DNA with

specific oligonucleotide probes fixed as parallel lines on a

membrane strip. This method was compared to a PCR-RFLP

procedure that is capable of differentiating 28 species of

clin-ically encountered mycobacteria (28, 29).

The LiPA MYCOBACTERIA test was easy to perform, and

the interpretation of the results was clear-cut and objective.

The LiPA assay identified 60 mycobacterium isolates from 59

patients. Six of seven of the isolates were

M. fortuitum

and one

was

M. szulgai, for which the assay does not have specific

probes; therefore, they were identified as

Mycobacterium

spe-cies in the LiPA assay. The assay correctly identified 50 of 53

isolates to the species level. The remaining three isolates were

identified as

M. avium-M. intracellulare

group isolates by LiPA

and were identified as

M. intracellulare

by PCR-RFLP analysis

and with the ACCUPROBE DNA probe. One culture was

found to contain two organisms by RFLP analysis,

M. fortuitum

and

M. abscessus. M. abscessus

was correctly identified in the

LiPA assay with the

M. chelonae

group probes. Since the

pres-ence of

M. fortuitum

would not be distinguishable from

M. che-lonae, we cannot determine whether both organisms were

am-plified and detected in the assay.

In a smaller study of 27 specimens from liquid culture (S. A.

Watterson, B. A. Hussein, and F. A. Drobniewski, Abstr. 99th

Gen. Meet. Am. Soc. Microbiol. 1999, abstr. U-29, p. 639, 1999),

there was agreement with standard methods of identification

for 26 of 27 of the isolates. The discrepant sample was

identi-fied by standard biochemical methods as

M. fortuitum

but as

M. chelonae

group III by the LiPA assay. However, a subsequent

sample from the same patient was identified as

M. chelonae

by

standard biochemical methods.

The main advantage of LiPA compared to testing with DNA

probes is that LiPA can identify a wide range of species in a

single assay instead of a technician performing a different test

for each species or waiting for growth on solid media to guide

the choice of DNA probe. The PCR-RFLP method has the

same advantage over the use of DNA probes, although the

PCR-RFLP results require more expertise to interpret than

the LiPA results. It was not surprising that the three

M. kan-TABLE 2. Summary of mycobacterial species identification

by all methods tested

Organism _isolatesNo. of

No. of isolates positive by:

LiPA M. tuber-culosis

PCR Probe RFLPanalysis Bio-chemical

testing

M. tuberculosis 26 26 26 26

M. kansasii 3 3 3 3

M. avium-M.

intracellu-larecomplex 15 15 15 15

M. avium 9 9 9 9

M. intracellulare 6 3b ₅c ₆

M. gordonae 4 4 4 4

M. fortuitum 7 7d,e ₇ ₇

M. chelonaegroupa ₅ ₅ ₅ ₅

M. szulgai 1 1d ₁ ₁

a_Includes_{M. chelonae}_and_{M. abscessus}_.

b_{All six were positive by the}_{M. avium-M. intracellulare}_{complex probe.} c_{All were positive by the}_{M. avium-M. intracellulare}_{complex probe}

(ACCU-PROBE).

d_{Identified as}_{Mycobacterium}_{species only.}

e_{One sample was positive for two different species by culture, but we were}

unable to determine if two species were present in the LiPA samples.

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sasii

isolates were all reactive with the

M. kansasii

group I

probe (MKA-1) and negative with the probes for group II

(MKA-2) and group III, IV, and V and

M. gastri

(MKA-3)

isolates since

M. kansasii

group I represents the most common

clinical isolate from humans (1). Group I has a PCR-RFLP

pattern distinguishable from those of other

M. kansasii

groups,

and in our laboratory all clinical isolates have been group I.

In our laboratory, 50% of mycobacterial isolates in 1998

were

M. tuberculosis; the majority of isolates were identified by

an

M. tuberculosis

complex PCR assay. An additional 34.7% of

our isolates were identified using DNA probes for

M. avium-M.

intracellulare

complex (29.7%),

M. kansasii

(3.3%), and

M. gordonae

(1.7%). The LiPA assay was able to identify these

isolates directly from the positive BACTEC 12B bottles with a

single assay and could also identify

M. chelonae, which

com-prised 5.7% of our isolates. In total, the LiPA assay could have

identified 90.6% of our isolates to the species level (1998 data).

The remaining 9.4% of isolates in our lab are composed mainly

of miscellaneous organisms, including

M. fortuitum, for which

the LiPA assay does not have a specific probe. In conclusion,

we found the LiPA MYCOBACTERIA assay to be an easy

test to perform in the clinical setting and one that provides

identification of a large variety of mycobacterial species in a

single test. As yet, the cost of the assay has not been set by the

manufacturer (Innogenetics NV).

The line probe assay technology has also been used for

detection of mutations in the

rpoB

gene of

M. tuberculosis

that

confer resistance to rifampin (12). The rate of concordance

with phenotypic rifampin susceptibility testing results was

92.2%. Another application of the line probe assay is to detect

and identify human papillomavirus (HPV) strains using a strip

with 28 specific probes for each of the 25 HPV genotypes (15).

Since HPV cannot be cultured efficiently, diagnosis of HPV

infection is based on cytology and molecular tools, which

makes this organism a perfect candidate for this type of assay.

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