Transcript TK SYSTEM ASM 107 GM TORONTO

U-057
Tanıl Kocagöz1, Sema Tiryaki2, Thomas Silier3, Cengiz Güney4
1Yeditepe
University Med. Sch., Istanbul, TURKEY, 2Turkish Innovative Biotechnology Organization, Istanbul, TURKEY, 3SALUBRIS, Inc., Woburn, MA, U.S.A., 4Ministry of Health, Sureyyapasa Pulmonary and Cardiovascular Diseases Education and Research Hospital, Istanbul, TURKEY.
ABSTRACT
Isolation of mycobacteria from clinical samples is the most sensitive and
definite way of tuberculosis diagnosis. When classical mycobacterial
culture media like Löwenstein Jensen (LJ) are used, it is required 3 to 6
weeks for the detection of growth. All previously developed rapid
mycobacterial culture systems use selective Middlebrook broth and
suffer from not being ready-to-use, since this medium requires the
addition of oleic acid, albumin, dextrose, catalase (OADC) and selective
antimicrobials, before inoculation. They also can not differentiate
mycobacterial growth from contamination prior to microscopic
examination since it is not possible to understand what causes turbidity
when there is growth in a liquid medium. We have previously developed
TK (Salubris, Inc.), a ready-to-use rapid culture medium which indicates
mycobacterial growth by changing its original red color to yellow and
growth of contaminating organisms by a color change to green. We have
tested the efficiency of TK and its selective type TK SLC, which contains
selective antimicrobials, in the isolation of mycobacteria from clinical
samples and compared it to Löwenstein Jensen and BACTEC MGIT 960
(Becton Dickinson). After decontamination and concentration of 261
samples by NaOH-NALC method using Mycoprosafe (Salubris, Inc.) we
have inoculated them to LJ, MGIT, TK and TK SLC. While the growth in LJ
was followed visually, MGIT tubes were followed in MGIT 960 instrument,
TK and TK SLC in automated incubator reader MYCOLOR TK.
Mycobacterial growth was detected in 71 samples (27%) in at least one of
the media inoculated. Contamination was observed in 1.53% of LJ, 1.15%
of MGIT, 2.68% of TK and 0.77% of TK SLC. The median for growth
detection time was 23 days for LJ, 10 days for MGIT, 14 days for TK and
TK SLC. Having the advantages of being ready to use, the ability of
differentiating mycobacterial growth from contamination and providing
growth results in average 40% earlier than LJ medium, TK culture system
proves to be a new effective culture system especially for laboratories
processing large number of samples.
INTRODUCTION
Tuberculosis continues to be one of the major health problems around the
world. The most important element in tuberculosis control is to find out
patients who have tuberculosis bacilli in their sputum and treat them before
they transmit the disease to healthy individuals. Culture is the gold
standard, most sensitive and specific method for the diagnosis of
tuberculosis. Unfortunately it takes 3 to 6 weeks to detect mycobacterial
growth in classical media like Löwenstein Jensen (LJ). Rapid automated
culture systems like BACTEC MGIT 960 (BD Diagnostic Systems, USA) and
BacT/Alert 3D (Biomerieux, France) are not used widely as classical media,
mainly because of their high cost, requirement for expensive
instrumentation and their media being not ready to use. All rapid
mycobacterial culture systems except TK use Middlebrook broth that require
the addition of oleic acid, albumin, dextrose, catalase (OADC) and selective
antimicrobials before inoculation of processed sample.
TK Medium is a ready to use, rapid mycobacterial culture medium. It enables
early detection of mycobacterial growth by changing its color. Its original
red color turns to yellow by mycobacterial growth. The color change occurs
before the colonies become visible. TK Medium has the advantage of
differentiating mycobacterial growth from the growth of most common
contaminants like fungi and Gram negative bacilli by changing to green
instead of yellow when these microorganisms grow (Figure 1). It is designed
as a solid medium to enable the visualization and isolation of individual
colonies. Pure cultures of mycobacteria can be obtained by subculturing
individual mycobacterial colonies if mixed organisms are obtained in the
original culture. Since the growth in TK Media is detected by color change,
it can be easily followed by visual evaluation and can be used in
laboratories that have a regular 37°C incubator. On the other hand it has a
very eloborate and inexpensive automated incubator reader, called Mycolor
TK (Salubris Technica) (Figure 2 and 3). Mycolor TK, provides growth
curves and its expert system predicts the type of growing microorganism,
without the need for expensive additional software. Mycolor TK also enables
the recording of all detailed information related to the sample and patient
including the result of microscopy. It provides easily statistical data like the
rate of culture positives in different samples, resistance rate for each
antimycobacterial drug and multi-drug resistant (MDR) isolates.
This study was done to investigate the performance and advantages of TK
culture system in daily use in a busy tuberculosis diagnostic laboratory.
MATERIALS AND METHODS
Samples submitted to the microbiology laboratory of Süreyyapaşa Pulmonary and Cardiovascular Diseases
Education and Research Hospital, İstanbul, Turkey were included in the study. A total of 261 sputum samples were
processed by NaOH-NALC decontamination and concentration method, using the ready to use kit Mycoprosafe
(Salubris Inc., USA). MGIT tubes were prepared by the addition of OADC and PANTA as suggested by the producer.
From processed samples 0.5ml was inoculated to MGIT and LJ tubes and 0.2ml to TK Medium and TK SLC. MGIT
tubes were incubated in BACTEC MGIT 960 instrument. TK Media were followed by automated incubator reader
Mycolor TK. LJ tubes were incubated in a regular 37°C incubator. LJ tubes were checked three times a week for
mycobacterial colonies. As soon as colonies were visible, this was recorded as growth detection time for LJ. The
growth detection time for MGIT and TK Media were recorded as indicated by the automated instruments. Smears
were prepared from any type of media that indicated growth, stained by Ziehl Neelsen method and checked by
microscopy for the presence of acid fast bacilli (AFB) and other contaminating organisms.
RESULTS
Among 261 samples, 40 were found to be AFB positive by microscopic
examination. Mycobacterial growth was detected in 71 samples (27%), in at
least one of the media inoculated. Some isolates grew only in some type of
media and did not grow in others. The microscopy results of culture positive
samples are shown in table 1. The growth of mycobacterial isolates according
to the type of media is shown in table 2. The median for growth detection time
was 23 days for LJ, 10 days for MGIT, 14 days for TK and TK SLC.
Contamination was observed in 1.5% of LJ, 1.2% of MGIT, 2.7% of TK and 0.8%
of TK SLC. The results showing the performance of each medium are shown in
table 3.
Table 1. Microscopy results of culture positive samples.
AFB
Negative
Number of culture
positive samples (71)
31
1+
2+
3+
4+
32
2
5
1
Table 2. Mycobacterial isolates obtained in different culture media.
Types of Media in which
mycobacterial isolates are obtained
Growth on LJ, MGIT, TK and TK SLC
Number of
mycobacterial isolates
59
MGIT, TK and TK SLC only
1
MGIT and LJ only
2
TK and TK SLC only
1
LJ and TK SLC only
1
MGIT only
4
LJ only
1
TK SLC only
Total
2
71
Figure 2. Mycolor TK
ASM General Meeting. May 21-24, Toronto, Canada
Table 3. The performance of culture media in the diagnosis of tuberculosis.
Culture Medium
MGIT
Mycobacteria
isolation %
Growth detection
time (median, days)
25.3
10
Contamination
%
Figure 1. TK Medium turns yellow by mycobacterial
growth and green by contamination
1.2
TK
23.0
14
TK SLC
23.8
14
0.8
LJ
23.8
23
1.5
2.7
DISCUSSION
Culture is the gold standard method in the diagnosis of tuberculosis. Although it
takes several weeks to detect mycobacterial growth in a classical culture medium,
its sensitivity and specificity is much better than microscopy. Rapid mycobacterial
culture systems developed so far did not manage to replace classical culture media
like LJ, because of several disadvantages among which their high cost, requirement
of additional instrumentation, and most important, being not ready to use can be
included. To overcome the disadvantages present in other rapid culture systems, we
have previously developed the TK culture system. This study was done to evaluate
the performance of TK culture system in the diagnosis of tuberculosis.
Our study site was a busy tuberculosis diagnostic laboratory with high percentage
of AFB positive samples. However the concentration of AFB in the samples was
pretty low. Among all 71 culture positive samples, 63 were either smear negative
(31) or AFB 1+ (32). The growth detection time depends on the concentration of
mycobacteria in the sample, especially in rapid culture systems that detect the
metabolic activity. Median growth detection time, obtained in this study, can be
considered very good for MGIT (10 days) and TK Media (14 days) since the
concentration of mycobacteria in the samples were pretty low. The contamination
rate was low in all types of media including TK Medium which does not contain
selective antimicrobials. This can be considered due to effective decontamination
and concentration by Mycoprosafe. This may have played also an important role in
speeding up the growth detection even in LJ which was 23 days in average in this
study. Although TK Media were slightly slower than MGIT, they provided culture
results 40% earlier than LJ medium. On the other hand it was much easier to use TK
and LJ media since they were ready to use and did not require preparatory work like
MGIT before inoculation of samples. TK culture system was also the easiest to
follow since the system can differentiate real mycobacterial growth from
contamination and all tubes showing growth or negative tubes that completed
incubation duration are indicated on the screen of Mycolor TK and it takes only a
few minutes every day to evaluate the results.
Having the advantages of being ready to use, the ability of differentiating
mycobacterial growth from contamination, providing growth results earlier than
classical culture media, and being very practical to follow the culture tubes, TK
culture system proves to be a new effective culture system in the diagnosis of
tuberculosis.
TK MEDİUM
MYCOBACTERIA
CONTAMINATION
Figure 3. Mycolor TK
Acknowledgement: This study was supported by
Foundation for Innovative New Diagnostics (FIND).