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Delay in the diagnosis of pulmonary tuberculosis, London, 1998-2000: analysis of surveillance data

BMJ 2003; 326 doi: https://doi.org/10.1136/bmj.326.7395.909 (Published 26 April 2003) Cite this as: BMJ 2003;326:909

Delays in diagnosis of tuberculosis?Let us use thyroxine supplemented culture medium for early lab-diagnosis.

A Research letter:
Delays in diagnosis of tuberculosis?
Let us use thyroxine supplemented culture medium for early Lab-diagnosis.
Prof (Dr) Jogenananda Pramanik.MD
Academic Co-ordinator & Foreign Expert,
Xinxiang Medical College, Henan Province,
Peoples’ Republic of China-453003.
E-mail: pramanik_dr@hotmail.com

Abstract: Tuberculosis is re-emerging as a dreaded
killer disease in recent years.
Due to delayed diagnosis, aggravation of this disease is a common feature.
Slow growth rate of tubercle bacilli in vitro is a bottleneck problem.
Several attempts have been made till recently to enrich the conventional L
-J medium for accelerating the bacillary growth rate in vitro. Thyroid
hormone induces cellular gene transcription and promotes protein
synthesis. Effect of thyroid hormone in acceleration of bacterial growth
rate was reported earlier. Thyroxine supplemented L-J medium was used in
our study for in vitro culture of tubercle bacilli. Possibilities of
contamination of bacillary seeds by rapid grower species of Mycobacteria
were eliminated after related biochemical tests. Tubercle bacillary growth
rate acceleration was observed in L-J medium and Sauton medium following
thyroxine supplementation (4ug/ml and 8ug/ml respectively).). Use of
thyroxine supplemented L-J medium for culture of tubercle bacilli may be
helpful for early laboratory diagnosis of suspected tubercular patients as
well as for antitubercular drug sensitivity tests.

Key words: L-J medium, Thyroxine, Mycobacterium tuberculosis.
Background:

In recent years, tuberculosis is re-emerging as a dreaded killer
disease worldwide. Multi drug resistant tuberculosis (MDR-TB) and Acquired
immune deficiency disease (AIDS) in tuberculosis are more life threatening
cases.

Early diagnosis of this disease is found to be difficult in third
world country set up due to lack of advanced laboratory facilities like
Bactec radiometric culture technique, PCR, Tubercular antigen and antibody
detection ELISA2 etc. Current reports from developed countries are also
narrating a number of factors for delayed diagnosis of tuberculosis and
related complications1.

The slow growth rate of tubercle bacilli in vitro seems to be one of
the major limitations in early laboratory diagnosis in tuberculosis.
Several attempts have been made to enrich the culture medium for
acceleration of tubercle bacillary growth rate in vitro.

Acceleration of tubercle bacillary growth was urgently needed, during
our research work on immunodiagnosis of tuberculosis using excretory-
secretory antigenic proteins released by the growing tubercle bacilli in
vitro2,4.

Effect of thyroid hormone in acceleration of different bacterial
growth rate was reported earlier3. thyroxine supplemented media for
tubercle bacillary culture4. .

Method & Materials:

Mycobacterium tuberculosis H37Ra( a gift from Tuberculosis Research
Centre,
Chennai,India) was used as bacillary seed. Lowenstein Jensen (L-J) medium
was used for seed culture and modified synthetic Sauton medium( containing
0.05 v/v Tween-80) was used as subculture medium. Thyroxine
(Eltroxin,Glaxo Lab.,India) was incorporated in culture media.

At a concentration of 10ug/ml,thyroxine was dissolved in mineral salt
solution during preparation of L-J medium. Following inspissations of L-J
medium,final concentration of thyroxine in the medium was
4ug/ml.Thyroxine supplemented Sauton medium was prepared with serially
increased concentrations of thyroxine ranging from 2ug/ml to 16ug/ml in
the medium. The test strain of tubercle bacilli was grown in thyroxine
supplemented and thyroxine free L-J slopes for a period of 2-4 weeks at
370C
in incubator(Prototech).

One loopful of bacilli(1x104 bacilli/ml) was scraped from the
respective L-J medium slope and inoculated in 50ml glass bottles(Borosil)
containing 10ml of thyroxine free Sauton medium.

The cultures were further incubated at 370C for there days, with
shaking on a rotary shaker for 2 hours twice a day.

All test and control cultures( 9 sets in triplicate ) were expanded
to 100ml volume in 500ml conical flasks(Borosil) and incubated for 7days
at 370C with intermittent shaking as before.Bacillary growth rate was
measured by spectrophotometric method. The optical density at 540nm was
recorded using spectronic-20 (Bausch and Lomb) immediately after expansion
to 100ml volume and at the end of 7 days of subculture. Necessary strict
aseptic measures were maintained during transfer bacilli from different
media using laminar flow, a biohazard safety hood(Klenzaids).

Before transferring the bacilli to different media, purity of the
culture was verified by bacteriological and biochemical methods.
Result:

In L-J media, visible tubercle bacillary colonies appeared at 10+3
days interval.

A dose dependent acceleration of tubercle bacillary growth rate was
observed with varying concentrations of thyroxine (2ug/ml-16ug/ml) in
liquid culture media .

An optimal concentration of 8ug/ml of thyroxine concentration in
Sauton medium showed maximum bacillary growth4.
Discussion:

It may not be always true that delay in diagnosis of tuberculosis is
primarily due to the fault of the health care providers1.

One of the well documented limitation in culture confirmation of
tuberculosis cases in most of the laboratories in third world countries,
is the extremely slow growth rate of tubercle bacilli in vitro.
Radiometric Bactec culture technique, PCR technique, Immunodiagnostic
techniques etc, are available in some of the sophisticated laboratories.
Therefore, an inexpensive rapid culture technique is the need of the hour
for early diagnosis of tuberculosis.

Mycobacterium tuberculosis grows much faster in vivo5. One of the
reasons for such accelerated growth may be due to the circulating hormonal
influences. Thyroxine stimulates cellular growth rate5 in cell culture
medium due to its enhancing effect on DNA transcription process through
its action on hormone response element. In persuasion to the earlier
report2, thyroxine was incorporated in L-J medium and Sauton medium for in
vitro culture of tubercle bacilli. Necessary bacteriological and
biochemical tests were performed to eliminate rapid grower species of
tubercle bacilli. A dose dependent acceleration of bacillary growth
response was observed4.Further molecular analytical studies are needed to
establish the basis of growth accelerating effect of thyroxine on slow
grower species of Mycobacterium tuberculosis5.

Concluding Remark:

Thyroxine supplemented culture media (Solid and Liquid media) may be
useful for early culture confirmation of suspected smear negative
tubercular cases which may also in culture sensitivity tests for
antitubercular drugs to exclude MDR-TB cases as well.

Use of this inexpensive rapid culture technique may be encouraged in
financially compromised laboratories at the peripheral health centers of
third world countries for control of this killer disease.
Note:
*This paper was presented by the author in 51st National conference on
Tuberculosis and allied chest diseases, held at Bangalore, India.( Nov’97
)
*This research work was funded by the Tropical Disease Research programme
for MD thesis work of the author,
Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra,
India.

References:
1.Alison Rodger, Shabbar Jaffar, Stuart Paynter, Andrew Hayward, Jacqui
Carless, and Helen Maguire
Delay in the diagnosis of pulmonary tuberculosis, London, 1998-2000:
analysis of surveillance data
BMJ 2003; 326: 909-910.

2.Dr.J.Pramanik et al., Detection of tubercular antibody and antigen
in sera of bone and joint tuberculosis.Ind.J.Clin.Bioch.2000,15(1),22-28.

3.Dr.S.K.Biswas, Effect of thyroxine on bacterial growth.
Lancet;1975, 2,716.

4.Dr.J.Pramanik et al., Increased yield of excretory-secretory
antigen with thyroxine supplementation in in vitro culture of tubercle
bacilli. Ind. J. Tub.1997,44, 185-190.

5. J.Robert,A. North Angelo, Izzo: Mycobacterial virulence. Virulent
strain of mycobacterium tuberculosis have faster in vivo doubling times
and are better equipped to resisting growth inhibiting functions of
macrophages in the presence and absence of specific immunity. J.Exp.Med:
1993, 177,1723.

Competing interests:
None declared

Competing interests: No competing interests

16 November 2003
Prof(Dr)Jogenananda Pramanik.MD
Academic Co-ordinator & foreign Expert
Xinxiang Medical College,Xinxiang City,Henan Province,Peoples' republic of China-453003