3Indian

2 Journal of Medical Microbiology, (2007) 25 (1):32-6

Original Article

USE OF BACTEC 460 TB SYSTEM IN THE DIAGNOSIS OF TUBERCULOSIS

*CS Rodrigues, SV Shenai, DVGAlmeida, MA Sadani, N Goyal, C Vadher, AP Mehta

Abstract
Purpose: To evaluate, the efficacy of BACTEC 460 TB system for the diagnosis of tuberculosis in a tertiary care hospital
in Mumbai, India. Methods: We compared 12,726 clinical specimens using BACTEC 460 TB system and conventional
method for detection of Mycobacterium tuberculosis over a period of six years. Result: The overall recovery rate was
39% by BACTEC technique and 29% using Lowenstein-Jensen (LJ) medium. An average detection time for BACTEC
460 TB system was found to be 13.3 days and 15.3 days as against 31.2 days and 35.3 days by LJ method for respiratory
and nonrespiratory specimens respectively. The average reporting time for drug susceptibility results ranged from 6-10
days for the BACTEC 460 TB system. Conclusions: The BACTEC system is a good system for level II laboratories,
especially in the diagnosis of extrapulmonary and smear negative tuberculosis.
m
Key words: Acid fast bacilli culture, BACTEC 460 TB system, Mycobacterium tuberculosis
rf o
Acid fast bacilli (AFB) culture is usually considered a gold Materials and Methodsa
d ns
lo analyzed
t io for six years from December
standard for the laboratory diagnosis of tuberculosis (TB).
The data has nbeen
a clinical specimens received were
to depend on conventional Lowenstein and Jensen (LJ) media 1998 to June w
Most of the mycobacteriology laboratories in India continue
2004. Allcthe
for culture, however, the time required and frequent negative subjectedd
o i
l microscopy by Ziehl-Neelsen (ZN)
b
to direct smear
u
results with paucibacillary specimens are important limitations.
e e
staining method.
P
Specimens,
).
which contain normal bacterial
It is well known that the use of liquid based culture system
r
flora,
NaOH
were
w
decontaminated
f the sediment
method. 1
m
Specimens
by
from
standard N-acetyl-L-cysteine­
sterile sites were centrifuged
improves the recovery with shortening of the time required to
o rand o o
was inoculated into the BACTEC 12 B vial
detect TB. Among the methods utilizing liquid media, semi-
automated radiometric BACTEC 460 TB system (Becton f k n
supplemented . c
with the antimicrobial mixture PANTA (Becton
Dickinson, Sparks, MD, USA) is the most widely accepted leas inoculated.
d
Dickinson,
o w
Sparks, MD, USA). One LJ slant was also
the reference standard in the developed nations.
a b The e1 C. Inoculatedn All the inoculated media were incubated at 37 ±
detection of mycobacterial growth in BACTEC 12Bilmedium isM k
o
LJ slants were incubated for 8-10 weeks and

v a liberated
14
y e d
checked everyday for growth for the first week after which

a presentd inband
carried out by quantitatively measuring of the CO 2,
14 they were inspected weekly for 10 weeks.
by the metabolism of C-labelled substrate
medium. The system gives results of culture
is e
the
.m All inoculated 12 B vials were tested twice a week for first
Antimycobacterial Drug Susceptibility Test (AST)
F s t within 10-wof three weeks and then once a week for remaining three weeks.
14 days. However, there are only about
D o wbeen Positive vials were subjected to smear microscopy. Final
37 installations
w
used frequently in our country.P
BACTEC 460 TB system in India, h (
suggesting it has not
identification of M. tuberculosis complex (MTB) was done by

is1998 and
BACTEC
it e 460 TB system was
the BACTEC NAP (r-nitro-α-acetylamino-β-hydroxy
h
launched at our institute in
s More recently a non- propiophenone)
we have processed a
differentiation test (Becton Dickinson, Sparks,
radiometric BACTEC T MGITa960 TB system has been index (GI) to high levels
large number of specimens since then.
MD, USA). Obvious turbidity or sudden increase in growth
indicated contamination. This was
introduced, which is better automated with no issues of
confirmed by Gram staining of smear as well as subculture on
radioactive disposal. As this new non-radiometric system is
blood agar medium. In addition, 0.2 mL of positive BACTEC
going to replace the radiometric TB system, we felt the need
broth was subcultured onto one more LJ slant. Growth on this
to analyze our data with BACTEC 460 TB liquid system to
LJ subculture was used to rule out mixed infection, for
evaluate its performance and its contribution to our TB
biochemical identification of MTB and non tuberculous
diagnosis before adopting another liquid culture system.
mycobacteria (NTM) strains.

Quality control of the instrument

*Corresponding author (email: <dr_crodrigues@hindujahospital.com>)
Department of Microbiology, PD Hinduja National Hospital and
Medical Research Center, Mumbai - 400 016, India
Received : 28-02-06
Accepted : 17-10-06
Performance test was run daily according to the
manufacturer’s instructions. Maintenance of the needle heater,
filters, media trap and UV light was strictly followed to prevent
cross-contamination. Needles were changed daily. Quality

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32 CMYK
January-March 2007 Rodrigues et al - Diagnosis of Tuberculosis 33

control of the 12 B growth medium and NAP disk was carried Table 1: Positivity rate of BACTEC 12B and LJ medium
out using standard H37 Rv strain of M. tuberculosis. (n = 12,726)
Antimicrobial susceptibility Test (AST) by BACTEC 460 TB Results by LJ Results by BACTEC 460 TB system
system method Culture positive Culture negative
n=5006 n=7720
AST for four primary first line and four second line anti- Culture positive 3551 (27.9%) 156 (1.2%)
TB drugs was performed only on request, using BACTEC 460 n=3707
TB system. The first line drugs were provided in a drug kit Culture negative 1455 (11.4%) 7564 (59.4%)
(Becton and Dickinson) and second line drugs were acquired n=9019
from local pharmaceuticals. Following concentrations were
used-Streptomycin (STR) 2.0 mg/mL, isoniazid (INH) 0.1 mg/ Overall isolation rate for BACTEC 460 TB system: 39%

Overall isolation rate for L.J media: 29%

mL, rifampicin (RIF) 2.0 mg/mL, ethambutol 2.5 mg/mL,

Combine isolation rate of liquid plus solid media: 40%

kanamycin (K) 5.0 mg/mL, ethionamide (ETA) 5.0 mg/mL, r Concordance (agreement) between the 2 systems: 87.6%

amino salicylic acid (PAS) 4.0 mg/mL and ofloxacin (OF) 2.0 Two-tailed P<0.00001
mg/mL. Drug susceptibility testing was done using standard Cornfield 95% confidence limits: 99.5155 and 140.7809
procedure. Briefly, O.1 mL of the appropriate drug solution was
o m
injected into labeled 12 B vials which resulted into the desired
fr
media showed a higher recovery rate of 40% (Figure).
concentration of a drug in the medium. This was followed by
inoculation of 0.1 mL of bacterial suspension from a positive
a
As shown in the figure, d majority
n sduring
of the clinical specimens
12B vial with a GI 500-800. For control, the bacterial inoculum
l
(73%) were positive by
o LJ took o
BACTEC
i first two weeks of
tLJ between four to Most
was diluted 1:100 before inoculation. The inoculated 12B vials
were incubated and read daily on the instrument till the GI of
n
incubation however,
specimens were positive by a
a longer time.
w ic for TB-BACTEC was found to be
of the
eight weeks.
An average o detection ltime
13.3 days d
the control reached >30. When growth from a solid medium
(excludingbthe time required for Nap differentiation)
to 31.2udays by LJ method (excluding the time
was used, a well-dispersed suspension (approximately
corresponding to McFarland no. 1 standard turbidity) was
e e
compared
P ).
prepared from a fresh culture. These suspensions were used
fr
required for species
w m
identification) in case of respiratory
in the same manner as a BACTEC positive vial described r o
specimens.
o
However, in case of nonrespiratory specimens an
above. Reference strain of M. tuberculosis H37Rv was used fo BACTECn
average
.
detection
c time was found to be 15.3 days for TB­
as a batch quality control on a weekly basis.
le dk ow compared to 35.3 days by LJ method.

a b e Tablen2 shows the analysis of recovery time and recovery

Results
l
i patients,M rate d k
according to the smear positivity. Smear-negative cases

v a
Of the total 12,726 clinical specimens from 9226
b y e to the time
were picked up in about 24 days by TB-BACTEC which
79% (7288) were received from out door (OPD)
a analysis .mOf the total 2254 smeartaken
1938 (21%) from hospital admitted patients. Furtherd
patients and compares by LJ in 4+ smear positive cases.

is thatte41% werew medium, 2212 were positive by BACTEC (98%) whereas only
negative culture positive cases by any
of all the OPD clinical specimens, revealed
consulted by our hospital physicians. F o
Anothers 30% wwere 1526 (68%) were positive by LJ.
PD thomes
referred to us by general practitioners
14% from private hospitals / nursing
or h
e (w from In Table 3 culture positive data was analyzed according
family physicians,
and 12%
government hospitals. Inisabout 3% i of cases the exact to the site of infection from where the clinical specimens were
s
Th Ofspecimens
information was not available.
7153 (56.2%) were respiratory athe 12,726and
clinical specimens
5573 (43.8%) 40%

35%
non-respiratory specimens. Overall, M. tuberculosis was 34%

isolated in 5162 specimens whereas NTM were isolated in 380 30%

28%
Recovery rate

27%
specimens by both the culture methods. 25%

20%
The overall recovery rate was 39% for BACTEC 12B and 17%
15% 15%
29% for LJ medium (Table 1). Compared to the conventional 11%
13% 13%
10%
LJ method, BACTEC 460 TB system detected a total of 1455 9%
5% 5% 6% 6%
(11.4%) additional positive cultures, which is statistically 3% 2% 3% 2% 2%
0% 0% 0% 0% 1% 1% 1% 0% 0% 0% 1%
0%
highly significant. Of the 156 BACTEC negative LJ positive
to
5 10 15 20 25 30 35 40 45 50 55 60 65 70
to to to to to to to to to to to to to
cases, 112 were clearly negative by BACTEC whereas in 44 0 6 11 16 21 26 31 36 41 46 51 56 61 66

cases NTM was isolated by BACTEC and M. tuberculosis No. of days

was isolated by LJ. A mixed growth of M. tuberculosis and TB - BACTEC L.J Medium
NTM was identified in two cases showing two different types
of colonies on LJ slant. A combination of liquid plus solid Figure: Recovery time in days by BACTEC 460 and LJ system

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33 CMYK
34 Indian Journal of Medical Microbiology vol. 25, No. 1

Table 2: Analysis of culture result according to the acid fast bacilli smear status
Smear results BACTEC 460 TB LJ media
n = 5006 n = 3707
Recovery time days No. of specimens Recovery time days No. of specimens
++++ (n = 732) ≤07 690 28 539
+++ (n =587) 09 506 36 423
++ (n = 756) 13 564 37 417
+ (n = 1936) 13 1034 42 802
Smear negative (n=2254) 24 2212 ≥42 1526
Two-tailed P for recovery time = 0.2587 (non-significant for df 4)

Table 3: Culture positivity according to the site of infection Of the total 130 culture isolates tested 100 were multi-drug
resistant (resistant to atleast rifampicin and INH with or
Specimens BACTEC 460 TB (%) LJ media (%) without resistance to other antimycobaterial drugs) and 30
n=12,726 n=5006 (39) n=3707 (29) were pansusceptibile (sensitive to all the drugs). Drug
m
rf o
Respiratory specimens susceptibility results of 2565 cultures using BACTEC 460 TB
Sputum (5026) 2978 (59.3) 2268 (45.0) system are summarised in Table 5.
BAL (1780)
946 (53)
Tracheal secretion (347) 63 (18.2)
655 (36.7)
47 (13.5) Discussion
a d ns
Total (7153) 3987 (56) 2970 (42) The efficacy of
n lothe BACTEC
tio 460 TB system in the
Non-respiratory specimens detection of mycobacteriaafrom clinical specimens has been
Aspirates (447) 70 (17.7) 54 (12.0) demonstratedo wa number
in
lic of field trials and clinical correlation
Pus (631) 286 (45.3) 223 (35.3) studies. d Analysis b
2-5
of our data revealed that BACTEC 460
FNAC 436
126 (28.9) 84 (19.3)
e for israpid
TB system
P u . superior than LJ medium in our
significantly
CSF (460)
40 (8.7) 18 (3.9)
f e
r was wellisolation
settings ) and identification of MTB from
Abscess/wound (622) 148 (23.8) 104 (16.7) asm
rof 5162 opositive cultures
respiratory non-respiratory specimens. With a total
o by any medium, 97% were positive
91 (9.4) fo by BACTEC
Pleural fluid (665) 70 (10.5) 46 (6.5)
Tissue/lymphnodes (960) 115 (12.0)
49 (6.0)e kn .
withc 28% positive in BACTEC only. On the other
Urine (822) 74 (9.0)
b l e d 3%
hand, 72%
o w
of the total cultures positive were positive by LJ
Others (530)
Total (5573)
90 (17.0)
1019 (18)
68 (12.8)
li a(13) M recovery
737
with onlyn were positive
k (P<0.00001) in recovery
on LJ only. Looking at the overall

va b y
Two-tailed P<0.000001 (statistically highly significant for df 11)
e d
difference
from specimens, a statistically highly significant
rate was demonstrated by
a d .mwas more prominent in smear-negative specimens.
BACTEC 460 TB system. The performance of BACTEC 460
collected. BACTEC was found to is be moreesensitive Of the

F s t w 2254 smear negative culture positive specimens MTB was

(statistically significant) in detecting more number
D o and w
of MTB
correctly isolated in 98% of the specimens compared
wpleural 68% by LJ. The system was found to be more advantageous, to only
from respiratory as well as non-respiratory
P
especially in cases of cerebrospinal fluid h
(CSF)
specimens
(
fluids.
is site in paucibacillary specimens, especially in case of extra-

T a
AST of first and secondh pulmonary cases like CSF, body fluids and fine needle
line drugs by BACTEC 460 TB aspiration cytology, where LJ media yielded very scanty
system and LJ proportion method has been shown in Table 4. growth or no growth.

Table 4: Antimycobacterial susceptibility results of first and second line drugs by BACTEC 460 TB system and LJ
proportion method (n=130)
BACTEC 460 TB system
STR INH RIF EMB KANA ETA PAS OF PZA
R S R S R S R S R S R S R S R S R S
R 60 2 96 4 97 3 45 2 15 1 22 5 13 13 17 03 5 0
L.J
S 1 67 0 30 0 30 2 81 0 114 2 101 0 104 0 110 1 124
Concordance 98% 97% 98% 97% 99% 95% 90% 98% 99%
(Agreement)
STR-Streptomycin, INH- Isoniazid, EMB- Ethambutol, KANA-Kanamycin, ETA- ethionamide, PAS-r amino salicylic acid, OF- Ofloxacin,
PZA, R- Resistant, R- Resistant

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34 CMYK
January-March 2007 Rodrigues et al - Diagnosis of Tuberculosis
Table 5: Drug susceptibility results of M. tuberculosis by
BACTEC 460 system (n = 2565)
Drugs (µg/mL) % resistant
Streptomycin (2.0) 48
Isoniazid (0.1) 68
Rifampicin (2.0) 61
Ethambutol (2.5) 31
Kanamycin (5.0) 18
Ethionamide (2.0) 23
PAS (4.0) 18
Ofloxacin (2.0) 26
Sensitive to all - 667 (26%), Monoresistant - 282 (11%), MDR ­
1616 (63%), Respiratory infection –2232 (87%), Non-respiratory
infection – 333 (13%)
A more careful analysis was done on those specimens,
which were positive by LJ and negative by BACTEC. Of the
156 only L.J positive MTB cases, BACTEC yielded no growth
in 112 cases while NTM were isolated in the remaining 44
cases instead of MTB. Most of the BACTEC negative LJ
positive strains were isolated from pus, abscesses or tissue
biopsies indicating; some strains of mycobacteria require high
protein egg-based media to grow. Of the 44 NTM positive in
BACTEC, mixed growth (two different types of colonies on
LJ) was observed in two cases, however, in 42 cases NTM
(mostly rapid growers) were possibly contaminants. There was
a possibility that growth of NTM in BACTEC had masked the
or o o
growth of MTB. Being a liquid medium, colonized f kn .c
mycobacterial flora or other mycobacterial contaminants
b le ed ow a CDC recommendation.10 The early availability of AST results
la M dkn
especially rapid growers, were also detected by the system.
i
Hence reports showing NTM infections should be clinically
a by e
correlated or confirmed by testing more number of specimens
v
a d .m
(at least 3).6 Apart from NTM, bacterial or fungal contamination
is te w
rates for BACTEC 460 TB system and LJ were found to be
3% and 8% respectively. Being an egg-based medium, LJ is
F os w
more prone to the fungal contamination resulting in total loss
PD te h (w
of the medium. The advantage of BACTEC 12 B medium over
LJ is that if it gets contaminated, the medium can be processed
is si
again for decontamination to recover mycobacteria.
h
The major advantage T of the aBACTEC 460 TB system is Certain concerns associated with the BACTEC 460 TB
the early availability of results. It was observed that as many system include the need for syringe and needle for inoculation,
as 73% yielded a positive result by BACTEC within 15 days
as against none by the conventional method. It was also
observed that with heavy smear positives (4+,3+) growth was
seen in <5 days compared to 24 days in smear negative
specimens. Contrast to the other studies,7 the mean recovery
time required to isolate MTB, for TB-BACTEC and LJ was
slightly higher in our lab. This could be attributed to the type
of patient population included. Being a tertiary referral center,
most of the clinical specimens we received were from the
patients receiving the anti-TB treatment. We do not have
complete data on the treatment status of the patients but on
an average about 53% patients were receiving anti-TB
treatment, which could increase the recovery time.
www.ijmm.org
35
Once a presumptive diagnosis of tuberculosis is
established, an early result on drug susceptibility is vital for
effective treatment of the patient, particularly when resistance
to one or more drugs is suspected. The LJ medium requires
four weeks for the AST results to become available. There are
other concerns in using LJ medium for AST. Since LJ drug
medium is made in the laboratory, there is less standrdisation
and quality control in prepation of the drug medium. It is also
possible that inspissation of LJ medium, binding of the drugs
to proteins and storage of the prepared medium and prolonged
incubation period could result in loss of potency of the drugs.8
Further, in the conventional methods, lack of standardisation
in methodology and definitions of resistance used may cause
errors in the interpretation and validity of the results.
o m
Susceptibility testing of MTB by BACTEC 460 TB system
is based on the modified proportion method and is FDA
fr
approved for first line anti-TB drugs. The medium and the
a d ns
procedure is standardized to increase the accuracy of the
results. Initially, we compared susceptibility results with LJ
lo tio
proportion method in 130 clinical isolates (unpublished data).9
w n a
The concordance ranged from 97 to 98% for first line drugs
o lic
and 90 to 99% for second line drugs (Table 4). We continued
d ub
with BACTEC 460 TB system, as there is no gold standard,
e
especially in case of second line drugs. Another advantage is
rf e w P m).
that the susceptibility results are available within 6-10 days
compared to two to four weeks by LJ. This means that we
could report complete results of isolation, identification and
AST of the isolated culture within an average of 4 weeks time,
would be especially beneficial to patients harboring multidrug
resistant organisms to enable them to receive effective
treatment with appropriate regimens. At our institute multidrug
resistance (MDR) TB was found to be as high as 65%, which
could be attributed to the fact that ours is a tertiary referral
center and most of the patients are more likely to have been
unresponsive to therapy or had relapse resulting in a bias
toward drug resistant isolates.11 In institutions dealing with
referral cases with high prevalence of drug resistance,
availability of a liquid media system is extremely important.
use of radio labeled products and its disposal and finally the
cost. BACTEC 460 TB system involves high initial investment
of 20 lakhs rupees and the high cost of culture medium.
Technical support for repairs and maintenance is adequate and
cost-effective. In India, an average cost for isolation and
identification using BACTEC 460 TB systems is Rs. 475 per
patient as compared to Rs. 40 using LJ medium. Similarly the
cost for AST to SIRE using BACTEC 460 TB system is Rs.
1000 vs Rs. 250 using LJ. Thus, in developing countries, such
as India, LJ is cost-effective, however, a careful analysis needs
to be done to look at time to report results and its cost benefit
in the long run. It takes four to six weeks for primary isolation
of TB bacilli using LJ. Once isolated, species identification or
35 CMYK
36 Indian Journal of Medical Microbiology vol. 25, No. 1

drug susceptibility testing can take another four to eight weeks
and during subculturing the isolate could be lost or get
contaminated. Even if, biochemical tests for identification are
inexpensive they are time-consuming and many times give
ambiguous results. In contrast, BACTEC 460 TB system,
though expensive, offers certain advantages like speed, higher
recovery rate and accurate drug susceptibility testing.
Moreover, cost of the BACTEC 460 TB system could be
partially offset by the ease of use and early availability of
reliable results. It could be extremely crucial in a region with
high prevalence rates of TB, particularly in the non-respiratory
and paucibacillary smear negative forms of TB.
Automated MGIT is a newer version state-of-the-art
machine, which would eliminate the concern of handling
radioactivity and needles. However, one has to evaluate this
mycobacteria from clinical specimens. J Clin Microbiol
1990;28:1288-91.
4. Morgan MA, Horstmeier CD, DeYoung DR, Roberts GD.
Comparison of radiometric method (BACTEC) and conventional
culture media for recovery of mycobacteria from smear negative
specimens. J Clin Microbiol 1983;18:384-8.
5. Kirihara JM, Hillier SL, Coyle MB. Improved Detection times
for Mycobacterium avium complex and Mycobacterium
tuberculosis with the BACTEC radiometric system. J Clin
Microbiol 1985;22:841-5.
6. Laszlo A, Siddiqi SH. Evaluation of rapid radiometric
differentiation test for the Mycobacterium tuberculosis complex
by Selective Inhibition with p-nitro-α-acetylamino-β-hydroxy­
propiophenone. J Clin Microbiol 1984;19:694-8.
7.
m
Krashow I, Wayne LG. Comparison of methods for tuberculosis

rf o
newer system as the identification of isolated mycobacteria is
bacteriology. Appl Microbiol 1969;18:915-7.
not fully standardized like NAP on BACTEC. The alternative
option of molecular based techniques (probe hybridization)
may be expensive in routine laboratories.
ad ns
8. Casal M. Laboratory approaches to mycobacterial susceptibility
to antibiotics (review). Rev Esp Quimioterap 1995;8:4-9.

Acknowledgement
nlo tio
9. Ajay K. Comparison of drug susceptibility pattern in non-

We would like to thank Dr. Salman Siddiqui for all his help
ow lica responding (MDR-TB) Patients by conventional L.J. method
and BACTEC method. Thesis submitted to the University of
and guidance. d ub Mumbai for Master of Science in Applied Biology. May 2003
e
rf e w P m).
References 10. Shinnick TM, Iademarco MF, Ridderhof JC. National plan for
reliable tuberculosis laboratory services using a systems
r
fo kno .co
1. Kent, Kubica GP. Public health mycobacteriology a guide for approach. Recommendations from CDC and the Association of
level III lab. Atlanta, GA: U.S. Department of health and human Public Health Laboratories Task Force on Tuberculosis

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services, Public health services. Center for disease control. 1985. Laboratory Services. MMWR Recomm Rep 2005;54:1-12.
p. 64-8.
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la M dkn
11. Almeida D, Rodrigues C, Udwadia ZF, Lalvani A, Gothi GD,
2.
i
Damato JJ, Collins MT, Rothlauf MV, McClatchy JK.
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Detection of mycobacteria by radiometric and standard plate
Mehta P, et al. Incidence of multidrug–resistant tuberculosis in
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3.
si ted w.m
Anargyros P, Astill DS, Lim IS. Comparison of Improved
Source of Support: Nil, Conflict of Interest: None declared.
F o s w
BACTEC and Lowenstein-Jensen media for culture of

PD te h (w
h is si
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• There is no data on follow-up of these patients.
Authors’ Reply: The follow up of patients have been included in the results section [Page 3, para 2]
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