Sensors and Actuators B 193 (2014) 715–722

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Sensors and Actuators B: Chemical

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Novel phage-piezoelectric sensor for rapid drug susceptibility testing

of Mycobacterium tuberculosis
Xianwen Mi a,b , Fengjiao He a,∗ , Meiyu Xiang a , Yan Lian a , Songlin Yi c
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
b
Huaihua Medical College, Huaihua 418000, Hunan, China
c
Hunan Institute of Tuberculosis Control, Changsha 410006, China

a r t i c l e i n f o a b s t r a c t

Article history: Novel phage-piezoelectric sensor was constructed for rapid drug susceptibility testing based on the pro-
Received 4 October 2013 posed method of phage amplified multichannel series piezoelectric quartz crystal sensor (PA-MSPQC) for
Received in revised form the detection of Mycobacterium tuberculosis and criterion of resistant strains by CLSI. Under the condition
19 November 2013
of anti-TB drugs, the resistant M. tuberculosis survived, but the sensitive strains can be killed. According to
Accepted 20 November 2013
Available online 11 December 2013
the criterion of resistant strains by CLSI, the 105 cfu of strains with frequency shift less than 121 Hz were
resistant to rifampin, isoniazid and ethambutol, 92 Hz to streptomycin. The sensitivity and specificity of
557 strains drug susceptibility test by proposed methods are 91% and 93%, respectively, compared with
Keywords:
Mycobacterium tuberculosis the mycobacteria growth indicator tube (MGIT) method. But the turnaround time of proposed method is
Mycobacterium smegmatis no more than 35 h, far less than that of MGIT 960 methods. It will find application in clinical test.
Phage D29 © 2013 Elsevier B.V. All rights reserved.
Drug susceptibility testing

1. Introduction polymorphism (PCR-SSCP) technology [6,7], LCD array technology

The reasons for the pandemic emergency of TB are AIDS
epidemic and the appearance of multi-resistance strains. WHO
recommended the directly observed therapy, short-course strat-
egy (DOTS) measurement for controlling TB in the whole world.
It is critical to DOTS strategy that the Mycobacterium tuberculosis
(M. tuberculosis) are rapidly detected and patients are rationally
administrated based on drug susceptibility testing [1].
So far, there are two main susceptibility testing methods of
M. tuberculosis, including molecular biology method and pheno-
type analysis method. The principle of the detection method for
drug susceptibility testing based on molecular biology is that: the
encoding gene mutation of the target molecule aimed by the drug
will leads to the lost sites of drug action, and resulted in resistant
stains to drugs. The resistant strains can be detected by deter-
mining the presence of drug-resistance mutation with molecular
biology method. The molecular biology method for drug sus-
ceptibility testing includes polymerase chain reaction–restriction
fragment length polymorphism (PCR-RFLP) method [2,3], nested
PCR amplification [4], denaturing high-performance liquid chro-
matography (DHPLC) method [5], PCR-single strand conformation
∗ Corresponding author. Tel.: +86 137 87799232/+86 731 88272269;
fax: +86 731 88808630/+86 731 88055818.
E-mail address: fengjiao87799232@hotmail.com (F. He).
with DNA sequencing method [8] and molecular beacons meth-
ods [9,10]. These methods were rapid and sensitive [11–15]. They
are specially suits the susceptibility testing of mycobacteria with
slow growth rate. However, their molecular mechanisms in anti-
mycobacterial agents have not been elucidated and it is difficult to
design a rapid diagnostic test to detect all resistant tubercle bacilli,
and then to develop more effective therapeutics method for treat-
ing multidrug-resistant M. tuberculosis [16–18]. The sensitivity and
specificity of the molecular biology method are affected by the state
of sample [7], and inhibitors and the amount of bacteria. These
will easily result in false-negative results. Meanwhile, non-specific
amplification products of polluting bacterial DNA decreased detec-
tion specificity.
The drug-resistant detection method of M. tuberculosis based
on phenotypes analysis include agar proportion method, mini-
mum inhibitory concentration (MIC) method [19,20], automated
mycobacterial culture systems and phage culture method. Most of
their detection time were limited by generation time of M. tuber-
culosis(18 h). Among them, agar proportion method is prescript
standard method for susceptibility of M. tuberculosis by CLSI, but
it is time-consuming (28 days), and complicated to operation, and
the test results are easily affected by artifacts. The MIC method is
based on the lowest concentration of an antimicrobial to inhibit the
visible growth of a microorganism (28 days). Automated mycobac-
terial culture systems such as BACTEC 460 TB, BACTEC MGIT 960
and ESP, showed good sensitivity and shortened the detection time

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716 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722
to 10 days for detection of M. tuberculosis in clinical specimens [21].
This cannot meet the clinical requirement for diagnosis. Phage cul-
ture method was based on the fact that the phage D29 can specifi-
cally infect the viable M. tuberculosis and Mycobacterium smegmatis,
and the detection of resistant M. tuberculosis was converted to the
detection of M. smegmatis by solid agar culture. So detection time
in phage culture method was shorted when compared with agar
proportion method and MIC method [19,20]. The detection speed
of M. smegmatis is much faster than that of M. tuberculosis. The
luciferase reporter phage method can improve the sensitivity of
the method. But the luciferase reporter phage (phAE142) requires a
special propagating strain of M. smegmatis (mc2 4502) as host cells
[22–24]. But this method was limited by solid agar culture [25–28],
time-consuming and complicated to operation.
Recently, Dickert fabricated the mass sensitive sensor combing
bio-mimetic imprinted surfaces with quartz crystal microbalances
for directly detection of the yeast and bacterial cells [29]. The
multi-channel series piezoelectric quartz crystal(MSPQC) sensor
technology has been applied to detecting microorganism based on
the changes of electrical parameters in the medium during the
growth process [30,31]. Ren developed a new method for rapid
detection and antibiotic susceptibility testing of M. tuberculosis,
and the turnaround time of M. tuberculosis detection has been
shortened to 7 days [32,33]. However, it is difficult to further
shorten the turnaround time due to long generation time (18 h)
for M. tuberculosis. In addition, the PQC sensor for the detection
of microorganism is lack of specificity. Our previous work pro-
posed a phage amplified–MSPQC method for the detection of M.
tuberculosis, which shortened the turnaround time to 30 h [34], and
improved the specificity of the MSPQC method.
In this paper, based on criterion of resistant strains of M.
tuberculosis proposed by CLSI and our previous work, a novel
phage-piezoelectric sensor for rapid drug susceptibility testing of
M. tuberculosis was proposed. In this method, the turnaround time
was shortened to 30 h. As the phage D29 can only specifically infect
the viable M. tuberculosis and M. smegmatis, it was more special than
MSPQC method. The drug susceptibility testing of 557 M. tubercu-
losis isolates were detected by proposed method and mycobacteria
growth indicator tube (MGIT) method at the same time. The results
showed that there was no significant difference between two meth-
ods. But the detection time was shorted to 35 h, far less 110 h of
MGIT method.
2. Experimental section
2.1. Reagents and isolates
M. tuberculosis (H37 Ra) was bought from National Institute for
the Control of Pharmaceutical and Biological Production. M. smeg-
matis (ATCC607, dry powder) and phage D29 (dry powder) were
purchased from Institute of Microbiology Chinese Academy of Sci-
ences. M. tuberculosis clinical isolates were collected from the
Hunan Institute of Tuberculosis Control.
Anti-tuberculosis drugs were purchased from Sigma. The con-
centrations of stock and working solution were prepared according
to the CLSI procedure. The concentration of stock solution of iso-
niazid, rifampicin, streptomycin and ethambutol were 2, 40, 40,
100 ␮g/mL, respectively, 20 times higher than that of the working
solution [20,35,36].
2.1.1. The preparation of M. smegmatis suspension
First, M. smegmatis was inoculated on L-J slants and incubated at
37 ◦ C. After 40 h, pure lawns were scraped from slants by sterile ring
vaccination and placed into a sterile porcelain mortar, ground and
mixed with the detection medium in the porcelain mortar. Finally,
108 cfu/mL of M. smegmatis suspension was made for use according
to McFarland’s turbidity.
2.1.2. Preparation of clinical bacterium suspension
The clinical isolates of positive M. tuberculosis were inoculated
on the L-J slants, incubated at 37 ◦ C. After 28 days, single colonies
were scraped from slants by sterile ring vaccination and placed into
a sterile porcelain mortar. M. tuberculosis suspension of 0.5 McFar-
land’s (1.5 × 108 cfu/mL) unit was made for susceptibility testing.
2.1.3. Preparation of phage solution
Phage D29 was streaked on the M7H9 plate containing M. smeg-
matis. After 24 h, the plaques were collected with M7H9 medium.
The phage solution was 10-fold dilution series. Then, 1.0 mL of
the different titer phage solution was mixed with M. smegma-
tis solution, and incubated 24 h at 37 ◦ C. The titer of phage D29
was calculated according to the plaque number on different M7H9
plates and dilution. The concentration of phage D29 was diluted to
107 pfu/mL before use.
2.2. Detection system
2.2.1. MSPQC system
The MSPQC system was self-designed product in our lab [30].
The block diagram of the MSPQC system is shown in Fig. 1. This
system consists of three major components. Part I is an array con-
sisting of 32 quartz crystals sensing growth signal of microbal.
Label 1 is detection cells, and label 2 is piezoelectric quartz crys-
tal. Part II is a signal processing system of the sensors; Part III is a
data processing system, which sets experimental parameters and
records the experimental data.
This system is sensitive to the change of electrical parameters in
solution [37]; the value of frequency shift can be expressed as Eq.
(1):
F02 Cq (42 F02 Cs2 Y + 4F0 Cs G − YG2 )
F =  2 × G (1)
42 F02 Cs (C0 + Cs ) − 2F0 C0 YG + G2
• F is the frequency shift (F0 is the fundamental frequency of the
piezoelectric crystal);
• C0 and Cp are static capacitance and motional capacitance of the
crystal, respectively;
• G and Cs are the conductance and capacitance of solution, respec-
tively. G = , Cs = ε + Cp , ( is the cell constant. X the specific
conductivity. ε the solution permittivity. Cp is the parasitic capac-
itance between the leading wires of the electrodes).
• Y is a parameter related to phase shift of the oscillator.
Eq. (1) could be simplified as Eq. (2) under the experimental
condition [34]:
F = −kG = −2.9G (2)
This is the basic principle of MSPQC analysis.
MGIT 960 was produced by Becton & Dickinson Corporation.
2.2.2. Experimental procedure
A volume of 1.0 mL M. tuberculosis positive clinical isolate
suspension was individually transferred to five reaction tubes
containing 0.1 ␮g/mL isoniazid, 2.0 ␮g/mL rifampicine, 2.0 ␮g/mL
streptomycin, 5 ␮g/mL ethambutol and a medium blank tube,
respectively. The mixed solutions were incubated for 48 h. A vol-
ume of 0.2 mL 107 pfu/mL phage D29 solution was added to reaction
tubes and infected viable M. tuberculosis for 1 h. FAS solution with
final concentration of 10 m mol/L was added to reaction tubes and
 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722
Fig. 1. Block diagram of a multichannel series piezoelectric quartz crystal sensor system.
reacted for 5 min, 10 mL of the M7H9 medium was then added for
susceptibility testing.
A volume of 1.0 mL sample solution was added to different
detection cells containing 4.0 mL of detection medium, 0.5 mL of
108 cfu/mL M. smegmatis solution, and then placed into the MSPQC
system for detection. For comparison, 1.0 mL of sample solution
was inoculated in a growth indicator tube of MGIT 960, and the
results were obtained after inoculated for 6 weeks in the MGIT 960
system.
All experiments described here were operated in Class II biolog-
ical safety cabinets, and all experiment wastes and utensils were
washed after autoclaving.
3. Results and discussions
3.1. Principle of phage-piezoelectric method for detection of drug
susceptibility testing of M. tuberculosis
3.1.1. The principle of the phage-piezoelectric sensor for detection
of M. tuberculosis
The turnaround time is mainly depends on bacterium gen-
eration time. The turnaround time of M. tuberculosis by the
piezoelectric sensor method is 7 days because of its 18 h generation
time [33]. To solve this problem, the phage-piezoelectric sensor
is constructed for the rapid detection of M. tuberculosis, and its
turnaround time are 36 h [34]. This method was based on the fact
that M. tuberculosis and M. smegmatis can all be infected by the
phage D29, and they can protect the phage D29 from being killed
by FAS. The phage which added to the sample was transferred into
the detection cells by the M. tuberculosis as a carrier. The amount
of phage D29 which transferred into the detection cell was propor-
tional to the concentration of M. tuberculosis in the sample. Then,
717
the progeny phage D29 was released by the creaking host cell M.
tuberculosis in detection medium. The progeny phage D29 under-
went a rapid cycle process of infection, replication and lysis in M.
smegmstis. The cycles of phage D29 constitutes the biological chain
amplification reaction, and causes complete inhibition of M. smeg-
matis growth. The number of phage D29 cycles to completely inhibit
the growth of the host cell was linear to the negative logarithm of
M. tuberculosis in the samples. So, the growth state of M. smegma-
tis is related to concentration of M. tuberculosis in the sample. The
conductive ingredients in M7H9 medium were consumed by M.
smegmatis for growth, which resulted in the conductivity change
of the medium. This change was monitored by phage-piezoelectric
sensor. The response curve of M. tuberculosis at different concen-
tration in M7H9 medium by phage-piezoelectric sensor was shown
in Fig. 2.
The phage-piezoelectric sensor was rapid, sensitivity, secu-
rity and economy. The turnaround time was greatly improved by
using MSPQC and the transformation of detection of M. tuber-
culosis into M. smegmatis and, in which phage D29 acted as a
bridge.
3.1.2. The construction of phage-piezoelectric sensor for rapid
drug susceptibility testing
The principle of the phage-piezoelectric sensor for the detec-
tion of resistant strains of M. tuberculosis was shown in Fig. 3.
Based on the advantages of the phage-piezoelectric sensor and
the characteristics of phage D29 only infected viable bacterium,
the phage-piezoelectric method for the drug susceptibility testing
was constructed according to the criterion of resistant strains of M.
tuberculosis proposed by CLSI. The basic idea was that: when the
clinical isolates are treated with drugs at a critical concentration
prescribed by CLSI, the resistant strains were survived from the
718 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722
Fig. 2. The response curve of M. tuberculosis at different concentration by phage-
piezoelectric method: (a) 0 cfu/mL; (b) 102 cfu/mL; (c) 103 cfu/mL; (d) 104 cfu/mL;
(e) 105 cfu/mL;
killing of drug and the sensitive strains were inactivated. The resis-
tant strains can be rapidly detected by phage-piezoelectric sensor.
The whole process can be divided into 5 steps: First, the suspen-
sion of M. tuberculosis (105 cfu) was exposed to the environment in
the presence of the anti-mycobacterial agents for 48 h. The resis-
tant strains were survived from the antibiotic killing and sensitive
strains were killed by drug (a, a ). Second, the viable M. tuberculosis
can be infected by the phage D29, thus protecting phage D29 from
killing by FAS (b); Being killed sensitive strains can’t infected by
the phage D29, phage D29 were killed by FAS (b ). Third, sample
solution was transferred to detection medium after 10-fold dilu-
tion to eliminate the role of FAS, and phage D29 in viable resistant
M. tuberculosis replicates and ultimately lyze their host cells(c); for
sensitive strains, no phage D29 in sample solution (c ). Forth, the
released phage infected M. smegmatis host and undergone a rapid
cycle process of infection, replication and lysis (d); For sensitive
strains, no phage was amplified and no M. smegmatis was infected
by phage D29 (d ). Finally, the phage-piezoelectric signal of resis-
tant strains was obtained because a large number of phage D29
Fig. 3. Flowchart of the phage-piezoelectric sensor for the detection of resistant strains of M. tuberculosis.
Fig. 4. The phage-piezoelectric curve of resistant strains and sensitive strains: (a) M.
smegmatis control; (b) sensitive strains; (c) resistant strains; (d) phage D29 control.
were proliferated, and M. smegmatis growth was inhibited (e); for
sensitive strains, M. smegmatis grown well (e ).
The phage-piezoelectric curves of drug susceptibility testing
were shown in Fig. 4. Curve a showed phage-piezoelectric response
curve of 0 cfu/mL M. tuberculosis with frequency shift more than
350 Hz, and it’s essentially the growth curve of M. smegmatis in
detection cells. curve b showed phage-piezoelectric response curve
of 105 cfu of sensitive M. tuberculosis with frequency shift more than
140 Hz, and it’s one of the sensitive strains that its frequency shift
is close to that of the criterion of clinical concentration; curve c
demonstrated phage-piezoelectric response curve of 105 cfu resis-
tant M. tuberculosis with the frequency shift of 30 Hz; curve d is
phage D29 control with concentration of 105 pfu/mL.
3.2. The effect of medium on the turnaround time and sensitivity
of detection method
M. smegmatis can grow in YC medium and M7H9 medium. To
obtain more sensitive and earlier piezoelectric signal, the response
 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722
Fig. 5. Growth curve of 105 cfu/mL M. smegmtis in YC and M7H9 medium
signals of piezoelectric sensor of M. smegmatis in different media
are analyzed. The piezoelectric responsive curves of M. smegmatis
in YC medium and M7H9 medium are shown in Fig. 5. The curve
a is the growth signal curve of M. smegmatis in YC medium. The
level stage of the curve a means M. smegmatis utilize the non-
conductance ingredients in anabolic. For the downward phase of
curve a reveals that M. smegmatis release the conductive ingre-
dients in their catabolism after 30 h, such as CO2 . Therefore, the
concentration of hydrogen ions caused by the saturated carbon
dioxide is 1.3 × 10−4 mol/L, as shown in Eq. (3).
   
H+ = Ka1 × C = 4.3 × 10−7 × 0.04 = 1.30 × 10−4 mol/L
Because the molar conductance of hydrogen ion in dilute solu-
tion is 350 S cm2 /mol, the conductivity of hydrogen ion in detection
cell is 4.55 × 10−5 (S/cm), as shown in Eq. (4):
C × 350 × 1.3 × 10−4
= = = 4.55 × 10−5 (S/cm)
1000 1000
The constant of the detection cell in this experiment is 0.978 cm,
and the conductance caused by hydrogen ion can be expressed as
Eq. (5):
A less than that of critical concentration. At 95% confidence interval
L= = 4.55 × 10−5 × 0.978 = 4.5 × 10−5 (S)
L
The frequency shift of a piezoelectric sensor caused by hydrogen
ion is shown by Eq. (6):
H = −2.9 × L = −2.9 × 45(S) = −130.5 (Hz)
The results calculated by the mentioned equations further
prove that the frequency shift (130 Hz) of the piezoelectric sen-
sor is obtained due to the saturated carbon dioxide resulted from
complete decomposition of carbohydrates in water during the
catabolism.
The growth state of M. smegmatis in M7H9 medium is show in
the curve b. The curve b including the rising phase, turning point (T),
and plateau phase. The rising phase indicates that the concentra-
tion of M. smegmatis is increased along M. smegmatis. Therefore, the
value of frequency shift rose up, and the speed of frequency shift-
up gradually increased from the point of inoculation to 30 h. The
reasons are that M. smegmatis can specifically utilize the conduc-
tive ingredients (e.g., citrate, calcium, and iron ion), thus resulting
in the increase frequency shift in anabolism process. As a mat-
ter of fact, the concentrations of citrate, calcium, and iron ion are
3.8 × 10−4 , 1.0 × 10−3 , and 8.2 × 10−5 mol/L in the M7H9 medium,
respectively, and the citrate, calcium, and iron ions consumed by
M. smegmatis can give rise to 43, 334, and 16 Hz frequency shift
719
according to the equations 3, 4, 5. Additionally, since the phos-
phate buffer system neutralized the hydrogen ion, the frequency
shift was not affected by hydrogen ion caused by CO2 in catabolism.
All the results imply that the curve b is a signal in piezoelectric
sensor indicated anabolism, not catabolism. The turning point in
curve b demonstrates that (1) the conductive ingredients were
entirely consumed in the only presence of M. smegmatis; (2) all
M. smegmatis were infected by phage D29 when M. smegmatis and
phage D29 coexisted in the detection medium. After turning point,
the frequency shift arrived at the maximum. Beyond the turning
point, medium conductance no longer changed, the frequency shift
seemed to level off even when the time is up to 45 h, and a steady
frequency shift value is obtained.
The turnaround time of curve b is 25 h, faster than that of the
curve a (50 h). In addition, the intensity of the curve b is 350 Hz,
and much more than that of the curve a (130 Hz). Based on the
comparison between the curves a and b, anabolism of M. smegma-
tis in M7H9 medium has earlier and more sensitive response than
that in YC, and the as-prepared M7H9 medium is more suitable for
piezoelectric detection of M. smegmatis.
3.3. The criterion for resistant M. tuberculosis
3.3.1. The criterion of resistant strains for rifapicin, isoniazid,
ethambutol
According to the agar proportional method for drug susceptibil-
ity testing for rifapicin, isoniazid, ethambutol proposed by the CLSI,
drug-resistance was defined as the growth on tubes containing
drug greater than 1% of that on drug-free medium for rifapicin, iso-
niazid, ethambutol [38]. For rifapicin, isoniazid, ethambutol, when
(3) the concentration of M. tuberculosis in drug free is 105 cfu/mL, the
critical concentration of the viable bacterium between the sensi-
tive strains and resistant strains is 103 cfu/mL after drug treatment;
the average of frequency shift and standard deviation of 11 paral-
lel critical concentration at 103 cfu/mL are 171 Hz (138, 148, 153,
160, 167, 172, 178, 186, 189, 195 and 199) and 20 Hz by phage-
(4) piezoelectric method, respectively. The frequency shift was linear
to the negative logarithm of M. tuberuculosis concentration [34].
The higher the concentration of M. Tuberculosis was, the lower the
frequency shift of page-piezoelectric method was. So, the isolate
can be judged as the resistant strain when its frequency shift is
(5) range (x̄ ± 2Sd , 131–213), it is defined as resistant strains when the
frequency shift of the sample is less than 131 Hz; while sensitive
strain more than 131 Hz.
(6) 3.3.2. The criterion of resistant strains for streptomycin
According to the agar proportional method for drug suscep-
tibility testing for streptomycin proposed by the CLSI, resistance
was defined as the growth on drug containing tubes greater than
10% of the growth on drug free medium for streptmycin [38]. For
streptomycin, the critical concentration of the viable bacterium
between the sensitive strains and resistant strains is 104 cfu/mL
M. tuberculosis after drug treatment. The average frequency shift of
11 parallel sample concentration at 104 cfu/mL M. tuberculosis was
122 Hz (101, 104, 105, 112, 118, 123, 126, 135, 136, 139 and 144 Hz),
and standard deviation was 15 Hz. When the frequency shift of the
sample is less than 92 Hz, it is defined as resistant strains at 95%
confidence interval range (x̄ ± 2Sd , 92–152); more than 92 Hz, as
sensitive strain.
3.4. The comparison of the results of drug susceptibility testing
for clinical isolates by phage-piezoelectric method and MGIT 960
The susceptibility testing of clinical strains, collected by Hunan
Institute of Tuberculosis Control (Changsha China), was performed
720 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722

Table 1 by the phage-piezoelectric method and MGIT 960 method. The

Comparison of susceptibility to rifampicine of clinical strains by the phage-
experimental procedure for the collection, numbering, detection
piezoelectric method and MGIT method.
and analysis were designed according to the principle of double
Phage-piezoelectric MGIT960 Total blind. Tables 1–4 show detection results of susceptibility testing of
S R clinical isolates to rifampicine, isoniazid, ethambutol and strepto-
mycin, respectively by two methods. In four drugs susceptibility
S 73(a) 4(b) 77
R 8(c) 49(d) 57 testing, the chi-square value of correlation and difference of two
detection results are shown in Table 5. The detection results are
Total 81 53 134
associated with each other at a = 0.05 level (P < 0.005), and there
are no significant difference between the results of two methods at
Table 2 a = 0.05 level (P > 0.25).
Comparison of susceptibility to isoniazid of clinical strains by the phage-
piezoelectric method and MGIT method.

Phage-piezoelectric MGIT960 Total 3.5. The comparison of turnaround time of the

S R phage-piezoelectric method and MGIT method for the
susceptibility testing
S 88(a) 2(b) 90
R 4(c) 45(d) 49
The turnaround times of the phage-piezoelectric method and
Total 92 47 139
MGIT 960 method for the susceptibility testing of M. tuberculo-
sis are stated in Table 6. The turnaround times for four first-line
Table 3
anti-tuberculosis drugs are all less than 35 h by phage-piezoelectric
Comparison of susceptibility to Ethambutol of clinical strains by the phage- method, but they are all more than 110 h by MGIT 960 method. The
piezoelectric method and MGIT method. reasons for this are stated as follows: first, the MGIT 960 method
MGIT960
was monitored the growth state of M. tuberculosis in liqium, and its
Phage-piezoelectric Total
generation time is 18 h. While the phage-piezoelectric method was
S R detected the growth process of M. smegmatis, its generation time is
S 112(a) 2(b) 114 4 h. Second, the detection of M. tuberculosis can be converted to the
R 6(c) 18(d) 24 detection of phage D29 in M. smegmatis cells in which resistant M.
Total 118 20 138 smegmatis was used as carrier of phage D29. Third, the burst size of
phage D29 in M. smegmatis was 15 much more than that the binary
fission of M. tuberculosis in MGIT 960 method, so the chain biomag-
Table 4 nifications of phage D29 in liquid medium can shorten the detection
Comparison of susceptibility to streptomycin of clinical strains by the phage-
time and distinguish the resistant and sensitive strain quickly, but
piezoelectric method and MGIT method.
the role of that doesn’t work in MGIT 960 method. Forth, the phage-
Phage-piezoelectric MGIT960 Total piezoelectric method can sensitively detect conductance changes
S R in M7H9 medium. So, the phage-piezoelectric method can quickly
complete detection and identification of susceptibility testing of M.
S 95(a) 3(b) 98
R 5(c) 43(d) 48 tuberculosis.

Total 100 46 146

Table 5
The chi-square analysis of comparison of clinical strain susceptibility results by the phage-piezoelectric method and MGIT method.

Drug Correlation Difference Sensitivity (%) Specificity (%)

2 p Conclusion 2 P Conclusion

Rifampicine 89.7 <0.005 Y 0.75 >0.25 N 91 93

Isoniazid 113 <0.005 Y 0.16 >0.50 N 96 96
Ethambutol 85 <0.005 Y 1.125 >0.50 N 95 96
Streptomycin 111 <0.005 Y 0.125 >0.25 N 95 94

Table 6
the comparison of turnaround time of drug susceptibility testing by the phage-piezoelectric method and MGIT method.

Drug Result Number of samples Phage-piezoelectric method MGIT 960 method

Turnaround time (h) Turnaround time (h)

Mean Range Mean Range

Rifampicine R 73 25 18–30 240 115–402

S 49 30 25–35 162 112–264
Isoniazid R 45 22 18–30 210 120–420
S 88 30 25–35 150 110–240
Ethambutol R 18 25 18–30 230 112–400
S 112 30 25–35 210 120–250
Streptomycin R 43 25 18–30 250 125–420
S 95 30 25–35 180 130–250
 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722
4. Conclusions
The phage-piezoelectric method for rapid drug susceptibility
testing of M. tuberculosis was successfully constructed and applied
in clinical strains. The turnaround time of the phage-piezoelectric
method was shortened to the 35 h, lower than that of MGIT 960
method (110 h). The sensitivity and specificity of the proposed
method were 91% and 93%, respectively, and consistent with that
of the MGIT 960 method. So the phage-piezoelectric method can
replace the MGIT 960 method in rapid drug susceptibility testing
of M. tuberculosis with practical clinical value. The proposed method
is rapid and specifical for the detection of resistant strains. The sen-
sitivity of described method was restricted by efficiency of plating
of phage D29 to M. tuberculosis. The detection and drug suscepti-
bility testing can be simultaneously completed if the efficiency of
plating is improved.
Acknowledgements
This research work was supported by the National Natural
Science Foundation of China (no. 21275042), and National High
Technology Research and Development Program of China (863 Pro-
gram) (no. 2011AA02A107).
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Biographies
Fengjiao He. She received her PhD degree in analytical chemistry at Hunan University
in 1994, PR China. She was a post-doctoral fellow at UCLA from 1997 to 1999. She is
now a professor in the department of Chemistry and Chemical Engineering of Hunan
University. She is interested in development and application of piezoelectric quartz
crystal biosensor in microorganism.
Xianwen Mi. He is a doctoral student of State Key Laboratory of Chemo/Biological
Sensing and Chemometrics, Hunan University, PR China. His main research interest
is in study, development and application of novel piezoelectric quartz sensor for
microorganism detection.
722 X. Mi et al. / Sensors and Actuators B 193 (2014) 715–722

Meiyu Xiang. She received her MS degree in State Key Laboratory of Chemo/Biological
Sensing and Chemometrics, Hunan University, PR China. She is interested in biolog-
ical and chemical sensors for applications.
Yan Lian. He is a doctoral student of State Key Laboratory of Chemo/Biological
Sensing and Chemometrics, Hunan University, PR China. His main research interest
is in study, development and application of novel piezoelectric quartz sensor for
microorganism detection.
Songlin Yi. He is assistant director technician in Hunan institute of tuberculosis con-
trol. He mainly engaged in detection and the improvement of detection method of
Mycobacterium tuberculosis.