* To whom all correspondence should be addressed. Tel.: +91 4162202546; Fax: +91 4162243092; E-mail: [email protected]
Evolution of
In vitro
Drug Susceptibility Testing of
Pyrazinamide
K. Reena1,2 and V. Shanthi1*
1School of Bio Sciences and Technology, VIT University, Vellore - 632 014, India 2National Tuberculosis Institute, Bangalore, India.
DOI: http://dx.doi.org/10.13005/bbra/1199
(Received: 06 November 2013; accepted: 14 December 2013)
Pyrazinamide is an important drug used in anti tubercular treatment which helps in killing old and semi dormant bacilli which are not metabolically active. Drug susceptibility tests are routinely performed before initiating multidrug drug resistant tuberculosis treatment. However, many Tuberculosis laboratories do not undertake drug susceptibility testing of Pyrazinamide as it is difficult to perform. In vitro drug susceptibility testing of Pyrazinamide requires acidic conditions which hinders the growth of TB bacilli. Hence, Nicotinamide a drug analog of Pyrazinamide was utilized for performing susceptibility testing. A literature review was undertaken and an inventory of in vitro tests for the drug susceptibility testing of Pyrazinamide was conducted. Media specifications and drug concentrations used for the susceptibility testing have also been described in chronological order. Solid culture method was used earlier, which gave consistent results but was time consuming. Liquid culture systems which had a shorter time of detection were also assessed and many studies were undertaken to evaluate their performance with respect to Pyrazinamide testing. The use of growth indicators and biochemical tests for Pyrazinamide testing has also been described.
Key words: Multi drug resistant tuberculosis , Pyrazinamide susceptibility testing, Nicotinamide, LJ Medium, MGIT 960.
Pyrazinamide (PZA) is an important first line anti Tuberculosis (TB) drug which is used along with Isoniazid, Rifampicin and Ethambutol in the DOTS as recommended by the World Health Organization (WHO) PZA is responsible for killing most of the persisting TB bacilli during the initial intensive phase of chemotherapy, allowing treatment to be shortened from 9 months to 6 months1. It is interesting to note that PZA is the most active synthetic analog of vitamin B3 (Nicotinamide) that had inhibitory activity against
Mycobacterium tuberculosis 2. Studies of the analogs of Nicotinamide also led to the discovery of another powerful anti TB drug Isoniazid (INH) and Ethionamide2.
nonsense mutations in the pncA structutral gene or in the putative promoter region of pncA gene have been identified to produce alterations in the active site of the PZase enzyme2.
PZA was first demonstrated to have anti tubercular activity in mouse by Krushner et al 3 in 1948 and by Malone etal 4in 1952. Tarshis & Weed5 in 1953, Shwartz & Mayo, Stenken & Wolvisky6 in 1954 showed that PZA was devoid of in vitro activity against Mycobacterium tuberculosis. However, later studies by McDermott & Tompsett in 1954 revealed that PZA had in vitro activity only in acid medium of Ph5 – 5.57.
Materials and Methods
In vitro methods used for Pyrazinamide susceptibility testing was searched using the internet. Key words such as “Pyrazinamide”, “PZA susceptibility testing” and “antitubercular drug susceptibility” testing were used for the search. Research articles which described novel methods of testing Pyrazinamide susceptibility testing were only reviewed.
in vitro susceptibility testing utilizing solid media
Based on previous studies which revealed the fact that PZA was active on Mycobacterium tuberculosis three acidic solid medium for drug susceptibility testing of PZA were conventionally being used. Steeken & Smiths acid medium (US veterans admin 1960), Lowenstein Jensen (LJ) medium acidified with citric acid and LJ medium acidified with Hydrochloric acid. All the three media had a pre inspissation Ph of 5.4-5.5. It was also found that the LJ medium acidified with Hydrochloric acid yielded the most satisfactory result8.
In 1967 Stottmeir, Beam & Kubica developed a method for determining the susceptibility of tubercle bacilli to PZA in 7H10agar medium in Petri dish9. The technique involved altering the contents of the buffer salts to provide a final Ph of 5.5. The plates were recommended to be read at 20 days of incubation to show the Minimal inhibitory concentration of PZA for Mycobacterium tuberculosis to be 18 – 22 micro perml. The study also showed that the acid environment of Ph 5.5 causes 50% inhibition in the colony count of Mycobacterium tuberculosis
and a considerable reduction in colony size when compared with growth at ph 6.8
In 1969 , G.Cannetti & others described the proportion sensitivity method of drug susceptibility testing for first and second line drugs10. The concentrations of the drugs and the innoculum used were described in detail. The LJ medium used for the PZA susceptibility testing was 4.9.Many strains grew well at this Ph. The innocula used for the PST was also 10 times more concentrated than the usually used for the other drugs. The results of the PZA susceptibility testing was found to be less reliable than that of the other drugs leading to mis classification of resistant strains as sensitive.
Brander in 1972, performed DST of PZA using Nicotinamide a drug analog of Pyrazinamide as suggested by Tan Thiam Hok in 19628. Nicotinamide did not require acidification of the medium for its activity and the growth of Mycobacterium tuberculosis was inhibited at 5mg/ml at neutral Ph. This was utilized to differentiate Mycobacterium tuberculosis from Non Tuberculous Mycobacteria by Kestle & Abbot and by Kubica in 1967. In Tan Thiam Hoks’s method LJ medium of pH around 7 was used and the concentration of Nicotinamide was 100 times greater than is needed for growth inhibition in the acid medium employing PZA8.
Butler & Kilburn in 1982 described a method employing the commercially available 7H10 medium at Ph 5.5 with the use of ADC instead of the routinely used OADC11. Oliec acid had some inhibitory effect on the growth of Mycobacterium tuberculosis, but avoiding OADC also did not always result in growth on some control plates. In 1954, McDermott and Tompsett studied the PZA activity in Tween Albumin and in Oleic acid – Albumin broth and determined the MIC macroscopically (visible bacterial growth in 2 weeks)7.
in vitro susceptibility testing utilizing liquid media
A rapid method to obtain growth of
t
able 1.
Performance of automated liquid culture systems
S.
Author
, publication
Media used
Drug &
No. of isolates
Results/ Major No year Concentration used tested findings 1.
1988, Max Salfinger
B
A
CTEC
vs
PZA 100µg/ml
428 Correlation for & Heifets 12 W ayne’ s test
susceptible 98.2%, for resistant 100%
2.
1995, Mark Miller
B
A
CTEC –
MIC of PZA
36
90% of strains
etal
13
Tw
o pH ;5.5
At pH 5.5 - 50 µg/ml
had an MIC of
&5.8
At pH 5.8 - 400µg/ml
50 µg/ml at pH 5.5 & 5.8
3. 1996, R.R.Cutler B A CTEC vs B A
CTEC- Ph 5.9
34
B
A
CTEC- 80%
etal 14 Biphasic LJ &100mg/l *RPV % with semi
Biphasic LJ- Ph 5.2 &
LJ – ^SPV
solid 66mg/l 66%’RPV 50% Krichners W ayne’
s test- 75%SPV
media & W ayne’ s test 4. 1999M.Mestdagh 15 B A CTEC vs
PZA 100µg/ml
62
92.8% of isolates which
W
ayne’
s test &
sho
wed PZase ne
gati
ve sho
wed
pncA mutations
resistance on B
A
CTEC.
5.
1982 Butler and
Middlebrook
PZA 25 µg/ml Ph5.5
76
90%of the strains gre
w at Kilb ur 16 and Cohn 7H10 medium Ph5.5 when ADC w
as used to pretest the
gro
wth supporti
ve ability .
6. 2004 W ayne’ s test B A
CTEC - 100µg/ml
130 Sensiti vity- A.Krishnamurth y vs
LJ - 100µg/ml
83.33%
etal
17
B
A
CTEC & LJ
Specificity- 100% proportion method 7 2007, P .Singh etal 18 B A CTEC vs B A
CTEC - 100µg/ml
107
LJ as gold standard; Sensiti
vity of
LJ
LJ - 100µg/ml
B
A
CTEC &
W
ayne’
s test- 100% &82.85%,
proportion
Specificity – 98.61%
method & W ayne’ s test 8. 2008, Shubhada MGIT 960
PZA 100µg/ml
33
Sequencing as gold standard
etal 19 vs W ayne’ s B A CTEC sensiti vity -97%,
test & pncA mutations
specificity- 100%,
W
ayne’
s
test- sensiti
vity 92%,specificity 81%
*RPV – resistance predicti
ve v
alue; ^SPV
-susceptible predicti
ve v
traditional “gold standard” for TB culture work which is LJ medium. Studies which have evaluated such automated liquid culture systems are given below in Table 1.
Calorimetric methods using nicotinamide to detect Pyrazinamide resistance
During recent years many studies have evaluated the accuracy and feasibility of calorimetric methods using different growth indicators for detecting resistance in Mycobacterium tuberculosis. These methods appear to be faster than the conventional DST method and are less expensive than the various automated systems available20. Calorimetric methods are based on the color change which occurs as a result of reduction of the colored indicator added to the culture medium. Resistance is detected as change of color in the medium. Different indicators have been evaluated, such as MTT-(3- 4,5- dimethylthiazol- 2-yl), XTT (2,3-bis2-methoxy-4-nitro-5-sulfophenyl- 2H-tetrazolium-5-carboxanilide), Alamar blue, Malachite green and Resazurin.20,31, The DST was performed in microtitre plates with turn around times between 7 and 14 days. The calorimetric assays are performed on culture isolates which means that extra time of 2- 6 weeks would be required for primary isolation.
Use of biochemical tests
The Nitrate reductase assay (NRA) is routinely performed as part of the biochemical identification of M.tuberculosis in most TB laboratories. The technique is based on the property of M.tuberculosis to reduce nitrate to nitrite which is revealed as a color change of the culture media using Griess method30. With the NRA it was possible to obtain results in 7-14 days. Martin etal
in 2005 have shown in a multicentric evaluation study that NRA may be utilized to conduct DST for anti TB drugs21. Based on the same the principle they also evaluated DST for PZA using Nicotinamide (1000, 500,250mg/L) at neutral pH22. The results were compared with MGIT960 which gave sensitivity and specificity of 91% and 94% respectively. Mirabal etal in 2010 also reported similar results when the NRA was used for PZA testing23.
Use of Mods
The Microscopic observation drug susceptibility assay (MODS) uses liquid media and is fast to perform. The method is based on
the principle that M.tuberculosis grows faster in liquid medium and its characteristic growth ‘cord formation’ can be observed through an inverted light microscope. Several studies have used this method to detect resistance to Isoniazid and Rifampicin. Ghiraldi etal in 2011, utilized the MODS assay for PZA susceptibility testing where concentrations of 6.25 – 3200mg/ml was used24. The specificity and sensitivity of MODS and Broth Microdilution method was 100%.
ConClUsion
There are no reliable tests available for the drug susceptibility testing of Pyrazinamide. This is because of the difficulty in developing a test with PZA concentrations equivalent to in vivo concentrations of 16-60µg/ml which are attainable in blood of tissue25. These low concentrations are active in vitro only at pH 5-5 – 5.6 ,and most TB bacilli are inhibited at such low Ph. Studies have shown that more than 10% of the isolates do not grow at this Ph25,35,36,37. In order to overcome this problem Salfinger etal suggested using a high concentration of PZA, of 300 or 400µg/ml at Ph 6.0 – 6.2 in 7H12 broth12. They also reported that some strains even grew well at this pH than at the pH normally used for TB bacilli cultivation. Since the in vitro concentration of PZA is very high, the test results cannot be correlated with pharmacokinetic parameters. Zhang etal have made theoretical assumptions based on Henderson –Hasselbach equations in regard to concentrations of PZA needed to inhibit growth of M.tuberculosis
ml or more employing an innoculum containing approximately 0.4mg(moist wt ) of bacilli per ml. and a 10-colony end point,and proportions of 20% or more on 25µg/ml , or 5% or more on 50µg/ml and 1% or more on 100µg/ml.The efficiency of the four definitions were of the same order28 .
In 2007, H.Zhang etal characterized Pyrazinamidase /Nicotinamidase and found that the monomeric protein had an optimal enzymatic activity at Ph 7.027. In contrast to earlier studies, they demonstrated that both the enzymes had similar hydrolytic activity for the substrates Nicotinamide & Pyrazinamide27.
To avoid the problem of acidic pH in PZA susceptibility testing, Brander in 1972 had developed a PZA susceptibility testing using high concentrations of Nicotinamide (500, 1000, 2000µg/ml) at neutral Ph in LJ medium8. Although this method gave reliable results it was not widely adopted as reported by Y.Zhang & D.Mitchison in 20032. They also pointed that it would be prudent to use the Brander technique in epidemiological studies for determining the prevalence of PZA resistance.
Although the MGIT 960 has been considered to be the “gold standard” for PZA susceptibility, the study by Pamela Chedore etal
points out that the system is prone to give erroneous results38. The false results are mainly due to the use of a lower drug concentration (100µg/ml) supplied by the manufacturer and variations in the size of the inoculu38,39.
In conclusion based on the studies which were reviewed , it is felt that PZA resistance may be determined by using a combination of two in vitro tests32,33,34,40, the Wayne’s PZase enzymatic assay29 and DST using Nicotinamide at high concentrations on LJ medium. A rapid alternative for the DST would be to use any of the calorimetric tests such as MTT, Alamar blue, Resazurin, or Malachite green in liquid medium.
aCknowledgMents
The authors express deep sense of gratitude to the management of Vellore Institute of Technology for all the support, assistance and constant encouragements to carry out this work
referenCes
1. World Health Organization, Treatment of
Tuberculosis: Guidelines, Fourth edition Geneva: World Health Organization Press, 2010.
2. Zhang, Y., and D.Mitchison, The curious
charataristics of Pyrazinamide: a review. Int J Tuberc Lung Dis , 2003; 7(1): 6-21.
3. Mc Kenzie D,N Malone L,Kushner S,Oleson JJ,
Subbarow Y. The effect of nicotinic acid amide on experimental Tuberculosis of white mice. J Lab Clin Med, 1948; 33:1249-1253.
4. Malone L,Schurr A, Lindh H, McKenzie D,Kiser
JS,Williams JH.The effect of Pyrazinamide (Aldinamide) on experimental tuberculosis in
mice. Am Rev Tuberc,1952; 65: 511-518.
5. Trashis MS,Weed WA.Lack of significant in
vitro sensitivity of Mycobacterium tuberculosis to pyrazinamide on three different solid media
.Am Rev Tuberc ,1953; 67: 391-395.
6. Steeken W Jr,Wolinsky E.The anti-tuberculous
activity o pyrazinamide in vitro and in guinea
pig.Am Rev Tuberc 1954; 70: 367-369.
7. Mc DermottW, Tompsett R.Activation of pyrazinamide and nicotinamide in acidic
environment in vitro. Am Rev Tuberc. 1954; 70:
748-754.
8. Brander, A simple way to detecting Pyrazinamide
resistance. Tubercle 1972; 53: 128-131.
9. Stottmeier K.D, Beam R.E, Kubica G.P,
Determination of drug susceptibility of Mycobacteria to Pyrazinamide in 7H10 Agar. Am Rev Resp Dis, 1967; 1072-1075.
10. Canetti G, Wallace Fox, Khomenko. A, Mahler
H.T, Menon.N.K et al, Advances in techniques of testing Mycobacterial Drug Sensitivity, and the use of sensitivity tests in Tuberculosis Control
Programmes. Bull. Wld Hlth Org.1969; 41:
21-43.
11. Butler W.R, Kilburn J.O, Improved method
for testing susceptibility of Mycobacterium
tuberculosis to Pyrazinamide. Journal of Clinical Microbiology ,1982; 1106-1109.
12. Max Salfinger and Leonid B Heifets,
Determination of Pyrazinamide MICs for Mycobacterium tuberculosis at different pHs by radiometric Method. Antimicrobial Agents and Chemotherapy,1988; 1002-1004.
13. Mark.A.Miller Louise Thibert etal, Testing of
Susceptibility of Mycobacterium tuberculosis to Pyrazinamide: Comparison of BACTEC method
with Pyrazinamide Assay. Journal of Clinical
Microbiology ,1995; 2468-2470.
14. Cutler R.R, Wilson P, Villarroel J, Clarke F.V.
of the susceptibility of mycobacteria to Pyrazinamide, conventional, radiometric BACTEC and two methods of Pyrazinamidase testing. Letters in Applied Microbiology, 1997;
24: 127-132.
15. Mestdagh M, Fonteyne P.A, Realini L, etal
Relationship between Pyrazinamide resistance, loss of Pyrazinamidase activity and mutations in the pncA locus in Multi drug resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother, 1999;43 (9):2317-2319.
16. Butler W.R, James O.Kilburn ,Antimcrobioal
Agents and Chemotherapy, 1982; 1106- 1109.
17. Krishnamurthy A, Almeida.D, Rodrigues C,
Mehta A.Comparison of Pyrazinamide drug susceptibility of M.tuberculosis by radiometric BACTEC and enzymatic Pyrazinamidase assay. Indian Journal of Medical Microbiology, 2004;
22(3):166-168.
18. Pushpendra Sing, Clement Wesley, G.P.S.
Jadaun, Sunil Kumar Malonia,R.Dass, P. Upadhyay, J. Faujdar, P.Sharma,P.Gupta etal. Comparitive evaluation of Lowenstein Jensen proportion method,BacT/ALERT 3D System ,and Enzymatic Pyrazinamidase Assay for Pyrazinamide susceptibility Testing of Mycobacterium tuberculosis. J Clin. Microbiol,
2007; 45(1): 76-80.
19. Shubhada Shenai,Camilla Rodrigues, Meeta
Sadani, Nesline Sukhadia and Ajita Mehta. Comparison of Phenotypic and genotypic methods for Pyrazinamide susceptibility testing. Indian Journal of Tuberculosis. 2009; 56:82-90
20. Anandi Martin, Francoise Portaels and Juan
Carlos Palomino.Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta analysis.Journal of Antimicrobiol Chemotherapy, 2007; 59: 175-183.
21. Anandi Martin, Howard Takiff, Peter Vandamme,
Jean Swings, Juan Carlos Palomino and Francoise Portaels. A new rapid and simple colorimetric method to detect Pyrazinamide resistance in Mycobacterium tuberculosis using Nicotinamide. Journal of Antimicrobiol Chemotherapy, 2006;
58: 327-331.
22. Anandi Martin, Andres Cubillos-Ruiz, Andrea
Von Groll, Patricia Del Portillo, Francoise Portaels and Juan Carlos Palomino. Nitrate assay for the rapid detection of pyrazinamide
resistance in Mycobacterium tuberculosis
using nicotinamide. Jurnal of Antimicrobiol
Chemotherapy. 2008; 61: 123-127
23. Niuris C. Mirabal, Scrgio L. Yzquierdo,
Dhihadenys Lemus, Mariela Madruga,Yoslaine Milian etal .Evaluation of Colorimetric methods using Nicotinamide for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis. Journal of Clinical Microbiology, 2010; 2729-2733.
24. GhiraldiL.D, CampanerutP.A.Z,Spositto
F.L.E,Sato.D.N,Leite C.Q.F,Hirata M.Hirata R.D.C and Cardoso R.F. Evaluation o the microscopic observation drug susceptibility assay
for detection of Mycobacterium tuberculosis
resistance to Pyrazinamide. Clin Microbiol
Infect, 2011; 17: 1792-1797.
25. Leonid Heifets , Susceptibility testing of
Mycobacterium tuberculosis to Pyrazianmide. J Med Microbiol ,2002; 51: 11-12
26. Ying Zhang, Sallie Permar and Zhonghe
Sun, Conditions that may affect the results
of susceptibility testing of Mycobacterium
tuberculosis to Pyrazinamide. J Med Microbiol,
2002; 51: 42-49.
27. Hua Zhang,Jiao – Yu Deng, Li-Jun Bi,Ya- Feng
Zhou,Zhi –Ping Zhang, Cheng –Gang Zhang,Ying Zhang and Xian-En Zhang. Charactarization of Mycobacterium tuberculosis nicotinamidase/
pyrazinamidase. The FEBS Journal, 2008; 275:
753-762.
28. Tripathy S.P, .Mitchison D.A, Nair N.G.K,
Radhakrishna. S. and Subbammal S, A comparison of various measures of sensitivity of M.tuberculosis to Pyrazinamide, Tubercle,
1970; 51:375-388.
29. Wayne L.G, Simple Pyrazinamidase and urease
tests for routine identification of Mycobacteria. Am Rev Resp Dis, 1974; 109: 147-151.
30. Humberto R Musa, Marta Ambroggi,Alejandro
Souto and Kristian Angeby K.A. Drug
susceptibility testing of Mycobacterium
tuberculosis by a Nitrate Reductase assay applied directly on microscopy positive sputum samples. Journal of Clinical Microbiology, 2005; 3159-3161.
31. Parissa Farnia, Mohammad Reza Masjedi,Foroozan
Mohammadi, Payam Tabarsei etal. Colorimetric detection of Multidrug –resistant or extensively drug resistant tuberculosis by use of Malachite
Green Indicator Dye. Journal of Clinical
Microbiology, 2007; 46(2): 796-799.
32. Wergren J, Sturgard E, Jureen P, Angeby K,
33. Ying Zhang,Kwok Chiu Chang,Chi-Chiu Leung,Wing Wai Yew,Brigitte Gicquel,Dorothy Fallows,Gilla Kapla, Richard E Chaisson and
Wenhong Zhang. ‘Zs – MDR-TB’ versus ‘Zs –
MDR-TB’ :improving treatment of MDR-TB by identifying pyrazinamide susceptibility. Emerging Microbes and Infections. 2012; 1: 1-4.
34. Ying Zhang.Wing Wai Yew and Micheal R. Barer.
Targeting persisters for Tuberculosis Control. Antimicorbiol Agents and Chemotherapy, 2012: 2223-2230.
35. Ying Zhang, Angelo Scorpio ,Hiroshi Nikaido
and Zhonghe Sun, Role of acid Ph and deficient Efflux of Pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to Pyrazinamide. Journal of Bacteriology, 1999; 2044-2049.
36. Glenn P Morlock, Jack T Crawford, W.Ray Butler
etal Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis Anti microbial Agents Chemotherapy, 2000; 2291-2295.
37. Lina Marcela Aragon, Montserrat Garrigio,
Carmen Moreno,Montserrat Espanol and Pere
Coll. Evaluation of the BacT/ALERT PZA kit in comparison with the BACTEC 460TB
PZA for testing Mycobacterium tuberculosis
susceptibility to Pyrazinamide. Journal of
Antimicrobiol. Chemotherapy. 2007; 60: 655-657.
38. Pamela Chedore,Lina Bertucci, Joyce
Wolfe,Meenu Sharma and Frances Jamieson. Potential erroneous results indicating resistance when using the Bactec MGIT960 system
for testing susceptibility of Mycobacterium
tuberculosis to Pyrazinamide. Journal of Clinical Microbiology.2010; 48(1): 300-301.
39. Babita Sharma, Nita Patel, Bharti Malhotra,Leela
Vyas and Suman Rishi. Comparison of MGIT960 & pyrazinamidase susceptibility testing of Mycobacterium tuberculosis. Indian J Med Res
2010; 132: 72-76.
40. Ernesto Montoro,Dihadenys Lemus,Niuris
Mirabal, Miguel Echemendio etal . Contributions
