|Year : 2017 | Volume
| Issue : 1 | Page : 69-73
Detection of ofloxacin resistance by nitrate reductase assay in Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis
Neeta Shrivastava, Reena Set, Sheetal Bankar, Jayanthi Shastri
Department of Microbiology, TNMC and B. Y. L. Nair Charitable Hospital, Mumbai, Maharashtra, India
|Date of Web Publication||16-Mar-2017|
314, 3rd Floor College Building, TNMC and B. Y. L. Nair Charitable Hospital, Mumbai - 400 008, Maharashtra
Source of Support: None, Conflict of Interest: None
Context: Increased use of fluoroquinolones to treat community-acquired infections has led to the decreased susceptibility to Mycobacterium tuberculosis. There is a paucity of data on ofloxacin (OFX) resistance detection by nitrate reductase assay (NRA). Hence, the present study was carried out to find the efficacy of NRA for detection of OFX resistance in M. tuberculosis isolated from extrapulmonary tuberculosis (EPTB) cases. Aims: (1) To compare sensitivity, specificity and median time required to obtain results by NRA with economic variant proportion method (PM) for detection of OFX resistance.(2) To determine the extent of OFX resistance in clinical isolates of M. tuberculosis. Settings and Design: Seventy-three M. tuberculosis isolates from cases of EPTB were subjected to economic variant of PM for isoniazid, rifampicin and OFX. NRA was done for detection of OFX resistance. Subjects and Methods: Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Drug susceptibility test was performed on Lowenstein–Jensen medium with and without drugs. Statistical Analysis Used: Of turnaround time was done by Mann–Whitney test on SPSS (version 19, released in 2010, IBM Corp, Armonk NY),P < 0.05. Results: OFX resistance was seen in nine isolates. The sensitivity and specificity of OFX resistance by NRA was 100% and 96.87%, respectively. Median time required to obtain results by NRA was 10 days as compared to 28 days by PM. Conclusions: NRA is a specific and sensitive method for detection of OFX resistance in resource-restricted settings.
Keywords: Multidrug-resistant tuberculosis, Mycobacterium tuberculosis, Pre-XDR, Proportion method
|How to cite this article:|
Shrivastava N, Set R, Bankar S, Shastri J. Detection of ofloxacin resistance by nitrate reductase assay in Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis. Indian J Med Microbiol 2017;35:69-73
|How to cite this URL:|
Shrivastava N, Set R, Bankar S, Shastri J. Detection of ofloxacin resistance by nitrate reductase assay in Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 26];35:69-73. Available from: http://www.ijmm.org/text.asp?2017/35/1/69/202338
| ~ Introduction|| |
Tuberculosis (TB) remains a major public health problem. Over the past few years, the increased use of fluoroquinolones (FQs) to treat community-acquired infections has led to their decreased susceptibility to Mycobacterium tuberculosis. The inadequate control of drug prescription has made it one of the most common drugs abused in India. Studies have shown that previous FQ use and multidrug-resistant TB (MDR-TB) were associated with FQ resistance in M. tuberculosis isolates. Therefore, it is crucial to maintain information about the FQ susceptibility in different patient populations to guide the selection of the most appropriate treatment. Pre-extensively, drug-resistant TB is defined as 'disease caused by a TB strain resistant to isoniazid and rifampicin and either a FQ or a second-line injectable drug, but not both.' Resistance to ofloxacin (OFX) is considered to be a risk factor for treatment failure and relapse in MDR-TB. Thus, there is a pressing need for early and proper detection of OFX resistant cases.
The proportion method (PM) as described by Canetti et al. requires several weeks to give results. The automated systems are rapid but expensive. For developing countries, there is a requirement of a simple and rapid test that could be performed at ease in all settings.
Nitrate reductase assay (NRA) is an alternative method which was initially developed at the Central TB Research Institute in Moscow, Russia, where it was called Griess method after Griess and Bemerkungen  who discovered the chemistry of this method. It is based on the ability of M. tuberculosis to reduce nitrate to nitrite which can be easily detected by specific reagents which produce a colour change. Hence, results can be obtained faster than by visual detection of growth colonies. The protocol has been standardised by Angeby et al.
A lot of studies on NRA have been done for first-line antitubercular drugs, but very few studies for the second-line drugs (SLDs) are available. In the scenario of an escalating drug resistance, the need for second-line testing has become a necessity. Most of the studies have dealt with pulmonary TB (PTB). Thus, there is a paucity of data on the FQ resistance in extra PTB (EPTB) cases. The present study was carried out to find the efficacy of NRA for detection of OFX resistance as compared to economic variant of PM. The objectives were to compare sensitivity, specificity and median time required to obtain results by NRA with economic variant of PM for detection of OFX resistance and to determine the extent of OFX resistance in isolates of M. tuberculosis.
| ~ Subjects and Methods|| |
The study was conducted in a tertiary health care set-up in Mumbai in 2013–2014 for a period of 1 year. Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Since the study was on isolates, a waiver of consent was used. The study was approved by the Institutional Ethics Committee.
All isolates were subcultured on Lowenstein–Jensen (LJ) medium for 4 weeks before being studied. The isolates were stained by Ziehl–Neelsen staining and tested by niacin accumulation, nitrate reduction and susceptibility to paranitrobenzoic acid. Drug susceptibility testing (DST) was performed in accordance with the standard method for economic variant of PM for isoniazid, rifampicin and OFX. The LJ medium slants with the critical concentration of the drugs were isoniazid 0.2 µg/ml, rifampicin 40 µg/ml and OFX 2 µg/ml (Himedia,) (Mumbai, India). With each batch of freshly prepared media, H37Ra (ATCC 25177) was tested with the drug-free as well as the drug-containing media. The PM was performed as per Canetti et al. NRA was performed for OFX only according to the protocol described by Angeby et al.
Standard LJ medium with 1000 μg/ml of potassium nitrate was used as a control medium. In the drug-containing slope, 0.2 μg/ml of OFX was added to the control medium along with potassium nitrate. Two loop-full of isolates from fresh cultures on LJ was dispersed in phosphate-buffered saline to obtain the approximate turbidity of McFarland No. 1. Part of the suspension was diluted 1:10 in PBS.
Incubation and reading
A volume of 0.2 ml of the undiluted suspension was inoculated into tubes containing LJ with KNO3 and antibiotics. A volume of 0.2 ml of the diluted suspension was inoculated into three drug-free tubes containing LJ with KNO3. These serve as growth controls. The tubes were incubated at 37°C.
We tested the nitrate reductase test with M. tuberculosis H37Rv (ATCC 27292) as the control strain which gave the positive NRA test on 10th day. Hence, we selected to read the test on 10, 14 and 21 days.
On 10th day, one control tube was selected and 0.5 ml of a mixture of three reagents (50% concentration hydrochloric acid, 0.2% sulphanilamide and 0.1% N-1-naphthyl ethylenediamine dihydrochloride) was added in a ratio of 1:2:2. If the clear reagent mix turned pink colour, the drug-containing tube was tested with the same reagents in the same proportion.
| ~ Results|| |
An isolate was considered resistant if the drug-containing tube produced a colour change more intense than the drug-free tube [Figure 1]. The result was classified as negative (no colour change) [Figure 2] or pale pink to deep red as positive.
|Figure 1: Drug susceptibility testing by nitrate reductase assay method showing Mycobacterium tuberculosis strain resistant to ofloxacin.|
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|Figure 2: Drug susceptibility testing by nitrate reductase assay method showing Mycobacterium tuberculosis strain sensitive to ofloxacin.|
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NRA for OFX resistance with M. tuberculosis H37Ra (ATCC 25177) as the control strain gave positive NRA test on 10th day. Hence, the test was read on 10, 14 and 21 days.
To calculate the difference between the turnaround time (TAT) by the two methods, SPSS software version 19 (released in 2010, IBM Corp, Armonk NY) was used. Mann–Whitney test was used to calculate the P value. P < 0.05 was considered statistically significant.
Seventy-three isolates of M. tuberculosis isolated from EPTB cases were selected. Maximum isolates of M. tuberculosis were obtained from pus (47.9%) followed by fine needle aspirates (36.9%) as seen in [Table 1]. The sensitivity and specificity of detection of OFX resistance by NRA in comparison to economic variant of PM in this study was 100% and 96.87%, respectively, as derived from [Table 2].
|Table 2: Sensitivity and specificity of nitrate reductase assay for ofloxacin in comparison with economic variant of proportion method (n=73)|
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In the present study, the median time for NRA for OFX resistance detection was 10 days which is more rapid than PM method which had a median of 28 days. The difference between the two was statistically significant (P < 0.05). Nearly 61.6% of the isolates gave positive results on the 10th day.
As depicted in [Table 3], 38/73 (52.05%) were sensitive to both isoniazid and rifampicin [Figure 3], 5/73 showed monoresistance to isoniazid, 10/73 (13.70%) showed resistance to rifampicin only and 20/73 isolates (27.39%) were MDR by economic variant PM [Figure 4]. OFX resistance was seen in 9/73 of the isolates. Amongst the twenty MDR-TB isolates, nine were OFX resistant as given in [Table 4] and [Figure 5].
|Table 3: Drug sensitivity of Mycobacterium tuberculosis to isoniazid and rifampicin (n=73)|
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|Figure 3: Drug susceptibility testing by economic variant of proportion method showing sensitive Mycobacterium tuberculosis strain.|
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|Figure 4: DST by economic variant of proportion method showing MDR Mycobacterium tuberculosis strain.|
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|Table 4: Drug sensitivity of Mycobacterium tuberculosis to ofloxacin (n=73)|
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|Figure 5: DST by economic variant of proportion method showing MDR+Ofloxacin Resistant Mycobacterium tuberculosis strain.|
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| ~ Discussion|| |
NRA is a simple and rapid test that could be performed at ease in all settings. Majority of the studies have been done on first-line drugs. We studied the efficacy of NRA for detection of OFX resistance. The sensitivity and specificity of detection of OFX resistance by NRA in comparison to economic variant of PM in our study was 100% and 96.87%, respectively.
The results in our study have correlated with the study of Devasia et al. who reported 100% sensitivity of NRA and 98.7% specificity of NRA as compared to PM for determining OFX resistance. The first study of detection of OFX resistance by NRA was done by Martin et al. in 2005 who found 100% agreement between NRA and PM on 7H11 agar.
In this study, NRA for OFX resistance detection had a median TAT of 10 days (range 10–21 days). Our study has shown similarity in results with most of the major studies of the world, in which the TAT range from 7 to 14 days. A study by Angeby et al. had TAT of 7 days.
The great advantage is that the NRA can be performed on LJ with nitrate incorporated into the medium. The presence of nitrite can be detected by addition of Griess reagent which changes the colour of the culture medium. Hence, results can be obtained faster than by visual detection of growth colonies. DST can be carried out by adding anti-TB drugs to the media at the time of media preparation. Moreover, it does not require any sophisticated instrument for visualisation of the results making it ideal for a resource-poor setting. Hence, laboratories do not have to change completely to another method since adaptation to a novel technology is not always easy for laboratories involved in routine diagnostic work. Furthermore, no extra biosafety concerns are required. Our study clearly confirms that NRA is a specific, sensitive and rapid method for the identification of FQ resistance. Considering the threat that drug-resistant TB poses for the global control of the disease, there is without question also a need for timely, reliable and easy to implement assay for anti-TB agents for a setting already using PM for DST for M. tuberculosis. With the intention of making the transition to NRA even easier, we recommend limiting the reading to two time points after the inoculation.
NRA method needs very little training as it differs only slightly from the conventional method of DST on LJ. Furthermore, it uses only simple reagents that are inexpensive and easily obtained, does not require maintenance of any specialised equipment and requires minimal laboratory spacing and staffing.
Our results in [Table 3], correlate with the study by Sethi et al. who has reported monoresistance to isoniazid in 4.4% and to rifampicin in 8% EPTB cases; 52.7% isolates being sensitive to first-line drugs and was also similar to a study done in Pakistan where 50.1% isolates were sensitive to first-line drugs.
MDR was seen in 27.39% of the isolates in our study [Table 3]. Dalal et al. carried out a study about the trend of drug resistance in TB amongst eight health care facilities in Greater Mumbai between 2005 and 2013, the overall MDR-TB cases were 29.4%. It was 39.4% in the period of 2005–2007 and 27.8% in 2011–2013 which is similar to our findings. Furthermore, a study in Pakistan showed 34.4% of the isolates to be MDR.
The high frequency of chronic cases may explain the high resistance rate in previously treated cases. Since our set-up is a tertiary care hospital in Mumbai, it caters to a large number of population in and around Mumbai as well as many immigrants from many parts of the country. The high population density of Mumbai and an overstretched public with unregulated private health sectors have made the scenario of TB in Mumbai exceptionally grim as compared to other parts of India. In 1991, Uplekar and Shepard  reported that 100 private physicians in Dharavi slums in Mumbai prescribed eighty different anti-TB regimens; amongst which most were both inappropriate and expensive. Agrawal et al. in Mumbai showed 33% MDR in 1995 which rose to 56.5% in 2004.
Nine out of 73 of extrapulmonary isolates showed resistance to OFX in our study [Figure 5]. Our study was close to the study of Pakistan which showed an overall OFX resistance to be 21.01%. There are varied data from all over the world showing overall FQs resistance amongst M. tuberculosis cases.
The mechanism underlying development of FQ resistance in M. tuberculosis appears to involve amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of the A or B subunit of DNA gyrA and several studies have been done on QRDRs of gyrB.
All the nine EPTB isolates which were OFX resistant by economic variant PM method were MDR. OFX resistance was not seen in isolates sensitive to isoniazid and rifampicin or those showing monoresistance to these two drugs. Our study differs from most of the larger studies throughout the world who have found varied degrees of OFX resistance in isolates sensitive to both isoniazid and rifampicin or monoresistant isolates. Huang et al. conducted a study to determine the trend of FQ resistance in M. tuberculosis in Taiwan amongst isolates susceptible to first-line drugs and MDR isolates and found it to be 1.6% and 15.2%, respectively, from 1995 to 2003.
Out of the twenty MDR-TB isolates, nine were OFX resistant as given in [Table 4]. This conforms with the study by Udwadia and Moharil  in a private tertiary care referral hospital of Mumbai in 2014 who have reported OFX resistance of 43.59% amongst MDR-TB cases. A study in Pakistan  conducted between 2005 and 2009 also showed 34.4% MDR of which 42.9% were OFX resistant. There is a paucity of data on the resistance level of FQs amongst EPTB cases, so we have compared it with studies on PTB cases or both PTB and EPTB wherever applicable.
Agrawal et al. in their study in Mumbai found an escalation of ofloxacin resistance amongst MDR from 11.3% in 1999 to 35.2% in 2004. During this period, MDR-TB rates also increased from 52% in 1999 to 56.5% in 2004. Verma et al. have reported 38.2% OFX resistance in MDR-TB and 14.9% in isolates sensitive to first-line drugs from New Delhi in 2011. Dalal et al. concluded that contrary to popular belief, Pre-XDR-TB was the most common form of drug-resistant TB in Mumbai population at approximately 57% of the entire cohort, as compared to MDR being 29.4%. The OFX resistance amongst MDR isolates was 69.1%. These figures clearly indicate the unchecked use of FQs in our country for a lot of bacterial infections. FQs are used as a monotherapy for the treatment of community-acquired pneumonia. It is, therefore, recommended that the testing of FQ should be considered as a part of first-line DST and should be included in rapid diagnostics for the detection of resistant strains. The use of FQs as empirical therapy for community-acquired infections should be brought under control. The fast emergence of resistant strains demand the use of cheaper and rapid diagnostic methods
| ~ Conclusions|| |
That NRA should be used in the testing of FQ drug resistance in TB as it is sensitive and specific and gives faster results. It is further recommended that research on the use of NRA for testing other SLDs should also be conducted.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]