|Year : 2018 | Volume
| Issue : 3 | Page : 369-375
Rapid detection of the commonly encountered carbapenemases (New Delhi metallo-β-lactamase, OXA-48/181) directly from various clinical samples using multiplex real-time polymerase chain reaction assay
Mubin Kazi, Rukhsar Khot, Anjali Shetty, Camilla Rodrigues
Department of Microbiology, P. D. Hinduja Hospital and Medical Research Centre, Mumbai, Maharashtra, India
|Date of Web Publication||14-Nov-2018|
Dr. Camilla Rodrigues
Department of Microbiology, P. D. Hinduja Hospital and Medical Research Centre, Veer Savarkar Marg, Mahim (West), Mumbai - 400 016, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Resistance due to New Delhi metallo-β-lactamase (NDM) and OXA-48/181 continues to emerge as a threat which is associated with nosocomial outbreaks and is a serious healthcare concern. Phenotypic detection being laborious and time-consuming requires rapid detection of NDM and OXA-48/181, which is achieved through real-time polymerase chain reaction (RT-PCR). Materials and Methods: In this study, RT-PCR assay was developed to simultaneously detect NDM and OXA-48/181. The assay was validated on 102 non-duplicate, phenotypically characterised clinical samples. Results: The assay showed a sensitivity and specificity of 97% and 100% for the detection of carbapenemases in comparison to conventional PCR. The in-house developed multiplex RT-PCR would help to rule-in the presence of NDM and OXA-48/181. Conclusions: Rapid detection of these carbapenemases would be assist in better patient management, in terms of accurate antimicrobial treatment, help in cohorting infected from uninfected patient to prevent spread.
Keywords: Carbapenemases, infection control, New Delhi metallo-β-lactamase, OXA-48-181, patient management, real-time polymerase chain reaction, rule-in
|How to cite this article:|
Kazi M, Khot R, Shetty A, Rodrigues C. Rapid detection of the commonly encountered carbapenemases (New Delhi metallo-β-lactamase, OXA-48/181) directly from various clinical samples using multiplex real-time polymerase chain reaction assay. Indian J Med Microbiol 2018;36:369-75
|How to cite this URL:|
Kazi M, Khot R, Shetty A, Rodrigues C. Rapid detection of the commonly encountered carbapenemases (New Delhi metallo-β-lactamase, OXA-48/181) directly from various clinical samples using multiplex real-time polymerase chain reaction assay. Indian J Med Microbiol [serial online] 2018 [cited 2019 Aug 18];36:369-75. Available from: http://www.ijmm.org/text.asp?2018/36/3/369/245391
| ~ Introduction|| |
Resistance due to carbapenemases continues to emerge as a threat and is also associated with nosocomial outbreaks that have led to a serious healthcare concern, worldwide. The increasing rate of mortality, associated due to carbapenem resistance resulting in therapeutic failure, is a major challenge. Resistance to carbapenem can be due to the production of carbapenemases, loss of outer membrane porins or efflux mechanisms. Outbreaks due to carbapenemases have been recorded in several countries around the globe; moreover, infections due to New Delhi metallo-β-lactamase (NDM) and OXA-48/181 carbapenemases have become endemic in countries such as India, Morocco, Tunisia, Turkey, Russia, Germany, France and Spain., Recent findings highlight the frequent occurrence of NDM and OXA or NDM plus OXA carbapenemases in Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp.,
Currently, laboratories still practice traditional method for phenotypic characterisation and antimicrobial susceptibility which may be occasionally equivocal for some resistant strains. In addition, the turn-around time is not less than 72 h. Moreover, with the increasing number of resistance mechanisms, identifying the most encountered resistance is crucial in a clinical setting. Thus, the need-of-the-hour is to detect the most encountered carbapenemases that cause serious resistance. In the last decades, molecular methods have been a boon in clinical microbiology, where it has bypassed the traditional method of growth. However, gel-based detection (conventional PCR) are less sensitive and laborious as compared to real-time polymerase chain reaction (RT-PCR). RT-PCR is a rapid, sensitive and non-laborious assay used in routine clinical microbiology laboratories for diagnosis of infectious diseases. Although the genetic approach is not always predictive of phenotypic resistances, in specific situations, it may help to optimise therapeutic management, implement infection control practices and prevent further transmission.
In this study, our focus was to develop and validate multiplex RT-PCR assay for detection of the most encountered carbapenemases (blaNDM and blaOXA-48/181) genes directly from clinical samples, avoiding the preliminary phenotypic tests and reducing the time to result. The assay was validated by comparing RT-PCR with conventional uniplex in-house molecular assay. The test can be implemented to 'rule-in' the presence of NDM or OXA-48/181 carbapenemases directly from clinical samples with a turn-around time of~150 min.
| ~ Materials and Methods|| |
Setting, study design and ethics statement
The study was approved by the Institutional Review Board of P. D. Hinduja National Hospital and Medical Research Centre and conducted at Microbiology Section. It is a single-centred study where the need for informed consent was waived off, as the study was performed on banked left-over samples that were anonymised and identified only by laboratory-generated numbers, with no traceability back to patients. All the patient details remained confidential.
Sample collection and processing
A total of 102 non-duplicates, phenotypically characterised, with a volume of ~400 μl samples were collected between May 2015 and January 2016 and stored at −80°C until further use. [Figure 1] highlights the sample-wise distribution of a variety of clinical samples. These samples were classified as carbapenem-resistant, non-carbapenem resistant (extended-spectrum β-lactamases [ESBLs], methicillin-resistant Staphylococcus aureus [MRSA], Enterococcus spp. and pan-susceptible) and culture negative. [Figure 2] highlights the genus-wise distribution of clinical samples depending on the bacterial identification and resistant to carbapenem drug. [Figure 3] highlights the workflow to detect NDM and OXA-48/181 post-sample reception.
|Figure 1: Distribution of clinical samples based on susceptibility to carbapenem drug|
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|Figure 2: Genus-wise distribution of clinical samples depending on the bacterial identification and resistant to carbapenem drug|
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Identification and susceptibility testing
Bacterial identification was performed by biochemical tests routinely performed. Susceptibility was performed using Vitek-2 device (BioMerieux, France) using card N280 for Enterobacteriaceae for antibiotics and N101 for fermenters as per the manufacturer's protocol. Results were interpreted according to the CLSI guidelines.,
Phenotypic detection of carbapenemases
The presence of carbapenemases was screened by employing modified Hodge test (MHT) as performed earlier. The test was performed on carbapenem-resistant bacterial isolates.
DNA extraction from various clinical samples (Method A)
DNA was extracted using QIAamp DNA mini kit (Qiagen) as per the manufacturer's protocol. In case of swabs, 300 μl of sterile distilled water was added and vortexed for 30 s and 200 μl of the sample was utilised for DNA extraction.
DNA extraction from bacterial colonies (Method B)
For all the culture-positive clinical samples and standard strains, DNA was extracted from fresh, well-isolated colonies by employing heat-boil method as described earlier.
Detection of carbapenemases from bacterial isolates
In-house uniplex PCR was performed using the primers enlisted in [Table 1] to detect the presence of NDM and OXA-48/181. Detecting blaNDM and blaOXA-48/181 from bacterial colonies was performed as it has well-established set of primers that have been employed in previous studies. The optimal cycling condition were 94°C for 3 min followed by 29 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s with a final extension of 72°C for 5 min. Band of appropriate size was detected on 2% agarose gel electrophoresis.
Multiplex real-time polymerase chain reaction
Multiplex RT-PCR was performed on ABI 7500 Fast Instrument (Applied Biosystems, USA). Each reaction consisted of 10 μl Premix Ex Taq ([2X, Probe qPCR] from Takara, Bio, Inc.), 0.2 μM of each primer, 0.1 μM of each TaqMan Probe and sterile distilled water to raise the volume to 20 μl. [Table 1] enlists the set of primers and probes employed for multiplex RT-PCR. The optimal cycling conditions were 20 s for 95°C and 40 cycles of 3 s at 95°C and 30 s of 60°C. Each batch was integrated with a positive controls for blaNDM, blaOXA-48/181, human β-globin gene (HBG) and one no template control (NTC).
Interpretation of real-time polymerase chain reaction assay results
For a run to be valid, following conditions were fulfilled: (i) negative controls were below the threshold, (ii) all positive controls (blaNDM and blaOXA-48/181) and HBG had a positive exponential amplification curve and Ct were <35. Unknown samples were positive for respective targets if (i) Ct value was between 10 and 35, (ii) there was an exponential curve, (iii) NTC had no Ct value and (iv) there was amplification of internal control target HBG which was incorporated into the assay to validate the run and also check the presence of inhibitors. Samples with Ct of >36 and <40 were repeated, those samples which showed band on 2% agarose gel electrophoresis would be reported as low positive and those on a repeat with no band on gel electrophoresis were reported as negative. [Figure 4] highlights amplifications plots for the respective targets.
|Figure 4: Amplification plots: (a) New Delhi metallo-β-lactamase, (b) OXA-48/181, (c) human β-globin. The plots above the threshold with exponential curves are positive for the respective targets and below the threshold are negative|
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Cross-reactivity with other bacterial pathogens
The specificity of the primers and probes was checked by employing BLAST program. blaNDM and blaOXA-48/181 were negative with DNA extracted from following: Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Enterococcus faecalis (ATCC 51299), Haemophilus influenzae (ATCC 35056), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC 9150) and Staphylococcus aureus (ATCC 25923). Likewise, the assay was tested on 12.7% (13/102) non-carbapenem-resistant (ESBL producers, MRSA, Gram-positive organism) and 10.7% (11/102) culture-negative clinical samples.
Sensitivity and specificity were calculated by comparing phenotypic and molecular assay using Meta Disc Software Ver 1.4 (Ramón y Caja, Spain). Conventional PCR from bacterial colonies was kept as a reference method to detect the presence of NDM-1 and OXA-48/181; whereas, MHT was performed to detect the presence of carbapenemases.
| ~ Results|| |
In this study of the 102 clinical samples, 76.4% (78/102) were carbapenem-resistant, 12.7% (13/102) were non-carbapenem resistant (ESBL producers [n = 3], MRSA [n = 2], pan-susceptible [n = 3] and Gram-positive organisms [n = 5]) and 10.7% (11/102) were culture negative. 79.4% (81/102) of the samples were monomicrobial producers, whereas 9.8% (10/102) were polymicrobial producers. Genus wise distribution of clinical samples depending on the bacterial identification and resistant to carbapenem drug. Each of the samples were tested against five to six groups of antibiotics and were found resistant to most of them, sparing colistin (100%) and tigecycline (89%).
By phenotypic analysis, among the carbapenem-resistant group, 84.6% (66/78) were carbapenemases producers by MHT and 15.3% (n = 12/78) did not produce carbapenemases.
By genotypic analysis, by Method A (RT-PCR on direct samples), 83.3% (65/78) were carbapenemases producers of which 21.7% (17) were NDM plus OXA producers, 28.2% (22) were OXA producers, 33.3%(26) were NDM producers and 16.6% (13) had none of the targeted mechanism. By Method B (conventional PCR on bacterial isolates), 85% (67/78) were carbapenemases producers of which 32% (25), 29.4% (23) and 24.3% (19) were NDM, OXA-48/181 and NDM plus OXA-48/181 producers, respectively, and 14.1% (11) had none of the targeted mechanism.
[Table 2] highlights the comparison of the target between RT-PCR (direct samples) versus conventional PCR (bacterial isolates) and phenotypic assay.
|Table 2: Comparison of target between real time-polymerase chain reaction (direct samples) versus conventional polymerase chain reaction (bacterial isolates) and phenotypic assay|
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The concordance of the RT-PCR assay for detection of carbapenemases by phenotypic assay was 82.8% (58/70), as 10.2% (n = 8) samples had none of the targeted mechanism detected by MHT (in comparison to conventional PCR). These eight samples that were MHT positive belonged to Acinetobacter spp. (n = 5), Pseudomonas spp. (n = 3) and Enterobacter spp. (n = 1) indicating the presence of different carbapenemases mechanism, that was beyond the scope of study.
The RT-PCR assay showed 97% sensitivity and 100% specificity for the detection of carbapenemases from direct clinical samples in comparison to conventional PCR on bacterial isolates (Method B). In addition, the concordance for detection of NDM, OXA-48/181 and NDM plus OXA-48/181 was 100%, 95% and 89%, respectively. In comparison to conventional PCR, there were three discrepant samples that harboured NDM + OXA (conventional PCR) among which RT-PCR assay failed to detect two samples that harboured OXA (however, detected NDM) and one sample that harboured NDM (however, detected OXA). Similarly, one of the discrepant samples harboured OXA but went undetected by conventional PCR. Among these four samples, two were sputum, one tracheal secretion and one pus sample; these sample type being mucoid might have led to some level of inhibition of the assay.
No false-positive results were observed in the detection NDM and OXA-48/181 targets among the samples that belonged to non-carbapenemases and culture-negative group.
The assay was tested for cross-reactivity with other bacterial pathogens which were negative for the enlisted standard strains.
| ~ Discussion|| |
In the past several years, there are increasing reports of the presence of carbapenemases (KPC, NDM, OXA, VIM and imipenem [IMP]). Among these, NDM and OXA have topped the list in countries such as India, Vietnam and China., In 2009, New-Delhi metallo-β-lactamases (MBLs) were identified since then it has been reported in 70 countries. Till date, 17 variants of NDM have been identified; the presence of NDM was restricted to Enterobacteriaceae; however, it has been reported in Acinetobacter spp. and Pseudomonas spp. as well. The presence of NDM carbapenemases is invariably associated with resistance to β-lactams, aminoglycosides, fluoroquinolones and other antibiotics, sparing aztreonam. In a global surveillance study performed between 2008 and 2012, the majority of NDM-carbapenemases, among the nine participating countries, were isolated from India, Vietnam and Serbia. On further molecular analysis of the NDM-positive isolates from India (n = 71), 94.3%, 2.8% and 1.4% harboured NDM-1, -4 and -6, respectively. In this study, 33.3% (26/78) of the carbapenem-resistant clinical samples harboured NDM alone; the designed [Table 1] probes and primers detects NDM-1 and its variants -3, -4, -5, -6, -7 and -16. OXA-48 carbapenemases, which was first identified in 2001 at Turkish Hospital, have spread widely. Till date, there are 11 variants of OXA-48 (OXA-48, 48b, 54, 162, 163, 181, 199, 204, 232, 244, 245 and 247) that have been detected in different bacterial species across the world. The presence of OXA-48 has shown reduced susceptibility to both carbapenem and broad-spectrum cephalosporins. In this study, 28.5% (22/78) among the carbapenem-resistant clinical samples harboured OXA-48/181 alone, the designed [Table 1] probes and primers detects OXA-48, 48b, 54, 162, 163, 181, 204, 232, 244, 245 and 247 and other variants from different bacterial species. Thus, detection of blaNDM and blaOXA-48/181 genes are crucial since the pathogens carrying these resistant have the potential to spread in hospital and cause nosocomial outbreaks resulting in high mortality rates.
Detection of carbapenemase-producing organism has been challenging, because some may express low levels of resistance. In certain cases, the correlation between minimum inhibitory concentrations (MICs) and the target gene is important as it has a direct impact on patient treatment. In a study, it has been reported that IMP-containing therapy failed to treat infection caused due to OXA-48 producer, and in our experience, it has been observed that the strains having lower MIC are often blaOXA-48/181 carriers. Furthermore, in this study, 19.2% (15/78) harboured that blaOXA-48/181 alone had MIC ranging from 0.5–2 μg/ml, >8 μg/ml and >16 μg/ml for IMP, ertapenem and meropenem, respectively. However, 6.4% (5/78) of the carbapenem-resistant clinical sample that harboured OXA-48/181 carbapenemases alone displayed an elevated MIC of 8–>16 μg/ml, >8 μg/ml and >16 μg/ml for IMP, ertapenem and meropenem, respectively. The elevated MIC levels can be due to co-production of OXA-48 along with other carbapenemases, ESBLs or AmpC or loss of porin or efflux mechanism, detection for which was beyond the scope of the study. Phenotypic reporting has remained same in the last 70 years, detecting carbapenemases through phenotypic assay is laborious, time-consuming and requires separate inhibitor-based confirmatory test (displays variable sensitivity and specificity) to identify each class of β-lactamases.,
NDM-carbapenemases belongs to Class B (MBLs) and OXA-carbapenemases belongs to Class D (carbapenem-hydrolysing class D β-lactamases [CHDLs]) of the Ambler class. Co-production of MBLs and CHDLs within a single isolate has been reported. In our previous studies performed on clinical isolates, among the carbapenemases producers, 15.3% (17/111) of the fermenters and 1.2% (1/81) the non-fermenters harboured NDM plus OXA. Co-production of multiple carbapenemases results in increased level of MIC that has been observed in one of our study, where 16.2% (12/74) of the NDM plus OXA producer had MIC >12 μg/mL which might be due to increased level of hydrolytic activity. In this study, 21.7% (17/78) of the carbapenem-resistant organism harboured NDM plus OXA with MIC levels >16 μg/mL for IMP and meropenem and >8 μg/mL for ertapenem. To phenotypically classify MBLs and CHDLs, several laborious confirmatory test is required which shows a varying rate of sensitivity and specificity towards the detection of Class B and Class D carbapenemases.
Molecular methods are rapid, non-laborious and have higher sensitivity and specificity as compared to phenotypic test and hence remain the reference method to identify carbapenemases gene., There are several assays to detect the big five carbapenemases, but most of them have been evaluated on clinical isolates or rectal swabs. The multiplex RT-PCR assay developed was able to detect 83.3% (65/78) of the most encountered carbapenemases genes among the carbapenem-resistant clinical samples, with a sensitivity of 97% and specificity of 100%. However, 16% (n = 13) of the samples did not harbour the targeted mechanism, which does not signify the organism is susceptible as there might be other mechanism/s under action. The sensitivity and specificity of the assay are in concordance with other studies that targeted the detection of NDM and other genes.,,,, Although the commercial assay such as older version of Xpert MDR detects the predominant carbapenemases, it had some issues in identification of OXA 48/181;,, whereas, assay such as FlimArray (BioFire Diagnostics) is not incorporated with the resistance detection target NDM, OXA, VIM and IMP. In addition, most of the RT-PCR assays are designed to target KPC and NDM; however, the occurrence of KPC is sporadic in India. The clinical utility of the genotypic assay often depends on the local susceptibility pattern and prevalent mechanisms of resistance. In a recent study performed at our institute on healthy individuals, 2.4% and 3.3% harboured NDM-1 and OXA-48/181, respectively, in the stool samples (unpublished data). Thus, it would be wise to screen patients for NDM-1 and OXA-48/181 in countries where their presence is endemic.
The widespread of carbapenemases producing organism is a challenge both for clinicians as well as a microbiologist. In countries, where NDM and OXA-48/181 carbapenemases have become endemic, it is prerequisite to identify a colonised or infected patient. The phenotypic assay having a longer turnaround time and molecular assays covering targeted mechanism does not resolve the issue. However, the present scenario does not allow choosing between phenotypic or a molecular assay, but a combination of both would help us identify carbapenem-resistance. Moreover, the in-house developed multiplex RT-PCR assay would detect NDM and OXA carbapenemases from a variety of clinical samples that would assist to 'rule-in' their presence. This would further help to select accurate antimicrobial therapy for good clinical outcome, cohort infected from uninfected ones and prevent further spread.
We thank the National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre, for their encouragement and support. We also thank the hospital staff of the Department of Microbiology for their support and co-operation.
Financial support and sponsorship
This study was funded by the National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]