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  Table of Contents  
SPECIAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 482-490
 

How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India


Departments of Microbiology and Clinical Hematology, Tata Medical Center, Rajarhat, Kolkata, India

Date of Submission14-Apr-2014
Date of Acceptance06-Jul-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
Sanjay Bhattacharya
Departments of Microbiology and Clinical Hematology, Tata Medical Center, Rajarhat, Kolkata
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167351

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 ~ Abstract 

Development of a reliable, cost-effective cytomegalovirus quantitative polymerase chain reaction (QPCR) is a priority for developing countries. Manufactured kits are expensive, and availability can be inconsistent. Development of an in-house QPCR kit that is reliable and quality assured requires significant effort and initial investment. However, the rewards of such an enterprise are manifold and include an in-depth understanding of molecular reactions, and expertise in the development of further low-cost molecular kits. The experience of an oncology centre in Eastern India has been shared. Hopefully, this would provide a brief roadmap for such an initiative. Staff with adequate understanding of molecular processes are essential along with vital infrastructure for molecular research and development.


Keywords: Cytomegalovirus, standardisation, troubleshooting, validation, viral load


How to cite this article:
Harishankar A, Chandy M, Bhattacharya S. How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India. Indian J Med Microbiol 2015;33:482-90

How to cite this URL:
Harishankar A, Chandy M, Bhattacharya S. How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Oct 19];33:482-90. Available from: http://www.ijmm.org/text.asp?2015/33/4/482/167351



 ~ Introduction Top


In recent years, molecular diagnosis has become the standard of care for the specific diagnosis, prognostication, monitoring of various infections. For polymerase chain reaction (PCR) based molecular diagnosis, two formats are generally used - the qualitative and the quantitative. Currently, both these assays are generally done using real-time PCR format. Real-time PCR equipment are costlier than conventional end-point PCR systems, reagents for real-time PCR (especially the probes) are more expensive, and the number of reactions that need to be done for a QPCR are more than that of an end point PCR. Quantitative or QPCR kits are available from various manufacturers (e.g., Qiagen Artus, R-gene bioMerieux, Cepheid, etc.). Some of these kits are Conformité Européenne (CE) marked and in-vitro diagnosis (IVD) approved.[1],[2],[3] However, these quality standards come at a price, and most commercial assays are found to be too expensive for routine use. For example, the price of cytomegalovirus (CMV) QPCR assay from two commercial stand-alone laboratory service providers are in excess of Rs. 7500 (source SRL Religare and Dr. Lal's Path Labs), a sum of money which is beyond the reach of most people in the developing world. It is noteworthy that in the contract the average monthly income of most Indians is less than the price of a commercial CMV PCR assay (source World Health Organization [WHO]: Gross National Income per capita). There is no doubt that there is a need for a reliable sensitive specific cost-effective assay in the context of resource-poor settings. The objective of this article is to share the experience of developing such an assay along with quality control requirements for accreditation. For the purpose of the article the example of the CMV QPCR assay has been used, but the approach may be used to develop other similar QPCR assays.


 ~ General Requirements for the Development of a Molecular Biology Laboratory Top


The following could be considered as essential for the development of an in-house molecular assay.[4] These equipment and infrastructure are present in the Tata Medical Centre molecular lab where the CMV PCR was developed.

  • Molecular biology laboratory (ideally four separate rooms for nucleic acid extraction, mastermix preparation, PCR, post-PCR analysis such as gel electrophoresis and sequencing)
  • Staff with molecular biology knowledge and experience
  • Biological safety cabinet-type II A2 with ducting especially in the sample handling/nucleic acid extraction room
  • Pipettes (of various dimensions) which are calibrated regularly ideally once in 6 months (once a year a third party external calibration is desirable)
  • Spectrophotometer or a nanodrop to measure the yield and quality of extracted nucleic acid (using 260/280 and 260/230 ratios)
  • Good microbiological practices and good laboratory practices
  • Freezer and fridges for storing samples, reagents, nucleic acid extracts. The fridges should be monitored regularly for temperature control
  • Uninterrupted power supply for PCR systems, biological safety cabinets. Back-up power supply for fridges and freezers
  • Reliable DNA/RNA extraction protocols - manual, semi-automated or automated
  • Real-time PCR system: E.g., ABI 7500 (Applied Biosystems), Rotorgene (Qiagen), Roche (LightCycler), etc
  • Endpoint PCR system (ABI Veriti, Bio-Rad, etc.)
  • Agarose gel electrophoresis system
  • PCR set up box-for mastermix preparation and PCR set-up (addition of mastermix to extract/target)
  • DNA sequencer-optional
  • Standard operating procedure
  • Quality management system: Training and competency assessment of staff, internal quality control, external quality assurance, assay standardisation and validation
  • Consumables for nucleic acid extraction and PCR: Primers, probes, mastermix, quantitative standards, positive controls, pipette tips, PCR tubes, etc., (we have used Sigma-Aldrich for the custom made primers and probes, and Qiagen blood mini kit for extraction).



 ~ Steps Followed in the Development of In-House Assays: Example of Cytomegalovirus Top


The charting of the genomic map of cytomegalovirus for the selection of the polymerase chain reaction target

The PCR target is one where the primers and probes bind. Since any pathogen or non-microbial disease may have multiple potential genetic targets for PCR, it is essential that some thought is given to the selection of the gene target. The target selection may be based on the presence of the target in clinically relevant strains, the conserved nature of the target, a number of copies of the target present in various species or growth stages, etc. Studies have shown human CMV (HCMV) to be a highly divergent species, especially in certain coding regions, and frequent recombination are known to occur.[5],[6],[7] Due to the strain variations seen, there are fewer conserved regions that can be exploited as target genes for detection. Among those which have been used are the envelope glycoproteins, immediate early antigen, DNA polymerase, and Thymidine kinase genes.[7],[8],[9],[10] In our case, we have used the gene target as used in the published article and which is also being used by the Clinical Virology Department at CMC Vellore.

Selecting primers, probes

Once the gene target has been selected, primers and probes for selectively amplifying this region have to be picked. They can be done through literature review and adopting from a previously published article, [Table 1] gives a detailed summary of targets and primer-probes targeting them.[10],[11],[12],[13],[14],[15],[16] Alternatively, they can be designed using commercial or free to use the online software (e.g., Primer 3, Primer Blast and Primer premier). The main criteria being mis-priming, G-C content, 3' G-C clamp and compatibility of annealing temperatures. Once primer-probes have been selected, they have to be verified for specificity towards gene target by using the Basic Local Alignment and Search Tool software.
Table 1: An example set of primers/probe sequences for amplifying CMV DNA

Click here to view


Selection of appropriate specimen (plasma or whole blood)

CMV is a cell associated virus, hence the viral loads done on whole blood are generally higher than plasma viral load However, in active infections the virus is released from leucocytes, endothelial and reticuloendothelial cells into the plasma, thus reflecting plasma load as an indicator for active infections. Moreover, the pre-engraftment phase of hematopoietic stem cell transplant (HSCT) is a period of leukopenia, in such cases plasma viral loads are more representative. Negative plasma viral load, on the other hand, does not rule out active CMV disease and delays detection of low levels of infection. Whole blood viral loads are generally higher, and the specimen is easier to process as no cell or plasma separation step is involved. But, the clinical relevance of enhanced detection of residual viraemia is minimal, and neither is it an accurate predictor of recurrence. As each type of sample has its virtues and woes, the question of which sample to use in a laboratory setting is contextual.[17],[18] Apart from blood, urine, respiratory samples (such as broncho-alveolar lavage), cerebrospinal fluid aqueous humour, etc., are also used for detecting congenital CMV, pneumonitis, encephalopathy, retinitis.[16] In our centre for all quantitative CMV PCR on blood samples, whole blood ethylenediaminetetraacetic acid is used collected in a BD vacutainer.

DNA extraction

One of the very important pre-analytical steps for detection of the virus is its DNA extraction. This step involves isolation of DNA, eliminating the undesirable and potentially PCR inhibiting substances such as heme, protein, salts, etc. Though basic principle involves cell lysis, precipitation of DNA, its purification and elution; certain modifications are done, based on the specimen used. DNA extraction for real-time PCR is done either using automated platforms or manual kit based methods. We have used Qiagen blood mini kit for DNA extraction. Automated systems (such as QIAcube, Qiagen Biorobot, EZ1 DNA extraction) may have high throughput and have a consistent and reproducible recovery. As sample manipulation is kept to a bare minimum, cross contaminations are virtually non-existent. Most systems are closed, which again protects from contaminations. Different automated systems have differential extraction efficiency.[19],[20] However, these systems require capital investment, and if not run to their full capacity, are not very economical. As far as manual extraction method is concerned, plenty of commercial kits are available, each varying in its methodology, cost and time required for extraction. While most kits effectively remove PCR inhibitors, sensitive in case of low copy numbers and are cost-effective, they are laborious, time-consuming and require specialised personnel for handling and processing.

Development of standards for the quantitative polymerase chain reaction assay

The Minimum Information for Publication of Quantitative Real-time PCR Experiments guidelines state that standards which form the curve in absolute quantification methods, should be any one of the following: Synthetic DNA constructs spanning the entire region of the target, amplicon or plasmid DNA construct or linear DNA of the virus suspended in specific biological sample or an internationally recognised biological reference standard.[21] Among the standards mentioned, plasmid constructs are easier to prepare and handle. The copy number of standard is calculated using the formula:[22]

Weight in daltons (g / mol)=(bp size of ds product)

(300 Da ×2nt / bp) Hence : (g / mol) / Avogadro's

number = g / molecule = copynumber (where :

bp = base pairs, ds = double - stranded, nt = nu - cleotides).

However, there may be over quantification, and gross shifts in standard curves by plasmids due to their variable conformations (super coiled, nicked circular, linear) obtained during their extractions. As absolute quantification is completely dependent on the standard curve, such variations could lead to measurement bias of the viral load.[23] WHO along with the National Institute of Biological Standards and Control, in 2010, proposed and established HCMV Merlin strain National Institute for Biological Standards and Control (NIBSC) ID: 09/162 as the first WHO international standard for CMV with a potency of 5 × 106 International units (IU)/mL of nuclease-free water. With the advent of this standard, the standardisation of viral load can be done across platforms. A sample spiked with a known concentration of genomic copy number is also run along with the assay each time. This serves as an assay load calibrator. We have used the WHO international standard as a calibrator for our in-house assay [Figure 1].
Figure 1: Levey-Jennings chart of the performance of the cytomegalovirus polymerase chain reaction assay with the National Institute for Biological Standards and Control calibrator

Click here to view


Real-time polymerase chain reaction to establish viral genomic load

Real-time PCR is the current standard technology for the performance of CMV QPCR. In the non-real-time PCRs traditional method of DNA amplification is followed by electrophoresis of the product in the presence of a DNA intercalating dye like ethidium bromide irradiated by ultra violet light. Southern blot techniques involve hybridization with the specific probe and separating them out in a solid matrix or PCR-ELISA combination wherein, the amplified products were captured on solid phase using digoxigenin or biotin labelled primers and probes. However, these methods have come to be replaced with 'real-time' PCR technique. Real-time PCR, as the name suggests, is the one where, the formation of the amplified product can be observed through its progress. This method dispenses with the need of post-PCR processing seen in the conventional methods. Other notable advantages include its relative rapidity, a greater degree of sensitivity and diminished chances of amplicon contamination, owing to the closed system. The disadvantages include the lack of universal compatibility of the fluorophores (probes) with PCR platforms, its high capital cost (equipment), which impedes its application in the low throughput and resource poor laboratories.

There exist several chemistries with respect to real-time PCRs. These include SYBR green based method, primer based technologies, dual-labelled probes. In our centre, we have used the Taqman based dual-labelled probe although the SYBR green based method has also been briefly described since it is a less expensive option and may be used in the initial phases of PCR development to look for adequacy of primer binding.

Sequence independent DNA labelling dyes (SYBR green I): The fluorophore - SYBR green binds to the minor groove of the double-stranded DNA. It is easy to optimise, less expensive but prone to non-specific binding.[24] In our setting, we have not used this method for CMV PCR assay development but have standardised the CMV PCR assay using the dual-labelled hydrolysis probe chemistry (Taqman). This technology involves the hybridisation of hydrolysis probes targeted within the primer amplification region. The probes are tagged with a reporter dye at their 5' end and a quencher at 3' end. Once the probes binds to the amplicon, the 5'-3' exonuclease activity of Taq polymerase causes its cleavage and hence separation of the quencher from the reporter with a concomitant raise in fluorescence. Taqman is the most extensively studied and used probe owing to its simplicity, ease of optimization and appreciable sensitivity and specificity.

In performing real-time PCR for a target load, two strategies are commonly employed-absolute quantification and relative quantification. Absolute quantification measures the absolute number of genomic copies, which are present in the sample against a set of standards, whereas relative measures the change in copy number of the target over a period of time against a reference matrix. While relative quantification is easier to optimise, absolute copy numbers are preferred as it gives a value that can be universally adopted by clinicians, scientists across different platforms and laboratories. However, standardisation and validation of absolute quantification requires assessment of the assay against international standards provided by the organisation such as the (NIBSC, United Kingdom). In our centre, we have used the Taqman chemistry and used the 1st WHO International Standard for (HCMV), NIBSC code 09/162 (2013 price £352, including packaging and transport). The CMV international standard has been assigned a concentration of 5 × 106 IU when reconstituted in 1 mL of nuclease-free water, based on the results of a worldwide study.

Analysis of cytomegalovirus quantitative polymerase chain reaction assay

The linear analysis of the exponential rise in fluorescence is the norm for analysis in QPCR. These 'calibration curves' measure the efficiency, sensitivity and thereby, the robustness of the assay and are simple and easy to reproduce. In absolute quantification, the curves are the function of the quantification cycle (i.e., PCR cycle wherein the fluorescence exceeds the threshold set), and copy numbers of the standards used.

Certain parameters have to be assessed before calculating the copy numbers in samples.

  • Polymerase chain reaction efficiency: This is defined as the rate of conversion of reagents into the target amplicon by the Taq polymerase present. An ideal reaction should see a two-fold raise in a number of amplicons/cycle; resulting in 100% efficiency. Low-efficiency reactions (<90%) are indicators of inactive/inhibited Taq polymerase activity, improper optimization of annealing, poorly designed primers and secondary structures in amplicons. High-efficiency reactions (>110%) denote primer dimer formation or non-specific amplification. Pipetting errors or improperly calibrated pipettes cause both low and high efficiency issues
  • Standard curve: Usually, standard curves are produced using at least three replicates of standards with ten-fold serial dilution, covering the analytical sensitivity of the assay. The equation of the straight line (with co-efficient of correlation r2 and slope) to the curve to evaluate PCR optimisation. Dilution series produce curves, which are evenly spaced. If exact doubling of amplicons occurs in each cycle, the spacing between the adjacent fluorescence curves is determined by the equation 2n = dilution factor, where n is the number of cycles between subsequent dilutions or the difference in quantification cycles between them. For a ten-fold dilution, n = 3.32. In the equation of the line, n represents the slope. R2 represents the ability of the standard-data points to fall on the line and is, therefore, a measure of the linearity of the curve. The co-efficient measures the variability among the standard replicates and whether the PCR amplification efficiency has been the same for all standards used. The value should be >0.98. Lesser values symbolise greater variability.


In absolute quantification reactions, the standard curve determines the viral load. The major deviation in one or all of the above parameters, therefore, causes gross quantification errors in the sample load measured.[22],[23],[24]

Prior to the institution of the first WHO standard for the quantification of CMV genome in samples, the load was measured and reported as absolute copies/mL of the sample used or log value of the absolute load. Due to lack of a standard unit, there was a great deal of variations in reporting across centres and even platforms and assays. Deviations higher than the accepted ±0.5 logs copies/mL were seen, which narrowed the adaptability of results and made monitoring of cases difficult. With the creation of reference standard, calibration of the assay has become easier as assays across centres and platforms can be normalised against it. The reporting format of the load has since changed to IU/mL of the sample used.[25]

Validation of cytomegalovirus quantitative polymerase chain reaction

Validation done during DNA extraction and polymerase chain reaction run: The samples are run every time, with at least one negative control (either reagent template control or a negative DNA control) and a calibrator of known concentration, that is, extract from NIBSC reference strain-09/162. An internal control gene such as GAPDH, Beta actin etc., are commonly used to ascertain extraction and PCR efficiency. Standards and samples are usually run in triplicates to measure repeatability and quality control measures like sending blinded samples to other laboratories and comparing viral loads aid in the reproducibility measures.

Validation was done during standardisation of quantitative polymerase chain reaction assay:

The major factors involved in are:

  • Analytical sensitivity: Expressed as the limit of detection (LOD) which is the lowest concentration that can be detected with some degree of certainty. It is calculated by serially diluting a known concentration of CMV DNA and the last concentration detected is made the LOD of the assay
  • Analytical specificity: The ability of the PCR to detect only the desired target without amplifying other undesirable regions of the genome or DNA of other species. The assay is tested against genomes of other viruses, bacteria, fungi, parasites and Human DNA, wherein amplification is not seen
  • Accuracy: This is the difference between the load obtained and the actual load present
  • Repeatability: This is defined as the precision with which the assay estimates load of same samples run in replicates or multiple runs
  • Reproducibility: This determines differences between assays across platforms or laboratories, when with same samples.[21]


Troubleshooting in quantitative polymerase chain reaction

PCR, as is common with any technique, has its share of malfunctions, which greatly reduces assay robustness. [Table 2] gives a list of common issues encountered and possible solutions.[23],[26]
Table 2: Common problems and troubleshooting methods in real-time PCR

Click here to view


Comparison of commercial or kit based assay with in-house assays

A list of commercial kits are available for CMV viral load estimation, not all of which are CE and/or IVD approved. A few examples are Cobas Amplicor Taqman kit by Roche Diagnostics and CMV Artus kit by Qiagen. The main advantages of kit based assays are they may be validated and certified, less labour intensive, does not require in-depth molecular biology skills and experience as is required for in-house assay development. On the negative side, they are expensive, lack universal compatibility with real-time PCR platforms. On the other hand, in-house assays are certainly more labour intensive, specifically at the standardisation stages, require rigorous validation protocols but are more useful when it comes to platform compatibility and relatively cheaper compared to kits. They are also devoid of the kit dependency issues. The choice between commercial kits and in-house assays is dependent on factors like platforms available and other associated infrastructure, expenditure, availability of trained personnel. In our centre, we had compared the performance of the in-house assay to a commercial assay (Qiagen Artus). The development of an in-house assay and its interpretation was possible because of the presence of a scientific officer and a consultant microbiologist trained in molecular virology, and for the institutional support (financial and scientific) received during the development of the PCR which took about a year. The results of the comparison are as follows

  • Number of samples tested in parallel by the in-house assay and the Artus assay: 18
  • Number of samples concordant qualitatively: 15/18 (83.3%)
  • Number of qualitatively discordant results: 3/18 (16.7%)
  • False positive rate of the in-house assay using the commercial Artus assay as the gold standard: 11.1% (2 out of 18 positive by in-house assay but negative by Artus assay)
  • False negative rate of the in-house assay using the commercial Artus assay as the gold standard: 5.6% (1/18 samples)
  • Viral load ranges of the samples tested and detected: Artus assay = 0–4.08 log10 copies/mL; in-house assay = 0–4.19 log10 copies/mL
  • Median viral load by the commercial Artus assay: 2.74 log10 copies/mL; median viral load by the in-house assay: 3.46 log10 copies/mL; median difference in viral load: 0.72 log10 copies/mL (in-house assay over quantitating)
  • Correlation co-efficient of the Artus and in-house assay: 0.93
  • Lower LOD: 3 log10 copies/mL (1000 copies/mL) by in-house assay and 360 copies/mL (2.56 log10 copies/mL) by Artus assay (also known as the analytical sensitivity of the assay)
  • Analytical specificity of the assay was checked using human DNA, Staphylococcus aureus,  Escherichia More Details coli, Candida albicans, hepatitis B virus (HBV), herpes simplex virus 1 and 2, Epstein–Barr Virus, CMV, and water, and was found to be specific.
  • Upper LOD of the in-house assay: 7.1 log10 copies/mL (1.27 × 107 copies/mL)
  • The linearity of the assay: R2 = 0.999.


The possible reasons for these observed differences could be as follows:

  • Recommended extraction methodology (as per Qiagen-Artus) kit could not be followed in this comparison (EZ1 DSP Virus Kit as per Qiagen Artus; extraction method followed for in-house assay was Qiagen blood mini kit using manual method)
  • Differences in methods:


    • Quantitative standards (plasmids used in-house after cloning)
    • Gene targets
    • Amplification platform: Rotorgene for Artus assay versus ABI 7500 for the in-house assay
    • Primers and probes used
    • Differences in ideal sample type: Plasma in Artus assay and whole blood in the in-house assay.


    Such differences have been shown to cause differences in viral load estimation by WHO, UK Health Protection Agency and United Kingdom National External Quality assurance Scheme (UK NEQAS). For example, WHO's expert committee on biological standardisation reported a difference of about 2 log10 copies/mL within assay types of a given sample tested for CMV viral load.[27] Refer to [Figure 2] for an example of the report.

    External quality assurance scheme for cytomegalovirus quantification

    For the last 1-year, we have participated in the UK NEQAS program for CMV DNA quantification. Although participation in the UK NEQAS is expensive by Indian standards (Rs. 50,400, 2014 price for three distributions in a year for 2 samples each), it has helped us in better quality assurance and enhanced satisfaction and confidence of the users (clinicians) of the CMV QPCR services. The summary of our results from three distributions is given in [Figure 2].
    Figure 2: Reports of the United Kingdom National External Quality Assurance Scheme-Tata Medical Centre, Kolkata, India. Bottom of the figure: Graphic showing performance from 1st distribution

    Click here to view



     ~ Conclusion Top


    Viral load measurements by real-time PCR has become the standard method for the management of wide range of conditions (HIV, HBV, HCV, post-transplant monitoring for CMV, Epstein–Barr Virus, Adenovirus and BK virus). Accurate estimation of CMV viral loads is essential for effective clinical management of many clinical situations which range from congenital CMV, HIV infection, post solid organ transplant and post-HSCT scenario. Technologies such as real-time PCR systems provide optimal tools to assess viral loads. However, accurate estimation requires not just good kits, but a host of other quality assurance measures. Development of in-house quantitative CMV PCR assays should be a priority for developing countries as CE/IVD approved kits are expensive and mostly beyond the economic reach in resource-poor settings. The development of these in-house assays will require investment and patience. There is also need of caution to take care of genomic variations that may affect assay sensitivity.[9],[10] But this is a worthy investment as the laboratory and the workforce will gain immensely from the experience, and acquire skills to develop other assays. Close collaboration between different laboratories in terms of expertise, training, sharing of standard operating procedure, standards is invaluable especially in the context of developing countries where resources are sparse. Participation in an external quality assurance programme is essential.

    Nothing that has been mentioned in this paper is fundamentally new or based on an original research. The emphasis of this paper was to describe in simple terms that modalities of development, standardisation, and validation of a real-time PCR using CMV viral load measurements as an example. In a country where import of technology both of capital equipment and kits is an expensive, difficult and contentious issue we hope that the approach cited in this paper will make some difference in the way PCR technology for the diagnosis of infections is taken forward.

    Acknowledgement

    We would like to acknowledge our gratitude to Dr. Asha Mary Abraham, MD, PhD, and Department of Clinical Virology, CMC Vellore for providing training and SOP development with regard to the CMV PCR. We are also grateful to Dr. Provash Sadhukhan PhD, and his team (Kallol Saha and Rushna Firdaus) at the ICMR Virus Unit in Kolkata for helping us with the cloning experiments for the development of CMV quantitative standards.

    Financial support and sponsorship

    Nil

    Conflicts of interest

    There are no conflicts of interest.

     
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        Figures

      [Figure 1], [Figure 2]
     
     
        Tables

      [Table 1], [Table 2]



     

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04