Current situation
Sri Lanka produces about 1035 million of medical drugs annually through the State Pharmaceutical Manufacturing Corporation (SPMC) [1], and another large quantity of pharmaceuticals are being imported to the country to cover the medical needs of about 20 million people. Although SPMC plans to double its pharmaceutical production with new equipment with Japan International Cooperation Agency aid [2], Sri Lanka still lacks an efficient, comprehensive microbial quality assurance technique to eliminate the non- sterile or potentially hazardous pharmaceutical products. Speaking to the Sunday Leader, on an issue regarding the Concerns over quality of Imported Drugs, SPMC Chairman Prof. S.D.Jayaratne has emphasized on the need for a quality assurance facility that can test the whole range of pharmaceutical products [3]. Currently, only a few batches of drugs are subjected to inspection due to high cost and limited availability of resources.
Current testing procedures are mainly based on chemical methods as well as on conventional microbiological techniques. In culture-based conventional microbiological techniques, tests is performed under various criteria such as total aerobic microbial count, total combined yeast/mold count, and for the absence of dosage form specific microorganisms such as Escherichia coli, Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans etc in order to determine product safety and sterility. Although used in routine practice, these tests are highly dependent on the operator’s expertise and may often lead to underestimation of contaminants due to poor identification facilities. In addition, detection may often be hampered by uncultivable species. As a test requires about a week for confirmation from start-to-end, conventional methods may not be best suited for immediate identification of microorganisms during a contamination event. The process can be costly when similar drug is subjected to testing under different criteria to determine the presence of each contaminant.
Solution
Sequencing based testing of 16s rRNA gene of bacteria and the ITS region of fungi are two of the most successful molecular detection techniques used to identify the whole spectrum of possible pharmaceutical contaminants. These techniques allow species-level identification, which is crucial in providing a definitive root cause as part of an investigation. Given the potential risks of bacterial and fungal contaminations, quality of the end products as well as the current Good Manufacturing Practices (cGMP) should be continuously assessed and assured. The goal of a thorough cGMP is to have precise data highlighting where incursion points may exist, and control those points to limit or prevent migration into the process flow [4].
In addition, this technology can be used to assess the microbial community structure of effluent treatment plants (ETPs) which represents microbial communities existing as dynamic consortia, in order to understand their constortial functions that changes with reactor operational conditions. Contribution of such communities towards overall degradation is expected to provide unprecedented control over the bioremediation of the effluents [5].
This sequence based approach of microbial identification can be applied as a comprehensive solution to address quality related problems in pharmaceuticals, since poor microbial quality of drugs is a definite threat to consumers, as well as to the manufacturers of the product.With sequencing of 16s rRNA gene of bacteria and the ITS region of fungi, rapid identification of contaminants up to species level has become possible which helps the manufacturer to understand and investigate potential physical and temporal sources of contamination. The technology can also differentiate between the intragenic variations among closely related species, while providing an idea about relative abundances of species which reflect relative pressures that shape diversity within biological communities [5]. The identification process only takes 48 hours of run time which makes this technology a rapid solution to all the contamination related problems. Another advantage of the technique is that it eliminates the need for cloning of amplified fragments, and the obstacles caused by unculturable phyla of microorganisms. Since the technology involves semi conductor based next generation sequencing, there is a massive increase of throughput with an unmatched accuracy.
To summarize, 16s rRNA Sequencing has following advantages compared to other techniques-
| Conventional techniques | 16s rRNA Sequencing |
| Longer turnaround times. A single test requires a minimum of 7 days | Lesser turnaround times. Extremely rapid with a run time of only 24 hours. |
| Test accuracy is dependent on operator expertise. The accuracy of testing Is ̴80%[7] | Accuracy is operator-independent. Highly accurate with an accuracy of 99.9% |
| Fastidious, unculturable organisms and unusual biochemical reactions impede the identification process. | Test method is culture independent; Growth requirements, biochemical reactions and culture failures do not affect the identification process. |
| Only a limited number of organisms can be identified. Important organisms in a test sample can be missed out. | Identifies the complete range of organisms present in a test sample. |
| The percentage of strains correctly identified to the species level is sometimes less thansatisfactory. | Permits identification of complete spectrum of organisms down to species level, their intragenic variations and relative abundances |
| Limited throughput, Sensitivity may vary. | Dramatic increase in throughput; High sensitivity |
References-
- spmclanka.lk/images/Expansion.docx
- http://www.colombopage.com/archive_13A/May08_1368024687JV.php
- http://www.thesundayleader.lk/2012/07/29/concerns-over-quality-of-imported-drugs/
- S. SIDHU, C.T. TYLER, G. MA, AND M. SAMADPOUR, MOLECULAR EPIDEMIOLOGY, INC., AND E.J. BRANDRETH, ALTHEA TECHNOLOGIES; Managing Objectionable Events in cGMP Cleanrooms: A Polyphasic Approach
- Asha Rani, Shalini Porwal, Rakesh Sharma, Atya Kapley, Hemant J. Purohit; Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches.
- Carlotti; – Identification of environmentally relative bacteria from pharmaceutical industries
- Michael Janda and Sharon L. Abbott; Bacterial Identification for Publication: When Is Enough Enough?
