Towards the real application of rapid microbiological methods in developing countries

One such need is the rapid detection of malaria. The World Health Organisation recommends diagnosis of malaria before treatments are begun, because drug-resistant varieties of Plasmodium are on the rise. And with more than 200 million cases and over 660,000 deaths reported annually, the need to develop a more reliable, inexpensive and effective tool to detect malaria without the need for trained personnel has never been higher. Fortunately, rapid diagnostic tests developed for malaria in recent years, including those that utilise polymerase chain reaction (PCR), have made it much easier to diagnose without using the conventional method of staining blood films and then examining them under a microscope.

Another global necessity is the rapid detection of Mycobacterium tuberculosis. Preliminary diagnosis of tuberculosis (TB) is often made by collecting a sample of lung secretions and examining the sample under a microscope to see if it contains the bacteria that cause TB. A sample is also sent to a laboratory so the bacteria can be cultured and identified. It may take as long as six weeks for the culture test to show a positive result. Because children have lower levels of infectious bacteria than adults do, it is more difficult to detect the bacteria under a microscope and to grow it in a culture. For this reason, accurately diagnosing TB in children, especially in developing countries, has been difficult. But researchers have been working on the next generation of detection platforms that are small in size and relatively easy to use. For example, one technology innovator has developed a portable device that combine microfluidic technology with nuclear magnetic resonance (NMR) to detect Mycobacterium tuberculosis and other pathogens (See http://blog.rapidmicromethods.com/2013/05/portable-nanodevice-provides-rapid.html).  The size and ease of use of these devices make them ideal for use in developing countries. In fact, the device’s scientific principle ensures that pathogen detection is very reliable, regardless of the quality of the initial sample, meaning that extensive purification, which would be difficult in a resource-limited setting, is not necessary. And the ability to diagnose tuberculosis (TB) in a matter of hours could allow testing and treatment decisions within the same clinic visit, which can be crucial to controlling the spread of TB in less-developed countries.

PCR technologies are also being used promote the rapid detection and identification of foodborne pathogens such as diarrheagenic E. coli. Children are at higher risk of contracting diarrhoea than adults due to their underdeveloped immune systems and they are likely to be affected for longer periods. In the Western world, most cases are easily treated but it is a different story in developing countries, where infection rates are higher and death is a common outcome. Recent advances in PCR and HPLC technologies have provided a molecular-based multiplex method that is capable of detecting various strains of diarrheagenic E. coli in a single procedure, permitting rapid detection and diagnosis and reducing the time before appropriate action can be taken (See http://blog.rapidmicromethods.com/2011/12/hplc-of-pcr-products-now-being-used-to.html).

With the adoption of RMMs within the global pharmaceutical community and acceptance by many regulatory authorities around the globe, one might expect that the implementation of new microbiology technologies would be consistent within our industry across all geographic regions. Unfortunately, this has not been the case, and conventional methods continue to be applied in almost every aspect of pharmaceutical microbiological testing in developing countries. But things are starting to change.

To promote the advancement of sterile product and pharmaceutical sciences in the Middle East, the Future University in Egypt hosted the 2012 International Conference on Pharmaceutical Technologies.  The conference included workshops on Advanced Drug Delivery Systems, Advances in Sterile Products, Tablets, Capsule Technologies and QC/QA. During the symposium on sterile product technologies, I had an opportunity to speak on the advancements and applications of RMMs. More importantly, I was able to meet with scientists from Middle Eastern pharmaceutical companies, as well as undergraduate and graduate students, who were engaged in research and validation studies associated with new microbiology technologies. My co-author, Suzan Mohammed Ragheb, is one of those scientists, and she has spent considerable effort promoting the use of molecular methods for pharmaceutical applications in her resident country, as well as other developing countries around the world. She shares her perspectives below.

The implementation of PCR for pharmaceutical applications in developing countries

In 1993, Kary Mullis was awarded the Nobel Prize in Chemistry for the invention of PCR.  PCR has been successfully used in several fields and various research groups worldwide have adopted PCR for the microbiological quality control of pharmaceuticals. Microorganisms that are detected using PCR include bacteria such as Salmonella spp., E. coli, S. aureus, P. aeruginosa, or B. cepacia, and fungi such as Asperigellus niger¹. Researchers have encouraged the application of PCR for the detection of Mycoplasma in biologics and this also represents an example of pharmacopeia and regulatory adaptation of molecular techniques². On the other hand, the microbiological assessment of pharmaceutical samples such as gelatin has also been performed by molecular techniques other than simple PCR. Molecular techniques such as repetitive-element genomic fingerprinting (rep-PCR) and 16S rRNA gene sequencing were applied by De Clerck et al. for detection of Bacillus and related endospore-forming genera³.

In developing countries, conventional methods are routinely applied in the microbiological assessment of pharmaceuticals. These methods consume both time and large amounts of microbiological media. In Egypt, as one of the largest Middle Eastern countries with a strong pharmaceutical industry, comprehensive studies have been performed which describe the microbiological quality control of pharmaceuticals and cosmetics using conventional methods⁴. However, the application of RMMs can save time and resources, allowing rapid corrective actions when required. But the acceptance of such techniques in the pharmaceutical industry, especially in developing countries, requires a delicate balance between meeting critical parameters and expectations, such as the correct estimation of contaminants, rapid time to result, having adequate resources and technical expertise, and the cost of implementation. As such, pharmaceutical companies operating in developing countries should consider several points when applying rapid technologies, including PCR.

Several considerations govern the application of PCR for microbiological quality control of pharmaceuticals including the manner in which the PCR laboratory is constructed (i.e., the separation of critical activities such that cross contamination is minimised) as well as the use of appropriate equipment and reagents. One of the most critical points is the utilisation of well-trained personnel, and this can be achieved by the provision of experts that can contribute adequate technical support.  Furthermore, the implementation of robust quality control / quality assurance systems and the generation of standard operating procedures that control each performed step in the PCR procedure are paramount to success.

Although the application of such technologies may face some obstacles as a result of cost, lack of adequate validation resources and technical support in developing countries, the industry can benefit from pharmaceutical standards and training organisations. For example, Parenteral Drug Association (PDA) Technical Report No. 33 provides guidance for the evaluation, validation, and implementation of new and alternative microbiological methods, including molecular techniques. The United States Pharmacopeia (USP) has outlined comprehensive discussions of rapid molecular methods, including nucleic acid basic techniques (Chapter 1125), extraction, detection and sequencing (Chapter 1126), amplification (Chapter 1127), microarrays (Chapter 1128), genotyping (Chapter 1129), detection of trace nucleic acids, such as residual DNA (Chapter 1130), Mycoplasma testing (Chapter 63) and validation of alternative microbiological tests methods (Chapter 1223). The European Pharmacopeia (Ph. Eur.) also provides guidance on molecular methods, including nucleic acid amplification techniques (Chapter 2.6.21), Mycoplasmas (Chapter 2.6.7) and validation of alternative methods for microbiological quality (Chapter 5.1.6).

The issues mentioned above will be addressed in a more comprehensive publication, which is currently under review. We demonstrated the validation strategies that should be adopted by pharmaceutical companies in addition to other important topics that should be further studied.

In addition, our previous work on PCR has been published, and is based on guidance documents and other resources⁵. Here, we optimised and applied the most suitable conditions for performing a successful PCR reaction for the detection of specified microorganisms in pharmaceutical excipients and non-sterile drug products produced at The Nile Company for Pharmaceuticals and Chemical Industries in Cairo, Egypt. In our study, the detection of the four main specified microorganisms according to the pharmacopeial recommendations, Salmonella spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, were optimised in different pharmaceutical dosage forms and raw materials. Uniplex PCR was performed for the detection of each microorganism individually targeting the conserved region in each bacterial genome. Further optimisations were done to perform duplex and multiplex PCR assays considering relative concentrations of competitor primers used in the reaction. The uniplex PCR amplicons were successfully sequenced, confirming the conservation of used primers. Other validation parameters such as specificity, sensitivity, and robustness were examined closely.

We appreciated the importance of such rapid methods in many situations such as limited raw material(s), and the need to have process controls, especially in the case of producing successive batches, allowing the potential for rapid corrective actions, when required.

Although we continue to utilise conventional methods, our previous published work and our current studies (that have been submitted for publication) are considered trials for encouragement in the use of molecular biology techniques within the pharmaceutical industry in Egypt and the Middle East, since there has been progress in demonstrating such techniques academically, and there is now a notable establishment of biotechnology departments in a number of Egyptian pharmaceutical companies such as Nile Company, MINAPHARM, SEDICO and others.

Summary

The detection of bacterial contaminants in pharmaceutical preparations has been revolutionised recently by the availability of techniques such as the use of PCR, which is gaining huge momentum across our industry. In the Nile Company for Pharmaceuticals and Chemical Industries, where one of the authors performed this work, the PCR technique for the detection of four specified bacterial contaminants that are usually tested for in oral or topical products have been enhanced. Optimisation of this technique, in one of the largest pharmaceutical companies in Egypt, can serve as a starting point for applying these same molecular tests in quality control departments across Egypt, the Middle East and other developing nations, with the resulting benefit of time savings accompanied by the rapid release of pharmaceutical products in these emerging international markets.

Biographies

Dr. Michael J. Miller

Michael J. Miller, President, Microbiology Consultants, LLC

Dr. Michael J. Miller is an internationally recognised microbiologist and subject matter expert in pharmaceutical microbiology and the design, validation and implementation of rapid microbiological methods.  He is currently the President of Microbiology Consultants, LLC. Over the course of 25 years, he has held numerous R&D, manufacturing, quality, and consulting and business development leadership roles at Johnson & Johnson, Eli Lilly and Company, Bausch & Lomb, and Pharmaceutical Systems, Inc. In his current role, Dr. Miller consults with multinational companies in providing technical, quality and regulatory solutions in support of RMMs, sterile and non-sterile pharmaceutical manufacturing, contamination control, isolator technology, validation and microbiological PAT. He also provides comprehensive training for his clients in the areas of rapid method validation and implementation.
 
Dr. Miller has authored more than 100 technical publications and presentations in the areas of rapid microbiological methods, PAT, ophthalmics, disinfection and sterilisation, is the editor of PDA’s Encyclopedia of Rapid Microbiological Methods, and is the owner of http://rapidmicromethods.com, a website dedicated to the advancement of rapid methods. He currently serves on the editorial board for European Pharmaceutical Review, is chairing the revision of PDA Technical Report #33: Evaluation, Validation and Implementation of New Microbiological Testing Methods, and routinely provides RMM training programs for the industry and professional organisations worldwide.
 
Dr. Miller holds a PhD in Microbiology and Biochemistry from Georgia State University (GSU), a B.A. in Anthropology and Sociology from Hobart College, and is currently an adjunct professor at GSU.  He was appointed the John Henry Hobart Fellow in Residence for Ethics and Social Justice, awarded PDA’s Distinguished Service Award and was named Microbiologist of the Year by the Institute of Validation Technology (IVT).

Suzan Mohammed Ragheb

Pharmacist Suzan Mohammed Ragheb holds a Bachelor degree in pharmaceutical science, The College of Pharmaceutical Science and Industrial Pharmacy, Misr University for Science and Technology (MUST), Egypt. She holds Master degree in Microbiology and Immunology, Faculty of Pharmacy- Cairo University, Egypt.

She worked at The Nile Company for Pharmaceuticals and Chemical Industries, Cairo, Egypt as in process control pharmacist from 2006 to 2012. In addition, she was a researcher at Microbiology and Biotechnology Departments. Currently, Ms. Ragheb is a quality assurance pharmacist in the same firm supervising manufacturing steps at sterile and nonsterile products departments in addition to revising related documents.

She has a previous published work on the subject of PCR⁵. A poster presentation related to the same point was presented in 2012 at the Future University International Conference on Pharmaceutical Technology, Cairo-Egypt and The Third International Scientific Conference of Faculty of Pharmacy Cairo University.

References

1. Jimenez L. Molecular applications to pharmaceutical processes and cleanroom environments. PDA J. Pharm. Sci. Technol. 2011. 65(3): 242-253
2. Volokhov, D.V.; Graham, L.J.;  Brorson, K.A.; Chizhikov, V.E. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Mol. Cell. Probes. 2011 25(2-3),69-77
3. De Clerck, E.; Vanhoutte, T.; Hebb, T.; JGeerinck, J.; Devos, J.; De Vos, P. Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts. Appl. Environ. Microbiol. 2004, 70(6), 3664-3672
4. Abdelaziz, A. A. and Ashour M. S. Microbial contamination of hexetidine mouth washes. Zentralbl. Bakteriol. Mikrobiol. Hyg. B. 1987.184(3-4), 262-268
5. Ragheb, S. M.; Yassin, A.S.; Amin M.A. The application of uniplex, duplex, and multiplex PCR for the absence of specified microorganism testing of pharmaceutical excipients and drug products. PDA J. Pharm. Sci. Technol. 2012. 66(4), 307-17