Screening for Novel Anti-cancer Bioactive Compounds Produced by Bacteria Found in Marine Sponges.
A Proposal for Novel Compound Discovery
H. A. Ward, Melbourne
Abstract
Introduction
With an estimated 7.6 million deaths reported in 2005, cancer is the second leading cause of death amongst the world’s population . Each year, several billion dollars is invested in cancer research in an attempt to understand the disease processes involved and to try to discover possible therapies to combat this debilitating disease. The potential market for immune drugs is huge and is a rapidly growing area in the biopharmaceutical arena. It is expected that there will be a US $15 billion worldwide market opportunity for therapeutic immune drugs by the year 2010 .
During the past 5 decades of research in anti-cancer drug discovery, about 100 products have been provided for clinical treatment of malignancy (Wagner-Dobler et al., 2002). Significant progress has been made in the chemotherapeutic management of hematologic malignancies, however, more than 50% of patients with tissue tumours either fail to respond or will die from the disease (Wagner-Dobler et al., 2002). Hence, the discovery of novel anti-cancer therapeutic agents remains critically important.
The tremendous biochemical diversity of marine microorganisms and their biotechnological potential, as extraordinary resources for the discovery of new anti-cancer compounds, is becoming more and more recognized by both microbiologists as well as the pharmaceutical industry (Schweder et al., 2005). Of these microorganism species, the microorganisms living in marine sponges have attracted significant attention as potential sources of bioactive compounds (Garcia Camacho et al., 2006). Because of their phenomenal potency, even very small quantities of these compounds can be of significant value in a commercial sense (Simmons et al., 2005).
Culture based techniques are inadequate for studying bacterial diversity from marine samples as many bacteria cannot be cultured using artificial laboratory conditions (Webster et al., 2001) and thus, is not accessible for detailed taxonomical and physiological characterizations. The tools of molecular biology and the phylogenetic framework, which is now available as 16S rDNA sequence alignments, allow a complementary strategy to be pursued. This is based on phylogenetic screening of marine isolates and an in-depth investigation of the biological activity and chemical diversity of selected phylogenetic groups in order to identify a new hotspot for the production of bioactive compounds (Schweder et al., 2005). Selected isolates can then be cultivated on a large scale and under a variety of cultivation conditions.
This report aims to provide an insight into the steps which are involved in screening for these novel anti-cancer bioactive compounds which are produced by marine bacteria.
Proposal
Searching for novel bioactive compounds is a multi-step procedure which begins with the selection of a suitable source to be investigated- in this case, it is a sample of sponge tissue which will contain numerous bacteria, producing bioactive compounds (Wagner-Dobler et al., 2002). The sponge tissue is collected by scuba-diving in the region where the marine sponges are located. The phylogenetic affiliation of the sponge-associated bacteria is assessed using16S rDNA analysis which is mainly based on the selective amplification of 16S rRNA gene sequences, of taxon-specific lengths, by the application of primers of conserved regions of the 16S rDNA in combination with PCR and gel electrophoresis (Schweder et al., 2005). Due to its highly conserved and variable sequence regions, the 16S rDNA sequence is used as a phylogenetic marker (Schweder et al., 2005). This method of direct isolation of DNA from the environment and cloning and sequencing of 16S rRNA genes enables identification and taxonomical affiliation of bacterial species in different natural marine habitats without the cultivation of the microbial cells.
Production of secondary metabolites is usually a strain-specific trait. Thus, typing of the isolated bacteria with a high resolution is necessary to assess the genetic diversity of the strains within a given phylogenetic group (Wagner-Dobler et al., 2002). This high resolution is obtained by using genomic fingerprint methods- in this case a RAPD (Random Amplified Polymorphic DNA) technique with arbitrary primers is used (Wagner-Dobler et al., 2002).
Additionally, phylogenetic data on microbial community composition in sponges can indicate possible nutritional requirements and physiological niches of many microbes based on information already available for known phylogenetic relatives (Webster et al., 2001). This may assist in the experimental manipulation of culture conditions to provide the correct growth environment for targeted bacteria (Webster et al., 2001).
One of the limitations associated with the construction of 16S rDNA clone libraries from total environmental DNA is that it requires the use of PCR, which precludes quantitative estimates of abundance for each organism (Webster et al., 2001). This can be overcome to some degree by the use of fluorescence in situ hybridization (FISH) probing. FISH with rRNA specific probes allows phylogenetic identification of bacteria in mixed assemblages and enables the cells to be visualized and semi-quantified (Webster et al., 2001).
The crude extracts are investigated to evaluate anti-bacterial, anti-fungal, phytotoxic or cytotoxic activity. Individual bacterial colonies are obtained by serially diluting the sample and spread-plating appropriate dilutions on agar plates containing a variety of marine media (Wagner-Dobler et al., 2002). Brine shrimp toxicity has a strong correlation with cytotoxicity and is therefore a good indicator for potential anti-cancer activity (Wagner-Dobler et al., 2002). Secondary metabolite production can only be assigned to the bacteria when synthesis has been demonstrated in cultures isolated from the host species (Webster et al., 2001).
The plates are then incubated at room temperature for up to 4 weeks. The growth of eukaryotes and protozoan grazing is prevented by the addition of the antibiotic cycloheximide (Wagner-Dobler et al., 2002). The isolated bacteria are then compared to the total community structure of the sample determined by the small subunit rDNA approach. Novel bacterial status is assigned to isolates after comparison of colony morphotype and microscopic appearance of gram-stained preparations with previously obtained isolates (Webster et al., 2001).
The strongly positive hits obtained using the serial dilution and agar diffusion tests are then screened further using tumour cell-line based screening (‘in-vitro’ testing) (Wagner-Dobler et al., 2002). In the current National Cancer Institute (NCI) anti-cancer screen, each extract is tested against 60 human tumour cell lines derived from several cancer types (Wagner-Dobler et al., 2002). The most active extracts are then selected for further testing for the following criteria: (i.) potency, (ii.) cell-type specificity, (iii.) unique structure and (iv.) unique mechanism of action (Wagner-Dobler et al., 2002). To obtain information on the mechanism of action, the most active extracts are subjected to cell-cycle analysis. Continuously dividing tumour cells go from one mitosis (M) to the next, passing through the G1-, S- and G2-phases (Wagner-Dobler et al., 2002). Potential anti-cancer compounds will alter the cell-cycle in a specific manner, as is shown below in Figure 1. Hence, cell-cycle analysis can be used as a first indicator to identify the mechanism of action of the new compounds produced by the bacteria (Wagner-Dobler et al., 2002).
Figure 1. Cell-cycle analysis showing the M-, G1-, S- and G2-phases
(Details not posted) Source: Advances in Biochemical Engineering/Biotechnology, Vol. 74
The results from this screening enable the selection of strains whose bioactive compounds can now be isolated and their structure determined. Since even modern methods for structure determination and evaluation require at least 10mg of every compound, a scale-up fermentation is necessary (Wagner-Dobler et al., 2002). Depending on the strain of bacteria, the fermentation conditions can be optimized to achieve maximum yield of metabolite and to increase the genetic stability of the bacteria.
Typically, small-scale 100ml shake flask experiments are initially used, after which the cultivation experiments can then be transferred to glass or stainless steel bio-reactors for cultivation on a large-scale (Wagner-Dobler et al., 2002). The bioreactor design, aeration and agitation are chosen accordingly, depending on the individual strain requirement. Depending on the strain, the cultivation time is generally in the range 24-72 hours. For downstream processing, cells and supernatant are separated by centrifugation and extracted separately using organic solvents such as chloroform/methanol or ethyl acetate (Wagner-Dobler et al., 2002). These experiments provide the material for structural elucidation. In addition, they provide significant quantities of the bioactive compounds in order to carry out the in-vivo anti-tumour tests as well as the pre-clinical pharmacokinetic and toxicological studies before proceeding to the Phase I-III clinical trials.
The isolation and structural determination of natural products is a time consuming and expensive process, hence, it is very important to recognize and exclude known compounds at the earliest possible stage by a process which is called dereplication (Wagner-Dobler et al., 2002) . For this, easily accessible properties of metabolites are compared with literature data. There are databases, such as the Dictionary of Natural Products, where substructures, NMR or UV data and a variety of other molecular description can be searched using computers. Also widely used is the comparison of UV or MS data and HPLC retention times with appropriate reference data collections (Wagner-Dobler et al., 2002).
The most common methods for structural elucidation is mass spectrometry, using electro spray ionization techniques or MALDI-TOF, and NMR spectrometry which are often combined with chromatographic methods (Schweder et al., 2005). The hyphenated techniques HPLC-NMR and HPLC-MS, which need only microgram amounts of compound and have a high resolution, are shown to be powerful tools in combination with databases (Wagner-Dobler et al., 2002).
The in-vivo anti-tumour tests are carried out by testing the bioactive compounds in mice bearing the tumour cell line which was shown to be most sensitive in the in-vitro screen (Wagner-Dobler et al., 2002). Compounds that show significant tumour growth inhibition are then selected for further in-vivo evaluation against more advanced-stage tumours.
Next, the pre-clinical pharmacokinetic studies (absorption, bioavailability, distribution and excretion) and pre-clinical toxicology studies are carried out. The vast majority of anti-cancer drugs are cytotoxic compounds which have significant side-effects and a very small therapeutic index. The objective of these studies is to find a safe initial dose for Phase I clinical studies and to define the qualitative and quantitative organ toxicities (Wagner-Dobler et al., 2002).
Once this data has been established, the potential anti-cancer drug needs to be extensively reviewed by a range of regulatory authorities and committees to determine whether the drug is safe for carrying out studies on humans. Once it has been approved, it is possible to start the Phase I-III clinical trials. If the drug proves to be safe and effective in the clinical studies and once full regulatory approval is granted then the drug can then be scaled up in order to be sold in the market3. It is critical to protect the intellectual property surrounding the drug through the acquisition of various patents that will prevent others from making, using or selling what is described in the patent.
The productivity of the past decade in terms of the discovery of new clinical anti-cancer leads from diverse marine bacteria should translate into a number of new treatments for cancer in the decades to come. Exploitation of the potential of these marine microorganisms as producers of bioactive metabolites, with a wide range of potential pharmacological activities, is only just beginning. With the recent advances in both molecular biology and marine biotechnology, it is indeed very promising to see that the marine microbial environment is likely to continue to be prolific source of novel natural bioactive compounds for many years to come. In the future, further innovations in media development (chemical engineering), bioreactor design (bioprocess engineering) and transgenic production (molecular engineering), coupled with efficient downstream processing and product recovery (Pomponi, 1999), will continue to further enhance both the discovery and bulk production of these novel marine bioactive compounds.
References
Garcia Camacho, F., Chileh, T., Ceron Garcia, M.C., Sanchez Miron, A., Belarbi, E.H., Chisti, Y. & Molina Grima, E. (2005). A bioreaction-diffusion model for growth of marine sponge explants in bioreactors. Applied Microbiology and Biotechnology (details missing)
National Cancer Institute: www.cancer.gov
Pomponi, S.A. (1999). The Potential for the Marine Biotechnology Industry. Trends and Future Challenges for U.S. National Ocean and Coastal Policy: Workshop Materials, pp.101-104. Retrieved from website:
http://oceanservice.noaa.gov/websites/retiredsites/natdia_pdf/17pomponi.pdf
Schweder, T., Lindequist, U. & Lalk, M. (2005). Screening for New Metabolites from Marine Microorganisms. Advances in Biochemical Engineering/Biotechnology, Vol. 96, pp.1-48
Simmons, T.L., Andrianasolo, E., McPhail,K., Flatt, P. & Gerwick, W.H. (2005). Marine natural products as anticancer drugs. Molecular Cancer Therapeutics 2005, Vol.4, No.2, pp.333-342
Virax : www.virax.com.au
Wagner-Dobler, I., Beil, W., Lang, S., Meiners, M. & Laatsch, H. (2002). Integrated Approach to Explore the Potential of Marine Microorganisms for the Production of Bioactive Metabolites. Advances in Biochemical Engineering/Biotechnology, Vol. 74, pp.208-238
Webster, N.S., Wilson, K.Y., Blackall, L.L., Hill, R.T. (2001). Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile. Applied and Environmental Microbiology, Vol.67, No.1, pp.434-444
World Health Organisation: Ten Statistical Highlights in Global Public Health Retrieved from: http://www.who.int/whosis/whostat2006_10highlights.pdf
H. A. Ward, Melbourne
Abstract
The chemical and biological diversity of the marine environment is immeasurable and is therefore an extraordinary resource for the discovery of novel anti-cancer compounds. Recent technological and methodological advances in structure elucidation, biological assays and organic synthesis have enabled the isolation and clinical evaluation of various novel anti-cancer agents from marine microorganisms. This report provides insight into the processes involved in the isolation and identification of novel marine microorganisms, the screening for novel bioactive anti-cancer compounds produced by the microorganisms, as well as the evaluation and characterization of their structure and bioactivity.
Introduction
With an estimated 7.6 million deaths reported in 2005, cancer is the second leading cause of death amongst the world’s population . Each year, several billion dollars is invested in cancer research in an attempt to understand the disease processes involved and to try to discover possible therapies to combat this debilitating disease. The potential market for immune drugs is huge and is a rapidly growing area in the biopharmaceutical arena. It is expected that there will be a US $15 billion worldwide market opportunity for therapeutic immune drugs by the year 2010 .
During the past 5 decades of research in anti-cancer drug discovery, about 100 products have been provided for clinical treatment of malignancy (Wagner-Dobler et al., 2002). Significant progress has been made in the chemotherapeutic management of hematologic malignancies, however, more than 50% of patients with tissue tumours either fail to respond or will die from the disease (Wagner-Dobler et al., 2002). Hence, the discovery of novel anti-cancer therapeutic agents remains critically important.
The tremendous biochemical diversity of marine microorganisms and their biotechnological potential, as extraordinary resources for the discovery of new anti-cancer compounds, is becoming more and more recognized by both microbiologists as well as the pharmaceutical industry (Schweder et al., 2005). Of these microorganism species, the microorganisms living in marine sponges have attracted significant attention as potential sources of bioactive compounds (Garcia Camacho et al., 2006). Because of their phenomenal potency, even very small quantities of these compounds can be of significant value in a commercial sense (Simmons et al., 2005).
Culture based techniques are inadequate for studying bacterial diversity from marine samples as many bacteria cannot be cultured using artificial laboratory conditions (Webster et al., 2001) and thus, is not accessible for detailed taxonomical and physiological characterizations. The tools of molecular biology and the phylogenetic framework, which is now available as 16S rDNA sequence alignments, allow a complementary strategy to be pursued. This is based on phylogenetic screening of marine isolates and an in-depth investigation of the biological activity and chemical diversity of selected phylogenetic groups in order to identify a new hotspot for the production of bioactive compounds (Schweder et al., 2005). Selected isolates can then be cultivated on a large scale and under a variety of cultivation conditions.
This report aims to provide an insight into the steps which are involved in screening for these novel anti-cancer bioactive compounds which are produced by marine bacteria.
Proposal
Searching for novel bioactive compounds is a multi-step procedure which begins with the selection of a suitable source to be investigated- in this case, it is a sample of sponge tissue which will contain numerous bacteria, producing bioactive compounds (Wagner-Dobler et al., 2002). The sponge tissue is collected by scuba-diving in the region where the marine sponges are located. The phylogenetic affiliation of the sponge-associated bacteria is assessed using16S rDNA analysis which is mainly based on the selective amplification of 16S rRNA gene sequences, of taxon-specific lengths, by the application of primers of conserved regions of the 16S rDNA in combination with PCR and gel electrophoresis (Schweder et al., 2005). Due to its highly conserved and variable sequence regions, the 16S rDNA sequence is used as a phylogenetic marker (Schweder et al., 2005). This method of direct isolation of DNA from the environment and cloning and sequencing of 16S rRNA genes enables identification and taxonomical affiliation of bacterial species in different natural marine habitats without the cultivation of the microbial cells.
Production of secondary metabolites is usually a strain-specific trait. Thus, typing of the isolated bacteria with a high resolution is necessary to assess the genetic diversity of the strains within a given phylogenetic group (Wagner-Dobler et al., 2002). This high resolution is obtained by using genomic fingerprint methods- in this case a RAPD (Random Amplified Polymorphic DNA) technique with arbitrary primers is used (Wagner-Dobler et al., 2002).
Additionally, phylogenetic data on microbial community composition in sponges can indicate possible nutritional requirements and physiological niches of many microbes based on information already available for known phylogenetic relatives (Webster et al., 2001). This may assist in the experimental manipulation of culture conditions to provide the correct growth environment for targeted bacteria (Webster et al., 2001).
One of the limitations associated with the construction of 16S rDNA clone libraries from total environmental DNA is that it requires the use of PCR, which precludes quantitative estimates of abundance for each organism (Webster et al., 2001). This can be overcome to some degree by the use of fluorescence in situ hybridization (FISH) probing. FISH with rRNA specific probes allows phylogenetic identification of bacteria in mixed assemblages and enables the cells to be visualized and semi-quantified (Webster et al., 2001).
The crude extracts are investigated to evaluate anti-bacterial, anti-fungal, phytotoxic or cytotoxic activity. Individual bacterial colonies are obtained by serially diluting the sample and spread-plating appropriate dilutions on agar plates containing a variety of marine media (Wagner-Dobler et al., 2002). Brine shrimp toxicity has a strong correlation with cytotoxicity and is therefore a good indicator for potential anti-cancer activity (Wagner-Dobler et al., 2002). Secondary metabolite production can only be assigned to the bacteria when synthesis has been demonstrated in cultures isolated from the host species (Webster et al., 2001).
The plates are then incubated at room temperature for up to 4 weeks. The growth of eukaryotes and protozoan grazing is prevented by the addition of the antibiotic cycloheximide (Wagner-Dobler et al., 2002). The isolated bacteria are then compared to the total community structure of the sample determined by the small subunit rDNA approach. Novel bacterial status is assigned to isolates after comparison of colony morphotype and microscopic appearance of gram-stained preparations with previously obtained isolates (Webster et al., 2001).
The strongly positive hits obtained using the serial dilution and agar diffusion tests are then screened further using tumour cell-line based screening (‘in-vitro’ testing) (Wagner-Dobler et al., 2002). In the current National Cancer Institute (NCI) anti-cancer screen, each extract is tested against 60 human tumour cell lines derived from several cancer types (Wagner-Dobler et al., 2002). The most active extracts are then selected for further testing for the following criteria: (i.) potency, (ii.) cell-type specificity, (iii.) unique structure and (iv.) unique mechanism of action (Wagner-Dobler et al., 2002). To obtain information on the mechanism of action, the most active extracts are subjected to cell-cycle analysis. Continuously dividing tumour cells go from one mitosis (M) to the next, passing through the G1-, S- and G2-phases (Wagner-Dobler et al., 2002). Potential anti-cancer compounds will alter the cell-cycle in a specific manner, as is shown below in Figure 1. Hence, cell-cycle analysis can be used as a first indicator to identify the mechanism of action of the new compounds produced by the bacteria (Wagner-Dobler et al., 2002).
Figure 1. Cell-cycle analysis showing the M-, G1-, S- and G2-phases
(Details not posted) Source: Advances in Biochemical Engineering/Biotechnology, Vol. 74
The results from this screening enable the selection of strains whose bioactive compounds can now be isolated and their structure determined. Since even modern methods for structure determination and evaluation require at least 10mg of every compound, a scale-up fermentation is necessary (Wagner-Dobler et al., 2002). Depending on the strain of bacteria, the fermentation conditions can be optimized to achieve maximum yield of metabolite and to increase the genetic stability of the bacteria.
Typically, small-scale 100ml shake flask experiments are initially used, after which the cultivation experiments can then be transferred to glass or stainless steel bio-reactors for cultivation on a large-scale (Wagner-Dobler et al., 2002). The bioreactor design, aeration and agitation are chosen accordingly, depending on the individual strain requirement. Depending on the strain, the cultivation time is generally in the range 24-72 hours. For downstream processing, cells and supernatant are separated by centrifugation and extracted separately using organic solvents such as chloroform/methanol or ethyl acetate (Wagner-Dobler et al., 2002). These experiments provide the material for structural elucidation. In addition, they provide significant quantities of the bioactive compounds in order to carry out the in-vivo anti-tumour tests as well as the pre-clinical pharmacokinetic and toxicological studies before proceeding to the Phase I-III clinical trials.
The isolation and structural determination of natural products is a time consuming and expensive process, hence, it is very important to recognize and exclude known compounds at the earliest possible stage by a process which is called dereplication (Wagner-Dobler et al., 2002) . For this, easily accessible properties of metabolites are compared with literature data. There are databases, such as the Dictionary of Natural Products, where substructures, NMR or UV data and a variety of other molecular description can be searched using computers. Also widely used is the comparison of UV or MS data and HPLC retention times with appropriate reference data collections (Wagner-Dobler et al., 2002).
The most common methods for structural elucidation is mass spectrometry, using electro spray ionization techniques or MALDI-TOF, and NMR spectrometry which are often combined with chromatographic methods (Schweder et al., 2005). The hyphenated techniques HPLC-NMR and HPLC-MS, which need only microgram amounts of compound and have a high resolution, are shown to be powerful tools in combination with databases (Wagner-Dobler et al., 2002).
The in-vivo anti-tumour tests are carried out by testing the bioactive compounds in mice bearing the tumour cell line which was shown to be most sensitive in the in-vitro screen (Wagner-Dobler et al., 2002). Compounds that show significant tumour growth inhibition are then selected for further in-vivo evaluation against more advanced-stage tumours.
Next, the pre-clinical pharmacokinetic studies (absorption, bioavailability, distribution and excretion) and pre-clinical toxicology studies are carried out. The vast majority of anti-cancer drugs are cytotoxic compounds which have significant side-effects and a very small therapeutic index. The objective of these studies is to find a safe initial dose for Phase I clinical studies and to define the qualitative and quantitative organ toxicities (Wagner-Dobler et al., 2002).
Once this data has been established, the potential anti-cancer drug needs to be extensively reviewed by a range of regulatory authorities and committees to determine whether the drug is safe for carrying out studies on humans. Once it has been approved, it is possible to start the Phase I-III clinical trials. If the drug proves to be safe and effective in the clinical studies and once full regulatory approval is granted then the drug can then be scaled up in order to be sold in the market3. It is critical to protect the intellectual property surrounding the drug through the acquisition of various patents that will prevent others from making, using or selling what is described in the patent.
The productivity of the past decade in terms of the discovery of new clinical anti-cancer leads from diverse marine bacteria should translate into a number of new treatments for cancer in the decades to come. Exploitation of the potential of these marine microorganisms as producers of bioactive metabolites, with a wide range of potential pharmacological activities, is only just beginning. With the recent advances in both molecular biology and marine biotechnology, it is indeed very promising to see that the marine microbial environment is likely to continue to be prolific source of novel natural bioactive compounds for many years to come. In the future, further innovations in media development (chemical engineering), bioreactor design (bioprocess engineering) and transgenic production (molecular engineering), coupled with efficient downstream processing and product recovery (Pomponi, 1999), will continue to further enhance both the discovery and bulk production of these novel marine bioactive compounds.
References
Garcia Camacho, F., Chileh, T., Ceron Garcia, M.C., Sanchez Miron, A., Belarbi, E.H., Chisti, Y. & Molina Grima, E. (2005). A bioreaction-diffusion model for growth of marine sponge explants in bioreactors. Applied Microbiology and Biotechnology (details missing)
National Cancer Institute: www.cancer.gov
Pomponi, S.A. (1999). The Potential for the Marine Biotechnology Industry. Trends and Future Challenges for U.S. National Ocean and Coastal Policy: Workshop Materials, pp.101-104. Retrieved from website:
http://oceanservice.noaa.gov/websites/retiredsites/natdia_pdf/17pomponi.pdf
Schweder, T., Lindequist, U. & Lalk, M. (2005). Screening for New Metabolites from Marine Microorganisms. Advances in Biochemical Engineering/Biotechnology, Vol. 96, pp.1-48
Simmons, T.L., Andrianasolo, E., McPhail,K., Flatt, P. & Gerwick, W.H. (2005). Marine natural products as anticancer drugs. Molecular Cancer Therapeutics 2005, Vol.4, No.2, pp.333-342
Virax : www.virax.com.au
Wagner-Dobler, I., Beil, W., Lang, S., Meiners, M. & Laatsch, H. (2002). Integrated Approach to Explore the Potential of Marine Microorganisms for the Production of Bioactive Metabolites. Advances in Biochemical Engineering/Biotechnology, Vol. 74, pp.208-238
Webster, N.S., Wilson, K.Y., Blackall, L.L., Hill, R.T. (2001). Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile. Applied and Environmental Microbiology, Vol.67, No.1, pp.434-444
World Health Organisation: Ten Statistical Highlights in Global Public Health Retrieved from: http://www.who.int/whosis/whostat2006_10highlights.pdf
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