New directions in molecular techniques for pathogen identification.

The comprehensive review by Paul Kellam in last month's issue of Trends Microbiol.[1xKellam, P. Trends Microbiol. 1998; 6: 160–166Abstract | Full Text | Full Text PDF | PubMed | Scopus (13)See all References[1]provides a thorough summary of molecular techniques currently used to identify unculturable viruses. A pathogen can now be discovered without in vitro cultivation solely from a fragment of its nucleic acid sequence, which in turn can lead directly to production of synthetic peptide antigens for serodiagnosis and subunit vaccines for disease prevention. By knowing the class of agent, anti-infective therapies might be tested and, eventually, sequence-based modeling might help in the rational design of specific and nontoxic small-molecule therapeutics. All of these events have already occurred or are achievable in the foreseeable future. In our own experience, identification of <1% of the Kaposi's sarcoma-associated herpesvirus genome[2xChang, Y. et al. Science. 1994; 265: 1865–1869CrossrefSee all References[2]by representational difference analysis (RDA) has led to whole virus genome sequencing, development of an appropriate virus culture system for antiviral testing, and synthesis of recombinant antigens in less than two years. The importance of finding only a few base pairs of unique sequence data from a new agent should not be underestimated.Several molecular pathogen detection techniques, such as RDA, now require intensive benchwork to isolate a pathogen genome from the human genome. In this regard, virologists have something to learn from their colleagues in bacteriology. Use of conserved 16S prokaryote consensus ribosomal gene primers for PCR has helped to resolve the long-standing riddle of Whipple's disease and the more-recent riddles of AIDS-related bacillary angiomatosis and cat-scratch disease[3xFredericks, D.N. and Relman, D.A. Clin. Microbiol. Rev. 1996; 9: 18–33PubMedSee all References[3]. The value of consensus primer PCR detection for new viruses has been demonstrated by the recent discoveries of primers amplifying the sin nombre hantavirus capsid gene[4xNichol, S.T. et al. Science. 1993; 262: 914–917Crossref | PubMedSee all References[4], panherpesvirus primers[5xRose, T.M. et al. J. Virol. 1997; 71: 4138–4144PubMedSee all References[5]and consensus primers to identify new papillomavirus types.Consensus primer amplification is not as tractable for viruses as it is for prokaryotes, and a serious, systematic search for useful virus family consensus primers has not been undertaken. This should be a primary goal for the emerging infectious disease initiative being developed by various public health agencies. Panels of consensus primers could prove invaluable as diagnostic aids during outbreak emergencies, particularly for unidentified arboviral and zoonotic infections occurring in remote regions lacking ready access to tissue-culture facilities.As high-throughput genomic sequencing and analysis improves, detection techniques may shift away from the current emphasis on physical isolation of pathogen nucleic acids. Chip and sequencing technologies [e.g. serial analysis of gene expression (SAGE)][6xVelculescu, V.E. et al. Science. 1995; 270: 484–487Crossref | PubMedSee all References[6]do not necessarily rely on known sequence data and have the potential to interrogate unique nucleic acids present in diseased tissues but not healthy tissues from the same individual. Although the current intellectual climate in genomics is geared towards characterizing known sequences faster and better, it might be a small step for current technologies to identify unique genes expressed by previously unknown viruses.Knowing where to look for a new pathogen is just as important as knowing how to look for it. As Kellam[1xKellam, P. Trends Microbiol. 1998; 6: 160–166Abstract | Full Text | Full Text PDF | PubMed | Scopus (13)See all References[1]points out, good epidemiology is absolutely essential for knowing which diseases are likely to have an infectious component and determining whether the newly found agent actually causes the disease in question. Unfortunately, most molecular biologists know little about epidemiological techniques and frequently dismiss epidemiological methodology. Similarly, epidemiologists are generally guilty of being unable to interpret and employ the revolutionary advances in molecular biology made in the past two decades. However, use of sophisticated molecular biology, together with fundamental and rigorous epidemiology, can be a potent combination for discovering the next generation of human pathogens.