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Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology
 
Thi-Nguyen-Ny Tran, Gérard Aboudharam, Didier Raoult, and Michel Drancourt
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Provisional recommendations for nonnucleotidic biomolecule analysis in paleomicrobiology

Several pitfalls in protein-based detection of ancient microbes must be addressed through the incorporation of proper controls before and during the analysis of ancient microbial proteins. Sampling mummified tissues for biological investigations prior to radiological investigations to prevent potential degradation of the ancient material and false-negative data has been advocated, but this recommendation lacks definitive evidence (31). False-negative data may result from the suboptimal extraction of proteins in concentrations below that detectable by current techniques. The amount of protein present should be measured prior to determining the identity of the protein, and fluorometric detection is suitable for such a purpose. False-positive data may result from the contamination of ancient material by modern materials. For example, the initial claim of MALDI-TOF MS detection of mycolic acids (69) was further invalidated by the observation that peaks were not mycolic acids; the samples were probably contaminated by the polyethylene glycol component of a mycolic acid standard (70). The possibility of contamination must be addressed by the parallel testing of several experimental blanks consisting of buffer without ancient material. In addition, false-positive results may result from a lack of microbe specificity of the test, as in the case of cross-reactivity of primary antibodies with host tissue or the nonspecific reaction of secondary antibodies with host tissue. For example, what was thought to be the initial detection of hemoglobin in 4500-year-old human bones (71) was later found to be a false result due to cross-reactivity of the primary antibody with nonhemoglobin bone proteins (72). The possibility of cross-reactivity must be addressed by the parallel testing of negative tissue controls, namely, a sample of tissue identical to or similar to the tested host tissues and collected from corpses devoid of any anthropological evidence of infection. In immunodetection, this control ensures that the primary and secondary antibodies lack nonspecific reactivity against the host tissue. Additionally, the tested tissue should be incubated with a primary, nonrelevant antibody to further ensure nonspecific antigenicity of the tested tissues. False-positive data may result from cross-reactivity between microbes and a lack of specificity of the primary antibody. Therefore, testing the primary antibody against a collection of relevant organisms is advocated prior to testing ancient specimens. As for the general experimental design, parallel detection of any other biomolecule using an unrelated technique (e.g., PCR sequencing of aDNA) is warranted. Controlling the reproducibility of the data and blind testing additional specimens in the same batch as negative controls are also warranted.

Perspectives

Even though the detection and characterization of aDNA is the most widely used approach for the study of ancient pathogens (73), the difficulty of obtaining ancient microbial DNA and the critical question of contamination by PCR-amplified DNA may limit this approach in paleomicrobiology. For example, the parallel investigation of Y. pestis aDNA by PCR and F1 antigen by the dipstick assay in the analysis of 17th century skeletal remains indicated that 83% of the samples contained too few aDNA molecules to start PCRs (48). Therefore, studying past infections by the detection of nonnucleotidic biomolecules is an approach that is being increasingly used in paleomicrobiology (Figure 4). As for the immunodetection technique and late serology, these techniques can only answer one question at a time. However, a versatile immunodetection technique referred to as autoimmunohistochemistry has been developed for the diagnosis of modern infectious diseases (43). Since it has been shown that antibodies can survive for centuries, this method can potentially be applied to ancient specimens to demonstrate the presence of microorganisms. The capacity of immunodetection to trace minute quantities of microbial materials can be further increased by the application of immuno-PCR, a technique combining the advantages of immunodetection with the logarithmic, PCR-based amplification of the signal (74). Immuno-PCR was first presented as a technique in which a specific antibody-DNA conjugate is used to detect antigen (74). Accordingly, immuno-PCR has a sensitivity greater than any existing antigen detection system because of the tremendous amplification capability and specificity of PCR (74). We are successfully developing immuno-PCR applications for the detection of Y. pestis in the dental pulp of past plague victims (D. Raoult, unpublished data). With the foreseeable developments of technology, such as MS and immuno-PCR, the analysis of nonnucleotidic biomolecules is becoming a standard approach to complement aDNA analysis in the field of paleomicrobiology.





Competing interests

T.-N.-N. T., D.R., and M.D. own a patent on the MALDI-TOF MS of the dental pulp for classification of mammals. G.A. declares no competing interests.

Correspondence
Address correspondence to Michel Drancourt, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Faculté de Médecine, Université de la Méditerranée, Marseille, France. e-mail: [email protected]

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