There are currently three types of in vitro diagnostic systems widely used for the detection of non-viral pathogens. These are direct culture of the pathogenic agent from the biological sample; immunological assays based on the detection of products or antigens of the infectious agent; and indirect immunological assays that can detect antibodies produced against the infectious agent during infection.
In the first system, the principal disadvantage is that the biological sample must be considered to be at risk for the transmission of the pathogenic agent. In the second and third systems, the disadvantages include sample retrieval that is often invasive and potentially infective sample when collected. In the third system, one major disadvantage is that there is often little possibility of discriminating between past and current infections.
More recently, molecular diagnostic methods have been developed based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays are generally much more sensitive than the immunological assays. However, they may require the presence of special equipment and qualified personnel. Furthermore, the biological samples—in the case of plasma, blood, or cell cultures—are difficult to store unaltered, except under controlled temperature conditions, and are considered to be biohazardous to personnel who handle them.
Recently, molecular diagnostic methods based on transrenal DNA (Tr-DNA) have been described for monitoring the progress of allogeneic transplants, to diagnose the sex of a fetus, and to detect the presence of tumor markers. (Botezatu et al. Clinical Chemistry 46(8):1078-84 (2000); Su et al. Ann. NY Acad. Sci. 1022:81-89 (2004)) For example, U.S. Pat. No. 6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant women. U.S. Pat. No. 6,287,820 describes a system aimed at the diagnosis of tumors, particularly of adenocarcinomas of the colon and pancreas. U.S. Pat. No. 6,492,144 teaches that the Tr-DNA nucleic-acid analysis method may be used to monitor the progress of allogeneic transplants. The presence of transrenal DNA in urine, in the form of nucleic-acid fragments of fewer than 1000 base pairs was also described in Al-Yatama et al. (2001), Prenat Diagn, 21:399-402; and Utting, M., et al. (2002), Clin Cancer Res, 8:35-40. Keiko Koide, et al., Prenat Diagn, 2005; 25: 604-607; Mengjun Wang, et al., Clinical Chemistry, 2004, 50: 211-213; Y.-H. Su, et al., J. Mol. Diagn., 2004, 6: 101-107.
The presence of transrenal DNA has been explained through the apoptosis phenomenon. During cell death most of the nuclear DNA is converted into nucleosomes and oligomers (Umansky, S. R., et al. 1982, Biochim. Biophys. Acta 655:9-17), which are finally digested by macrophages or neighboring cells. However, a portion of this degraded DNA escapes phagocytic metabolism, and can be found in the bloodstream (Lichtenstein, A. V., et al. 2001, Ann NY Acad Sci, 945:239-249), and, as confirmed in the above-indicated patents, also in urine.
The application of this system to pathogenic microorganism infections has never been studied. Previously, it was only known that prokaryotic DNA could be isolated from urine sediment that contained bacteria (Frasier, et al. 1992, Acta Virol, 36:83-89). During a pathogenic infection, prokaryotes and parasites are generally ingested by the cells of the immune system, such as macrophages and dendritic cells. The prokaryotes are then dissolved by the phagolysosome vesicles. The prokaryotic DNA is then released by the cell and a portion of this DNA enters the bloodstream in either of two ways. Either the ingesting cell becomes apoptotic and breaks apart (Navarre, W. V. 2000; Cell Microbiol 2:265-273); or the phagolysosome vesicles release the fragments of the prokaryote (including the fragmented DNA) into the bloodstream (Friedlander, A. M. 1978, Infect Immune 22:148-154). However, these fragmented nucleic acids have never been detected in the urine of the infected subject.
The instant invention describes a method of detecting the presence of non-viral pathogens in a subject through the detection of DNA sequences from those pathogens in the urine of the subject.