Diagnosis of infection frequently involves a determination of the causative pathogen. In the case of bacterial infection, a knowledge of the pathogen can lead to an informed choice of antibiotic.
Numerous methods have been devised for clinical diagnosis. Among these are culture techniques, immunoassay, microscopy, analytical chemistry and nucleic acid hybridization.
By far, the most widely used method is culture, which involves inoculating a nutrient medium with a sample and then determining the extent of organism growth. Culture techniques are time consuming and are restricted in that they are capable of revealing the presence of only live organisms. Successful diagnosis relies upon selecting the appropriate culture media and conditions which will foster the growth of the etiologic agent. Fastidious organisms such as Mycoplasma spp., Neisseria spp., Haemophilus spp., and all obligate anaerobic organisms for example, require extraordinary culture conditions and extended periods of time to grow. If appropriate growth conditions are not met, these fastidious etiologic agents are likely to be missed when subjected to standard culture screening procedures.
Immunoassay, while promising the potential for rapidity of diagnosis, frequently lacks the sensitivity required to detect microorganisms which, during 30 to 50% of all septicemia, are present at less than or equal to one colony forming unit (CFU) per milli-liter of blood. [Kiehn, T. E. et al., J. Clin. Micro. 18, 300-304 (1983).]
Microscopy, analytical, chemical and hybridization methods frequently require sophisticated instrumentation and sample preparation and are therefore quite labor intensive.
It is known that phagocytes engulf numerous microbial pathogens. Some organisms reside within the phagocytes as intracellular parasites, while others are at a minimum, rendered nonviable by the internal degradative process of the phagocyte and, at a maximum, digested by these degradative processes.
Zierdt et al. have shown that lysis-filtration blood culture was a more sensitive culture technique than a non-lysis blood culture technique for the detection of pathogenic bacteria during septicemia in rabbits. The authors postulate that the lyses solution (a mixture of Tween-20 and Rhozyme) lyses phagocytes, thereby releasing engulfed pathogens whose viability has not been destroyed by the internal degradative processes of the phagocyte. These authors detected the presence of pathogens using conventional culture techniques. [Zierdt et al., J. Clin. Micro., 15, 74-77 (1982).] These conventional techniques can, however, only detect viable organisms not soluble products of phagocytic degradation.
The lysing solution used by Zierdt et al. is a mixture of a mild, nonionic detergent (Tween-20), and a protease including some lipase and nuclease (Rhozyme). [Zierdt, J. Clin. Micro., 15, 172-174 (1982). ]
While the lysing solution used by Zierdt et al. does lyse phagocytes to release viable pathogens for subsequent culturing, the presence of protease and nuclease in the lysing solution could present problems in other detection systems such as immunoassay or nucleic acid hybridization. It might be expected that these degradative enzymes would destroy the specific reagents required. Similarly, the degradation enzymes of the phagocyte are also released by lysis and are another source of potential degradation of the specific detection reagents.
According to Zierdt et al., the release of viable pathogen from phagocytes is desirable because the total number of viable organisms available for growth in a culture-based detection system increases.
Slama, T. G. et al. reported that the Gc2 polysaccharide derived from lipopolysaccharide of N. gonorrhoeae cell wall antigens was immunochemically detectable in PMN-containing fluids 2 to 6 hours after the bacterial cells had been phagocytized. Since unlysed PNM were used, the lipopolysaccharide detected was external to the PMN and there is no report by the authors of lysis of the PMN to increase the concentration of the antigens to be detected. After 24 hours, the same PMN-containing fluids did not react with specific antisera. Attempts to culture the bacteria from PMN-containing fluids 6 hours after phagocytosis indicated that the fluids were sterile. Results showed that both culture and immunochemical techniques for assay of N. gonorrhoeae using PMN-containing fluids with intact PMN cells were applicable, but only on a transient basis over a relatively short period. [Slama, T. G. et al., Sex Transm Dis., 9, 70-73 (1982).]
Friedlander, Infection and Immunity, Volume 22, 148-154 (1978), reports an assay for measurement of microbial killing by phagocytes. This method assumes that release of soluble radioactively labelled DNA from the microbe is direct evidence of cell death. As noted by Friedlander, since the standard assay condition did not include lysis of the phagocytes, all radioactivity detected must have been external to the phagocytes. When the phagocytes were lysed prior to testing for soluble radioactivity, no additional radioactivity was detected. This indicated that the soluble degraded DNA did not accumulate in the phagocytes, but rather was immediately released.
Richards et al., U.S. Pat. No. 4,581,331, issued Apr. 8, 1986, report a method for detection of virus or viral antigens by lysing phagocytes, separating the soluble fraction containing the virus or viral antigen and then detecting the virus or viral antigen. The only specific lytic agent reported is purified saponin. Detector systems reported include tissue culture, immunoassay and nucleic acid hybridization.
It has been found that phagocyte lysates yielding nonviable products of pathogens subjected to the internal degradative processes of the phagocytes can be used to advantage in clinical diagnosis if one uses an immunoassay or hybridization based pathogen detection system rather than a culture based detection system. The degradative processes of the phagocyte have been found in at least two examples not to degrade antigens to the point where they can no longer be recognized by their complementary antibodies.