Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-01-02
2004-02-10
Lankford, Jr., Leon B (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S030000, C435S031000, C435S032000, C435S008000
Reexamination Certificate
active
06689577
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention is a reagent and a microscopy-based procedure for the detection of pathogens, especially spirochetes, from body fluids.
Provided that sterility is observed, the reagent according to the invention makes it possible to examine human body fluid samples under a dark-field microscope without staining.
As early as in 1683, the Dutch scientist, Anthonij van Leeuwenhoek discovered the spirochetes through his microscope and informed the British Royal Society about his discovery. Two centuries later, József Fodor was the first to discover that the blood of healthy individuals does not contain germs.
In comparison to serological and PCR techniques, the independence of the morphological examination (microscopy) of antigenicity and other changes (which—among spirochetes—is most typical of Borreliae) as well as of the emergence of new subspecies is still considered an advantage. The genetic make-up and the phenotype of these pathogens can even change during the illness of a single person, and some of these changes may not be detectable using the current techniques. After excluding infections with incompatible clinical presentations and higher local probabilities, morphological identification of the causative agent is diagnostic.
Despite the fact that the existence of spirilla was unequivocally demonstrated with the use of the microscope, even the well-known and widely recognized studies of Leeuwenhoek were forgotten—only to be re-discovered centuries later.
Indeed, conventional staining can hardly make these extremely thin and long pathogens visible; the tedious and most sophisticated technique of silver impregnation is still the only way to detect them in smears and slides. Cultivation is still problematic.
Since 1909, however, dark-field microscopy has made it possible to firmly diagnose spirochetoses from native, not fixed slides before the appearance of antibodies, provided the samples are taken from a patient who shows certain well-defined symptoms.
This means that the diagnosis can be made at an early stage, when treatment is expected to be most effective.
It was microscopy that proved that from the portal of entry, pathogens reach all organs (including the CNS) via the bloodstream and the lymphatic system.
The laboratory diagnosis of borrelioses with different clinical presentations is based primarily on the detection of spirochetes from blood samples. This is easily accomplished in recurrent fever because normally, there is a large number of
B. recurrentis
present. Besides, other morphological properties of this pathogen (shown in the table enclosed) and the fact that this pathogen is easy to stain also make its detection easier. There are mild cases, however, when the symptoms suggest the diagnosis of recurrent fever but the cell count is too low for the conventional methods to detect the causative agent. To solve this problem, the technique of microhematocrit concentration (double centrifugation of blood samples) has been used since 1972. Microscopy is superior in that the test result is not affected by the changing antigenicity of Borreliae. [Goldsmid, J M. Mohamed: The use of Microhematrocit Technic for the Recovery of
Borrelia duttonii
from the Blood, Am. J. Clin. Pathol. 58:165-169 (1972)].
Sample concentration has also been used to enhance conventional microscopy in parasitology. This way, pathogens can be detected more efficiently.
The diagnosis of Lyme borreliosis and the identification of its causative agent emerged as a new problem.
It is known that, in the seventies there was an outbreak of arthritis cases among the children who lived around the town of Lyme, Conn., USA. Ticks were soon identified as the vectors but the causative agent of the mysterious Lyme disease was not identified until much later.
After the unsuccessful investigations using top technologies, Willy Burgdorfer discovered the causative agent with a microscope and identified it as a new spirochete. This pathogen is referred to as
Borrelia burgdorferi sensu lato
[(Burgdorfer, W, Barbour, A. G., Hayes, S., F., Benach, J. L, Grunwaldt, E., Davis, J. P: Lyme Disease—a tick-borne spirochetosis? Science, 216(4552): 1317-9, Jun. 18, 1982)].
As mentioned above, morphological examination has long been included in the laboratory diagnosis of spirochetoses. If dark-field microscopy is employed, concentrated fluid samples do not need to be stained, which means that the long and thin spirochetes are not washed off the slides, which, in turn, increases sensitivity.
Soon after the introduction of this technique, which has now been used for decades, it was noted that pseudospirochetes (also known as myeloid figures), which are formed mainly during the degradation of red blood cells, can mislead the examiner whether it is human or animal blood that is examined. These artifacts are most likely to be present when stored samples or the stomach contents of blood-sucking insects (louse or ticks) are examined for infection. This issue is addressed in the following articles:
Chamber, H.: A new spirochaeta found in human blood; Lancet 1913; 1: 1728-1729;
Brecher, G.; Bessis, M: Present status of spiculed red cells and their relationship to the discocyte, echinocyte transformation: a critical review. Blood 1972; 40: 333-344;
Smith, T F.; Wold, A D.; Fairbanks, V F.; Washington, J A 2nd; Wilkowske, C J.: Pseudospirochetes a cause of erroneous diagnoses of leptospirosis.
Am. J.Clin. Pathol.
1979; 72: 459-63;
Greene,R T.; Walker,R L.; Greene,C E.: Pseudospirochetes in animal blood being cultured for
Borrelia burgdoiferi.
J.Vet. Diagn.Invest. October 1991; 3(4): 350-2;
Mursic,V P.; Wanner,G; Reinhardt,S; Wilske,B; Busch,U; Marget,W: Formation and cultivation of
Borrelia burgdorferi
spheroplast-L-form variants. Infection. 1996; 24: 218-26.
At the same time, however,
Borrelia burgdorferi sensu lato
even has several different morphological patterns in cultivation. Degenerative forms are also present if antibiotics are added to the culture (Aberer,E; Duray,P H: Morphology of
Borrelia burgdorferi
: structural patterns of cultured borreliae in relation to staining methods. J.Clin.Microbiol. 1991; 29(4): 764-72;
Barbour,A G; Todd,W J; Stoenner,H G: Action of penicillin on
Borrelia hermsii.
Antimicrob.Agents.Chemother. May; 21, 1982(5): 823-9;
Kersten,A; Potschekm,C; Rauch,S; Aberer,E: Effects of penicillin, ceftriaxone, and doxycycline on morphology of
Borrelia burgdorferi.
Antimicrob.Agenst.Chemother. 1995; 39(5): 1127-33.)
Today the diagnosis of spirochetoses is not problematic, except for borrelioses, which still present a challenge worldwide.
It is the direct identification of
Borrelia burgdorferi sensu lato
that can unambiguously verify Lyme disease. This is a tedious and ineffective process because the causative agent is difficult to cultivate and the low cell count of tissue and body fluid samples makes microscopy unworkable. Not even the newer genetic tests are sensitive enough in this case.
The indirect techniques used in the diagnosis of Lyme borreliosis are discussed below.
In this approach, host antibodies produced in response to
Borrelia burgdorferi sensu lato
infection are detected; the idea behind it is that nothing else but the infection can cause the antibody titers to rise. During the first stage of this infection, antibody production is slower than usual. Antibodies do not appear until weeks after the infection and are only rarely present throughout the whole course of the disease because titers keep changing and—after some time—they may become normal without any intervention. This makes it difficult to define the threshold titer. There is no clinically applicable threshold that could make a clear-cut distinction between those who are infected and those who are not. Besides, generation cycles of the causative agent cause a fluctuation of the early—IgM type—antibody titers. As far as we know, this is the only disease in which the causative agent blocks the production of the more specific and more effective IgG type antibodies, whic
Albert Janos
Bozsik Bela Pal
Dufla Ilona
Bozsik Bela P.
Davis R
Lankford, Jr. Leon B
Morris LLP Duane
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