Equipment, kit and method for microbiological diagnosis

Details Equipment, kit and method for microbiological diagnosis Equipment, kit and method for microbiological diagnosis

1999-10-18

2003-03-25

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S975000, C435S283100, C435S968000, C435S848000, C435S849000

Reexamination Certificate

active

06537772

ABSTRACT:

TECHNICAL SECTOR. BACKGROUND OF THE INVENTION
The present invention relates to microbiological diagnosis, and more specifically to a method for rapid microbiological diagnosis, and the equipment in the form of a kit, which is used to perform this diagnosis. This diagnosis method has applications in human and veterinary clinical medicine.
PRIOR ART
Microbiological diagnosis is based on physical, chemical and biological methods that have been widely developed in previous prior art.
For example U.S. Pat. No. 3,506,544 describes an electrochemical method for detecting bacteria through measurement of the decrease in polarographic content of the oxygen which circulates through an electroanalytical cell that contains two different electrodes immersed in an inoculated culture medium. This method uses great quantities of culture medium (15-18 ml) for its analysis, thus making handling of the samples difficult on a routine level.
Another method that has been used to detect microbial growth is a method using a voltaic cell source, which is based on the use of an appropriate medium with electrodes of noble metals and predetermined volumes, that generate a potential which drops at the moment of growth of the bacterium. Other equipment using this principle has been described in the scientific literature. For example, the equipment described by patent GB 83-17685 uses the same procedure. This patent describes a method of detection that uses the variation of the potential between electrodes that are in contact with fluid samples. Thus lower potentials are measured with implied higher impedance at the opening which cause a change in the measured signal due to undesirable and unavoidable noises. In most cases the system uses electrodes of noble metals or no recoverable gold plated electrodes.
An efficient and simple method for detecting microbial growth is based on the measurement of conductometric changes that occur in a suitable culture medium due to the microbial growth. According to pertinent literature, ionic movements produce a signal of conductivity measurement of the solution in a cell that indicates the conductometric value of the solution.
It is known that conductivity cells do not have total lineal behavior in their baseline scale. In addition, analysis depends on temperature. In U.S. Pat. No. 4,482,967 a detector and a method to measure the conductivity that corrects these defects is described. This reference shows equipment of high accuracy and complexity with special provisions to measure absolute values of conductivity in a gas chromatograph, with conventional cells and large volumes.
It is known that microbial growth can be detected in fluid samples using different methods, for example, by using a turbidimetric method in which the bacteria growth produces turbidity that is read by the system detector and compared with established standards. This system requires conventional optical sensors, with high quality optical receivers, sample containers of complex design to work with samples that include visual solids. (for example, antibiotic discs) A disadvantage of this method is that is does not analyze impure samples, i.e. it requires homogeneous optical samples, because of the optical complexity of the apparatus system. U.S. Pat. Nos. 3,832,532, 3,895,661 and 3,889,011 describe methods and apparati based on these principles. U.S. Pat. Nos. 4,021,120 and 3,714,445 describe devices (turbidimeters) which measure the turbidity of microorganisms in liquid mediums.
U.S. Pat. Nos. 4,021,120 and 3,714,445 describe devices based on turbidimetric principle to measure the growth of microorganisms in liquid mediums. U.S. Pat. No. 4,021,120 describes a device to monitor the growth of microorganisms in a liquid medium that contains gas. Samples are taken from the medium, using a pump that carries the sample to a degassing chamber, eliminating gas bubbles. The sample is then introduced into a calibrated chamber through which a light ray passes. The light ray strikes a photoelectric cell, producing a current that is increased by an amplifier. This indicates growth of the microorganisms. The magnitude of this current will depend on the intensity of the light ray and will be influenced by the turbidity of the medium. The sample is then pumped back to the receiving vessel to be analyzed. This method of measurement, as well as one described in U.S. Pat. No. 3,714,445, is complex from an optical and mechanical perspective; in addition, the measurement chamber and the pumps and ducts used to transfer the samples should be sterilized frequently, this makes its use difficult in routine diagnostic methods.
Patent GB 2 221 986 and U.S. Pat. Nos. 3,819,278 and 4,725,148 refer to turbidmeters that directly measure the microbial growth using the same principle of previous methods. They present optically and mechanically complex systems that need sterilization between each batch of microorganisms.
On the other hand, U.S. Pat. No. 3,832,532, also uses an conventional optically device that includes a cuvette of spectrophotometric quality, the device takes measurements using antibiotic discs included in its design as an interconnected bi-lobed chamber. After the incubation is completed, the liquid must pass to the other chamber for measurement, in order to avoid presence of the antibiotic disc during the reading step.
Thus the invention of U.S. Pat. No. 3,832,532 presents a system of operative and technical complexity and in addition has economical implications.
The present trends of microbiology make use the search of procedures that allow rapid identification of microorganisms (between 2-4 hours) in biological samples. To accomplish this, different strategies have been used, among them the use of specific enzyme markers.
According to the present state of the art, most of the biological samples cannot be used directly; isolation and growth of the microorganism must be completed before the sample can be identified, requiring 24-48 hours of laboratory time.
Infections of the urinary tract are considered one of the most prevalent among infectious illnesses.
The classic technique for detecting bacterial infections in urine requires cultivation on plates for at least 24 hours in order to discard all negative samples and to select positive ones.
Only 20% of the urine samples that arrive at the lab are positive, and from these 70% correspond to infections provoked by
E.coli
. Identification of
E.coli
saves time and resources, as only 30% of the positive samples would be isolated for identification.
According to the state of the art, identification of
E.coli
is accomplished mainly by two specific enzyme markers for this bacterium, &bgr;-D-glucuronidase and tryptophanase enzymes, through Indol formation (Kovacks, N. Eine vereinfachte Methode zum der Nachweis der Indolbildung durch Bakerien. Z. Immunitatsforsch., 55; 311-315, 1928). 94% of all
E.coli
, a few Salmonellas and Shigullas show positive reaction towards &bgr;-D-glucuronidase. Indol formation is positive for 99% of all
E.coli
; thus combination of both tests allows unmistakable identification of this microorganism.
Presently, different tests are being marketed, like BACTIDENT-
E.coli
and different culture mediums like FLUOROCULT-MUG, (both from MERCK DIAGNOSTICA). They are based on the above principle. In order to use them, an isolated colony from a previous isolation of the microorganism used to make the BACTIDENT identification must be taken; or the sample can be inoculated into the culture medium and grown for 24 hours; it is only possible to detect associated changes to the specific substrate transformation (FLUOROCULT-MUG).
A solid culture medium for simultaneous detection of coliform bacteria and/or
E.coli
in water samples and in foods is reported in the patent application No. WO 95/03424. 24 hours of incubation is required after inoculation of the plate with the sample to be evaluated. Similar procedures are followed in the Diagnostic Kit URILINE ID and the culture medium CPS ID, both from BIOMERIEUX, France. The incubation of the samples on sol

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