Detection of candida

An alkaline solution containing a complex of C.sub.30 H.sub.31 ClN.sub.6 is used as a specific indicator for detecting the presence of Candida spp. When Candida yeast bodies are introduced into the indicator solution, the yeast bodies are stained and form a blue or violet precipitate.

This invention relates to detection of Candida. More particularly, it 
relates to materials and methods for detecting and providing immediate 
colorimetric indication of the presence of as well as representative 
infection levels of resident colonies of Candida, particularly C. albicans 
and C. tropicalis. 
BACKGROUND OF THE INVENTION 
Although many fungal genera have been identified as etiologic opportunistic 
infections, it is known that Candida. constitute the majority of the 
pathogens involved in these infections. Candida is unique among 
opportunistic pathogens because it is a resident fungus found in the 
normal flora of mucosa and skin of many animals, including humans. 
Although there are numerous species of Candida, the majority of infections 
are caused by C. albicans and C. tropicalis. 
Immunosuppression, wherein fungal growth attains rapid and extensive 
colonization, is attributable to numerous causes, some of which are 
lifestyle related. It is known, for example, that changes in flora pH 
increases the likelihood of Candida growth. Fermentation of resident 
lactobacilli, with the subsequent formation of acid, favors yeast growth, 
Additionally, pregnancy and diabetes may incite growth since the level of 
glycogen increases in the vagina. Broad spectrum antibiotics causing 
changes in flora are probably the greatest contributing factor in 
observational increases of Candida infections. Lifestyle clothing has been 
given much consideration because restrictive, non-aerable garments are 
commensal in incubation of fungal genera. Additionally, commonly-used 
feminine hygiene products may contribute to the condition. 
Laboratory diagnosis is currently the accepted and most definitive method 
for determining the presence of infection levels of Candida. However, 
these methods are time-consuming because they generally require culturing, 
sub-culturing and clinical evaluation. Therefore, the patient and 
physician are relegated to simply collecting a sample and waiting while 
the sample is submitted to a clinical laboratory for appropriate 
diagnosis. Although methods for colorimetric determination of Candida 
exist, these procedures still require extended time for culturing prior to 
the diagnosis. Accordingly, a time period of approximately forty-eight 
(48) hours is required for diagnostic evaluation using conventional 
methods. 
SUMMARY OF THE INVENTION 
The present invention permits rapid detection and evaluation of Candida 
yeast bodies without employing culturing, incubation, sub-culturing or 
microscopic examination. In accordance with the invention, a specimen is 
collected and introduced into a solution containing a specific indicator 
which is selectively responsive to the presence of Candida yeast bodies. 
Within minutes after exposure to the indicator, Candida yeast bodies 
assume a distinct color and appear as a violet-colored suspension of 
precipitate in the solution. The color can be concentrated and the 
relative amount of Candida bodies can be determined by centrifuging the 
solution. Color density and speed of formation are indicative of Candida 
concentration. Because of simplicity of use and immediate indication, the 
method of the invention may be self-administered or physician-administered 
by simply collecting a specimen; introducing the specimen into an alkaline 
solution containing specific indicator; allowing the indicator to mark the 
fungal bodies; and observing the color change representative of the 
presence of Candida yeast bodies. 
DESCRIPTION OF THE PREFERRED EMBODIMENT 
In the preferred practice of the invention, a specimen suspected of 
containing Candida is mixed with a solution of potassium hydroxide and an 
indictor of the formula 
EQU C.sub.30 H.sub.31 ClN.sub.6 
which is known as Janus Green B and is described as 3 
(diethylamino)-7-[p-(dimetyhlamino)phenylazo]-5-phenylphenazinium 
chloride. Without being bound by any theories, it is believed that the 
Janus Green compound, when dissolved in an alkaline solution, forms a 
complex and is highly specific to phospholipid concentrations in the 
fungal bodies because its diethyl-dimethyl structure readily attaches to 
and becomes chemically bound with the lipid groups within the Candida 
bodies. The highly alkaline solution eliminates all other existing 
microbial bodies and solids. The following examples are demonstrative of 
specific test results.

EXAMPLE I 
A 0.5 mM C.sub.30 H.sub.31 ClN.sub.6 indicator solution was formed by 
dissolving Janus Green B in 2.5 ml of a 25% KOH solution in a 
conical-bottom centrifuge test tube. A 0.1 ml specimen taken from a 
cultured colony of Candida was introduced into the indicator solution and 
agitated by hand until no solids were visible (about 15 seconds). The 
solution was then centrifuged for five minutes. A dark purple precipitate 
was amassed in the lower tip of the test tube. 
The precipitate was extricated and, under microscopic comparison, exhibited 
exact morphology with a control colony culture. The extracted precipitate 
was smeared onto Sabouraud and Nickerson medium and incubated for 
twenty-four hours. No colonization was observed, thus evidencing 
destruction of Candida activity. 
EXAMPLE II 
The procedure of Example I was duplicated using a 10% KOH solution. The 
observed results were essentially identical except that colonization of 
Candida was evident after incubation of the precipitate for twenty-four 
hours. 
EXAMPLE III 
The procedure of EXAMPLE I was duplicated using a 1.0 mM C.sub.30 H.sub.31 
ClN.sub.6 concentration of Janus Green B. The observed results were 
essentially identical except that the solution above the amassed 
precipitate exhibited slight coloration. 
EXAMPLE IV 
The procedure of EXAMPLE I was duplicated using a 0.1 ml vaginal secretion 
specimen known to contain infectious levels of Candida. The observed 
results were essentially identical. 
EXAMPLE V 
The procedure of EXAMPLE I was duplicated using a 1.0 ml vaginal secretion 
specimen from an infection-free candidate. No precipitate was formed. The 
test solution exhibited no discernible coloration. 
EXAMPLE VI 
The procedure of EXAMPLE I was duplicated using a 1.0 ml vaginal secretion 
specimen from an infection-free candidate and 1.0 mM C.sub.30 H.sub.31 
ClN.sub.6. No precipitate was formed but the solution exhibited a faint 
bluish coloration. 
In addition to the foregoing specific examples, various tests were made to 
determine the effect of variations in concentration of the alkaline 
solution. It was discovered that the concentration of KOH could be varied 
from less than 5% to about 35% without appreciable effect on the rapidity 
of the staining or precipitation of Candida bodies. However, stained 
Candida precipitate taken from indicator solutions containing KOH 
concentrations of less than about 15% exhibited a definite tendency to 
produce viable colonies of yeast when incubated. Since clinical preference 
dictates destruction of infectious bodies in test procedures, the 
concentration of KOH should preferably be in the range of about 25% to 
30%. 
Potassium hydroxide has been demonstrated to be particularly useful as the 
alkaline solution. However, other solutions having a high degree of 
alkalinity (such as sodium hydroxide and the like) may be used in similar 
manner and produce similar results. 
The Janus Green B compound appears to be a specific indicator for Candida 
as described above. Various tests using various solutions of other known 
biological indicators failed to produce any discernible marking of Candida 
yeast bodies. 
The indicator solution of the invention may be prepared by simply adding a 
measured amount of water to a prepared dry mix of KOH and C.sub.30 
H.sub.31 ClN.sub.6 in appropriate amounts and proportions. Since the 
indicator solution is specific to Candida and produces essentially 
immediate visual results, the dry mix (or separate pre-measured amounts of 
caustic and Janus Green B) may be marketed as a unit with simple 
instructions for either self-administered or 
physician/technician-administered applications. The test results are not 
only specific, but essentially immediately available and produce nothing 
more than a disposable caustic solution containing dead yeast bodies. The 
invention thus provides an inexpensive, safe and rapid means for detecting 
the presence of Candida and an indication of the relative infection level 
thereof. 
Although the invention has been described herein with particular reference 
to specific embodiments thereof, it is to be understood that the forms 
described in detail are to be taken as preferred embodiments. Various 
changes, modifications, combinations and variations may be resorted to 
without departing form the spirit and scope of the invention as defined by 
the appended claims.