Sputum analysis method

BACKGROUND OF THE INVENTION
This invention relates to the quantitative analysis of human sputums for the presence of microbial pathogens. In another aspect, this invention relates to a novel technique of liquefying sputum specimens with a saponin mucolytic agent.
The condition of pneumonia in a human patient generally comprises an acute inflammatory condition of a lung or lungs which can be caused by microbial pathogens such as bacteria or viruses as well as chemical irritants or foreign bodies. In order to determine the causal agent in a patient with a presumptive diagnosis of pneumonia, samples of lung fluid are required. The ideal specimens of lung fluid are homogeneous samples which can be obtained by surgical intervention, for example, transtrachael aspiration. As is typical, surgical intervention techniques are time consuming and present a risk to the patient. Fatal reactions have been reported resulting from transtrachael aspiration. For example, hypotoxic patients and those suffering from debilitating diseases such as blood dyscrasia are especially prone to serious complications from transtrachael aspiration.
As a consequence, most physicians rely on the early morning couph sputum specimen as the means of obtaining a lung fluid specimen. This technique is simple and very common. Unfortunately, the cough sputum specimen can be readily contaminated by the normal flora in the mouth, nose, posterior pharynx, and stomach. Furthermore, sputums from patients with pneumonia are very viscous and heterogeneous in nature and therefore difficult to disperse in a reproducible manner. Quantitative analysis of sputum cannot be accomplished on the very viscous heterogeneous sputums because it is generally necessary in quantitative analysis of specimens like sputum to dilute the specimen and thereafter plate minor portions of the specimen on various growth media and then determine the type and number of colonies of microbial organisms which result on the media. Generally, the maximum number of colonies which can be effectively counted on a petri dish is about 300. Furthermore, it has been accepted that normal sputum samples can contain contaminating microorganisms in quantites up to 10.sup.5 per milliliter, whereas, as causative organisms of pneumonia microorganisms are generally present in quantities greater than about 10.sup.6 per milliliter. Therefore, before a sputum specimen can be quantitatively analyzed, it must be diluted in a manner such that the microbial pathogens are uniformly dispersed in the resulting diluted sample. Accordingly, if a minor portion of this diluted sputum specimen is quantitatively analyzed for the type and number of microbial pathogens, the accurate number of microbial pathogens per milliliter of the sputum sample can be accurately calculated.
Thus, in order to reduce the problem of external contamination and heterogeneity of the sputum sample and provide a quantitative sputum analysis, several attempts have been made to digest the sputum specimen with enzymatic and chemical digestants. Several such digestants have been theretofore tried and all of the digestants have one or more of the following disadvantages: expensive; short shelf life; temperature sensitive; toxic to some or many pathogenic organisms; and require long digestion time. For example, a number of proteolytic enzymes have been tested in both purulent and mucoid sputum. Of such enzymatic materials, trypsin, elastase, and chymotrypsin appear the most effective, and enzymes such as bromelain, ficin or papain were only effective at extreme high concentrations, while plasmin has no detectable effect on sputum viscosity. All such proteolytic enzymes appear to be more effective with mucoid sputums than with purulent sputums. The most widely used digestants for quantitative sputum analysis are aqueous solutions of N-acetylcysteine and Cleland's reagent (1,4-dithio-mesoerithritol). In general, Cleland's reagent exhibits greater mucolytic activity than N-acetylcysteine at lower concentrations, but Cleland's reagent generally loses its mucolytic activity in relatively short periods of time in aqueous solution. Furthermore, both of these reagents are somewhat toxic to microbial pathogens at concentrations needed for rapid digestion of sputum.
Consequently most sputum specimens are processed by a nonquantitative streaking technique, which generally involves streaking the heterogeneous sputum specimen on various growth media. These techniques lead to a substantial number of false positive cultures, and in many instances, overgrowth of the pathogenic organisms by contaminating microorganisms.
SHORT STATEMENT OF THE INVENTION
According to the invention, I have discovered that certain purified saponins exhibit mucolytic activity and will effectively degrade sputum samples including both purulent and mucoid sputums and uniformly disperse microbial pathogens therewithin without harming the pathogens.
According to one embodiment of the subject invention, a novel technique for the quantitative analysis of sputums is provided which includes the steps of contacting the sputum specimen after it is collected with an effective mucolytic portion of a nontoxic saponin to convert the sputum specimen to a substantially uniform viscosity and mixing the specimen such that any pathogens are uniformly distributed therewithin and thereafter conducting quantitative analysis techniques on the resulting specimen.
DETAILED DESCRIPTION OF THE INVENTION
The nontoxic saponin which is utilizable within the scope of the subject invention can be purified by the method disclosed in U.S. Pat. No. 3,883,425 which patent is herein incorporated by reference into this specification.
Saponins are glycosides widely distributed in plants and are capable of forming oil-in-water emulsions, and act as protective colloids. Each saponin molecule consists of a sapogenin which constitutes the aglucone moiety of the molecule, and a sugar.
The sapogenin is either a triterpenoid (usually a pentacyclic structure, such as quillaic acid), or a stearoid structure (usually having a spiro acetal side chain as in diosgenin). The sugar portion of the saponin glucoside includes one or more sugars such as glucose, sucrose, xylose, a pentose or methyl pentose, or other sugars. On hydrolysis, the saponins yield the sapogenin and one or more of these sugars.
Commercially available saponins comprise a white to brown amorphous powder which is pungent, and has a disagreeable taste and odor. This powder is very soluble in water and foams very strongly when shaken with water.
Commercial saponins are prepared by extracting plant tissue with water and/or other organic solvents, such as alcohol and recovering saponin by precipitation, recrystallization and the like. Saponins are widely distributed in plants. For example, saponins are very widely distributed in the plant family caryophyllaceae. Specific examples of saponin sources include soap bark, panama wood, soap berry, liquorice, and the like. A specific example of a process for producing commercial saponin is described in Kingzette's Chemical Encyclopedia, D. H. Hey, 9th Edition, (1966) which includes either extracting powdered soap bark (Quillaria saponaria) with hot alcohol or by boiling powdered dry aqueous extract of such bark with alcohol, and allowing saponin to crystallize from the alcohol upon cooling.
Saponins are practically nontoxic to humans upon oral ingestion, but act as powerful hemolytics when injected into the bloodstream, thereby dissolving the red blood corpuscles. Because of this characteristic, saponins have been conventionally used in hemotology laboratories for lysing the red cells whenever their presence interferred with other procedures, e.g., hemoglobin determinations and white cell counts.
As set forth in said patent, U.S. Pat. No. 3,883,425, a technique is provided for removing microbial toxins from saponin which is extracted from a plant source by removing consitutents from an aqueous solution of said saponin which have an apparent molecular weight of less than about 600, e.g., a molecular size in aqueous solution between about 140 to about 600. One technique disclosed in said patent is to form an aqueous solution of a commercial saponin extract from plants and thereafter pass the solution through a microporous filter membrane which has a mean actual pore size no smaller than about 11 angstroms in diameter and no larger than about 24 angstroms in diameter. The aqueous solution which passes through the microporous filter membrane will contain the antimicrobial toxin, and the filter will exclude the saponin material.
I have now found that such purified saponin plant extracts are effective mucolytic agents in that they will efficiently degrade all types of sputum and disperse microbial pathogens therewithin without harming the pathogens.
In carrying out the method of the subject invention, the sputum sample is collected by any conventional technique. Thereafter the sputum is admixed with a minor but effective mucolytic amount of the nontoxic saponin described above. Preferably the saponin is in aqueous solution when admixed with the sputum. The aqueous solution can be of any convenient concentration such that when the desired amount of solution is admixed with the sputum specimen, sufficient saponin will be present to degrade the same. Generally, it is necessary from about 0.1% by weight to about 20% by weight and preferably from about 1.0% by weight to about 10% by weight and even more preferably from about 5% by weight to about 10% by weight of saponin be contained in the resultant mixture of saponin and sputum.
In addition, it is preferable to add to the resultant mixture of saponin and sputum from about 0.1% by weight to about 20% by weight and more preferably from about 5% by weight to about 10% by weight of an antifoamant which is nondeleterious to microbial pathogens. Suitable such antifoam agents include materials sold under the trademarks of Dow X Antifoam B and Dow X H 10. These agents contain chemical polymers of dimethylsiloxane and a sterilizable, e.g., autoclavable, nonionic emulsifier such as the type sold under the trademark of Triton X. The material sold under the trademark of Dow X H 10 may be preferred since it is very stable to sterilization by autoclaving. The saponin effectively degrades all types of sputum both mucoid and purulent very rapidly to form a mixture of uniform viscosity.
The sputum sample is then thoroughly admixed to uniformly disperse the microorganisms therewithin. If desired, portions of the resulting degraded sputum sample can be plated directly on various media to obtain an improved qualitative sputum analysis which is not subject to the substantial number of false-positive cultures and overgrowth of pathogenic flora by contaminating microorganisms as is the conventional qualitative technique which does not utilize degraded sputum.
When conducting an improved quantitative analysis of sputum in accordance with the subject invention, the saponin treated sputum is diluted as desired, e.g., 1:100-1:1000 with phosphate buffered normal saline, pH 7.2 or any other suitable diluent which will not interfere in the analysis technique and is nontoxic to the microbial pathogens. The resulting diluted specimen is thoroughly admixed to assure uniform distribution of the microbial pathogens throughout the solution. Next, samples of the diluted solution are added to conventional growth media and allowed to incubate. Next, any resulting colonies on the media can be identified and counted and by knowing the extent of dilution of the sputum specimen, the total quantity of different types of microbial pathogens can thereby be easily calculated. Alternately, if desired, the nondiluted specimen can be subjected to microscopic examination.
The following examples are presented to more specifically illustrate the subject invention.

EXAMPLE I
In this Example, the toxicity of various known mucolytic agents was compared to that of saponin. In each instance, the strains of bacteria shown in Table 1 below were grown overnight in the growth media indicated in Table 1 and the turbidity of the resultant growth media was adjusted with sterile saline to a barium sulfate standard to approximately 10.sup.8 bacteria per milliliter. Thereafter the resulting broth was diluted 1:200 in nutrient broth. Next, a 40 weight percent solution of saponin which was purified in accordance with the procedure set forth in U.S. Pat. No. 3,883,425 (to thereby remove toxic contaminants therefrom which have an apparent molecular weight of less than about 600 in aqueous solution) was added to the type of growth broth for the particular bacteria as indicated in Table 1. Thereafter, 1 milliliter of each bacterial-growth broth suspension was added to 1 milliliter of saponin solution described above to give a final concentration of 20% by weight saponin, and then was incubated overnight at 37.degree. C. A control and test suspension was also streaked on rich agar containing 1 weight percent glucose, or blood agar.
The other compounds tested for toxicity were Cleland's reagent (1,4-dithio-mesoerithritol), N-acetylcysteine, and sodium lauryl sulfate. These mucolytic agents were prepared in the concentrations and in the growth broth for each of the microorganisms as indicated in Table 1 and tested for toxicity in the same manner as the saponin described above.
If the particular bacteria grew in the broth containing the indicated quantity of mucolytic agent, the run was repeated to see if growth could be duplicated in a second run. If growth was not obtained either the first or second time, a third run was made in an attempt to grow the bacteria. In some instances, a third run was made even though bacteria grew in the prior two runs. Therefore, in the table a plus indicates a successful growth run and a minus indicates a negative growth run. More specifically:
+++ = grew three out of three times
--- = did not grow once out of three times
--+ = grew one out of three times
-++ = grew twice out of three times
The results are set forth in Table 1 below:
Table 1__________________________________________________________________________ Mucolytic Agent and Percent Concentration Type of Saponin Cleland's N-acetylcysteine Sodium lauryl sulfateOrganism Growth Broth* 20% 0.1% 0.5% 0.5% 5.0% 2.5% 10.0%__________________________________________________________________________Escherichia coli I 1 ++ +++ -++ +-+ --- +++ +++Staphylococcus XIII 1 ++ +++ --- ++- --- --- ---Pseudomonas species 1 ++ +++ --- ++- --- +-+ +-+Candida species 1 ++ +++ -++ +++ --- +-- ---Torulopsis glabrata 1 ++ +++ -+- +++ --- --- ---Streptococcus pyogenes 2 ++ ++ -- +- +- -- --Streptococcus pneumoniae 3 ++ ++ -- -- -- -- --Citrobacter freundii 1 ++ +++ +++ -++ --- +++ +++Klebsiella pneumoniae 1 ++ +++ -++ +++ --- +++ +++Enterobacter aerogenes 1 ++ +++ -++ +++ --- +++ +++Salmonella cholerasuis 1 ++ +++ -++ --+ --- +++ +++Proteus mirabilis 1 ++ +++ -++ +++ --- +++ +++Listeria monocytogenes 4 ++ +++ ++- +-- --- --- ---Neisseria meningitidis 4 ++ - - - - - -Brucella suis 4 ++ -+ -- +- -- -- --Bacteroides fragilis 5 ++ ++ +- +- -- -- --Clostridium perfringins 5 ++ ++ +- +- - - -Propionibacterium shermanii 5 ++ +- +- -- - - -- --Mycoplasma hominis 6 ++ NT NT NT NT NT NT__________________________________________________________________________ *1 grew in rich broth containing 1 wt % glucose. 2 grew in Todd - Hewett broth and thereafter streaked on blood. 3 grew in rich broth containing 1 wt % glucose and thereafter streaked on blood. 4 grew in rich broth containing 1 wt % glucose and thereafter streaked on blood in a CO.sub.2 jar. 5 grew in a peptone yeast glucose maltose broth and thereafter streaked o blood in an anaerobic jar. 6 originally grew in a PPLO broth and thereafter streaked on PPLO agar in a CO.sub.2 jar. NT not tested.
As can be seen from Table 1 above, the relatively high concentration of the purified saponin did not kill or inhibit the growth of any of the micoorganisms tested. However, each of the other mucolytic agents tested prevented growth of several of the microorganisms at much lower concentration than the saponin. It should also be noted that even at recommended use concentration of presently used mucolytic agents (0.5% N-acetylcysteine and 0.1% Cleland's reagent) toxicity against several organisms was noted.
EXAMPLE II
Clinical analyses of several sputum samples was conducted after the samples had been dissolved in nontoxic saponin and the results compared to the routine sputum analysis that is conventionally conducted in a typical hospital. Initially, an early morning sputum specimen was collected and a wet prep and smear were made to grade the sputum and to observe characteristics thereof such as spores. The sputums were graded 1 through 4 according to the following protocol:
Grade 1: saliva -frothy, clear, colorless, mostly epithelial cells on a 40X field.
Grade 2: mucoid -colorless, clear to translucent, majority of eosinophils and macrophages, polymorphonuclear cells less than 60%, and less than 1/2 of a 40X field having cells.
Grade 3: mucopurulent - colorless to greenish, slightly translucent, and approximately 1/2 of the field filled with cells.
Grade 4: purulent - yellow or greenish, cloudy, polymorphonuclear cells more than 70%, and greater than 1/2 of the field covered with cells.
In each instance, the cellular density was determined by observing what percentage of a 40X field is filled with cells on a wet mount sample. The cytology is determined by staining (Papanicolaou) a slide, counting 200 cells of the sample and identifying the polymorphonuclear cells (PMN), eosinophils, basophils, macrophages, monocytes, and lymphocytes.
After each sputum sample was graded, a routine analysis was run thereof which includes making 6 semi quantitative streaks with part of the sputum sample on a blood agar plate (BA); on a chocolate agar plate (CHOC); on a eosin methylene blue plate (EMB); on a mannitol salts plate (MAN); on a mannitol salts containing penicillin plate (MAN-PEN); and on a Sabourand's + gentamicin plate (SDA-GENT), and allowing any bacteria to grow on the plate and thereafter identifying the same. The remaining part of each specimen was thereafter digested with an equal amount of 10 weight percent saponin (which has been purified in accordance with U.S. Pat. No. 3,833,425) and 0.1 m sodium citrate, and an antifoaming agent (Antifoam B Emulsion - Dow Corning) to give a final concentration of 5% by weight saponin therein. Thereafter the saponin-sputum mixture is vortexed for at least 30 seconds.
Next, the original digested sputum was streaked onto the following plates: BA; CHOC; EMB; MAN; MAN-PEN; and SDA-GENT. Thereafter, two 1:100 dilutions were made from a portion of the digested sputum into saline bottles to give 10.sup.2 and 10.sup.4 dilutions, e.g., 0.1 milliliters sputum was placed into a 9.9 milliliters saline for the 10.sup.2 dilution, and 0.1 milliliters from the 10.sup.2 dilution was placed into 9.9 milliliters of saline to make the 10.sup.4 dilution. Next, a 0.01 milliliter loop was used to streak from the 10.sup.4 dilution bottle on to the following plates: BA; CHOC; EMB; MAN; MAN+PEN; and SDA+GENT, to give a final definite dilution of 10.sup.6. Thereafter, another 0.01 milliliter sample was streaked onto SDA gentamicin from the 10.sup.2 dilution bottle to give a definite dilution of 10.sup.4. All the plates were incubated and read in 24 and 48 hours. The SDA plates were also read after one week. The plates were interpreted as follows:
a. an interpretation of normal flora (normal level and type of microorganisms found in nasopharyngeal passages) for Alpha streptococcus and Neissera and diphtheroids occurring in the 10.sup.6 and 10.sup.7 range.
b. positive infection for all other nonyeast organisms (other than set forth in (a) above) occurring in the 10.sup.6 and greater range.
c. 10.sup.5 was interpreted as 1-5 organisms found randomly on a set of plates as a possible positive.
10.sup.3 and upon which is 1 to 5 organisms found randomly on a set of plates is a possible positive for Candida Albicans, and a positive for all other yeasts.
The results are set forth in Table 2 below.
Table 2__________________________________________________________________________CLINICAL RESULTS OF SPUTUM ANALYSIS BYROUTINE AND QUANTITATIVE ANALYSISCase Grade QuantitativeNo. Sputum Routine Analysis Conclusion Analysis Conclusion__________________________________________________________________________1 2 Light Enterococcus, light Kleb- Normal Flora Normal Flora Normal Flora siella pneumoniae, light Staphylococcus epidermidis, light Pseudomonas2 4 Light Staphylococcus aureus Normal Flora 2 .times. 10.sup.7 Possible positive seria perflava for Neisseria perflava3 1 Moderate Serratia marcescens, Significant 1 .times. 10.sup.6 Positive for light Pseudomonas growth Serra- monas aeruginosa, Pseudomonas aeru- tia marcescens 1 .times. 10.sup.5 ginosa, possible marcescens positive for Serratia marcescens4 2 Normal Flora Normal Flora Normal Flora Normal Flora5 2 Normal Flora Normal Flora Normal Flora Normal Flora6 2 Normal Flora Normal Flora Normal Flora Normal Flora7 1 Light Pseudomonas, light Candida Normal Flora 1 .times. 10.sup.5 Possible positive albicans monas aerugin- for Pseudomonas osa aeruginosa8 4 Heavy Klebsiella pneumonia, Positive for 2.5 .times. 10.sup.5 Positive for light Candida tropi- Klebsiella (Candida krusei yeast, Kleb- calis, light Enterococcus pneumoniae and Torulopsis siella pneumoniae glabrata), 1 .times. 10.sup.8 and Enterococcus Klebsiella pneu- (D) monia, 4 .times. 10.sup.7 Enterococcus9 4 Moderate Candida albicans Positive for TNTC Hansenula Positive for yeast, Candida albicans polymorpha, 1 .times. 10.sup.5 possible positive Klebsiella pneu- for Klebsiella moniae pneumoniae10 1 Normal Flora Normal Flora 1 .times. 10.sup.5 Normal Flora lococcus aureus11 3 Moderate Klebsiella pneumoniae, Positive for 1 .times. 10.sup.6 Positive for Candida light Candida albicans Klebsiella pneumoniae, 1 .times. 10.sup.5 albicans, possible pneumoniae Candida albicans positive for Kleb- siella pneumoniae12 1 Light Pseudomonas fluorescens, Normal Flora 1 .times. 10.sup.6 Positive for Candida light Candida albicans albicans, 1 .times. 10.sup.6 albicans and Pseu- Pseudonomas fluo- domonas fluorescens, rescens, 1 .times. 10.sup.5 possible positive Klebsiella pneu- for Klebsiella pneu- moniae moniae13 2 Normal Flora Normal Flora Normal Flora Normal Flora14 3 Moderate Klebsiella pneumoniae, Positive for Kleb- 1 .times. 10.sup.6 Positive for Candi- light Candida albicans siella pneumoniae albicans da albicans15 2 Light Staphylococcus aureus, Normal Flora 1 .times. 10.sup.6 Positive for Candida light Candida albicans albicans, 1 .times. 10.sup.6 albicans and Staphy Staphylococcus lococcus aureus aureus16 2 Candida albicans Positive for Can- 1 .times. 10.sup.3 Possible positive dida albicans albicans for Candida albi- cans17 2 Light Beta streptococcus, light 2 .times. 10.sup.5 Positive for Toru- Staphylococcus aureus, light sis magii, 4 .times. 10.sup.5 lopsis magii and Candida albicans and Candida trop- Candida albicans Candida albicans icalis18 4 Haemophilus influenzae Positive for 6 .times. 10.sup.8 Positive for Haemo- Haemophilus philus influenzae, philus influenzae, influenzae 1 .times. 10.sup.3 possible positive glabrata for Torulopsis gla- brata19 1 Normal Flora Normal Flora Normal Flora Normal Flora20 3 Heavy Enterobacter cloacae, heavy Positive for Enter- >10.sup.8 Klebsiella Positive for Kleb- Klebsiella pneumoniae, heavy bacter cloacae, pneumoniae, >10.sup.8 siella pneumoniae, Staphylococcus epidermidis Klebsiella pneu- Enterobacter Enterobacter cloa- moniae and Staphy- cloacae, 10.sup.5 cae, Candida krusei, lococcus epidermi- dida krusei, 10.sup.5 Torulopsis glabrata dis Torulopsis glab- and Candida tropi- rata, 10.sup.4 Candida calis tropicalis21 3 Normal Flora Normal Flora 1 Penicillium Normal Flora humulii22 2 Normal Flora Normal Flora Normal Flora Normal Flora23 2 Normal Flora Normal Flora Normal Flora Normal Flora24 2 Light Candida tropicalis Normal Flora 2.5 .times. 10.sup.4 Positive for Candi- tropicalis, 1.5 .times. da tropicalis and 10.sup.4 Torulopsis Torulopsis glabrata glabrata25 4 Light Klebsiella pneumoniae, 3 Normal Flora .about. 10.sup.5 Possible positive col. Staphylococcus aureus pneumoniae for Klebsiella pneumoniae26 3 Normal Flora Normal Flora Normal Flora Normal Flora27 4 Light Candida tropicalis Normal Flora 10.sup.6 Escherichia Possible positive coli, 10.sup.3 Toru- for Escherichia lopsis glabrata coli and Torulop- sis glabrata28 4 Normal Flora Normal Flora 6 .times. 10.sup.7 Possible positive philus, 1 .times. 10.sup.3 for Haemophilus Torulopsis gla- and Torulopsis brata glabrata29 2 Normal Flora Normal Flora Normal Flora Normal Flora30 3 Moderate Acinetobacter calcoaceti- Positive for 2 .times. 10.sup.8 Positive for cus (H) Acinetobacter bacter calcoa- Acinetobacter calcoaceticus ceticus (H), calcoaceticus (H) 1 .times. 10.sup.8 and Haemophilus philus31 4 Normal Flora Normal Flora 1 .times. 10.sup.8 Positive for coccus Diplococcus32 3 Normal Flora Normal Flora Normal Flora Normal Flora33 2 Candida albicans Positive for Can- 1 .times. 10.sup.5 Positive for Can- dida albicans albicans dida albicans34 2 Normal Flora Normal Flora Normal Flora Normal Flora35 3 Light Enterobacter cloacae Normal Flora 10.sup.6 Pseudomonas Possible positive aeruginosa, 10.sup.5 for Pseudomonas Proteus mirabilis aeruginosa and Pro- teus mirabilis36 3 Light Proteus vulgaris Normal Flora 1 .times. 10.sup.6 Possible positive vulgaris for Proteus vul- garis37 3 Light Candida albicans Normal Flora Normal Flora Normal Flora38 2 Normal Flora Normal Flora 1 .times. 10.sup. 3 Possible positive albicans for Candida albi- cans39 3 Moderate Klebsiella pneumoniae, Positive for 10.sup.5 Klebsiella Positive for Candi- light Enterococcus and Candida Klebsiella pneumoniae, 10.sup.5 da albicans, possi- tropicalis Enterococcus 10.sup.5 ble positive for Candida albicans Enterococcus and Klebsiella pneu- moniae40 3 Heavy Candida albicans, light Positive for 10.sup.9 aeruginosa, Klebsiella Positive for Kleb- Klebsiella pneumoniae Candida albicans pneumoniae, >10.sup.5 siella pneumoniae Candida albicans Candida albicans41 4 10.sup.6 Haemophilus Possible positive for Haemophilus42 2 Normal Flora Normal Flora Normal Flora Normal Flora43 4 Light Candida albicans Normal Flora 10.sup.5 Candida Positive for Candi- cans da albicans144 4 HeavyCandida albicans Positive for Can- >10.sup.6 Candida Positive for Candi- dida albicans cans, 10.sup. 5 Ser- da albicans, possi-- ratia liquefa- ble positive for ciens, 10.sup.5 Pseudo- Serratia liquefa- monas ciens and Pseudo- monas45 2 Normal Flora Normal Flora Normal Flora Normal Flora46 4 Proteus mirabilis and gram positive Positive for Pro- 10.sup.6 Proteus Possible positive cocci (unable to isolate) teus mirabilis mirabilis for Proteus mira- bilis47 4 Light Klebsiella pneumoniae Normal Flora 10.sup.7 Klebsiella Positive for Kleb- pneumoniae, siella pneumoniae Basidiomycetes48 3 Staphylococcus aureus Normal Flora Normal Flora49 4 Heavy Klebsiella pneumoniae Positive for 1 .times. 10.sup.8 Positive for Kleb- Klebsiella pneu- pneumoniae siella pneumoniae moniae50 2 Normal Flora Normal Flora Normal Flora Normal Flora51 3 Light Klebsiella pneumoniae, light Positive for Citro- 1 .times. 10.sup.6 Possible positive Beta Streptococcus, moderate Citro bacter freundii monas putida for Pseudomonas bacter freundii putida52 3 Normal Flora Normal Flora Normal Flora Normal Flora53 4 Moderate Candida albicans Positive for Candi- 2 .times. 10.sup.5 Positive for Candi da albicans albicans and da albicans and Candida tropicalis Candida tropicalis54 3 Light Candida albicans Normal Flora 1 .times. 10.sup.3 Possible positive albicans for Candida albi- cans55 2 Heavy Klebsiella pneumoniae, light Positive for Kleb- 1 .times. 10.sup.8 Possible positive Escherichia coli siella pneumoniae species, 1 for Neisseria Fusarium species species56 3 Light Klebsiella pneumoniae, light Normal Flora .about.3 .times. 10.sup.5 Positive for Candi- Candida tropicalis para-psilosis da para-psilosis57 3 Light Haemophilus, light Candida Normal Flora 1 .times. 10.sup.3 Positive for albicans albicans, 1 .times. 10.sup.8 Haemophilus possi- Haemophilus ble positive for Candida albicans58 4 Moderate Haemophilus Positive for 1.0 .times. 10.sup.8 Positive for Haemophilus philus Haemophilus59 2 Heavy Klebsiella pneumoniae, light Positive for Kleb- 1 .times. 10.sup.6 Possible positive Escherichia coli siella pneumoniae pneumoniae for Klebsiella pneumoniae60 4 Light Haemophilus Normal Flora >3 .times. 10.sup.8 Positive for philus Haemorphilus61 2 Light Klebsiella pneumoniae Normal Flora 1 .times. 10.sup.6 Positive for Kleb- pneumoniae, 1 .times. 10.sup.3 siella pneumoniae, Candida albicans possible positive for Candida albicans62 4 Normal Flora Normal Flora Normal Flora Normal Flora63 4 Haemophilus Positive for 1 .times. 10.sup.7 Positive for Haemophilus philus Haemophilus64 2 Light Serratia liquefaciens Normal Flora Normal Flora Normal Flora65 4 Normal Flora Normal Flora Normal Flora Normal Flora66 3 Normal Flora Normal Flora Normal Flora Normal Flora67 3 Heavy Klebsiella pneumoniae, light Positive for 1 .times. 10.sup.7 Positive for Enterococcus Klebsiella pneu- coccus (D), 3 .times. Enterococcus (D) moniae 10.sup.8 Klebsiella and Klebsiella pneumoniae pneumoniae68 2 Normal Flora Normal Flora Normal Flora Normal Flora69 2 Moderate Staphylococcus aureus, Positive for 1 .times. 10.sup.7 Positive for light Alpha streptococcus Staphylococcus coccus aureus Staphylococcus aureus aureus70 2 Heavy Serratia liquefaciens, Positive for >10.sup.8 Serratia Positive for Ser- light Alpha Streptococcus Serratia lique- liquefaciens ratia liquefa- faciens ciens71 2 Normal Flora Normal Flora Normal Flora Normal Flora72 3 Moderate Enterobacter aerogenes Positive for Enter- 1 .times. 10.sup.7 Positive for Enter- obacter aerogenes bacter aerogenes, obacter aerogenes, 1 .times. 10.sup.6 possible positive liquefaciens for Serratia lique- faciens73 2 Normal Flora Normal Flora 1 .times. 10.sup.7 Positive for Ser- liquefaciens ratia liquefaciens74 4 Light Klebsiella pneumoniae, Normal Flora Normal Flora Normal Flora light Enterococci75 2 Enterobacter, Candida albicans 1 .times. 10.sup.4 Positive for Candi- albicans da albicans76 4 Enterobacter aerogenes, Candida >1 .times. 10.sup.8 Positive for Enter- albicans bacter cloacae, obacter cloacae and 1.5 .times. 10.sup.5 Candida albicans da albicans77 4 Light Klebsiella pneumoniae (2) Normal Flora >1 .times. 10.sup.8 Positive for Haemo- philus, 1 .times. 10.sup.6 philus, possible Klebsiella pneu- positive for Kleb- moniae and siella pneumoniae Hormodendrum and Pasteurella species, 1 .times. 10.sup.6 Pasteurella78 3 Normal Flora Normal Flora Normal Flora Normal Flora79 2 Normal Flora Normal Flora Normal Flora Normal Flora80 3 Alpha streptococcus, Neisseria Normal Flora 1 .times. 10.sup.8 Positive for Haemo- philus, 1 .times. 10.sup.7 philus and Kleb- Klebsiella siella pneumoniae pneumoniae__________________________________________________________________________
As can be seen in Table 2, the routine clinical method and the quantitative method (which utilized the saponin digestion step) were carried out on 80 sputum specimens, and 29 results differed. In 26 cases, the quantitative method found a positive when the routine clinical lab method did not, and in 3 cases, the routine clinical lab method found a positive whose numbers were not high enough to be considered a positive by quantitative standards. It is particularly noted that in 7 patients Haemophilus influenzae was found in the quantitative method on chocolate plates greater than 10.sup.7 per milliliter and was not detected by the routine clinical lab because of overgrowth. This particular organism has great clinical significance in pneumonia. Also, on four occasions, other organisms which constituted double infections were overgrown and not detected by the routine clinical laboratory analysis. Most of the differences in the results set forth in the table were in yeasts shown as positive by the quantitative method, and either not detected or considered light grown by the routine clinical laboratory method.
Next, to correlate laboratory findings with patient history, Table 3 shows a few correlations from the throat and lung autopsies. The results of these correlations is set forth in Table 3 below:
Table 3______________________________________PatientNo. Culture Results______________________________________I Throat Light Candida albicans Sputum* 1 .times. 10.sup.5 Candida albicans, 1 .times. 10.sup.5 Proteus and Pseudomomas aeruginosaII Throat Normal Flora Sputum 10.sup.8 Haemophilus, 10.sup.6 Klebsiella pneu- moniae, 10.sup.6 PasturellaIII Sputum 1 .times. 10.sup.9 Klebsiella pneumoniae Throat Moderate Candida albicansIV Throat Light Pseudomonas aeruginosa Sputum 1 .times. 10.sup.5 Pseudomonas aeruginosaV Throat Staphylococcus, Streptococcus Beta, light Klebsiella pneumoniae Sputum 10.sup.5 Klebsiella pneumoniae Lung autopsy Heavy Klebsiella pneumoniaeVI Throat Light Candida albicans Sputum >10.sup.6 Candida albicans, 10.sup.5 Serratia Liquefaciens, 10.sup.5 PseudomonasVII Throat Enterococcus, Escherichia coli Sputum Normal FloraVIII Sputum Normal Flora Lung autopsy Enterbacter aerogenesIX Mouth Heavy Klebsiella pneumoniae and Candida tropicalis Throat Heavy Klebsiella pneumoniae and Candida tropicalis Tongue Heavy Klebsiella pneumoniae and Candida tropicalis Sputum 10.sup.8 Klebsiella pneumoniae, 10.sup.7 Entero- coccusX Throat Lactobacillus and Candida albicans Sputum 10.sup.8 Klebsiella pneumoniae, 10.sup.8 Entero- bacter cloacae, 10.sup.5 Candida kruseii, 10.sup.4 Candida tropicalis, 10.sup.5 Torulopsis glabrataXI Throat Moderate Staphylococcus aureus Sputum 1 .times. 10.sup.7 Staphylococcus aureusXII Throat Heavy Klebsiella pneumoniae and Entero- bacter aerogenes, Candida albicans Sputum 10.sup.8 Enterobacter aerogenes and 10.sup.5 Candida albicans Blood autopsy Enterobacter aerogenes and PseudomonasXIII Throat Normal Flora Sputum 10.sup.4 Candida tropicalis, 10.sup.4 Torulopsis glabrata______________________________________ *quantitative sputum analysis
As can be seen from Table 3, in some cases the throat has the same organisms at the sputum which means that the patient was either colonized or there was perhaps oral contamination in the sputum sample. Repeat cultures and observations on changing counts would possibly enable the physician to differentiate between these two latter possibilities. If colonization is observed, precautions could then be taken to prevent the establishment of a lung infection. Furthermore, in many instances, the quantitative sputum analysis reveals heavy infection wherein the throat analysis of the throat specimen does not. In this latter case, the data would support the conclusion that there is minimal nasopharyngeal contamination and that a lung infection in this case is highly probable.
EXAMPLE III
This Example is presented to show the mucolytic action (ability to degrade viscosity) of saponin on a clinical sputum specimen. The sputum specimen was 2 milliliters of a grade 2 sputum as defined in Example II above. To measure the viscosity of the sample, a small viscometer was built such as described in "A Simple Method of Measuring Sputum Viscosity", A. O. Jenssen, Scand. J. Resp. Dis.; 54, 290-296 (1973). This viscometer comprises a single plastic block, the bottom face of which was ground plane. In this bottom face, a canal, semicircular in section, was drilled out, ending in a vertical bore adapted to top to a Y fitting, one branch being connected via plastic hose to an air flow regulator, and the other branch of the Y fitting was connected to a water nonometer via plastic tube. An air escape port was drilled into the vertical bore just above the canal. Changes in pressure were measured as a decrease in the height of the water column in the nonometer. The time required to void the viscometer was recorded in seconds. To measure the viscosity of a given sample, the sputum was placed on a glass plate. The viscometer, with constant air flow, was placed on the sputum filling the channel of the viscometer. An air escape port on the side of the viscometer was closed. The time required to empty the viscometer and the maximum deflection of the height of the water column were recorded. The change in height in milliliters of the water column was converted to millilmeters of mercury (pressure) by multiplication by 0.00881. Next, according to the Jenssen article, pressure x seconds x K = viscosity, where K is a calibration constant for the viscometer. The viscometer was calibrated against an Oswald viscometer using glycerol as a primary standard. For this viscometer, K = 13.5 .times. 10.sup.3. The viscosities obtained with grade 2 sputum with and without addition of the purified saponin described in Example I and II are shown in Table 4 below:
Table 4______________________________________Mucolytic Action of Saponin on aClinical Sputum SpecimenNature of sputum: 2 ml of a grade 2.0 sputum. Centipoise______________________________________A. Viscosity of 5 different 881 portions before addition of 1,332 saponin: 14,295 19,929 6,962 X-- = 8678 s.d. = 8306B. Viscosity of 4 different 166 portions after the addition 177 of 0.1 ml of 13% saponin: 161 172 X-- = 169 s.d. = 7______________________________________
As can be seen, the action of the saponin effectively digested the sputum and resulted in a substantially uniform viscosity of all portions.
While the invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will now be apparent to one of ordinary skill in the art upon reading this specification and it is intended to cover such modifications as fall within the scope of the appended claims.

Number	Name	Date	Kind
3445339	Controni et al.	May 1969
3883425	Dorn	May 1975

Sputum analysis method

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