The present invention relates to a method and composition for a transport-growth medium, particularly adapted to permit a specimen of pathogenic Neisseria to be taken from the patient and transported to a remote laboratory for analysis.
This invention is particularly adapted to utilization with N. gonorrhoeae and N. meningitidis and is particularly useful to combat the wave of untreated and drug-resistant strains of these organisms by identification in central laboratories remote from a patient located in rural or semi-urban territories.
In the prior art, one major problem in developing a successful growth medium which will harbor the gonococcus and meningicoccus has been that in the culture method utilized there developed an overgrowth or contamination with other bacteria which surpassed the slowly developing gonococci, for example, and make identification difficult or impossible. In the methodology of these mediums, the present inventor has been known as a co-developer in the so-called Thayer-Martin medium of which the present invention is a refinement and in its optimum variety, specially targeted towards overcoming the problem of competing bacteria in the medium. The literature prior art into which this invention falls is reflected by the following:
1. James D. Thayer and John E. Martin, "A Selective Medium for the Cultivation of N. gonorrhoeae and N. meningitidis," PHR 79, pages 49-57, 1964.
2. James D. Thayer and John E. Martin, "Improved Medium Selective for Cultivation of N. gonorrhoeae and N. meningitidis," PHR 81, pages 559-561, 1966.
3. Martin et al., "Primary Isolation of N. gonorrhoeae with a New Commercial Medium," PHR 82, pages 361-363, 1967.
The previous compositions, such as the basic Thayer-Martin medium, although containing other additive nutrients, were based upon an agar concentration which was about 1 percent by weight. In this concentration, it was found that the agar was insufficient in quantity for optimum nutrition of the gonococci and, further, that when the agar set in a semi-gel it easily permitted the insertion of the swab into the body of the agar rather than encouraging the physician to roll the specimen swab along the top surface for better results. Therefore, in the present compositions the agar percentage has been raised to about 2.0-2.5 percent and the improved results achieved have also been increased by addition of a specific but minor amount of dextrose, i.e., 0.25 percent by weight.
Finally, in order to screen out other gram-positive and gram-negative bacteria, principally from the Proteus family, the present invention utilizes a 2,4-diamino-5-benzyl pyrimidine to screen out the offending Proteus causing overgrowth. More particularly, the present invention utilizes two related compounds, namely 2,4-diamino-5-(3'4'-dimethoxybenzyl)-pyrimidine which is Diaveridine (Burroughs Wellcome and Company) and 2,4-diamino-5-(3'4'5'-trimethoxybenzyl)-pyrimidine which is Trimethoprim (Burroughs Wellcome and Company).
THE SUBJECT MICROORGANISM
The microorganisms which this invention is designed to transport and grow (TRANS-GROW) as a medium are N. gonorrhoeae (the gonococcus) and N. meningitidis (the meningicoccus) which are species of Neisseria, and both are rather small gram-negative diplococci characterized by a cell having somewhat the shape of a coffee bean. The Neisseria family are aerobic, and grow well at 34°-37°C. Frobisher observes, in his "Fundamentals of Microbiology," VII, 1965, page 449 (Saunders), that the two varieties are very much alike and one is probably a minor variant of the other. Additionally, both are extremely fragile in the outer world and grow best upon an agar with 5-10% CO2, especially a blood agar or chocolate agar.
Thus, in recognition of the preferred CO2 blanket over the nutrient, a preferred modus for transportation consists of a 1-ounce flat specimen bottle with an air-tight rubber top which has been filled with 10% CO2 /90% air mixture. A convenient method of providing a CO2 atmosphere for the specimen may be in accordance with the procedure of U.S. Pat. No. 2,706,702, Carski.
THE COMPETING MICROORGANISMS
Although in the case of N. gonorrhoeae various other microorganisms present in the g.u. tract may introduce growth overlay complications, it has been found that the principal source of difficulty lies in the genus Proteus which are soil and water saprophytes found in the human intestine and in infections in the urinary and intestinal tract. In this genus, the "H. and O." colonies of many strains of Proteus are distinctive in that they usually spread rapidly over the moist surface of solid media forming a thin, grey, almost transparent film. Two species, P. vulgaris and P. mirabilis, produce hydrogen sulfide rapidly and swarm on moist agar media (from Smith Conant, Overman, Microbiology, XIII, page 618, 1964, Appleton). It is to be noted that the frequency of Proteus in the urinary tract is pointed up by the fact that Proteus, next to E. coli, produce the most infections in the urinary tract.
Agar in the art generally refers to a strongly gelling seaweed hydrocolloid characterized by low ester sulfate content. The basic repeating unit of agar consists of alternating 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α-L-galactopyranose units. This substance has been utilized as a solid culture medium since about 1882.
In the development of culture media since the latter part of the 19th Century, many substances have been added to agar to ameliorate or change its growth potential. The so-called chocolate agar and variations were made from dried blood, and dried and processed hemoglobin has been substituted for the blood content of the chocolate agar. Dorland defines "chocolate agar" as follows: "agar to which fresh blood has been added and which is then heated, the red blood changing to a chocolate brown color." (Dorland's Illustrated Medical Dictionary, XXIV, 1965, Saunders). A chocolate agar is most preferred of the agars utilizable in the present invention.
It has been found that an increase in the agar concentration from about 1 percent as previously utilized to about 2-2.5 percent preferred for the present invention is further assisted by the addition of a minor but essential amount of dextrose--about 0.25 percent by weight.
It is noted that in the Thayer-Martin description, at least two agar base media are utilized with additives, and in particular PHR 82, page 361, 1967 ante, is incorporated by reference, together with the two base media utilized, namely Difco Chocolate Medium, Difco Laboratories, Detroit, Michigan, and BBL Chocolate Medium, Baltimore Biological Laboratories, Baltimore, Maryland.
It is noted that in the original Thayer-Martin description, circa 1964, that overgrowth by some gram positive and some other gram-negative bacteria and yeast was prevented because the medium contained 3.0 units of Vancomycin, 7.5 μg of Colistimethate sodium, and 12.5 units Nystatin, per ml. Additionally, the medium contained multi-vitamin growth promoters such as Isovitalex (Bioquest) or Supplement B (Difco).
The Proteus Screening Agent
It has been known for some time that the Proteus microorganism was the chief miscreant which created optical films on the nutrient to forbid layer analysis of the desired N. gonorrhoeae and N. meningitidis. Recently, attention has been directed to the 2,4-diamino-5-benzyl pyrimidines for this purpose, and more particularly directed to two anti-bacterial compounds which have given superior results for screening out the offending Proteus. These compounds are 2,4-diamino-5-(3'4'5'-trimethoxybenzyl)-pyrimidine, Trimethoprim (Burroughs Wellcome, U.S. Pat. No. 3,049,544, Stenbuck et al.) and 2,4-diamino-5-(3'4'-dimethoxybenzyl)-pyrimidine, Diaveridine (Burroughs Wellcome, U.S. Pat. No. 2,624,732, Hitchings et al.). The concentration of the compound is quite specific in the medium, for example, for Trimethoprim a concentration of 5 micrograms per milliliter has been found optimum and for the Diaveridine a concentration of about 25 micrograms per milliliter has been found optimum. It is known that selective media for the isolation of a single bacterial species, e.g., gonococcus, from contaminated specimens, are seldom absolute. However, in this case, the growth of the desired organism is favored and the undesired Proteus is inhibited to a point where pure cultures may be much more easily isolated.
One thousand 1 oz. specimen bottles with rubber caps were utilized as a starting glass material. Into each bottle was inserted 10 ml. of a 3 percent solution of BBL chocolate agar together with a 0.25 weight per cent dextrose, c.p. The bottles were stored in racks upright, and passed under a small gas injection machine which fed 10% CO2 and 90 percent air to fill the bottles. At the next index of a rotating machine, an antiseptic swab was taped to the bottle for specimen-gathering purposes and the growth medium was shipped to point of use with instructions to maintain the temperature of the bottle at 34°-37°C. in an incubator for 20 hrs. at the central diagnostic laboratory. At the laboratory, the agar surface was examined for growth of typical gonococcal and meningococcal colonies and identification was confirmed by conventional oxidase test and gram stain.
Five hundred 1 oz. flat specimen bottles, previously sterilized and with rubber tops, were utilized as glass material. Into each bottle a 3 percent solution of Difco chocolate agar, 0.25 percent by weight dextrose and 5 micrograms Trimethoprim were added. The bottles were subsequently individually filled with a 10 percent CO2 blanket, capped, labeled, and provided with specimen applicators. A warning legend noted that the physician should keep the specimen bottle upright while obtaining a specimen to preserve the CO2 blanket and the laboratory should maintain the specimen at 34°-37°C. for 20 hrs. to incubate the colonies.
In the same manner as above noted in Example II, an additional unit of 500 bottles was prepared for shipment to user, with the exception that 25 micrograms per milliliter of Diaveridine were substituted for the 5 micrograms per milliliter of Trimethoprim utilized in Example 2.
Preparation and Use of Transgrow Medium Materials and Methods
All media and supplements used throughout this study were those of Difco and Bioquest.
The stock cultures of bacteria used were obtained locally. Suspensions of stock organisms were prepared by washing off the 20-hours' growth with Trypticase Soy Broth (TSB) and diluting to give a 45 percent transmittance of light in a spectrophotometer (B & L spectronic 20, wavelength 530). The stock suspensions were diluted to 10-3 for testing.
Secretions from patients suspected of gonorrheal infection were obtained from the Fulton County Health Department, Atlanta, Georgia, and from the Norfolk City Health Department, Virginia. Each specimen was taken by cotton swab and placed in 1.0 ml of sterile TSB. The swabs were rotated and the excess fluid expressed against the side of the tube. To promote homogeneity of the suspension, the specimen was mixed by using a Vortex shaker. Two swabs were placed into the mixture, and one swab used to inoculate each of the media tested.
Twenty-five recent isolates of meningococci were furnished through Third U.S. Army Laboratory, Fort McPherson, Georgia.
The experimental medium for transport and growth (Transgrow) of Neisseria gonorrhoeae and Neisseria meningitidis was prepared as follows:
Mixture I GC Medium Base Agar 36.0 gms. Agar 10.0 gms. Distilled Water 500.0 ml. Mixture 2 Hemoglobin 10.0 gms. Distilled water 500.0 ml.
After the two mixtures were autoclaved (21°C, 15 minutes) they were allowed to cool to 56°C. Then, to Mixture 1 was added 10.0 ml of an enrichment [Supplement B (Difco) or Isovitalex (Bioquest)], 10 ml of an antibiotic supplement, and 6.0 ml of a sterile 25 percent dextrose solution. After thorough mixing, Mixture 1 was combined with Mixture 2.
Eight ml of the liquid Transgrow medium was then poured into a horizontally positioned 1-ounce prescription bottle (Brockway Glass Company). A rubber-lined screw cap was loosely applied, and the medium allowed to cool with the bottle remaining horizontal until solidification. A CO2 atmosphere was introduced into the bottle by placing a group of bottles, containing solidified medium, upright in a vacuum jar, partially exhausting the air with a vacuum pump, and refilling the chamber with a 10% CO2 90 percent filtered air mixture (obtained commercially) until the chamber returned to atmospheric pressure. Screw caps were then tightly fastened.
The medium was inoculated by briefly removing the screw cap and rolling the swab from side to side over the surface of the agar. During the inoculation procedure the mouth of the bottle was kept elevated to minimize the loss of CO2. The caps were then replaced and tightened.
Following inoculation the bottles were placed in a urethane container (Phillips-Foscus Company) and mailed to the laboratory. Transit time varied from 48-96 hours. Upon receipt, the bottles were incubated for 16 hours and 35°C. Oxidase-test reagents were applied to suspected colonies. Samples of oxidase-positive colonies were taken for gram-staining and subculturing for analysis by sugar fermentation. N. gonorrhoeae was said to be present if typical oxidase-positive colonies contained gram-negative diplococci and fermented glucose but not maltose.