Process for detection of biological activity

[57] ABSTRACT

A sample of blood to be tested for the presence of biological activity is added to a suitable growth medium which includes C¹⁴ containing carbon source fermentable to produce gaseous C¹⁴O₂. After a suitable incubation period, a portion of the gaseous atmosphere above the medium is analyzed for the presence of radioactivity. In the improvement herein described, a growth medium containing at least about 5 percent by weight of a material selected from the group consisting of sucrose, raffinose and glycylglycine is employed in order to reduce the incidence of high background readings due to sterile blood.

Claims, No Drawings

3,854,041

PROCESS FOR DETECTION OF BIOLOGICAL ACTIVITY

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No. 353,952 filed Apr. 24, 1973 and entitled “Process for Detection of Biological Activity.”

DESCRIPTION OF THE INVENTION

This invention is directed to an improved means for the detection of biological activity in blood.

Biological activity in blood, such as bacteremia (bacteria in blood), in the past, presented a difficult detection problem. Culturing was done manually and, of course, was a tedious procedure that required considerable periods of time until the physical presence of bacteria, fungi, or other microorganisms could be detected.

More recently, a radiometric technique for the detection of biological activity in the blood has undergone clinical testing and has been adopted for commercial practice. In that method samples of blood are inoculated into a suitable growth medium that includes a C¹⁴containing carbon source, the inoculated medium is incubated for a suitable period, and a portion of the gaseous atmosphere is analyzed for C^I4O₂ while in the gaseous state. Such process is described, inter alia, in U.S. Pat. No. 3,676,679 issued July 11, 1972; and in the articles “Early Detection of Bacterial Growth, with Carbon¹⁴ Labeled Glucose,” Radiology, 92, No. 1, pp. 154-5 (Jan. 1969); “Automated Radiometric Detection of Bacterial Growth in Blood Cultures,” J. Labs. Clin. Med., 75, No. 3, pp. 529-34 (March 1970); and “Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures,” Applied Microbiology, 22, No. 5, pp. 846-849 (Nov. 1971). A commercial instrument for the practice of a rapid, automated process is available under the trademark Bactec (Johnston Laboratories, Inc.).

In practicing the process for the gaseous detection of the C¹⁴O₂, a general background reading of about five units is frequently encountered. It has also been generally observed when the gas from a sample inoculated with blood is tested, the background reading is generally increased by 10 to 15 units. In other words, even when the blood sample is from a patient who has no microbial infection, the radioactivity will read, on the average, between about 15 and 20 units. The gas from the blood sample of a patient with bacteremia or the like, of course, provides an even higher reading. Generally, a reading of about 30 units or more is used as an indication of microorganisms in the blood sample. On occasion the blood background reading can exceed 30 units making it very difficult to distinguish a true positive caused by microbial infection from the background reading.

Reference to units, herein, is to units of the Bactec instrument. One hundred units are equal to 0.025 microcuries of C¹⁴ activity.

While the described system has proven effective for the rapid detection of biological activity in the blood and is in commercial use, it is desirable to reduce the background readings due to blood and particularly to reduce the incidence of high background readings produced by sterile blood.

It is an object of this invention to reduce the incidence of high background readings due to sterile blood.

It is another object of this invention to reduce the background reading in the gas measurement of the Cⁱ⁴O₂ when blood is screened for the presence of biological activity.

This invention is directed to the process of detecting biological activity in the blood wherein:

a. a growth medium including a C¹⁴ containing carbon source which is fermentable to produce carbon dioxide is inoculated with a blood sample;

b. the inoculated sample is exposed to conditions conducive to the occurrence of normal metabolic processes for a period of time sufficient to cause production of carbon dioxide by the fermenting of said source; and

c. at least a portion of the gaseous atmosphere from said fermentation is measured for radioactivity to determine the presence of C^I4O2. The improvement, according to the present invention, comprises employing a growth medium containing at least about 5 percent by weight of a material selected from the group consisting of sucrose, raffinose and glycylglycine, whereby the high background readings for the measurement of gaseous Cⁱ⁴O2 are reduced.

The precise reason for the increased background reading due to components in the blood is not fully understood. Whatever the cause, it has been discovered that the background reading is decreased if the growth medium contains at least about 5 percent of the additives of this invention. The mechanism by which such additives decrease the background reading is not clear. While the additives of this invention increase osmotic pressure, other agents which also increase osmotic pressure, such as dextran, lactose, sodium chloride, or potassium chloride, do not materially reduce the background reading caused by blood.

In tests employing this invention, the background reading due to the blood has generally been decreased somewhat. Importantly, however, anomalously high readings due to sterile blood may be eliminated so that such samples produce only normal readings. Therefore the accuracy of the technique is increased.

The process to which this invention relates and the apparatus for carrying out the process are described, inter alia, in U.S. Pat. No. 3,676,679; Radiology, 92, No. 1, pp. 154—5 (Jan 1969); J. Labs. Clin. Med., 75, No. 3, pp. 529-34 (March 1970); Applied Microbiology, 22, No. 5, pp. 846-849 (Nov. 1971); Johnston Laboratories, Inc. Technical Data Application Notes JLI 512, JLI 604, JLI 609A, JLI 610A, JLI 611, and JLI 612. These publications are incorporated herein by reference.

Generally, the process employs a nutrient medium that contains water, a suitable C¹⁴ containing carbon source, a nitrogen source, minerals and trace elements. Typical C¹⁴ containing carbon sources may be glucose, fructose, galactose, mannose, rhamnose, or the like, phenylalanine, lysine, arginine or the like, glycerol, urea, or carboxylic acids such as citric acid or the like. Glucose, which is readily available, constitutes a preferred C¹⁴ containing material. Generally the levels of radioactivity will vary from about 0.1 to about 10 microcuries per 10 ml. The assimilable nitrogen source may be either organic or inorganic, such as nitrates, nitrites, ammonia, urea, amino acids, or the like, while

3,854,041 minerals such as the chlorides, sulfates or phosphates of calcium, sodium, potassium, magnesium or the like and trace elements such as manganese, iron, zinc, cobalt or the like, may also be employed. Vitamins, cofactors or other enrichment agents such as anti-coagulants may also be added if desired. Finally, the medium may also include a buffer for pH adjustment and maintenance. The atmosphere above the culture medium can be air, oxygen, or the like if aerobic tests are being conducted, whereas nitrogen, CO₂, or the like can be employed if anaerobic tests are being conducted.

Commercial media (except for the additive of this invention) are described in detail in the above publications and particularly in JLI 611, and JLI 612. A broad selection of possible components that may be included in the media is also set out in U.S. Pat. No. 3,676,679. While that patent indicates that up to 20 percent or more carbohydrate may be employed in the medium, preferred media are said to contain only up to about 0.001 percent carbohydrate and commercial media do contain such small amounts of carbohydrate added as such. In addition, if the media contains peptone, yeast extract, or the like, an additional 1 percent or so of carbohydrates may be present as a component of this additive.

Vials containing media for aerobic cultures, designated No. 6A, and vials containing media for anaerobic cultures, designated No. 7A, are commercially available from Johnston Laboratories, Inc. A medium suitable for aerobic fermentation (6A) may contain tryptic soy broth, hemin, menadione, sodiumpolyanethol sulfonate, and C¹⁴-labelled substrates, while a medium suitable for anaerobic culture (7A) may contain tryptic soy broth, yeast extract, hemin, menadione, L-cysteine, sodium polyanethol sulfonate and C¹⁴-labelled substrates. The 50 ml vials containing 30 ml of medium have a radioactivity of about 1.5 microcuries. Commercial media have a pH of about 7.3.

Most frequently the sample of blood will be added to the medium in amounts ranging from about 0.5 to about 10 percent by volume and the inoculated medium can be cultured under agitation at temperatures between about 20°C and about 60°C and most often between about 33° and about 39°C. The length of time for the culture will depend, inter alia, on the amount of inoculum employed and the particular microorganism involved. Tests have demonstrated that microorganisms generally will create a positive response in time periods ranging from 1 to 24 hours.

After a suitable period of time, e.g., 8 hours, the gaseous atmosphere over the culture medium is tested for radioactivity. Readings of about 30 units or more generally have indicated the presence of microbial activity in the blood. The threshold reading will, of course, depend in part on the background reading due to the general surroundings. Such noise can readily be determined by testing the atmosphere from a medium that has not been inoculated. The reading used to indicate the presence of microorganisms may, of course, vary somewhat depending on the level of general background noise. When the threshold level has been reached, the sample is considered positive. Negative samples are further incubated and tested again to insure against the presence of slow growing microbes.

In accordance with the improvement of this invention, a material selected from the group consisting of sucrose, raffinose, and glycylglycine is incorporated in the medium in amounts of at least about 5 percent by weight and preferably at least about 10 percent by weight. Amounts of 30 percent by weight or more may be employed. Sucrose is a particularly preferred additive of this invention. Because the additives of this invention particularly reduce the high background readings due to sterile blood, the threshold reading to indicate the presence of microbes may be reduced to about 20 or about 25.

The following examples are included in order to illustrate the practice of this invention. These examples are included for illustrative purposes only and in no way are intended to limit the scope of the invention.

EXAMPLES 1-7

Stock culture bacteria were grown overnight on chocolate agar slants at 35°C. The organisms tested were Diplococcus pneumoniae, Haemophilus sp., Pseudomonas aeruginosa, Pseudomonas diminuta, and Streptococcus pyogenes. The growth on the slants was suspended with 1 ml tryptic soy broth, diluted with tryptic soy broth to 1:10,000 of the original concentration, and 1 ml of each suspension inoculated into the test media. For the Haemophilus culture, 1 ml of whole blood was also added to the culture media. The control medium was a 50 ml JLI No. 6A blood culture vial (Johnston Laboratories, Cockeysville, Maryland) containing 30 ml of enriched tryptic soy broth with 1.5 microcuries of C^I4-labeled substrates and a magnetic stirring bar. The hypertonic media consisted of various agents added to the control medium. These agents were either 1% NaCl, 3% NaCl, 10% sucrose, 5% dextran (Sigma Chemical Company, St. Louis, Mo., approximate average molecular weight = 15,000 - 20,000), 10% dextran, or 15% dextran. All percentages are expressed on a weight to volume basis.

The evolution of C¹⁴O2 by these cultures was monitored by using the automated BACTEC Model 225 system (Johnston Laboratories). This radiometric technique has been demonstrated to be very sensitive in detecting C¹⁴O2 production by all levels of bacterial inocula and is used in clinical laboratories for routine blood culturing. The culture vials were incubated at 35°C while providing continuous agitation through the magnetic stirring bar. Samples were analyzed for C¹⁴O₂ production every 2 hours using 90% air - 10% CO₂ as the flushing and culture gas.

In Example 1, 1 ml of whole blood obtained from a healthy donor was used instead of bacteria. These blood cultures were tested as described above except on a 4-hour cycle.

Detection time of the cultures was taken when the Growth Index reached a value of 20 or higher. The Growth Index (GI) is an arbitrary scale of 0 to 100 which is linearly proportional to the amount of C¹⁴ detected. A full-scale reading of 100 corresponds to 0.025 microcurie of C¹⁴ liberated. The maximum GI indicated in the data is the highest GI produced by a culture after its detection.

Table 1 provides some chemical characteristics of the agents and the resultant media, assuming 100 percent dissociation of NaCl.

3,854,041

<td></td><td></td><td></td><td colspan="2"> TABLE 1</td><td></td><td></td><td rowspan="2"> the sample was inoculated into t JLI 6A medium containing 10 maximum Growth Index values are reported in Table 4.</td><td rowspan="2"> t vial having 30 ml of percent sucrose. The for 103 clinical tests</td>

<td> Ex</td><td> Agent</td><td> %w/v</td><td> Molarity</td><td> moles particles/ liter</td><td> pH of medium</td><td rowspan="2"> 5</td>

<td rowspan="2"> 1 2 3 4 5 6 7</td><td rowspan="2"> Control NaCI NaCI sucrose dextran dextran dextran</td><td rowspan="2"> 1 3 10 5 10 15</td><td> 0.17 0 51</td><td> 0.34 1.02</td><td> 7.4 7.35 7.3</td><td> TABLE 4</td><td></td>

<td> 0.29 0.0025 0.005 0.0075</td><td> 0.29 0.0025 0.005 0.0075</td><td> 7.3 7.25 7.15 7.05</td><td> 10</td><td> Max. GI 0-20</td><td> Max. Max, GI GI 2 1-30 3 1 or more</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> Control 85 Control + 10% sucrose 103</td><td> 15 3 0 0</td>

Table 2 reflects the effect of each of the agents on whole blood from a healthy patient. As this table demonstrates, only the addition of 10% sucrose affected the maximum Growth Index.

TABLE 2 20

<td> Ex</td><td> Medium</td><td> Max. GI</td>

<td> 1</td><td> Control</td><td> 17 25</td>

<td> 2</td><td> Control +10% sucrose</td><td> 8</td>

<td> 3</td><td> Control + 1% NaCI</td><td> 15</td>

<td> 4</td><td> Control+ 3% NaCI</td><td> 15</td>

<td> 5</td><td> Control + 5% dextran</td><td> 20</td>

<td> 6</td><td> Control + 10% dextran</td><td> 20</td>

<td> 7</td><td> Control + 15% dextran</td><td>²² 30</td>

The median maximum GI for the 103 control samples was 16.5 while the median maximum GI for the 10 percent sucrose-containing medium was 13.5. The median maximum GI for the 85 control samples in the 0-20 category was 15 while the median maximum GI for the same 85 samples in 10 percent sucrose-containing medium was 13.5.

EXAMPLE 9

The procedure of Examples 1-7 was repeated except that hemin and menadione were omitted from the medium and 3 ml of sterile blood was used as inoculum. The results (mean of 3 replicates) of the control, the control containing + 10 percent sucrose, and the control containing + 10 percent glycylglycine are shown in Table 5 below.

Table 3 represents the effects of the various agents on the test bacteria.

TABLE 5

<td> Medium</td><td colspan="2"> 1 2</td><td> 3</td><td> 4 (days)</td>

<td> Control</td><td> 17</td><td> 18</td><td> 16</td><td> 15</td>

<td> Control 4- 10*2 sucrose</td><td> 13</td><td> 10</td><td> 9</td><td> 8</td>

<td> Control + 10% glycylglycine</td><td> 17</td><td> 14</td><td> 9</td><td> 6</td>

TABLE 3

<td colspan="2" rowspan="2"> Ex</td><td colspan="2"> D. nneumoniae</td><td colspan="2"> P. aerucinosa</td><td colspan="2"> P. diminuta</td><td colspan="2"> Haemophilus</td><td colspan="2"> S. pyogenes___</td>

<td> detection time (hrs.)</td><td> max. GI</td><td> detection time (hrs.)</td><td> max. GI</td><td> detection time (hrs.)</td><td> max. GI</td><td> detection time (hrs.)</td><td> max. GI</td><td> detection time (hrs.)</td><td> max. GI</td>

<td> 1</td><td> Control</td><td> 6</td><td> 93</td><td> 2</td><td> 100</td><td> 14</td><td> 49</td><td> 6</td><td> 100</td><td> 4</td><td> 32</td>

<td> 2</td><td> Control+ 1% NaCI</td><td> 8</td><td> 76</td><td> 2</td><td> 100</td><td> 18</td><td> 40</td><td> 10</td><td> 65</td><td> 4</td><td> 34</td>

<td> 3</td><td> Control+ 3% NaCI</td><td> NG</td><td> —</td><td> 4</td><td> 100</td><td> PG</td><td> —</td><td> NG</td><td> —</td><td> 6</td><td> 37</td>

<td> 4</td><td> Control + 10% sucrose</td><td> 10</td><td> 55</td><td> 4</td><td> 100</td><td> 18</td><td> 39</td><td> 12</td><td> 33</td><td> +</td><td> 12</td>

<td> 5</td><td> Control+ 5% dextran</td><td> 8</td><td> 100</td><td> 4</td><td> 100</td><td> 16</td><td> 45</td><td> 6</td><td> 66</td><td> 6</td><td> 37</td>

<td> 6</td><td> Control + 10% dextran</td><td> 8</td><td> 100</td><td> 4</td><td> 100</td><td> 18</td><td> 40</td><td> 6</td><td> 61</td><td> 6</td><td> 28</td>

<td> 7</td><td> Control + 15% dextran</td><td> 10</td><td> 100</td><td> 4</td><td> 100</td><td> 18</td><td> 44</td><td> 6</td><td> 67</td><td> 8</td><td> 28</td>

NG, no growth; PG, poor growth; +, good growth, no detection

EXAMPLE 8

EXAMPLES 10-16

A 6 ml sample of blood was drawn from a patient and 3 ml of the sample was inoculated into a vial containing 30 ml of JLI 6A medium while the remaining 3 ml of

In the following examples, the control medium was tryptic soy broth containing 0.025 percent sodium polyanethol sulfonate and C'⁴ substrates to provide

3,854,041 about 1.5 microcuries per 30 cc of medium. In these examples, 50 cc vials containing 30 cc of the control medium were employed. The chemical and physical characteristics of the media are shown in Table 6 below.