Tube&e, Lond., (1969), 50. 299

A SPUTUM CETRIMIDE

SWAB CULTURE METHOD FOR TUBERCLE BACILLI USING COMPARED WITH TWO OTHER SWAB CULTURE METHODS AND THE CONCENTRATION CULTURE METHOD*

By S. JOSEPH, N. G. K. NAIR, and P. R. J. GANGADHARAMt

from the Tuberculosis Chemotherapy Centre, Madras 3 I, Zndias

SUMMARY

A simple sputum swab culture method using cetrimide as the sole reagent was compared with two other swab culture methods (one employing sulphuric acid and the other oxalic acid) as well as with the sodium hydroxide concentration method. In smear-positive specimens, the cetrimide method was as sensitive as the other methods in detecting tubercle bacilli, but in smear-negative specimens it was appreciably less sensitive than the concentration method, although at least as sensitive as the other two swab methods. The incidence of contamination was least with the cetrimide method.

Une mtthode simple pour la culture a partir d’ecouvillons de crachats utilisant le cetramide comme seul reactif a et6 comparte a deux autres methodes de culture a partir d’tcouvillons (une utilisant l’acide sulfurique et l’autre I’acide oxalique) de m&me qu’a la methode de concentration a la soude. Dans les Cchantillons positifs a l’examen direct, la methode au cetramide s’est r&&e aussi sensible que les autres mtthodes pour deceler les bacilles tuberculeux, mais dans les Cchantillons negatifs a l’examen direct cette methode s’est revtlee nettement moins sensible que la mtthode par concentration, bien qu’au moins aussi sensible que les deux autres methodes de culture a partir d’ecouvillons. La frtquence des contaminations a Ctt moindre avec la mtthode au cetramide.

RESUMEN Se compararon 10s metodos de cultivo de una sola muestra de esputos empleando centri- mide coma unico reactive, con otros metodos en 10s cuales se emplearon acido sulftirico. acido oxalico y el metodo de concentration con hidroxido de sodio. En las muestras positivas, el metodo con centrimide fuC tan sensible coma 10s demas para demostrar la presencia de bacilos tuberculosos, pero en 10s extendidos negativos fuC menos sensible que el metodo de la concentration y analogo a 10s otros dos metodos. La incidencia de con- taminacion fuC menor con el metodo de cetrimide.

*This paper is also being published in the Indian Journal of Medical Research. tPresent address: Baylor University College of Medicine, Houston, Texas, U.S.A. §The Centre is under the joint auspices of the Indian Council of Medical Research, the Tamil Nadu (Madras State)

Government, and the World Health Organization in collaboration with the Medical Research Council of Great Britain.

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ZUSAMMENFASSUNG Ein Kulturverfahren mit einfachem Ausstrich des Sputums unter Verwendung von Cetrimid als alleiniges Reagens wurde mit anderen gleichartigen Kulturverfahren, jedoch unter Verwendung von Schwefeldure und, im anderen Fall, mit Oxals2ure verglichen, aui3erdem mit dem Anreicherungsverfahren mittels Natronlauge. In Proben mit positivem Ausstrich war das Cetrimid-Verfahren genau so empfindlich wie die anderen Methoden, aber in Proben mit negativem Ausstrich war es dem Anreicherungs- verfahren unterlegen, den beiden anderen Verfahren immer noch gleichwertig. Verunreini- gungen fanden sich am seltensten bei der Ceterimidmethode.

Introduction Sputum swab culture technique is a simple method for culturing tubercle bacilli. The technique is

employed with several combinations of reagents, the oxalic acid-sodium citrate method (Nassau, 1954) being the most common. At this Centre a similar method is in use for processing laryngeal swabs, the reagents employed being sulphuric acid and sodium hydroxide (Tuberculosis Chemo- therapy Centre, Madras, 1959). In these and other methods, two or more reagents are invariably used, including some which are appreciably lethal to tubercle bacilli. Some workers have reported on culture methods employing quarternary ammonium compounds like ‘Desogen’ (Saxholm, 1954; 1955), ‘Zephiran’ (Hirsch, 1954) and cetrimide (Sikand & Ranga Rao, 1958), which are relatively harmless to tubercle bacilli but highly lethal to other bacteria. Hitherto, however, these compounds have been used at specific pH and in combination with digestants, and appear to depend on prolonged contact for effective action,

Encouraged by the results of a preliminary study with ‘cetrimide’ at this Centre, a simple method employing cetrimide alone was evolved for processing sputum swabs for culturing tubercle bacilli. This paper presents the findings of a controlled comparison of this method with (a) two other swab culture methods, namely, the sulphuric acid method (Tuberculosis Chemotherapy Centre, Madras, 1959) and the oxalic acid method (Nassau, 1954) and (b) the sodium hydroxide concentration method, which is in use in this laboratory (Tuberculosis Chemotherapy Centre, Madras, 1959).

Specimens Materials and Methods

The sputum specimens used in this investigation were chosen from among those received in the laboratory for routine bacteriological examinations, which had more than the quantity of sputum required for such purposes, namely 5 ml. Smear-positive and smear-negative specimens were selected in equal proportions to ensure adequate numbers for separate analysis of the two groups. The specimen bottles were shaken by hand, and the excess sputum poured into another bottle for the preparation of swabs. Preparation of Swabs

Tight cotton swabs, wound on the corrugated end of stainless steel wire, were sterilised in bulk in hot air oven and stored. After moistening with sterile distilled water the swab was dipped in the sputum and rotated and transferred to a labelled test-tube. Six swabs were made from each specimen of sputum and randomly allocated in pairs to the three methods. Medium

Lawenstein-Jensen medium (Cruickshank, 1965) was used. Smear Examination

Direct smears were examined by fluorescence microscopy (Hoist, Mitchison. & Radhakrishna, 1959).

SPUTUM SWAB CULTURE 301

Culture Methods Cetrimide Method-Each swab was kept in a test-tube half-filled with sterile 1% aqueous solution of cetrimide B.P.* for 60 minutes. It was then drained free of the fluid and smeared firmly over the entire surface of a Lbwenstein-Jensen medium slope, the swab being rotated to ensure maximum transfer of the test material to the slope. Sulphuric Acid Method-Each swab was kept in a test-tube half-filled with 4% sulphuric acid for 10 minutes, and then transferred to another test-tube half-filled with sterile 1% sodium hydroxide for five minutes for neutralisation. The swab was then smeared over a Liiwenstein-Jensen medium slope, as described above. Oxalic Acid Method-Each swab was kept in a test-tube half-filled with sterile 5 % oxalic acid for 25 minutes and then in another test-tube half-filled with sterile 5 % trisodium citrate solution for 10 minutes. The swab was then smeared over a Lowenstein-Jensen medium slope, as described above. Concentration Method-Five ml. of sputum were treated with twice the volume of sterile sodium hydroxide and mechanically shaken in the incubator (37” C.) for 20 minutes. It was then centrifuged for 15 minutes, and the deposit suspended in sterile distilled water and recentrifuged. A loopful of the sediment was inoculated on each of two slopes of Lowenstein-Jensen medium using 5 mm. diameter loop.

The processing of the swabs by the three methods and the setting-up of the corresponding cultures were all done by one of us (S.J.). Cultures by the concentration method were, however, set up along with routine specimens by two laboratory technicians on a rota basis. Reading of Cultures

All pairs of slopes (four pairs per specimen) set up in a week were rearranged in random order, so that, for any individual pair, the reader was unaware of the culture method employed or the identity of the sputum specimen. The slopes were incubated at 37” C. and examined weekly for eight to nine weeks for growth of tubercle bacilli or for presence of contamination. The growth was graded, when first seen, as 3 + if it was confluent, as 2+ if there were more than 100 colonies and as 1 + if there were 20-100 colonies: the actual number of colonies was recorded if this was less than 20.

Results The analyses below are based on the findings in 498 specimens, 251 with a positive smear and 247

with a negative smear. Culture Positivity Smear-Positive Specimens-Of the 251 specimens with a positive smear, 231 (92.0%) yielded a positive culture with the cetrimide method, 222 (88.4%) with the sulphuric acid method, 222 (88.4%) with the oxalic acid method and 235 (93.6%) with the concentration method (Table I). Thus, there is a suggestion that the cetrimide method was slightly more sensitive than the other two swab methods. Further, growth emerged earlier with the cetrimide method, the proportion of specimens with a positive culture by two weeks being 41.4% with this method as compared with 35.5 % by the sulphuric acid method (P = O-03), and 34.3 % by the oxalic acid method (P < 0.01). However, there was little difference between the three methods in the degree of growth.

The difference in culture positivity between the cetrimide method and the concentration method was small (l-6 ‘A) and not significant (P = O-4). Furthermore, there was little evidence that positivity on both slopes occurred less often with the cetrimide method than with the concentration method, the proportions being 77.3% and 80.5% respectively- a non-significant difference (P = O-3). However, growth had emerged earlier and was more intense with the concentration method; thus, it occurred by two weeks for 52.6 % of the specimens by the concentration method and for 41.4 % by the cetrimide method (P <OGOl), and was 2t- or more in 76.1% and 58.2% respectively (P < O*OOl).

*Marketed as ‘Cetavlon’ by I.C.I.

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TABLE I.-CULTURE POSITWTY BY THE FOUR METHODS

Percentage of‘positive cultures among:

Method Smear-positive Smear-negative specimens specimens

Cetrimide Sulphuric acid Oxalic acid Concentration

92.0 8.5 88.4 7.7 88.4 X.1 Y3.6 14.2

Total specimens 251 247

Smear-negative Specimens-Of the 247 specimens with a negative smear, 21 (8.5%) were positive on culture by the cetrimide method, 19 (7.7%) by the sulphuric acid method, 20 (8.1%) by the oxalic acid method and 35 (14.2%) by the concentration method (Table I). The differences between the cetrimide method and the other two swab methods were small and not significant, but that between the cetrimide method and the concentration method was appreciable (5.7 %) and significant (P < 0.01). (As the numbers of positive cultures were small, the speed of emergence of growth and the degree of growth were not compared.) Contamination

In general, there was a tendency for contamination, whether of a single slope or of both slopes, to occur less frequently with the cetrimide method than with the other methods (Table II). This is summarized by the figures in the last row of the Table II, which sets out the incidence of contami- nation in individual slopes, For instance, of 502 slopes from 251 smear-positive specimens, 7.6% were contaminated with the cetrimide method, 10.6 % with the sulphuric acid method, 9-O % with the oxalic acid method and 8.6 % with the concentration method. However, apart from the differ- ence between the cetrimide method and the sulphuric acid method (P = 0*04), none of the other differences was significant (P > 0.3). Considering next the smear. negative specimens, the incidence of contamination was 9.6 ‘A, 16.6 %, 15-2 % and 16.2 ‘A by the four methods respectively; the contrast between the cetrimide method and each of the other three methods was significant (P < 0.001).

TABLE II.-INCIDENCE OF CONTAMINATION WITH THE FOUR METHODS __ ~~~ .._.. .~___ -..

Smear-negative specimens ( %)

contaminated “z? 1 ‘2” (Concentration

74.5 1 , 74.1 75.3 178 I 21.5 i 17.8

7.7 i 4.5 / 7.3

/ 247 I 247 247

Percentage of I I I

I I I slopes that ’ / , ‘1

were contaminated 1

7.6 ~ 10.6 9.0 : 8.6 9.6 ; 16.6 15.2 / 16.2 I 1, ~~~__~~ ~~~ ~_~ __ _~_~

SPUTUM SWAB CULTURE 303

Statistical Note When assessing the statistical significance of differences between the various methods in the

incidence of culture positivity (or contamination), cognisance was taken of the fact that the same sputum specimens had been tested by all the methods. This was done by analysing the data as a randomised block experiment, after assigning a score of 1 for a positive and 0 for a negative culture (or 1 for contamination and 0 for no contamination).

Discussion This paper describes a simple sputum swab culture method for tubercle bacilli employing cetri-

mide as the sole reagent. The method was compared concurrently with two other swab culture methods, one employing sulphuric acid (Tuberculosis Chemotherapy Centre, Madras, 1959) and the other employing oxalic acid (Nassau, 1954) in processing about 500 sputum specimens. The cetrimide method was found to be at least as sensitive as the other two swab methods in detecting tubercle bacilli; moreover, in smear-positive specimens, there was evidence that growth emerged earlier with the cetrimide method.

It is reasonable to suppose that failure of the decontaminating reagent(s) will usually lead to contamination of both slopes set up from a specimen. The occurrence of double-slope contamina- tion was similar for all the three swab methods, indicating that, as a decontaminant, cetrimide was as efficient as the other reagents. The incidence of single-slope contamination was least with the cetrimide method, the differences being appreciable in smear-negative specimens. Extraneous factors being common to all the three swab methods, it is possible that these differences are due to the fact that the cetrimide method involves a single-step manipulation whereas the other methods employing more than one reagent involve multiple steps.

In a concurrent comparison the cetrimide method was found to be almost as sensitive as the sodium hydroxide concentration method in detecting tubercle bacilli in smear-positive specimens, but significantly less sensitive in smear-negative specimens. The relatively low sensitivity of the cetrimide method in the smear-negative specimens may be due to low bacterial populations present in the sputum and the fact that only a small quantity of sputum is used in any swab method. Support for this explanation is derived from the finding that, of 14 smear-negative specimens which were positive on culture by the concentration method but negative by the cetrimide method, as many as 11 (79 %) yielded growth of less than 20 colonies.

The incidence of contamination was lower with the cetrimide method than with the concentration method, especially in smear-negative specimens. The higher incidence of contamination with the concentration method may have been due to the fact that, unlike the swab methods, it was set up along with the routine culture work, though it is more likely to have been caused, as stated above for the other swab methods, by the multiple steps in the method.

The concentration method, which is the most sensitive for detecting tubercle bacilli, may not be within the means of most workers in developing countries, as centrifuges require a high capital outlay and are difficult and expensive to maintain, especially in peripheral laboratories. In such circumstances, the cetrimide method described in this paper appears to be a satisfactory alternative. It is at least as sensitive as the other swab methods investigated (although less sensitive than the concentration method), carries a lower risk of contamination and is simple to perform.

The authors are grateful to Mrs. S. Subbammal for reading the cultures, and to Mr. C. Alexander for technical assistance.

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