Improving Sensitivity of Direct Microscopy for Detection of Acid-Fast Bacilli in Sputum: Use of Chitin in Mucus Digestion

In order to try to improve the results of direct smear microscopy,we used the mucus-digesting quality of chitin in tuberculosis(TB) laboratories. For this purpose, a total of 430 sputum specimenswere processed by the N-acetyl-L-cysteine concentration, sodiumhypochlorite (NaOCl) liquefaction, chitin sedimentation, anddirect microscopy methods. Then, the smear sensitivity for acid-fastbacillus detection by chitin-treated sputum was compared withthe sensitivity of smears prepared by other methods. Our resultsshowed that the chitin solution took less time to completelyhomogenize the mucoid sputum than did the N-acetyl-L-cysteineand NaOCl methods. The N-acetyl-L-cysteine concentration methoddemonstrated sensitivity and specificity levels of 83 and 97%,respectively. In comparison, the sensitivity of chitin sedimentationwas 80%, with a specificity of 96.7%. The NaOCl liquefactionmethod showed a sensitivity of 78%, with a specificity of 96%.Finally, the sensitivity of direct microscopy was lower thanthose of the other tested methods and was only 46%, with a specificityof 90%. The chitin and NaOCl liquefaction methods are both easyto perform, and they do not require additional equipment (centrifuges).Also, our results demonstrated that the chitin method is lesstime-consuming than the NaOCl method, since only 30 min of incubationis required to bring complete sedimentation of bacilli in chitin-treatedsputum whereas the NaOCl method needs 10 to 12 h to give thesame results in the same sputum specimens. Therefore, the chitinliquefaction and sedimentation method may provide better resultsin TB laboratories of developing countries than the N-acetyl-L-cysteineconcentration, NaOCl overnight sedimentation, and direct smearmicroscopy methods.

INTRODUCTION

Top

Introduction

The microscopic examination of acid-fast bacilli (AFB) remainsthe main bacteriological tool for tuberculosis (TB) detection(1, 10). Previous studies showed that the technique sensitivitymay vary depending on smearing, staining, and smear reading(5, 16). For proper smearing, N-acetyl-L-cysteine (CH₃-CO-NH-CH-COOH-CH₂SH)with 2% sodium hydroxide (NaOH) is considered the best (15).N-acetyl-L-cysteine (NaLc) acts as a strong mucus digester,and the processed smear has less debris and a greater concentrationof AFB (15). However, since the method is costly and time-consumingand requires special equipment, it is not applicable in thevast majority of TB laboratories in developing countries (2,11). In these countries, limited resources allow only directmicroscopy (unconcentrated sputum) as the best available optionfor TB diagnosis (5, 11, 15). The technique has a low sensitivity,because it requires meticulous preparation of smears and islabor-intensive (14). In recent years, interest in improvingthe direct smear microscopy technique in developing countrieshas arisen (7, 13). For example, Miorner et al. demonstratedthat the simple liquefaction and overnight sedimentation ofsputum with sodium hypochlorite (NaOCl) would significantlyaugment smear sensitivity, up to 70% (13). In the present study,we attempted to improve the efficiency of direct smear microscopythrough liquefaction and sedimentation of sputum by chitin.Chitin, with a formula of (C₈H₁₃O₅N)_n, where n may range from100 to 10,000, is a polysaccharide formed by repeating unitsof N-acetyl-D-glucosamine (3, 9, 17). It has a high molecularweight (1.04 x 10⁶), and it is considered the second-most-abundantnatural polymer after cellulose (8, 18).

In fact, chitin molecules resemble cellulose molecules, exceptthat the hydroxyl (OH) groups in position C-2 have been replacedby acetylamine groups (NH-CO-CH3). A closer look at N-acetyl-L-cysteineand chitin reveals that both molecules have an acetylamine groupin their structure. And since NH-CO-CH3 is responsible for themucolytic effect of cysteine, we thought that chitin might inducethe same effect in sputum. Therefore, for the first time wetried to evaluate the usefulness of chitin in TB laboratories.In this regard, the effects of chitin on mucoid sputum weredetermined. Then, the efficiency of AFB concentration from chitin-treatedsputum was compared with that obtained with the N-acetyl-L-cysteinemethod.

Regarding the applicability of chitin-treated sputum in anyperipheral TB laboratories, we compared the sensitivity of smearmicroscopy in chitin-treated sputum with those obtained withthe NaOCl sedimentation and direct microscopy methods.

MATERIALS AND METHODS

Top

Materials and Methods

Preparation of chitin. Experiments were performed by using both commercially availableand home-made chitin. However, since the results obtained werealmost similar, the data presented here are those obtained withchitin (C-3641) purchased from Sigma Chemical Company, St. Louis,Mo.

(i) Liquefying solution made with ready-made chitin. For each experimental setup 20 mg of chitin was dispersed in10 ml of hexafluoroisopropanol (CF₃)₂CHOH and stirred slowlyfor 18 h (18). It was allowed to stand at room temperature untilneedle-like solid materials disappeared. Then, the solutionwas filtered through sterile gauze and the clear solution wasused as a liquefying agent

(ii) Home-made chitin. The preparation followed the method suggested by Seiichi etal. (20). Five hundred milligrams of prawn shell (exoskeleton)was ground by a coffee grinder. The ground shell was put in700 ml of 1 N NaOH solution and was heated at 90°C for 1h. Once the solution had cooled down, 700 ml of 2N HCl was addedto it. The solution was kept stirring slowly for 6 to 8 h atroom temperature.

After being washed with water (four to five times), the obtainedmaterial was dried in an oven at 50°C for 4 h. The leftovermaterial was approximately 50 to 70 mg. To prepare the chitin-liquefyingsolution, the same procedure was followed as that describedabove.

Preparation of slides. Four hundred thirty sputum specimens were collected from suspectedtuberculosis patients hospitalized or referred to the NationalResearch Institute of Tuberculosis and Lung Disease, Tehran,Iran. Slides for direct microscopy were prepared by taking asmall portion of the purulent part of sputum with a sterileloop (11).

The remaining sputum specimen was divided into 3 equal partsin 10-ml conical screw-cap tubes. The first part of sputum wasprocessed by a standard N-acetyl-L-cysteine with 2% NaOH digestionand decontamination method (15).

Briefly, to an NaLc-2% NaOH solution was added an equal amountof sputum, and the mixture was vortexed for 20 to 30 s. To effectdecontamination, the tubes were kept at room temperature for15 min. Then, the tubes were filled with phosphate buffer (pH6.8) and concentrated by centrifugation at 3,000 x g for 15min. The supernatant was poured off, the resulting sedimentwas resuspended in 1 to 2 ml of phosphate-buffered saline, andthe suspension was used to prepare the concentrated smears.

The second part of sputum was processed by the NaOCl liquefactionand sedimentation method (13). To an equal volume of sputum,NaOCl was added, and the mixture was vortexed for 1 to 3 min(or 10 to 15 min by handshaking), and then the tube was lefton the bench at room temperature overnight (12 to 15 h).

Finally, to the third part of sputum specimen (2 ml), approximately0.5 ml of chitin solution was added, and after being vortexedfor 5 to 6 s (or shaken by hand for 5 to 8 min), the tube wasleft on the bench at room temperature for 30 min. (To checkthe effect of chitin, in a separate set of experiments 0.5 mlof hexafluoroisopropanol alone was added to 2 ml of mucoid sputum,but it took 20 to 25 min of shaking by hand to incompletelyhomogenize the sputum).

The supernatant of each tube was carefully poured off, the sedimentwas mixed well with the remaining fluid, and 1 to 2 drops weretransferred, with a sterile pipette, to a slide. The slideswere coded, air dried, heat fixed, and stained by the Ziehl-Neelsentechnique. The stained slides were examined under oil immersion(1,000x lens objective), and they were reported negative whenno AFB were seen in at least 100 microscopic fields. Smearswere reported positive (11) for any of the following observations:when 1 to 9 AFB were seen in 100 microscopic fields (scoredas few bacilli), when 10 to 99 AFB were seen in 100 fields (scoredas 1+), when 1 to 10 AFB were seen per field in at least 50fields (2+), and when more than 10 AFB were seen per field inat least 20 fields (3+). The coded slides were examined by twotechnicians, and their observations were cross-checked by asenior technician. Statistical analysis was performed with thet and paired t tests.

RESULTS

Top

Results

The results of smear microscopy are summarized in Table 1. Of430 samples, 69(16%) were positive with the N-acetyl-L-cysteineconcentration method, 65 (15.1%) were positive with the chitin-treatedsputum method, and 63 (14%) were positive with the NaOCl liquefactionmethod. In comparison, the AFB-positive smear samples detectedby direct sputum microscopy amounted only to 41 (9.5%).

View this table:
[in this window]
[in a new window]
TABLE 1. Results^a of Ziehl-Neelsen staining of smears prepared by different methods

The results shown in Table 2 demonstrate that the sensitivityof AFB detection in sputa that contain a high number of bacilli(i.e., scoring 3+ and 2+) would be almost similar to the oneobtained with a different smearing method. Of 69 smears detectedas positive, 39 (56%) scored 3+ and 2+ by N-acetyl-L-cysteineconcentration methods. Interestingly, the same number of positivesmears, 39, was detected by NaOCl and by chitin sedimentationmethods. The number was only 34 (49%) in direct smear microscopy,the difference being not statistically significant (P > 0.005).

View this table:
[in this window]
[in a new window]
TABLE 2. Correlation of sensitivity to smear preparation and AFB concentration

In a series of 30 specimens having a low number of bacilli (withthe N-acetyl-L-cysteine concentration method), 24 (80%) and26 (86%) by NaOCl and chitin sedimentation methods, respectively,could be detected whereas the number detected by direct microscopydecreased to 7 of 30. The difference was statistically significant(P < 0.05).

Overall, the sensitivity and specificity of the N-acetyl-L-cysteineconcentration method were 83 and 97%, respectively. In comparison,the sensitivity of chitin sedimentation was 80%, with a specificityof 96.7%. The NaOCl liquefaction method showed a sensitivityof 78%, with a specificity of 96%. Finally, the sensitivityof direct microscopy was lower than the other tested methodsand was only 46%, with a specificity of 90% (Table 3).

View this table:
[in this window]
[in a new window]
TABLE 3. Sensitivities and specificities of AFB detection by different smear preparations

DISCUSSION

Top

Discussion

Proper identification of cases is the pillar of tuberculosiscontrol programs (10). Currently, this is accomplished mostlyby microscopic examination of stained sputum. Thus, technicalaccuracy and proficiency are of paramount importance (1, 2,4). Among the methods suggested for smear preparation, the N-acetyl-L-cysteinemethod has been shown to be a sensitive (28 to 87%) and reliablemethod for microscopy and culture (4, 5). However, due to limitationsin funds and equipment, this technique is not being performedin TB laboratories of countries with limited resources (10,14). In most parts of these countries direct smear microscopywith low sensitivity (25 to 50%) is the only available methodfor the diagnosis of tuberculosis (6, 7, 10, 22).

In our study, of 430 specimens, 69 (16%) smear-positive caseswere detected by the N-acetyl-L-cysteine method, whereas withdirect microscopy the number of AFB smears detected (with thesame sputum specimens examined by the same technical staff)was reduced to 41 (9.5%) from 69. It means that 28 AFB-positivepatients were missed when the direct microscopy method was used(Table 1).

Further analysis of the data (Table 2) showed that of the 28missed smears, 23 (82%) belonged to patients with low numbersof bacilli in their sputa. In other words, fewer bacilli inthe sputa would automatically increase the rate of false-negativeresults by direct microscopy. Therefore, as it has been suggestedby others (19, 21), the direct microscopy is a technique oflow sensitivity, so there is an urgent need to improve the procedure,especially the steps involved in preparation.

Many reports have suggested that liquefaction and overnightsedimentation of sputum with NaOCl would significantly increasethe smear sensitivity, up to 70% (6, 7, 13). Our findings demonstratedthe same results (Table 3). The number of AFB-positive smearsincreased from 41 by direct microscopy to 63 by NaOCl-treatedsputum. Indeed, of 28 false-negative reports by direct smearmicroscopy, 22 (78%) turned out to be positive by the NaOClmethod. The increased sensitivity of the NaOCl method is attributedto clearer microscopic fields and higher bacillus concentrations(13, 21).

Chitin and its derivatives, especially deacetylated chitin,called chitosan, are finding increasing uses in different fieldssuch as wound-healing adjuvant, metal ion scavengers, wastewatertreatment, and food-processing compounds (12, 17, 18). Chemically,chitin resembles cellulose, except that the hydroxyl group inposition C-2 is replaced by an acetylamine (HN-CO-CH3) group(8, 9). This new nitrogen group could be responsible for diverseproperties of chitin (18, 20).

The presence of NH-CO-CH3 suggested that chitin might be usedas a mucus digester. Therefore, we added chitin to the denserpart of the mucoid portion of sputum and noticed the rapid liquefactionof sputum. In fact, this method took less time to homogenizethe mucoid sputum than did the N-acetyl-L-cysteine and NaOClmethods. The quality of the smears was as good as that of thesmears prepared by the N-acetyl-L-cysteine concentration method.

Overall, 65 (15.3%) of 430 specimens were detected as smearpositive by the chitin-treated sputum. This number was slightlyless than that obtained with the N-acetyl-L-cysteine (69/430)concentration method, but the difference was not statisticallysignificant (P > 0.05). However, the main advantage of thechitin-treated sputum method over the N-acetyl-L-cysteine concentrationmethod is that it does not require centrifugation.

The number of smears detected as positive by the chitin-treatedsputum method (65/430) was slightly greater than that obtainedby the NaOCl liquefaction method (63/430), but the differencewas not significant. However, it is important to note that dueto the high molecular weight of chitin, only a maximum of 30min was required to achieve complete sedimentation of bacilli,whereas with the NaOCl liquefaction method at least 10 to 12h was needed for bacillus sedimentation. Therefore, the chitinmethod is considerably less time-consuming than the NaOCl method.

Finally, the sensitivity of the chitin-treated sputum methodwas significantly increased (80%) in comparison to that of directsputum microscopy (46%). Overall, both the chitin-treated sputumand the NaOCl liquefaction methods are easy to perform and theyoffer greater sensitivity than that of direct microscopy. Inaddition, because they don't require any special equipment theycan be easily used under existing conditions of TB laboratoriesin developing countries.

ACKNOWLEDGMENTS

We thank the tuberculosis laboratory technicians at NRITLD.In addition, the excellent laboratory support provided by A.Baharami and Bostan Ali in the Animal Research Unit of NRITLDis appreciated.

Financial support from NRITLD is gratefully acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: Dept. of Mycobacteriology, National Research Institute of Tuberculosis and Lung Disease (NRITLD), P. O. Box 19575/154 Shaheed Bahonar Ave., Daradad, Tehran 19556, Iran. Phone: 982-122-97-075. Fax: 982-122-85-777. E-mail: pfarnia{at}hotmail.com.

REFERENCES

Top