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Journal of Clinical Microbiology, July 2005, p. 3460-3462, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3460-3462.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improved Sensitivity of Sputum Smear Microscopy after Processing Specimens with C₁₈-Carboxypropylbetaine To Detect Acid-Fast Bacilli: a Study of United States-Bound Immigrants from Vietnam

K. F. Laserson,^1* N. T. N. Yen,² C. G. Thornton,³ V. T. C. Mai,² W. Jones,^1,4 D. Q. An,² N. H. Phuoc,² N. A. Trinh,² D. T. C. Nhung,¹ T. X. Lien,⁵ N. T. N. Lan,⁵ C. Wells,¹ N. Binkin,¹ M. Cetron,¹ and S. A. Maloney¹

Centers for Disease Control and Prevention, Atlanta, Georgia,¹ Cho Ray Hospital, Ho Chi Minh City, Vietnam,² Integrated Research Technology, LLC, Baltimore, Maryland,³ International Organization of Migration, Ho Chi Minh City, Vietnam,⁴ Institute Pasteur, Ho Chi Minh City, Vietnam⁵

Received 17 November 2004/ Returned for modification 10 January 2005/ Accepted 3 March 2005

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The goal of this study was to evaluate the effect of the specimen-processing method that uses the detergent C₁₈-carboxypropylbetaine (CB-18) on the sensitivity of acid-fast bacillus (AFB) staining. Vietnamese immigrants with abnormal chest radiographs provided up to three sputum specimens, which were examined for acid-fast bacilli by use of direct auramine and Ziehl-Neelsen staining. The remaining sputum was split; half was cultured, and the other half was incubated with CB-18 for 24 h, centrifuged, and examined for AFB by both staining methods. CB-18 processing improved the sensitivity of AFB staining by 20 to 30% (only differences in auramine sensitivity were statistically significant) but reduced specificity by 20% (P < 0.05). These findings have direct utility for overseas migrant tuberculosis screening programs, for which maximizing test sensitivity is a major objective.

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Sputum smear microscopy to detect acid-fast bacilli (AFB) is a rapid, inexpensive, and highly specific tool for identifying persons with active tuberculosis (TB) and represents a critical component of overseas screening for immigrants. The sensitivity of the AFB smear method is moderate (1, 2, 8, 10); a recent study of overseas TB screening for United States immigrant visa applicants reported the sensitivity of AFB smears to be approximately 34% (9). The goal of this study was to evaluate the effects of C₁₈-carboxypropylbetaine (CB-18) (10, 16) specimen processing on AFB smear sensitivity and specificity for detecting TB disease (i.e., Mycobacterium tuberculosis complex [MTBC] culture-positive TB disease) among United States-bound immigrants from Vietnam.

A prospective cohort of United States-bound adult (18 years) Vietnamese immigrants with abnormal chest X rays (CXRs) suggestive of TB was enrolled at Cho Ray Hospital in Ho Chi Minh City, Vietnam, between May 2000 and June 2001. Applicants with CXR findings suggestive of TB disease provided up to three sputum specimens (5); only specimens of 10 ml or greater were eligible for analysis.

Specimens were examined by direct staining by use of both auramine and Ziehl-Neelsen (ZN) staining techniques according to recommended procedures (7). After staining, equal volumes of 50 mM NaOH containing 0.5% N-acetyl-L-cysteine (NALC) were used to homogenize the specimens, and specimens were split approximately equally. One half of each specimen was transferred to the Institute Pasteur in Ho Chi Minh City, where culture facilities are available, and was digested by using the standard oxalic acid (OxAC) procedure (7). After centrifugation at 3,046 x g for 15 min, specimens were decanted and the residue neutralized by using 4% NaOH containing phenol red. Portions of each specimen were analyzed by culture (BACTEC 12B with PANTA Plus [Becton Dickinson, Sparks, MD] and Lowenstein-Jensen slants).

Positive 12B bottles underwent ZN staining to confirm the presence of AFB. Lowenstein-Jensen slants were incubated at 35°C to 37°C for two months and inspected weekly for growth. Tubes were examined macroscopically; only white or buff-colored colonies were examined microscopically after ZN staining. If confirmed as AFB, the colonies were classified as M. tuberculosis complex or Mycobacterium avium complex colonies (pigmented mycobacteria were not identified to the species level). Mycobacterial isolates were identified by standard procedures (7, 11) and by DNA probes (AccuProbe; Gen-Probe, Inc., San Diego, Calif.).

The other half of each specimen was processed with CB-18 at Cho Ray Hospital. The first portion taken from each specimen for culture and the portion taken for CB-18 processing were switched on alternate days. A modified version of CB-18 processing previously described was used (14). Briefly, buffered CB-18 (4 mM CB-18, 50 mM Tris-HCl, 12.5 mM citrate, pH 6.0, 1.5 mM NaCl, 0.3% NALC) was added to each specimen to a final volume of 30 ml, vortexed, and then incubated at room temperature for 24 h prior to centrifugation at 1,818 x g for 15 min at room temperature. All processed specimens were decanted, resuspended in the remaining supernatant backwash, and transferred to duplicate microscope slides for staining.

Participants with 1 positive AFB smear were considered smear positive, and those with all negative AFB smears were considered smear negative. Any participant with 1 culture positive for MTBC by either culture method was considered MTBC positive. Participants with all negative cultures were considered MTBC negative. Exact binomial 95% confidence intervals were calculated. Analysis was performed using STATA 7.0 (version 7.0; STATA Corporation, College Station, Texas).

A total of 338 patients (989 specimens) were enrolled; 138/338 patients had cultures positive for MTBC (40.8%). Only 12 of 138 individuals (9%) reported TB symptoms, and 8 (6%) reported a history of TB disease.

The direct and CB-18-processed smear results of the 138 culture-positive and 200 culture-negative participants were compared (Table 1). CB-18 processing significantly increased auramine smear sensitivity by 33.9% (P < 0.05), from 38.4% to 51.4%, and decreased auramine smear specificity by 21.9% (P < 0.05), from 96.0% to 75.0% (Table 2). A comparison of the direct and CB-18 ZN smear results showed that CB-18 processing also increased ZN smear sensitivity by 23%, from 34.8% to 42.8%; however, this increase was not statistically significant. ZN smear specificity also decreased, from 97.5% to 80.5%, a 17.4% decrease (P < 0.05).

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TABLE 1. Comparison of smear methods among both MTBC culture-positive and culture-negative patients

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TABLE 2. Summary of diagnostic values of the various smear methods

We observed that 81.7% and 85.5% of all positive smear results following CB-18 processing by auramine and ZN staining, respectively, had either a ± (1 to 9 AFB) or 1+ (10 or more AFB) value (Table 3). In contrast, only 51.6% and 63.0% of positive direct auramine and ZN results, respectively, had either a ± or 1+ value. The increase in smear-positive results following CB-18 processing was due exclusively to the increased number of results with ± or 1+ smear values.

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TABLE 3. Per-specimen comparison of direct-smear results and smear following CB-18 processing among both MTBC culture-positive and culture-negative specimens, by both auramine and Ziehl-Neelsen stainings

Fifty-two culture-negative participants had 1 positive smear results following CB-18 processing using either auramine or ZN staining. Nine of these participants reported previous treatment for TB, two people also reported symptoms, and one reported a history of TB in the family. One additional participant also reported TB symptoms ( = 19%).

In overseas TB immigrant screening, maximizing sensitivity takes precedence over maximizing specificity in order to prevent potential transmission during travel and to ensure timely identification and treatment of TB among United States-bound immigrants. We demonstrated that CB-18 processing increased the sensitivities of auramine and ZN stainings by 34% and 23%, respectively. Increased sensitivity following CB-18 processing may be the result of improved recovery of M. tuberculosis isolates during the centrifugation step: it has been hypothesized that CB-18 alters the buoyant density of the bacilli, thereby facilitating recovery by centrifugation (10, 16, 17). CB-18 processing also significantly reduced the specificities of both staining techniques by approximately 20%. Loss of specificity is unusual for acid-fast staining methods, especially when appropriate quality control measures are in place (12, 15). The fact that the specificities following direct smear analyses were 96% suggests that these measures were in place.

The loss of specificity may be due a combination of four possible reasons. First, acid-fast staining cannot distinguish between tuberculous and nontuberculous mycobacteria. However, previous studies suggest that the maximum incidence of nontuberculous mycobacteria in this population is no greater than 8% (9) Second, OxAC processing was used to minimize contamination caused by the high incidence of Pseudomonas spp. in this population. While OxAC processing is an efficient means of decontaminating specimens (7), it is also efficient at killing tuberculous mycobacteria (3). Indeed, a higher-than-expected number of negative cultures (23%) was also observed in a previous study in this same population (9). Third, some United States visa applicants may have taken anti-TB antibiotics to avoid detection during screening. Fourth, the CB-18 smear-positive and culture-negative specimens may actually reflect true positives. An attempt to resolve these results demonstrated that approximately 19% of these patients, all of whom had CXR findings suggestive of TB disease, had other clinical or demographic characteristics suggestive of current or previous TB disease.

An increase in smear sensitivity must come from borderline smear-positive cases. If CB-18 processing is enhancing the number of bacilli recovered, then discordant specimens that were reported as direct smear-negative but CB-18 smear-positive will be those that did not reach the approximately 10,000-bacilli-per-ml threshold needed for detection by direct staining (6). Consistent with this was the observation that all of the 116 discrepant auramine smear specimens had low smear values. It is this group of specimens that would also be the most susceptible to sterilization by OxAC processing and/or represents the low-bacillary-load patients who would most efficiently sterilize their sputa with antibiotics.

There were two main limitations to this study. First, we were unable to perform smear analysis following OxAc processing, which would have allowed the measurement of the individual effect of centrifugation on the observed increased sensitivity. Smear analysis following acidic processing methods has been considered to be difficult, owing to the presence of extraneous cellular debris that is difficult to decolorize (13). Second, participants may have underreported prior histories of previous or current TB treatments, the presence of TB symptoms, or other TB-related factors in an attempt to improve their chances of being allowed to immigrate.

These data should be considered in future policy recommendations. Appropriate use of CB-18 processing has the potential to significantly increase TB case detection globally, thereby facilitating the achievement of World Health Organization target goals (4).

 
We thank the following individuals for their support and contribution to this study: Hoang Hoa Hai, Truong Van Viet, Le Thien Huong Loan, Tran Thi Tin (Cho Ray Hospital); Nguyen Thi Thu (Institute Pasteur); Frank Seawright, Micheal Iademarco, Beverly Metchock, and Thomas Shinnick (Centers for Disease Control and Prevention).

This study was supported by the Centers for Disease Control and Prevention and by Integrated Research Technology, LLC.

 
* Corresponding author. Mailing address: Division of Tuberculosis Elimination, Centers for Disease Control and Prevention, 1600 Clifton Road, MS E-10 Atlanta, GA 30333. Phone: (404) 639-5334. Fax: (404) 639-1566. E-mail: klaserson{at}cdc.gov.

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Journal of Clinical Microbiology, July 2005, p. 3460-3462, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3460-3462.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Maloney, S. A., Fielding, K. L., Laserson, K. F., Jones, W., Yen, N. T. N., An, D. Q., Phuoc, N. H., Trinh, N. A., Nhung, D. T. C., Mai, V. T. C., Seawright, M. F., O'Rourke, T., Lien, T. X., Lan, N. T. N., Binkin, N., Cetron, M. S. (2006). Assessing the Performance of Overseas Tuberculosis Screening Programs: A Study Among US-Bound Immigrants in Vietnam. Arch Intern Med 166: 234-240 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Laserson, K. F. Articles by Maloney, S. A. Search for Related Content PubMed PubMed Citation Articles by Laserson, K. F. Articles by Maloney, S. A.