Clinical Evaluation of Gen-Probe’s Amplified Mycobacterium Tuberculosis Direct Test for Rapid Diagnosis of Mycobacterium Tuberculosis in Egyptian Children at Risk for Infection

By Zaki, Maysaa El-Sayed Hassan, Samir Abou-El

Context.-Diagnostic detection of tuberculosis (TB) has improved considerably. Available, standardized, nucleic acid-based amplification techniques have been shown to yield reliable results within 4 to 7 hours of sample processing. Objective.-To study the diagnostic performance of Gen-Probe’s technique for direct detection of Mycobacterium tuberculosis in comparison with BACTEC 460 TB culture for both positive and negative Ziehl-Neelsen smears in Egyptian children at risk for TB infection.

Design.-We prospectively evaluated 50 children from families with a positive history of TB. All patients were referred from outpatient clinics of the Mansoura University Children’s Hospital, Egypt. The children had a positive tuberculin skin test with an induration diameter of more than 10 mm and had scars from a bacille Calmette- Guerin vaccination within the past 2 years. Three consecutive sputum samples were taken from each patient. The samples were examined to detect M tuberculosis by means of the Gen Probe technique, direct smear microscopy, and bacterial culture by BACTEC 460 TB.

Results.-Of the 50 cases, 30 (60%) had sputum samples that were positive for TB by BACTEC 460 TB culture, and 29 cases (58%) were positive by the Gen-Probe technique. Sensitivity and specificity of Ziehl-Neelsen smears was 83.3% and 100%, respectively, with overall accuracy of 90%. Sensitivity and specificity of the Gen-Probe technique were 96.7% and 100%, respectively, with overall accuracy of 98%.

Conclusions.-The results of this study suggest that the Gen- Probe technique is an accurate method for rapid detection of M tuberculosis complexes in respiratory samples from children at risk for TB. It can be used for diagnosis of smear-negative cases that are suspect for TB.

(Arch Pathol Lab Med. 2008;132:244-247)

Childhood tuberculosis (TB) has its highest incidence among children in contact with bacilliferous adults.1,2 The present study was motivated by the increase in the number of TB cases that are being observed in children.

Traditional laboratory diagnosis of mycobacterial infections by culture usually requires 2 to 8 weeks. The recent increase in new cases of TB has shown the need for rapid, specific, diagnostic assays for Mycobacterium tuberculosis.3 With the development of novel techniques in molecular biology, this delay might be shortened. Most of the nucleic acid amplification assays are rapid and specific.4

Despite their theoretical ability to detect even a single mycobacterial cell, nucleic acid amplification tests (NAATs) are not sufficiently reliable to replace conventional diagnostic methods for detecting TB. Inherent test characteristics and errors in testing procedures may ac count for their inaccuracy.5 Furthermore, the presence in respiratory secretions of enzymes capable of inhibiting amplification reactions accounts for an additional 3% to 25% of false-negative results.6

On the other hand, false-positive results arise most often from contamination of negative samples with either organisms or target DNA from samples containing large numbers of mycobacteria or from amplicons contaminating the laboratory room.5,6

To overcome these problems, automated commercial systems were developed that were made more robust by the use of standardized procedures and reagents for sample processing, amplification, and detection.

Various automated systems, based on amplification and detection techniques, have been devised for the detection of M tuberculosis in clinical samples. The systems include the polymerase chain reaction- based COBAS AMPLICOR Mycobacterium system (Roche Diagnostics, Basel, Swit-zerland) 7; the transcription-mediated, amplification-based Amplified Mycobacterium Tuberculosis Direct Test (AMTD; Gen-Probe, Inc, San Diego, Calif)8; the strand displacement amplification- based BD ProbeTec ET system (Becton Dickinson and Company, Franklin Lakes, NJ)9; and the ligase chain reaction-based Abbott LCx M tuberculosis assay system (Abbott Laboratories, North Chicago, Ill).10 The AMTD by Gen-Probe detects specific RNA by using an isothermal transcription-mediated amplification method.11,12

The aim of this work was to study the diagnostic performance of Gen-Probe’s technique for direct detection of M tuberculosis in comparison to BACTEC 460 TB culture for both positive and negative Ziehl-Neelsen (ZN) smears in Egyptian children at risk for TB infection.

MATERIALS AND METHODS

Patient Selection

This study prospectively evaluated 50 children ranging from 10 to 15 years of age from families with a positive history of TB. All patients were referred from outpatient clinics of the Man-soura University Children’s Hospital, Egypt. Children were symptomatic with productive coughs. They had positive tuberculin skin test with a diameter of the induration of more than 10 mm and had scars from a previous bacille Calmette-Gue?rin (BCG) vaccination received within the past 2 years. Informed written consent was obtained from all parents of the participants, and the ethics committee of the Mansoura University Hospital approved the study.

All patients underwent a standardized interview, physical examination, and chest radiographs in posteroanterior and lateral views. Three consecutive sputum samples were taken from each patient, lumped together, and treated as 1 sample.

Methods

Processing of Sputum Samples. All sputum samples were subjected to decontamination by the NaOH and N-acetyl-L-cys-teine method and then concentrated by centrifugation for 20 minutes at 10 000g. The sediments were resuspended in sterile normal saline and subjected to ZN smear and culture.

Culture Using Radiometric BACTEC 460 TB System. BAC-TEC 460 TB system and BACTEC 12B vials (Becton Dickinson and Company) were used for radiometric culture of M tuberculosis. BACTEC 12B vials were prepared by addition of antimicro-bial agents, which include polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half milliliter of concentrated sputum sediments was inoculated into each BACTEC 12B vial, kept at 37[degrees]C, and tested 3 times a week. Positive cultures, as indicated by BACTEC 460 TB culture, were subjected to further identification by P-nitro- acetyl amino hydroxyl-proionophenone test for the presence of typical M tuberculosis.

Gen-Probe’s AMTD for Detection of TB. This system amplifies a specific ribosomal RNA target of M tuberculosis via DNA intermediates, and the amplified ribosomal RNA sequence (am-plicon) was detected by hybridization with chemiluminescent ac-ridinium ester-labeled probes. The N-acetyl-L-cysteine and NaOH-treated sample (50 mL) and 200 mL of the sample buffer were placed in a lysing tube, and the mixture was sonicated in a water bath sonicator for 15 minutes at room temperature. For amplification, 25 mL of the reconstituted amplification reagent was placed in a reaction tube and covered with 200 mL of the oil, and 50 mL of the lysate was added below the oil layer. The mixture was heated at 95[degrees]C for 15 minutes and then cooled at 42[degrees]C for 5 minutes. After adding 25 mL of the enzyme reagent, the mixture was incubated at 42[degrees]C for 2 hours. To terminate amplification, 20 mL of the termination reagent was added to the tube, and the mixture was incubated at 42[degrees]C for 10 minutes. For detection of the amplicon, 100 mL of the reconstituted probe was added, and the tube was incubated at 60[degrees]C for 15 minutes. The selection reagent (300 mL) was added and the mixture was rein-cubated at 60[degrees]C for 10 minutes to hydrolyze the acridinium ester on the unhybridized probe.

After cooling at room temperature for 10 minutes, the tube was read in a luminometer. Samples with values of more than 30 000 relative light units were considered positive, and those with values of less than 30 000 relative light units were considered negative.

Statistical Analysis

Sensitivity, specificity, and accuracy of the Gen-Probe test and the ZN smears were calculated using the BACTEC 460 TB system as the gold standard method.

RESULTS

The study prospectively evaluated 50 children (43 boys and 7 girls). The children were from families with a positive history of TB. Suspected lymphadenopathy was present in 20 patients (40%) by x- ray.

Sputum samples were culture positive by BACTEC 460 TB in 60% of the cases, ZN smears were positive in 50% of the cases, and Gen- Probe’s AMTD was positive in 58% of the cases (Table 1).

Of the 50 total samples, 30 were culture positive, and 25 of them were ZN positive. The Gen-Probe test reported 29 culture-positive samples as positive, whereas 1 case was culture positive and reported negative by the Gen Probe test (Table 2). Four samples of acid-fast bacilli (AFB)-negative smears were positive by culture and AMTD. Patients with positive x-ray also had positive culture (data not shown).

Sensitivity and specificity of ZN were 83.3% and 100%, respectively, with overall accuracy of 90%. For the Gen Probe test, sensitivity was 96.7%, specificity was 100%, positive predictive value was 100%, negative predictive value was 95.2%, and overall accuracy was 98% (Table 3). Sensitivity of the Gen-Probe test for ZN- negative samples was 80%.

COMMENT

Patients in our study were chosen from children at risk for TB and with a history of BCG vaccination. Tuberculin reactions may be because of infection by M tuberculosis, BCG vaccination, or infection by other mycobacteria. It is believed, however, that the possibility of a cross-reaction decreases when the diameter of the induration exceeds 10 mm.13 From the 50 samples studied, 60% of them were positive by culture. All 30 children with disease had received a dose of BCG vaccine at birth. Despite this high coverage, it must be considered that, even though immunized, individuals exposed to bacilliferous patients are considered at high risk for infection and that recently infected individuals have a greater probability of developing the disease. 14 Beyers et al15 reported that 34% of children younger than 5 years who were exposed to bacilliferous adults at home had the disease, and 14% were infected, despite 98.7% of them having received the BCG vaccination. Also, the TB control strategy of vaccinating newborns with BCG is currently undergoing reevaluation in Canada, as Long et al16 reported outbreaks of TB in both vaccinated and unvaccinated children.

In the United States, Dewan et al17 reported 11 TB outbreaks in child care centers because of the presence of adults with TB. Close interaction with family members who had a delayed diagnosis was the primary means of TB transmission to children. The increase in pediatric TB likely reflects improved clinical diagnostic capacity. Programmatic improvements in TB control and targeted outreach to high-risk immigrant populations may increase pediatric and adult source case detection and reduce M tuberculosis transmission.18 In Egypt, there are trials to install an effective programmatic approach for diagnosis and treatment of TB.

An interesting finding in the present study was that all patients with positive x-ray also had positive culture. However, x-ray abnormalities were found in only 40% of patients. Similarly, Swingler et al19 reported that the accuracy of diagnosis of TB in children with detection of mediastinal lymphadenopathy is questionable. Therefore, we have to choose more accurate methods so as not to misdiagnose those patients.

When evaluating the performance of ZN smear as a rapid and cheap method for TB diagnosis, specificity was 100% and sensitivity was 83.3%, with overall accuracy of 90%. Although culture for TB is considered the gold standard method for detection of viable M tuberculosis,20,21 microscopy is rapid enough to provide a result within 24 hours; however, the latter lacks sensitivity and is unable to distinguish tubercle bacilli from other mycobacteria.22

For results of AMTD compared with culture, 29 cases were Gen- Probe positive and culture positive, and 1 case was negative by Gen- Probe and culture positive. Four samples of AFB smears were positive by culture and Gen Probe.

Generally, differences between cutoff values of positive and negative controls and specimens were broad enough to permit easy discrimination. Negative results obtained by AMTD for culture- positive specimens may be explained by unequal distribution of a small number of my-cobacteria. 23 It is clear that the Gen-Probe technique can be used for the confirmation of TB in a percentage of those providing AFB samples. A similar conclusion was reported by Greco et al23 for most automated systems.

The impact of the NAATs on patient outcome varies based on the result of the AFB smear. In smear-positive patients, public health and hospital infection-control resources are predominantly affected. The potential for influencing patient outcome is much greater when the AFB smear is negative. In smear-negative patients, the NAAT could provide more rapid diagnosis of TB and subsequent initiation of therapy; this would eliminate the need for invasive diagnostic procedures, which are costly and pose an added risk to the patient and allow for earlier discharge of hospitalized patients.25

Therefore, in a developing country such as Egypt, we can restore the use of NAAT in suspect cases with AFB samples to decrease the cost of diagnosis. The cost of culture versus smear examinations is around $25, whereas the cost of the Gen-Probe per case is around $50.

The data presented here show the outstanding sensitivity and specificity of the Gen-Probe test. Sensitivity of the Gen-Probe test was 96.7%, specificity was 100%, positive predictive value was 100%, negative predictive value was 95.2%, and overall accuracy was 98%. Sensitivity was 80% for smear-negative samples. Wang and Tay25 similarly reported that sensitivity of AMTD was 98.4%, specificity was 100%, and positive predictive value was 100%.

In the present study, the Gen-Probe test proved to be valuable as a rapid and accurate method for diagnosis of pulmonary TB in children at risk for TB. The results can be available within 4 hours, whereas the culture results may be obtained within 14 days. We cannot depend on ZN alone as a rapid method because of its reduced sensitivity.

There are numerous difficulties in controlling TB contacts in the Egyptian scenario. Although official procedures recommend that all contacts of TB patients be screened, the lack of diagnostic strategies prevents the early diagnosis of a large number of TB cases, thereby increasing disease transmission. We can suggest the use of combinations of simple tests, such as chest x-ray and ZN stain, for children at risk for TB. For those with negative results, we can proceed to culture or NAAT, according to the situation.

The results of this study suggest that the Gen-Probe test is an accurate method for rapid detection of M tuberculosis complexes in respiratory samples from children at risk for TB. It can be used for smear-negative cases that are suspect for TB.

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Maysaa El-Sayed Zaki, MD; Samir Abou-El Hassan, MD

Accepted for publication September 17, 2007.

From the Departments of Clinical Pathology (Dr El-Sayed Zaki) and Pediatrics (Dr Abou-El Hassan), Faculty of Medicine, Mansoura University Egypt.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Maysaa El-Sayed Zaki, MD, Egypt-Mansoura University, Faculty of Medicine, Department of Pathology, Mansoura 65 Egypt (e- mail: may_s65@hotmail.com).

Copyright College of American Pathologists Feb 2008

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