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J Clin Microbiol. Nov 2011; 49(11): 3967–3970.
PMCID: PMC3209093

Xpert MTB/RIF for Rapid Diagnosis of Tuberculous Lymphadenitis from Fine-Needle-Aspiration Biopsy Specimens [down-pointing small open triangle]

Abstract

This study demonstrates the excellent diagnostic accuracy of the Xpert MTB/RIF test in patients with tuberculous lymphadenitis. The test sensitivity and specificity were 96.7% (95% confidence interval [CI], 86.6 to 100%) and 88.9% (95% CI, 69.6 to 100%), respectively, and it correctly identified 6/6 (100%) of the cytology smear-negative/culture-positive cases and 1 of 2 (50%) rifampin-resistant cases.

TEXT

Tuberculous lymphadenitis is the most common extrapulmonary manifestation of tuberculosis (TB) (4, 8), and the majority of cases have no active lung involvement. Fine-needle aspiration biopsy (FNAB) offers a feasible and safe option for specimen collection (11, 15). The use of cytology together with the confirmation of acid fastness by Ziehl-Neelsen (ZN) staining and Papanicolaou stain-induced fluorescence microscopy as well as mycobacterial detection by culture offers excellent yields (13, 14) but remains limited by the absence of species confirmation, slow turnaround times, and/or lack of drug resistance guidance. Conventional microbiological culture and drug susceptibility testing are not always available and in rare instances may take 6 weeks or longer (10).

The World Health Organization (WHO)-endorsed Xpert MTB/RIF combines sample processing and real-time PCR in a fully automated platform and detects Mycobacterium tuberculosis complex and rifampin resistance in less than 2 h (2, 3, 9). Xpert MTB/RIF has been used successfully on various extrapulmonary specimens, including urine and stool (6), but has not been rigorously evaluated with the use of tissue specimens, such as FNAB specimens.

To determine the diagnostic utility of the Xpert MTB/RIF, FNAB specimens were collected from 50 consenting patients by aspirating two passes of a 23- or 25-gauge needle attached to a 10-ml syringe. Two smears were prepared from each aspirate, one fixed with commercial cytology fixative for Papanicolaou staining and evaluation by fluorescence microscopy and the other air dried for Giemsa and subsequent ZN staining. Smears were evaluated for adequacy and for a morphological diagnosis, and cases were excluded from the analysis if either one or both passes had inadequate cellular material on smears. Both ZN and Papanicolaou stain-induced fluorescence microscopy evaluations were performed for direct detection of mycobacteria on all specimens. Residual material from one of the aspirates was rinsed in a mycobacterial growth indicator tube (MGIT 960; Becton Dickinson) by aspirating a small volume of fluid into the syringe and expressing it back into the MGIT 960 tube, followed by incubation in a MGIT 960 instrument for mycobacterial culture. Positive cultures were identified as belonging to the Mycobacterium tuberculosis complex, and genotypic drug susceptibility testing was done using the genotype MTBDRplus assay (Hain Lifescience, Germany) (1).

The residual material from the remaining aspirate was rinsed as described above into 0.7 ml sterile phosphate-buffered saline (PBS) in a 10-ml headspace glass vial sealed with a TFE/Sil Septa and aluminum open-top seal. Sample preparation buffer was then added to the vial in a 2:1 ratio, incubated at room temperature, and subsequently processed for Xpert MTB/RIF testing as previously described (2).

Performance calculations, including test sensitivity, specificity, and predictive values, were done using Statistica version 8 to compare the diagnostic performance of the Xpert MTB/RIF test to the reference standard (as positive cytology [cytomorphology consistent with mycobacterial infection and direct visualization of the organism on ZN and/or Papanicolaou stain-induced fluorescence microscopy] and/or culture positive for M. tuberculosis) (11, 12).

Of the 50 patients recruited, 48 cases had adequate smears for diagnosis (see Fig. 1 for the patient recruitment flow diagram). In total, cytomorphological features associated with TB were seen in 32 (66.7%) patients, nonspecific reactive nodes were identified in 10 (20.8%), acute bacterial lymphadenitis was seen in 1 (2.1%), malignancy was seen in 4 (8.3%), and an epithelial inclusion cyst was seen in 1 (2.1%) (Table 1).

Fig. 1.
Flow diagram of all patients referred for fine-needle aspiration biopsy with possible mycobacterial lymphadenitis.
Table 1.
Demographics and diagnostic outcomes of patients referred for fine-needle aspiration biopsy with possible mycobacterial lymphadenitis

Compared to the reference standard, Xpert MTB/RIF correctly identified 29 out of 30 TB cases (sensitivity, 96.7%; 95% CI, 86.6 to 100) (Table 2). The possible false-negative result had a prolonged transit interval of 9 days before Xpert MTB/RIF testing, which may have affected the result. Xpert MTB/RIF was positive in two cases, with negative cytomorphology and culture (specificity, 88.9%; 95% CI, 69.6 to 100). The cytomorphology from one of the false-positive results showed a necrotizing suppurative lymphadenitis, which is consistent with TB. However, no organisms could be identified on microscopy or culture. The cytomorphology of the other false-positive result showed an epithelial inclusion cyst, and the reason for this false-positive result remains unknown. One case had a negative culture result with positive cytology (including mycobacterial identification) and a positive Xpert MTB/RIF test. This patient had been on TB treatment for 1 month at the time of specimen collection, which provides the likely explanation for this discrepant result.

Table 2.
Diagnostic accuracy of the Xpert MTB/RIF test versus various reference standardsa

The Xpert MTB/RIF test was positive in all 6 smear-negative culture-positive cases and correctly identified 1 of the 2 rifampin-resistant cases. The average time to results for microbiological culture was 18.5 days (range, 9 to 55 days), while the Xpert MTB/RIF test result was available within 2 h of commencing the test. This represents a substantial reduction in diagnostic delay, thereby permitting real-time decision making and planning of treatment (5).

A recent study by Hillemann et al. demonstrated the effectiveness of Xpert MTB/RIF on extrapulmonary tissue (6). In that study, the combined sensitivity and specificity of 77.3% and 98.2% were reported, respectively. Our study is the first to evaluate the performance of Xpert MTB/RIF in diagnosing tuberculous lymphadenitis through the use of FNAB specimens. Study limitations include the small number of rifampin-resistant cases identified and the fact that the research was conducted in a referral center, as ideally the technique is suited to use in peripheral laboratories to be effective in controlling the disease. A positive aspect of the study is that the patient population is representative of patients presenting with peripheral lymphadenopathy in most areas where TB/HIV are endemic. It is unlikely that our patient cohort had exacerbated disease compared to patients presenting at primary health care clinics, as these patients are routinely referred from the primary health care clinic to the referral center for FNAB.

In conclusion, FNAB is a simple procedure which can be performed in an outpatient setting by clinicians or nursing staff after a short training period (7, 15). It is ideal for use in resource-limited settings, including more remote and rural areas (15). Specimen collection is simple and safe. With the use of a transport vial, virtually no sample preparation is required and there is minimal risk of contamination. Furthermore, the transmission risk to the operator may also be reduced. Combining FNAB and rapid genotypic diagnosis using automated systems should greatly improve access to appropriate diagnosis and treatment for patients with tuberculous lymphadenitis.

Acknowledgments

We thank Justin Harvey (Stellenbosch University) for statistical assistance.

We report no conflicts of interest.

Footnotes

[down-pointing small open triangle]Published ahead of print on 31 August 2011.

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