Smear-negative Specimens Research Articles

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Utility of Line Probe Assay for diagnosis of extrapulmonary tuberculosis

IntroductionIn 2008, the World Health Organization (WHO) approved a rapid molecular test known as Line Probe Assay (LiPA) for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) in pulmonary specimens. Due to lack of available data, its use in extra-pulmonary tuberculosis (EPTB) specimens has not been determined yet. Recommendations for the use of Xpert MTB/RIF in EPTB have been issued, but alternative rapid molecular tests for EPTB diagnosis need to be evaluated. MethodLiPA (GenoType® MTBDRplus 2.0 Hain Lifescience) was performed on 97 specimens of extrapulmonary origin at the clinical laboratory at Aga Khan University (Dec 2012–Jan 2014). The results were compared with TB cultures in solid and liquid medium. Results97 specimens were tested simultaneously for culture and LiPA, including pleural fluid (35), CSF (22), pus (17), tissue (10) and urine (3). Concentrated smear was positive for 7 while 14 were culture positive for MTB. All 7/7 smear-positive specimens were LiPA positive, while 6/7 were culture positive. Amongst the smear-negative specimens, 8/90 were culture positive and 9/90 were LiPA positive. The overall sensitivity and specificity of LiPA for the detection of MTB in the EPTB specimens was 71.4% (95% CI 41.9–91.4) and 92.8% (95% CI 84.9–97.3), respectively. The highest sensitivity (100%) was seen in urine as 2 of 3 which were culture positive were also LiPA positive. Pus samples showed sensitivity of 85.7% and specificity of 70%. ConclusionThe study shows that LiPA has good overall sensitivity and specificity compared with culture. Although the number of samples was very small, the applicability appears to be most useful in urine and pus specimens and should be explored further as a diagnostic tool in these cases.

Evaluation of the implementation of the Xpert® MTB/RIF assay in Fiji.

All Xpert® MTB/RIF tests performed in the three TB (tuberculosis) treatment centres in Fiji from June 2012 to February 2013. To determine 1) the number of Xpert tests performed in each centre, 2) the association between sputum quality and Xpert results, 3) the agreement of Xpert with acid-fast bacilli (AFB) smear microscopy and TB culture and 4) error rates. Retrospective review of records. A total of 415 Xpert tests were performed in the study period. Mycobacterium tuberculosis was detected in 69 (16.6%) samples. No rifampicin resistance was detected. M. tuberculosis was detected from 60 (18.7%) good-quality sputum samples. A total of 43 (10.4%) errors occurred during this period. M. tuberculosis was detected in 10 (2.9%) smear-negative specimens. There was a substantial and an almost perfect agreement between Xpert and AFB microscopy (κ = 0.793) and culture results (κ = 0.818), respectively. Although a good correlation between Xpert and the two tests were shown in the study, Xpert should not replace the routine first-line TB diagnostic tests used in Fiji for reasons related to logistics and sustainability. A further evaluation of the assay's performance is required over a longer time period to gauge its diagnostic value in detecting smear-negative, Xpert-positive cases in Fiji.

English

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The Gene Xpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary specimens obtained from possible tuberculosis patients. Aim of the study is to assess diagnostic usefulness of Gene Xpert MTB/RIF assay technique in management of tuberculosis. This is an Observational Study. Study is performed in tertiary teaching hospital, department of pulmonary medicine, maharajah's institute of medical sciences, Vizianagaram between June 2012 and December 2013. Two hundred five Sputum samples were obtained from TB suspects. All samples were tested on Gene Xpert for MTB/RIF detection after AFB microscopy. 108(52.68%) sputum samples were AFB smear positive and 96 (47.32%) were negative. In MTB/RIF assay 144 (70.24%) were MTB positive and 61 (29.76%) were negative. Chi-Square test was applied; P value is <0.001. All results are highly significant. The MTB/RIF assay also detected 4 RIF-resistant specimen and 140 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. It helps to avoid injudicious use of anti-tuberculosis drug.

Multicenter evaluation of Seegene Anyplex TB PCR for the detection of Mycobacterium tuberculosis in respiratory specimens.

Culture is the gold standard for diagnosis of tuberculosis, but it takes 6 to 8 weeks to confirm the result. This issue is complemented by the detection method using polymerase chain reaction, which is now widely used in a routine microbiology laboratory. In this study, we evaluated the performance of the Seegene Anyplex TB PCR to assess its diagnostic sensitivity and specificity, and compared its results with the Roche Cobas TaqMan MTB PCR, one of the most widely used assays in the world. Five university hospitals located in the Chungcheong area in South Korea participated in the study. A total of 1,167 respiratory specimens ordered for acid-fast bacilli staining and culture were collected for four months, analyzed via the Seegene Anyplex TB PCR, and its results were compared with the Roche Cobas TaqMan MTB PCR. For detection of Mycobacterium tuberculosis, the diagnostic sensitivity and specificity of the Anyplex TB PCR were 87.5% and 98.2% respectively, whereas those of the Cobas TaqMan were 92.0% and 98.0% respectively (p value > 0.05). For smear-positive specimens, the sensitivity of the Anyplex TB PCR was 95.2%, which was exactly the same as that of the Cobas TaqMan. For smear-negative specimens, the sensitivity of the Anyplex TB PCR was 69.2%, whereas that of the Cobas TaqMan TB PCR was 84.6%. For detection of MTB, the Seegene Anyplex TB PCR showed excellent diagnostic performance, and high sensitivity and specificity, which were comparable to the Roche Cobas TaqMan MTB PCR. In conclusion, the Anyplex TB PCR can be a useful diagnostic tool for the early detection of tuberculosis in clinical laboratories.

Optimization of the microscopic observation drug susceptibility assay for four first-line drugs using Mycobacterium tuberculosis reference strains and clinical isolates

ObjectiveThe aim of this study is to determine the appropriate cut-off value and turnaround time of the microscopic observation drug susceptibility assay (MODS) for isoniazid (INH), rifampicin (RMP), streptomycin (STR), and ethambutol (EMB). DesignA total of 39 Mycobacterium tuberculosis strains with confirmed drug susceptibility (reference strains) were tested with a range of drug concentrations to determine the optimal cut-off values for INH, RMP, STR, and EMB by MODS. Standard drug susceptibility testing (DST) results were evaluated relative to the Löwenstein–Jensen (L–J) proportion method. Following which, the performance of MODS was evaluated again using 36 sputum samples from patients with tuberculosis (TB) using the cut-off values determined in the aforementioned process. ResultsWith 39 reference strains, DST identified the following cut-off values: 0.8μg/ml INH (sensitivity, 96.0%; specificity, 92.9%), 2.0μg/ml RMP (sensitivity, 100%; specificity, 95.5%), 4.0μg/ml STR (sensitivity, 90.5%; specificity, 93.8%), and 4.0μg/ml EMB (sensitivity, 100%; specificity, 91.7%). When these cut-off values were used to analyze the 36 clinical isolates, the sensitivity and specificity of MODS were 100% and 93.1% for INH, 100% and 93.8% for RMP, 87.5% and 96.4% for STR, and 100% and 88.2% for EMB, respectively. The turnaround time for these clinical specimens was 9.0days by MODS (95% CI: 5.3–12.7), compared with 11.7days (95% CI: 9.5–13.9) for smear negative specimens. ConclusionOur study identified the optimal cut-off values of the four first-line drugs for MODS based on a wide concentration range. With the optimal cut-off values determined in this study, MODS showed high discriminatory efficiency for DST. This study also demonstrated that MODS is useful for rapid diagnosis of drug-resistant TB even for a smear negative specimen, despite the fact that it generally uses smear positive specimens as direct DST.

Evaluation of Fluorotype MTB for detection of Mycobacterium tuberculosis complex DNA in clinical specimens from a low-incidence country

BackgroundWith Fluorotype MTB (FT MTB, HAIN Lifesciences, Germany) a new semi-automated assay for detection of M. tuberculosis complex (MTBC) in clinical specimens has been introduced. In a prospective study, we evaluated the diagnostic performance of FT MTB in a routine diagnostic setting in a low-incidence country.MethodsA total of 1039 respiratory specimens received for routine mycobacteriology diagnostics were analysed by FT MTB. Results were compared to those of culture, microscopy and clinical diagnosis. 61 specimens were excluded from further analysis due to bacterial contamination of cultures.ResultsFT MTB detected 52 of 59 TB specimens (45 culture-positive with MTBC, 7 with clinical diagnosis of TB). With 902 of 912 non-TB specimens (884 culture-negative, 18 with growth of non-tuberculous mycobacteria) FT MTB was negative; discrepant positive FT MTB results were found with 10 specimens. Overall sensitivity, specificity, positive and negative predictive values were 88.1%, 98.9%, 83.8% and 99.2%. Sensitivity rates for smear-positive and smear-negative TB specimens were 100% and 56.3%, respectively. Seven of 978 samples (0.7%) yielded invalid FT MTB results.ConclusionsFT MTB is a new accurate, half automated assay for rapidly diagnosing TB and suitable for larger series of samples. Performance characteristics were found to be similar to those of other commercial NAATs. Its sensitivity in paucibacillary, smear-negative specimens and its utility for TB diagnostics in high-incidence settings needs to be addressed in further studies.

Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis, Tuberculosis (TB) Drug Resistance, and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV(+)) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.

Effect of Relative Centrifugal Forces and Centrifugation Times on Recovery of Mycobacterium Tuberculosis

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Tuberculosis (TB) is one of the major cause of morbidity and mortality worldwide, affecting one third of world's population and the incidence is much higher in South-East Asia (India and China together account for nearly 40% of the global TB cases)&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Objectives:&lt;/strong&gt; The current study was designed to evaluate the efficacy of relative centrifugal forces (RCFs) and various centrifugations times (CTs) on recovery of Mycobacterium tuberculosis (MTB) from sputum specimens pre-inoculated with MTB.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Materials and Methods:&lt;/strong&gt; For this study, direct acid fast bacilli (AFB) smear negative sputum specimens were randomly collected, which were further subjected to sterilization (by autoclaving at 121°C for 15 min) and then seeded with MTB. Afterwards, these sputum specimens were subjected to centrifugation (in a cold centrifuge) at various RCFs and CTs. After centrifugation, supernatants and sediments were inoculated on LJ media and incubated at 37°C with daily monitoring for 8 weeks.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; In the current study, as the RCFs (6000 × g) and CTs (25 min and 20 min) were increased, the growth detection time exponentially decreased (9 and 11 days) and culture grades (4+) were increased when cultured from the sediments. However, the recovery rates of MTB from the supernatant fluids were constantly decreased as RCFs and CTs increased.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; The current study concluded that a higher RCF of 6000 × g and CTs of 20 and 25 min could effectively detect the tubercle bacilli from the sputum specimens more efficiently than other lower RCFs and CTs.&lt;/p&gt;

Is a composite reference standard (CRS) an alternative to culture in assessment and validation of a single tube nested in-house PCR for TB diagnosis?

IntroductionDelay in diagnosis of paucibacillary extra pulmonary tuberculosis (TB) and of smear negative TB has hampered the efforts taken by Control Programs to curb its spread. Better efforts to control spread of TB require more accurate and rapid diagnostic test. AimsTo facilitate early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of a single tube in-house nested PCR in comparison against culture and composite reference standard (CRS). MethodsSingle tube nested PCR was performed using primers targeting Insertion Sequence (IS) 6110 of Mycobacterium tuberculosis complex. Microbiological techniques includes AFB smear microscopy, and cultivation on solid egg-based medium (Löwenstein–Jensen [LJ]) and on liquid culture medium using BACTEC MGIT 960 system, BD Microbiology Systems. ResultsThe sensitivity and specificity of PCR against culture was observed to be 89.7% [95% CI: 84.1–93.5] and 73.1% [95% CI: 67.4–78.1] respectively and that against CRS criteria was 80.2% [95% CI: 75.1–84.5] and 97.1% [95% CI: 92.9–98.9] respectively. PCR showed 100% [111/111, 95% CI: 97–100] sensitivity for smear positive specimens. For smear negative specimens sensitivity and specificity of PCR against culture was observed to be 78.4% [69/88, 95% CI: 68.4–86.5] and 77.3% [204/264, 95% CI: 71.7–82.2] respectively and that against CRS was 68.1% [124/182, 95% CI: 60.8–74.8] and 97.1% [165/170, 95% CI: 93.3–99] respectively. ConclusionCRS criteria were observed to be better than culture for assessing the diagnostic accuracy of PCR test.

Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens

The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.

The accuracy of the Xpert®MTB/RIF assay compared to smear microscopy and culture for detecting pulmonary tuberculosis and rifampicin resistance in adults

The evidence summary1 of the Cochrane Review2 is timely, as the questions addressed are important in the light of the fact that in December 2010 the World Health Organization (WHO) endorsed the use of the Xpert®MTB/RIF assay after reviewing available evidence through a wider consultation process. The WHO came out with a strong recommendation to use this as an initial diagnostic test in individuals suspected of having MDR-TB or HIV-associated TB, and a conditional recommendation to use Xpert® as a follow-on test to microscopy in settings where MDR-TB and/or HIV are of lesser concern, especially in smear-negative specimens.

Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.

Improved Ziehl-Neelsen Microscopy: Bleach Sputum Smear Negative Specimens after Centrifugation

Background: Direct ZN (Ziehl-Neelsen) sputum smear microscopy for diagnosis of TB (tuberculosis) has low sensitivity, especially in TB/HIV co-infected patients. Sputum concentration by bleach (NaOCl) with sedimentation has been used to increase the sensitivity of sputum smear microscopy in many settings but with varying results. Objective: To determine whether bleach plus centrifugation significantly improves the detection of AFB (acid-fast bacilli) in ZN smear-negative sputum specimens. Methods: Three hundred and seventy sputum specimens were collected from new TB suspects attending a Nairobi referral district hospital and processed for direct microscopy using ZN technique and culture on Lowenstein Jensen Media. All smear-negative specimens were treated with 3.5% bleach and left to stand for 30 min before centrifugation. The bleach treated smears were processed and examined using ZN technique. Results: Of the 370 specimens, 200 (54%) were positive culture. The number of sputum samples that were smear-positive by direct ZN was 138 (37.2%), with a sensitivity of 66%. After treatment of direct ZN smear-negative specimens with 3.5% bleach and centrifugation, the total number of AFB smear-positive samples increased to 171 with an increase in sensitivity of 66% to 81.1% (15.1%). Conclusion: In this study, bleach with centrifugation significantly increased the yield of sputum smear microscopy. Further evaluation of these techniques in routine programmes is required especially in settings where the burden of TB/HIV is high.

Rapid Diagnosis of Mycobacterium tuberculosis Infection and Drug Susceptibility Testing

The global control of tuberculosis remains a challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. This is an area of special concern to the health of women and children, particularly in regions of the world with high infant mortality rates and where women have limited access to health care. Because treatment can only be initiated when infection is detected, and is guided by the results of antimicrobial susceptibility testing, there recently has been a marked increase in the development and testing of novel assays designed to detect Mycobacterium tuberculosis complex, with or without simultaneous detection of resistance to isoniazid and/or rifampin. Both nonmolecular and molecular assays have been developed. This review will summarize the current knowledge about the use of rapid tests to detect M tuberculosis and drug resistance. Review of the most recent World Health Organization Global Tuberculosis Report, as well as selected publications in the primary research literature, meta-analyses, and review articles. To a large extent, nonmolecular methods are refinements or modifications of conventional methods, with the primary goal of providing more rapid test results. In contrast, molecular methods use novel technologies to detect the presence of M tuberculosis complex and genes conferring drug resistance. Evaluations of molecular assays have generally shown that these assays are of variable sensitivity for detecting the presence of M tuberculosis complex, and in particular are insensitive when used with smear-negative specimens. As a group, molecular assays have been shown to be of high sensitivity for detecting resistance to rifampin, but of variable sensitivity for detecting resistance to isoniazid.

The Health-System Benefits and Cost-effectiveness of Using Mycobacterium Tuberculosis Direct Nucleic Acid Amplification Testing to Diagnose Tuberculosis Disease in the United States

The utility of Mycobacterium tuberculosis direct nucleic acid amplification testing (MTD) for pulmonary tuberculosis disease diagnosis in the United States has not been well described. We analyzed a retrospective cohort of reported patients with suspected active pulmonary tuberculosis in 2008-2010 from Georgia, Hawaii, Maryland, and Massachusetts to assess MTD use, effectiveness, health-system benefits, and cost-effectiveness. Among 2140 patients in whom pulmonary tuberculosis was suspected, 799 (37%) were M. tuberculosis-culture-positive. Eighty percent (680/848) of patients having acid-fast-bacilli-smear-positive specimens had MTD performed; MTD positive-predictive value (PPV) was 98% and negative-predictive value (NPV) was 94%. Nineteen percent (240/1292) of patients having smear-negative specimens had MTD; MTD PPV was 90% and NPV was 88%. Among patients suspected of tuberculosis but not having MTD, smear PPV for lab-confirmed tuberculosis was 77% and NPV 78%. Compared with no MTD, MTD significantly decreased time to diagnosis in patients with smear-positive/MTD-positive specimens, decreased respiratory isolation for patients having smear-positive/MTD-negative/culture-negative specimens, decreased outpatient days of unnecessary tuberculosis medications, and reduced resources expended on contact investigation. While MTD generally cost more than no MTD, incremental cost savings occurred in patients with human immunodeficiency virus (HIV) or homelessness to diagnose or to exclude tuberculosis, and in patients with substance abuse having smear-negative specimens to exclude tuberculosis. MTD improved diagnostic accuracy and timeliness and reduced unnecessary respiratory isolation, treatment, and contact investigations. It was cost saving in patients with HIV, homelessness, or substance abuse, but not in others.

For better or worse, microscopy is here to stay….

In this issue of PHA, Shah and colleagues reaffirm the utility of Xpert® MTB/RIF as a sensitive tool for the laboratory diagnosis of pulmonary tuberculosis.1 Their study found that 38 of 560 (6.8%) smear-negative samples were Xpert-positive, almost doubling the percentage of samples that were smear-positive by traditional brightfield microscopy. Importantly, Shah and colleagues reaffirm the cost implications and sustainability of Xpert in settings such as Pakistan, and by inference, the necessity of prioritising patients for Xpert testing. The policy statement by the World Health Organization for Xpert made a strong recommendation that ‘Xpert MTB/RIF should be used as the initial diagnostic test in individuals suspected of MDR-TB or HIV-associated TB’, and a conditional recommendation, recognising major resource implications, ‘as a follow-on test to microscopy in settings where MDR and/or HIV is of lesser concern, especially in smear-negative specimens’.2 For Pakistan, and for most other high TB burden countries, sputum smear microscopy is, and will remain, the primary tool for the laboratory diagnosis of TB. In the present study, sputum smear microscopy detected 46/606 (7.6%). Although this much maligned diagnostic tool may miss nearly half of all cases, an optimised and quality assured smear microscopy network can approach ‘point-of-care testing’ for all TB suspects. It all starts with patient education and sputum collection. A study in Rawalpindi, Pakistan, found that for women, guidance on sputum collection increased smear positivity from 8% (control group) to 13% for the intervention group. In part, the intervention group were also more likely to submit multiple specimens.3 An Indonesian study found that simple instructions increased smear positivity by 15.1%.4 The microscopy laboratory involved in the Indonesian study continues to the present day to perform brightfield microscopy at a consistently high standard (multiple personal observations from 2003 to 2013). A good quality specimen handled by a good quality smear microscopy laboratory will identify the majority of patients at greatest risk of transmitting TB in the community, and may be useful in following up patients on treatment. Fortunately, in this study only one TB suspect with presumptive pulmonary TB was identified as rifampicin-resistant by Xpert MTB/RIF. Depending upon the patient’s history, the result may have created a dilemma for the clinician and the TB programme.5 Is the result true? As always, a careful taking of patient history is critical, especially regarding previous treatment(s) with anti-tuberculosis drugs in the public and/or private sector. Where the prevalence of rifampicin resistance is less than 5% among patients who have never previously been treated for TB, such ‘naive’ TB patients with rifampicin resistance detected by Xpert require a confirmatory test (by line probe assay or phenotypic testing) to confirm the diagnosis.5 National TB programmes in high burden settings need both smear microscopy and Xpert MTB/RIF. Prioritising the testing of good quality samples using either technology in quality assured laboratories will achieve the best outcomes for the patient and the programme.

Epidemiology of Smear ‐ Negative Tuberculosis in Ibadan, Nigeria

Inadequate case detection has been identified as one of the reasons for high burden of tuberculosis (TB) in the world especially in poor resourced countries of Africa and Asia. This retrospective laboratory study involving the review of specimens processed at the TB laboratory of the Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan, Nigeria was carried out over a period of five years (January 2006-December 2010) to access the epidemiology of smear-negative TB. Of the 3468 specimens processed, 2,175 (62.7%) were from males while a lower percentage (37.3%)1293 were from females, giving a M:F = 1:0.37. Over half of the specimens, 2,046 (59.0%) were from patients aged 21 to 60 years, 392 (11.3%) from 11 to 20 years, 825 (23.8%) from 60 years and above while 205 (5.9%) were from age 1-10 years. Most of the 2,663 (76.8%) specimens processed were sputum while 201 (5.8%) were gastric washings. Three hundred and nine (8.9%) were smear positive while 392 (11.3%) out of the 3468 specimens processed were culture positive. However, 83 (2.6%) of the 3159 smear-negative specimens were culture positive (false negative) while 66 (21.4%) of the 309 smear-positive specimens were negative for culture (false positive). The majority, 3010 (86.8%) were smear and culture negative while 309 (8.9%) were positive for both tests. Of the 83 false negative specimens, 51 were sputum samples representing (61.4%), 19 (22.9%) were gastric washings while 13 (15.7%) were from extra-pulmonary sites (CSF, aspirates, ascitic fluids, etc). The findings of 2.6% smear-negative but culture positive (false negative) specimens in this study reveals that culture of specimens in addition to smear microscopy from suspected cases is necessary as a diagnostic /confirmatory tool for tuberculosis.

Evaluation of the Fluorotype MTB Assay for the Rapid and Reliable Detection of Mycobacterium Tuberculosis in Respiratory Tract Specimens

We performed the first evaluation study of the new FluoroType MTB assay, a HyBeacon based nucleic acid amplification test for the direct detection of Mycobacterium tuberculosis in respiratory tract specimens. Using mycobacterial culture as a reference, 661 specimens were tested. Sensitivity and specificity were 95.1% and 96.4% for all specimens, sensitivity was 100% for smear-positive and 84.6% for smear negative specimens, respectively. The FluoroType MTB assay showed fast and accurate results within 3 hours, including DNA extraction.

Evaluation of BACTEC Micro MGIT with Lowenstein Jensen Media for Detection of Mycobacteria in Clinically Suspected Patients of Extra Pulmonary Tuberculosis in a Tertiary Care Hospital at Mullana (Ambala)

Background and objectives: Tuberculosis continues to be an important public health problem in India and globally. Extra pulmonary mycobacterial infections are more often smear negative than pulmonary cases which makes the diagnosis difficult. With this in background, this study aimed at the isolation of Mycobacteria from clinical specimens of patients suspected of extra pulmonary tuberculosis using BACTEC Micro MGIT, Lowenstein Jensen (LJ) media and Direct acid fast bacilli smear examination. Methods- A total of 200 samples were processed for direct AFB smear examination, and culture on M960 and LJ media. Acid fast staining of the specimens was done using the Ziehl-Neelsen method. Results and Interpreation - Of the 24 positive specimens, the highest rate of Mycobacterial recovery was by MGIT (91.6%) as compared to LJ media (58.3%).From smear negative samples LJ and MGIT media detected 6.06 % and 10.10 % isolates respectively. For smear-positive specimens, the mean turnaround time was 8 days by MGIT whereas on LJ medium, it was 36 days. For smear-negative specimens, the same was 18 days for MGIT and 40 days for LJ medium. Conclusion-The MGIT system is a good system for, especially in the diagnosis of extra pulmonary samples. The MGIT system was found to be more sensitive in detecting mycobacterium in smearnegative samples. The MGIT system detected mycobacterium significantly earlier than the LJ medium.