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1.  LED Fluorescence Microscopy for the Diagnosis of Pulmonary Tuberculosis: A Multi-Country Cross-Sectional Evaluation 
PLoS Medicine  2011;8(7):e1001057.
This study, nested within a clinical trial, by Luis Cuevas and colleagues finds that LED-FM microscopy has higher sensitivity but lower specificity than Zn microscopy for detecting tuberculosis in sputum samples.
Background
The diagnosis of tuberculosis (TB) in resource-limited settings relies on Ziehl-Neelsen (ZN) smear microscopy. LED fluorescence microscopy (LED-FM) has many potential advantages over ZN smear microscopy, but requires evaluation in the field. The aim of this study was to assess the sensitivity/specificity of LED-FM for the diagnosis of pulmonary TB and whether its performance varies with the timing of specimen collection.
Methods and Findings
Adults with cough ≥2 wk were enrolled consecutively in Ethiopia, Nepal, Nigeria, and Yemen. Sputum specimens were examined by ZN smear microscopy and LED-FM and compared with culture as the reference standard. Specimens were collected using a spot-morning-spot (SMS) or spot-spot-morning (SSM) scheme to explore whether the collection of the first two smears at the health care facility (i.e., “on the spot”) the first day of consultation followed by a morning sample the next day (SSM) would identify similar numbers of smear-positive patients as smears collected via the SMS scheme (i.e., one on-the-spot-smear the first day, followed by a morning specimen collected at home and a second on-the-spot sample the second day). In total, 529 (21.6%) culture-positive and 1,826 (74.6%) culture-negative patients were enrolled, of which 1,156 (49%) submitted SSM specimens and 1,199 (51%) submitted SMS specimens. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. Using two LED-FM or two ZN smears per patient was 72.8% (385/529, 95% CI 68.8%–76.5%) and 65.8% (348/529, 95% CI 61.6%–69.8%) sensitive (p<0.001) and 90.9% (1,660/1,826, 95% CI 89.5%–92.2%) and 98% (1,790/1,826, 95% CI 97.3%–98.6%) specific (p<0.001). Using three LED-FM or three ZN smears per patient was 77% (408/529, 95% CI 73.3%–80.6%) and 70.5% (373/529, 95% CI 66.4%–74.4%, p<0.001) sensitive and 88.1% (95% CI 86.5%–89.6%) and 96.5% (95% CI 96.8%–98.2%, p<0.001) specific. The sensitivity/specificity of ZN smear microscopy and LED-FM did not vary between SMS and SSM.
Conclusions
LED-FM had higher sensitivity but, in this study, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. Performance was independent of the scheme used for collecting specimens. The introduction of LED-FM needs to be accompanied by appropriate training, quality management, and monitoring of performance in the field.
Trial Registration
Current Controlled Trials ISRCTN53339491
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis is a global public health problem. Every year, about 1.7 million people die from this contagious bacterial infection, and about 9 million new cases occur, mainly in low- and middle-income countries. Mycobacterium tuberculosis, which causes tuberculosis, is spread in airborne droplets when people with the disease cough or sneeze, and usually infects the lungs (pulmonary tuberculosis). Symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Because tuberculosis is easily transmitted and potentially deadly, it is important that it is diagnosed quickly and accurately and immediately treated. The “gold standard” diagnostic test for tuberculosis is mycobacterial culture (in liquid or solid medium), in which laboratory technicians try to grow M. tuberculosis from sputum (mucus brought up from the lungs by coughing). However, this test is expensive, so most patients suspected of having pulmonary tuberculosis in resource-limited countries are investigated using sputum smear microscopy. In this cheaper but less sensitive test, sputum samples are “smeared” onto microscope slides, stained with Ziehl-Neelsen (ZN) dye, and then examined with a microscope for the presence of M. tuberculosis.
Why Was This Study Done?
With smear microscopy, multiple samples have to be examined to increase the test's sensitivity (the proportion of patients with culture-positive tuberculosis that the test detects). Because each smear examination takes up to 10 minutes, tuberculosis diagnosis with ZN smear microscopy creates a large laboratory workload. A variant form of smear microscopy—light-emitting-diode fluorescence microscopy (LED-FM)—could reduce this workload. With LED-FM, smears stained with a fluorescent dye can be examined in a quarter of the time it takes to examine ZN smears. In this study, the researchers evaluate the sensitivity and specificity (the proportion of people with a negative smear among people without tuberculosis; a high specificity indicates a low false-positive rate) of LED-FM using samples collected in a trial undertaken in four resource-limited countries (Ethiopia, Nepal, Nigeria, and Yemen) to investigate two schemes for sputum sample collection. In the spot-morning-spot (SMS) scheme, patients provide an on-the-spot specimen at their initial consultation, a specimen collected at home the next morning, and a second on-the-spot sample when they deliver their morning specimen. In the spot-spot-morning (SSM) scheme, patients provide two on-the-spot samples during their first clinic visit and a sample collected at home the next morning.
What Did the Researchers Do and Find?
In the main trial, the researchers collected sputum samples using the SMS or SSM scheme from 6,627 patients with a cough lasting more than two weeks. For their investigation of LED-FM, they examined nearly 2,400 samples (half SSM and half SMS specimens, about a quarter of which were tuberculosis culture-positive) with both ZN smear microscopy and LED-FM and determined the sensitivity and specificity of both tests—with one, two, or three sputum samples per patient—relative to mycobacterial solid culture. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. The sensitivities of two LED-FM and two ZN smears were 72.8% and 65.8%, respectively; the specificities of these tests were 90.9% and 98.0%. The sensitivities of three LED-FM and three ZN smears were 77% and 70.5%, respectively; the specificities of these tests were 88.1% and 96.5%. The sensitivity and specificity of both tests was similar for samples collected using the SMS and the SSM schemes.
What Do These Findings Mean?
These findings show that in the resource-limited countries included in this trial, LED-FM has a higher sensitivity but lower specificity than ZN smear microscopy. The researchers calculate that in this study the accuracy of three LED-FM examinations was 85% (2,017 out of 2,355 patients were correctly classified as infected or uninfected), whereas the accuracy of three ZN smears was 91.8%. Thus, although LED-FM should identify more people with tuberculosis than ZN smear microscopy, because of its lower specificity, its use might also lead to more people without tuberculosis being needlessly treated for the disease. Nevertheless, provided that the introduction of LED-FM is accompanied by appropriate training and performance monitoring, LED-FM is an attractive potential tool for the laboratory diagnosis of tuberculosis that, together with a move towards the collection of two on-the-spot smears in a single clinic visit, could ensure that poor patients have access to timely tuberculosis diagnosis and prompt treatment.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001057.
Details of the parent trial in which the samples used in this study were collected are available in a PLoS Medicine Research Article by Cuevas et al.
The World Health Organization provides information on all aspects of tuberculosis, including information on tuberculosis diagnostics; recent WHO policy statements on diagnosis of tuberculosis are available; the Stop TB Partnership provides information on global tuberculosis control (some information in several languages)
The US Centers for Disease Control and Prevention has information about tuberculosis, including information on the diagnosis of tuberculosis disease
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
A new Web site dedicated to the discussion and optimization of smear microscopy has recently been launched
doi:10.1371/journal.pmed.1001057
PMCID: PMC3134458  PMID: 21765809
2.  Rapid Diagnosis of Tuberculosis with the Xpert MTB/RIF Assay in High Burden Countries: A Cost-Effectiveness Analysis 
PLoS Medicine  2011;8(11):e1001120.
A cost-effectiveness study by Frank Cobelens and colleagues reveals that Xpert MTB/RIF is a cost-effective method of tuberculosis diagnosis that is suitable for use in low- and middle-income settings.
Background
Xpert MTB/RIF (Xpert) is a promising new rapid diagnostic technology for tuberculosis (TB) that has characteristics that suggest large-scale roll-out. However, because the test is expensive, there are concerns among TB program managers and policy makers regarding its affordability for low- and middle-income settings.
Methods and Findings
We estimate the impact of the introduction of Xpert on the costs and cost-effectiveness of TB care using decision analytic modelling, comparing the introduction of Xpert to a base case of smear microscopy and clinical diagnosis in India, South Africa, and Uganda. The introduction of Xpert increases TB case finding in all three settings; from 72%–85% to 95%–99% of the cohort of individuals with suspected TB, compared to the base case. Diagnostic costs (including the costs of testing all individuals with suspected TB) also increase: from US$28–US$49 to US$133–US$146 and US$137–US$151 per TB case detected when Xpert is used “in addition to” and “as a replacement of” smear microscopy, respectively. The incremental cost effectiveness ratios (ICERs) for using Xpert “in addition to” smear microscopy, compared to the base case, range from US$41–$110 per disability adjusted life year (DALY) averted. Likewise the ICERS for using Xpert “as a replacement of” smear microscopy range from US$52–$138 per DALY averted. These ICERs are below the World Health Organization (WHO) willingness to pay threshold.
Conclusions
Our results suggest that Xpert is a cost-effective method of TB diagnosis, compared to a base case of smear microscopy and clinical diagnosis of smear-negative TB in low- and middle-income settings where, with its ability to substantially increase case finding, it has important potential for improving TB diagnosis and control. The extent of cost-effectiveness gain to TB programmes from deploying Xpert is primarily dependent on current TB diagnostic practices. Further work is required during scale-up to validate these findings.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB) is a bacterial disease that infects one-third of the world's population. The disease is caused by Mycobacterium tuberculosis, a bacterium that most commonly infects the lungs (known as pulmonary TB) and is transmitted from person to person when an infected individual coughs, sneezes, or talks. The symptoms of TB include chest pain, weight loss, fever, and a persistent cough that sometimes contains blood. Only 5%–10% of people who are infected with TB become sick or infectious, but people with weakened immune systems, such as individuals who are HIV-positive, are more likely to develop the disease. TB is estimated to have killed 1.7 million people in 2009 and is currently the leading cause of death among people infected with HIV.
Why Was This Study Done?
Although TB can be treated with a six-month course of antibiotics, effectively diagnosing TB is not always straightforward and drug resistance is becoming an increasing problem. One of the most common and simple methods to diagnose TB is a technique called sputum smear microscopy, which involves examining matter from the lungs under a microscope for the presence of TB-causing bacteria. However, despite being cheap and relatively simple, the test does not always detect active TB (smear-negative) and cannot determine whether the TB-causing bacteria are resistant to antibiotics. The World Health Organization has recently endorsed a new rapid test, called Xpert MTB/RIF (referred to as Xpert), for the initial diagnosis of TB. The test uses DNA amplification methods to reliably and quickly detect TB and whether infecting bacteria are resistant to the antibiotic rifampicin. The new test is expensive so there are concerns that the test might not be cost-effective in low- and middle-income countries.
What Did the Researchers Do and Find?
The researchers used a technique called modeling to simulate the outcome of 10,000 individuals with suspected TB as they went through a hypothetical diagnostic and treatment pathway. The model compared the costs associated with the introduction of Xpert to a base case for two different scenarios. In the base case all individuals with suspected TB had two sputum smear microscopy examinations followed by clinical diagnosis if they were smear-negative. For the different scenarios Xpert was either used in addition to the two sputum smear microscopy examinations (if the patient was smear-negative) or Xpert was used as a replacement for sputum smear microscopy for all patients. Different input parameters, based on country-specific estimates, were applied so that the model reflected the implementation of Xpert in India, South Africa, and Uganda.
In the researcher's model the introduction of Xpert increased the proportion of TB-infected patients who were correctly diagnosed with TB in any of the settings. However, the cost per TB case detected increased by approximately US$100 in both scenarios. Although the cost of detection increased significantly, the cost of treatment increased only moderately because the number of false-positive cases was reduced. For example, the percentage of treatment costs spent on false-positive diagnoses in India was predicted to fall from 22% to 4% when Xpert was used to replace sputum smear microscopy. The model was used to calculate incremental cost effectiveness ratios (ICERs—the additional cost of each disability-adjusted life year [DALY] averted) for the different scenarios of Xpert implementation in the different settings. In comparison to the base case, introducing Xpert in addition to sputum smear microscopy produced ICERs ranging from US$41 to US$110 per DALY averted, while introducing Xpert instead of sputum smear microscopy yielded ICERs ranging from US$52 to US$138 per DALY averted.
What Do These Findings Mean?
The findings suggest that the implementation of Xpert in addition to, or instead of, sputum smear microscopy will be cost-effective in low- and middle-income countries. The calculated ICERs are below the World Health Organization's “willingness to pay threshold” for all settings. That is the incremental cost of each DALY averted by introduction of Xpert is below the gross domestic product per capita for each country ($1,134 for India, $5,786 South Africa, and $490 for Uganda in 2010). However, the authors note that achieving ICERs below the “willingness to pay threshold” does not necessarily mean that countries have the resources to implement the test. The researchers also note that there are limitations to their study; additional unknown costs associated with the scale-up of Xpert and some parameters, such as patient costs, were not included in the model. Although the model strongly suggests that Xpert will be cost-effective, the researchers caution that initial roll-out of Xpert should be carefully monitored and evaluated before full scale-up.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001120.
The World Health Organization provides information on all aspects of tuberculosis, including tuberculosis diagnostics and the Stop TB Partnership (some information is in several languages)
The US Centers for Disease Control and Prevention has information about tuberculosis, including information on the diagnosis of tuberculosis disease
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001120
PMCID: PMC3210757  PMID: 22087078
3.  Additional role of second washing specimen obtained during single bronchoscopy session in diagnosis of pulmonary tuberculosis 
BMC Infectious Diseases  2013;13:404.
Background
Flexible bronchoscopy with bronchial washing is a useful procedure for diagnosis of pulmonary tuberculosis (TB), when a patient cannot produce sputum spontaneously or when sputum smears are negative. However, the benefit of gaining serial bronchial washing specimens for diagnosis of TB has not yet been studied. Therefore, we conducted a retrospective study to determine the diagnostic utility of additional bronchial washing specimens for the diagnosis of pulmonary TB in suspected patients.
Methods
A retrospective analysis was performed on 174 patients [sputum smear-negative, n = 95 (55%); lack of sputum specimen, n = 79 (45%)] who received flexible bronchoscopy with two bronchial washing specimens with microbiological confirmation of pulmonary TB in Samsung Medical Center, between January, 2010 and December, 2011.
Results
Pulmonary TB was diagnosed by first bronchial washing specimen in 141 patients (81%) out of 174 enrolled patients, and an additional bronchial washing specimen established diagnosis exclusively in 22 (13%) patients. Smear for acid-fast bacilli (AFB) was positive in 46 patients (26%) for the first bronchial washing specimen. Thirteen patients (7%) were positive only on smear of an additional bronchial washing specimen. Combined smear positivity of the first and second bronchial washing specimens was significantly higher compared to first bronchial washing specimen alone [Total cases: 59 (34%) vs. 46 (26%), p < 0.001; cases for smear negative sputum: 25 (26%) vs. 18 (19%), p = 0.016; cases for poor expectoration: 34 (43%) vs. 28 (35%), p = 0.031]. The diagnostic yield determined by culture was also significantly higher in combination of the first and second bronchial washing specimens compared to the first bronchial washing. [Total cases: 163 (94%) vs. 141 (81%), p < 0.001; cases for smear negative sputum: 86 (91%) vs. 73 (77%), p < 0.001; cases for poor expectoration: 77 (98%) vs. 68 (86%), p = 0.004].
Conclusions
Obtaining an additional bronchial washing specimen could be a beneficial and considerable option for diagnosis of TB in patients with smear-negative sputum or who cannot produce sputum samples.
doi:10.1186/1471-2334-13-404
PMCID: PMC3765986  PMID: 24059248
Bronchial washing; Bronchoscopy; Diagnosis; Tuberculosis
4.  Commercial Serological Antibody Detection Tests for the Diagnosis of Pulmonary Tuberculosis: A Systematic Review 
PLoS Medicine  2007;4(6):e202.
Background
The global tuberculosis epidemic results in nearly 2 million deaths and 9 million new cases of the disease a year. The vast majority of tuberculosis patients live in developing countries, where the diagnosis of tuberculosis relies on the identification of acid-fast bacilli on unprocessed sputum smears using conventional light microscopy. Microscopy has high specificity in tuberculosis-endemic countries, but modest sensitivity which varies among laboratories (range 20% to 80%). Moreover, the sensitivity is poor for paucibacillary disease (e.g., pediatric and HIV-associated tuberculosis). Thus, the development of rapid and accurate new diagnostic tools is imperative. Immune-based tests are potentially suitable for use in low-income countries as some test formats can be performed at the point of care without laboratory equipment. Currently, dozens of distinct commercial antibody detection tests are sold in developing countries. The question is “do they work?”
Methods and Findings
We conducted a systematic review to assess the accuracy of commercial antibody detection tests for the diagnosis of pulmonary tuberculosis. Studies from all countries using culture and/or microscopy smear for confirmation of pulmonary tuberculosis were eligible. Studies with fewer than 50 participants (25 patients and 25 control participants) were excluded. In a comprehensive search, we identified 68 studies. The results demonstrate that (1) overall, commercial tests vary widely in performance; (2) sensitivity is higher in smear-positive than smear-negative samples; (3) in studies of smear-positive patients, Anda-TB IgG by enzyme-linked immunosorbent assay shows limited sensitivity (range 63% to 85%) and inconsistent specificity (range 73% to 100%); (4) specificity is higher in healthy volunteers than in patients in whom tuberculosis disease is initially suspected and subsequently ruled out; and (5) there are insufficient data to determine the accuracy of most commercial tests in smear microscopy–negative patients, as well as their performance in children or persons with HIV infection.
Conclusions
None of the commercial tests evaluated perform well enough to replace sputum smear microscopy. Thus, these tests have little or no role in the diagnosis of pulmonary tuberculosis. Lack of methodological rigor in these studies was identified as a concern. It will be important to review the basic science literature evaluating serological tests for the diagnosis of pulmonary tuberculosis to determine whether useful antigens have been described but their potential has not been fully exploited. Activities leading to the discovery of new antigens with immunodiagnostic potential need to be intensified.
Based on a systematic review, Madhukar Pai and colleagues conclude that none of the commercial immune-based tests for pulmonary tuberculosis so far evaluated perform well enough to replace sputum smear microscopy.
Editors' Summary
Background.
Tuberculosis (TB) is, globally, one of the most important infectious diseases. It is thought that in 2005 around 1.6 million people died as a result of TB. Controlling TB requires that the disease is correctly diagnosed so that it can then be promptly treated, which will reduce the risk of infection being passed on to other individuals. The method normally used for diagnosing TB disease in poor countries (where most people with TB disease live) involves taking a sample of mucus coughed up from the lungs; this mucus is then spread thinly onto a glass slide, dyed, and viewed under the microscope. The bacteria responsible for TB take up the dye in a particular pattern and can be clearly seen under the microscope. Although this test (sputum smear) is relatively straightforward to carry out even where facilities are basic, it is not particularly good at identifying TB disease in children or amongst individuals who are HIV-positive. Finally, the sputum smear test is also not very sensitive; that is, many people who have TB disease may not give a positive reading. Therefore, there is an urgent need to develop and evaluate new tests that are suitable for use in poor countries, which will accurately diagnose TB disease, especially amongst children and people who are HIV-positive.
Why Was This Study Done?
New tests for TB have become available which detect whether an individual has raised antibodies against particular proteins and other substances present on the surface of the TB bacterium. These tests are carried out on blood samples, once blood cells and other factors have been taken out. These antibody tests are often quite simple to carry out, so in principle they could be suitable for use in developing countries. Since the tests are available on the market and can be freely used in some developing countries without any need for government regulatory bodies to approve them, it is important to know how good these tests are at diagnosing TB disease. The researchers here wanted, therefore, to evaluate all of the available data relating to the accuracy of antibody detection tests for diagnosis of TB disease.
What Did the Researchers Do and Find?
In order to evaluate all of the information available on commercial antibody detection tests for diagnosis of TB disease of the lungs, the researchers carried out a systematic review. First, they searched biomedical literature databases using specific terms to identify studies for inclusion. A study was included in their analysis if the commercial test was compared against one of two other standard tests (sputum smear microscopy, or growth of TB bacteria in culture). One researcher from the team then pulled out specific pieces of information from each published study: these included the type of study design; information on study participants; the type of test; what the test was compared against; and finally the results of evaluation of the test. A second researcher pulled out pieces of information from several of the same studies. The researchers then compared the information to ensure that it was recorded correctly. Each study was also assigned a quality rating, based on four distinct criteria. For each type of test, the researchers used the data in the published studies to work out the test's accuracy, both in terms of its ability to give a positive reading for people who have TB disease as well as its ability to give a negative reading for people who do not have TB disease.
The researchers found 27 papers meeting their criteria. These papers reported the results of 68 original studies. Nine different commercial tests were examined in the studies. Overall, the studies seemed to be of relatively poor quality, with only 25% of them meeting all four of the researchers' criteria for a good-quality study. The different studies appeared to produce varying results for the accuracy of these commercial tests. In particular, the tests seemed to be less accurate at detecting TB disease amongst people who had a negative sputum smear than amongst people with a positive sputum smear. When all the data for these different studies were combined, the statistics indicated that the commercial tests, overall, were only modestly accurate for diagnosis of TB disease. None of the studies had been carried out in children or in HIV-positive people.
What Do These Findings Mean?
The results of this systematic review suggest that the commercial antibody detection tests considered here are not particularly useful in diagnosis of TB disease as compared to other tests, such as sputum smear and bacterial culture. Some people are concerned that there is pressure in certain developing countries to start using these tests, but the current data do not support greater use. This systematic review also highlights the fact that many studies evaluating commercial TB tests are of poor quality, and that further research needs to be done to evaluate the accuracy of different TB tests amongst children and HIV-positive patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040202.
World Health Organization Stop TB Department website. Information about the current Stop TB strategy, data and factsheets about TB, and other resources are available
Questions and Answers about Tuberculosis provided by the US Centers for Disease Control and Prevention
Information about TB tests from Médicins sans Frontières (MSF). Links to MSF reports on new diagnostic tests are also available
Wikipedia entry on Systematic Reviews (Note: Wikipedia is an internet encyclopedia anyone can edit)
doi:10.1371/journal.pmed.0040202
PMCID: PMC1891320  PMID: 17564490
5.  Insufficient quality of sputum submitted for tuberculosis diagnosis and associated factors, in Klaten district, Indonesia 
Background
Sputum smear microscopy is the standard diagnostic method for detection of smear positive pulmonary tuberculosis (TB). Insufficient quality of sputum might result in missing cases. In this study we aimed at assessing the quality of sputum in a district in Central Java and determining patient and health worker factors associated with submission of three good quality sputum samples.
Methods
In 16 health centers information was collected on the quality of sputum submitted by TB suspects, i.e. volume, color, and viscosity. TB suspects were interviewed to assess their knowledge of TB, motivation to provide sputum and whether they were informed why and how to produce a sputum sample. Health workers were interviewed to assess what information they provided to TB suspects about the reason for sputum examination, methods to produce sputum and characteristics of a good quality sputum sample. All health worker and patient factors were evaluated for association with sputum quality.
Results
Of 387 TB suspects, 294 (76.0%) could be traced and interviewed, and of 272 (70.3%) information about sputum quality was available. Of those 203 (74.6%) submitted three samples, 90 (33.1%) provided at least one good sample, and 37 (13.6%) provided three good quality sputum samples. Of the 272 TB suspects, 168 (61.8%) mentioned that information on the reason for sputum examination was provided, 66 (24.3%) remembered that they were informed about how to produce sputum and 40 (14.7%) recalled being informed about the characteristics of good quality sputum. Paramedics reported to provide often/always information on the importance of sputum examination, and when to produce sputum. Information on how to produce sputum and characteristics of a good sputum sample was less often provided. None of the studied patient characteristics or health worker factors was associated with providing good quality sputum.
Conclusion
A considerable number of TB suspects did not provide three sputum samples and a large number of sputum samples were of insufficient quality. Training of health workers in providing health education to the TB suspect about the reason for sputum examination and how to produce a good quality sputum sample should be a priority of the TB program.
doi:10.1186/1471-2466-9-16
PMCID: PMC2689165  PMID: 19426477
6.  Comparison of Xpert MTB/RIF with Other Nucleic Acid Technologies for Diagnosing Pulmonary Tuberculosis in a High HIV Prevalence Setting: A Prospective Study 
PLoS Medicine  2011;8(7):e1001061.
In this prospective, real-world cohort study nested within a national screening program for tuberculosis, Lesley Scott and colleagues compare the performance of Xpert MTB/RIF on a single sputum sample with different TB sputum detection technologies.
Background
The Xpert MTB/RIF (Cepheid) non-laboratory-based molecular assay has potential to improve the diagnosis of tuberculosis (TB), especially in HIV-infected populations, through increased sensitivity, reduced turnaround time (2 h), and immediate identification of rifampicin (RIF) resistance. In a prospective clinical validation study we compared the performance of Xpert MTB/RIF, MTBDRplus (Hain Lifescience), LightCycler Mycobacterium Detection (LCTB) (Roche), with acid fast bacilli (AFB) smear microscopy and liquid culture on a single sputum specimen.
Methods and Findings
Consecutive adults with suspected TB attending a primary health care clinic in Johannesburg, South Africa, were prospectively enrolled and evaluated for TB according to the guidelines of the National TB Control Programme, including assessment for smear-negative TB by chest X-ray, clinical evaluation, and HIV testing. A single sputum sample underwent routine decontamination, AFB smear microscopy, liquid culture, and phenotypic drug susceptibility testing. Residual sample was batched for molecular testing. For the 311 participants, the HIV prevalence was 70% (n = 215), with 120 (38.5%) culture-positive TB cases. Compared to liquid culture, the sensitivities of all the test methodologies, determined with a limited and potentially underpowered sample size (n = 177), were 59% (47%–71%) for smear microscopy, 76% (64%–85%) for MTBDRplus, 76% (64%–85%) for LCTB, and 86% (76%–93%) for Xpert MTB/RIF, with specificities all >97%. Among HIV+ individuals, the sensitivity of the Xpert MTB/RIF test was 84% (69%–93%), while the other molecular tests had sensitivities reduced by 6%. TB detection among smear-negative, culture-positive samples was 28% (5/18) for MTBDRplus, 22% (4/18) for LCTB, and 61% (11/18) for Xpert MTB/RIF. A few (n = 5) RIF-resistant cases were detected using the phenotypic drug susceptibility testing methodology. Xpert MTB/RIF detected four of these five cases (fifth case not tested) and two additional phenotypically sensitive cases.
Conclusions
The Xpert MTB/RIF test has superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy and other molecular methodologies in an HIV- and TB-endemic region. Its place in the clinical diagnostic algorithm in national health programs needs exploration.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial infection that mainly affects the lungs—is a global public health problem. In 2009, 9.4 million people developed TB, and 1.7 million people died from the disease; a quarter of these deaths were in HIV-positive individuals. People who are infected with HIV, the virus that causes AIDS, are particularly susceptible to TB because of their weakened immune system. Consequently, TB is a leading cause of illness and death among people living with HIV. TB is caused by Mycobacterium tuberculosis, which is spread in airborne droplets when people with the disease cough or sneeze. Its characteristic symptoms are a persistent cough, night sweats, and weight loss. Diagnostic tests for TB include sputum smear analysis (the microscopic examination of mucus brought up from the lungs by coughing for the presence of M. tuberculosis) and mycobacterial liquid culture (in which bacteriologists try to grow M. tuberculosis from sputum samples and test its drug sensitivity). TB can usually be cured by taking several powerful drugs daily for at least six months.
Why Was This Study Done?
Mycobacterial culture is a sensitive but slow way to diagnose TB. To halt the disease's spread, it is essential that TB—particularly TB that is resistant to several treatment drugs (multidrug-resistant, or MDR, TB)—is diagnosed quickly. Recently, several nucleic acid amplification technology (NAAT) tests have been developed that rapidly detect M. tuberculosis DNA in patient samples and look for DNA changes that make M. tuberculosis drug-resistant. In December 2010, the World Health Organization (WHO) endorsed Xpert MTB/RIF—an automated DNA test that detects M. tuberculosis and rifampicin resistance (an indicator of MDR TB) within two hours—for the investigation of patients who might have TB, especially in regions where MDR TB and HIV infection are common. TB diagnosis in HIV-positive people can be difficult because they are more likely to have smear-negative TB than HIV-negative individuals. In this prospective study, the researchers compare the performance of Xpert MTB/RIF on a single sputum sample with that of smear microscopy, liquid culture, and two other NAAT tests (MTBDRplus and LightCycler Mycobacterium Detection) in adults who might have TB in Johannesburg (South Africa), a region where many adults are HIV-positive.
What Did the Researchers Do and Find?
The researchers evaluated adults with potential TB attending a primary health care clinic for TB according to national guidelines and determined their HIV status. A sputum sample from 311 participants underwent smear microscopy, liquid culture, and drug susceptibility testing; 177 samples were also tested for TB using NAAT tests. They found that 70% of the participants were HIV-positive and 38.5% had culture-positive TB. Compared to liquid culture, smear microscopy, MTBDRplus, LightCycler Mycobacterium Detection, and Xpert MTB/RIF had sensitivities of 59%, 76%, 76%, and 86%, respectively. That is, assuming that liquid culture detected everyone with TB, Xpert MTB/RIF detected 86% of the cases. The specificity of all the tests compared to liquid culture was greater than 97%. That is, they all had a low false-positive rate. Among people who were HIV-positive, the sensitivity of Xpert MTB/RIF was 84%; the sensitivities of the other NAAT tests were 70%. Moreover, Xpert MTB/RIF detected TB in 61% of smear-negative, culture-positive samples, whereas the other NAATs detected TB in only about a quarter of these samples. Finally, although some TB cases were identified as drug-resistant by one test but drug-sensitive by another, the small number of drug-resistant cases means no firm conclusions can be made about the accuracy of drug resistance determination by the various tests.
What Do These Findings Mean?
Although these findings are likely to be affected by the study's small size, they suggest that Xpert MTB/RIF may provide a more accurate rapid diagnosis of TB than smear microscopy and other currently available NAAT tests in regions where HIV and TB are endemic (i.e., always present). Indeed, the reported accuracy of Xpert MTB/RIF for TB diagnosis—85% sensitivity and 97% specificity—has the potential to save more than 400,000 lives per year. Taken together with the results of other recent studies (including an accompanying article by Lawn et al. that investigates the use of Xpert MTB/RIF for screening for HIV-associated TB and rifampicin resistance), these findings support the WHO recommendation that Xpert MTB/RIF, rather than smear microscopy, should be the initial test in HIV-infected individuals who might have TB.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001061.
This study is further discussed in a PLoS Medicine Perspective by Carlton Evans; a related PLoS Medicine Research Article by Lawn et al. is also available
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report
WHO also provides information about tuberculosis and HIV
The US National Institute of Allergy and Infectious Diseases has detailed information on tuberculosis and HIV/AIDS
The US Centers for Disease Control and Prevention also has information about tuberculosis, including information on the diagnosis of and on tuberculosis and HIV co-infection
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on HIV-related tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001061
PMCID: PMC3144192  PMID: 21814495
7.  Multiple Sampling in One Day to Optimize Smear Microscopy in Children with Tuberculosis in Yemen 
PLoS ONE  2009;4(4):e5140.
Background and Aim
The diagnosis of pulmonary Tuberculosis (TB) in children is difficult and often requires hospitalization. We explored whether the yield of specimens collected for smear microscopy from different anatomical sites in one visit is comparable to the yield of specimens collected from a single anatomical site over several days.
Methodology and Principal Findings
Children with signs/symptoms of pulmonary TB attending a reference hospital in Sana'a Yemen underwent one nasopharyngeal aspirate (NPA) the first day of consultation and three gastric aspirates (GA) plus three expectorated/induced sputa over 3 consecutive days. Specimens were examined using smear microscopy (Ziehl-Neelsen) and cultured in solid media (Ogawa). Two hundred and thirteen children (aged 2 months–15 years) were enrolled. One hundred and ninety seven (93%) underwent nasopharyngeal aspirates, 196 (92%) GA, 122 (57%) expectorated sputum and 88 induced sputum. A total 1309 specimens were collected requiring 237 hospitalization days. In total, 29 (13.6%) children were confirmed by culture and 18 (8.5%) by smear microscopy. The NPA identified 10 of the 18 smear-positives; three consecutive GA identified 10 and induced/expectorated sputa identified 13 (6 by induced, 8 by expectorated sputum and one positive by both). In comparison, 22 (3.7%) of 602 specimens obtained the first day were smear-positive and identified 14 (6.6%) smear-positive children.
Conclusion/Significance
The examination of multiple tests the first day of consultation identified a similar proportion of smear-positive children than specimens collected over several days; would require half the number of tests and significantly less hospitalization. Optimized smear microscopy approaches for children should be explored further.
doi:10.1371/journal.pone.0005140
PMCID: PMC2663055  PMID: 19357770
8.  Prospective Cross-Sectional Evaluation of the Small Membrane Filtration Method for Diagnosis of Pulmonary Tuberculosis 
Journal of Clinical Microbiology  2014;52(7):2513-2520.
Smear microscopy has suboptimal sensitivity, and there is a need to improve its performance since it is commonly used to diagnose tuberculosis (TB). We prospectively evaluated the diagnostic accuracy of the small membrane filtration (SMF) method, an approach that uses a vacuum manifold and is designed to concentrate bacilli onto a filter that can be examined microscopically. We enrolled hospitalized adults suspected to have pulmonary TB in Kampala, Uganda. We obtained a clinical history and three spontaneously expectorated sputum specimens for smear microscopy (direct, concentrated, and SMF), MGIT (mycobacterial growth indicator tube) 960 and Lowenstein-Jensen (LJ) cultures, and Xpert MTB/RIF testing. We performed per-specimen (primary) and per-patient analyses. From October 2012 to June 2013, we enrolled 212 patients (579 sputum specimens). The participants were mostly female (63.2%), and 81.6% were HIV infected; their median CD4 cell count was 47 cells/μl. Overall, 19.0%, 20.4%, 27.1%, 25.2%, and 25.9% of specimens tested positive by direct smear, concentrated smear, MGIT culture, LJ culture, and Xpert test, respectively. In the per-specimen analysis, the sensitivity of the SMF method (48.5%; 95% confidence interval [CI], 37.4 to 59.6) was lower than those of direct smear (60.9%; 51.4 to 70.5 [P = 0.0001]) and concentrated smear (63.3%; 53.6 to 73.1 [P < 0.0001]). Subgroup analyses showed that SMF performed poorly in specimens having a low volume or low bacterial load. The SMF method performed poorly compared to standard smear techniques and was sensitive to sample preparation techniques. The optimal laboratory SMF protocol may require striking a fine balance between sample dilution and filtration failure rate.
doi:10.1128/JCM.00642-14
PMCID: PMC4097702  PMID: 24808236
9.  False-Positive Gen-Probe Direct Mycobacterium tuberculosis Amplification Test Results for Patients with Pulmonary M. kansasii and M. avium Infections 
Journal of Clinical Microbiology  1999;37(1):175-178.
The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test has been approved for use in the United States for the rapid diagnosis of pulmonary tuberculosis in patients with acid-fast smear-positive sputum samples since 1996. Four patients infected with human immunodeficiency virus and one chronic pulmonary-disease patient seen in our institutions with abnormal chest radiographs and fluorochrome stain-positive sputa were evaluated for tuberculosis, including performance of the MTD test on expectorated sputum samples. Three of these five patients’ sputa were highly smear-positive (i.e., more than 100 bacilli per high-power field), while two patient’s sputa contained 1 to 10 bacilli per field. MTD results on sputum specimens from these patients ranged from 43,498 to 193,858 relative light units (RLU). Gen-Probe has defined values of at least 30,000 RLU as indicative of a positive test, i.e., the presence of Mycobacterium tuberculosis RNA. Four of the patients’ sputum cultures yielded growth of M. kansasii within 6 to 12 days, and the fifth produced growth of M. avium only. One patient’s culture contained both M. kansasii and M. avium, but none of the initial or follow-up cultures from these five patients revealed M. tuberculosis. However, subsequent cultures from three of the patients again revealed M. kansasii. During the period of this study, in which MTD tests were performed on smear-positive sputum specimens from 82 patients, four of seven patients with culture-proven M. kansasii pulmonary infections yielded one or more false-positive MTD tests. The MTD sensitivity observed in this study was 93.8%, and the specificity was 85.3%. Five cultures of M. kansasii (including three of these patients’ isolates and M. kansasii ATCC 12478), and cultures of several other species were examined at densities of 105 to 107 viable CFU/ml by the MTD test. All five isolates of M. kansasii and three of three isolates of M. simiae yielded false-positive test results, with readings of 75,191 to 335,591 RLU. These findings indicate that low-level false-positive MTD results can occur due to the presence of M. kansasii, M. avium, and possibly other Mycobacterium species other than M. tuberculosis in sputum. Low-level positive MTD results of 30,000 to 500,000 RLU should be interpreted in light of these findings. It remains to be determined if the enhanced MTD test (MTD 2) recently released by Gen-Probe will provide greater specificity than that observed in this report with its first-generation test.
PMCID: PMC84200  PMID: 9854086
10.  SIMPLE MEASURES ARE AS EFFECTIVE AS INVASIVE TECHNIQUES IN THE DIAGNOSIS OF PULMONARY TUBERCULOSIS IN MALAWI 
Setting
Detection of smear-positive pulmonary tuberculosis (PTB) cases is vital for tuberculosis control. Methods to augment sputum collection are available but their additional benefit is uncertain in resource-limited settings.
Objective
To compare the diagnostic yields using five methods to obtain sputum from adults diagnosed with smear-negative PTB in Malawi.
Design
Self-expectorated sputum was collected under supervision for microscopy and mycobacterial culture in the study laboratory. Confirmed smear-negative patients, provided physiotherapy-assisted sputum and induced sputum followed, the next morning, by gastric washing and bronchoalveolar-lavage samples.
Results
150 patients, diagnosed with smear-negative PTB by the hospital service, were screened. 39 (26%) were smear-positive from supervised self-expectorated sputum examined in the study laboratory. The remaining 111 confirmed smear-negative patients were enrolled; 89% were HIV positive. Seven additional smear-positive cases were diagnosed using the augmented sputum collection techniques. No differences were observed in the numbers of cases detected using the different methods. 44 (95.6%) of the 46 smear-positive cases could be detected from self-expectorated and physiotherapy-assisted samples
Conclusions
For countries like Malawi, the best use of limited resources to detect smear-positive PTB cases would be to improve the quality of self-expectorated sputum collection and microscopy. The additional diagnostic yield using bronchoalveolar-lavage after induced sputum is limited.
PMCID: PMC2873674  PMID: 19105886
Induced sputum; gastric washings; physiotherapy; BAL; HIV
11.  Diagnosing sputum/smear-negative pulmonary tuberculosis: Does fibre-optic bronchoscopy play a significant role? 
Background:
Diagnosis of sputum/smear-negative pulmonary tuberculosis patients can be both challenging and time consuming with many patients being put on empirical anti-tubercular treatment. Fibreoptic bronchoscopy may provide a confirmative and early diagnosis in such patients.
Aims:
To assess the role of fibreoptic bronchoscopy in the diagnosis of sputum /smear-negative pulmonary tuberculosis.
Materials and Methods:
The study was conducted on 75 suspected sputum / smear-negative pulmonary tuberculosis cases attending Pulmonary Medicine Department of Mamata Medical College and Hospital, Khammam, AP. Fibreoptic bronchoscopy was performed; culture of sputum and bronchial washings for Mycobacterium tuberculosis was done by BACTEC method.
Results:
A final diagnosis of sputum /smear-negative pulmonary tuberculosis was made in 60 patients. Bronchial washings smear for acid-fast bacilli (AFB) was positive in 21 patients while culture of bronchial washings was positive in 39 patients. In 29 patients, smear or culture of bronchial washing alone contributed to the final diagnosis. Total yield of bronchoscopy in diagnosis of sputum smear negative pulmonary tuberculosis was 83.33% (50/60); bronchoscopy was the only diagnostic method in 66% cases (40/60) with bronchial washings being the only diagnostic method in 48.33%. Bronchial washings smear for AFB and histopathological evidence of caseating granuloma made immediate diagnosis possible in 48.33% (29/60) patients.
Conclusion:
Our study suggests that fibreoptic bronchoscopy can provide excellent material for diagnosis of suspected cases of Pulmonary Tuberculosis in whom smears of expectorated sputum do not reveal mycobacteria.
doi:10.4103/0970-2113.63607
PMCID: PMC2893426  PMID: 20616936
Bronchial washings; fibreoptic bronchoscopy; pulmonary tuberculosis; sputum smear negative
12.  Serological Testing Versus Other Strategies for Diagnosis of Active Tuberculosis in India: A Cost-Effectiveness Analysis 
PLoS Medicine  2011;8(8):e1001074.
This cost-effectiveness study shows that sputum smear microscopy is the most cost-effective test for active tuberculosis (TB) in India, and liquid culture plus microscopy is more cost-effective for TB diagnosis than serological tests.
Background
Undiagnosed and misdiagnosed tuberculosis (TB) drives the epidemic in India. Serological (antibody detection) TB tests are not recommended by any agency, but widely used in many countries, including the Indian private sector. The cost and impact of using serology compared with other diagnostic techniques is unknown.
Methods and Findings
Taking a patient cohort conservatively equal to the annual number of serological tests done in India (1.5 million adults suspected of having active TB), we used decision analysis to estimate costs and effectiveness of sputum smear microscopy (US$3.62 for two smears), microscopy plus automated liquid culture (mycobacterium growth indicator tube [MGIT], US$20/test), and serological testing (anda-tb ELISA, US$20/test). Data on test accuracy and costs were obtained from published literature. We adopted the perspective of the Indian TB control sector and an analysis frame of 1 year. Our primary outcome was the incremental cost per disability-adjusted life year (DALY) averted. We performed one-way sensitivity analysis on all model parameters, with multiway sensitivity analysis on variables to which the model was most sensitive.
If used instead of sputum microscopy, serology generated an estimated 14,000 more TB diagnoses, but also 121,000 more false-positive diagnoses, 102,000 fewer DALYs averted, and 32,000 more secondary TB cases than microscopy, at approximately four times the incremental cost (US$47.5 million versus US$11.9 million). When added to high-quality sputum smears, MGIT culture was estimated to avert 130,000 incremental DALYs at an incremental cost of US$213 per DALY averted. Serology was dominated by (i.e., more costly and less effective than) MGIT culture and remained less economically favorable than sputum smear or TB culture in one-way and multiway sensitivity analyses.
Conclusions
In India, sputum smear microscopy remains the most cost-effective diagnostic test available for active TB; efforts to increase access to quality-assured microscopy should take priority. In areas where high-quality microscopy exists and resources are sufficient, MGIT culture is more cost-effective than serology as an additional diagnostic test for TB. These data informed a recently published World Health Organization policy statement against serological tests.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year, about 2 million people develop tuberculosis in India—a fifth of the global incidence of this highly contagious bacterial infection. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is spread in airborne droplets when people with the disease cough or sneeze and usually infects the lungs although it can also infect other organs. The characteristic symptoms of tuberculosis are a persistent cough, weight loss, and night sweats. Diagnostic tests for tuberculosis include sputum smear microscopy (microscopic analysis of mucus brought up from the lungs by coughing), culture (growth) of M. tuberculosis from sputum samples in liquid media (using, for example, a commercial product called the mycobacteria growth indicator tube or MGIT), and nucleic acid amplification tests (which detect the bacterium's genome in patient samples) such as the Xpert MTB/RIF system. Tuberculosis can usually be cured by taking several powerful antibiotics daily for at least 6 months.
Why Was This Study Done?
In India, as elsewhere, undiagnosed and misdiagnosed tuberculosis drives the tuberculosis epidemic by increasing the transmission of M. tuberculosis. Unfortunately, sputum smear microscopy, the current mainstay of tuberculosis diagnosis worldwide, detects only half of tuberculosis cases, mycobacterial culture can take weeks to provide a diagnosis, and rapid techniques such as nucleic acid amplification require infrastructure that is often not available in developing countries. Consequently, in India and other developing countries, serological tests are widely used for the diagnosis of tuberculosis. Serological tests detect antibodies against M. tuberculosis in the blood (antibodies are proteins made by the immune system in response to infections). Serological tests are fast and simple to perform, but they are not recommended for clinical use, and the available evidence suggests that they do not diagnose tuberculosis accurately. Even so, and in the absence of information about the cost and impact (cost-effectiveness) of serological testing, about 1.5 million serological tests for tuberculosis are conducted every year in India at a cost of more than US$15 million. Here, the researchers analyze the cost-effectiveness of serological tests compared to other diagnostic tests from the perspective of tuberculosis control in India.
What Did the Researchers Do and Find?
The researchers used “decision analysis” to estimate the cost-effectiveness of sputum smear microscopy, microscopy plus liquid culture using the MGIT system, and serological testing using the widely used anda-tb ELISA commercial test in a hypothetical group of 1.5 million people suspected of having tuberculosis. Decision analysis formally assesses the decision-making process by using models that evaluate outcomes under different scenarios. By feeding data on the costs and accuracy of different diagnostic tests into their decision-analysis model, the researchers estimate that, over a year, serology would generate 14,000 more tuberculosis diagnoses than sputum microscopy. However, it would also generate 121,000 more false-positive diagnoses and 32,000 more tuberculosis transmissions to other people (secondary transmissions), and avert 102,000 fewer disability-adjusted life years (DALYs; a DALY is a year of healthy life lost because of premature death or disability) at four times the incremental cost of sputum microscopy. MGIT culture added to sputum smear microscopy would avert 130,000 DALYs at an incremental cost of US$213 per DALY averted. Finally, sensitivity analyses (reruns of the decision-analysis model using different values for test costs and accuracy) identified no scenario in which serology was either less costly or more effective than sputum smear microscopy alone or in which serology plus sputum microscopy was more cost-effective than MGIT culture plus sputum microscopy.
What Do These Findings Mean?
These findings identify sputum smear microscopy as the most cost-effective existing diagnostic test for tuberculosis in India. Moreover, they suggest that in areas where high-quality microscopy is available, resources are sufficient, and infrastructure to effectively use culture exists, the addition of MGIT culture to sputum smear microscopy would be more cost-effective than the addition of serology. Importantly, these findings suggest that, if used as an initial test for tuberculosis in India, serology would result in more DALYs, more secondary infections, and more false-positive diagnoses than sputum smear microscopy while increasing per-patient costs to the Indian tuberculosis control sector. Given these findings and the results of a recent updated systematic review on the accuracy of serological tests, the World Health Organization's Strategic and Technical Advisory Group for Tuberculosis recently advised against the use of currently available serological tests for the diagnosis of tuberculosis. The WHO negative policy against serological tests must now be implemented in India.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001074.
Details of the recent systematic review of serological tests for tuberculosis diagnosis are available in a PLoS Medicine Research Article by Steingart et al.
The World Health Organization provides information on all aspects of tuberculosis, including tuberculosis diagnostics and the Stop TB Partnership (some information is in several languages); its Strategic and Technical Advisory Group for Tuberculosis recommendations on tuberculosis diagnosis are available
The Evidence-based TB Diagnosis Web site by the Stop TB Partnership's New Diagnostics Working Group provides evidence syntheses on various TB tests, along with guidelines, resources, and training materials
The US Centers for Disease Control and Prevention has information about tuberculosis, including information on the diagnosis of tuberculosis disease
The US National Institute of Allergy and Infectious Diseases also has information on all aspects of tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001074
PMCID: PMC3153451  PMID: 21857810
13.  Xpert MTB/RIF assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility 
BMC Microbiology  2015;15:220.
Background
Xpert MTB/RIF assay is considered as a great advance over conventional smear and culture in the diagnosis of TB and MDR-TB by simultaneously detecting M.tuberculosis and rifampicin resistance bacilli. However, very little information regarding the performance characteristics of Xpert MTB/RIF assay is available in Ethiopia. Therefore, the purpose of this study was to evaluate the performance of Xpert MTB/RIF assay compared to conventional sputum smear and culture methods for the diagnosis of pulmonary tuberculosis in remote health care facility.
Methods
A paired expectorated sputum samples were obtained from 227 consecutively recruited patients with signs and symptoms suggestive of tuberculosis at Karamara hospital during December 2013 to May 2014. One of the sputum specimen was tested directly by Ziehl-Neelsen staining and Xpert MTB/RIF assay without NALC-NaOH decontamination. The other of pair of sputa specimen was cultured for isolation of TB bacilli by conventional methods. Diagnostic performance of Xpert MTB/RIF assay and AFB smear microscopy were calculated against culture as the gold standard.
Results
Overall 25.5 % (58/227) samples were positive for Mycobacterium tuberculosis complex (MTBC) by MGIT and/or LJ media of which 36.2 % (21/58) and 65.5 % (35/58) were positive by AFB smear microscopy and Xpert MTB/RIF respectively. The sensitivity, specificity, as well as the positive and negative predictive value of Xpert MTB/RIF assay were 65.5 % (95 % CI: 53.3–77.7 %), 96.3 % (95 % CI: 93.4–99.2 %), 86.4 % (95 % CI: 76.2–96.5 %), and 88.6 % (95 % CI: 83.9–93.3 %) respectively. Eighteen of 58 (31 %) cases that were smear microscopy negative, were positive by Xpert MTB/RIF assay.
Conclusions
Although Xpert MTB/RIF assay demonstrated high sensitivity in detecting MTBC in sputum specimens compared with conventional AFB smear microscopy, it demonstrated suboptimal sensitivity in smear negative patients compared to conventional culture.
doi:10.1186/s12866-015-0566-6
PMCID: PMC4615882  PMID: 26483194
NPV; PPV; Sensitivity; Specificity; Xpert MTB/RIF assay; Remote health facility
14.  A Multi-Country Non-Inferiority Cluster Randomized Trial of Frontloaded Smear Microscopy for the Diagnosis of Pulmonary Tuberculosis 
PLoS Medicine  2011;8(7):e1000443.
Luis Cuevas and colleagues report findings from a multicenter diagnostic clinical trial in tuberculosis, showing that the sensitivity and specificity of a “front-loaded” diagnostic scheme is not inferior to that of a standard diagnostic scheme.
Background
More than 50 million people around the world are investigated for tuberculosis using sputum smear microscopy annually. This process requires repeated visits and patients often drop out.
Methods and Findings
This clinical trial of adults with cough ≥2 wk duration (in Ethiopia, Nepal, Nigeria, and Yemen) compared the sensitivity/specificity of two sputum samples collected “on the spot” during the first visit plus one sputum sample collected the following morning (spot-spot-morning [SSM]) versus the standard spot-morning-spot (SMS) scheme. Analyses were per protocol analysis (PPA) and intention to treat (ITT). A sub-analysis compared just the first two smears of each scheme, spot-spot and spot-morning.
In total, 6,627 patients (3,052 SSM/3,575 SMS) were enrolled; 6,466 had culture and 1,526 were culture-positive. The sensitivity of SSM (ITT, 70.2%, 95% CI 66.5%–73.9%) was non-inferior to the sensitivity of SMS (PPA, 65.9%, 95% CI 62.3%–69.5%). Similarly, the specificity of SSM (ITT, 96.9%, 95% CI 93.2%–99.9%) was non-inferior to the specificity of SMS (ITT, 97.6%, 95% CI 94.0%–99.9%). The sensitivity of spot-spot (ITT, 63.6%, 95% CI 59.7%–67.5%) was also non-inferior to spot-morning (ITT, 64.8%, 95% CI 61.3%–68.3%), as the difference was within the selected −5% non-inferiority limit (difference ITT = 1.4%, 95% CI −3.7% to 6.6%). Patients screened using the SSM scheme were more likely to provide the first two specimens than patients screened with the SMS scheme (98% versus 94.2%, p<0.01). The PPA and ITT analysis resulted in similar results.
Conclusions
The sensitivity and specificity of SSM are non-inferior to those of SMS, with a higher proportion of patients submitting specimens. The scheme identifies most smear-positive patients on the first day of consultation.
Trial Registration
Current Controlled Trials ISRCTN53339491
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year, nearly 10 million people develop tuberculosis—a contagious bacterial infection that usually affects the lungs (pulmonary tuberculosis)—and about 1.7 million people die from the disease. Mycobacterium tuberculosis, which causes tuberculosis, is spread in airborne droplets when people with the disease cough or sneeze. Thus, to control tuberculosis, it is essential that infected individuals are rapidly identified and treated. The “gold standard” diagnostic test for tuberculosis is mycobacterial culture, in which laboratory staff try to grow M. tuberculosis from sputum (mucus brought up from the lungs by coughing). However, although this test is sensitive (it detects most patients with tuberculosis) and has a high specificity (a low rate of false-positive results), it is too slow to produce results and too complex for routine use in the low- and middle-income countries where tuberculosis mainly occurs. In these countries, patients are usually investigated using direct sputum smear microscopy, a cheaper but less sensitive test in which multiple sputum samples treated with the acid-fast Ziehl-Neelsen stain are examined for the presence of M. tuberculosis bacilli.
Why Was This Study Done?
In most national tuberculosis control programs, patients provide an “on the spot” specimen during their initial consultation, a specimen collected at home the next morning, and another on-the-spot specimen when they bring their morning specimen to the clinic (a “spot-morning-spot,” or SMS, collection scheme). Unfortunately, patients often fail to return with their morning sample. Furthermore, the examination of three samples strains the limited laboratory resources of developing countries. Based on several recent reviews, the World Health Organization recently recommended that only two samples need be examined, a policy change that reduces the laboratory workload but does not avoid the problems of collecting a morning sample and patient drop-out during the diagnostic process. In this non-inferiority, cluster randomized trial, the researchers compare the sensitivity and specificity of a spot-spot-morning (SSM; two on-the-spot specimens collected during the first clinic visit an hour apart, and a third specimen collected at home the next morning) scheme for tuberculosis diagnosis with those of the standard SMS scheme. A non-inferiority trial investigates whether an intervention is not worse than a control intervention; a cluster randomized trial randomly assigns groups of patients rather than individual patients to the test and control interventions.
What Did the Researchers Do and Find?
The researchers enrolled 6,627 patients in Ethiopia, Nepal, Nigeria, and Yemen who had had a cough for more than two weeks (a characteristic symptom of tuberculosis). A quarter of the patients had culture-positive tuberculosis. The centers participating in the study were randomly assigned each week for a year to use either the SMS or the SSM sample collection scheme. Compared to mycobacterial culture, the sensitivities of the SSM and SMS schemes were 70.2% and 65.9%, respectively, which indicates that the new scheme was non-inferior to the SMS scheme. Similarly, the specificity of SSM (96.9%) was non-inferior to that of SMS (97.6%). Importantly, the sensitivity of diagnosis using just the first two samples collected in the SSM scheme was also non-inferior to the sensitivity of diagnosis using the first two samples collected in the SMS scheme (63.6% versus 64.8%; the researchers defined non-inferiority of SSM as a difference in its sensitivity compared to that of SMS of less than −5%). Finally, patients tested using the SSM scheme were more likely to provide the first two samples than patients tested using the SMS scheme (98% versus 94.2%).
What Do These Findings Mean?
These findings suggest that a sputum collection scheme in which two samples are collected one hour apart followed by a morning specimen could identify as many smear-positive patients as the standard SMS scheme. Importantly, they also indicate that examination of the first two specimens alone identifies most smear-positive patients independently of which scheme is used. These findings suggest that the SSM scheme might be more suitable for tuberculosis diagnosis than the SMS scheme in locations where patients are likely to drop out of the diagnosis process (for example, in low- and middle-income countries, where patients often live a long way from clinics). However, for an SSM scheme to work effectively, an on-site laboratory with a same-day turn-around service will be essential, and tuberculosis clinics will need to minimize contact between patients waiting to provide their second on-the-spot specimen.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000443.
A related PLoS Medicine Research Article by Cuevas et al. uses LED fluorescence microscopy for the diagnosis of pulmonary tuberculosis
The World Health Organization provides information on all aspects of tuberculosis, including information on tuberculosis diagnostics and on the recommendation to reduce the number of smears for diagnosis to two; the Stop TB Partnership provides information on global tuberculosis control (some information in several languages)
The US Centers for Disease Control and Prevention has information about tuberculosis, including information on the diagnosis of tuberculosis disease
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
A new Web site dedicated to the discussion and optimization of smear microscopy has recently been launched
doi:10.1371/journal.pmed.1000443
PMCID: PMC3134460  PMID: 21765808
15.  Screening for HIV-Associated Tuberculosis and Rifampicin Resistance before Antiretroviral Therapy Using the Xpert MTB/RIF Assay: A Prospective Study 
PLoS Medicine  2011;8(7):e1001067.
In a prospective study, Stephen Lawn and colleagues find that pre-ART screening with Xpert MTB/RIF increased tuberculosis case detection by 45% compared to smear microscopy in HIV-positive patients at high risk of TB risk. AE competing interests must also pull through to the proof. “The Academic Editor, Madhukar Pai, declares that he consults for the Bill & Melinda Gates Foundation (BMGF). The BMGF supported FIND which was involved in the development of the Xpert MTB/RIF assay. He also co-chairs the Stop TB Partnership's New Diagnostics Working Group that was involved in the WHO endorsement of the Xpert assay.” Linked: Scott pmed.1001061; Evans pmed.1001064; Dowdy pmed.1001063
Background
The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown.
Methods and Findings
Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102–236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%).
Conclusions
In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial infection that mainly affects the lungs—is a leading cause of illness and death among people who are infected with HIV, the virus that causes AIDS by destroying the immune system, which leaves infected individuals susceptible to other infections. TB is caused by Mycobacterium tuberculosis, which is spread in airborne droplets when people with the disease cough or sneeze. Its symptoms include a persistent cough, weight loss, and night sweats. Diagnostic tests for TB include chest X-rays, sputum smear analysis (microscopic examination of mucus coughed up from the lungs for M. tuberculosis bacilli), and mycobacterial liquid culture (the growth of M. tuberculosis from sputum and determination of its drug sensitivity). TB can be cured by taking several drugs daily for six months, although the recent emergence of multidrug-resistant TB (MDR-TB) is making the disease increasingly hard to treat.
Why Was This Study Done?
TB is a major problem in clinics that provide antiretroviral therapy (ART) for HIV-positive people in resource-limited settings. Not only is it a major cause of sickness and mortality in those affected by it, but TB (especially MDR-TB) can also spread to other patients attending the same clinic for health services. Rapid diagnosis and appropriate treatment are very important to reduce these risks. Unfortunately, sputum smear analysis—the mainstay of TB diagnosis in resource-limited settings—only detects about a fifth of TB cases when used as a screening tool before initiating ART. Chest X-rays are costly and don't always detect TB, and liquid culture—the gold standard method for TB diagnosis—is costly, technically difficult, and slow. Consequently, the World Health Organization (WHO) recently endorsed a new test for the investigation of patients suspected of having TB, especially in regions where HIV infection and MDR-TB are common. Xpert MTB/RIF is an automated DNA test that detects M. tuberculosis and DNA differences that make the bacteria resistant to the drug rifampicin (an indicator of MDR-TB) within 2 hours. In this study, the researchers investigate whether Xpert MTB/RIF could be used as a routine screening test to increase TB detection among HIV-positive people initiating ART.
What Did the Researchers Do and Find?
The researchers collected sputum from HIV-infected adults with no current TB diagnosis enrolling at an ART clinic in a South African township where HIV infection and TB are both common. They then compared the diagnostic accuracy of Xpert MTB/RIF (performed at a centralized laboratory) with that of several other tests, including liquid culture (the reference test). Nearly a fifth of the patients had culture-positive TB. Xpert MTB/RIF identified three-quarters of these patients (a sensitivity of 73.3%). By contrast, the sensitivity of smear microscopy was 28%. The new test's specificity (the proportion of patients with a negative Xpert MTB/RIF result among patients without TB) was 99.2%. That is, Xpert MTB/RIF had a low false-positive rate. Notably, Xpert MTB/RIF detected all cases of smear-positive, culture-positive TB but only 43.4% of smear-negative, culture-positive cases from a single sputum sample; it detected 62.3% of such cases when two sputum samples were analyzed. Finally, Xpert MTB/RIF correctly identified rifampicin resistance in all four patients who had MDR-TB but incorrectly identified resistance in three patients with drug-sensitive TB.
What Do These Findings Mean?
In this population of HIV-positive patients with a high TB risk, pre-ART screening with Xpert MTB/RIF increased case detection by 45% compared to smear microscopy, a finding that needs confirming in other settings. Importantly, Xpert MTB/RIF reduced the delay in diagnosis of TB from more than 20 days to two days. This delay would be reduced further by doing the assay at ART clinics rather than at a centralized testing facility, but the diagnostic accuracy of point-of-care testing needs evaluating. Overall, these findings (and those of an accompanying article by Scott et al. that examines the performance of Xpert MTB/RIF in an area where HIV infection is common) support the replacement of smear microscopy with Xpert MTB/RIF for pre-ART TB screening (provided misdiagnosis of rifampicin resistance can be reduced). These findings also suggest that routine screening with Xpert MTB/RIF could reduce the risk of MDR-TB outbreaks in HIV care and treatment settings and improve outcomes for HIV-positive patients with MDR-TB who currently often die before a diagnosis of TB can be made.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001056.
This study is further discussed in a PLoS Medicine Perspective by Carlton Evans; a related PLoS Medicine Research Article by Scott et al. is also available
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report
WHO also provides information about tuberculosis and HIV
The US National Institute of Allergy and Infectious Diseases has detailed information on tuberculosis and HIV/AIDS
The US Centers for Disease Control and Prevention also has information about tuberculosis, including information on the diagnosis of and on tuberculosis and HIV co-infection
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV-related tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001067
PMCID: PMC3144215  PMID: 21818180
16.  Accuracy of the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in Cape Town, South Africa: a descriptive study 
The Lancet. Infectious diseases  2011;11(11):819-824.
Summary
Background
WHO recommends that Xpert MTB/RIF replaces smear microscopy for initial diagnosis of suspected HIV-associated tuberculosis or multidrug-resistant pulmonary tuberculosis, but no data exist for its use in children. We aimed to assess the accuracy of the test for the diagnosis of pulmonary tuberculosis in children in an area with high tuberculosis and HIV prevalences.
Methods
In this prospective, descriptive study, we enrolled children aged 15 years or younger who had been admitted to one of two hospitals in Cape Town, South Africa, with suspected pulmonary tuberculosis between Feb 19, 2009, and Nov 30, 2010. We compared the diagnostic accuracy of MTB/RIF and concentrated, fluorescent acid-fast smear with a reference standard of liquid culture from two sequential induced sputum specimens (primary analysis).
Results
452 children (median age 19·4 months, IQR 11·1–46·2) had at least one induced sputum specimen; 108 children (24%) had HIV infection. 27 children (6%) had a positive smear result, 70 (16%) had a positive culture result, and 58 (13%) had a positive MTB/RIF test result. With mycobacterial culture as the reference standard, MTB/RIF tests when done on two induced sputum samples detected twice as many cases (75·9%, 95% CI 64·5–87·2) as did smear microscopy (37·9%, 25·1–50·8), detecting all of 22 smear-positive cases and 22 of 36 (61·1%, 44·4–77·8) smear-negative cases. For smear-negative cases, the incremental increase in sensitivity from testing a second specimen was 27·8% for MTB/RIF, compared with 13·8% for culture. The specificity of MTB/RIF was 98·8% (97·6–99·9). MTB/RIF results were available in median 1 day (IQR 0–4) compared with median 12 days (9–17) for culture (p<0·0001).
Interpretation
MTB/RIF testing of two induced sputum specimens is warranted as the first-line diagnostic test for children with suspected pulmonary tuberculosis.
Funding
National Institutes of Health, the National Health Laboratory Service Research Trust, the Medical Research Council of South Africa, and Wellcome Trust.
doi:10.1016/S1473-3099(11)70167-0
PMCID: PMC4202386  PMID: 21764384
17.  Impact of Replacing Smear Microscopy with Xpert MTB/RIF for Diagnosing Tuberculosis in Brazil: A Stepped-Wedge Cluster-Randomized Trial 
PLoS Medicine  2014;11(12):e1001766.
Betina Durovni and colleagues evaluated whether implementation of Xpert MTB/RIF increased the notification rate of laboratory-confirmed pulmonary tuberculosis and reduced the time to tuberculosis treatment initiation in 14 Brazilian primary care laboratories.
Please see later in the article for the Editors' Summary
Background
Abundant evidence on Xpert MTB/RIF accuracy for diagnosing tuberculosis (TB) and rifampicin resistance has been produced, yet there are few data on the population benefit of its programmatic use. We assessed whether the implementation of Xpert MTB/RIF in routine conditions would (1) increase the notification rate of laboratory-confirmed pulmonary TB to the national notification system and (2) reduce the time to TB treatment initiation (primary endpoints).
Methods and Findings
We conducted a stepped-wedge cluster-randomized trial from 4 February to 4 October 2012 in 14 primary care laboratories in two Brazilian cities. Diagnostic specimens were included for 11,705 baseline (smear microscopy) and 12,522 intervention (Xpert MTB/RIF) patients presumed to have TB. Single-sputum-sample Xpert MTB/RIF replaced two-sputum-sample smear microscopy for routine diagnosis of pulmonary TB. In total, 1,137 (9.7%) tests in the baseline arm and 1,777 (14.2%) in the intervention arm were positive (p<0.001), resulting in an increased bacteriologically confirmed notification rate of 59% (95% CI = 31%, 88%). However, the overall notification rate did not increase (15%, 95% CI = −6%, 37%), and we observed no change in the notification rate for those without a test result (−3%, 95% CI = −37%, 30%). Median time to treatment decreased from 11.4 d (interquartile range [IQR] = 8.5–14.5) to 8.1 d (IQR = 5.4–9.3) (p = 0.04), although not among confirmed cases (median 7.5 [IQR = 4.9–10.0] versus 7.3 [IQR = 3.4–9.0], p = 0.51). Prevalence of rifampicin resistance detected by Xpert was 3.3% (95% CI = 2.4%, 4.3%) among new patients and 7.4% (95% CI = 4.3%, 11.7%) among retreatment patients, with a 98% (95% CI = 87%, 99%) positive predictive value compared to phenotypic drug susceptibility testing. Missing data in the information systems may have biased our primary endpoints. However, sensitivity analyses assessing the effects of missing data did not affect our results.
Conclusions
Replacing smear microscopy with Xpert MTB/RIF in Brazil increased confirmation of pulmonary TB. An additional benefit was the accurate detection of rifampicin resistance. However, no increase on overall notification rates was observed, possibly because of high rates of empirical TB treatment.
Trial registration
ClinicalTrials.gov NCT01363765
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Each year, about 8.6 million people develop active tuberculosis and at least 1.3 million people die from the disease, mainly in resource-limited countries. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of tuberculosis include cough, weight loss, and night sweats. Diagnostic tests for tuberculosis include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs), the growth (culture) of M. tuberculosis from sputum samples, and molecular tests (for example, the Xpert MTB/RIF test) that rapidly and accurately detect M. tuberculosis in sputum and determine its antibiotic resistance. Tuberculosis can be cured by taking several antibiotics daily for at least six months, although the emergence of multidrug-resistant tuberculosis is making the disease increasingly hard to treat.
Why Was This Study Done?
Quick, accurate diagnosis of active tuberculosis is essential to reduce the global tuberculosis burden, but in most high-burden settings diagnosis relies on sputum smear analysis, which fails to identify many infected people. Mycobacterial culture correctly identifies more infected people but is slow, costly, and rarely available in resource-limited settings. In late 2010, therefore, the World Health Organization recommended the routine use of the Xpert MTB/RIF assay (Xpert) for tuberculosis diagnosis, and several resource-limited countries are currently scaling up the use of Xpert in their national tuberculosis control programs. However, although Xpert works well in ideal conditions, little is known about its performance in routine (real-life) settings. In this pragmatic stepped-wedge cluster-randomized trial, the researchers assess the impact of replacing smear microscopy with Xpert for the diagnosis of tuberculosis in Brazil, an upper-middle-income country with a high tuberculosis burden. A pragmatic trial asks whether an intervention works under real-life conditions; a stepped-wedge cluster-randomized trial sequentially and randomly rolls out an intervention to groups (clusters) of people.
What Did the Researchers Do and Find?
The researchers randomly assigned 14 tuberculosis diagnosis laboratories in two cities to switch at different times from smear microscopy to Xpert for tuberculosis diagnosis. Specifically, at the start of the eight-month trial, all the laboratories used smear microscopy for tuberculosis diagnosis. At the end of each month, two laboratories switched to using Xpert, so that in the final month of the trial, all the laboratories were using Xpert. During the trial, 11,705 samples from patients with symptoms consistent with tuberculosis were examined using smear microscopy (baseline arm), and 12,522 samples were examined using Xpert (intervention arm). The researchers obtained the results of these tests from a database of all the diagnostic tests ordered in the Brazilian public laboratory system, and they obtained data on tuberculosis notifications during the trial period from the national notification system. In total, 9.7% and 14.2% of the tests in the baseline and intervention arm, respectively, were positive, and the laboratory-confirmed tuberculosis notification rate was 1.59 times higher in the Xpert arm than in the smear microscopy arm. However, the overall notification rate (which included people who began treatment on the basis of symptoms alone) did not increase during the trial. The time to treatment (the time between the laboratory test date and the notification date, when treatment usually starts in Brazil) was about 11 days and eight days in the smear microscopy and Xpert arms, respectively.
What Do These Findings Mean?
The findings indicate that, in a setting where laboratory diagnosis for tuberculosis was largely restricted to sputum smear examination, the implementation of Xpert increased the rates of laboratory-confirmed pulmonary (lung) tuberculosis notifications and reduced the time to treatment initiation, two endpoints of public health relevance. However, implementation of Xpert did not increase the overall notification rate of pulmonary tuberculosis (probably because of the high rate of empiric tuberculosis treatment in Brazil), although it did facilitate accurate and rapid detection of rifampicin resistance. The accuracy of these findings may be limited by certain aspects of the trial design, and further studies are needed to evaluate the possible effects of Xpert beyond diagnosis and the time to treatment initiation. Nevertheless, these findings suggest that replacing smear microscopy with Xpert has the potential to increase the confirmation (but not detection) of pulmonary tuberculosis and to reduce the time to treatment initiation at the population level in Brazil and other resource-limited countries.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001766.
The World Health Organization (WHO) provides information (in several languages) on tuberculosis, on tuberculosis diagnostics, and on the rollout of Xpert; further information about WHO's endorsement of Xpert is included in a Strategic and Technical Advisory Group for Tuberculosis report; the “Global Tuberculosis Report 2013” provides information about tuberculosis around the world, including Brazil
The Stop TB Partnership is working towards tuberculosis elimination and provides patient stories about tuberculosis (in English and Spanish); the Tuberculosis Vaccine Initiative (a not-for-profit organization) also provides personal stories about tuberculosis
The US Centers for Disease Control and Prevention provides information about tuberculosis and its diagnosis (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
More information about this trial is available
doi:10.1371/journal.pmed.1001766
PMCID: PMC4260794  PMID: 25490549
18.  Should Sputum Smear Examination Be Carried Out at the End of the Intensive Phase and End of Treatment in Sputum Smear Negative Pulmonary TB Patients? 
PLoS ONE  2012;7(11):e49238.
Background
The Indian guidelines on following up sputum smear-negative Pulmonary tuberculosis (PTB) patients differ from the current World Health Organization (WHO) guidelines in that the former recommends two follow up sputum examinations (once at the end of intensive phase and the other at the end of treatment) while the latter recommends only one follow up sputum smear microscopy examination, which is done at the end of the intensive phase. This study was conducted to examine if there was any added value in performing an additional sputum smear examination at the end of treatment within the context of a national TB program.
Methods
This study was a descriptive record based review conducted in nine tuberculosis (TB) units in Delhi, India. All consecutive new sputum smear-negative PTB patients registered in these nine TB units from 1st January 2009 to 31st December 2009 were included in the study.
Results
Of 2567 new sputum smear-negative TB patients, 1973 (90%) had sputum specimens examined at the end of the intensive phase, of whom 36 (2%) were smear-positive: the majority (n = 28) successfully completed treatment with either the same or a re-treatment regimen. At treatment completion, 1766 (85%) patients had sputum specimens examined, of whom 16 (0.9%) were smear-positive: all these were changed to a re-treatment regimen. Amongst the sputum-positive patients identified as a result of follow up (n = 52), four were diagnosed with multi-drug resistant TB (MDR-TB), three of whom were detected after smear examination at the end of treatment.
Conclusions
Given the high burden of TB in India, a 0.9% additional yield of smear-positive sputum smears at the end of treatment translates to 3,297 cases of smear-positive PTB. End-of-treatment smear is a low-yield strategy for detection of smear-positive TB cases, although further studies are needed to determine its population-level impact and cost, particularly in relation to other TB control interventions.
doi:10.1371/journal.pone.0049238
PMCID: PMC3494682  PMID: 23152880
19.  Acceptability of Sputum Specimens for Diagnosing Pulmonary Tuberculosis 
Journal of Korean Medical Science  2015;30(6):733-736.
The evaluation of the quality of a sputum specimen prior to bacterial culture has been an accepted practice. However, optimal sputum criteria for pulmonary tuberculosis (TB) are not well established. We investigated indicators for sputum acceptability in tuberculosis cultures and acid-fast bacilli (AFB) smear. A post-hoc analysis of a randomized trial with 228 sputum specimens from 77 patients was conducted. In the trial, pulmonary TB suspects were requested for collecting three sputum specimens. We performed both TB study (AFB smear and M. tuberculosis culture) and Gram staining in each specimen. By using generalized estimating equations, the association between sputum characteristics and positive TB testings were analyzed. Although acceptable specimens for bacterial pneumonia showed higher TB-culture positive rates than unacceptable specimens (adjusted odds ratio [aOR]=1.66; 95% confidence interval [CI]=1.11-2.49), a specimen with ≥25 white blood cells/low-power field was the better predictor for positive M. tuberculosis cultures (aOR=2.30; 95% CI=1.48-3.58) and acid-fast bacilli smears (aOR=1.85; 95% CI=1.05-3.25). Sputum leukocytosis could be an indicator of sputum acceptability for diagnosing pulmonary tuberculosis.
Graphical Abstract
doi:10.3346/jkms.2015.30.6.733
PMCID: PMC4444473  PMID: 26028925
Acceptable Sputum; Tuberculosis, Pulmonary; Sputum WBC
20.  Induced sputum and bronchoscopy in the diagnosis of pulmonary tuberculosis 
Thorax  2002;57(12):1010-1014.
Background: Previous studies suggest that bronchoscopy and a single induced sputum sample are equally effective for diagnosing pulmonary tuberculosis.
Methods: In a prospective study of subjects with possibly active pulmonary tuberculosis, the diagnostic yield of three induced sputum tests was compared with that of bronchoscopy. Subjects either produced no sputum or (acid fast) smear negative sputum. Bronchoscopy was only performed if at least two induced sputum samples were smear negative.
Results: Of 129 subjects who completed all tests, 27 (21%) had smear negative and culture positive specimens, 14 (52%) on bronchoscopy and 26 (96%) on induced sputum (p<0.005). One patient was culture positive on bronchoscopy alone compared with 13 on induced sputum alone; 13 were culture positive on both tests. Induced sputum positivity was strikingly more prevalent when chest radiographic appearances showed any features of active tuberculosis (20/63, 32%) than when appearances suggested inactivity (1/44, 2%; p<0.005). Induced sputum costs were about one third those of bronchoscopy, and the ratio of costs of the two tests per case of tuberculosis diagnosed could be as much as 1:6.
Conclusions: In subjects investigated for possibly active or inactive tuberculosis who produce no sputum or have smear negative sputum, the most cost effective strategy is to perform three induced sputum tests without bronchoscopy. Induced sputum testing carries a high risk of nosocomial tuberculosis unless performed in respiratory isolation conditions. The cost benefits shown could be lost if risk management measures are not observed.
doi:10.1136/thorax.57.12.1010
PMCID: PMC1758793  PMID: 12454293
21.  Diagnostic accuracy of same-day microscopy versus standard microscopy for pulmonary tuberculosis: a systematic review and meta-analysis 
The Lancet infectious diseases  2012;13(2):10.1016/S1473-3099(12)70232-3.
Summary
Background
Sputum smear microscopy is the most widely available diagnostic test for pulmonary tuberculosis in countries with a high burden of the disease. Improving its accuracy is crucial to achievement of case-detection targets established by the Millennium Development Goals. Unfortunately, many patients are unable to submit all of the specimens needed for examination or to return for treatment because standard sputum collection and reporting requires several clinic visits. To inform policy recommendations by a WHO-convened Expert Group, we aimed to assess the accuracy of sputum smear examination with strategies for obtaining sputum on 1 day compared with strategies for obtaining sputum over 2 days.
Methods
We did a systematic review and meta-analysis of research articles comparing the accuracy of front-loaded or same-day microscopy and standard sputum smear microscopy for diagnosis of culture-confirmed pulmonary tuberculosis. We searched Medline, Embase, Biosis, and Web of Science for articles published between Jan 1, 2005, and Feb 14, 2012. Two investigators identified eligible articles and extracted data for individual study sites. We generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when four or more studies were available.
Findings
We identified eight relevant studies from five articles enrolling 7771 patients with suspected tuberculosis in low-income countries. Compared with the standard approach of examination of two smears with Ziehl-Neelsen light microscopy over 2 days, examination of two smears taken on the same day had much the same sensitivity (64% [95% CI 60 to 69] for standard microscopy vs 63% [58 to 68] for same-day microscopy) and specificity (98% [97 to 99] vs 98% [97 to 99]). We noted similar results for studies employing light-emitting diode fluorescence microscopy and for studies examining three smears, whether they were compared with two-smear strategies or with one another.
Interpretation
Same-day sputum smear microscopy is as accurate as standard smear microscopy. Data from tuberculosis programmes are needed to document the changes required in the health system to successfully implement the strategy and understand its effects.
doi:10.1016/S1473-3099(12)70232-3
PMCID: PMC3836432  PMID: 23099183
22.  Direct microscopy versus sputum cytology analysis and bleach sedimentation for diagnosis of tuberculosis: a prospective diagnostic study 
BMC Infectious Diseases  2010;10:276.
Background
Diagnostic options for pulmonary tuberculosis in resource-poor settings are commonly limited to smear microscopy. We investigated whether bleach concentration by sedimentation and sputum cytology analysis (SCA) increased the positivity rate of smear microscopy for smear-positive tuberculosis.
Methods
We did a prospective diagnostic study in a Médecins Sans Frontières-supported hospital in Mindouli, Republic of Congo. Three sputum samples were obtained from 280 consecutive pulmonary tuberculosis suspects, and were processed according to WHO guidelines for direct smear microscopy. The remainder of each sputum sample was homogenised with 2.6% bleach, sedimented overnight, smeared, and examined blinded to the direct smear result for acid-fast bacilli (AFB). All direct smears were assessed for quality by SCA. If a patient produced fewer than three good-quality sputum samples, further samples were requested. Sediment smear examination was performed independently of SCA result on the corresponding direct smear. Positivity rates were compared using McNemar's test.
Results
Excluding SCA, 43.2% of all patients were diagnosed as positive on direct microscopy of up to three samples. 47.9% were diagnosed on sediment microscopy, with 48.2% being diagnosed on direct microscopy, sediment microscopy, or both. The positivity rate increased from 43.2% to 47.9% with a case definition of one positive smear (≥1 AFB/100 high power fields) of three, and from 42.1% to 43.9% with two positive smears. SCA resulted in 87.9% of patients producing at least two good-quality sputum samples, with 75.7% producing three or more. Using a case definition of one positive smear, the incremental yield of bleach sedimentation was 14/121, or 11.6% (95% CI 6.5-18.6, p = 0.001) and in combination with SCA was 15/121, or 12.4% (95% CI 7.1-19.6, p = 0.002). Incremental yields with two positive smears were 5/118, or 4.2% (95% CI 1.4-9.6, p = 0.062) and 7/118, or 5.9% (95% CI 2.4-11.8, p = 0.016), respectively.
Conclusions
The combination of bleach sedimentation and SCA resulted in significantly increased microscopy positivity rates with a case definition of either one or two positive smears. Implementation of bleach sedimentation led to a significant increase in the diagnosis of smear-positive patients. Implementation of SCA did not result in significantly increased diagnosis of tuberculosis, but did result in improved sample quality. Requesting extra sputum samples based on SCA results, combined with bleach sedimentation, could significantly increase the detection of smear-positive patients if routinely implemented in resource-limited settings where gold standard techniques are not available. We recommend that a pilot phase is undertaken before routine implementation to determine the impact in a particular context.
doi:10.1186/1471-2334-10-276
PMCID: PMC2946302  PMID: 20858253
23.  Population Health Impact and Cost-Effectiveness of Tuberculosis Diagnosis with Xpert MTB/RIF: A Dynamic Simulation and Economic Evaluation 
PLoS Medicine  2012;9(11):e1001347.
Nicolas Menzies and colleagues investigate the potential impact and cost-effectiveness of implementing Xpert MTB/RIF for diagnosing tuberculosis in five southern African countries.
Background
The Xpert MTB/RIF test enables rapid detection of tuberculosis (TB) and rifampicin resistance. The World Health Organization recommends Xpert for initial diagnosis in individuals suspected of having multidrug-resistant TB (MDR-TB) or HIV-associated TB, and many countries are moving quickly toward adopting Xpert. As roll-out proceeds, it is essential to understand the potential health impact and cost-effectiveness of diagnostic strategies based on Xpert.
Methods and Findings
We evaluated potential health and economic consequences of implementing Xpert in five southern African countries—Botswana, Lesotho, Namibia, South Africa, and Swaziland—where drug resistance and TB-HIV coinfection are prevalent. Using a calibrated, dynamic mathematical model, we compared the status quo diagnostic algorithm, emphasizing sputum smear, against an algorithm incorporating Xpert for initial diagnosis. Results were projected over 10- and 20-y time periods starting from 2012. Compared to status quo, implementation of Xpert would avert 132,000 (95% CI: 55,000–284,000) TB cases and 182,000 (97,000–302,000) TB deaths in southern Africa over the 10 y following introduction, and would reduce prevalence by 28% (14%–40%) by 2022, with more modest reductions in incidence. Health system costs are projected to increase substantially with Xpert, by US$460 million (294–699 million) over 10 y. Antiretroviral therapy for HIV represents a substantial fraction of these additional costs, because of improved survival in TB/HIV-infected populations through better TB case-finding and treatment. Costs for treating MDR-TB are also expected to rise significantly with Xpert scale-up. Relative to status quo, Xpert has an estimated cost-effectiveness of US$959 (633–1,485) per disability-adjusted life-year averted over 10 y. Across countries, cost-effectiveness ratios ranged from US$792 (482–1,785) in Swaziland to US$1,257 (767–2,276) in Botswana. Assessing outcomes over a 10-y period focuses on the near-term consequences of Xpert adoption, but the cost-effectiveness results are conservative, with cost-effectiveness ratios assessed over a 20-y time horizon approximately 20% lower than the 10-y values.
Conclusions
Introduction of Xpert could substantially change TB morbidity and mortality through improved case-finding and treatment, with more limited impact on long-term transmission dynamics. Despite extant uncertainty about TB natural history and intervention impact in southern Africa, adoption of Xpert evidently offers reasonable value for its cost, based on conventional benchmarks for cost-effectiveness. However, the additional financial burden would be substantial, including significant increases in costs for treating HIV and MDR-TB. Given the fundamental influence of HIV on TB dynamics and intervention costs, care should be taken when interpreting the results of this analysis outside of settings with high HIV prevalence.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
In 2010, about 9 million people developed tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—and about 1.5 million people died from the disease. Most of these deaths were in low- and middle-income countries, and a quarter were in HIV-positive individuals, who are particularly susceptible to TB. Mycobacterium tuberculosis, the bacterium that causes TB, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of TB are a persistent cough, weight loss, fever, and night sweats. Diagnostic tests for TB include sputum smear analysis (microscopic examination of mucus coughed up from the lungs for the presence of M. tuberculosis) and mycobacterial liquid culture (growth of M. tuberculosis from sputum and determination of its drug sensitivity). TB can be cured by taking several antibiotics daily for at least six months, although the recent emergence of multidrug-resistant TB (MDR-TB) is making the disease increasingly hard to treat.
Why Was This Study Done?
To reduce the global TB burden, active disease must be diagnosed quickly and accurately. In most high-burden settings, however, TB diagnosis relies on sputum smear analysis, which fails to identify some people (especially HIV-infected individuals) who have TB. Mycobacterial culture correctly identifies more infected people but is slow and costly, and many high-burden settings lack the infrastructure for high-volume culture diagnosis of TB. Faced with these diagnostic inadequacies, the World Health Organization (WHO) recently recommended the use of Xpert MTB/RIF for initial diagnosis in patients suspected of having MDR-TB or HIV-associated TB. This new, automated DNA test detects M. tuberculosis and DNA differences that make the bacteria resistant to the drug rifampicin (an indicator of MDR-TB) within two hours. Many countries are moving toward adopting Xpert for TB diagnosis, so it is essential to understand the population health impact and cost-effectiveness of diagnostic strategies based on this test. Here, the researchers use a calibrated, dynamic mathematical model of TB to investigate the consequences of Xpert MTB/RIF implementation in five southern African countries where both TB-HIV coinfection and MDR-TB are common.
What Did the Researchers Do and Find?
The researchers used their mathematical model, which simulates the movement of individuals through different stages of TB infection, to investigate the potential health and economic consequences of implementing Xpert for initial TB diagnosis in Botswana, Lesotho, Namibia, South Africa, and Swaziland. In the modeled scenarios, compared to an diagnostic approach based on sputum smear (the “status quo”), implementation of Xpert averted an estimated 132,000 TB cases and 182,000 TB deaths in southern Africa over the ten years following its introduction, reduced the proportion of the population with TB by 28%, and increased health service costs by US$460 million. Much of this cost increase reflected increased antiretroviral therapy costs for TB/HIV-infected individuals who survived TB infection because of better case-finding and treatment. Finally, relative to the status quo, over ten years, Xpert implementation in southern Africa cost US$959 for every DALY (disability-adjusted life-year) averted. Cost-effectiveness ratios in individual countries ranged from US$792 per DALY averted in Swaziland to US$1,257 per DALY averted in Botswana.
What Do These Findings Mean?
These findings suggest that Xpert implementation in southern Africa could substantially reduce TB illness and death through improved case-finding and treatment, but that the impact of Xpert on long-term transmission dynamics may be more limited. Although the additional financial burden associated with Xpert roll-out is likely to be substantial, these findings suggest that using Xpert for TB diagnosis offers reasonable value given its cost. WHO considers any intervention with a cost-effectiveness ratio less than the per-capita gross domestic product (GDP) highly cost-effective—in 2010, the per-capita GDP ranged from US$7,000 in South Africa and Botswana to US$982 in Lesotho.
These findings may not be generalizable to regions with different HIV infection rates, and their accuracy is likely to be affected by the quality of the data fed into the mathematical model and by the structure of the model. Thus, it is essential that the impact of Xpert-based TB diagnosis be carefully evaluated as the approach is rolled out, and that the information generated by these evaluations be used to improve the accuracy of model-based estimates of the long-term effects of this new strategy for TB diagnosis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001347.
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the roll-out of the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report; WHO also provides information about tuberculosis and HIV
The Stop TB Partnership is working towards tuberculosis elimination; patient stories about TB-HIV coinfection are available
The US Centers for Disease Control and Prevention has information about tuberculosis, and about TB diagnosis
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
The Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001347
PMCID: PMC3502465  PMID: 23185139
24.  Oral Antimicrobial Rinse to Reduce Mycobacterial Culture Contamination among Tuberculosis Suspects in Uganda: A Prospective Study 
PLoS ONE  2012;7(7):e38888.
Rationale
Contamination by bacterial or fungal organisms reduces the effectiveness of mycobacterial culture for diagnosis of pulmonary tuberculosis (TB). We evaluated the effect of an anti-microbial and an anti-fungal oral rinse prior to expectoration on culture-contamination rates.
Methods
We enrolled a consecutive random sample of adults with cough for ≥2 weeks and suspected TB admitted to Mulago Hospital (Kampala, Uganda) between October 2008 and June 2009. We randomly assigned patients to oral rinse (60 seconds with chlorhexidine followed by 60 seconds with nystatin) vs. no oral rinse prior to initial sputum collection. Uganda National Tuberculosis Reference Laboratory technicians blinded to the method of sputum collection (with or without oral rinse) processed all sputum specimens for smear microscopy (direct Ziehl-Neelsen) and mycobacterial culture (Lowenstein-Jensen media).
Results
Of 220 patients enrolled, 177 (80%) were HIV-seropositive (median CD4-count 37 cells/uL, IQR 13–171 cells/uL). Baseline characteristics were similar between patients in the oral-rinse (N = 110) and no oral-rinse (N = 110) groups. The proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group (4% vs. 15%, risk difference −11%, 95% CI −18 to −3%, p = 0.005). Oral rinse significantly reduced the proportion of contaminated cultures among HIV-infected patients (3% vs. 18%, risk difference −14%, 95% CI −23 to −6%, p = 0.002) but not HIV-uninfected (6% vs. 4%, risk difference 2%, 95% CI −12 to +15%, p = 0.81) patients. However, the proportion of smear-positive specimens (25% vs. 35%, p = 0.10) and culture-positive specimens (48% vs. 56%, p = 0.24) were lower in the oral-rinse compared to the no oral-rinse group, although the differences were not statistically significant.
Conclusions
Oral rinse prior to sputum expectoration is a promising strategy to reduce mycobacterial culture contamination in areas with high HIV prevalence, if strategies can be devised to reduce the adverse impact of oral rinse on smear- and culture-positivity.
doi:10.1371/journal.pone.0038888
PMCID: PMC3395623  PMID: 22808020
25.  Comparison of Xpert MTB/RIF Assay and the Conventional Sputum Microscopy in Detecting Mycobacterium tuberculosis in Northern Thailand 
Background. Despite low sensitivity in detection of Mycobacterium tuberculosis, sputum acid-fast smear remains the main diagnostic method. This study aimed to compare the diagnostic performance of Xpert MTB/RIF assay versus conventional sputum acid-fast smear. Materials and Methods. A cross-sectional study was conducted at Chiang Mai University Hospital, Thailand. Patients who were ≥15 years old and had clinically suspected pulmonary tuberculosis were included. Results. 109 specimens from 57 patients were included. Using MGIT sputum culture as a reference standard, the sensitivity (SEN) and specificity (SPEC) for Xpert were 95.3% (95% CI, 84.2%, 99.4%) and 86.4% (95% CI, 75.7%, 93.6%). The SEN and SPEC for sputum acid-fast smear were 60.5% (95% CI, 44.4%, 75.0%) and 98.5% (95% CI, 91.8%, 100%). Xpert had significantly higher sensitivity (p value < 0.001) and lower specificity (p value = 0.022) than sputum acid-fast smear. Among 43 culture-proven M. tuberculosis specimens, sensitivity of Xpert was 100% (95% CI, 86.7%, 100%) in acid-fast positive smears (n = 26) and 88.2% (95% CI, 63.5%, 98.5%) in acid-fast negative smears (n = 17). Conclusions. The good sensitivity and specificity of Xpert assay in detecting M. tuberculosis from sputum specimens may help in early diagnosis and treatment of pulmonary tuberculosis, particularly among patients who had acid-fast negative sputum smear.
doi:10.1155/2015/571782
PMCID: PMC4430669  PMID: 26064681

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