Smear-negative Sputum Samples Research Articles

Evaluation of Xpert MTB/RIF Assay, MTB Culture and Line Probe Assay for the Detection of MDR Tuberculosis in AFB Smear Negative Specimens.

The global burden of tuberculosis (TB), particularly with multidrug resistance (MDR), is escalating and has become a major health challenge. It is well known that acid-fast bacilli (AFB) smear-negative TB patients are the major source of spreading TB to healthy individuals when left untreated. Early diagnosis of TB and rapid detection of drug resistance are important for the proper management of drug-resistant TB (DR-TB). Therefore, a laboratory based cross-sectional study was conducted from July to December 2019 at the National Tuberculosis Centre, Thimi, Nepal, with the objective of evaluating the diagnostic performance of Xpert MTB/RIF assay, Mycobacterium tuberculosis (MTB) culture and line probe assay (LPA) for the detection of MDR-TB in AFB smear-negative sputum samples. We evaluated a total of 222 AFB smear-negative sputum specimens, of which 21.6% (n = 48) showed MTB positive with Xpert MTB/RIF assay and, while culturing on Lowenstein-Jensen (LJ) media, 21.2% (n = 47) were MTB culture positive. The sensitivity, specificity, PPV and NPV at 95% confidence interval of Xpert MTB/RIF assay on diagnosing M. tuberculosis from smear-negative specimens were 73% (57-84), 92% (87-96), 71% (59-81) and 93% (89-95), respectively. In addition, the sensitivity of Xpert MTB/RIF assay and LPA in detecting rifampicin resistance was 75% (42-94, 95% CI) and 91.67% (62-99, 95% CI), respectively. The current study also assessed a significant association between the occurrence of pulmonary tuberculosis with different age group, TB history and alcohol consumption. These findings indicate that Xpert MTB/RIF assay and LPA are appropriate methods for early detection and accurate diagnosis of TB and RIF mono-resistant cases.

Role of GeneXpert MTB/RIF assay in detection of Mycobacterium tuberculosis in smear-negative sputum samples

Role of GeneXpert MTB/RIF assay in detection of Mycobacterium tuberculosis in smear-negative sputum samples

Diagnostic performance of GeneXpert MTB/RIF in detecting MTB in smear-negative presumptive TB patients

BackgroundTuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Control of TB is lingering by the lack of diagnostic tests that are simple, rapid, yet accurate. Thus, smear-negative pulmonary TB often misses the diagnosis. The study evaluated the performance of GeneXpert MTB/RIF assay for the detection of Mycobacterium tuberculosis (MTB).MethodsThe study was carried out from June to December 2016 in Nepal Tuberculosis Center, Bhaktapur, Nepal. A total of 173 sputum samples were collected and processed by microscopy [Auramine-O staining and Ziehl–Neelsen (ZN) staining], followed by GeneXpert MTB/RIF assay and culture in Lowenstein-Jensen (LJ) medium.ResultsOf 173 sputum samples, 162 (93.6%) were smear-negative. Of 162 smear-negative sputum samples, 35 (21.6%) were confirmed to have MTB by culture, and 31 (19.1%) by GeneXpert MTB/RIF assay. Of 31 GeneXpert-positive samples, 25 (80.6%) were susceptible, 4 (12.9%) were resistant, and 2 (6.45%) were intermediate to rifampicin. The sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. The GeneXpert MTB/RIF has a substantial diagnostic agreement of 90.91% with culture (Cohen’s Kappa coefficient = 0.73).ConclusionThe diagnostic performance of GeneXpert MTB/RIF assay was almost on par with culture, and thus can be relied upon for MTB detection in smear-negative sputum samples.

Prevalence and Clinical Profiling of Dysglycemia and HIV Infection in Persons With Pulmonary Tuberculosis in Brazil.

BackgroundThere are scarce data on the prevalence and disease presentation of HIV in patients with tuberculosis (TB) and dysglycemia (diabetes [DM] and prediabetes [PDM]), especially in TB-endemic countries.MethodsWe assessed the baseline epidemiological and clinical characteristics of patients with culture-confirmed pulmonary TB, enrolled in a multicenter prospective cohort in Brazil (RePORT-Brazil) during 2015–2019. Dysglycemia was defined by elevated glycated hemoglobin and stratified as PDM or DM. Additionally, we used data from TB cases obtained through the Brazilian National Notifiable Diseases Information System (SINAN), during 2015–2019. In SINAN, diagnosis of diabetes was based on self-report. Logistic regression models were performed to test independent associations between HIV, dysglycemia status, and other baseline characteristics in both cohorts.ResultsIn the RePORT-Brazil cohort, the prevalence of DM and of PDM was 23.7 and 37.8%, respectively. Furthermore, the prevalence of HIV was 21.4% in the group of persons with TB-dysglycemia and 20.5% in that of patients with TBDM. In the SINAN cohort, the prevalence of DM was 9.2%, and among the TBDM group the prevalence of HIV was 4.1%. Logistic regressions demonstrated that aging was independently associated with PDM or DM in both the RePORT-Brazil and SINAN cohorts. In RePORT-Brazil, illicit drug use was associated with PDM, whereas a higher body mass index (BMI) was associated with DM occurrence. Of note, HIV was not associated with an increased risk of PDM or DM in patients with pulmonary TB in both cohorts. Moreover, in both cohorts, the TBDM-HIV group presented with a lower proportion of positive sputum smear and a higher frequency of tobacco and alcohol users.ConclusionThere is a high prevalence of dysglycemia in patients with pulmonary TB in Brazil, regardless of the HIV status. This reinforces the idea that DM should be systematically screened in persons with TB. Presence of HIV does not substantially impact clinical presentation in persons with TBDM, although it is associated with more frequent use of recreational drugs and smear negative sputum samples during TB screening.

Comparison of Polymerase Chain Reaction With Fluorescent Microscopy for Detecting Pulmonary Tuberculosis in Smear-Negative Sputum Samples in Quetta, Pakistan

Background: One of the common clinical manifestations of extra-pulmonary tuberculosis is tuberculous meningitis (TBM). New methods, which are simple and effective, are necessary for early diagnosis of TBM. Objective: This study aimed to assess the use of fluorescence microscopy and polymerase chain reaction (PCR) techniques targeting pncA and IS6110. PCR is a fast and reliable technique for diagnosing tuberculosis (TB), particularly in paucibacillary specimens such as smear-negative sputum for diagnosing patients with Mycobacterium tuberculosis. Materials and Methods: The pncA and IS6110 multiplex-PCR methods were applied to analyze sputum using a specific pair of primers designed to amplify the insertion sequence, pncA, and IS6110 in the M. tuberculosis genome. A total of 200 sputum samples were collected from patients with TB. Out of 200 patients, 54.0% (n=108) and 46.0% (n=92) were males and females at Fatima Jinnah General and Chest Hospital, Quetta, respectively. Results: M. tuberculosis was detected as 15 (16.3%) and 18 (16.7%) for pncA and IS6110 using PCR and fluorescence microscopy, respectively. M. tuberculosis DNA detected by PCR and smear-negative fluorescence microscopy was 16.50% (n=33) and 14.70% (n=18), respectively. In other words, PCR assay detected the increased prevalence of M. tuberculosis. Risk factors revealed high exposure to receiving TB infection which was associated with urban areas (OR=1.07, 95% CI: 0.34–3.35), TB in family (OR=1.22, 95% CI: 0.38-3.88), over crowdedness (OR=2.93, 95% CI: 0.37-23.7), malnutrition (OR: 1.39; 95% CI: 0.52-3.74), and rural areas (OR: 1.54, 95% CI: 0.72-3.31). Conclusion: A wide range of molecular assays are now being developed and reviewed for detecting TB. We conclude that the use of pncA and IS6110 PCR assays is beneficial in the quick diagnosis of TB meningitis.

Fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in Ethiopia.

BackgroundDespite its low sensitivity, microscopy remains the main method for the diagnosis of pulmonary tuberculosis in most laboratories in Ethiopia. Few studies have evaluated the performance of light-emitting diode fluorescent microscopy (LED-FM) in bleach-concentrated smear-negative sputum specimens.ObjectiveThis study aimed to evaluate the diagnostic performance of LED-FM for smear-negative pulmonary tuberculosis in Ethiopia.MethodsA total of 194 adult patients with a cough lasting for more than two weeks, and who had three direct smear-negative sputum tests for Mycobacterium tuberculosis by Ziehl-Neelsen light microscopy, were included. All direct Ziehl-Neelsen-stained smear-negative sputum samples were cultured and were also visualised by LED-FM. Smears for LED-FM were performed from bleach-concentrated sputum sediment. The diagnostic performance of the LED-FM was compared to the culture method (the reference standard).ResultsOf the 194 smear-negative sputum specimens analysed, 28 (14.4%) were culture-positive and 21 (10.8%) were LED-FM-positive for M. tuberculosis. However, only 11 of the 21 (52.4%) LED-FM-positive patients had a confirmed tuberculosis diagnosis by culture. Light-emitting diode fluorescence microscopy (FM) had a sensitivity of 39.3% (95% confidence interval: 21.2–57.4) and specificity of 93.9% (95% confidence interval: 90.4–97.6). Ten LED-FM-positive specimens were culture-negative, and all of these specimens had scanty grading (1–19 bacilli per 40 fields on LED-FM).ConclusionThis study showed that implementation of LED-FM on bleach pre-treated and concentrated sputum can significantly improve the diagnosis of smear-negative pulmonary tuberculosis. However, all scanty grade, positive smears by LED-FM need to be confirmed by reference culture method.

Diagnosis of tuberculosis from smear-negative presumptive TB cases using Xpert MTB/Rif assay: a cross-sectional study from Nepal

BackgroundIn most developing countries, smear-negative pulmonary TB (SNPT) often gets missed from the diagnosis of consideration, though it accounts 30–65% of total PTB cases, due to deficient or inaccessible molecular diagnostic modalities.MethodsThe cross-sectional study enrolled 360 patients with clinical-radiological suspicion of SNPT in Tribhuvan University Teaching Hospital (TUTH). The patient selection was done as per the algorithm of Nepal’s National Tuberculosis Program (NTP) for Xpert MTB/RIF testing. Participants’ demographic and clinical information were collected using a pre-tested questionnaire. The specimens were collected, processed directly for Xpert MTB/RIF test according to the manufacturer’s protocol. The same samples were stained using the Ziehl-Neelsen technique then observed microscopically. Both findings were interpreted; rifampicin-resistant, if obtained, on Xpert testing was confirmed with a Line Probe Assay.ResultOf 360 smear-negative sputum samples analyzed, 85(23.61%) found positive while 3(0.8%) of them were rifampicin resistance. The infection was higher in males, i.e. 60(25.3%) compared to female 25(20.3%). The age group, > 45(nearly 33%) with median age 42 ± 21.5, were prone to the infection. During the study period, 4.6% (515/11048) sputum samples were reported as smear-positive in TUTH. Consequently, with Xpert MTB/RIF assay, the additional case 16.5% (n = 85/515) from smear-negative presumptive TB cases were detected. Among the most occurring clinical presentations, cough and chest pain were positively associated with SNPT. While upper lobe infiltrates (36.4%) and pleural effusion (40.4%) were the most peculiar radiological impression noted in PTB patient. 94 multi-drug resistant(MDR) suspected cases were enrolled; of total suspects, 29(30.8%) samples were rifampicin sensitive, 1(1.06%) indeterminate, 3(3.19%) rifampicin-resistant while remaining of them were negative. 2(2.2%) MDR cases were recovered from the patient with a previous history of ATT, of total 89 previously treated cases enrolled However, a single rifampicin-resistant from the new suspects.ConclusionWith an application of the assay, the additional cases, missed with smear microscopy, could be sought and exact incidence of the diseases could be revealed.

Evaluation of Pot’s Technique and Sodium Hypochlorite Concentration Technique in Smear Negative Sputum Samples for Increased Sensitivity of Sputum Microscopy

Evaluation of Pot’s Technique and Sodium Hypochlorite Concentration Technique in Smear Negative Sputum Samples for Increased Sensitivity of Sputum Microscopy

Feasibility of a streamlined tuberculosis diagnosis and treatment initiation strategy.

SUMMARYOBJECTIVE:To assess the feasibility of a streamlined strategy for improving tuberculosis (TB) diagnostic evaluation and treatment initiation among patients with presumed TB.DESIGN:Single-arm interventional pilot study at five primary care health centers of a streamlined, SIngle-saMPLE (SIMPLE) TB diagnostic evaluation strategy: 1) examination of two smear results from a single spot sputum specimen using light-emitting diode fluorescence microscopy, and 2) daily transportation of smear-negative sputum samples to Xpert® MTB/RIF testing sites.RESULTS:Of 1212 adults who underwent sputum testing for TB, 99.6% had two smears examined from the spot sputum specimen. Sputum was transported for Xpert testing within 1 clinic day for 83% (907/1091) of the smear-negative patients. Of 157 (13%) patients with bacteriologically positive TB, 116 (74%) were identified using sputum smear microscopy and 41 (26%) using Xpert testing of smear-negative samples. Anti-tuberculosis treatment was initiated in 142 (90%) patients with bacteriologically positive TB, with a median time to treatment of 1 day for smear-positive patients and 6 days for smear-negative, Xpert-positive patients.CONCLUSION:The SIMPLE TB strategy led to successful incorporation of Xpert testing and rapid treatment initiation in the majority of patients with bacteriologically confirmed TB in a resource-limited setting.

Evaluation of genotype MTBDRplus VER 2.0 line probe assay for the detection of MDR-TB in smear positive and negative sputum samples

BackgroundMulti drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples.MethodA cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant.ResultsThe sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples.The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance.ConclusionThe diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.

Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis

BackgroundTuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death from infectious diseases worldwide. Sputum smear microscopy remains the most widely available pulmonary TB diagnostic tool particularly in resource limited settings. A highly sensitive diagnostic with minimal infrastructure, cost and training is required. Hence, we assessed the diagnostic performance of Loop-mediated isothermal amplification (LAMP) assay in detecting M.tuberculosis infection in sputum sample compared to LED fluorescent smear microscopy and culture.MethodA cross-sectional study was conducted at the University of Gondar Hospital from June 01, 2015 to August 30, 2015. Pulmonary TB diagnosis using sputum LED fluorescence smear microscopy, TB-LAMP assay and culture were done. A descriptive analysis was used to determine demographic characteristics of the study participants. Analysis of sensitivity and specificity for smear microscopy and TB-LAMP compared with culture as a reference test was performed. Cohen’s kappa was calculated as a measure of agreement between the tests.ResultsA total of 78 pulmonary presumptive TB patients sputum sample were analyzed. The overall sensitivity and specificity of LAMP were 75 and 98%, respectively. Among smear negative sputum samples, 33.3% sensitivity and 100% specificity of LAMP were observed. Smear microscopy showed 78.6% sensitivity and 98% specificity. LAMP and smear in series had sensitivity of 67.8% and specificity of 100%. LAMP and smear in parallel had sensitivity of 85.7% and specificity of 96%. The agreement between LAMP and fluorescent smear microscopy tests was very good (κ = 0.83, P-value ≤0.0001).ConclusionsTB-LAMP showed similar specificity but a slightly lower sensitivity with LED fluorescence microscopy. The specificity of LAMP and smear microscopy in series was high. The sensitivity of LAMP was insufficient for smear negative sputum samples.

Rapid detection of extensively drug-resistant (XDR-TB) strains from multidrug-resistant tuberculosis (MDR-TB) cases isolated from smear-negative pulmonary samples in an Intermediate Reference Laboratory in India

BackgroundDirect sputum smear microscopy is commonly used for diagnosing tuberculosis (TB). The objectives of the study were first, to determine the recovery of Mycobacterium tuberculosis in smear-negative sputum samples through liquid culture (using MGIT 960) and solid culture (using LJ slant) and second, to screen multidrug-resistant isolates through line probe assay and further third, to identify XDR isolates through MGIT second-line DST from these positive MDR cultures in Delhi region. MethodsIn this study, the sample size was 717 (sputum smear AFB negative and culture positive for M. tuberculosis complex by both solid and liquid culture methods) MDRTB suspects who were enrolled from January 2014 to December 2014 at the Intermediate Reference Laboratory in New Delhi Tuberculosis Centre, New Delhi. Rapid line probe assay was performed on all culture-positive samples, which were direct smear-negative specimens, and LPA-confirmed MDR samples were tested on MGIT 960 second-line DST for identification of XDR strains. ResultsAn overall increase in the culture positivity (9.4%) among these smear-negative cases shows a good sign of recovery from M. tuberculosis infection in these samples. 717 (9.4%) positive cultures (MGIT+LJ) were subjected to line probe assay. Out of these 717 cultures, 9 (1.2%) were confirmed as NTM, 50 (7%) were MDR, 4 (0.6%) were mono-rifampicin resistant and 654 (91.2%) cultures were sensitive to both drugs Rif and Inh, respectively. Out of these 54 (50 MDR +4 mono-RIF resistant) cultures as screened by LPA, 1 (1.8%) was XDR, 10 (18.6%) were mono-ofloxacin resistant and 1 (1.8%) was mono-Kanamycin resistant. Sensitivity to both drugs KAN and OFX was seen in 42 (77.8%) cultures. ConclusionsSince the bacterial load in direct smear-negative suspected MDR samples is less, it is important to recover mycobacteria by rapid liquid culture method in such samples. Initial screening for MDRTB is to be done in such cases by performing rapid molecular genotypic drug susceptibility test such as LPA. Baseline second-line DST is also done to rule out the XDR cases among them for rapid and better management of XDRTB patients.

Increased detection of smear-negative pulmonary tuberculosis by GeneXpert MTB/RIF® assay after bleach concentration

Objective/backgroundThe GeneXpert MTB/RIF assay (Xpert) was endorsed as the initial diagnostic tool in people suspected of human immunodeficiency virus-associated or drug-resistant tuberculosis (TB). However, information regarding the performance of Xpert for diagnosing smear-negative TB in high burden settings remains limited. We evaluated the diagnostic accuracy of Xpert and the impact of bleach concentration on the performance of Xpert using smear-negative sputum samples from human immunodeficiency virus-negative patients. MethodsOne spot and one morning smear-negative sputum samples per patient were examined using Xpert and culture at the Mycobacteriology Research Center of Jimma University, Ethiopia. The sputum culture on both Löwenstein–Jensen and/or Mycobacteria Growth Indicator Tube was the gold-standard. ResultsOf 185 smear-negative presumptive pulmonary TB cases, 19 (10.3%) had culture-proven TB. The sensitivity of Xpert on spot and morning sputum was similar (63.2%). Testing two specimens per patient insignificantly increased the sensitivity of Xpert. Bleach concentration and pelleting improved the sensitivity of Xpert over unprocessed sputum in paired samples (73.8% vs. 63.2%) without affecting the specificity (95%). Bleach concentration and pelleting allowed an additional seven cases of TB (missed on the first and second direct Xperts) to be detected, five of which were from culture-negative cases. ConclusionTesting of a single sputum sample by Xpert can reach reasonable sensitivity and results would be available on the same day, avoiding loss of patients and treatment delay. The sensitivity of Xpert was improved after bleach concentration and pelleting, although its added value needs further study on a larger scale.

Implementing rapid testing for tuberculosis in Mozambique.

ProblemIn Mozambique, pulmonary tuberculosis is primarily diagnosed with sputum smear microscopy. However this method has low sensitivity, especially in people infected with human immunodeficiency virus (HIV). Patients are seldom tested for drug-resistant tuberculosis.ApproachThe national tuberculosis programme and Health Alliance International introduced rapid testing of smear-negative sputum samples. Samples were tested using a polymerase-chain-reaction-based assay that detects Mycobacterium tuberculosis deoxyribonucleic acid and a mutation indicating rifampicin resistance; Xpert® MTB/RIF (Xpert®). Four machines were deployed in four public hospitals along with a sputum transportation system to transfer samples from selected health centres. Laboratory technicians were trained to operate the machines and clinicians taught to interpret the results.Local settingIn 2012, Mozambique had an estimated 140 000 new tuberculosis cases, only 34% of which were diagnosed and treated. Of tuberculosis patients, 58% are HIV-infected.Relevant changesFrom 2012–2013, 1558 people were newly diagnosed with tuberculosis using sputum smears at intervention sites. Xpert® detected M. tuberculosis in an additional 1081 sputum smear-negative individuals, an increase of 69%. Rifampicin resistance was detected in 58/1081 (5%) of the samples. However, treatment was started in only 82% of patients diagnosed by microscopy and 67% of patients diagnosed with the rapid test. Twelve of 16 Xpert® modules failed calibration within 15 months of implementation.Lessons learntUsing rapid tests to diagnose tuberculosis is promising but logistically challenging. More affordable and durable platforms are needed. All patients diagnosed with tuberculosis need to start and complete treatment, including those who have drug resistant strains.

A first insight into high prevalence of undiagnosed smear-negative pulmonary tuberculosis in Northern Ethiopian prisons: implications for greater investment and quality control.

BackgroundTuberculosis (TB) transmission in prisons poses significant risks to inmates as well as the general population. Currently, there are no data on smear-negative pulmonary TB cases in prisons and by extension no data on the impact such cases have on TB incidence. This study was designed to obtain initial data on the prevalence of smear-negative cases of TB in prisons as well as preliminary risk factor analysis for such TB cases.MethodsThis cross-sectional survey was conducted in November 2013 at eight main prisons located in the state of Amhara, Ethiopia. Interviews using a structured and pretested questionnaire were done first to identify symptomatic prisoners. Three consecutive sputum samples were collected and examined using acid fast bacilli (AFB) microscopy at the point of care. All smear-negative sputum samples were taken for culture and Xpert testing. Descriptive and multivariate analysis was done using SPSS version 16.ResultsOverall the prevalence of smear-negative pulmonary TB cases in the study prisons was 8% (16/200). Using multivariate analysis, a contact history to TB patients in prison, educational level, cough and night sweating were found to be predictors of TB positivity among smear-negative pulmonary TB cases (p≤ 0.05).ConclusionsIn the studied prisons, high prevalence of undiagnosed TB cases using AFB microscopy was documented, which is an important public health concern that urgently needs to be addressed. Furthermore, patients with night sweating, non-productive cough, a contact history with TB patients and who are illiterate merit special attention, larger studies are warranted in the future to assess the associations more precisely. Further studies are also needed to examine TB transmission dynamics by patients with smear-negative pulmonary TB in a prison setting.

Editorial Commentary: Molecular Laboratory Testing for Tuberculosis: Innovators, Early Adopters, or Laggards?

Despite the recent progress toward meeting the Millennium Development Goal target to halt and to reverse the tuberculosis epidemic by 2015, the global burden of tuberculosis remains significant. In 2012, there were an estimated 8.6 million new cases of tuberculosis and 1.3 million died from the disease—5 deaths every 2 minutes! Among these deaths, an estimated 170 000 were caused by multidrug-resistant (MDR) tuberculosis, a relatively high total compared with 450 000 incident MDR tuberculosis cases. Worldwide and in most countries with a high burden of MDR tuberculosis, less than one-third of the tuberculosis patients estimated to have MDR tuberculosis were actually detected in 2012 [1]. In 2012, a total of 9945 new tuberculosis cases were reported in the United States, representing a 5.4% decrease from 2011 and 62.7% decrease from 1992 when the tuberculosis resurgence peaked at 26 673 cases. While this is indeed an accomplishment, the fact that 17 states reported increased case counts from 2011 to 2012 is concerning. Primary MDR tuberculosis is reported in 1.1% of tuberculosis cases and fluctuated between 0.9% and 1.3%— stable but no progress. Thirty-one states reported ≥50% of tuberculosis cases in foreign-born persons and 11 states even reported≥70%. The top 5 countries of origin of foreign-born persons with tuberculosis during 2007–2012 were Mexico, the Philippines, India, Viet Nam, and China [2]. According to the World Health Organization (WHO), more than half of the global MDR tuberculosis cases are from India, China, and the Russian Federation [1]. Today, laboratory testing in the mycobacteriology field is experiencing more changes than ever before. Determining what assay will be most useful to the healthcare provider is a challenge, and rapid acceptance of the new technology by the medical community an even greater one. In August 2013, the US Food and Drug Administration permitted marketing of the Xpert MTB/RIF (Xpert) assay (Cepheid, Sunnyvale, California) to detect DNA of the Mycobacterium tuberculosis complex and genetic mutations associated with resistance to rifampin (RIF) in unprocessed sputum and concentrated sputum sediments. In this issue of Clinical Infectious Diseases, Sohn and colleagues [3] from the Montreal Chest Institute, Canada, evaluated the Xpert assay in a low-incidence, high-resource ambulatory setting, specifically, in a university hospital tuberculosis clinic for the detection of pulmonary tuberculosis on induced sputum samples, using mycobacterial cultures as the reference standard. During the duration of the study, 924 patients were evaluated for tuberculosis and 502 consecutive patients were enrolled in the study (most with abnormal chest radiographs; only 18% symptomatic). Reasons for not enrolling included language barrier and refusal of the patient. Four induced sputum samples were collected. The second samplewas subjected to the Xpert assay and the other 3 samples were sent to the clinical microbiology laboratory for acid-fast bacilli (AFB) smear microscopy and culture using one liquid medium (Mycobacterium Growth Indicator Tube, BD). If the AFB smear was positive, a reflex nucleic acid amplification test (NAAT) was performed. A NAAT was performed on AFB smear-negative sputum samples by request only. Xpert results were not made available for clinical decision making. Twenty-four subjects were identified with active tuberculosis by culture. Xpert had an overall sensitivity of 46% and a specificity of 100%. The sensitivity was 86% in the 7 AFB smear-positive subjects and 29% in the 18 AFB smearnegative, culture-positive subjects. Most participants with culture-positive tuberculosis had minimal disease: only 7 of 25 (28%) culture-positive subjects were AFB smear-positive, only 12 (44%) had symptoms at presentation, and 2 subjects Received 29 December 2013; accepted 07 January 2014. Correspondence: Max Salfinger, MD, Mycobacteriology Laboratory, K 420, National Jewish Health, 1400 Jackson St, Denver, CO 80206 (salfingerm@njhealth.org). Clinical Infectious Diseases © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. permissions@oup.com. DOI: 10.1093/cid/ciu024

Evaluation of in-house PCR for diagnosis of smear-negative pulmonary tuberculosis in Kampala, Uganda

BackgroundNucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture.ResultsSeventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively. One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up. Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094).ConclusionIn-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.

Implementation of the Thin Layer Agar Method for Diagnosis of Smear-Negative Pulmonary Tuberculosis in a Setting with a High Prevalence of Human Immunodeficiency Virus Infection in Homa Bay, Kenya

The objective of this study was to evaluate the performance of a low-cost method, the thin layer agar (TLA) method, for the diagnosis of smear-negative patients. This prospective study was performed in Homa Bay District Hospital in Kenya. Out of 1,584 smear-negative sputum samples, 212 (13.5%) were positive by culture in Löwenstein-Jensen medium (LJ) and 220 (14%) were positive by the TLA method. The sensitivities of LJ and TLA were 71% and 74%, respectively. TLA could become an affordable method for the diagnosis of smear-negative tuberculosis in resource-limited settings, with results available within 2 weeks.