Evaluation of combined LED-fluorescence microscopy and bleach sedimentation for diagnosis of tuberculosis at peripheral health service level

Background

Sputum microscopy is the only diagnostic for tuberculosis (TB) available at peripheral levels of health service in resource-poor countries. Its sensitivity is reduced in high HIV-prevalence settings. Sodium hypochlorite (NaOCl) specimen sedimentation prior microscopy and light-emitting diode (LED)-fluorescence microscopy (FM) can individually improve performance of microscopy. This study aimed to evaluate the performance of combined LED-FM and NaOCl sputum sedimentation for TB detection at peripheral level of health services.

Methods

A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from consecutive TB suspects. Smears were prepared and stained with auramine O and Ziehl-Neelsen (ZN) methods. Bleach (3.5%) was added to the remaining specimen before overnight sedimentation at room temperature. Auramine O staining was performed on smears of sediment. A 4^th specimen was collected for TB culture. Auramine smears were read under the same microscope as used for ZN smears, but equipped with the LED FluoLED™ fluorescence illuminator.

Results

497 patients were included, and 1394 specimens collected. The yield of positive specimen was significantly increased after NaOCl sedimentation (24.9%) compared to direct LED-FM (20.6%) and direct ZN (20.3%). In detecting smear-positive patients, sensitivity was 78.5% for LED-FM after NaOCl sedimentation compared to 73.2% and 72.0% for direct LED-FM (P = 0.06) and direct ZN (P = 0.06), respectively. Specificity was 87.8% for LED-FM after NaOCl sedimentation compared to 96.7% and 95.9% for direct LED-FM (P<0.01) and direct ZN (P<0.01), respectively. Inter-reading agreement (kappa = 0.7) and technicians'' acceptability were good.

Conclusion

NaOCl sedimentation did not improve the performance of LED-FM in the diagnosis of pulmonary TB at peripheral health service level.     

LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

New Policies,New Technologies: Modelling the Potential for Improved Smear Microscopy Services in Malawi

Background

To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.

Methodology/Principal Findings

An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.

Conclusions/Significance

Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment.     

Implementation of LED Fluorescence Microscopy for Diagnosis of Pulmonary and HIV-Associated Tuberculosis in a Hospital Setting in Indonesia

Background

Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients.

Method

We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard.

Results

Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of ≥2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23±0.78 vs 5.82±1.60 minutes, P<0.01), while costs per slide were similar.

Conclusion

High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories.     

LED Fluorescence Microscopy for the Diagnosis of Pulmonary Tuberculosis: A Multi-Country Cross-Sectional Evaluation

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     

The Risk and Timing of Tuberculosis Diagnosed in Smear-Negative TB Suspects: A 12 Month Cohort Study in Harare,Zimbabwe

Background

Cases of smear-negative TB have increased dramatically in high prevalence HIV settings and pose considerable diagnostic and management challenges.

Methods and Findings

Between February 2006 and July 2007, a cohort study nested within a cluster-randomised trial of community-based case finding strategies for TB in Harare, Zimbabwe was undertaken. Participants who had negative sputum smears and remained symptomatic of TB were follow-up for one year with standardised investigations including HIV testing, repeat sputum smears, TB culture and chest radiography. Defaulters were actively traced to the community. The objectives were to investigate the incidence and risk factors for TB. TB was diagnosed in 218 (18.2%) participants, of which 39.4% was bacteriologically confirmed. Most cases (84.2%) were diagnosed within 3 months, but TB incidence remained high thereafter (111.3 per 1000 person-years, 95% CI: 86.6 to 146.3). HIV prevalence was 63.3%, and HIV-infected individuals had a 3.5-fold higher risk of tuberculosis than HIV-negative individuals.

Conclusion

We found that diagnosis of TB was insensitive and slow, even with early radiography and culture. Until more sensitive and rapid diagnostic tests become widely available, a much more proactive and integrated approach towards prompt initiation of ART, ideally from within TB clinics and without waiting for TB to be excluded, is needed to minimise the risk and consequences of diagnostic delay.     

Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi

Background

Sputum microscopy is the only tuberculosis (TB) diagnostic available at peripheral levels of care in resource limited countries. Its sensitivity is low, particularly in high HIV prevalence settings. Fluorescence microscopy (FM) can improve performance of microscopy and with the new light emitting diode (LED) technologies could be appropriate for peripheral settings. The study aimed to compare the performance of LED-FM versus Ziehl-Neelsen (ZN) microscopy and to assess feasibility of LED-FM at a low level of care in a high HIV prevalence country.

Methods

A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from suspected TB patients. Each sample was processed with Auramine O and ZN methods and a 4^th specimen was collected for TB culture reference standard. Auramine smears were read using the same microscope, equipped with the FluoLED™ fluorescence illuminator. Inter-reader agreement, reading time and technicians'' acceptability assessed feasibility.

Results

497 patients were included and 1394 specimens were collected. The detection yields of LED-FM and ZN microscopy were 20.3% and 20.6% (p = 0.64), respectively. Sensitivity was 73.2% for LED-FM and 72% for ZN microscopy, p = 0.32. It was 96.7% and 95.9% for specificity, p = 0.53. Inter-reader agreement was high (kappa = 0.9). Mean reading time was three times faster than ZN microscopy with very good acceptance by technicians.

Conclusions

Although it did not increase sensitivity, the faster reading time combined with very good acceptance and ease of use supports the introduction of LED-FM at the peripheral laboratory level of high TB and HIV burden countries.     

Can Follow-Up Examination of Tuberculosis Patients Be Simplified? A Study in Chhattisgarh,India

Background

Each follow-up during the course of tuberculosis treatment currently requires two sputum examinations. However, the incremental yield of the second sputum sample during follow-up of different types of tuberculosis patients has never been determined precisely.

Objectives

To assess the incremental yield of the second sputum sample in the follow-up of tuberculosis patients under the Revised National Tuberculosis Control Programme (RNTCP) in Chhattisgarh, India.

Methodology

A record review of tuberculosis (TB) patients registered in 2009 using a structured proforma from two sources, Tuberculosis and Laboratory Register, was undertaken in the six districts of Chhattisgarh, India.

Results

In smear positive cases, of 10,048 follow-up examinations, 45 (0.5%) were found to be smear positive only on the second sputum when the result of the first sample was negative. In smear negative pulmonary and extra pulmonary TB patients, of 6,206 follow-up smear examinations, 11(0.2%) were found to be smear positive.

Conclusions

The incremental yield of a second smear examination was very low, indicating that examination of one sputum sample is enough during follow-up among TB patients. There is insufficient yield to support sputum smear microscopy for monitoring smear negative pulmonary TB and extra pulmonary TB patients. These results indicate that the follow-up smear microscopy can be substantially simplified with favourable resource implications.     

Performance of three LED-based fluorescence microscopy systems for detection of tuberculosis in Uganda

Background

Direct smear microscopy using Ziehl-Neelsen (ZN) staining is the mainstay of tuberculosis (TB) diagnosis in most high burden countries, but is limited by low sensitivity in routine practice, particularly in high human immunodeficiency virus (HIV) prevalence settings.

Methods

We compared the performance of three commercial light emitting diode (LED)-based microscopy systems (Primostar™ iLED, Lumin™ and AFTER®) for fluorescent detection of Mycobacterium tuberculosis with ZN microscopy on slides prepared from sputum of TB suspects. Examination time for LED-based fluorescent microscopy (LED FM) and ZN slides was also compared, and a qualitative user appraisal of the LED FM systems was carried out.

Results

LED FM was between 5.6 and 9.4% more sensitive than ZN microscopy, although the difference was not statistically significant. There was no significant difference in the sensitivity or specificity of the three LED FM systems, although the specificity of Fraen AFTER was somewhat lower than the other LED FM methods. Examination time for LED FM was 2 and 4 times less than for ZN microscopy. LED FM was highly acceptable to Ugandan technologists, although differences in operational performance of the three systems were reported.

Conclusions

LED FM compares favourably with ZN microscopy, with equivalent specificity and a modest increase in sensitivity. Screening of slides was substantially quicker using LED FM than ZN, and LED FM was rated highly by laboratory technologists. Available commercial systems have different operational characteristics which should be considered prior to programmatic implementation.     

Comparative Yield of Different Diagnostic Tests for Tuberculosis among People Living with HIV in Western Kenya

Background

Diagnosis followed by effective treatment of tuberculosis (TB) reduces transmission and saves lives in persons living with HIV (PLHIV). Sputum smear microscopy is widely used for diagnosis, despite limited sensitivity in PLHIV. Evidence is needed to determine the optimal diagnostic approach for these patients.

Methods

From May 2011 through June 2012, we recruited PLHIV from 15 HIV treatment centers in western Kenya. We collected up to three sputum specimens for Ziehl-Neelsen (ZN) and fluorescence microscopy (FM), GeneXpert MTB/RIF (Xpert), and culture, regardless of symptoms. We calculated the incremental yield of each test, stratifying results by CD4 cell count and specimen type; data were analyzed to account for complex sampling.

Results

From 778 enrolled patients, we identified 88 (11.3%) laboratory-confirmed TB cases. Of the 74 cases who submitted 2 specimens for microscopy and Xpert testing, ZN microscopy identified 25 (33.6%); Xpert identified those plus an additional 18 (incremental yield = 24.4%). Xpert testing of spot specimens identified 48 (57.0%) of 84 cases; whereas Xpert testing of morning specimens identified 50 (66.0%) of 76 cases. Two Xpert tests detected 22/24 (92.0%) TB cases with CD4 counts <100 cells/μL and 30/45 (67.0%) of cases with CD4 counts ≥100 cells/μl.

Conclusions

In PLHIV, Xpert substantially increased diagnostic yield compared to smear microscopy and had the highest yield when used to test morning specimens and specimens from PLHIV with CD4 count <100 cells/μL. TB programs unable to replace smear microscopy with Xpert for all symptomatic PLHIV should consider targeted replacement and using morning specimens.     

Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study

Background

Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.

Methods

We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.

Results

Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28–51) and that of secA1 was 24% (95% CI 15–35). Both tests had specificities of 95% (95% CI 90–98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.

Conclusions

The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.     

Assessing the Quality of Tuberculosis Evaluation for Children with Prolonged Cough Presenting to Routine Community Health Care Settings in Rural Uganda

Background

Improving childhood tuberculosis (TB) evaluation and care is a global priority, but data on performance at community health centers in TB endemic regions are sparse.

Objective

To describe the current practices and quality of TB evaluation for children with cough ≥2 weeks'' duration presenting to community health centers in Uganda.

Methods

Cross-sectional analysis of children (<15 years) receiving care at five Level IV community health centers in rural Uganda for any reason between 2009–2012. Quality of TB care was assessed using indicators derived from the International Standards of Tuberculosis Care (ISTC).

Results

From 2009–2012, 1713 of 187,601 (0.9%, 95% CI: 0.4–1.4%) children presenting to community health centers had cough ≥ 2 weeks'' duration. Of those children, only 299 (17.5%, 95% CI: 15.7–19.3%) were referred for sputum microscopy, but 251 (84%, 95% CI: 79.8–88.1%) completed sputum examination if referred. The yield of sputum microscopy was only 3.6% (95% CI: 1.3–5.9%), and only 55.6% (95% CI: 21.2–86.3%) of children with acid-fast bacilli positive sputum were started on treatment. Children under age 5 were less likely to be referred for sputum examination and to receive care in accordance with ISTC. The proportion of children evaluated in accordance with ISTC increased over time (4.6% in 2009 to 27.9% in 2012, p = 0.03), though this did not result in increased case-detection.

Conclusion

The quality of TB evaluation was poor for children with cough ≥2 weeks'' duration presenting for health care. Referrals for sputum smear microscopy and linkage to TB treatment were key gaps in the TB evaluation process, especially for children under the age of five.     

Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms

Background

We conducted a tuberculosis (TB) prevalence survey and evaluated the screening methods used in our survey, to assess if screening in TB prevalence surveys could be simplified, and to assess the accuracy of screening algorithms that may be applicable for active case finding.

Methods

All participants with a positive screen on either a symptom questionnaire, chest radiography (CXR) and/or sputum smear microscopy submitted sputum for culture. HIV status was obtained from prevalent cases. We estimated the accuracy of modified screening strategies with bacteriologically confirmed TB as the gold standard, and compared these with other survey reports. We also assessed whether sequential rather than parallel application of symptom, CXR and HIV screening would substantially reduce the number of participants requiring CXR and/or sputum culture.

Results

Presence of any abnormality on CXR had 94% (95%CI 88–98) sensitivity (92% in HIV-infected and 100% in HIV-uninfected) and 73% (95%CI 68–77) specificity. Symptom screening combinations had significantly lower sensitivity than CXR except for ‘any TB symptom’ which had 90% (95%CI 84–95) sensitivity (96% in HIV-infected and 82% in HIV-uninfected) and 32% (95%CI 30–34) specificity. Smear microscopy did not yield additional suspects, thus the combined symptom/CXR screen applied in the survey had 100% (95%CI 97–100) sensitivity. Specificity was 65% (95%CI 61–68). Sequential application of first a symptom screen for ‘any symptom’, followed by CXR-evaluation and different suspect criteria depending on HIV status would result in the largest reduction of the need for CXR and sputum culture, approximately 36%, but would underestimate prevalence by 11%.

Conclusion

CXR screening alone had higher accuracy compared to symptom screening alone. Combined CXR and symptom screening had the highest sensitivity and remains important for suspect identification in TB prevalence surveys in settings where bacteriological sputum examination of all participants is not feasible.     

Rapid Molecular Testing for TB to Guide Respiratory Isolation in the U.S.: A Cost-Benefit Analysis

Background

Respiratory isolation of inpatients during evaluation for TB is a slow and costly process in low-burden settings. Xpert MTB/RIF (Xpert) is a novel molecular test for tuberculosis (TB) that is faster and more sensitive but substantially more expensive than smear microscopy. No previous studies have examined the costs of molecular testing as a replacement for smear microscopy in this setting.

Methods

We conducted an incremental cost–benefit analysis comparing the use of a single negative Xpert versus two negative sputum smears to release consecutive adult inpatients with presumed TB from respiratory isolation at an urban public hospital in the United States. We estimated all health-system costs and patient outcomes related to Xpert implementation, diagnostic evaluation, isolation, hospitalization, and treatment. We performed sensitivity and probabilistic uncertainty analyses to determine at what threshold the Xpert strategy would become cost-saving.

Results

Among a hypothetical cohort of 234 individuals undergoing evaluation for presumed active TB annually, 6.4% had culture-positive TB. Compared to smear microscopy, Xpert reduced isolation bed utilization from an average of 2.7 to 1.4 days per patient, leading to a 48% reduction in total annual isolation bed usage from 632 to 328 bed-days. Xpert saved an average of $2,278 (95% uncertainty range $1582–4570) per admission, or $533,520 per year, compared with smear microscopy.

Conclusions

Molecular testing for TB could provide substantial savings to hospitals in high-income countries by reducing respiratory isolation usage and overall length of stay.     

Yield of Two Consecutive Sputum Specimens for the Effective Diagnosis of Pulmonary Tuberculosis

Background

From long instances, it is debatable whether three sputum specimens are required for the diagnosis of pulmonary tuberculosis (TB) or TB can be diagnosed effectively using two consecutive sputum specimens. This study was set out to evaluate the significance of examining multiple sputum specimens in diagnosis of TB.

Methods

We retrospectively reviewed the acid-fast bacillus (AFB) smear and culture results of three consecutive days’ sputum specimens from 413 confirmed TB patients which were detected as part of a larger active case finding study in Dhaka Central Jail, the largest correctional facility in Bangladesh.

Results

AFB was detected from 81% (n = 334) patients, of which 89% (n = 297) were diagnosed from the first and additional 9% (n = 30) were from the second sputum specimen. M. tuberculosis growth was observed for 406 patients and 85% (n = 343) were obtained from the first sputum and additional 10% (n = 42) were from the second one. The third specimen didn’t show significant additional diagnostic value for the detection of AFB by microscopy or growth of the M. tuberculosis.

Conclusions

We concluded from our study results that examining two consecutive sputum specimens is sufficient enough for the effective diagnosis of TB. It can also decrease the laboratory workload and hence improve the quality of work in settings with high TB burden like Bangladesh.     

Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis

Background

Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples.

Methodology/Principal Findings

In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as Löwenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively.

Conclusions/Significance

The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient.     

Diagnosis of Tuberculosis in the Wild Boar (Sus scrofa): A Comparison of Methods Applicable to Hunter-Harvested Animals

Background

To obtain robust epidemiological information regarding tuberculosis (TB) in wildlife species, appropriate diagnostic methods need to be used. Wild boar (Sus scrofa) recently emerged as a major maintenance host for TB in some European countries. Nevertheless, no data is available to evaluate TB post-mortem diagnostic methods in hunter-harvested wild boar.

Methodology/Principal Findings

Six different diagnostic methods for TB were evaluated in parallel in 167 hunter-harvested wild boar. Compared to bacteriological culture, estimates of sensitivity of histopathology was 77.8%, gross pathology 72.2%, PCR for the MPB70 gene 66.7%, detection of acid-fast bacilli (AFB) in tissue contact smears 55.6% and in histopathology slides 16.7% (estimated specificity was 96.7%, 100%, 100%, 94.4% and 100%, respectively). Combining gross pathology with stained smears in parallel increased estimated sensitivity to 94.4% (94.4% specificity). Four probable bacteriological culture false-negative animals were identified by Discriminant Function Analysis. Recalculating the parameters considering these animals as infected generated estimated values for sensitivity of bacteriology and histopathology of 81.8%, gross pathology 72.7%, PCR for the MPB70 gene 63.6%, detection of AFB in tissue contact smears 54.5% and in histopathology slides 13.6% (estimated specificity was 100% for gross pathology, PCR, bacteriology and detection of AFB in histopathology slides, 96.7% for histopathology and 94.4% for stained smears).

Conclusions/Significance

These results show that surveys for TB in wild boar based exclusively on gross pathology considerably underestimate prevalence, while combination of tests in parallel much improves sensitivity and negative predictive values. This finding should thus be considered when planning future surveys and game meat inspection schemes. Although bacteriological culture is the reference test for TB diagnosis, it can generate false-negative results and this should be considered when interpreting data.     

Tuberculosis contact screening and isoniazid preventive therapy in a South Indian district: operational issues for programmatic consideration

Background

Under India''s Revised National Tuberculosis Control Programme (RNTCP), all household contacts of sputum smear positive Pulmonary Tuberculosis (PTB) patients are screened for TB. In the absence of active TB disease, household contacts aged <6 years are eligible for Isoniazid Preventive Therapy (IPT) (5 milligrams/kilogram body weight/day) for 6 months.

Objectives

To estimate the number of household contacts aged <6 years, of sputum smear positive PTB patients registered for treatment under RNTCP from April to June''2008 in Krishna District, to assess the extent to which they are screened for TB disease and in its absence initiated on IPT.

Methods

A cross sectional study was conducted. Households of all smear positive PTB cases (n = 848) registered for treatment from April to June''2008 were included. Data on the number of household contacts aged <6 years, the extent to which they were screened for TB disease, and the status of initiation of IPT, was collected.

Results

Households of 825 (97%) patients were visited, and 172 household contacts aged <6 years were identified. Of them, 116 (67%) were evaluated for TB disease; none were found to be TB diseased and 97 (84%) contacts were initiated on IPT and 19 (16%) contacts were not initiated on IPT due to shortage of INH tablets in peripheral health centers. The reasons for non-evaluation of the remaining eligible children (n = 56, 33%) include no home visit by the health staff in 25 contacts, home visit done but not evaluated in 31 contacts. House-hold contacts in rural areas were less likely to be evaluated and initiated on IPT [risk ratio 6.65 (95% CI; 3.06–14.42)].

Conclusion

Contact screening and IPT implementation under routine programmatic conditions is sub-optimal. There is an urgent need to sensitize all concerned programme staff on its importance and establishment of mechanisms for rigorous monitoring.     

Sensitivity of the Quantiferon-Gold In-Tube Assay in Sputum Smear Positive TB Cases in Indonesia

Background

As part of a formal evaluation of the Quantiferon-Gold in-tube assay (QFT-IT) for latent TB infection we compared its sensitivity to the tuberculin skin test (TST) in confirmed adult TB cases in Indonesia. Smear-positive TB disease was used as a proxy gold standard for latent TB infection.

Methods and Findings

We compared the sensitivity of QFT-IT and TST in 98 sputum smear and chest x-ray positive TB cases and investigated risk factors for negative and discordant results in both tests. Both tests showed high sensitivity; (QFT-IT; 88.7%: TST; 94.9%), not significantly different from each other (p value 0.11). Very high sensitivity was seen when tests were combined (98.9%). There were no variables significantly associated with discordant results or with a negative TST. For QFT-IT which particular staff member collected blood was significantly associated with test positivity (p value 0.01). Study limitations include small sample size and lack of culture confirmation or HIV test results.

Conclusions

The QFT-IT has similar sensitivity in Indonesian TB cases as in other locations. However, QFT-IT, like the TST cannot distinguish active TB disease from LTBI. In countries such as Indonesia, with high background rates of LTBI, test specificity for TB disease will likely be low. While our study was not designed to evaluate the QFT-IT in the diagnosis of active TB disease in TB suspects, the data suggest that a combination of TST and QFT-IT may prove useful for ruling out TB disease. Further research is required to explore the clinical role of QFT-IT in combination with other TB diagnostic tests.