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     

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.     

Performance of Frontloading for Smear Microscopy in the Diagnosis of Pulmonary Tuberculosis: A Cross-Sectional Study at a Referral Hospital in Uganda

Objective

To compare the performance of frontloading and the standard WHO method for diagnosis of pulmonary TB at Mulago Hospital in order to validate the technique in this setting.

Methods

This was a cross-sectional study in which 229 adult (≥18 years) TB suspects were consecutively enrolled. Suspects submitted three sputum samples as follows: at initial presentation, one hour after the first sample, and the next morning. The first and next morning samples formed the standard WHO method, while the first and the one hour later samples formed the frontloading method. Sample processing was by the standard N-acetyl L-cystein (NALC)-NaOH method, and fluorescent microscopy was done for both methods, while cultures of the first sample on Lowenstein-Jensen slants acted as a gold standard. The sensitivity, specificity and predictive values for the WHO standard and frontloading methods were compared.

Results

The sensitivity of both the frontloading and standard schemes was 91.1% while their specificities were 86.2% and 91.7% respectively. There was excellent agreement between the diagnostic capacity of the two methods (kappa statistic = 0.87, P<0.0001). The positive predictive value for the frontloading scheme was 87.2% and that for the standard approach was 91.9%, while the negative predictive values were 90.4% and 90.9%, respectively. Among the HIV positive patients, frontloading identified 59/79 (74.7%) culture positive samples while the standard approach identified 55/79 (69.6%). In the HIV sero-negative category, on the other hand, front-loading identified 48/110 (43.6%) culture positive samples compared to 45/110 (40.9%) by the standard approach.

Conclusion

Frontloading based on smear examination of two same-day sputum samples has a similar performance to the current standard method and would not be associated with any significant missed diagnosis. It may therefore be advocated for use in our setting so as to reduce time to completion of diagnosis and patient loss to follow-up.     

Diagnosis of Tuberculosis in a General Hospital

In a survey of 71 new cases of tuberculosis diagnosed in a general hospital the average interval between admission and diagnosis of tuberculosis (the diagnostic interval) ranged between 10 days for intrathoracic tuberculosis and 20 days for genitourinary tuberculosis. The average diagnostic interval was 10·9 days when tuberculosis was included in the initial differential diagnosis, and 22·8 days when other diagnoses were made. Undue delay in diagnosis occurred in 17 patients (24%). In eight this was due to failure to include tuberculosis in the initial differential diagnosis. Earlier diagnosis might have saved three of the five patients who died.In 21 patients (30%) a history of predisposing factors or associated illness was obtained. Ten of these had suffered from previous tuberculosis.The vital factor in diagnosis of tuberculosis in general hospital patients is consideration of this condition in the diagnosis of any unexplained illness, especially where a history of previous tuberculosis or a recognized predisposing factor is obtained.     

A Multi-Country Non-Inferiority Cluster Randomized Trial of Frontloaded Smear Microscopy for the Diagnosis of Pulmonary Tuberculosis

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     

Surveillance of Elderly Hospital Patients for Pulmonary Tuberculosis

A restrospective study of pulmonary tuberculosis in a general hospital showed that the diagnosis had been frequently overlooked in the middleaged or elderly because the patient also suffered from a more acute condition which preoccupied the attention of the doctor. The commonest error was to discount chest x-ray abnormalities by omitting sputum culture or serial radiography.Surveillance was carried out on all patients aged 60 or over admitted to a large general hospital whose routine chest radiograph showed signs of possible pulmonary tuberculosis whether apparently active or inactive. Three sputum samples from each patient were examined for Mycobacterium tuberculosis without reference to the clinical presentation. In a nine-month period six out of 81 patients proved to have active pulmonary tuberculosis (7·4%). It is suggested that this may be a useful method of screening the elderly hospital population for pulmonary tuberculosis.