The number of cases of PTB continues to increase worldwide despite major international control initiatives 
, and at least 50 million individuals with chronic cough are screened for TB each year. Although it is widely accepted that the early identification of cases through inexpensive point-of-care diagnostics is pivotal for effective control of TB, the diagnosis of PTB often relies on sputum smear microscopy, especially in resource-limited settings.
Sputum smear microscopy services are the most decentralized of the TB diagnostic services, allowing patients to be screened for tuberculosis at relatively basic health care facilities. In most LMICs, however, these centres are less numerous than treatment centres, and many patients still need to travel to submit sputum specimens for diagnosis, incurring considerable personal costs, and this often leads patients to abandon the process. The diagnosis of TB therefore is particularly difficult for poor and marginalised patients, who face multiple challenges to accessing a TB diagnosis 
, and the improvement of smear microscopy services remains necessary to increase patients' access to treatment 
. Furthermore, smear microscopy is known to be less sensitive in HIV-associated tuberculosis, and interventions which could increase this sensitivity are needed.
LED-FM holds multiple advantages over both the conventional mercury vapour lamp FM and standard light microscopy. LEDs are relatively inexpensive, can be powered by mains or batteries, and have an effective lifespan of thousands of hours 
. The longer lifespan of the LED systems, as well as facilitating their implementation, may result in better quality FM than conventional systems since mercury vapour and halogen lamps are often used after their effective lifespan, with diminished power, to cause fluorescence. The lower capital and maintenance costs of LED-based systems, the faster grading of smears than with ZN smear microscopy, and FM's reputed higher sensitivity makes LED-FM devices potentially more suitable for LMIC laboratories. Despite this potential, there have been very few well-designed field evaluations using culture as the reference standard 
to assess whether LED-FM performance varies in locations with no or limited experience with FM.
LED-FM in this study had higher sensitivity but lower specificity than ZN smear microscopy, which resulted in a significantly higher number of patients with PTB being treated, but lower overall diagnostic accuracy, as illustrated in Text S3
. Although most previous studies are small 
, they have reported increased sensitivity and similar specificity of LED-FM compared with ZN smear microscopy. Recently, however, Cattamanchi et al. suggested that LED-FM had lower specificity than conventional ZN smear microscopy in patients co-infected with HIV in Uganda 
, suggesting that the loss of specificity was more evident in patients who had scanty AFB in sputum. The investigators, however, had processed different specimens for ZN smear microscopy and FM; thus, the interpretation of this study is difficult 
, and further studies are needed. If these findings are corroborated by further studies, however, it is still important to consider that if a trade-off has to be made between sensitivity and specificity, it is preferable to err on the side of increased sensitivity and to treat a small number of patients who do not have TB. There are real consequences to a false-positive result: patients incorrectly diagnosed with TB have the cost and inconvenience of taking a six-month course of treatment, and troublesome side effects are common, but life-threatening treatment-related events (hepatitis and Stevens-Johnson syndrome) are infrequent (<5% incidence). It is also necessary to discuss whether solid culture, or liquid culture, can function as an adequate comparator, as neither is a perfect reference standard, and the underlying assumption that either culture system has higher specificity than smear microscopy might be debatable. The WHO manual for laboratory services in TB control states that the probability of obtaining a positive culture is related to the number of AFB in the specimen, with only about 50% of cultures of specimens with 1–2 AFB per 100 fields being identified as positive, increasing to 80% and 96.7% for specimens with “scanty” (1–9 AFB per 100 fields) and “+” AFB grades, respectively. The analysis of our smear-positive, culture-negative results indicated that more than 98% of these were scanty smears with a median of one AFB per smear, and 23.7% had a second positive smear by either ZN smear microscopy or LED-FM. It is thus highly likely that these patients either had paucibacillary tuberculosis in which M. tuberculosis
failed to grow in culture, or were infected/colonised by non-tuberculous mycobacteria. This is unfortunately an inherent weakness of solid culture (and, to a lesser extent, liquid culture) as a diagnostic reference standard. Judgements on the performance of new diagnostic tests must be done with considerations of these limitations. The “false positive” LED-FM results are thus most likely due to technician error, but we cannot discount the possibility that these were cases with TB or non-tuberculous mycobacteria with a false-negative solid medium result that would have been detected using liquid medium. There might also be cases of TB that would have been missed by both culture technologies (because of delayed processing/destruction of marginally viable bacilli during decontamination or bacterial overgrowth).
Unfortunately, HIV testing was not done systematically as part of this study. Overall, the vast majority of patients across the sites did not know their HIV status. The exception was the Nigerian site, where around half of patients reported knowing their HIV status. Only patients who volunteered this information were categorised as HIV positive or negative. Any sub-analysis of the results by HIV status would be underpowered, subject to self-selection bias, and confounded by the duration since last test and the methods used for testing.
Clinical laboratories in our study had considerable experience with ZN smear microscopy but limited experience with mercury vapour lamp FM, and their conventional FM microscopes were neither functioning nor in recent use at the time of the study. Although technicians received 2-d refresher training at the bench, were supervised for a further day to monitor performance, and had photographic bench-aids available, ZN smear microscopy had consistently higher specificity.
Interestingly, the rechecking of scanty smears and comparison against culture suggests that LED-FM also had higher sensitivity but lower specificity than conventional FM. These differences could have been due to the devices' performance characteristics, as reference laboratories are equipped with high-quality microscopes with greatly improved optics, and to the novelty of LED-FM, with laboratory technicians conducting more careful examinations and spending more time reading the smears to identify paucibacillary smears, while lacking experience to distinguish fluorescing artefacts from scanty AFB. Furthermore, technicians in the reference laboratories routinely use dark rooms, which facilitate visualizing the AFB in conventional FM but are not required with LED-FM, and may have been more cautious to declare smears with one or two bacilli as positive. Also, the smears may have faded, and AFB could have been washed off during re-staining.
Although the comparison of LED-FM and conventional FM was based on the verification of a partial selection of smears and needs to be replicated, national TB control programmes considering introducing LED-FM devices may wish to bear in mind that their performance could vary with the service setting, staff experience, and the type of devices used. In contrast to the findings of this study, other studies conducted at established research centres have found similar specificity in conventional FM and LED-FM. Our lower specificity may thus be the upside of the trade-off implicit in the larger amount of operational data collected in this study, at the cost of a less-than-ideal reference standard. The LED-FM technology, however, is likely to be widely implemented, and the study still gives valuable insights into operational issues. The performance of LED-FM, and indeed any diagnostic test, depends critically on proficiency of test operators. Adequate and appropriate training is thus required. Current expert opinion is that only moderate training (3 d) is required to make laboratory technicians already proficient in ZN smear microscopy proficient in LED-FM. This training was provided to study staff, involved instruction in specimen processing and staining techniques, and included 2 d of supervised routine microscopy during which an expert helped resolve any doubtful results in order to instil confidence in workers. All staff reported familiarity and confidence in their ability to perform the technique. We believe that this kind of training is most likely sufficient for the induction of staff in laboratories where the technique is well established and there are experienced staff who can provide continued mentoring. However, as stand-alone training, after which laboratory staff are expected to maintain performance without access to a second opinion, it is most likely inadequate, and the development of structured and standardised training packages and supervisory schemes is needed to support scale up. The introduction of LED-FM therefore needs to be accompanied by careful training, appropriate quality control, and monitoring of the performance of LED-FM in the field. Implementers may need to consider whether trade offs between higher sensitivity and lower specificity are acceptable.
The study also indicates that the sensitivity and specificity of LED-FM is independent of whether the sputum specimens are collected as SSM or SMS. There is hardly any evidence that a particular sequence of sputum collection increases the probability of finding TB bacilli, and we have recently demonstrated that this is not so 
. Although morning specimens have a higher yield (and single-smear sensitivity), examining two spot specimens identifies equivalent numbers of smear-positive patients as examining one spot specimen followed by one morning specimen. SMS requires that all patients return to provide the morning sample, while the SSM would identify most smear-positive cases in one day. Thus, what is crucial is the potential shortening of the process. Although many clinicians are reluctant to rely on spot specimens due to the higher sensitivity of the morning specimens, the 10% higher sensitivity of a single morning specimen becomes redundant once multiple specimens are used. The WHO has recently recommended using spot specimens (indeed spot-spot) to allow the screening of patients in one day. However, the change in policy was based on the evaluation of the method using ZN smear microscopy. We now provide evidence that a frontloaded LED-FM approach that uses two spot specimens results in the same yield as approaches using a conventional SMS LED-FM scheme. This seemingly small change may hold the key to improving current microscopy services. If two smears are collected and examined on the first day and one of them is positive, as was the case in the majority of smear-positive cases in this study, patients would not have to return the next day to complete the sputum submission process. If health systems are modified to provide results on the same day, patients could save the costs of one more visit in the diagnostic process, the majority of smear-positive patients could be referred for treatment on the day of consultation, and dropout rates may be reduced.
Moreover, it is important to consider the role that LED-FM can play in the recent WHO endorsement of the new rapid, automated nucleic acid amplification test, Xpert. It is unlikely, in the short term, that Xpert can be scaled up and decentralized sufficiently to replace smear microscopy as the initial diagnostic test worldwide, even in areas with high rates of multi-drug-resistant TB or HIV-associated TB, where Xpert is recommended as the initial screening test. Furthermore, the WHO recommends continuing to use smear microscopy for treatment monitoring, even in areas served by new technologies such as TB culture, line probe assays, and the Xpert assay. In areas without high rates of multi-drug-resistant TB or HIV-associated TB, cost-effectiveness considerations favour the use of smear microscopy as the initial diagnostic tool, and thus the improved performance of direct smear microscopy (and indeed a technique that facilitates faster examination of smears) is an important step for improving diagnostic services in LMICs, one that would reduce laboratory workloads and facilitate the faster identification of patients with negative smear microscopy who could undergo further tests.
Despite the reduced specificity of LED-FM, this approach still has operational advantages that make it an attractive tool for TB laboratory diagnosis. National control programmes introducing new LED-FM services should monitor the performance of the method under operational conditions, as training needs of staff may be greater than anticipated. This study has shown that LED-FM can play a key role in reaching WHO targets for TB detection, reducing laboratory workloads, and ensuring poor patients' access to TB diagnosis and prompt treatment.