FIND study trials at a glance: Primo Star iLED microscope

December 2008

Over the past four years, FIND has assessed a number of potential platforms for improving TB microscopy through advances in instrumentation. A recent technical advance, the advent of low-cost ultrabright light emitting diodes (LEDs), made feasible the development of inexpensive LED-illuminated microscopes that could be used in place of expensive mercury-vapor bulb solutions. A good technical summary was published by Ely Silk in 2002 (LED Fluorescence Microscopy, The Microscope, 2002;50:101-118).

Policy support for expanded use of fluorescence microscopy came from an expert meeting at WHO co-organized by FIND in September 2005. This meeting was organized around three systematic reviews on different aspects of microscopy commissioned by TDR (the WHO Special Programme on Research and Training for Tropical Diseases) and FIND. The systematic review of fluorescence microscopy (FM) included over 50 publications in the peer reviewed literature and provided convincing data that in experienced hands, FM was faster and an average of 10% more sensitive than conventional Ziehl-Neelsen (ZN) microscopy, without a loss of specificity. Recommendations on expanded use of FM microscopy outside of high-throughput were restrained by lack of information on how it would perform outside of reference centers.

The Primo Star iLED microscope

The feasibility of using LEDs to adapt existing expensive fluorescent microscopes was proven by Anthony and colleagues and published in 2006 (Int J Tuberc Lung Dis. 2006:10:1060-1062). In the meantime, FIND made the decision that the most useful approach would be the creation of a complete solution: a new dual-use microscope that could be easily switched to perform both fluorescent and brightfield applications. FIND required that the microscope be of the highest quality but also affordable for high-burden countries, and that the manufacturer have worldwide distribution capacity. Zeiss, a worldwide leader in optics and microscopy, and with high-end LED-based fluorescent systems already in development, was a natural partner. After signing an agreement with Carl Zeiss MicroImaging, FIND began co-development of the Primo Star iLED microscope in 2006, with some of the following product specifications:

  • high-grade optics
  • reflected rather than transmitted blue light for fluorescent applications
  • battery power on AC failure
  • easy switching between fluorescent and brightfield viewing
  • utility outside a darkroom
  • >10,000 hour bulb life
  • inexpensive bulb change
  • LED and filter sets that would allow use of Auramine O and acridine orange for TB and parasitologic examinations

Early prototypes of the microscope were developed by the end of 2007 and exhibited at the IUATLD World Congress in Cape Town. A design-locked manufacture version was ready in the second quarter of 2008 and was evaluated in time for the preparation of a poster for the 2008 IUATLD meeting in Paris.

During the process of development of the iLED, several other LED-based options for TB detection with fluorescent microscopy entered development or became commercially available. In order to harmonize to the degree possible the strategies for evaluating the performance and impact of these different microscopes or microscope adaptors, FIND held a meeting on LED Fluorescence Microscopy on 26 March 2008 in Geneva with a number of scientists, clinicians, and public health specialists working in this field. At that meeting, the preliminary plans for FIND Demonstration projects were shared with other investigators in the hopes that groups might take up some of the technical questions being answered through the studies and that similar approaches might be used across sites so that a harmonized data set would eventually be available for review by the Stop TB Department of WHO and its expert committees.

Since that time, final study protocols for Evaluation and Demonstration have been completed as well as training packages to accompany those studies. These materials are posted here on the FIND website, along with a brief overview of the evaluation process for the Primo Star iLED microscope developed by FIND and Zeiss.

Feasibility study
The feasibility of using the Primo Star iLED prototype for TB detection was determined in two laboratories experienced in conventional fluorescence microscopy. A set of study slides with 30-50% positivity rate was generated prospectively from patients undergoing diagnostic evaluation and stored until culture results were available. These slides were used in blinded studies assessing several questions, including:
 

  1. the performance of the iLED microscope in comparison to the more expensive standard FM scope
  2. the necessity of a darkroom
  3. the suitability of the iLED excitation wavelength for alternative stains (Auramine or Rhodamine) and counterstains (KMnO4 or Methylene blue)
  4. the reading time per slide
  5. the speed of fading of stored slides
  6. the appraisal of the laboratory technicians of the usability and technical suitability of the microscopes.

That protocol, and all others described here are available by hyperlink through the list below.

Evaluation study
Once feasibility targets were met, the final Primo Star iLED manufacture series of the microscope was evaluated for performance in the detection of TB in sputum specimens in a laboratory-based, blinded, multi-center study. Panels of slides with specific grading of positivity were created by a single facility and shipped to 6 reference laboratories with prior experience in fluorescence microscopy that carried out the study. The endpoints of the study were:
 

  1. Sensitivity of iLED in smear positive panel slides compared to light microscopy and conventional fluorescence microscopy
  2. Specificity in smear negative slides compared to light microscopy and conventional fluorescence microscopy
  3. Assessment of technicians’ appraisal of FIND/Zeiss microscope in terms of ease of use, maintenance, design and comfort, robustness, contrast, brightness, etc.
  4. Assessment of the adequacy of 200x vs. 400x magnification for slide screening.

The protocol for this study, the associated standard operating procedures, and the methodology for slide preparation are listed in the hyperlink below under Evaluation. Results of the Evaluation are shown in the Poster presentation also listed as a link.

Demonstration study
Once performance targets were met through the evaluation study, a Demonstration project was initiated in coordination with National TB Control Programs in several countries. FIND selected countries for the study where there was agreement at national level (MOU with NTP and/or MOH), with a high-burden of TB and settings representative of the global TB situation, with a ranking as low or middle income, and with the local presence of FIND or an implementing partner. At least two countries were selected that had a high prevalence of HIV. There were 36 primary sites and 20 extension sites chosen for this large study, with site selection based on the rate of smear-positivity, the training of microscopists, the volume of work, the reliability of AC power (sites intentionally selected with intermittent power supply), interest in the project, and accessibility of study sites to supervisory visits. At least 20 centers were included without any prior experience with fluorescent microscopy to determine the performance of LED FM in such settings.

For all sites, baseline performance data were collected using the standard ZN procedures over a month long pre-study period. Following a 5 day training session on the Primo Star iLED microscope, sites initiate a validation phase. During this phase, all slides are screened with the iLED microscope and then confirmed on a daily basis using a conventional fluorescent microscope by a supervisory site. Patient care during this phase is based on the results of conventional FM examination. Proficiency testing panels are also used to assess the study sites. Sites progress to demonstration after the validation phase only if there is 95% results concordance between the demonstration site and the supervisory site, if the quality of the Auramine stains was found acceptable in 100% of panel slides examined, and if there was an insignificant number of false results in the proficiency testing panel.

Sites that show acceptable performance in the validation phase continue onto a 3 month phase in which the iLED microscope is used as the primary screening tool, used in the context of NTP norms. Rechecking of stored slides continues during this phase, and is compared with the error rate with ZN microscopy in the pre-trial period. In some sites additional operational questions are being answered. The primary and additional study objectives are listed below.

Primary Objectives (Basic Protocol)

  1. To assess the feasibility of implementing Primo Star iLED for TB diagnosis at microscopy centers without prior experience with fluorescence microscopy in low- to moderate-income settings and to identify barriers to implementation
  2. To determine the false positivity and negativity rate of LED fluorescence reading compared to a ZN baseline and to results from the supervisory site
  3. To determine the development of false positivity and negativity rates of LED fluorescence reading over time (with increasing experience)
  4. To assess the impact of this implementation on daily workload and case detection rates for low, middle and high-volume settings
  5. Determine lab technicians’ appraisal of Primo Star iLED
  6. To evaluate detailed costs associated with LED-based fluorescence microscopy in comparison with conventional methods

Secondary Objectives (Extended protocol - FIND partners)

  1. In collaboration with other groups and in preparation for WHO STAG submission, establish comparative performance data for alternative LED-based approaches
  2. To identify minimal training needs and develop training modules accordingly
  3. To identify the optimal fluorescence staining method and ensure continuous and affordable supply
  4. To assess effects of fading speed on external quality assurance by rechecking
  5. To assess whether combining LED fluorescence microscopy with concentration methods such as bleach sedimentation further increases sensitivity

The study protocol and associated standard operating procedures are listed in the hyperlinks below. The full 9-module training manual is also linked. The size and complexity of the Demonstration study reflects the importance of having true performance data from sites without prior experience in fluorescence microscopy. LED-based FM is likely to see markedly expanded use in the coming few years, and it will be critical to understand the training and supervisory needs for peripheral microscopy sites using this technology as compared with conventional brightfield examination of ZN-stained slides. Data from these Demonstration projects, as well as other published and unpublished data will be submitted to WHO expert committees for review prior to the drafting of international policy guidelines. The intention is to execute that review in the second quarter of 2009.