This study evaluated the usefulness of QAF in the screening process of patients using systemic CQ/HCQ medication. Our results demonstrate increased QAF values in patients with or without BEM, as compared to age- and sex-matched controls. The increased QAF values are only minimally correlated with retinal thickness. QAF can be used as a biomarker for CQ/HCQ intake; however, our presented data does not proof QAF as a screening tool for CQ/HCQ maculopathy at this time. Furthermore, QAF analysis in CQ/HCQ patients requires adjustable analysis tools because CQ/HCQ-related lesions are beyond the common QAF analysis grids.
CQ/HCQ related maculopathy is a well-known complication in systemic CQ/HCQ therapy, which could affect up to 20% of all patients after 20 years of intake. There is a continuing trend of increasing prescriptions and also re-evaluating CQ/HCQ for new therapeutic indications such as anti-infectious disease and adjunct antineoplastic therapy or application in diabetes mellitus and heart disease.
1,39 Only recently, the use of chloroquine is intensively discussed as a treatment option in COVID-19 disease,
40 and numerous studies are ongoing or in preparation examining the protective effect of hydroxychloroquine in corona virus disease.
41 This means that an even larger patient population might be exposed to CQ/HCQ in the future and, if intake is long enough, at risk for potential retinal toxicity and sight-threatening maculopathy, especially if concomitant pathologic conditions are present.
16 The importance of continuing ophthalmic examinations and maculopathy screening has been discussed extensively and is well accepted nowadays.
42,43 In real life, however, not all CQ/HCQ patients have access to or are even aware of the necessary of retinal screening.
In the revised 2016 version of recommendations on screening for chloroquine and hydroxychloroquine retinopathy the AAO recommends the use of several tests in the screening of CQ/HCQ patients, including structural and functional tests.
16 Although there is still debate about which screening tool is the best to detect CQ/HCQ related maculopathy,
44 FAF has been considered being sensitive enough to detect those changes.
16,18,19
Within the last years, FAF analysis methods have markedly improved, and FAF imaging is far beyond sole qualitative analysis. Delori and coworkers
24 introduced a modified FAF camera in 2011, which has an internal well-defined fluorescent probe to quantify FAF signal. This probe emits light that is simultaneously captured during regular fundus autofluorescence imaging and serves as the basis for quantification of FAF intensities. The use
26,27,34 and repeatability of QAF
24,31,45 has successfully been demonstrated in several hereditary and degenerative retinal disease studies. The significantly increased QAF signals in CQ/HCQ patients leaves room for debate.
Our proposed hypothesis is that the drug itself or its metabolic by-products are autofluorescent
46 and stored within retinal cells or adjacent tissue, supported in several ways by findings in animal models. First, early studies found that chloroquine deposition is related to pigmented tissue (iris, RPE, choroid), although absent in nonpigmented tissue and albino animals.
47–49 This finding has been confirmed in rhesus monkeys.
50 The retinal drug deposition (probably at the RPE level, see above) is supported by the finding of generally increased QAF intensities in CQ/HCQ patients, still maintaining the typical FAF pattern (high parafoveal AF and less AF at the fovea). Second, the long-term effect of CQ/HCQ traceability: in animals, the drug levels in the retina were high even years after cessation, while absent in other tissues of the body.
47,51 This result is in line with increased QAF in our patients with high levels of QAF even more than five years and up to at least 12 years after termination of CQ/HCQ use (see
Figs. 2C–2E). Furthermore, the early onset of increased QAF (in our study about six months after treatment start) is also supported by animal models showing high retinal chloroquine levels immediately after drug administration and drug traceability even weeks after a single injection.
47 Bernstein et al.
47 summarized these findings in animals as “extensive [retinal] tissue accumulation and prolonged retention”—a model probably transferrable to the human retina and now trackable with QAF. QAF increase and persistent high QAF levels not related to the duration of intake and cumulative dose would also support these findings.
Other explanations for increased QAF levels include changes in lipofuscin, an age-related rather than toxic granule,
52 within the retinal pigment epithelium.
53 Lipofuscin granules comprise a variety of mostly unknown fluorophores that possess spectral emission features
54 and accumulate with age in healthy RPE cells,
55 clinically confirmed in several QAF studies.
24,31 This age effect of increasing QAF is also detectable in our study cohort, both in patients and controls. Also, the AF distribution pattern across the posterior pole (highest autofluorescence at the parafovea
24,31) in CQ/HCQ patients is similar to the healthy controls. Increased fundus QAF levels have been observed in some hereditary retinal diseases,
26,27 but none of our patients had any history or clinical signs of hereditary retinopathy.
The increased QAF levels in CQ/HCQ patients could also reflect increased metabolic activity in the photoreceptor/RPE system, enforced by the drug or its metabolites. RPE cells are in close interaction with both cones and rods (shedding and digestion of photoreceptor outer segments, accumulation of nondegradable lipofuscin).
56,57 Whereas the normal FAF distribution follows the rods rather the cones,
58,59 although cones also contribute to the total FAF signal, the overall increased QAF levels at the whole posterior pole in CQ/HCQ patients suggests that both the rod and cone system is affected. The continued use of drugs such as CQ/HCQ might have toxic effects on photoreceptors and lead to a shortening of photoreceptor outer segments resulting in increased autofluorescence from the RPE
60 as speculated for flood plain hyperautofluorescence in neovascular age-related macular degeneration.
61 However, our structural SD-OCT did not reveal any significant thinning of the outer retina (except: patients with existing BEM nor show any correlation between autofluorescence increase and retinal thickness in all eyes without BEM.
The rationale behind the fact that photoreceptors and RPE at the parafovea are clinically and histologically most affected by the toxic effects of CQ/HCQ and whether RPE or photoreceptors are the primary site of degeneration remains unclear. The multimodal detectable
62–65 CQ/HCQ maculopathy pattern that normally starts at the parafovea might be somehow related to the photoreceptor distribution with highest rod density 3 to 5 mm outside the fovea.
66
Of note, the increased QAF intensities in our patients without BEM was not accompanied by obvious pathologies in any of the AAO recommended functional and structural screening tests. Neither common 488 nm FAF, visual fields, color vision, electrophysiology, or SD-OCT scans revealed any abnormalities. Furthermore, there was no obvious correlation between increased QAF intensities and retinal thicknesses in our patient cohort (exception: patients with BEM), as determined in QAF measurements and parallel SD-OCT scans.
SD-OCT is considered to be an important screening and classification tool in the management of CQ/HCQ induced maculopathy
16,17 and several groups showed thinning of outer nuclear layer, loss of inner segment/outer segment junctions, as well as loss of RPE.
23,38,67,68 These changes can progress even years after cessation of the medication.
21,64 In our non-BEM patients, there was no obvious general reduction in retinal layer thickness measurable (selective measurements at eleven points across a horizontal line at the posterior pole), in line with other studies.
68 In addition, changes in SD-OCT reflectivity at the parafoveal ellipsoid zone and loss of a continuous interdigitation zone
69 and increased reflectivity in the outer nuclear layer
63 might be early signs of maculopathy and precede findings in any of the other screening modalities. Loss of reflectivity at the photoreceptor level could lead to decreased light-blocking phenomena of photoreceptor pigment and consequently to an increase of the FAF signal. However, because the aforementioned described SD-OCT changes are restricted to the parafovea, this SD-OCT finding doesn´t explain ubiquitous increased QAF at the posterior pole.
Emerging FAF imaging techniques like fluorescence lifetime imaging ophthalmoscopy (FLIO) might definitely add to QAF in screening for CQ/HCQ induced maculopathy. Recently, several groups reported prolonged lifetimes at the parafoveal region in patients with HCQ retinal toxicity.
46,70 However, whether FLIO could be a screening tool for early detection of CQ/HCQ induced maculopathy is still controversially discussed.
46,70
So far, in QAF studies, QAF8 (eight segments from a projected analysis grid, centered on the fovea, as introduced by Greenberg et al.
31) was used for analysis. As shown in our study, especially in the maculopathy patients, QAF8 analysis does not capture the affected areas and more flexible analysis patterns (narrowing of the analysis field, flexible dimensions, free hand tools) are warranted to specifically analyze the respective areas.
37,71
Limitations of this study include an imbalance according to gender, which is due to prevalence and incidence of the rheumatologic disorders and CQ/HCQ drug indications. Furthermore, cumulative dose and intake duration are mostly based on patient recall and were only partially available from documented patient charts, which might have led to unprecise cumulative doses in patients with long-lasting intake. Also, number of patients with drug induced maculopathy are not equally distributed due to limited recruitment possibilities. Documentation regarding macula status before the start of CQ/HCQ treatment was not available for all patients. However, our documentation (history, available images) for the five CQ/HCQ maculopathy patients revealed no signs of maculopathy based on causes other than CQ/HCQ (i.e., no history or signs of hereditary retinal disease, no signs of age-related macular degeneration). Because of the technical limitations of the device only the QAF features of the posterior pole have been examined and it is not clear whether QAF in the periphery or at the ora serrata would show similar results. Finally, our healthy control group did not have any signs of systemic rheumatologic disorders, which could per se (without any CQ/HCQ treatment) lead to increased QAF levels. In a few patients whom we examined before CQ/HCQ treatment start, QAF levels were comparable to the healthy controls (data not shown).
QAF is still in the experimental stage and depends on good imaging conditions, with clear optic media being the most important one. Therefore QAF values from phakic patients over 60 years (although biomicroscopically clear lens) should be critically reviewed. However, including subjects over age 60 years led to significant QAF differences between the patients without maculopathy and controls, probably because of a physiological QAF decline in normal aging whereas QAF in CQ/HCQ patients remained high (data not shown). Furthermore, well-trained technical assistance is required to guarantee reliability and repeatability of QAF imaging. Previous studies in our cohorts showed mean variability of about 8% in normal subjects
37 and <12% in patients with maculopathy, comparable to results from other groups.
31
In conclusion, this study is a prospective preclinical report on increased quantitative short wavelength FAF during CQ/HCQ treatment, compared to QAF of healthy controls. Although SD-OCT might be useful in detection of early maculopathy, QAF proves its clinical benefit in CQ/HCQ screening as the first tool that might indicate CQ/HCQ intake in general. Whether increased QAF in CQ/HCQ patients imperatively results in higher risk for the development of outer retinal atrophy or BEM needs further evaluation in larger cohorts and long-term follow-up studies, which are currently recruiting.