Fosfomycin is a broad-spectrum antibiotic used as empirical treatment for uncomplicated urinary tract infections (UTIs), of which Escherichia coli is the most common cause. To rapidly detect fosfomycin-resistant E. coli isolates and consequently improve patients’ treatment and management, we have developed the Rapid Fosfomycin/E. coli NP test, a rapid, easy-to-perform, specific and sensitive diagnostic test.

by Dr Linda Mueller, Dr Laurent Poirel and Prof. Patrice Nordmann

Introduction
Fosfomycin, a phosphonic acid-derived bactericidal antibiotic discovered in 1969, is now of renewed interest, especially for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. This antibiotic is water-soluble and has a low molecular weight, allowing high diffusion at the tissue level [1]. Its features such as broad-spectrum activity, safety and efficacy make fosfomycin as one of the first-line antibiotics used for uncomplicated urinary tract infections (UTIs) treatment [2]. More than 75% of UTIs are due to Escherichia coli [3].

Fosfomycin enters the bacterial cell by the transport proteins GlpT (glycerol-3-phosphate transporter) and UhpT (hexose-6-phosphat:phosphate antiporter); once in the cytosol it binds and inactivates MurA (UDP-N-acetylglucosamine enolpyruvyl transferase), the enzyme involved in the first step of peptidoglycan biosynthesis. Hence, it inhibits bacterial cell wall synthesis [4].

Because of its unique structure and mechanism of action, cross-resistance with fosfomycin and other bacterial agents has not been observed. Fosfomycin as a single agent works well for treating most of UTIs. Additionally, synergistic effects of fosfomycin with several unrelated molecules, such as gentamicin, carbapenems, aztreonam and aminoglycosides, have been observed when treating clinically-relevant MDR Gram-negative bacteria [5].

One of the main concerns with antibiotic resistance in E. coli corresponds to the acquisition of extended-spectrum β-lactamases (ESBL) leading to resistance to expanded-spectrum cephalosporins. ESBL-producing E. coli are mostly community-acquired and may represent 10 to 20% of E. coli isolates in several countries including in the US [6]. Those strains are often co-resistant to several aminoglycosides, to trimethoprim, cotrimoxazole and fluoroquinolones, leaving few therapeutic options available including fosfomycin [7].

Both wild-type susceptible E. coli and ESBL-producing E. coli show an overall high susceptibility rate to fosfomycin (>90%) [8]. However, a Spanish study monitoring fosfomycin resistance during 5 years, showed an increased use of fosfomycin [from 0.122 defined daily dose per 1000 inhabitants per day (DID) in 2004 to 0.191 DID in 2008] and an increased fosfomycin resistance rate in E.coli (from 1.6% to 3.8%) as well as in ESBL-producing E. coli (from 2.2% to 21.7%) [9].

The mechanisms of resistance to fosfomycin described in E. coli are either non-transferable or transferable. The non-transferable and chromosome-encoded resistance involve reduced permeability, resulting from mutations in glpT and uhpT genes, encoding for fosfomycin transporters, and amino acid mutations in the active site of the MurA target. Plasmid-mediated fosfomycin resistance mechanisms in E. coli correspond to production of fosfomycin-inactivating metallo-enzymes (encoded by the fosA genes) [10]. Among the plasmid-borne fosA variants described so far, fosA3 remains the most widespread resistance determinant among both human and animal isolates, those latter being either recovered from pets or livestock [11, 12]. Moreover, a study performed in Taiwan reported the transmission of FosA3-producing E. coli between companion animals and respective owners [13]. Importantly, the fosA3 gene is often identified onto conjugative plasmids along with CTX-M-type ESBL encoding genes, thus leading to acquired resistance to both fosfomycin and broad-spectrum cephalosporins [14, 15]. As fosfomycin is being used as an empiric treatment against UTIs, it was of great interest to develop a rapid test to evaluate the efficacy of this antibiotic.

Rapid Fosfomycin/E. coli NP test
Currently the reference technique recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to evaluate fosfomycin susceptibility is agar dilution, a fastidious technique requiring 18±2 h to get the results [16]. According to EUCAST, an isolate of E. coli is categorized as susceptible or as resistant when minimum inhibitory concentrations (MICs) are ≤32 and >32 mg/L, respectively.

Alternatively, disk diffusion and gradient strips, although exhibiting some discrepancies with the reference agar dilution method, might be used [17]. To accelerate the process of fosfomycin resistance detection, we have developed the Rapid Fosfomycin/E. coli NP test that allows detection of resistance within 1 h 30 min of fosfomycin-resistant E. coli isolated from culture plates.

This user-friendly technique is based on carbohydrate hydrolysis, detecting bacterial growth of fosfomycin-resistant isolates in the presence of a defined concentration (40 mg/L) of fosfomycin. Of note, fosfomycin-resistant isolates are detected independently of the molecular mechanism of resistance.

Briefly, the technique includes the preparation of a bacterial suspension (109 CFU/mL; 3–3.5 McFarland) that is poured on a 96-well polystyrene microplate. This culture is made in the Rapid Fosfomycin NP solution supplemented with 25 mg/L glucose-6-phosphate with or without 40 mg/L fosfomycin. The plate is incubated for 1 h 30 min at 35±2 °C and colour changes are detected by visual inspected. Fosfomycin-resistant isolates grow in the presence and absence of fosfomycin, triggering a colour switch from orange to yellow in both wells, a test result which is, therefore, considered as positive (Fig. 1). When dealing with fosfomycin-susceptible isolates, the well supplemented with fosfomycin does not exhibit any bacterial growth and remains orange; the test is, therefore, considered as negative. This test was evaluated with 100 strains including 22 fosfomycin-resistant isolates. It showed a sensitivity and a specificity of 100% and 98.7% respectively.

Conclusion
The Rapid Fosfomycin/E. coli NP test is rapid (1 h 30 min), specific (98.7%) and sensitive (100%). It is easy to perform, cost-effective, and may be used worldwide, regardless of the technical capabilities of the lab. Ongoing work aims to evaluate its performances directly from urine samples, which would represent significant added-value in terms of diagnostic rapidity.

The speed of this test allows a saving of at least 16 h when compared to the traditional agar dilution method. It is a potentially useful clinical test for first-step screening of fosfomycin resistance in E. coli.

Even though a low level of resistance to fosfomycin is currently observed among E. coli, the fact that we usually observe an increased fosfomycin clinical use, meaning an increased selective pressure, argues for a likely increased occurrence of fosfomycin-resistant isolates in the future. Since the principle of this test is based on a rapid culture, it may be used to detect any fosfomycin resistance trait that may be either chromosomally or plasmid-encoded. Fosfomycin is an old antibiotic that is very useful for the treatment of uncomplicated UTIs. On the one hand, even after extensive use for such an indication, the prevalence of resistance remains low, likely due to the fitness cost of the chromosomal mutations needed for acquired resistance, and also as a consequence of a high urinary drug concentration. On the other hand, the worldwide spread of fosfomycin-modifying enzymes should be monitored, as the biological cost of this emerging mechanism of resistance is much lower than that induced by chromosomal mutations [18] and the co-occurrence of fosA-like genes on plasmids with other resistance genes is commonly observed, meaning that co-selection can occur quite frequently.

References
1. Dijkmans AC, Zacarias NVO, Burggraaf J, Mouton JW, Wilms EB, van Nieuwkoop C, et al. Fosfomycin: pharmacological, clinical and future perspectives. Antibiotics (Basel) 2017; 6(4): pii: E24.
2. Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 2011; 52(5): e103–120.
3. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015; 13(5): 269–284.
4. Castaneda-Garcia A, Blazquez J, Rodriguez-Rojas A. Molecular mechanisms and clinical impact of acquired and intrinsic fosfomycin resistance. Antibiotics (Basel) 2013; 2(2): 217–236.
5. Falagas ME, Vouloumanou EK, Samonis G, Vardakas KZ. Fosfomycin. Clin Microbiol Rev 2016; 29(2): 321–347.
6. Castanheira M, Farrell SE, Krause KM, Jones RN, Sader HS. Contemporary diversity of beta-lactamases among Enterobacteriaceae in the nine U.S. census regions and ceftazidime-avibactam activity tested against isolates producing the most prevalent beta-lactamase groups. Antimicrob Agents Chemother 2014; 58(2): 833–838.
7. Wiedemann B, Heisig A, Heisig P. Uncomplicated urinary tract infections and antibiotic resistance-epidemiological and mechanistic aspects. Antibiotics (Basel) 2014; 3(3): 341–352.
8. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum β-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis 2010; 10: 4–-50.
9. Oteo J, Orden B, Bautista V, Cuevas O, Arroyo M, Martinez-Ruiz R, et al. CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin. J Antimicrob Chemother 2009; 64(4): 712–717.
10. Silver LL. Fosfomycin: mechanism and resistance. Cold Spring Harb Perspect Med 2017; 7(2): pii: a025262.
11. Alrowais H, McElheny CL, Spychala CN, Sastry S, Guo Q, Butt AA, et al. Fosfomycin resistance in Escherichia coli, Pennsylvania, USA. Emerg Infect Dis 2015; 21(11): 2045–2047.
12. Xie M, Lin D, Chen K, Chan EW, Yao W, Chen S. Molecular characterization of Escherichia coli strains isolated from retail meat that harbor blaCTX-M and fosA3 genes. Antimicrob Agents Chemother 2016; 60(4): 2450–2455.
13. Yao H, Wu D, Lei L, Shen Z, Wang Y, Liao K. The detection of fosfomycin resistance genes in Enterobacteriaceae from pets and their owners. Vet Microbiol 2016; 193: 67–71.
14. Benzerara Y, Gallah S, Hommeril B, Genel N, Decre D, Rottman M, et al. Emergence of plasmid-mediated fosfomycin-resistance genes among Escherichia coli isolates, France. Emerg Infect Dis 2017; 23(9): 1564–1567.
15. Yang X, Liu W, Liu Y, Wang J, Lv L, Chen X, et al. F33: A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosA3 and bla CTX-M-55/-14/-65 in Escherichia coli from chickens in China. Front Microbiol 2014; 5: 688.
16. Performance standards for antimicrobial susceptibility testing, 28th edn. Clinical and Laboratory Standards Institute (CLSI) document M100-S28 2018.
17. Hirsch EB, Raux BR, Zucchi PC, Kim Y, McCoy C, Kirby JE, et al. Activity of fosfomycin and comparison of several susceptibility testing methods against contemporary urine isolates. Int J Antimicrob Agents 2015; 46(6): 642–647.
18. Cattoir V, Guérin F. How is fosfomycin resistance developed in Escherichia coli? Future Microbiol 2018; 13(16): 1693–1696.

The authors
Linda Mueller*^1,2 PhD; Laurent Poirel^1,2,3 PhD; Patrice Nordmann^1,2,3,4 MD, PhD
¹Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Faculty of Science and Medicine, ^{University of Fribourg, Fribourg, Switzerland
2}Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), University of Fribourg, Fribourg, Switzerland
³INSERM European Unit (IAME, France),University of Fribourg, Fribourg, Switzerland
⁴University Hospital Center and University of Lausanne, Lausanne, Switzerland

*Corresponding author
E-mail: Linda.mueller@unifr.ch

For decades, attempts to biopsy or obtain fluid from eyes with retinoblastoma had been contraindicated, however recent changes in the management of retinoblastoma have allowed for safe sampling of the aqueous humour (AH) during therapy. Use of the AH as a liquid biopsy enables tumour biomarker analysis in these eyes; this has potential to dramatically alter the management of this pediatric cancer.

by Dr Benjamin K. Ghiam, Dr Liya Xu and Dr Jesse L. Berry

Introduction
Retinoblastoma (Rb) is the most common intraocular cancer in children, comprising 4 % of all pediatric malignancies [1, 2]. This potentially fatal malignancy often goes undiagnosed until the tumour is advanced and has damaged intraocular structures. Survival rates for Rb are in excess of 90 % in developed countries, though a critical, and often challenging, focus of Rb therapy is globe and vision preservation [3]. Throughout decades of ocular medicine and surgery, any attempt to biopsy these tumours, or even obtain fluid from Rb eyes had been fervently contraindicated for risk of tumour seeding and dissemination. Thus, much of the diagnosis and management of Rb is dependent on information gathered by the ophthalmologist through careful eye examination, and without histopathologic evidence.
In 2012, Munier et al. described a safety-enhanced protocol for intravitreal chemotherapy injections in the eyes of patients with Rb; this protocol requires an initial paracentesis [4]. As described by the authors of the study, a volume of 0.1  ml of aqueous fluid is aspirated to induce transient hypotony before the intravitreal injection as a safety measure to prevent reflux to the injection site. This protocol for intravitreal injection of chemotherapy has now been widely adopted worldwide and the risk of extraocular spread is considered extremely low (zero reported cases with the safety-enhanced procedure) [5]. This demonstrated safety record paved the way for aqueous humour (AH) extraction in eyes with Rb undergoing active therapy.
AH is the clear intraocular fluid produced by the ciliary processes that fills the front part of the eye (anterior chamber). The AH functions to maintain intraocular pressure, provide nutrients to the cornea, and remove waste products. It has also been shown to be a rich source of information for intraocular disease, including Rb [6]. Researchers have long sought to evaluate AH for the presence of biomarkers which may correlate with features of intraocular disease and provide diagnostic and prognostic value. However, before 2017, any evaluation of the AH was only done on eyes after enucleation. Now that the AH can be safely extracted during therapy, we hypothesized that previous evaluations of AH biomarkers (post-enucleation) may now be clinically applicable for the diagnosis, prognosis and/or management of Rb. This article excerpts our recently published systematic review, titled “Aqueous Humor Biomarkers for Retinoblastoma, a review” in the journal Translational Vision Science and Technology [7].
Lactate dehydrogenase
Lactate dehydrogenase (LDH) is an enzyme found in nearly all cells that acts as a regulator of metabolism; it has been used clinically as a non-specific marker found within body fluids in various pathological conditions, including malignant tumours.
In the early 1970s, Dias et al. examined LDH levels in the AH from enucleated Rb eyes [8]. Early reports demonstrated a significant increase in the levels of LDH within the AH of enucleated eyes with Rb when compared to patients without Rb, such that levels >1000  U/L strongly support the diagnosis of Rb (Table 1). Multiple studies on LDH levels in the AH from enucleated eyes were done between the years 1971 and 2008 which found that LDH levels were significantly elevated compared to controls, and more elevated in advanced eyes with delayed diagnosis; however, these levels did not correlate with other clinical features or outcomes. Elevation in AH LDH have been described in patients with other ocular conditions, including primary open angle glaucoma and Coats’ disease. Although LDH was the first described marker of tumour activity in the AH, the lack of specificity and correlation with patient or tumour features limits its use clinically. Owing to this lack of correlation this research was previously abandoned.
Enolase/neuron-specific enolase
Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase; it is highly specific for neurons and peripheral neuroendocrine cells. Increased body fluid levels of NSE occur with malignant proliferation and thus have been of value in the diagnosis and characterization of neuroendocrine tumours, including small cell lung cancer and retinoblastoma [9].
Evaluation of the isoenzyme patterns of enolase in the AH of enucleated Rb eyes demonstrated that NSE levels were elevated in AH Rb, whereas enolase was not detectable in the AH from controls (Table 1) [10–12]. Elevated levels of NSE significantly correlated with inflammation and tumour invasion into the anterior chamber [13]. NSE levels did not correlate with histological tumour parameters (tumour necrosis, calcification, optic nerve/choroidal invasion) as well as clinicopathological parameters (sex, enucleation age, presentation age, family history, previous treatment, and metastatic disease). Moreover, NSE levels were found to be within the control range in children more than 5 years after active therapy [14]. This suggests that NSE may be used clinically to indicate remission status. Although obtaining serial AH NSE measurements may have a significant role in determining tumour status in Rb patients in the future, additional evidence is required to further substantiate the use of this tumour marker clinically.
Surviving and transforming growth factor beta-1
Survivin is a protein that inhibits apoptosis. It has garnered significant interest as a diagnostic and prognostic factor in human neoplasms, including Rb. Elevated survivin levels are found in many human neoplasms, and it is used as a prognostic factor in several human neoplasms, including lung and colorectal cancers [15, 16]
Survivin expression in the AH from enucleated eyes of children with Rb was found to be significantly elevated, when compared to patients with non-malignant ophthalmic disease, such as congenital cataracts and glaucoma [17, 18]. AH survivin levels correlated with tumour stage and histopathologic post laminar optic nerve involvement.
Transforming growth factor beta-1 (TGF-β1) expression in the AH of enucleated Rb eyes was associated with poor differentiation of the tumour [17]. The authors demonstrated high sensitivity and specificity of these AH proteins which makes them promising markers for Rb, particularly of more aggressive pathologic features.

Uric acid and xanthine
During cell turnover, nucleic acids and nucleotides are degraded into xanthine and uric acid. Elevated levels of serum uric acid have been associated with many malignancies, as well as after rapid destruction such as after treatment with chemotherapy or radiation.
Elevated concentrations of uric acid and xanthines were found in the AH of children with Rb compared with control eyes (Table 1) [19]. Elevated levels of xanthine and uric acid in AH may support the diagnosis of Rb in children suspected of having the disease, however further studies are necessary to establish optimal cut-offs, explore clinicopathological correlations, and compare Rb levels to lesions simulating Rb (Coats’ disease and persistent fetal vasculature).
Protein content
Normally, the AH is virtually protein-free to ensure a clear optical media between the cornea and the lens. An increase in globulin content and an albumin/globulin ratio < 1 has been found in enucleated eyes with Rb [20]. Concentrations of interleukin (IL)-6, IL-7, IL-8, interferon gamma (IFN-γ), placental growth factor 1 (PlGF-1), vascular endothelial growth factor A (VEGF-A), beta-nerve growth factor (β-NGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) were significantly higher in the AH of patients with Rb than those in the control group [21]. Additionally, significantly decreased protein concentration was demonstrated in Rb eyes following treatment with selective intra-arterial chemotherapy (melphalan injection in the ophthalmic artery) that were subsequently enucleated after attempts at salvage, compared to primarily enucleated eyes [22].

Nucleic acids
Recent studies from Berry et al. demonstrated the presence of tumour-derived nucleic acids (DNA, RNA, miRNA) in the AH of Rb eyes [23]. Because of this, the authors suggest that the AH may be a rich source of tumour DNA and, thus, could be used as a liquid biopsy in children with Rb, without undergoing enucleation. A subsequent analysis by Berry et al. in 2018 showed that evaluation of the cell-free DNA (cfDNA) in the AH for chromosomal alterations has potential prognostic value as in indicator of aggressive disease [24]. Specifically, there was a significant increased odds of an eye failing therapy and requiring enucleation due to persistent and/or progressive cancer activity if gain of chromosome 6p was found in the AH cfDNA. Further research is required before this can be applied clinically, however this holds potential as a prognostic biomarker for Rb.

Conclusion
Despite significant investigation into tumour biomarkers for Rb spanning more than four decades, currently there are no active uses for the AH in a clinical setting. Diagnosis is made on the basis of examination and ancillary imaging findings without a biopsy, and molecular tumour markers are presently not used for diagnosis, prognosis, or to monitor therapeutic response. This is due in large part to the contraindication to biopsy in Rb; therefore, previously neither tumour nor AH or other ocular fluids were evaluated outside of specimens from enucleated eyes; clearly this limited the ability to correlate these markers with meaningful clinical outcomes. However, with recent advances in local therapy for Rb, paracentesis with extraction of the AH has now been shown to be safe in eyes being actively treated. This opens the door to for an AH liquid biopsy and thus there is renewed interest in these potential disease biomarkers.

Acknowledgement
This article excerpts our recently published systematic review, titled “Aqueous Humor Biomarkers for Retinoblastoma, a review” in the journal Translational Vision Science and Technology [7].
References
1. Shields, JA. Management and prognosis of retinoblastoma. In: Intraocular tumors: a text and atlas, pp377–391. WB Saunders 1992. ISBN 978-0721642680.
2. Shields JA, Shields CL. Intraocular tumors: an atlas and textbook, p574. Lippincott Williams & Wilkins 2008. ASIN B00XWR8WM6.
3. Pavan-Langston D. Manual of ocular diagnosis and therapy, p533. Lippincott Williams & Wilkins 2008. ISBN 978-0781765121.
4. Munier FL, Soliman S, Moulin AP, et al. Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track. Br J Ophthalmol 2012; 96(8): 1084–1087.
5. Smith SJ, Smith BD, Mohney BG. Ocular side effects following intravitreal injection therapy for retinoblastoma: a systematic review. Br J Ophthalmol 2013; 98(3): 292–297.
6. Macknight AD, McLaughlin CW, Peart D, et al. Formation of the aqueous humor. Clin Exp Pharmacol Physiol 2000; 27(1-2): 100–106.
7. Ghiam BK, Xu L, Berry JL. Aqueous humor markers in retinoblastoma, a review. Transl Vis Sci Technol 2019; 8(2): 13.
8. Dias PL, Shanmuganathan SS, Rajaratnam M. Lactic dehydrogenase activity of aqueous humour in retinoblastoma. Br J Ophthalmol 1971; 55(2): 130–132.
9. Kivelä T. Neuron-specific enolase in retinoblastoma. Acta Ophthalmol 2009; 64(1): 19–25.
10. Wu Z, Yang H, Pan S, et al. Electrophoretic determination of aqueous and serum neuron-specific enolase in the diagnosis of retinoblastoma. Yan Ke Xue Bao 1997; 13(1): 12–16.
11. Shine BS, Hungerford J, Vaghela B, et al. Electrophoretic assessment of aqueous and serum neurone-specific enolase in retinoblastoma and ocular malignant melanoma. Br J Ophthalmol 1990; 74(7): 427–430.
12. Nakajima T, Kato K, Kaneko A, et al. High concentrations of enolase, alpha- and gamma-subunits, in the aqueous humor in cases of retinoblastoma. Am J Ophthalmol 1986; 101(1): 102–106.
13. Abramson DH, Greenfield DS, Ellsworth RM, et al. Neuron-specific enolase and retinoblastoma. Clinicopathologic correlations. Retina 1989; 9(2): 148–152.
14. Comoy E, Roussat B, Henry I, et al. Neuron-specific enolase in the aqueous humor. Its significance in the differential diagnosis of retinoblastoma Ophtalmologie 1990; 4(3): 233–235 [in French].
15. Andersen MH, Svane IM, Becker JC, et al. The universal character of the tumor-associated antigen survivin. Clin Cancer Res 2007; 13(20): 5991–5994.
16. Rohayem J, Diestelkoetter P, Weigle B, et al. Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res 2000; 60(7): 1815–1817.
17. Shehata HH, Abou Ghalia AH, Elsayed EK, et al. Clinical significance of high levels of survivin and transforming growth factor beta-1 proteins in aqueous humor and serum of retinoblastoma patients. J AAPOS 2016; 20(5): 444.e1–444.e9.
18. Shehata HH, Abou Ghalia AH, Elsayed EK, Z et al. Detection of survivin protein in aqueous humor and serum of retinoblastoma patients and its clinical significance. Clin Biochem 2010; 43(4-5): 362–366.
19. Mendelsohn ME, Abramson DH, Senft S, et al. Uric acid in the aqueous humor and tears of retinoblastoma patients. J AAPOS 1998; 2(6): 369–371.
20. Dias PL. Postinflammatory and malignant protein patterns in aqueous humour. Br J Ophthalmol 1979; 63(3): 161–164.
21. Cheng Y, Zheng S, Pan C-T, et al. Analysis of aqueous humor concentrations of cytokines in retinoblastoma. PLoS One 2017; 12(5): e0177337.
22. Hadjistilianou T, Giglioni S, Micheli L, et al. Analysis of aqueous humour proteins in patients with retinoblastoma. Clin Experiment Ophthalmol 2012; 40(1): e8–15.
23. Berry JL, Xu L, Murphree AL, et al. Potential of aqueous humor as a surrogate tumor biopsy for retinoblastoma. JAMA Ophthalmol 2017; 135(11): 1221–1230.
24. Berry JL, Xu L, Kooi I, et al. Genomic analysis of aqueous humor cell-free dna in retinoblastoma predicts eye salvage: the surrogate tumor biopsy for retinoblastoma. Mol Cancer Res 2018; 16: 1701–1712.
25. Kabak J, Romano PE. Aqueous humour lactic dehydrogenase isoenzymes in retinoblastoma. Br J Ophthalmol 1975; 59(5): 268–269.
26. Piro PA Jr, Abramson DH, Ellsworth RM, et al. Aqueous humor lactate dehydrogenase in retinoblastoma patients. Clinicopathologic correlations. Arch Ophthalmol 1978; 96(10): 1823–1825.
27. Abramson DH, Piro PA, Ellsworth RM,  et al. Lactate dehydrogenase levels and isozyme patterns. Measurements in the aqueous humor and serum of retinoblastoma patients. Arch Ophthalmol 1979; 97(5): 870–871.
28. Dias PL. Correlation of aqueous humour lactic acid dehydrogenase activity with intraocular pathology. Br J Ophthalmol 1979; 63(8): 574–577.
29. Dias PL. Prognostic significance of aqueous humour lactic dehydrogenase activity. Br J Ophthalmol 1979; 63(8): 571–573.
30. Das A, Roy IS, Maitra TK. Lactate dehydrogenase level and protein pattern in the aqueous humour of patients with retinoblastoma. Can J Ophthalmol 1983; 18(7): 337–339.
31. Dias PL. Electrolyte imbalances in the aqueous humour in retinoblastoma. Br J Ophthalmol 1985; 69(6): 462–463.
32. Dayal Y, Goyal JL, Jaffery NF, et al. Lactate dehydrogenase levels in aqueous humor and serum in retinoblastoma. Jpn J Ophthalmol 1985; 29(4): 417–422.
33. Singh R, Kaurya OP, Shukla PK, et al. Lactate dehydrogenase (LDH) isoenzymes patterns in ocular tumours. Indian J Ophthalmol 1991; 39(2): 44–47.
34. Mukhopadhyay S, Ghosh S, Biswas PN, et al. A cross-sectional study on aqueous humour lactate dehydrogenase level in retinoblastoma. J Indian Med Assoc 2008; 106(2): 99–100.
35. Cheng Y, Meng Q, Huang L, et al. iTRAQ-based quantitative proteomic analysis and bioinformatics study of proteins in retinoblastoma. Oncol Lett 2017; 14(6): 8084–8091.
36. Cheng Y, Zheng S, Pan C-T, et al. Analysis of aqueous humor concentrations of cytokines in retinoblastoma. PLoS One 2017; 12(5): e0177337.

The authors
Benjamin K. Ghiam1 MD, Liya Xu2 PhD, Jesse L. Berry, MD*3,4 MD
1Oakland University, William Beaumont School of Medicine, Rochester, MI, USA
2Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA, USA
3The Vision Center at Children’s Hospital Los Angeles, Los Angeles, CA, USA
4USC Roski Eye Institute, Keck School of Medicine of USC, University of Southern California (USC), Los Angeles, CA, USA

*Corresponding author
E-mail: Jesse.Berry@med.usc.edu