The authors have no financial interest in this work

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Riboflavin/ UVA Combination For Acanthamoeba
Isolates. Evaluation of in Vitro Cysticidal
Antiamoebic Efficacy .

• J. A. Cristóbal, M. A. del Buey, P. Casas, E.
  Mínguez, L. Lavilla, E. Lanchares, C. Palomino.
• Clinical University Hospital, Zaragoza, Spain.

The authors have no financial interest in this
work
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INTRODUCTION
Acanthamoeba spp. has two morphological forms in
its life cycle, an amoeba or trophozoite stage,
that feeds and divides, and a resistant cyst
stage, that protects the amoeba from adverse
environmental conditions.
Cysts of Acanthamoeba.spp The cyst is the
resistance form, can be polygonal shaped and
shows a double wall and a nucleus.
Trophozoite of Acanthamoeba spp The trophozoite
is the mobile form, nucleus (N), contractile
vacuole (vc) and acanthopodia (A) can be seen in
the picture above.
Acanthamoeba Keratitis (AK) has always presented
a medical challenge for most ophthalmologists
because it is a frustrating and often devastating
infection. Recent in vitro studies have shown an
antimicrobial effect of riboflavin and
ultraviolet-A (UVA) collagen cross-linking (CXL)
on a number of bacterial and fungal pathogens,
but their effectiveness in vitro against protozoa
has not been proven in published studies.
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The aim of this work is to evaluate the in vitro
viability of two different strains of
acanthamoeba after UVA-CLX application. We
designed a method of assessing the growth of the
amoebae in a solid medium, which allows us to
reproduce the conditions of in vivo treatment.
METHODS

• Two different strains of Acanthamoeba were
  identically tested.
• Acanthamoeba sp 65 was used as an environmental
  amoeba isolated from superficial water.
• Acanthamoeba sp 7376 was used as a clinical
  strain isolated via corneal scraping for a
  patient with keratitis.

• Four groups of treatment were considered
• GROUP 1 0.1 riboflavin, 30-minute UVA
  irradiation.
• GROUP 2 0.1 riboflavin, 60-minute UVA
  irradiation.
• GROUP 3 (Control) no riboflavin, no UVA
  exposition.
• GROUP 4 0.1 riboflavin, no UVA exposition.

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METHODS
The experiments were performed three times with
each group.
The application of UVA was performed with the
same parameters used for in vivo corneal
collagen cross-linking.

The diameter of application in all cases
exceeded the limits of seeding of protozoa.

• The seeding plates for both species of amoebae
  for each group (including the control group) were
  evaluated after 24 hours of incubation at 30ºC
  after the treatment .
• If amoebae were observed around the seeding area,
  the migration radius (from the point of
  application of the amoeba to the point where
  trophozoites had migrated) would be measured to
  quantify the results for the different groups.

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RESULTS

• In all groups and for both isolates studied
  (environmental and pathogenic strains), cysts and
  trophozoites were detected at distances from the
  point of application greater than 5 mm after
  incubation at 30ºC for 24 hours, indicating the
  viability of the amoebae tested.
• Trophozoites were found at a similar distance
  from the point of application for all treatment
  groups and for the control group for both strains
  of amoebae (14-15 mm) the only exception was the
  group exposed to CXL and Riboflavin for 60 min
  (GROUP 2), for which the distance traveled was
  slightly lower (12 mm). After 72 hours, the
  trophozoites had spread across the agar in all
  groups.
• The morphology of the trophozoites at 24 hours
  after treatment was similar for the control and
  treatment groups. However, after 60 minutes of
  CXL application (GROUP 2), the degradation of
  some cysts was observed.

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RESULTS

• A) Point of Acanthamoeba application, diameter
  less than 5 mm.
• B) Riboflavin application and UVA radiation, 9 mm
  of diameter.
• C) After incubation at 30ºC for 24 hours,
  Acanthamoeba detection at distances from the
  application point greater than 5 mm (12-15 mm
  depending on group).

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RESULTS

• A) Untreated cysts of Acanthamoeba 7376
• B) Untreated trophozoites of Acanthamoeba 7376.
• C) Cysts of Acanthamoeba 7376 after treatment
  with rivoflavine and 60 minutes of Cross-linking.
  Note the degradation of some cysts with respect
  to the control.
• D) Trophozoites of Acanthamoeba 7376 after
  treatment with rivoflavine and 60 minutes of
  Cross-linking. Note the similar morphology to
  untreated control.

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DISCUSSION

• The efficacy of UV light as a disinfectant in
  contact lens, water or blood concentrates has
  been investigated in recent years. The
  eradication of E. Coli with UV-A requires much
  higher doses and application time than UV-B and
  UV-C. Nevertheless, UV-B (320-290 nm) and UV-C
  (290-200 nm) are highly damaging to DNA and are
  associated with a high risk of causing serious
  damage to cells and tissues. Thus, they are not
  used in humans.
• Our study was performed using the criteria for
  the UVA-riboflavin treatment of keratoconus
  described by Wollensak et al in 2003. Our results
  are consistent with those presented by Maya et
  al., who conclude that the total inactivation of
  amoebae requires higher UV doses than are
  necessary for bacteria.
• We must keep in mind that our study only assesses
  in vitro antiamoebicidal activity under levels of
  UVA radiation used in human therapy it does not
  assess the effectiveness of the same treatment
  with biological tissue that exhibits secondary
  changes such as edema or corneal melting.

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CONCLUSIONS

• In conclusion, given that 30 or 60 minutes of UVA
  exposure (3 mW/cm2, 370 nm) or riboflavin therapy
  is not effective against the two different
  acanthamoeba strains examined in our study
  (environmental acanthamoeba spp 65 and pathogenic
  acanthamoeba spp 7376), it seems reasonable to
  suppose that a longer exposure period could
  achieve the desired effect. However, such a
  treatment is not advisable due to toxicity and
  risk of tissue damage.
• In vitro results are not always indicative of in
  vivo efficacy thus, future studies should
  further evaluate this treatment for AK. Further
  studies using Acanthamoeba cultures on corneas
  may provide more information.

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REFERENCES

• Wollensak G, Spoerl E, Seiler T. Stress-strain
  measurements of human and porcine corneas after
  riboflavin-ultraviolet-A-induced cross-linking. J
  Cataract Refract Surg 2003291780-1785.
• Martins SA, Combs JC, Noguera G, et al.
  Antimicrobial efficacy of riboflavin/UVA
  combination (365 nm) in vitro for bacterial and
  fungal isolates a potential new treatment for
  infectious keratitis. Invest Ophthalmol Vis Sci
  2008493402-3408.
• Iseli HP, Thiel MA, Hafezi F, Kampmeier J, Seiler
  T. Ultraviolet A/riboflavin corneal cross-linking
  for infectious keratitis associated with corneal
  melts. Cornea 200827590-594.
• Maya C, Beltrán N, Jimenez B, Bonilla P.
  Evaluation of the UV disinfection process in
  bacteria and amphizoic amoeba inactivation. Water
  Sci Tech 20033285-291.
• Makdoumi K, Mortensen J, Crafoord S. Infectious
  keratitis treated with corneal crosslinking.
  Cornea 2010291353-1358.
• Khan YA, Kashiwabuchi RT, Martins SA, et al.
  Riboflavin and ultraviolet light A therapy as an
  adjuvant treatment for medically refractive
  acanthamoeba keratitis report of 3 cases.
  Ophthalmology 2011118 324-31.
• Ubomba-Jaswa E, Navntoft C, Polo-López MI,
  Fernandez-Ibáñez P, McGuigan KG. Solar
  disinfection of drinking water (SODIS) an
  investigation of the effect of UV-A dose on
  inactivation efficiency. Photochem Photobiol Sci
  20098587-595

• The authors gratefully acknowledge the research
  support of the Spanish Ministry of Education and
  Science through the research project
  DPI2008-02335.

madelbuey_at_telefonica.net
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