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ACTA
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MICROBIOLOGY
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MAILING ADDRESS OF JOURNAL: ACTA MICROBIOLOGICA HELLENICA


G. KATSANIS, K. TZANETOU, ASCENT, 77 VAS. SOFIAS AVE, GR-115 21 ATHENS, GREECE,
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OFFICIAL PUBLICATION OF THE GREEK SOCIETY OF MICROBIOLOGY
ACTA
MICROBIOLOGICA
HELLENICA
Contents
BIMONTHLY JOURNAL
OF THE REVIEWS
GREEK SOCIETY OF E. Xylouri Fragkiadaki: Avian influenza. Review of the recent
MICROBIOLOGY epizootic outbreak and its importance for public health.................................. 403

E. Kontakioti, E. Diza-Mataftsi: Epidemiology of syphilis,


gonorrhea and chlamydial infections............................................................... 434
ORIGINAL PAPER
. tZIMOGIANNI, .. ZATZARAKIS, . xAGORARI, . cHARVALOU,
cH. lIAPI, p. gALANOPOULOU, . pETRIKKOU, . KARAGEORGOU,
. ALKANI, g. pETRIKKOS: Water-soluble nystatin complexes:
ntifungal activity against Candida spp. and cellular toxicity......................... 449

INSTRUCTIONS TO AUTHORS . ............................................................. 455

VOLUME
51
NUMBER
6
November
December
2006






. :
........ 403
. , . -: ,
............................................. 434


. , .. , . , . ,
. , . , . , . ,
. , . :
. In vitro
............... 449

.......................................................... 455


51

6


2006
( 2006)



2006. ,
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from Vaccinated Animals), .
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404 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

. ,

51, -
.
2006, 51 (6): 403-433

,
,

, , 7

(. 2, 3).
. -
.8
(), La
Grippe, ,
gripan,
. (influenza) -
.1 ,
,
,
. , influence
, .
1918.9
,
412-414 .., -
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.
.

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19
51, 6 ( - 2006) 405

1. ,
Capua Mutinelli, 200151
/
1959 2 A/chicken/Scotland/59 (H5N1)
1963 29.000 A/turkey/England/63 (H7N3)
1966 8.100 A/turkey/Ontario/7732/66 (H5N9)
()
1976 25.000 A/chicken/Victoria/76 (H7N7)
() 17.000, 16.000
1979 1 600.000 , 80 A/chicken/Germany/79 (H7N7
1979 3 A/turkey/England/199/79 (H7N7)
1983- 17 452 A/chicken/Pennsylvania/1370/83
1985 ()* , (H5N2)

1983 800 A/turkey/Ireland/1378/83 (H5N8)
8.640, 28.020,
270.000
1985 24.000 , 27.000 A/chicken/Victoria/85 (H7N7)
() , 69.000
, 118.418

1991 8.000 A/turkey/England/50-92/91 (H5N1)
1992 12.700 , 5.700 A/chicken/Victoria/1/92 (H7N3)
()
1994 Queensland 22.000 A/chicken/Queensland/667-6/94
() (H7N3)
1994- * 360 A/chicken/Puebla/8623-607/94 (H5N2)
1995
1994 * 3,2 A/chicken/Pakistan/447/95 (H7N3)

1997 1,4 A/chicken/Hong Kong/220/97 (H5N1)
()
1997 New South 128.000 , A/chicken/New South Wales/1651/97
Wales 33.000, 261 (H7N4)
()
1997 6.000 , , A/chicken/Italy/330/97 (H5N2)
, , , ,
,
1999- * 413 14 A/turkey/Italy/99 (H7N1)
2000
2002- * , , , , A/chicken/East Asia/2003-2005
2005 , , , , (H5N1)
, 150 .

2002 A/chicken/Chile/2002 (H7N3)
2003 * : 255, 30 . A/chicken/Netherlands/2003 (H7N7)
: 8, 3.
: 1, 80.000
2004 (B.C.)* 53 , 17. A/chicken/Canada-BC/ 2004 (H7N3)
2004 () 6.600 A/chicken/USA-TX/2004 (H5N2)
2004 23.700 , 5.000 A/ostrich/S.Africa/2004 (H5N2)
* , . -
.
406 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

16
9

1.
.

2. -
.
: Avian Influenza in Humans-2004, Silvio Pitlik,
(http://abush.health.gov.il/download/pages/shapahat_of_adam.ppt)

20 , 1890-1892 . 20
1918 , 500
.11. ,
1918-1919, . ,
- 80%
51, 6 ( - 2006) 407

2. (H5N1) (felidae
civets). WHO, 2006

1970 & 1980 H3N2 ,
73 77 ,

.
2003
. 51
.
.
2004 3 51 -
Kasetsart University, . 15
14 .
2004 H5N1
.
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51,
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2006 51 .
28 2006 H5N1 Ruegen ()
, 100 .

3.
1 2 3 4 5 6 7 8 9
1 / / / / / /
2 / / /
3 / / / / / / / / / / /
4 / / / / /
5 / / / / /
6
7 / / / / / / / /
8
9 / / /
10 /
11
12
13 / /
14
15
16
*=, =, =, =, =, = , = ,
=
408 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

,
. 11,
1957. ,
. ,
, 1878 Per-
roncito, , , 16 (. 2).
,
-Fowl Plaque. , , , -
1955, , , , , ,
.16
.
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77.

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100 ( 1918, 1957
1968) 5 , (A, B, C),

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12,13 (. 2). ), -
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, , . ,
14
,
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H5N1
Guangdong ,
1996 A/goose/Guangdong/1/96
RNA , (H5N1).17 ,
Orthomyxoviridae ()
influenza virus. Hong Kong A/HK/156/97 (H5N1).15
R/ss: 7-8 5/L- : Se/E:V/R.10
(myxovirus)
1955 , ( ) ( -
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. :
()
(Orthomyxoviridae), .
(Paramyxoviridae), O
,
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Pathogen Index, IVPI).
, , , :
, .. - 4-8 , -
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(. 1). , ,
51, 6 ( - 2006) 409


.

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.18

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3. (,
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, , [
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500
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4. .
RNA (negative stranded : Avian Influenza in Humans-2004, Silvio Pitlik,
4.
(http://abush.health.gov.il/download/pages/shapahat_of_
RNA viruses). m-RNA
RNA adam.ppt)
RNA-
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glutinin Antigen, HA )
. (Neuraminidase Antigen, NA ).
C 16
(, 1-16) 9 (,
.16 1-9) (. 3,4).
410 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

, ,
. - pH, ,
. .
,
5 .
Sodium Dodecyl Sulphate (SDS)
RNA Sodium Desoxycholate (SDC).
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412 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

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66 29/11/2005
65 23/11/2005 , -

64 16/11/2005 -
63 9/11/2005 -
62 26/10/2005 -
61 24/10/2005 -
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59 23/10/2005 -
58 20/10/2005 -
57 20/10/2005 -
56 20/10/2005 -
55 19/10/2005 -
54 15/10/2005 -
53 13/10/2005 -
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43 21/7/2005 -
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41 8/6/2005 -
40 4/5/2005 1 -
39 30/4/2005 -

414 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

/
38 12/4/2005 1 -
37 29/3/2005 1 -
36 2/2/2005 -
35 6/1/2005 2 -
34 30/12/2004 1 -
33 2004 -
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30 2004 -
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26 11/10/2004 - 147

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416 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

. , ,
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,

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civetta, Viverra zibetha) .

418 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

77, .76
.68 ,
, - .

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, 51
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420 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

,
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422 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

,
(NEW- -
FLUBIRD, Network for early warning for influenza .
viruses in migratory birds in Europe)
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( ) 0,1% sodium
silicate



2% w/v 10






0,2% w/v 30 pH



2% w/v 10-30 ,
pH 9


424 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

5. -
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6. - .
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7. ( . ,
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426 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

Reyes. . ,
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430 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

summary
Avian influenza. Review of the recent epizootic outbreak and its importance for public health
E. Xylouri Fragkiadaki
Dept of Anatomy & Physiology of Farm Animals, Faculty of Animal Science, Agricultural University of Athens,
Athens, Greece
In this review article, a detailed description of avian influenza is presented, concerning both its
natural hosts and related susceptible animal species, and emphasizing its effect n public health
and its protection. The etymology of influenza, the historic background of the disease and the
aetiological agent of the disease are analyzed (i.e., nomenclature of influenza viral strains, viral
morphology and structure, environmental resistance, the characteristic antigenic drift and shift
of the influenza virus and its effect on the host immune response). The recent epizootic avian
influenza outbreak of 1996-2006 in Asia is comprehensively presented. Epizootic data on both
the global and the national (Greek) distribution of H5N1 and the other type A influenza infec-
tions in all susceptible animal species are provided. The clinical signs, macroscopic lesions
and laboratory diagnostic measures are described and preventive-surveillance measures for the
control of the disease in animal populations are presented (New-FLUBIRD programme, bios-
ecurity measures, bird euthanasia). Special reference is made to vaccination strategies against
avian influenza and to DIVA (Differentiating Infected from Vaccinated Animals) vaccines,
targeting influenza eradication in the avian population. The importance of avian influenza on
public health is emphasized, by setting questions and triggering thought about the possibility
of influenza transmission from birds to humans. Reference is made to the relationship between
avian influenza and human influenza cases observed during the recent epizootic outbreak, to
the protective measures and hygiene recommendations for farm and slaughterhouse employees
and for consumers. The biosecurity measures for all poultry-derived food (eggs, poultry meat)
and products (raw chicken products, products derived from birds vaccinated against influenza)
are presented, and the consumption of well-done cooked poultry-derived products is strongly
recommended. Conclusions are presented, along with the four Good Practice golden rules,
formulated by the World Health Organization, for consumers to protect themselves from influ-
enza H5N1 viral infection.
Acta Microbiologica Hellenica 2006, 51 (6): 403-433

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434 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

,

. , . -

-
340 12
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1943 1985 (11,4 66,4 -
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436 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

13,3/100.000 ,11 -
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2. , 1941-2003.*
438 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

1998 ( 15%) -
.16 19 (. 3).
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*Hopkins, S et al. Sex Transm Infect 2001;77:316-318

5. 1997-2001.*
440 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

3 .22 -
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1997-1998
(. 1).
442 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

1. 1973-
1998*


1973 551 418 133 285 255 30
1974 373 294 79 256 229 27
1975 318 232 86 93 84 9
1976 450 319 131 57 46 11
1977 426 319 107 56 51 5
1978 392 304 88 32 26 6
1979 437 360 77 33 29 4
1980 397 329 68 16 15 1
1981 360 283 77 16 13 3
1982 444 318 126 14 13 1
1983 290 225 65 15 10 5
1984 297 212 85 9 9 -
1985 286 228 58 12 9 3
1986 293 220 73 10 9 1
1987 222 158 64 14 10 4
1988 191 133 58 13 8 5
1989 169 115 54 3 2 1
1990 204 131 73 11 7 4
1991 199 117 82 6 3 3
1992 191 114 77 7 3 4
1993 215 125 90 4 3 1
1994 182 120 62 6 4 2
1995 186 110 76 5 3 2
1996 231 139 92 25 19 6
1997 257 - - 24 - -
1998 309 - - 32 - -
* , 1973-1989

16,4% , 8,7% ,
6,5% ,
0,8% . , - ,
34 600.000 .33
22 100 16-19
/100.000 , 25-34 ,
79% 1998. -
( 7,4%) 34 (. 2).
( 10,5%)
( 11,3%,
.
13,5%, 15,9%,

17%
19,8%).32 1985-1996 -

.7 - . 1985-1990
13% 1990-1996 10%

. ,
1990-1996,
.32 15%.17 1994
51, 6 ( - 2006) 443

, , 2000
, . 10 /100.000
. , , , -
1991-1996
87% , , 600 /100.000
18% , 17
100% -
51% . - ,

, , ,
, 30 (. 2).
.
( 90%)
.
( 52%).35

1997
1962 1976 -
1998-1999.
19971998
170% 46%. .
- 20-29
102% 1995-2000 .26
1999-2000, 1974-1981
16-19 ,
178% 133%. 1996.36,37
154% 1995-2000,
,
ADS
1998-1999. . 98,6%
.15 18-39 1,43%

2.


1985-1990 1990-1995
-17,3 -12,5
-16,0 -15,2
-14,3 -2,4
-16,6 -9,8
-13,5 -15,1
-13,5
-13,6 -13,6
-14,3 -11,4
-17,0 -12,9
-9,5 -7,8
-2,6 +15,0
-14,8
-17,0 -12,6
-1,4 -6,7
(-)
(+)
444 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

. 7,3% .
14,4%
. 1981-1984


1980
-
.7
, ,
1987-1995

281% (
.36 1985
47,8 182,2 /100.000 )
25 100.000 28
-
1986-1989
22 48.
-

.37
1988 1995
1990
. 1995 182,2 -
-
/100.000 .
.38
46,4 622 100.000
1989 1996


.
5.831 -
4% < 14 ,
369 , 355
46% 15-19 , 33% 20-24
34 ( 6,3%
17% >25 .
STDs).

-
(screening tests)
.
.


, -
.
.29 1997



,
.39 1996

500.000 ,
AIDS,
3
.38
. 1997 -

2,5-3,2
1973 1996 -
10-44 7 (. 2).


(. 1). .

-

,

.

-
20-25 . -

4 .
,


-
1990.1
,

.6

51, 6 ( - 2006) 445

- 1990, 2001-2002
4-6 14%. 84,2% -
. 1995 1996,
2001 245
. , 1996 1649 2001. 2002

.17 276/100.000 ( 160/100.000
1991-1996 - 1997) 2001-2002
2% 11%.1 2003
9% 384.000 (25% )
- 13% 15-29 .40
.17
1988
, -
. 1 ( 7).
15-29
,
.


.40 1989
350 100.000 -
1993-1997 160 /100.000
. . -
1986-1998
1989-1995 1989 1996
3-4/100.000
. 6.4% STDs (375
5.831 , 312 63
7.
1989-2003*
100.000



-
*Low N. Eurosurveillance 2004;8(41)
7. 1989-2003.*
446 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

).
. .42 1998
2004
17.869 :
. , 16.834 18-55 1.035 18-65
.
3,5% (4,1%
. 2,9% )
- 11,2%
.29 1989 .
268 18-35 , 193 88% 30 , 71%
.
91%
7,2% . -
10,6% .41
1991 14%
81,5% -
43,5% .43

Summary
The epidemiology of syphilis, gonorrhoea and chlamydial infections
E. Kontakioti, E. Diza-Mataftsi
A Dept of Microbiology, Medical School, University of Thessaloniki, Greece
Sexually transmitted infections (STI) are a major public health problem. It is estimated
that 340 million new cases of curable STIs occur annually, 12 million of which
concern syphilis. In the USA the incidence of primary and secondary syphilis declined
between 1943 and 1985 from 66.4 to 11.4 cases/100,000 population. Since 1987 an
increase has been reported, which peaked in 1990 with 20 cases/100,000. The upsurge
presented a specific socio-geographic distribution, affecting especially urban areas,
southern areas and black heterosexuals. During the period 1990-2000 the incidence
of syphilis decreased, but then increased again between 2001 and 2002 from 2.2 to 2.4
cases/100,000, with a 3.5% higher increase among men than women, which suggests
that mainly heterosexuals are affected. In Europe the reports of STIs are characterized
by heterogeneity. Generally the incidence of syphilis remained stable through 1990
(in Germany 2 cases/100,000, in England 100 new diagnoses per year and in France
approximately 40 cases per year). Since 1999 an upsurge has been observed, affecting
homosexuals. In England the new cases annually in 1995 to 2000 increased from 132 to
326, in France new cases increased from 37 in 2000 to 428 in 2003, while in Germany
they increased from 1,687 to 2,932. In Athens the prevalence of Syphilis remained stable
from 1974 to 1991, followed by a drop since 1996. Cases of Gonorrhoea are estimated
to be 70-200 million worldwide. During the period 1970-1980 in the USA the incidence
of gonorrhoea decreased, and between 1981 and 1996 the rate declined by 71.3%, then
a slight increase was observed between 1997 and 1998. In Europe the incidence of
51, 6 ( - 2006) 447

gonorrhoea declined during 1985-1996 in most developed countries, but an increase


has been observed since 1997. During 1997-1998 in France newly diagnosed cases
increased 170% while between 1995 and 2000 cases increased 102% in England and
Wales and 154% in Sweden. In Athens during 1974-1981 the numbers of new cases
were stable and have continued a gradual decline since 1996. Chlamydial infections
are the most prevalent bacterial STI. In the USA during 1987-1995 their annual rate
increased 281% (from 47.8 to 182.2 cases/100,000) while in 1996 500,000 new cases
were reported, although it is estimated that the actual number reached 3 million. The
reporting of chlamydial infections is not mandatory in most EU countries. Since the
end of 1990 the cases of chlamydial infections decreased in Sweden, Norway, Ireland,
and England and Wales, followed by an accelerating increase during 2001-2002.
Acta Microbiologica Hellenica 2006, 51 (6): 434-448

ondary syphilis---United States, 1981-1990. MMWR


1991, 40(19):314,321-23
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21. Hopkins S, Lyons F, Mulcahy F, Bergin C. The great MMWR 2000, 49(24): 538-543
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Hopkins S, OLorcain P et al. The epidemiology of 2(3):188-189
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surveillance 2004, 9(12):9-10 Stroobant A, and the European Study Group Trends
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lance and trends of the syphilis epidemic in Germany 1991 6. Sex Transm Inf 2000, 76:110-116
since the mid-90s. Eurosurveillance 2004, 9:9-10 36. Georgala S, Schulpis K, Georgala C, Karikas G. Gon-
24. Fenton KA, Imrie J. Increasing rates of sexually orrhoea control programme in Athens, 197498. Sex
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160 Flemetakis A, Frangouli E. Current characteristics of
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syphilis and chancroid in attendees at a sexually 42. Kalogeropoulou A, Frantzidou F, Klearchou N, Diza
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Dermatology 2003, 42:876- 81 chomatis in infertile Greek women. A serologic and
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51, 6 ( - 2006) 449


.
In vitro

. ,1 .. ,2 . ,3 . ,1 . ,4
. ,4 . ,5 . ,5 . ,1 . 5

-
.1,2
-
.
, . - -


Candida Aspergillus.

,
, 4
(PVP)
, 10 kDa (NC1), 24 kDa (NC2), 40
, - kDa (NC3) 360 kDa (NC4), -
. .3
, 0,25% w/w -
1:3338 mole :base mole
( ). PVP 0,5% w/w 1:1669
, - mole : base mole PVP
LD50~200 mg/kg .
.

-


, , -
1
, , 10 ppm. , -
, , ,2 -, ... ,4 pH tris buffer
, ,3 pH=7,
, ,4

.. 75%
, 5 25%
450 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

pH.
6 C -30 C 61 . O NCCLS7 (National
Committee for Clinical Laboratory Standards) M27-
. A2 (2002): Candida krusei ATCC 6258, Candida
parapsilosis ATCC 90018, Candida albicans ATCC
90028, Candida parapsilosis ATCC 22019.

In vitro
()
-
.5 NC3 (40 kDa)
-
.6 Candida albicans p 4,5
- ( ).
J774
. in vitro (MIC),
Candida, .6
1 2 g/ml. Sabouraud broth
RAW264.7 2 x 104 CFU/ml 24 .
,
, -
, 2,5, 5, 10, 20 .
DMEM
(10% ) 37 C 5% CO2
6 . 1 % -
10 g pH 4 pH 7. -
37 C 1 . 24 kDa
570 nm. 40 kDa.

J774 J774
H LDH
J774 , - J774, -
LDH 40 g/ml
, 40 g/ml
.5 NC1 (10kDa)
, 37 C NC3 (40 kDa) (. 1).
95% air/ 5% CO2, RPMI 1640 6 -
FCS (foetal calf serum). .

In vitro
(MIC)
T -
(MFC) -
. ,
4 ,
12 Candida spp - .
.
51, 6 ( - 2006) 451

pH~7 %%

61
61 30-30oC
C
pH~4
70 58,3 56,0
60
%

50
40
30 20,1
20 13,0
4,8 4,9 5,3 6,5
%

10 4,0 1,3
0
Nystatin Nys-PVP 1 Nys-PVP 2 Nys-PVP 3 Nys-PVP 4
(10 kDa) (24 kDa) (40 kDa) (360 kDa)
1. % 61
-30 C pH.

1. LDH SD J774,

NC1 NC2 NC3


LDH%SD 31.0335.864 14.8473.774 11.179 5.161 10.5740.912 7.2265.904

In vitro
.
4
MIC.
-PVP (. 2).
(%)

2,5 5 10 20
(mg/l)
2. -PVP: NC1, NC2, NC3, NC4 , -
.
452 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

MIC CFU/ml
Candida albicans 4.7
2 . MFC x105 CFU/ml. p 4,5, 24
-
MIC (. 2). 2,0 x 104 CFU/ml, 40 kDa
- 1,9 x 104 CFU/ml (. 3). 2, 4
8
In vitro
40 kDa .
(MIC) NC3
p
(40 kDa)
.
40 kDa
p 7,0 ,
MIC. 24

- 1,8 x 104 H -

2. MIC MFC ( g/ml)


Candida (Candida spp)
NC1 NC2 NC3 NC4
MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC
C. krusei ATCC 6258 4 4 16 16 4 8 4 8 2 4
C. parapsilosis ATCC 90018 4 4 16 16 4 8 4 8 16 16
C. albicans ATCC 90028 4 4 16 16 4 8 4 8 2 4
C. parapsilosis ATCC 22019 4 4 16 32 4 8 4 8 2 8
C. albicans 6451 4 4 16 16 4 8 32 32 2 4
C. albicans 6508 4 4 16 32 4 16 32 32 32 32
C. albicans 6401 >32 >32 32 32 8 32 32 32 32 32
C. albicans 6413 4 4 16 16 8 8 32 32 2 4
C. albicans 6738 4 4 16 32 8 8 8 8 4 8
C. albicans 6617 4 8 16 32 8 8 8 8 4 8
C. albicans 6625 4 8 16 32 8 8 8 8 4 8
C. albicans 6645 4 8 16 32 8 8 8 16 8 8
C. glabrata 5140 4 8 16 32 8 8 8 8 2 8
C. krusei 54554 4 8 16 32 8 8 8 8 2 8
C. lusitaniae 58622 4 4 16 16 4 8 4 8 2 4
C. parapsilosis 54286 4 8 16 32 8 8 8 16 4 16

3. NC3 (40kDa) Candida albicans 24 p 7


4x MIC
pH 7 0 (cfu/ml) 24 (cfu/ml)
(control) 1,8 x 104
4,8 x 106
1,8 x104 0
NC3 1,8 x 104 0
pH 4,5 0 24
(cfu/ml) (cfu/ml)
2 x 104 4,4 x 104
2 x 104 0
NC3 2 x 104 2 x 10
6
51, 6 ( - 2006) 453

, ,
-
.8 .
, .

,
. ,
- NC3 (40kDa),
Candida
- PVP 24 kD 40 kD spp. .
-
J774 . -

RAW264.7
- ,
-
. MIC .

Summary
Water-soluble nystatin complexes: ntifungal activity against Candida spp. and cellular toxicity
. tZIMOGIANNI,1 .. ZATZARAKIS,2 . xAGORARI,3 . cHARVALOU,1 cH. lIAPI,4
p. gALANOPOULOU,4 . pETRIKKOU,5 . KARAGEORGOU,5 . ALKANI,1 g. pETRIKKOS5
Technical Occupational Institution, Dept. of Medical Sciences1 Center for Toxicological Sciences and
Research, Medical School, University of Crete, Herakleion, Crete2 Hematology Clinic, General Hospital
Papanikolaou, Thessaloniki,3 Laboratory of Experimental Pharmacology, Medical School, University
of Athens,4 Laboratory for Research of Infections and Antimicrobial Chemotherapy G.K. Daikos, A
Propedeutic Clinic, Medical School, University of Athens, Athens, Greece
Four types of water-soluble polyvinylpyrrolidone (PVP) complexes of nystatin were prepared and
their biological activity was compared with that of free nystatin against 16 strains of candida
(C. albicans, C. glabrada, C. krusei, C. lusitania and C. parapsilosis). The prepared nystatin
complexes were pure substances, free from contaminants, with relatively simple, fast and cheap
production process and they were soluble in aqueous solutions. The molecular weight of nystatin
formulations NC1, NC2, NC3 and NC4 were 10 kDa, 24 kDa, 40 kDa and 360 kDa respectively.
Susceptibility testing for yeasts was performed according to the National Committee for Clini-
cal Laboratory Standards guidelines (NCCLS MA 7A 1997). MICs of nystatin complexes were
equal or slightly higher compared to those of nystatin, (with NC2 showing the lowest MICs
and MFCs) against Candida spp. Cytotoxicity was evaluated by measuring the release of LDH
from J774 macrophages after 4 hours incubation with 40 ug/ml of each compound. J744 cells
were cultured at 37C in 95%air/5%CO2 atmosphere in RPMI 1640 medium supplemented with
10% foetal calf serum. LDH release was considerably lower (up to 60% lower compared to
nystatin) for the complexes than for nystatin in J774 macrophages. MTT-formazan test showed
that the cytotoxic activities of the complexes on RAW264.7 cells at low levels of the antifungal
454 Acta Microbiologica Hellenica Volume 51, Number 6 (November - December 2006)

(1-2 ug/ml) did not affect the viability of the cells more than the liposomal amphotericin. The
haemolytic activities of nystatin and of the two complexes 24 kDa and 40 kDa were increasing
by increasing the concentration whereas the activity of liposomal amhotericin B did not. We
conclude that the complexes 24 kDa, 40 kDa represent potential antifungals for superficial
mycoses and for the prevention of gastrointestinal clonisation from Candida. Extended studies
on pharmacokinetic and pharmacodynamic properties of the complexes represent an important
task to accomplish.
Acta Microbiologica Hellenica 2006, 51 (6): 449-454
The present is co-funded by the European Social Fund and National Resources - (EPEAEK Archimedes)

5. Charvalos E, Tzatzaraki M, Van Bambeke F, Tulkens


PM, Tsatsakis A, Tzanakakis A et al. New water-solu-
1. Alifragis J, Rizos AK, Tsatsakis AM, Tzatzarakis M, ble amphotericin B-polyvinylpyrrolidone complexes
Shtilman M. New polymeric systems with controlled with maintained antifungal activity against Candida
release action: A light scattering investigation. J Non- spp. and Aspergillus spp. and reduced cytotoxicity
Crystall Solids 2002, 307-310: 882-886 toxicity towards J774 macrophages. JAC January
2. Charvalos E, Tsatsakis A, Tzatzarakis MN, Dolapsakis 2006
G, Stiakakis J. ew nystatin polymeric complexes 6. Espuelas S, Legrand P, Loiseau PM, Bories C, Barratt
and their in vitro antifungal evaluation in a model G, Irache JM. In vitro reversion of amphotericin B
study with Fusarium oxysporum. Mycopathologia resistance in Leishmania donovani by poloxamer 188.
2001a, 153(1):15-19 Antimicrob Agents Chemother 2000, 44(8):2190-2
3. Charvalos E, Liapi C, Tsatsakis A, Tzatzarakis M, 7. National Committee for Clinical Laboratory Stan-
Galanopoulou P, Koutis C. New nystatin polymeric dards. M27-A. Reference method for broth dilution
complexes and their in vitro antifungal evaluation in antifungal susceptibility testing of yeasts. Approved
a model study with Fusarium oxysporum. Abstract standard M27-A. National Committee Laboratory
136.10. XIVth World Congress of Pharmacology, Standards, Wayne, Pa 1997
Meeting Abstracts, Pharmacologist 2002, (Supple- 8. Tsatsakis AM, Tzatzarakis MN, Charvalos E, Samonis
ment 1) Vol 44, (2) G. Slow release antifungal agents: Novel water soluble
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G. Controlled release of water soluble polymeric amphotericin B. Patent, (Greek Organization
complexes of sorbic acid with antifungal activities. Industrial Property), 2000, Athens, Application No
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