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Mycoplasma System plus: Description and Comparative Evaluation with
Conventional Methods for Identification and Susceptibility Testing of urogenital
mycoplasmas

DR. BROCCO S., DR. BROCCO F., DR.SSA DI PASQUALE A.
Laboratory “Clini.Lab.” - Roseto degli Abruzzi (Te) – Italy DR.SSA DEMETRIO F.
Liofilchem srl - Roseto degli Abruzzi (Te) – Italy INTRODUCTION
Mycoplasma hominis and Ureaplasma urealyticum are the most common isolates from the genital tract. They may be present as normal
flora in up to 40% of asymptomatic males and females. Together, M. hominis and U. urealyticum, have been implicated in pelvic
inflammatory disease, infertility, non-gonococcal urethritis, vaginitis, cervicitis, amnionitis, pyelonephritis, post-partum septicemia,
neonatal pneumonia, neonatal conjunctivitis, Reiter's syndrome, peritonitis, wound infections (C-section), low birth weight infants, and
premature rupture of membranes.
Furthermore susceptibility testing of mycoplasmas by traditional methods and of broth and agar dilution is non standardized and
extremely labor-intensive because the required material are not readily available from commercial sources.
To the aim at providing a complete outline of the presence of mycoplasmas in genital system of patients and diagnosing in the shortest
possible time the onset of pathogenic microorganism infections, a compact bacteriological screening test called MYCOPLASMA
SYSTEM plus was developed by LIOFILCHEM BACTERIOLOGY PRODUCTS (Roseto degli Abruzzi - Te - Italy).
That 24 well test, containing dry biochemical substratum and antibiotics, permits detection, presumptive identification and susceptibility
test of microorganisms in genital specimens such as vaginal swabs, urethral secretions and seminal fluid in 24-48 hours.
To evaluate the accuracy and utility of Mycoplasma System plus, we have compared this system with conventional methods in
identification and susceptibility testing of 247 clinical sample analyzed in patients coming to the Laboratory CLINI.LAB. srl (Roseto Degli
Abruzzi - TE – Italy) in a period of about one year, from January 2007 to December 2007.
MATERIAL AND METHODS
Clinical samples: 172 Vaginal swabs; 40 Urethral swabs; 35 Seminal liquid.
Bacterial strains: Ureaplasma urealyticum ATCC 27618; Mycoplasma hominis ATCC 23114;
Candida albicans ATCC 10231; Trichomonas vaginalis ATCC 30001
Each sample was tested concurrently in the Mycoplasma System plus and with traditional methods.
ycoplasma System p
lus : identification, semiquantitative count and susceptibility testing method.
The Mycoplasma System plus is a 24-well system containing desiccated biochemical and antibiotic substrates .
MYCOPLASMA SYSTEM Plus allows the detection, semi-quantitative count, presumptive identification and susceptibility test of Mycoplasma hominis and Ureaplasma urealyticum isolated from clinical samples and the detection and presumptive identification of the microorganisms most frequently isolated from vaginal and urethral swabs and seminal fluid, such as Trichomonas vaginalis and Candida spp. The 24 tests are indicated in the table n.1. Growth of mycoplasmas (102 < titre < 104 CFU/mL) Growth of mycoplasmas (104 < titre < 105 CFU/mL) Growth of mycoplasmas (titre > 105 CFU/mL) 6-TR/YE
Trichomonas vaginalis / Candida spp. The tray was inoculated according to the manufacturer's instructions.
All the well except 6-TR/YE well are covered with three drops of Vaseline oil for microbiological use and then incubated for 24-48 hours at 36+/-1°C.
The results are read following the indications indicated in the table n.2 Well colour
COUNT AND IDENTIFICATION OF MICOPLASMAS / UREAPLASMAS
Positive reaction
Negative reaction
Growth of mycoplasmas (102 < titre < 104 CFU/mL) Growth of mycoplasmas (104 < titre < 105 CFU/mL) Growth of mycoplasmas (titre > 105 CFU/mL) Arginine Test: identifies Mycoplasma hominis Urea Test: identifies Ureaplasma urealyticum DETECTION OF T. VAGINALIS AND CANDIDA spp.
Microscopic observation (40x)
T. vaginalis: mobile ciliated trophozoite Trichomonas vaginalis / Candida spp. Candida spp.: chlamydospores and hyphae SUSCEPTIBILITY TEST OF
Well colour
MYCOPLASMAS / UREAPLASMAS
IDENTIFICATION: Traditional methods
Mycoplasma test (ref.89005 LIOFILCHEM) is used to test the presence of Mycoplasma hominis and Ureaplasma urealyticum
(IDENTIFICATION).
Trichomonas broth (ref.20115 LIOFILCHEM) is used to test the presence of Thricomonas spp. and Candida spp.(IDENTIFICATION)
watching at the microscope 40x for the presence of mobile ciliated trophozoite (Trichomonas vaginalis) and for chlamydospores and
hyphae (Candida spp.).
SUSCEPTIBILITY TESTING: Broth microdilution method
The broth microdilution method was based on dilutions of antibiotics in Urea broth (SANOFI PASTEUR) for Ureaplasma urealyticum
and Arginine broth (SANOFI PASTEUR) for Mycoplasma hominis in a 96-well microtitre plate and inoculated with clinical sample of
organisms which were then incubated and observed for growth for 2-5 days.
Tetracycline, Pefloxacin, Ofloxacin, Doxycycline, Erythromycin , Clarithromycin, Minociclyne, Clindamycin, Azithromycin powders were
used to prepare fresh stock solutions for each assay.
The final concentrations are indicated in the table n.3.
Table n.3
Each clinical samples was suspended in Physiological Solution, and 104 UFC/mL bacterial suspension of Ureaplasma urealyticum ATCC 27618 and Mycoplasma hominis ATCC 23114 were prepared . 200 µL aliquots of these cultures were used to inoculate the wells of the microtitre plates. The inhibition concentration is the antibiotic concentration that doesn't allow the growth of mycoplasmas, and doesn't cause any change of medium colour.
SEMIQUANTITATIVE COUNT
The evaluation of semiquantitative count has been performed inoculating the system with several dilution of known concentration of
Mycoplasmas hominis ATCC 23114 and Ureaplasma urealyticum ATCC 27618 strains.
The tests have been performed in three different batch of the system Mycoplasma System plus.
POSITIVE WELLS IN
INOCULUM CONCENTRATION
MYCOPLASMA SYSTEM PLUS
Mycoplasmas hominis ATCC 23114 2X101UFC/mL Mycoplasmas hominis ATCC 23114 2X102UFC/mL Mycoplasmas hominis ATCC 23114 2X103 UFC/mL Mycoplasmas hominis ATCC 23114 2X104 UFC/mL Mycoplasmas hominis ATCC 23114 2X105 UFC/mL Mycoplasmas hominis ATCC 23114 2X106UFC/mL Ureaplasma urealyticum ATCC 27618 2X101UFC/mL Ureaplasma urealyticum ATCC 27618 2X102UFC/mL Ureaplasma urealyticum ATCC 27618 2X103UFC/mL Ureaplasma urealyticum ATCC 27618 2X104UFC/mL Ureaplasma urealyticum ATCC 27618 2X105UFC/mL Ureaplasma urealyticum ATCC 27618 2X106UFC/mL Ur. urealyticum ATCC 27618/ Myc. hominis ATCC 23114 4X103UFC/mL Ur. urealyticum ATCC 27618/ Myc. hominis ATCC 23114 4X104UFC/mL Ur. urealyticum ATCC 27618/ Myc. hominis ATCC 23114 4X105UFC/mL The obtained results have allowed to establish these ranges of mycoplasmas population: Growth of mycoplasmas (102 < titre < 104 CFU/mL) Growth of mycoplasmas (104 < titre < 105 CFU/mL) Growth of mycoplasmas (titre > 105 CFU/mL) IDENTIFICATION
VAGINAL SWABS
VAGINAL SWABS
MYCOPLASMA
MICRORGANISMS ISOLATED FROM POSITIVE VAGINAL SWABS
TRADITIONAL METHOD
SYSTEM plus
Ureaplasma urealyticum+ Mycoplasma hominis The results obtained with Mycoplasma System plus agree with those obtained using traditional culture methods URETHRAL SWABS
URETHRAL SWAB
MICRORGANISMS ISOLATED FROM POSITIVE URETHRAL SWABS
TRADITIONAL
MYCOPLASMA SYSTEM plus
Ureaplasma urealyticum+Mycoplasma hominis The results obtained with Mycoplasma System plus agree with those obtained using traditional culture methods.
SEMINAL LIQUID
SEMINAL LIQUID
TRADITIONAL
MYCOPLASMA
MICROORGANISMS ISOLATED FROM LIQUID SEMINAL
SYSTEM plus
Ureaplasma urealyticum+Mycoplasma hominis U reaplas ma urealy -tic um+My c oplas ma The results obtained with Mycoplasma System plus agree with those obtained using traditional culture methods.
CONTROL STRAINS
The microorganims Ureaplasma urealyticum ATCC 27618, Mycoplasma hominis ATCC 23114, Candida albicans ATCC 10231and
Trichomonas vaginalis ATCC 30001 have been tested with traditional methods and in three different batches of Mycoplasma system
plus. The results obtained with Mycoplasma System plus agree with those obtained using traditional culture methods.
SUSCEPTIBILITY TESTING
The clinical samples previous identified, have been also tested for susceptibility testing with traditional method and in Mycoplasma system plus. In the table n° 5 are indicated the resistance data . Table n°5 RESISTANCE DATA OBTAINED WITH TRADITIONAL METHOD AND MYCOPLASMA SYSTEM PLUS n.samples
86
18
17
121
RESISTANCE
3,3 2,47 3,3 2,47
8,26 1,65 0,82 39,6 37,2 35,5 32,2 0,82 0
4,1 29,7 26,4
PERCENTAGE %
RESISTANCE
3,3 2,47 3,3 2,47
8,26 1,65 0,82 39,6 37,2 35,5 32,2 0,82 0
4,1 29,7 26,4
PERCENTAGE %
CONTROL STRAINS
The results obtained with Mycoplasma System plus agree 100% with those obtained using traditional culture methods.
CONCLUSION
The results obtained with Mycoplasma System plus match perfectly with those obtained with traditional methods; these considerations make this system simple to perform, easy to handle, a suitable alternative for identification and susceptibility testing of urogenital mycoplasmas.
References
1) Santacroce G., Santacroce F., Prevalenza di Mycoplasma hominis ed Ureaplasma urealyticum in una popolazione afferente alla
AUSL n.5 della regione Marche
. 1997, Microbiologia Medica n.3 Vol.12/97.
2) Ciannamea B., Leo G., Megha M., Miragliotta G., Incidenza di infezioni urogenitali da micoplasmi in pazienti ambulatoriali AUSL LE/I,
Lecce
. AMCLI 2003, Poster M 087.
3) Daghetta L., Ricci S., Ureaplasma urealyticum: epidemiologia ed antibiotico-resistenza. AMCLI 2003, Poster M 057.
4) Masala L., Baghi G., Floris B., Infezioni da micoplasmi in campioni genito-urinari:epidemiologia ed antibiotico-resistenza. AMCLI
2003, Poster M 039.
5) Bruno R., Negro C., Ghigonetto T., Neri R., Incidenza di micoplasmi nel secreto cervico-vaginale. 1998, Microbiologia Medica n.2
Vol.13/98.
6) Restelli A., Scararazatti M., Beghi G., Casalone A., Clerici P., Frequenza di isolamento e sensibilita' agli antibiotici dei micoplasmi
urogenitali. 1998, Microbiologia Medica n.2 Vol.13/98.
7) Forbes B.A., Sahm D.F., Weissfeld A.S., Bailey & Scott's Diagnostic Microbiology. 2002, Mosby, pp. 290-304.
8) Murray P.R., Manual of Clinical Microbiology. Sixth Edition, 1995, American Society for Microbiology, pp. 273-274, 374-375, 652-662.
.
15.10.2008

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