Tài liệu Prevalence of antimicrobial resistance of neisseria gonorrhoeae in national hospital of dermatology and venereology in 2017 – Le Van Hung: JMR 116 E3 (7) - 2018 125
JOURNAL OF MEDICAL RESEARCH
PREVALENCE OF ANTIMICROBIAL RESISTANCE OF NEISSERIA
GONORRHOEAE IN NATIONAL HOSPITAL OF DERMATOLOGY
AND VENEREOLOGY IN 2017
Le Van Hung, Nguyen Phuong Thuy, Le Ha Long Hai
Hanoi Medical University
The antimicrobial resistance of Neisseria gonorrhoeae is a complicated problem that causes many diffi-
culties for diagnosis and treatment. Thus, more research into all aspects of antimicrobial resistance of N.
gonorrhoeae is necessary. Objectives: 1. To determine the prevalence of N. gonorrhoeae infections in pa-
tients with urethral and/or vaginal discharge syndrome, who attended National Hospital of Dermatology and
Venereology in 2017. 2. To describe the antimicrobial resistant levels of isolated N. gonorrhoeae. Methods:
This was a cross sectional study. N. gonorrhoeae was isolated from pus or discharge of 1582 patients with
urethral and/or vaginal discharge syndromes. Isolates were determined by using Gram stain...
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JMR 116 E3 (7) - 2018 125
JOURNAL OF MEDICAL RESEARCH
PREVALENCE OF ANTIMICROBIAL RESISTANCE OF NEISSERIA
GONORRHOEAE IN NATIONAL HOSPITAL OF DERMATOLOGY
AND VENEREOLOGY IN 2017
Le Van Hung, Nguyen Phuong Thuy, Le Ha Long Hai
Hanoi Medical University
The antimicrobial resistance of Neisseria gonorrhoeae is a complicated problem that causes many diffi-
culties for diagnosis and treatment. Thus, more research into all aspects of antimicrobial resistance of N.
gonorrhoeae is necessary. Objectives: 1. To determine the prevalence of N. gonorrhoeae infections in pa-
tients with urethral and/or vaginal discharge syndrome, who attended National Hospital of Dermatology and
Venereology in 2017. 2. To describe the antimicrobial resistant levels of isolated N. gonorrhoeae. Methods:
This was a cross sectional study. N. gonorrhoeae was isolated from pus or discharge of 1582 patients with
urethral and/or vaginal discharge syndromes. Isolates were determined by using Gram staining, culture, and
identification test, followed by antimicrobial susceptibility method. Minimum inhibitory concentration (MIC)
was performed to differentiate the between resistant and susceptible isolates. Results: The prevalence of N.
gonorrhoeae in patients with urethral and/or vaginal discharge syndrome was 12.45%; the isolates of N.
gonorrhoeae showed resistance to ciprofloxacin (97.97%), nalidixic acid (85.79%), penicillin (45.69%) and
tetracycline (85.28%). N. gonorrhoeae was susceptible to spectinomycin (100%), azithromycin (99.49%),
cefotaxime (99.49%), cefixime (99.49%) and ceftriaxone (98.98%). As for MIC results: 7/12 isolates showed
its resistance to ciprofloxacin (MIC ≥ 1 àg/ml), 7/10 to tetracycline (MIC ≥ 2 àg/ml), 5/10 to penicillin (MIC ≥ 2
àg/ml), 2/17 to ceftriaxone (MIC ≤ 0,25 àg/ml) và 100% of isolates were susceptible to spectinomycin (MIC ≤
32 àg/ml). Conclusion: Ceftriaxone, spectinomycin, azithromycin, cefixime and cefotaxime were recom-
mended for N. gonorrhoeae treatments while ciprofloxacin, cefuroxime, tetracycline and penicillin were not.
Keywords: Neisseria gonorrhoeae, antimicrobial resistance
Corresponding author: Le Van Hung, Hanoi Medical
University
Email: levanhungvdl@yahoo.com
Received: 9/7/2018
Accepted: 22/11/2018
I. INTRODUCTION
Neisseria gonorrhoeae infection is a com-
mon sexually transmitted disease (STD). Ac-
cording to The World Health Organization re-
port, it was estimated there were 106 million
new cases of gonorrhea in 2012 [1]. Each year
at the National Hospital of Dermatology and
Venereology, more than 20% of all STD cases
are diagnosed as gonorrhea. Gonorrhea may
spread within a community and cause many
different genitourinary tract complications,
even infertility, if it is not correctly diagnosed
and treated in time. The antimicrobial resis-
tance of Neisseria gonorrhoeae is a major
topic of interest due to the appearance of the
multiple drug resistant species that can resist
current antibiotics used for treatment, which
remains a major public health concern world-
wide [2]. In Vietnam, using antibiotics without
prescription or antibiotics abuse in community
and health stations also may increase the an-
timicrobial resistance of N. gonorrhoeae,
which cause the difficulties for diagnosis and
treatment. Thus, we performed this study to
investigate the prevalence of N. gonorrhoeae
infections in patients with urethral and/or vagi-
nal discharge syndrome who attended Na-
126 JMR 116 E3 (7) - 2018
JOURNAL OF MEDICAL RESEARCH
tional hospital of Dermatology and Venereol-
ogy in 2017 and to describe the antimicrobial
resistant levels of isolated N. gonorrhoeae.
II. METHODS
1. Subjects
This study included all patients with ure-
thral and/or vaginal discharge syndrome who
visited the National Hospital of Dermatology
and Venereology, Hanoi, Vietnam in 2017.
Patients with AIDS or using antibiotics within 7
days were excluded from this study. A total of
1582 male and female patients agreed to par-
ticipate in this study.
2. Materials
Thayer-Martin agar (Oxoid) was used for
Neisseria gonorrhoeae isolation. This agar
medium is a selective medium used for the
isolation of Neisseria gonorrhoeae due high
nutritional content suitable for N. gonorrhoeae
growth. This medium includes the supplemen-
tary (Iso Vitalex) and the inhibitors (VCN-
vancomycin, colistin, nystatin) which can sup-
press other gram-negative diplococci, gram-
negative bacilli, gram-positive organisms, and
yeast.
Remel BactiCard Neisseria test kit (Thermo
Scientific) were used for N. gonorrhoeae iden-
tification. This test uses enzyme technology
for identification of pathogenic Neisseria spp.
(including N. gonorrhoeae, N. meningitis and
N. lactamica) and Moraxella catarrhalis iso-
lated on selective media.
- Antibiotic disks (Oxoid) were used for
determination of antimicrobial susceptibility.
- E-test (Abbiodisk, Sweden), a quantitative
technique, was used to determine the mini-
mum inhibitory concentration (MIC).
3. Methods: A cross-sectional study
Sample collection: (patients must be in-
formed and explained prior sample collection).
In women, samples were collected from
urethra, cervix, 2 sides of Bartholin’s Gland,
and 2 sides of Skene’s Gland. For cervical
samples, distilled water was used to pre-wet
the vaginoscope. Next, the vaginoscope was
inserted into the vagina to open the cervix,
then samples were obtained by inserting a
sterilized swab approximately 2 - 3 cm in the
cervix and keeping for 5 - 10 seconds to ab-
sorb cervical discharge. The swab was pulled
out without touching vaginal wall, followed by
making smear for gram staining, and culturing
in Thayer-Martin agar with VCN inhibitors.
Male patients were required to block
urination 2 - 3 hours before sample collection.
Sodium Chloride 0.9% was used to pre-wet a
sterilized swab. Sample was collected by
inserting the swab approximately 1.5 - 2 cm in
urethral meatus, rubbing against the inner wall
and keeping for 5 - 10 second to absorb
discharge. The swab was pulled out, followed
by making smear for gram staining and
culturing in Thayer-Martin agar with VCN
inhibitors.
Cultured plates were incubated at 36 -
37
0
C in a humid atmosphere (70% humidity)
containing 3 - 10% carbon dioxide (CO2) for
18 - 24 hours.
As for N. gonorrehoeae identification,
cultured colony could be visualized in an S
form, 1mm in diameter, mucus, gray and
twinkle. Next, the cultured colony underwent
gram staining and showed negative gram
results, with a coffee bean shape, in and
outside of neutrophils in the microscope field.
Finally, a cultured colony was confirmed by
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JOURNAL OF MEDICAL RESEARCH
using oxidase test and Remel BactiCard
Neisseria test kit [3].
For antibiotic sensitivity testing, N. gonor-
rhoeae colony was cultured in Thayer-Martin
agar without inhibitors, then incubated at 36 -
37
0
C in > 70% humidity containing 3 - 10%
CO2 for 18 - 24 hours. Colony purification was
rechecked by Gram staining. A suspension of
0.5 McFarland turbidity was made by mixing
colonies with Sodium Chloride 0.9%, then cul-
tured as monolayer in Thayer-Martin agar
without inhibitors. The agar surface was kept
dry up in room temperature. Next, 5 antibiotic
disks were put on each 9cm diameter plate
with a distance of 2 cm from plate wall and
kept at room temperature for 10 minutes for
the antibiotic diffusion. Cultured plates were
incubated at 36 - 37
o
C in > 70% humidity con-
taining 3 - 10% CO2 for 18 - 24 hours. The
zone of inhibition was measured by using mm
ladder and interpreted according to The Clini-
cal & Laboratory Standards Institute (CLSI)
guideline in 2015 [4].
Minimum inhibitory concentrations (MICs)
was performed by using E-test (Abbiodisk,
Sweden). MIC is the lowest concentration of
an antibiotic, which prevents visible growth of
bacterium. In our study, MIC was performed
for only N. gonorrhoeae with the zone of inhi-
bition close to sensitive or resistant level. For
example, Ceftriaxone ≥ 35mm was sensitive
result, thus, those strains with the inhibition
zone of 34 - 35 mm were examined for MICs
test.
III. RESULTS
1. The prevalence of N. gonorrhoeae infections in patients with urethral and/or vaginal
discharge syndrome (n = 1582)
Table 1. The prevalence of N. gonorrhoeae infections in patients with urethral and/or
vaginal discharge syndrome
Test results Gram staining (%) Culture (%) p value
Positive 190 (12.01) 197 (12.45)
0.71
Negative 1392 (87.99) 1385 (87.55)
Of 1582 patients with urethral and/or vaginal discharge syndrome, 197 (12.45%) were positive
for N. gonorrhoeae. As compared to gram staining method, the detection rate of N. gonorrhoeae
using culture method was higher (12.45% vs 12.01%) thought not significantly significant
(p = 0.71).
2. Describe the antimicrobial resistant level of isolated N. Gonorrhoeae
Of 197 N. gonorrhoeae isolates, 97.97% were resistant to ciprofloxacin, 85.79% to nalidixic
acid, 85.28% to tetracycline, 45.69% to Penicillin and 1.02% to Ceftriaxone. No resistance to
Spectinomycin was detected among isolates (Table 2).
Of 197 N. gonorrhoeae isolates, 99.49% were sensitive to Azithromycin, Cefotaxim and
Cefixim (Table 3).
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JOURNAL OF MEDICAL RESEARCH
Antibiotics Abbreviation
Sensitive Intermediate Resistant
n % n % n %
Penicillin PG 0 0 107 54.31 90 45.69
Spectinomycin SPT 197 100 0 0 0 0
Ciprofloxacin CIP 0 0 4 2.03 193 97.97
Ceftriaxon CRO 195 98,98 0 0 2 1.02
Nalidixic acid NAL 0 0 28 14.21 169 85.79
Tetracyclin TE 1 0.51 28 14.21 168 85.28
Tetracycline TE TRNG: 97(49.24%)
Table 2. The resistant levels against different antibiotics from main group (n = 197)
TRNG: tetracycline resistant Neisseria gonorrhoeae is the term used to describe isolates with
inhibition zone of Tetracycline being less than or equal to 20 mm.
Table 3. The resistant levels against antibiotics from additional groups
Antibiotics Abbreviation
Sensitive Intermediate Resistant
n % n % n %
Azithromycin AZM 196 99.49 0 0 1 0.51
Cefotaxim CTX 196 99.49 0 0 1 0.51
Cefixim CFM 196 99.49 0 0 1 51
Table 4. MIC value within several antibiotics to N. gonorrhoeae
Antibiotics Strains
MIC (àg/ml)
Sensitive Intermediate Resistant
Penicillin 10
≤ 0.06 0.12 - 1 ≥ 2
5 5
Spectinomycin 11
≤ 32 64 ≥ 128
11
Ciprofloxacin 12
≤ 0.06 0.12 - 0.5 ≥ 1
5 7
Ceftriaxon 17
≤ 0.25 - -
12 2
Tetracyclin 10
≤ 0.25 0.5 - 1 ≥ 2
3 7
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The MIC value showed that 5/10 strains resisted to penicillin (MIC ≥ 2 àg/ml), 7/12 strains to
ciprofloxacin (MIC ≥ 1 àg/ml), 7/10 to tetracyclin (MIC ≥ 2 àg/ml) and 2/17 to ceftriaxone (MIC >
0,25 àg/ml). No strain showed its resistance to spectinomycin.
IV. DISCUSSION
Since 2017, the World Health Organization
has supported five hospitals in Vietnam, in-
cluding the National hospital of Dermatology
and Venereology, to perform the project of
surveillance of antimicrobial resistance in
Neisseria gonorrhoeae. Therefore, specimens
from patients with urethral and/or vaginal dis-
charge syndrome in the hospital were proc-
essed in a clinical laboratory for bacterial cul-
ture, identification and antimicrobial suscepti-
bility test. Of 1582 patients with urethral and/or
vaginal discharge syndrome who attended
National hospital of Dermatology and
Venereology in 2017, 197 (12.45%) were posi-
tive for N. gonorrhoeae. This finding was
higher than previous studies. Nguyen Huu Sau
reported that of 20,260 patients who attend
National hospital of Dermatology and
Venereology for STDs from 2006 to 2010,
1946 (9.6%) were positive for N.gonorrhoeae
[5]. From November 2006 to April 2007 at Na-
tional hospital of Dermatology and Venereol-
ogy, Nguyen Dinh Ha reported that the preva-
lence of N. gonorrhoeae in STDs patients was
11.08%. Nguyen Hong Hinh reported that
prevalence of N. gonorrhoeae was 10.16%
among STDs patients in their study from
1/2001 to 12/2003. However, the real number
of gonorrhea patients in the community may
be higher because patients usually decide to
be examined in private clinics or even to treat
themselves without a prescription from a
health care professional. On the other hand,
the prevalence of N. gonorrhoeae also de-
pends on several factors, such as the experi-
ence of technicians and quality of laboratory
tests.
The antimicrobial resistance of N. gonor-
rhoeae becoming more widespread across
different classes of antibiotics, including peni-
cillin, tetracyclin, fluoroquinolone, erythromy-
cin, and azithromycin. Eventually, some N.
gonorrhoeae strains have shown the resis-
tance to broad-spectrum cephalosporin in vitro
which is recommended for treatment in many
countries [6]. In this study, we showed that
97.97% of N. gonorrhoeae isolates were resis-
tant to ciprofloxacin. According to the results
of Le Hong Hinh, there was an upward trend
of the resistant rate of N. gonorrhoeae to
ciprofloxacin with 42.26% in 2001; 46.01% in
2002 and 50% in 2003. Remarkably, in 2014,
the resistant rate of 96.33% was shown in Le
Van Hung’s study [7]. WHO has reported that
prevalence of antimicrobial resistance of N.
gonorrhoeae was 85.2% in China, 79.9% in
Hong Kong and 40% in Japan [8]. This preva-
lence was 4.89% in England 4.89%, 7.8% in
Scotland 7.8% and 0.9% in Papua New
Guinea [9]. The antimicrobial resistance of N.
gonorrhoeae has increased rapidly due to the
usage of quinolone not only for gonorrhea
treatment but also for some other infections
like urinary tract infections and injuries. Thus,
quinolone has not been recommended for
gonorrhea treatment.
As for tetracycline, our study showed that
the resistant rate to this antibiotic was 85.28%.
Of these tetracycline resistant isolates,
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JOURNAL OF MEDICAL RESEARCH
49.24% was TRNG with inhibition zone being
less than and equal to 20mm. . The study con-
ducted at The National Hospital of Dermatol-
ogy and Venereology in 2000 showed that the
resistant rate of N. gonorrhoeae to tetracycline
was 42.7% [7]. The results from study of Le
Hong Hinh showed that this resistant rate had
a downward trend as follows: 40.47% in 2001;
26.29% in 2002 and 27.9% in 2003. In Asia
Pacific, this rate ranged from 25% to 70% [9].
Thus, this resistant rate in Vietnam showed an
decreasing trend. This may be explained by
the multiple-dose regimens of tetracycline – an
old antibiotic, for the treatment of urethritis and
cervicitis due to N. gonorrhoeae and C. tra-
chomatis infections.
As for penicillin, the first class of antibiotics
to be discovered, the resistance rate to this
drug was 45.69% in our study. In 2014, Le
Van Hung et al reported this rate was 47.7%.
According to WHO report, in Asia Pacific
countries, the resistant rate was more than
90% in Korea and the Philippines; 80% in
China and 60% in Singapore [8]. Currently,
WHO recommended penicillin to not be used
for gonorrhea treatment among countries
where this resistant rate was high.
As for ceftriaxone, our study showed that
N. gonorrhoeae was sensitive (98.98%), how-
ever, we detected two resisted strains with
MIC > 0.25 àg/ml. This finding was consistent
with previous studies of Le Van Hung and
other authors in Vietnam and Asia Pacific
countries [7; 10]. However, a study reported
that 5% of N. gonorrhoeae strains showed the
resistance to ceftriaxone in Vietnam in 2011
[11].
Regarding spectinomycin, 100% of isolates
were sensitive to this drug, which was also
reported from other research groups in Viet-
nam. Therefore, this antibiotic is still recom-
mended for treatment. However, it was re-
ported that spectinomycin-resistant strains
have appeared with 0.47% in other country.
The additional antibiotic group which is se-
lected by the particular condition of each na-
tion showed the high sensitive result (99.49%)
in our study, including azithromycin, cefotaxim
and cefixim. These results were consistent
with study of Ana Paula Ramalho da Costa-
Lourenỗo et al in 2017 [12].
E-test was used for the identification of
MIC of N. gonorrhoeae. However, due to the
high costs, MIC was performed only for strains
which showed the difficult interpretation with
antibiotic sensitivity results closed to sensitive,
intermediate or resistance with five antibiotics
of main group.
MIC results showed 5/10 strains resistant
to penicillin (MIC ≥ 2 àg/ml), 7/12 strains resis-
tant to ciprofloxacin (MIC ≥ 1 àg/ml), 7/10
strains resistant to tetracycline (MIC ≥ 2 àg/
ml), 2/17 strains resistant to ceftriaxone (MIC
> 0.25 àg/ml). No strain showed the resistance
to spectinomycin. These finding were consis-
tent with other reports. According to Le Van
Hung et al in 2014, N. gonorrhoeae resisted to
penicillin, ciprofloxacin, tetracycline at 47.7%;
96.3% and 83.4%, respectively. Coinciden-
tally, N. gonorrhoeae showed its high sensitiv-
ity to ceftriaxone and spectinomycin [6]. In
2011 at National Hospital of Dermatology and
Venereology, Pham Thi Lan reported that the
sensitivity and resistance of N. gonorrhoeae to
ceftriaxone with 95% and 5%, respectively,
and 100% of N. gonorrhoeae strains were
sensitive to spectinomycin [10].
JMR 116 E3 (7) - 2018 131
JOURNAL OF MEDICAL RESEARCH
V. CONLUSION
Ciprofloxacin, tetracycline and penicillin
should not be used for N. gonorrhoeae treat-
ment due to the high resistance of N. gonor-
rhoeae to these antibiotics.
Spectinomycin, ceftriaxon, azithromycin,
cefotaxim and cefixim should be used for the
treatment of N. gonorrhoeae infection.
ACKNOWLEDGEMENTS
We acknowledge the support of Prof. Tran
Hau Khang, Assoc.Prof. Nguyen Van Thuong,
Bsc. Ninh Thi Dan and Bsc. Le Phuong Thao
for this study.
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