Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by

Duplex PCR among Female Inmates in Lubuk Pakam Prison

Indonesia

Tetty Aman Nasution

, Rina Yunita

and Cherry Siregar

Microbiology Department, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia

Clinical Microbiology Laboratory, H.Adam Malik-General Hospital, Medan, Indonesia

Keywords: female inmates, duplex PCR, Chlamydia trachomatis, Neisseria gonorrhoeae.

Abstract: Incarcerated women are at high risk for sexually transmitted infections (STI).Chlamydia and gonorrhea are

most commonly found in females less than 25 years of age, both in the general population and among

incarcerated women. This is the first study using a duplex PCR assays for C. trachomatis and N.

gonorrhoeae to identify both microorganisms from female inmates in Lubuk Pakam prison.The objectiove

of the study is to detect multiple microbial targets simultaneously. From 41 subjects, endocervical swabs

were taken and sent to the Laboratory for duplex CT-GC PCR identification. The result found that detection

rate for Chlamydia trachomatis is16(39%) and Neisseria gonorrhoeae is 5(12%) and for both found in 3

subjects (7%) It was concluded that duplex CT-GC PCR are potentially useful for rapid from female

detection C. trachomatis and N.gonorrhoeae from female genital secretions

1 INTRODUCTION

Incarcerated women are at high risk for sexually

transmitted infections (STI), which if left untreated

may lead to adverse complications such as preterm

delivery, infertility, pelvic inflammatory diseases,

ectopic pregnancy, and even increased HIV

transmission (Nijhawan, 2012) (Caviness, 2012). In

some population of American women, chlamydial

infection, trichomoniasis, and gonorrhea are the

most common STIs, who are generally affected by

STI at higher rates than men (Plitt, 2012). Two of

these infections (chlamydia and gonorrhea) are most

commonly found in females less than 25 years of

age, both in the general population and among

incarcerated women (Hardick, 2003). Chlamydia

trachomatis infection rate in the general population

in the West is 1–10% in both genders (Datta, 2012)

(Cecil, 2001) (Montagne, 2004). The risk of

acquiring C. trachomatis is associated with several

socio-demographic and behavioral factors (Gollub,

2010). Particularly, incarcerated persons are at a

high risk for sexually transmitted infections (STIs).

Inmates were reported multiple behaviors which

increased the risk of STIs such as C. trachomatis,

including sex with multiple partners, unprotected sex

and inconsistent condom use, and substance use

disorders (Freudenberg, 2007) (Warner, 2006).

Genital infections caused by C. trachomatis

closely parallel those owing to N. gonorrhoeae in

terms of clinical manifestations. Both organisms

preferentially infect columnar or transitional

epithelium of the urethra, with extension to the

epididymis; the endocervix, with extension to the

endometrium, salpinx, and peritoneum; and the

rectum. Both organisms can produce extensive

subepithelial inflammation, epithelial ulceration, and

scarring. In rare cases, both organisms can produce

systemic manifestations. C. trachomatis tends to

produce less exudates and a lower concentration of

segmented neutrophils than N. gonorrhoeae;

however these criteria are not always sufficient to

separate the two diseases. In developing countries,

traditional methods for diagnosing STIs are

laborious, often not very sensitive, and have a long

turnaround time with most recent commercially

available diagnostic tests targeting one or, at most,

two of these STIs at a time. However, studies have

shown that 45.7% of persons infected with N.

Nasution, T., Yunita, R. and Siregar, C.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Duplex PCR among Female Inmates in Lubuk Pakam Prison Indonesia.

DOI: 10.5220/0010092508250828

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

825-828

ISBN: 978-989-758-449-7

825

gonorrhoeae, are coinfected with C. trachomatis

(Ahmad, 2015).

PCR method combines high levels of sensitivity,

specificity, and accuracy, with rapid turnaround

times, making for a more appealing alternative to

culture-based testing methods which are often

limited by long culture times and low sensitivity

with certain sample types. PCR-based assays, on the

other hand, are compatible with several sample types

that can be used directly for DNA extraction without

the need for culture (Ahmad, 2015) Multiplex PCRs

are designed for the simultaneous detection of

multiple microbial targets. These assays have been

increasingly used for the detection of common

etiologic agents in genital discharge and genital

ulcers (Mahony, 1995) (Nasution, 2007) This is the

first study using a duplex PCR assays for C.

trachomatis and N. gonorrhoeae to identify both

microorganisms from female inmates in Lubuk

Pakam prison.

2 METHODS

This study was a cross-sectional study design. There

were 41 subjects involved in this study. They were

female inmates in Lubuk Pakam correctional facility

in North Sumatera. Sampling method was total

sampling which taken from all female inmates in

Lubuk Pakam prison on November 2016. The

inclusion criteria for this study are female inmates

whom age 20-50 years old, already married or have

been sexually active with partners and agree to

participate in the study by signing the informed

consent. The exclusion criteria were female inmates

whom refused to do the examination. Samples were

taken by swabbing endocervix and put inside

M4RT® Microtest Tube® (http://www.remel.com)

and directly send to Collaborative Laboratory in

Faculty of Medicine Universitas Sumatera Utara.

2.1 DNA Extraction

Extracted DNA (4µl) were purified using Presto TM

Buccal Swab gDNA Extraction Kit following the

manufacturer’s instructions

(http://www.geneaid.com)

2.2 Detection of Chlamydia trachomatis

and Neisseria gonorrhoeae by

Duplex PCR

To amplify rDNA from genomic DNA, a duplex

PCR was carried out. Duplex PCR was conducted by

using two sets of primer which are KL1 (5’-

TCCGGAGCGAGTTACGAAGA-3’) and KL2 (5’-

AATCAATGCCCGGGATTGGT-3’) for C.

trachomatis (Mahony, 1992); HO1 (5’-

GCTACGCATACCCGCGTTGC-3’) and HO3 (5’-

CGAAGACCTTCGAGCAGACA-3’) for N.

Gonorrhoeae (Ho, 1992). The amplification mixture

was carried out in 12,5 l master mix PCR which

consists of Taq polymerase enzyme, MgSO

, and

dNTP (Go Taq® PCR Core System, Promega); 7,5

l nuclease-free water and 4 l DNA template

(https://worldwide.promega.com) PCR was

performed in a thermocycler (Verity 96-well

Thermal Cycler, AppliedBiosystems) with an initial

denaturation of 95°C for 5 min, followed by 35

cycles of 1 min at 95°C, 1 min at 55°C, and 2 min at

72°C. All PCR products were analyzed by

electrophoresis in a 2% (wt/vol) agarose gel

(Promega®) by standard procedures. The result

showed DNA fragment size for C. trachomatis 241

bp and N. gonorrhoeae 390 bp

Table 1: Detection rates from duplex CT-GC PCR

No n %

1. Chlamydia trachomatis 61 39

2. Neisseria gonorrhoeae 5 12

3. C.trachomatis and N.gonorrheae 3 7

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

826

Figure 1: Duplex PCR Results.

Figure 1. M (100bp marker); N (Negative Control); P

(Positive Control); Lane 2: CT (+); Lane 3: GC (+); Lane

9: CT (+); Lane 10: CT&GC (+); Lane 12: CT&GC (+);

Lane 15: CT(+); Lane 17: GC(+); Lane 18: CT(+); Lane

19:CT(+); Lane 20 CT&GC (+); Lane 22: CT(+); Lane 23:

CT(+); Lane 25: CT(+); Lane 27: CT(+); Lane 31: CT(+);

Lane 35: CT(+); Lane 38: CT(+); Lane 41: CT(+)

3 RESULTS AND DISCUSSION

This study found that 16 female (39%) were positive

for C.trachomatis. This study showed that

chlamydial infection was the most common

infection observed. This result supported the results

of other studies (Parvez, 2013). The overall

prevalence of chlamydial infection varied according

to age, social behaviors, sexual activities, and

geographic location of the patients (WHO, 2001).

The high prevalence of chlamydia infection in our

studied group underscored the importance of

Chlamydia screening in the detained women in this

area. Hence, the study also found that 5 female

(12%) were positive for N.gonorrhoeae. The

prevalence of gonorrhea has been reported to range

from 0.2% to 17% among female prisoners (CDC,

2001). Furthermore, 20% of males and 40% of

females with gonorrhea are co-infected with C.

trachomatis (Holmes, 1994) The concomitant

infections were the main reason for using this duplex

CT-GC PCR for this study.

4 CONCLUSIONS

Duplex CT-GC PCR is potentially useful for rapid

detection C.trachomatis and N.gonorrhoeae from

female genital secretions.

REFERENCES

Ahmad N. Abou Tayoun, Paul R. Burchard, Angela M.

Caliendo, Axel Scherer, Gregory J. Tsongalis., 2015.

A multiplex PCR assay for the simultaneous detection

of Chlamydia trachomatis, Neisseria gonorrhoeae, and

Trichomonas vaginalis. Experimental and Molecular

Pathology 98, p.214-218

Caviness CM, Anderson BJ, Stein MD., 2012. Prevalence

and predictorsof sexually transmitted infections in

hazardously-drinking incarceratedwomen. Women

Health.; 52(2), p.119

Cecil JA, Howell MR, Tawes JJ, Gaydos JC, McKee KT

Jr, Quinn TC, Gaydos CA, 2001. Features of

Chlamydia trachomatis and Neisseria gonorrhoeae

infection in male Army recruits. J Infect Dis,184,

p.1216–1219.

Datta SD, Torrone E, Kruszon-Moran D, Berman S,

Johnson R, Satterwhite CL, Papp J, Weinstock H,

2012. Chlamydia trachomatis trends in the United

States among persons 14 to 39 years of age, 1999–

2008. Sex Transm Dis, 39, p.92–96.

Division of STD Prevention. Sexually Transmitted

Disease Surveillance, 2000. Atlanta: US Dept of

Health and Human Services, Public Health Services,

Centers for Disease Control and Prevention; 2001.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Duplex PCR among Female Inmates in Lubuk Pakam Prison Indonesia

827

Freudenberg N, Moseley J, Labriola M, Daniels J, Murrill

C, 2007. Comparison of health and social

characteristics of people leaving New York City jails

by age, gender, and race/ethnicity: implications for

public health interventions. Public Health Rep, 122,

pp.733–743.

Gollub EL, Armstrong K, Boney T, Mercer D, Chhatre S,

Fiore D, Lavalanet A, Mackey K, 2010. Correlates of

trichomonas prevalence among street-recruited, drug-

using women enrolled in a randomized trial. Subst Use

Misuse, 45, pp. 2203–2220.

Hardick J, Hsieh YH, Tulloch S, Kus J, Tawes J, Gaydos

CA, 2003. Surveillance of Chlamydia trachomatis and

Neisseria gonorrhoeae infections in women in

detention in Baltimore, Maryland. SexTransm

Dis.;30(1), pp. 64–70.

Ho BS, Feng WG, Wong BK, Egglestone SI., 1992.

Polymerase chain reaction for the detection of

Neisseria gonorrhoeae in clinical samples. J Clin

Pathol;45, pp. 439-42.

Holmes, K.K., P. Frederick Sparling, Per-Anders Mardh,

Stanley M. Lemon, Walter E. Stamm, Peter Piot,

Judith N. Wasserheit, (1994b) Sexually Transmitted

Diseases, In Walter E. Stamm, Chlamydia trachomatis

infections of the adult, USA: Mc Graw Hill. (p.407 –

13).

Instruction Manual ver 02.10.17 Presto

Buccal Swab

gDNA Extraction Kit [Internet]. Geneaid Biotech Ltd

[cited 2018 July 31]. Available from

http://www.geneaid.com/sites/default/files/GSK14_0.p

LaMontagne DS, Fenton KA, Randall S, Anderson S,

Carter P., 2004. Establishing the National Chlamydia

Screening Programme in England: results from the

first full year of screening. Sex Transm Infect, 80, pp.

335–341.

Mahony JB, Luinstra KE, Sellors JW, Jang D, Chernesky

MA., 1992. Confirmatory polymerase chainreaction

testing for Chlamydia trachomatis in first void urine

from asymptomatic and symptomatic men. J Clin

Microbiol;30, pp. 2241-5.

Mahony JB, Luinstra KE, Tyndall M, Sellors JW, Krepel

J, Chernesky M., 1995.Multiplex PCR for detection of

Chlamydia trachomatis and Neisseria gonorrhoeae in

genitourinary specimens. J Clin Microbiol;33, pp.

3049-53.

Nasution TA, Cheong SF, Lim CT, Leong EWK,Ngeow

YF., 2007.Multiplex PCR for the detection of

urogenital pathogens in mothers and newborns.

Malaysian J Pathol; 29(1), pp. 19 – 24.

Nijhawan AE, Chapin KC, Salloway R, Andrea S,

Champion J, Roberts M, et al., 2012. Prevalence and

predictors of trichomonas infection in newly

incarcerated women. Sex Transm Dis.;39(12), pp. 973–

Parvez F, Katyal M, Alper H, Leibowitz R, Venters H.,

2013. Female sex workers incarcerated in New York

City jails: prevalence of sexually transmitted infections

and associated risk behaviors. SexTransm Infect.;89(4),

pp. 280–4.

Plitt SS, Garfein RS, Gaydos CA, Strathdee SA, Sherman

SG, Taha TE., 2005. Prevalence and correlates of

Chlamydia trachomatis, Neisseria gonorrhoeae,

Trichomonas vaginalis infections, and bacterial

vaginosis among a cohort of young injection drug

users in Baltimore, Maryland. Sex Transm Dis.;32(7),

pp. 446–53.

Remel Microbiology Products [Internet] Thermo Fisher

Scientific[cited 2018 July 31].Available from

http://www.remel.com/Clinical/Virology/MicroTest.as

Technical Bulletin GoTaq® PCR Core Systems[Internet].

Wisconsin: Promega Corporation [cited 2017

September 4]. Available from:

https://worldwide.promega.com/-

/media/files/resources/protocols/technical-

bulletins/0/gotaq-pcr-core-systems-protocol.pdf

Warner L, Stone KM, Macaluso M, Buehler JW, Austin

HD., 2006. Condom use and risk of gonorrhea and

Chlamydia: a systematic review of design and

measurement factors assessed in epidemiologic

studies. Sex Transm Dis, 33(1), pp. 36–51.

World Health Organization . Global prevalence and

incidence of selected curable sexually transmitted

diseases: Overview and estimates. Geneva: WHO;

2001.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

828