Serotype and serovar distribution of Neisseria gonorrhoeae isolated from high-risk populations in Bangladesh

Neisseria gonorrhoeae, the causative agent of gonococcal infection, is known to frequently change their characteristics to evade host immune mechanism. Characterization of the clinical isolates of the organism can lead to identification of the circulating strains and often a sexual network in a community to help in designing the control strategy. Keeping in mind the above consideration, a total of 239 N. gonorrhoeae, isolated from high-risk populations, were characterized for serotypes and serovars by monoclonal antibodies against protein I of the organism. Majority of the serotypes were serotype B (142, 59.4%). Majority of the isolates showing resistance to at least one of the antibiotics tested were also serotype B (139, 59.2%), whereas, majority of the isolates showing resistance to any three of the antibiotics (multidrug resistant, MDR) (63%) was serotype A. A total of 41 different serovars were also identified and five of which (Arst, Bropt, Bopt, Arost, and Brop) included the highest percent (49.3%) of the isolates. Many serovars (23/41, 56.1%) were new emergent and included 58 (24.3%) of the isolates investigated. All of the new serovars were resistant to at least one of the antibiotics tested and the highest rate (40/102, 39.2%) was MDR. Serotyping and serovar determination was found contributory to understand the microepidemics of the N. gonorrhoeae isolates. Further studies including antibiogram and contact tracing can efficiently help in control of the disease.


Introduction
Neisseria gonorrhoeae, the aetiological agent of gonococcal infection or gonorrhoea, is pathogenic for humans only and adapts easily to the moist mucous membranes of genital tract and other similar structures.It is highly capable in resisting immune attack by the antigenic variability to evade host defenses, thus persisting and often causing asymptomatic (and undetected) infections 1 .In addition, the organism possesses some unique ability to develop resistance to antimicrobials and rapidly become ineffective to the commonly used antibiotics 2 .Characterization of the clinical isolates of the organism, including serotypes and serovars distribution in a community, can exactly identify the circulating strains to design the effective control strategies.
For the purpose of characterization, the information on the structural components of N. gonorrhoeae is very essential.The organism possesses a typical Gram-negative outer membrane composed of several proteins in addition to other structures.Three proteins are present in large amounts and these have been named proteins I, II, and III (PI, PII, and PIII respectively).Protein I (PI) is the predominant protein species of the gonococcal outer membrane and functionally is a porin protein 3 .There is extensive heterogeneity of gonococcal PI proteins between strains and this property makes PI an excellent marker for strain classification and epidemiological studies.Immunological and biochemical data have determined that there are two distinct structural variants of the porin protein, IA and IB that are further subclassified into serovars based on reactivity to a panel of PI-specific monoclonal antibodies.This information forms the basis of a serological typing system for the gonococcus 4 .The gonococcal serogrouping system has proved to have important epidemiological and clinical potentials.Serogroup PIA was found to correlate with resistance to the bactericidal action of normal serum, disseminated gonococcal infection (DGI) and asymptomatic gonorrhoea 5 .Whereas, serogroup PIB was found to correlate with resistance to several antimicrobial agents and with homosexually acquired infection 6 .Few studies with N. gonorrhoeae epidemiology suggest that some of the strains are likely to spread within the high-risk populations like commercial sex workers and their clients 7 .
In Bangladesh, situation of the disease is equally difficult and its epidemiological aspect has not been purposefully explored and gonococcal serotypes and serovar patterns have not been reported to date.The present study was, therefore, carried out to examine the diversity and distribution of N. gonorrhoeae serotypes and serovars of the previously isolated, identified and partially characterized N. gonorrhoeae strains from high risk populations in Bangladesh.

Materials and Methods
A total of 239 strains of Neisseria gonorrhoeae were selected randomly from ICDDR,B pool of the organisms isolated from high-risk populations by the then RTI/STI laboratory during 2003-2006.The high-risk populations from whom the isolates were collected included female commercial sex workers (CSWs)-hotel-and street-based at Dhaka City (CSWs-Floating); CSWs-brothel-based at Gualanda, Faridpur (CSWs-bb); male individuals attending out-patients department at Dhaka Medical College Hospital (male-OPD, Dhaka); male individuals attending OPD at Sylhet MAG Osmani Medical College Hospital (male-OPD, Sylhet); long distant male truckers (male truckers) by Paricharjya, Dhaka; and male individuals having sex with male (MSMs) at Sylhet.Each of the selected isolates of N. gonorrhoeae, harvested from pure subculture on GC agar medium into phosphate buffered saline (pH 7.2), was boiled for 10 minutes for antigen preparation.The serotype and serovar of the isolates were determined using an equal volume of the prepared antigen suspensions and monoclonal antibodies using Pharmacia (Ph) panel (Boule Diagnostics AB, Huddinge, Sweden) consisting of five Protein IA specific (Ar, Ao, As, At, Av) and nine Protein IB specific (Br, Bo, Bp, By, Bu, Bv, Bs, Bt, Bx) reagents.
Serotype of a strain of N. gonorrhoeae was defined as the type of reagents with which it reacted, as "type A" if reacted with PIA reagents and as "type B" if reacted with PrIB reagents.A serovar was assigned as the pattern of reactivity of a test strain with a given set of monoclonal antibodies specific for either PrIA or PrIB 8 .For example, when a N. gonorrhoeae strain reacted with PIB set with the antisera "r", "o", "p", "u" & "t", then the serovar of the strain was recorded as "Broput".
The selected isolates of N. gonorrhoeae were previously tested for antimicrobial susceptibility and plasmid profile by the same investigators and these results were used to correlate serotypes and serovars 9 .
Many of the serovars identified (23/41, 56.1%) were not reported previously and are therefore, designated as newly emerged serovars.The new serovars included 58 (24.3%) of the isolates investigated and among them "Ast" as a single new emergent included highest number of isolates (12, 5.0%).(Table V) All of the new serovars were resistant to at least one of the antibiotics tested and the highest number of them (40/58, 69.0%) were resistant to any three of the antimicrobial agents (MDR).(Table VI) Many of the serovars (23/41, 56.1%) have not been reported previously and therefore, were designated as new emergent.All of the newly emerging serovars were found resistant to any one of the antibiotics tested and majority of them was multidrug-resistant (MDR).These findings suggest that the circulating strains of N. gonorrhoeae among high-risk populations are changing their characteristics to evade antimicrobial challenge.
The NG serovars predominating among the commercial sex workers (CSWs) were not found consistently predominating among other populations which are supposed to have acquired from the CSWs.For this ambiguity, contact tracing might be one of the important mechanisms in the line of control of gonococcal infection.Contact tracing to identify the source and route of transmission as well as antimicrobial susceptibility surveillance are recommended.

Conclusion: N. gonorrhoeae serotype Protein IB
was predominating in the present study and as these strains were also found to be more resistant to the antimicrobial agents, treatment modality for gonococcal infection might be difficult in Bangladesh.Although serotyping & serovar determination was found contributory in understanding the microepidemics, these tests require other information, especially antibiogram of the circulating strains of the organism to have effective role in control of the disease.Therefore, surveillance for antimicrobial susceptibility of the gonococcal isolates along with contact tracing to understand route of transmission can help much more for designing an effective control measure in Bangladesh.

Table I :
Distribution of Neisseria gonorrhoeae (NG) serotypes among high-risk populations Considering relationship between the serotypes and antimicrobial resistance of the N. gonorrhoeae isolates, it was found that majority showing resistance to any one or two of the antibiotics tested was serotype B (59.3% of the isolates showing resistance to any one the antibiotics and 83.3% ofDistribution of NG serotypes

Table III :
Distribution of N. gonorrhoeae (NG) serotypes among plasmid-mediated resistant isolates

Table IV :
Major Neisseria gonorrhoeae (NG) serovars and their distribution among the source population Note: CSW-Floating: Commercial Sex Workers engaged in unlawful commercial sex in hotels and streets, CSW-bb: Brothel-based CSWs; Male-OPD, Dhaka: Symptomatic males attending outpatients department of Dhaka Medical College Hospital; Male-OPD, Sylhet: Symptomatic males attending OPD of Sylhet MAG Osmani Medical College Hospital, MSM-Male individuals having sex with male

Table VI :
Relationship between new serovars and antimicrobial resistance of the N. gonorrhoeae (NG) isolates