Prevalence of microorganisms associated with Pelvic inflammatory disease in reproductive aged women in Onitsha North, Anambra state, Nigeria

The prevalence of pelvic inflammatory disease (PID) and its associated microbes among reproductive aged women in Onitsha north, Anambra state, Nigeria, were investigated. A total of 500 reproductive aged women between the ages of 10 - 50 years were examined; where 300 of them showed positive results. A total of 640 microorganisms were isolated. Nine (9) microbial genera were recovered consisting of seven bacterial genera; one yeast sp. and one protozoan isolate. Monomicrobial growth was recorded in 53 (7.17%), polymicrobial growth in 23 (7.7%) and bacterio-fungal growth in 10 cases (33%). Staphylococcus aureus accounted for 150 (50%) cases; followed by Escherichia coli 125 (41.7%), Streptococcus pyogenes 15 (5%), Klebsiella pneumonia 55 (18.3%), Proteus mirabilis 25 (8.3 %), Pseudomonas aeruginosa 64 (21.3%), Neisseria gonorrhoeae 62 (20.7%), Candida albicans 56 (18.7%), and Trichomonas vaginalis 88 (29.3%), respectively. Frequency of occurrence was predominant with the age groups of 21-30 and 31-40 years; conversely was least in ages of 10-20 and those age >51 years; respectively. There was significant statistical difference between microbial infection and the age-group (p<0.05). PID is a major public health problem, thus needs to be prevented and controlled. post-gynecological


Introduction
Pelvic inflammatory disease (PID) is a polymicrobial infection and inflammatory disorder of the upper female genital tract; that primarily affects young sexually active women. These disorders may include; cervicitis, endometritis, salpingitis, parametritis, oophoritis, tubo-ovarian abscess (Banikarim, 2005;Crossman, 2006), and pelvic peritonitis (Buchan et al., 1993). PID may results from sexually transmitted infection; infection due to post-gynecological procedure or rarely from Novel Research in Microbiology Journal, 2018 hematological spread (Risch and Howe, 1995). Most cases of PID are presumed to occur in 2 stages. According to Audu, (2004), the first stage was acquisition of a vaginal or cervical infection; which was often sexually transmitted and may be asymptomatic. The second stage was direct ascent of microorganisms from the vagina or cervix to the upper genital tract; accompanied with infection and inflammation of these structures. The mechanism(s) by which microorganisms ascend from the lower genital tract involve multiple factors. Although cervical mucus provides a functional barrier against upward spread of microbes; however, the efficacy of this barrier may be decreased by vaginal inflammation and by hormonal changes that occur during ovulation and menstruation (Ehoton-Vlasak, 2000).
In addition, antibiotic treatment of sexually transmitted infections can disrupt the balance of endogenous microflora in the lower genital tract; causing nonpathogenic microorganisms to overgrow and ascend. Opening of the cervix during menstruation along with retrograde menstrual flow; may also facilitate ascent of microorganisms. Intercourse may contribute to the ascent of microbes through rhythmic uterine contractions occurring during orgasm. Bacteria may also be carried along with sperms into the uterus and fallopian tubes (Cohen and Brunham, 1999). The principal complications of PID were chronic pelvic pain, infertility and ectopic pregnancy.
In the upper tract, a number of microbial and host factors appear to influence the degree of inflammation that occurs, and thus the amount of subsequent scarring that develops. Emele et al., (2004) added that infection of the fallopian tubes initially affects the mucosa, but inflammation may rapidly become transmural. Inflammation may extend to uninfected parametrical structures including the bowel. In reference to Kwanwendo and Forslin, (1998), microorganisms that were implicated in PID were thought to spread in three ways: First: intra-abdominally, traveling from the cervix to the endometrium through the salpinx. Second: through the peritoneal cavity (causing endometritis, salpingitis, tubo-ovarian abscess, or pelvic peritonitis). Third: through the lymphatic systems including infections of the parametrium from an intrauterine device (IUD) (Mahon et al., 2005).
PID rarely occurs in pregnancy; however, chorioamnionitis can occur during the first 12 weeks of gestation, before the mucous plug solidifies and seals off the uterus from ascending bacteria. Fetal loss may thus result (Das et al., 2016). Concurrent pregnancy influences the choice of antibiotic therapy for PID; and demands that an alternative diagnosis of ectopic pregnancy be included (Ross et al., 2017). Genetically mediated variation in immune response plays an important role in susceptibility to PID (Simms and Stephenson, 2000). Variations in the genes that regulate toll-like receptors (TLRs; an important component in the innate immune system), have been associated with an increased progression of Chlamydia trachomatis infection to PID (Taylor et al., 2012).
PID is a polymicrobial infection which may begin as single infection due to Neisseria gonorrhoeae or C. trachomatis; which causes inflammation of the upper genital tract, thus facilitates the involvement of other pathogens (anaerobes; facultative anaerobes, and other bacteria). These pathogenic microorganisms were increasingly isolated as inflammation increased and as abscesses formed (Bevan et al., 1995). Microorganisms involved in PID include; Gardnerella vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Herpes simplex virus 2 (HSV-2), Trichomonas vaginalis, Cytomegalovirus (CMV), Haemophilus influenza, Streptococcus agalactiae, enteric gramnegative rods, (e.g.; E. coli) and Enterococcus sp. (Ross et al., 2017). Women with human immunodeficiency virus (HIV) infection also have an increased risk of progression to PID (Barbosa et al., 1997). Younger ages have been found to be Novel Research in Microbiology Journal, 2018 associated with an increased risk of PID. Likely reasons include; increased cervical mucosal permeability, larger zone of cervical ectopy, lower prevalence of protective anti-chlamydial antibodies, and increased risk-taking behaviors (Taylor et al., 2012).
The diagnosis of PID is based primarily on clinical evaluation, because of disease potentials for significant consequences (Ness et al., 2005). Since these infections are polymicrobial; broad-spectrum antimicrobial agents were recommended to treat the most likely pathogens. The first option for oral treatment includes a one-time 250-mg intramuscular dose of ceftriaxone (Rocephin); and 100 mg of doxycycline used orally twice per day for 14 days. Hence, it is pertinent to investigate the microbial effects of this type of disease. Therefore, the aim of this study was to investigate the prevalence of microorganisms associated with pelvic inflammatory diseases in reproductive aged women in Onitsha North, Anambra State, Nigeria.

Study Area
This study was carried out within Onitsha North, a major city in Anambra state, Southern Nigeria.

Collection of Specimens
High vaginal swabs were collected from 500 female patients at General hospital Onitsha, Anambra State, using sterile swab stick; and were then sent aseptically to the microbiological laboratory for bacteriological, fungal and protozoan analysis.

Microbial isolation
Specimens were inoculated on Nutrient agar (NA), Blood agar (BA), Thayer Martin agar medium, Chocolate agar and Sabouraud Dextrose agar (SDA) plates using spread plate method (Cheesbrough, 2006). Plates were incubated at 35°C for 24 h, whereas, SDA plates were incubated for 96 h. Recovered bacterial and yeasts isolates were purified; and then subcultured on agar slants for further studies.

Characterization of bacteria
The bacterial isolates were characterized on the basis of their colony; microscopical and biochemical characteristics according methods described previously by Cheesbrough, (2006).

Microscopy of the yeast isolates
A small loopful from growing yeast slant was placed on the microscope slide containing a drop of lactophenol blue. They were teased and mixed together and then coverslip was placed on it. The yeast cells were observed with x10 and x40 objective lenses for budding; size of each yeast cell, size of daughter cells and thickness of the cell wall.

Germ tube test
Pasteur pipette was used to dispense 3 drops of fresh pooled human serum into test tubes. With sterile inoculating loop, yeast loopful was picked, dropped into the serum and mixed. The suspended yeast cells were incubated for 2-3 h at 37°C (Warren and Shadomy, 2011). Then a drop of this suspension was placed on a clean microscopic slide and examined with a microscope using x10 and x40 objective lenses.

Growth on chromogenic agar
Chromogenic agar medium (Oxoid, UK) was prepared as reported by Akter et al., (2014); poured in Petri dish and then allowed to solidify. A loopful from 24 h SDA slants was removed and then spread onto the surface of chromogenic agar medium with a sterile straight inoculating wire. Plates were Novel Research in Microbiology Journal, 2018 incubated for 24 h at 37°C and colors of colonies were recorded.

Growth on corn meal agar
According to the method of Warren and Shadomy, (2011); a sterile inoculating needle was used to take a loopful from 48 h yeast slant and used to streak an "X" shape in the middle on one half of a corn meal agar plate. The arms of the "X" shape were about 2 cm long. Using the same procedure, a duplicate of "X" shape was made in the middle of the other half of the agar plate. Sterile forceps was used to place a sterile cover slip over the cross of one of the "X" patterns. Plates were then incubated for 48 h at 37°C. The "X" shape without the coverslip served as a growth control. After incubation; plates were examined for development of chlamydospores, blastospores, and pseudophae using low (×10) and high power (×40) objective lenses.

Characterization of protozoa
For characterization of protozoa; two sterile swab sticks were used to collect high vaginal specimens from each woman. The first swab was placed in 2 ml of sterile saline solution (0.85 % NaCl in sterile dist. water) for direct wet mount preparation (Nourian et al. 2013). Briefly, a single drop of well homogenized vaginal swab content in normal saline was placed on a clean microscopic glass slide. The slide was initially scanned for motile flagellates at (x10) lens; subsequently at (x100) lens to confirm the parasite motility, flagella movement and morphological features of the organism. Moreover, other microscopical findings such as; red blood cells, pus cells and epithelial cells were also examined. The second vaginal swab content was cultivated on Trichomonas medium (Oxiod, UK); supplemented with 8 % heat inactivated horse serum following the manufacturer's instructions. To suppress bacterial and fungal growth, 0.05 mg/ ml of streptomycin and 0.05 mg /ml of chloramphenicol were supplemented to this medium; respectively. Inoculated cultures were incubated at 37°C; and followed up microscopically for the presence of motile trophozoites after 24, 48 and 72 h of incubation.

Antibiotic susceptibility testing
Susceptibility testing was carried out by disc diffusion method according to the Clinical Laboratory Standard Institute (CLSI. 2011); using the following antibiotics discs: clindamycin, doxycycline, cefotetan streptomycin, tetracycline, trimethoprim sulfamethoxazole, ampicillin, gentamycin, erythromycin, nystatin, and augmentin. Zones of inhibition of these discs were measured after incubation.

Statistical analysis
Statistical analysis of results was done using SPSS (statistical package for social sciences) version 21.0. The data collected were analyzed using chisquare test. Results were tested at significance level of 0.05.

Results and Discussion
Findings of this study have established the existence of PID among the reproductive aged women in Onitsha north; particularly in Anambra state, eastern Nigeria, with high prevalence of 60 %. PID constitutes a major public health problem in both developed and developing countries. Although it was one of the most commonly reported infectious diseases; however, the number of infected cases increases daily. Based on the colonial; microscopical and biochemical characteristics, all isolates were identified as shown in Tables (1 and 2). Out of 500 high vaginal swabs specimens analyzed; 300 (60 %) of specimens were positive for microbial infections. Thus 7 bacterial genera, 1 yeast sp. and 1 protozoan organism were isolated and identified. A total of 640 microorganisms were isolated as clear in Table  (3     S. aureus accounted for 150 (50 %) of cases; followed by E. coli 125 (41.7 %), S. pyogenes 15 (5%), K. pneumonia 55(18.3%), Proteus mirabilis 25(8.3%), P. aeruginosa 64 (21.3%), N. gonorrhoeae in 62 (20.7 %), C. albicans in 56 (18.7 %), and T. vaginalis in 88 (29.3 %). S. aureus and E. coli were the predominant bacterial isolates in this study, as these pathogens were mostly isolated from the lower genital tract; and were responsible for significant proportions of sexually transmitted diseases in Nigeria. The dominance of these bacterial pathogens and their existence in the female genital tract confirmed being predisposing factors in acquisition of PID.
As evident in this study, the lower frequency of occurrence of N. gonorrhoeae might be attributed to; variation in the studied population, methods of microbial investigation, variations in the severity of diseases, sampling technologies, and sites of sampling. Technically; N. gonorrhoeae was highly fastidious fragile microorganisms, its isolation depended on; viability of the microbe in the specimen, prompt delivery to the laboratory, and suitability of isolation medium. T. vaginalis with prevalence of 29.3 % posed significant public health problems; because of close association of trichomoniasis with HIV infection. T. vaginalis was an irritating protozoa causing sexually transmitted diseases worldwide (Swygard et al., 2004). Buve et al., (2001) reported that trichomoniasis incidence was higher in cities where there were higher numbers of HIV positive individuals. High prevalence of trichomoniasis and candidiasis recorded in this study; basically revealed close association of poor personal hygienic conditions among the low socio-economic class, and diseases transmitted sexually. This was particularly obvious in cases of multiple sex partners, with high probability of PID infection.
Table (4) showed that the frequency of occurrence of infections was predominant with the ages groups of 21-30 and 31-40 years, and was least in 10-20, and >51 years; respectively. There was significant statistical difference between microbial infection and the age-group (p<0.05). This finding simply confirmed previous reports of Bucham et al., (1993) that highest rate of infection was recorded in the age group of 16-24 years. Furthermore, PID accounted for approximately 60% of gynecological problems in women aged less than 25 years. High prevalence of PID episodes in this sexually active age group emphasized the correlation between coexistence of etiological agents in the genital tract of the females, and acquisition of PID.
The prevalence rate of PID observed in the current study agreed with those findings of previous workers (Banikarim and Chacko, 2005). A number of reasons have been attributed by researchers for the rising prevalence of PID including; increased moral laxity among young people, lack of sexual education in schools and homes, and poor hygienic conditions (WHO. 2000;Das et al., 2016;Ross et al., 2017). The in-vitro antimicrobial susceptibility pattern of bacterial and yeast isolates revealed high zones of inhibition observed particularly with clindamycin, cefotetan and nystatin; conversely, low zones of inhibition were recorded with trimethoprim sulphamethoxazole and ampicillin. These antibiotic susceptibility patterns were similar to those of Kayode-Isa et al. (2010). The reduced susceptibility of antibiotics such as; ampicillin and trimethoprim sulphamethoxazole, might be attributed to the abuse of these antimicrobial agents through selfmedication practice, which was a common phenomenon in towns/cities of most developing countries. Clindamycin, cefotetan, and nystatin showed acceptable in-vitro susceptibility pattern; thus could serve as drugs of choice in PID treatment/management. Antibiotics susceptibility patterns and zones of inhibition of bacterial and yeast isolates are shown in Table (5).