Introduction
Streptococci are gram positive cocci arranged in chains and are part of normal flora of humans and animals. Streptococcus pyogenes is a beta-hemolytic bacterium belonging to Lancefield serogroup A, also known as the group A streptococci (GAS). GAS are known to be a cause of vulvo-vaginitis in women. It is a widely carried pathogen in the female genital tract and causes rare but serious infectious conditions in women: Toxic Shock-Like Syndrome (TSLS) which is associated with early onset shock and multi-organ system failure, necrotizing fasciitis (NF) which involves local necrosis of subcutaneous soft tissues and skin, and bacteremia which can cause focal infections including meningitis, pneumonia, peritonitis, osteomyelitis, septic arthritis, myositis, postoperative wound infections and puerperal sepsis, the latter being a major cause of maternal mortality. Incidence of these invasive infections is very low, around 1–8 per 100,000 population per year, but has a potential case fatality rate ranging from 5% to 20%, and in the case of septic shock up to 30–45%.1, 2, 3, 4 The risk of invasive GAS infection is very high for postpartum patients, with a case-fatality rate of 3.5%.5 There is an increased risk of GAS colonized women developing invasive infections when compared to males (female/male ratio 4:1).6 The risk of developing invasive GAS infections will increase in pregnancy being 20-fold higher than in non-pregnant women.7
Group B Streptococcus (GBS, Streptococcus agalactiae) is frequently found in the normal microbiota of the gastrointestinal and vaginal tracts of women.8, 9 GBS are a well-recognized cause of maternal sepsis in the puerperium and can cause life-threatening infections in neonates, with maternal colonization being the principal route of transmission. Early-onset GBS disease (EOD) results from ascending infection of the womb or by neonatal acquisition during vaginal passage and manifests on days 0–6 of life with pneumonia or respiratory distress commonly advancing to sepsis. Late-onset disease (LOD) is classified with onset from days 7 to 90, arising from maternal, nosocomial, or community sources, and presents with bacteremia with a high complication rate of meningitis.10, 11 In non-pregnant adults, the incidence of infection is continuously increasing.12, 13 Elderly and immunocompromised persons with underlying conditions, such as diabetes mellitus and cancer, are at increased risk of invasive GBS disease.14, 15 Skin and soft-tissue infection, primary bacteraemia and urinary tract infection are the most frequent clinical manifestations of invasive GBS disease in the elderly.16, 17
Group C Streptococcus (GCS) and group G Streptococcus (GGS) of human origin are now considered to constitute a single subspecies, Streptococcus dysgalactiae subsp equisimilis (SDSE).18 SDSE have been identifed as part of the normal fora of the human respiratory, gastrointestinal, and female genital tracts. Thus, for many years colonization by these bacteria has been considered harmless. However, the pathogenicity of SDSE has now been better recognized. Recent epidemiological studies have shown that SDSE causes a variety of diseases similar to those caused by S. pyogenes such as skin and soft tissue infections range from superfcial wound infections to severe invasive infections, life-threatening Streptococcal toxic shock syndrome (STSS) and bacteremia.19, 20, 21
The present study is carried out to determine the prevalence and risk factors for the carriage of beta-haemolytic streptococci among women visiting Dr. VRK Women’s Teaching Hospital & Research Centre, Hyderabad.
Materials and Methods
This cross sectional study was carried out in the Dr VRK Women’s Teaching hospital & Research Centre, a tertiary care hospital, at Aziznagar from March 2014 to June 2015. High vaginal swabs were collected from 250 patients attending outpatient department (OPD) of Dr. VRK Women’s Teaching hospital. Patients were divided into 6 clinical groups.
Patients attending family planning OPD for copper T insertion and post insertion follow-up.
Patients visiting Gynaecology OPD with history of white discharge and irregular menstrual periods
Pregnant women attending antenatal OPD
Patients from postnatal septic wards
Women in labour
Prostitutes
Sample collection and processing
Vaginal swabs were collected 250 female patients belonging various clinical groups. The demographic profile was taken from each case. Vaginal swabs were put into Amie’s transport medium and transported at 4°C in a temperature monitored cooler to the Clinical Microbiology Laboratory, where they were processed within 2 hours. Swabs were inoculated onto 5% sheep blood agar plates and incubated for 24 h at 37°C in a candle jar, which can provide an atmosphere of 5% CO2. Culture plates negative for β-haemolytic colonies were incubated for additional 24 hours to allow the growth of slow growers. Beta-haemolytic streptococci (BHS) isolates were phenotypically identified by standard microbiological techniques: which include β-haemolytic activity on sheep’s blood agar, Gram stain revealing Gram positive cocci, and negative catalase test.
All the isolates presumptively identified as BHS were tested for Bacitracin susceptibility. BHS strains inoculated on sheep blood agar plates. Test was performed using 0.04 units Bacitracin discs (Himedia Laboratories, Mumbai, India) as per standard protocol. Bacitracin disc was placed over the medium with the help of sterile forceps and the plates were incubated at 370C overnight. Isolates with a zone of inhibition ≥ 15 mm diameter were considered as sensitive. Isolates with no zone of inhibition or < 15 mm diameter were considered as resistant. Sensitive isolates were presumptively identified as GAS and resistant isolates were identified as non-group A BHS (NGABHS). Presumptively identified GAS & NGABHS isolates were serogrouped by Lancefield grouping using a commercially available StreptexTM Rapid kit (Thermo Fisher Scientific, India).
Results
In this study, a total of 250 female patients belonging to six different clinical groups attending outpatient department of Dr VRK Women’s Teaching hospital were screened for BHS. BHS were isolated from 31 (12.4%) of 250 patients (Table 1). Occurrence of BHS was more in patients with intrauterine contraceptive device (IUCD) i.e. 18% followed by antenatal cases (12%), prostitutes (10%), postnasal sepsis (8%) and patients visiting Gynaecology OPD (7.5%) (Table 1). Majority of the BHS, 18 (58%) of 31 were isolated from 15-25 years age group followed by 26-35 years age group (35.48%). Similar trend was observed for NGABHS (Table 2).
Table 1
Occurrence of BHS in female patients belonging to six different clinical groups
Table 2
Age wise distribution of BHS & NGABHS
As many as 12 (38.7%) of 31 BHS isolates were susceptible to Bacitracin and identified as GAS by serogrouping. Nineteen (61.3%) of 31 isolates were resistant to Bacitracin and presumptively identified as NGABHS. By Lancefield grouping, 10 (32.2%) were identified as group B Streptococci (GBS), 4 (12.9%) were identified as group C Streptococci (GCS) and 5 (16.12%) were identified as group G Streptococci (GGS) (Table 3). As many as 7 (38.9%) of 18 BHS isolated from IUCD group were identified as GBS (Table 3). In postnatal sepsis 1 (50%) of 2 BHS was identified as GBS (Table 3). Two (40%) of five BHS isolated from prostitutes were identified as GBS (Table 3). GCS were isolated from IUCD users and antenatal cases (Table 3). GGS were isolated from IUCD users and prostitutes (Table 3).
Table 3
Lancefield grouping of BHS isolated from female patients belonging to various clinical groups
We have also studied the prevalence of GAS, GBS, GCS and GGS in 100 women using IUCD. IUCD users were divided into two groups’ viz., pre insertion & post insertion. Results of our study showed that prevalence of NGABHS was more among post insertion (18%) IUCD group compared to pre insertion (8%) IUCD group (Table 4). However, there is not much difference in the prevalence of GAS among post insertion and pre insertion IUCD groups (Table 4).
Discussion
GAS are known to be a cause of vulvo-vaginitis in women. It also causes rare but serious infectious conditions in women viz., TSLS, NF and bacteremia. GBS is frequently found in the normal microbiota of the gastrointestinal and vaginal tracts of women and is a well-recognized cause of maternal sepsis in the puerperium and can cause life-threatening infections in neonates. Other NGABHS like GCS and GGS were responsible for a variety of diseases such as skin & soft tissue infections, life-threatening STSS and bacteremia. Hence it is important for clinician to identify the serogroup of BHS and to record the changes in their epidemiological patterns.
The presumptive identification of GAS is usually done by testing for susceptibility to bacitracin even though there are reports about the occurrence of bacitracin resistant strains. 22 Many laboratories use this as the sole test for diagnosing GAS infections. 23 In this study, all the 12 BHS isolates susceptible to bacitracin were identified as GAS by latex agglutination kit and all the 19 NGABHS isolates were resistant to bacitracin. Hence, bacitracin susceptibility test, along with the Lancefield antigen A test, can be used for greater specificity in the identification of S. pyogenes.
Previous studies reported a very low prevalence of GAS.24, 25 The prevalence of GAS carriage in the genital tract is lower than GBS carriage. In this study, S. pyogenes were isolated from 4.8% of the samples. The higher prevalence of GAS in this study could be due to the inclusion of different clinical groups (6) for sample collection. Among six clinical groups, the prevalence of GAS is highest i.e. 7.5% in female patients visiting Gynaecology OPD with history of white discharge. Previous studies also showed that GAS carriage in genital tract is likely to cause symptoms, the commoner being: vaginal soreness and irritation, and discharge associated with inflammation of the vaginal mucosa. 26
Severe peripartum GAS infections, a scourge of the pre-antibiotic area, virtually disappeared during the mid-20th century, but have been reported sporadically since the early 1990s. Though still infrequent, such infections must be recognized and treated aggressively to prevent severe morbidity or mortality. In this study, S. pyogenes was isolated from 1 (2.86%) of 35 postnatal sepsis cases. Most of the studies also reported 1-2 unrelated cases of postnatal sepsis caused by GAS. 27, 28, 29
The prevalence of NGABHS among BHS isolated from female patients is 61.29% which is in agreement with the previous work. 30 In the present study, prevalence of NGABHS was more among post insertion (18%) IUCD group compared to pre insertion (8%) IUCD group (Table 4). This result is in agreement with the previous report, 31 who found that incidence of NGABHS was more in post insertion group. This could be due to change in the vaginal flora by the presence of foreign body like copper T.
S. agalactiae (GBS) were isolated from 4% of samples. This result is in agreement with the previous studies, where the prevalence of GBS colonization of the lower genital tract of 'normal' women has been reported as 4-18%.32, 33 In this study, GBS were isolated from 7% of samples from IUCD group (Table 3). Previous studies also reported slightly higher prevalence of GBS in IUCD users compared to other women.31, 34 GBS were isolated from 4% of samples from prostitutes. A research study by Cools et al. 201635 also suggested that working as a female sex worker (FSW) is a risk factor for vaginal GBS colonization.
Conclusion
This study reports the prevalence of BHS among women visiting Dr. VRK Women’s Teaching Hospital & Research Centre, a tertiary care hospital in Hyderabad. This study also identified certain risk factors such as IUCD usage and working as a FSW are associated with the increased prevalence of NGABHS especially GBS.
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