Microorganisms associated with intraamniotic infection among women with preterm birth at Ruhengeri Referral Hospital, Rwanda: A case-control study

Preterm birth could worldwide open well-being It 15 million are and 40% A total of 40 women were selected. Of the 40 women, 20 had a premature birth, and the remaining 20 had a full-term birth. 120 Swab samples were collected from the placenta, amniotic uids, and fetal membrane immediately after birth. The sterile cotton swab was used to collect samples and put into swabs Stuart plastic to avoid sample contamination. Samples were transported to the clinical microbiology laboratory at INES Ruhengeri for microbiological investigation. Gram staining, culture, and biochemical tests were performed. The independent t-test was used to test for signicant difference between means of the two groups, while the chi-square test (x 2 ) was used to test for signicant association with microorganisms and intraamniotic infections.


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Background Preterm birth is a birth that comes earlier than 37 weeks of the full gestation period. Existing data shows that every year 15 million babies are born prematurely around the world. Preterm delivery is the leading cause of neonatal death in the developed world [1]. The health consequences of preterm delivery commonly occur to neonatal periods but can be long-lasting across the life course. Spontaneous development of the fetus or medical intervention provided for pregnancy could be the leading cause of preterm birth to women [2]. The speci c known factors of spontaneous preterm birth include infections, placental abruption, hormonal conditions, multiple gestations, etc. Intrauterine infection (intraamniotic infection) is the cause of around 40% of preterm deliveries globally [3]. It has been di cult to determine whether infections cause preterm delivery of women, but evidence shows that infection in the uterus could cause preterm birth. In ammatory diseases on gestational tissues caused by infections are the major cause of preterm delivery [4]. This is evidenced by a relatively higher rate of microbial colonization and in ammatory conditions in the intraamniotic uid of preterm labor patients compared to those lasting full term [5] There is a lack of calci cation on the rupture of membranes(ROM). The rupture of the membrane is a healthy sign of labor. Sometimes happens before onset of labor begins, it is considered as premature rupture of membrane(PROM) [6]. On the other hand, when the rupture of the membrane happens before 37 weeks, it is known as the preterm premature rupture of membrane(PPROM) which is a sign of intraamniotic infections in some cases [7]. Bacterial intrauterine infections are the major cause of preterm birth related infections. The amniotic cavity is considered to be free from infections, less than 1% of women in preterm labor have bacteria in their amniotic uids [8]. It has been di cult to isolate bacteria of amniotic uids before birth because of the risk posed by secondary complications from pregnancy. Most of the intraamniotic microbial colonization is not clinical and cannot be detected without microbiological analysis of amniotic uid [9]. It has been estimated that 12.8% of positive amniotic uid culture to occur to women with premature labor with an intact membrane. Some 22% of positive amniotic uid was reported to women that had experienced premature labor with the intact membrane but who delivered preterm babies [10].
The rate of positive amniotic uids was found to be 32.4% among women experiencing preterm premature rupture membranes(PPROM). During labor, more than 75% of these women suffer from the microbial invasion of the amniotic cavity (MIAC) due to the ruptured membrane which plays a protective role [11]. Women with microbial invasion of the amniotic cavity are at high risk to deliver a preterm baby, and to develop clinical chorioamnionitis [12].
Recent ndings reported that the rate of preterm birth in Rwanda stood at 10%, despite efforts by the government of Rwanda to mitigate the same [13]. However, there is a lack of studies on intraamnioticrelated infections and their in uence on preterm birth. This study, therefore, investigated major microorganisms that contribute to intraamniotic uid invasion among women with preterm birth at Ruhengeri referral Hospital.

Study setting
This study was carried out at Ruhengeri Referral Hospital located in Musanze District, Northern Province of Rwanda.

Study design
This was a case-control study. Data were collected from October 2018 to January 2019.

Study population and sample size
Cases consisted of women whose delivery was preterm and controls were selected from women with fullterm delivery. Non-probability purposive sampling was used and 20 women for both preterm and term delivery were selected.

Sample processing and collection
To compare the presence of microorganisms and intraamniotic infection between cases and controls, swab samples were collected from amniotic uid, placental, and fetal membrane. 3 samples were collected from each woman. Samples were collected immediately after delivery in the regional referral Hospital, Musanze. This was done using a sterile cotton swab and put in specimen bags (Stuart plastic) to avoid sample contamination. Samples were transported to INES Ruhengeri clinical microbiology laboratory for microbiological analysis. Microbiological analysis techniques including gram staining, culture, and biomedical tests were performed to isolate and identify microorganisms in amniotic uid, placental membrane, and fetal membrane samples.

Gram Staining
Gram stain technique was done to classify and differentiate the bacteria into Gram-Positive and Gram-Negative, microorganisms. After, smear preparation and application of different dyes, the slides were examined with the aid of light microscopy. Gram-positive microorganisms retain the primary dye Crystal violet and Gram-negative microorganisms take the color of the counterstain Safranin O.

Culture media preparation
The blood agar (Fluka®70133-500G) and MacConkey agar (QB-39-2707), culture media was used to isolate bacteria from the placental, fetal membrane, and amniotic uid samples. This was done based on Gram stain results. The samples were inoculated into prepared plates with solidifying culture media, the inoculated plates were then incubated at 37 ˚C for 24 hours. Therefore, the plates with growth colonies were morphologically observed and examined for biochemical tests.

Biochemical Tests
Different biochemical tests were performed to differentiate bacterial species. The catalase and coagulase tests were used to differentiate Staphylococcus species and Streptococcus species gram-positive bacteria. For the identi cation of Gram-negative bacteria isolates different biochemical culture media were used to test different biochemical parameters. Simon's citrate agar was used to test the microorganism's ability to utilize citrate as a source of energy. Motility was con rmed when a wide lament like-form appeared in the SIM medium. An appearance of a red ring-like formed into its surface after adding in two to three drops of Kovac's reagent after 24 hours of incubation indicates indole positive. Urease positive was proved by a color change in pink for urea broth. Furthermore, Kligler iron agar (KIA) permitted differentiation of Gram-negative bacilli by their ability to ferment glucose or lactose was also used. Red color changes from yellow due to the PH indicator in response to acid production of the fermentation of the sugar.

Yeast and molds identi cation
Sabouraud Dextrose agar (TM 387/500 g) selective media for both yeast and molds was used. Prepared Sabouraud agar plates were inoculated by streaking, as with standard bacteriological media. Molds were typically incubated at room temperature (22 to 25 °C for 2 to 5 days) and yeasts were incubated at 37 °C for 24 h. The growing yeast colonies (creamy whitish) were con rmed by Germ tube to differentiate Candida albicans and candida other species, whereas, molds grew as lamentous colonies of various colors were identi ed morphologically and by using lactophenol cotton blue stain.

Statistical analysis
We analyzed the signi cant difference in the mean between the two groups and the association with isolated microorganisms and intraamniotic infection among women with preterm delivery. SPSS version 22 was used. Both t-test and chi-square tests were carried out to test for statistical associations. The level of signi cance was α = 0.05.

Ethical consideration
The research ethics committee of INES Ruhengeri Institute of applied sciences provided a letter sanctioning the research to be conducted, but also an ethical approval letter was given by Ruhengeri Referral Hospital for swab sample collection. No names or any human identi cation data were recorded except the code number of the patient.

Results
Age distribution of the study participants Table 1 shows the age distribution of the study population. 50% of participants in both preterm and fullterm women were in the age range of 24-29years, and 20% aged 30-35 years. Pre-term women compared with full-term were aged 36-41 years. 15% compared with 10% and less likely to be aged 18-23 years. Comparative pro les of microorganisms isolated from amniotic uid Table 2 shows the percentages of isolated microorganisms from amniotic uid in both women with preterm and full-term birth. The predominantly isolated microorganisms among women with preterm birth were mold (28.8%) and non-Albicans Candida (28.8%). The non-Albicans Candida (66.7%) and molds (33.3%) were also predominant among women with full-term delivery in different proportions. No other microorganisms isolated from amniotic uid were observed of women with full-term delivery. Escherichia coli (9.6%), Klebsiella species (3.8%), Streptococcus species (13.4%), Staphylococcus species (5.7%), and Candida Albicans (9.6%) were only isolated among women with preterm birth. The signi cance of the difference between the means of the two groups was done using a t-test. The difference between the means of the two groups was statistically signi cant (t = 4.023, P = 0.006522).
Comparison of microorganisms isolated from placental membrane samples Comparison of microorganisms isolated from placental membrane samples Table 3 shows the percentages of isolated microorganisms from the placental membrane samples among women with preterm and full-term delivery. Isolated microorganisms from placental membrane taken from women with preterm birth were predominated by mold (25.8%) and Yeast (25.8%), the same condition was observed among women with full-term delivery where the predominant microorganisms were non-Albicans Candida (50%) and mold (30%). Staphylococcus species were also isolated from women with full-term birth and stood at 20%. Other microorganisms isolated from women with preterm delivery including Staphylococcus species (13.7%), Streptococcus species (12%), Candida albicans (8.6%), Escherichia coli (5.6%), and Klebsiella species (5.2%). These last microorganisms were uniquely observed among women with preterm birth excluding Staphylococcus species. The means difference (t = 7.17, P = 0.000372) of the two groups was statistically signi cant. Comparison of microorganisms isolated from fetal membrane samples Table 4 reveals microorganisms isolated from fetal membrane samples of women with preterm and fullterm delivery. Mold (26.3%) and non-albicans Candida (26.3%) were the isolated predominant microorganisms among women with preterm birth. These microorganisms also were predominant among women with term birth to 33.3% and 55.5% respectively. Staphylococcus species were observed among both preterm and full-term birth next to mold and non-albicans Candida with 14% and 11.1% respectively. Streptococcus species (12%), Candida albicans (8.7%), Escherichia coli (8.7%), and Klebsiella species (3.5%) were only observed among women with preterm delivery. The difference between the means of the two groups was statistically signi cant (t = 6.7, P = 0.000537).

Discussion
Intrauterine infection is also known as Intraamniotic infection or clinical chorioamnionitis. It is a combination of infection in amniotic uids, fetal membranes, and placental membranes [14]. This study analyzed the microbiological differences in amniotic uid, placental membranes, and fetal membranes. Escherichia coli, Streptococcus species, non-albicans Candida, and Candida albicans were associated with intraamniotic infections and were observed in all three types of samples ( Table 2,3&4). The study on the intraamniotic infection reported that isolated microorganisms from amniotic uid samples were the normal ora of the vagina including Escherichia coli and Streptococcus species [15]. The ascendance of vaginal ora to the uterus can cause intrauterine infection which is a precursor to preterm birth and fetal deaths. A similar nding from a systematic review of candida chorioamnionitis among pregnant women revealed a high prevalence of Candida albicans among candida chorioamnionitis patients [16]. Candida albicans is the leading cause of most vaginal yeast infection among women. Its ascendancy to the uterus could lead to preterm delivery, birth impairment, and fetal death. Some microorganisms were isolated from women with preterm birth and excluded from women that had carried their pregnancy to a full-term birth. Similar ndings reported that Escherichia coli and Streptococcus species were isolated from women with intraamniotic infections [17]. The current study compared the microbial differences between women with preterm and women with full-term delivery. The mean difference between the two samples was statistically signi cant (  [20]. Peng et al indicate the importance of these biomarkers in making a diagnosis for intrauterine infection or chorioamnionitis and instituting antibiotic management early to prevent preterm birth. However, the possibility of an antepartum diagnosis of these infections may be a challenge given the invasiveness of amniocentesis for obtaining an amniotic uid sample [21].
These ndings did not study which microbe was associated with intraamniotic infection among women with preterm labor and which organisms were associated with maternal or neonatal in ammatory symptoms such as fever. Molds, Klebsiella, and Staphylococcus spp were not associated with intraamniotic infection and past studies did not report any of them to contribute to clinical chorioamnionitis.

Conclusion
The invasion of microorganisms towards the uterus could lead to intraamniotic infection or clinical chorioamnionitis among pregnant women, and this could lead to preterm birth. There was a difference in microbial fauna among preterm and full-term birth regimes among women in all placental membranes, amniotic uid, and fetal membrane samples. The mean difference between the two groups was statistically signi cant. Escherichia coli, Streptococcus species, non-albicans Candida, and Candida albicans were the major cause of intraamniotic infection or clinical chorioamnionitis among women with preterm birth. Although intraamniotic infections may be subtle, pregnant women should seek immediate medical care when they show signs such as pre-labor draining of uids or are at higher or risk of intraamniotic infections. to be carried out, but also Ruhengeri Referral hospital provided ethical approval for sample collection in a maternity setting. Samples were obtained externally from the mothers, and the verbal informed consent was collected prior to delivery. The verbal informed was authorized because not all women know to read, and those who know to read were not in good mood to read the consent, they were exposed to birth process.

Consent for publication
Not applicable

Availability of data and materials
All about the dataset are available via the corresponding author

Competing interests
There is no competent of interest declared