Iron Sucrose

A prospective non-randomized before-and-after anaemia screening protocol implementation study was conducted at Emek Medical Center, a university-affiliated hospital in Israel, from June 29, 2015 to January 27, 2016 (ClinicalTrials.gov Identifier: NCT02434653, date of registration: 28/04/2015). This study was authorized by the local review board of the Emek Medical Center (EMC 112-14) and was performed in accordance with relevant guidelines and regulations of our review board. Participants provided written informed consent.
Women who intended to or eventually delivered vaginally (spontaneous or by vacuum extraction) were tested for eligibility at the labour and delivery, maternal foetal medicine, or maternity wards. Inclusion criteria were women who delivered vaginally and age ≥ 18 years. Exclusion criteria were women under the age of 18 or who had known allergy to iron sucrose. We also excluded women who delivered by caesarean section because they have routine full blood count as part of their care, and women with pre-eclampsia with severe features, since Hb level is taken every 8 hours according to the departmental protocol. Recruitment took place in the delivery unit before delivery or after delivery at the maternity ward in two consecutive periods in which different intervention was performed for each (listed below in the following section) until the calculated sample size was obtained.

Campylobacter spp. isolation was carried out using the Campylobacter isolation kit incorporating a membrane filter method (ZC-CAMPY-002, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China). Briefly, 1 mL stool specimen suspension was transferred into 4 mL enrichment buffer which was provided in the kit. The principle component of the enrichment buffer was the modified Preston broth which was described in the manual book. The enriched suspension was incubated at 42°C for 24 h in a microaerophilic atmosphere consisting of 5% O₂, 10% CO₂, and 85% N₂. Three hundred μL cultured enrichment suspension was then spotted on to the surface of the filter pasted on to the double medium plates, which contained Karmali and Columbia agar, respectively. The medium plates were incubated in a microaerophilic atmosphere at 42°C for 48 h. The suspected colonies were picked and identified by Gram stain and biochemical tests. Multiple PCR tests were used to confirm and identify the species according to the previous study (Wang et al., 2002 (link)).
Conventional culture methods were used for Salmonella, Shigella, DEC, and V. parahaemolyticus. One mL stool sample was inoculated into 5 mL selenite brilliant green broth and enriched at 36 ± 1°C for 18–24 h (Wang et al., 2015 (link)). After selective enrichment, a loop of the culture was streaked on Chromagar and incubated at 36 ± 1°C for 18–24 h. More than one presumptive Salmonella colony (usually 3–5 colonies) on the selective agar plate were inoculated on to triple sugar iron slant and incubated at 36 ± 1°C for 24 h. Isolates with typical Salmonella phenotypes were confirmed by systemic chemical tests followed by serotyping with serological test (Salmonella antisera, SSI, Denmark) (Brenner et al., 2000 (link)).
One loop of the stool sample was directly inoculated on to xylose lysine desoxycholate (XLD) medium and MacConkey (MAC) plates and cultured at 36°C for 24 h. Two non-fermented lactose colonies and four lactose-fermenting colonies were picked from the MAC plates. The colorless and transparent colonies were picked from the XLD plates. For each selected colony, the pure culture was amplified using the Tryptose Soya agar medium for 24 h. The pure culture for each suspected colony was identified using VITEK^® 2 COMPACT (BioMerieux) based on the biochemical test. The isolates identified as Shigella were further identified by serological typing (Japanese Health Research Serum). Six of the suspected colonies were further identified using real-time PCR according to previous study for eaeA, stx₁ and stx₂ genes of EHEC; eaeA and escV of EPEC; ipaH of EIEC; lt, sth, and stp of ETEC; and aggR of EAEC. The PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. A positive reaction was identified as the Ct value was less than 35 (Chen et al., 2014 (link)).
Simultaneously, another loop of stool sample was directly inoculated into 5 mL 3% Sodium Chloride Alkaline Peptone Water and enriched at 36°C for 24 h. Then, one loopful of enriched culture was streaked on to Chromagar and incubated at 36°C for 24 h. Three to five colonies of suspected V. parahaemolyticus colonies on the selective agar plate were transferred onto triple sugar iron slant and incubated at 36°C for 24 h. Isolates with typical V. parahaemolyticus phenotypes were confirmed by systemic chemical tests followed by serotyping with a commercial kit (V. parahaemolyticus antisera, Denka Seiken, Japan).

The techniques used to isolate and identify Salmonella were recommended by the International Organization for Standardization (ISO-6579, 2002) and the World Health Organization [27 ]. Global foodborne infections network (formerly WHO global Salmonella Surveillance) [27 ]. In a nonselective liquid medium (buffered peptone water (BPW) (Oxoid CM509, Basingstoke, England), a 10 ml milk sample was mixed with 90 ml of pre-enrichment, and the sample mixture was thoroughly shaken before being incubated at 37°C for 24 hours. Following incubation, the culture was mixed, and a portion (0.1 ml) was transferred to a tube containing 10 ml of selective enrichment liquid medium (Rappaport Vassiliadis (RV)) broth and incubated at 41.5°C for 24 hours. A 10 µl of loop full inoculum from selective enrichment media was streaked onto Xylose Lysine Deoxycholate (XLD) (Oxoid CM0469, Basingstoke, England) agar and Salmonella Shigella (SS) agar plates prepared on petri-dishes and incubated at 37 ± 1°C for 24 ± 3 hours. After proper incubation, the plates were examined for the presence of typical Salmonella colonies. The typical colonies of Salmonella grown on Xylose Lysine Deoxycholate agar medium produce black centers with distinct red colonies due to the color change of phenol red in medium and colorless transparent colonies on Salmonella Shigella agar. The presumptive Salmonella colonies on the XLD (Oxoid CM0469, Basingstoke, England) and SS agar medium were transferred onto the surface of predried nutrient agar plates in a manner that allow isolated colonies to develop and incubated at 37°C for 24 hours in further confirmation with biochemical tests. Thus, all suspected Salmonella colonies were picked from the nutrient agar and inoculated into the biochemical test including Triple Sugar Iron (TSI) agar (Oxoid CM0277, Basingstoke, England) for the TSI test, Simmons's Citrate agar (Oxoid CM53, Basingstoke, England) for the citrate utilization test, Tryptone Soya Broth (Becton Dickinson, USA) for the indole test and Methyl red-Voges Proskauer (MR-VP) (Micromaster Thane, India) for methyl red and Voges Proskauer test and incubated for 24 or 48 hours at 37°C. Colonies producing an alkaline (red) slant, with acid (yellow) but on TSI with blackening or hydrogen sulfide production, negative for Tryptophan utilization on indole test (yellow-brown ring), positive for Methyl red (produce red color on the surface of medium), negative for Voges–Proskauer (yellow color), and positive for Citrate utilization (deep blue slant) were consider to be Salmonella positive [28 (link), 29 ].

A total of 104 isolates of E. coli obtained from patients hospitalized at Imam Reza hospital, an 800-bed training hospital in Tabriz city (in period of June to September 2021) were studied. The clinical samples from which bacterial strains were isolated included urine, wound, blood and tracheal aspirates. Bacterial isolates were identified to species level using conventional biochemical methods including IMViC tests (indole test, methyl red test, Voges-Proskauer reaction, citrate utilization test), urease test, motility, ONPG (O-nitrophenyl-beta-D-galactopyranoside), reactions observed on Triple Sugar Iron (TSI) agar (H2S and gas production, carbohydrate utilization pattern) [32 ].

Confirmative test for Salmonella for the experiments mentioned above was conducted according to FDA-BAM method (Bacteriological Analytical Manual). In short, buffered peptone water from the pre-enrichment of each treatment sample, 1.0 mL and 0.1 mL, was transferred to 10 mL of Rappaport-Vassiliadis (RV; BD Difco, Sparks, MD, USA) and tetrathionate (TT; BD Difco, Sparks, MD, USA) broths, respectively, and incubated at 42 °C for 24 h for the selective enrichment of Salmonella. From each RV and TT broth tube, one loopful was streaked onto xylose lysine deoxycholate (XLD) agar plates in duplicate. Inverted plates were incubated at 37 °C for 24 h. Presumptive positive Salmonella colonies appeared as pink colonies with or without black centers, with most positive Salmonella-producing colonies having large, glossy black centers or being almost completely black. Presumptive Salmonella-positive colonies from XLD plates were then inoculated into triple sugar iron agar (TSI; BD Difco, Sparks, MD, USA) slants by streaking the slant and stabbing the butt and the lysine iron agar (LIA; BD Difco, Sparks, MD, USA) slants by stabbing the butt twice and then streaking the slant. The TSI and LIA slants were incubated at 37 °C for 24 h. The presumptive Salmonella-positive TSI reactions had alkaline (red) slants and acid (yellow) butts, while the LIA reactions had an alkaline (purple) butt with acidic (yellow) reaction negative for Salmonella. All cultures with an alkaline butt in LIA, regardless of TSI reaction, were retained as potential Salmonella isolates. Presumed positive TSI and LIA slant cultures were inoculated into TSB and incubated at 37 °C for 24 h, from which the cells were harvested, DNA extracted, and confirmed as Salmonella based on molecular analysis [24 (link)].