Criterios preanalíticos en la toma de muestras de hemocultivo en el área de Microbiología del Hospital General Portoviejo J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 https://doi.org/10.5281/zenodo.14602136 ISSN: XXXX-XXXX ORIGINAL ARTICLE Preanalytical criteria for collecting blood culture samples in the Microbiology area at Portoviejo General Hospital  Ivón Howland ivon.howland@utm.edu.ec Received: 11 September 2023 / Accepted: 17 December 2023 / Published online: 31 January 2024 © The Author(s) 2024 Carlos E. Mera 1 · Jean P. Sanclemente 2 · Ivón Howland 3 Abstract The General Hospital of Portoviejo serves a high demand of patients with suspected infections, bacteremia, or sepsis, highlighting the importance of performing high-qual- ity blood cultures. This study characterized errors in the pre- analytical sample collection phase for the microbiological diagnosis of bacteremia through blood cultures. Existing laboratory protocols were analyzed and compared with na- tional and international standards, focusing on asepsis, blood volume, biosafety, and techniques. Information was gath- ered through surveys conducted with healthcare personnel involved in sample collection, identifying key issues such as the need for more specific protocols, inadequate contain- ers for sample transportation, and the presence of person- nel in training. Although more than half of the respondents followed the correct sample collection and transport proce- dures, a significant proportion still needed to meet the stan- dards. As a result, a standardized operational procedure was developed based on good laboratory practices in Ecuador and internationally to improve the quality of blood cultures in the hospital. Keywords blood cultures, preanalytical phase, preanalyt- ical errors, good clinical laboratory practices. Resumen El Hospital General Portoviejo atiende una alta demanda de pacientes con sospecha de infecciones, bacterie- mia o sepsis, lo que resalta la importancia de realizar hemo- cultivos de calidad. Este estudio caracterizó los errores en la fase preanalítica de la toma de muestras para el diagnósti- co microbiológico de bacteriemias mediante hemocultivos. Se analizaron los protocolos existentes en el laboratorio y se compararon con estándares nacionales e internacionales, considerando aspectos como asepsia, volumen de sangre, bioseguridad y técnicas utilizadas. La información se reco- piló mediante encuestas al personal de salud involucrado, identificando como principales problemas la falta de proto- colos específicos, la carencia de recipientes adecuados para el transporte de muestras y la presencia de personal en for- mación. Aunque más de la mitad de los encuestados sigue correctamente los procedimientos de toma y envío, una pro- porción significativa no cumple con los estándares. Como re- sultado, se desarrolló un procedimiento operativo estandari- zado basado en buenas prácticas de laboratorio ecuatorianas e internacionales para mejorar la calidad de los hemocultivos en el hospital. Palabras clave hemocultivos, fase preanalítica, errores preanalíticos, buenas prácticas de laboratorio clínico. How to cite Mera, C. E., Sanclemente, J. P., & Howland, I. (2024). Preanalytical criteria for collecting blood culture samples in the Microbiology area at Portoviejo General Hospital. Journal of Advances in Education, Sciences and Humanities, 2(1), 11-18. https://doi.org/10.5281/zenodo.14602136 1 Laboratorio Clínico Crisvem, Portoviejo, Ecuador. 2 Quality Labs, Riochico, Portoviejo, Ecuador. 3 Facultad de Ciencias de la Salud, Universidad Técnica de Manabí, Portoviejo, Ecuador.

J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 12 Introduction Bloodstream infections represent a significant cause of morbidity and mortality, particularly in critically ill patients. Despite advancements in antimicrobial therapy and micro- biological diagnostic techniques, the incidence of sepsis remains a major challenge in modern medicine (Duncan et al., 2021). Timely initiation of appropriate treatment is cru- cial for reducing associated mortality and improving patient prognosis, provided there is no antimicrobial resistance. When a pathogen invades the bloodstream, clinical ma- nifestations can arise that may become fatal. Therefore, in addition to accurate clinical diagnosis, effective laboratory methods are required to rapidly identify the causative mi- croorganism, determine its antimicrobial susceptibility, and optimize clinical management (Huemer et al., 2020). Blood cultures are an essential tool in diagnosing bactere- mia and sepsis. Their accurate execution depends on standar- dized processes and strict adherence to protocols at all sta- ges, emphasizing the preanalytical phase (Lamy et al., 2016). The General Hospital of Portoviejo receives a high volume of local and referred patients suspected of severe infections, facing the challenge of ensuring the quality of performed blood cultures. To date, no prior research has been conducted to evaluate preanalytical criteria in this hospital, representing an opportunity to identify areas for improvement and enhan- ce patient care. The preanalytical phase is the stage of the process where most errors in clinical laboratories occur, with reported fre-quencies ranging from 17 to 84%, depending on the stu-died variables. These errors include issues related to sample quality, such as hemolysis, lipemia, insufficiency, or conta-mination, as well as failures in aseptic procedures, transpor-tation, and administrative records. Additionally, studies hi-ghlight the importance of having trained personnel and clear protocols to ensure compliance with good clinical laboratory practices (Alcantara et al., 2022). Implementing quality management systems in clinical la- boratories and using performance indicators enables tracking and evaluating each stage of the process, from the analysis request to the issuance of the result report (Chaudhry et al., 2023). This approach not only facilitates the identification of errors and delineation of responsibilities but also guides the implementation of improvements that directly impact clinical decision-making and, consequently, patient quality of life. This study aimed to characterize errors in the preanalytical phase of blood culture sample collection used in the micro- biological diagnosis of bacteremia in the microbiology de- partment of the General Hospital of Portoviejo. Methodology A qualitative, prospective, and cross-sectional study was conducted between June and September 2020 in the General Hospital of Portoviejo laboratory. Documents related to the quality management system of the hospital’s microbiology department were included. A survey was administered to 16 professionals involved in blood culture sample collection. The survey addressed key aspects such as knowledge of procedures, aseptic criteria, blood volume per blood cultu- re set, and biosafety measures. Data collection for the sur- vey, applied via Google Forms, was conducted directly by the researchers. The data were processed using Microsoft Excel and analyzed using descriptive statistics, calculating frequencies and percentages. Prior to the development of the research, approval was obtained from the Bioethics Committee of the Faculty of Health Sciences and the Presidency of the General Hospital of Portoviejo to review quality documents, collect blood cul- ture data, and implement the survey with personnel involved in this procedure. Results and discussion The results of blood cultures performed between January and October 2020 were reviewed, and a total of 1,084 tests were observed. Table 1 shows that nearly 50% of the blood culture tests were performed on patients over 61 years old, with a greater focus on patients over 71 years old. Pedia- tric patients accounted for 22% of the blood cultures, a high percentage compared to the 13% reported by Rodríguez et al. (2017) in a similar study conducted at the San Francisco Hospital in Quito. The blood culture was identified as the diagnostic method of choice in suspected bacteremia, being one of the most re- quested microbiological techniques in pediatrics. The detec- tion of bacteremia was essential, as it was associated with high morbidity and mortality, especially in immunocompro- mised patients, those with intravascular catheters, and those receiving broad-spectrum antibiotics. This situation favored the emergence of bacteremias caused by microorganisms that were previously considered rare or contaminants. Fur- thermore, the growing frequency of antibiotic-resistant mi- croorganisms was highlighted, a public health issue that has gained greater relevance today. Similar results were reported by Rodríguez et al. (2017) for the predominance of male gender (Table 1). In that study,

J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 13 60% of the patients who had blood cultures were male, and 13.48% of the blood cultures were positive for bacterial growth; this last value was similar to the 14.2% reported in the literature (Rodríguez et al., 2017). Table 1. Distribution of age, gender, and blood culture re- sults in the microbiology department of the General Hospital of Portoviejo Indicator Frequency Percentage Age (years) < 1 131 12.10 1 – 10 74 6.83 11 – 20 56 5.17 21 – 30 42 3.88 31 – 40 46 4.25 41 – 50 72 6.65 51 – 60 137 12.65 61 – 70 247 22.81 > 71 278 25.67 Gender Female 433 39.98 Male 651 60.02 Blood culture result Positive 146 13.48 Negative 938 86.52 Contamination rate Contaminated blood cultures 16 1.47 The staff responsible for conducting and analyzing the blood cultures indicated that, on occasion, results that could have been caused by germs proliferating due to contamina- tion were reported as positive cases. Not all blood cultures performed during 2020 were recorded, which was related to atypical characteristics due to the COVID-19 pandemic, which may have influenced the reported data. Table 2 shows that the most frequently isolated microorga-nism in blood cultures was Klebsiella pneumoniae, followed by Escherichia coli. Potential contaminant microorganisms such as Staphylococcus epidermidis, Staphylococcus sapro-phyticus, and Enterococcus faecalis were also identified. The frequency of appearance of potential contaminants in positi-ve blood cultures was 10.95%, with coagulase-negative Sta- phylococcus being the most common contaminant (7.53%), representing 68.75% of all contamination cases. Rodríguez et al. (2017) reported an overall contamination rate of 7.0%, higher than the rate recommended by interna- tional standards. They also found that 49.8% of all positive blood cultures were contaminated, with coagulase-negative staphylococci being the predominant contaminant microor- ganisms, representing 92.4% of contamination cases. On the other hand, Paniagua et al. (1988) reviewed 3,227 blood cultures processed over eight months. They found 249 Table 2. Microorganisms in blood cultures performed in the microbiology department of the General Hospital of Portoviejo Microorganism Frequency Percentage Most frequent Klebsiella pneumoniae 32 21.92 Escherichia coli 17 11.64 Acinetobacter lwoffii 11 7.53 Enterobacter aerogenes 10 6.85 Pseudomonas aeroguinosa 10 6.85 Klebsiella oxytoca 9 6.16 Staphylococcus aureus 8 5.48 Contaminant Staphylococcus epidermitis 6 4.11 Streptococcus bovis 6 4.11 Staphylococcus saprophyticus 5 3.42 Enterococcus faecalis 5 3.42 Serratia marcescens 5 3.42 Klebsiella aerogenes 4 2.74 Staphylococcus haemolyticus 4 2.74 Proteus mirabilis 4 2.74 Staphylococcus warneri 3 2.05 Acinetobacter baumannii 3 2.05 Enterobacter cloocae 1 0.68 Pseudomona alcaligenes 1 0.68 Achromobacter xylosoxidans 1 0.68

J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 14 positive blood cultures for coagulase-negative Staphylococ- cus sp., of which 30% corresponded to septicemia and 70% were considered contaminants based on clinical criteria and alterations in the blood count. Most septicemia cases were in neonates. They observed that the probability of isolating coagulase-negative Staphylococcus sp. from a blood culture was 7.7%. Coagulase-negative staphylococci are generally harmless inhabitants of the skin but can become pathogenic under certain conditions. The laboratory at the General Hospital of Portoviejo did not have a specific procedure for sample collection for this type of laboratory test, which is crucial in the treatment of sepsis. According to the Clinical Laboratory Operation Re- gulations approved by the Ministry of Public Health (MSP) of Ecuador in 2012, clinical laboratories must obtain an Annual Operating Permit, meeting the requirements of a Licensing Certificate, Quality and Biosafety Manuals, and certifications for waste management and staff training in the Technical Standard for Clinical Laboratories. ISO 15189 (2022), considered the Technical Standard for Clinical La- boratories, sets the requirements for the quality management system and technical aspects, including personnel, facilities, equipment, procedures, and quality assurance. This standard implies that laboratories must have specific procedures for their activities and define testing methods based on their characteristics and needs. Implementing ISO 15189 (2022) is necessary for laboratory accreditation and ensures quality and technical competence, benefiting patients by ensuring proper execution of tests and avoiding iatrogenic errors. Regarding international protocols for blood culture sample collection, the most commonly used include the M47A do- cument from the Clinical and Laboratory Standards Institute (Wilson et al., 2007) and the procedural manual from the Spanish Society of Infectious Diseases and Clinical Micro- biology (Rodríguez et al., 2007). These protocols agree on several key points to reduce contamination and false positi- ves, such as proper skin antisepsis with alcohol-based chlor- hexidine, phlebotomist training, the use of sterile gloves, cle- aning the blood culture bottle caps, and collecting samples during separate fever episodes (Tompkins et al., 2023). Using a Vacutainer® system with a reflux prevention me- chanism, adequate blood volume for children and adults, and collecting samples from multiple venipuncture sites are re- commended (Ombelet & Rico, 2019). They emphasize the need for bottles with resin for patients on antibiotic therapy and the implementation of monitoring programs that include contamination and positivity indicators (Lamy et al., 2016). The survey was conducted with staff dedicated to blood culture sample collection at the General Hospital of Porto- viejo. It was observed that there were no differences between the number of men and women, but the sample size was sma- ll. In some countries in the region, 75.8% of those enrolled are women (Alarcón, 2019). According to MSP data, 60% of health professionals in Ecuador are women, with 19,014 women out of a total of 37,930 general physicians and 9,351 women out of 19,444 specialists. The nursing field is domi- nated by women (Pérez-Sánchez et al., 2021). Half of the respondents were between 30 and 35 years old, 25% were between 36 and 40, and the remaining quarter were over 40 (Table 3). Age is considered an important fac- tor in blood culture sample collection, as older individuals are believed to have more experience with the procedure. Pearse & Scott (2023) indicated that clinical laboratory gra- duates should have experience gained by performing various procedures. Thus, older individuals achieve better results, greater concentration ability, and higher skill in executing what they have learned. Table 3. Sociodemographic characteristics of the surveyed healthcare staff Indicator Frequency Percentage Age (years) 30 - 35 8 50 36 - 40 4 25 > 40 4 25 Gender Female 8 50 Male 8 50 Profession 7 43.75 Bachelor's Degree in Clinical Laboratory Bachelor's Degree in Nursing 9 56.25 The majority (56%) of the surveyed healthcare staff had a degree in nursing, and the remaining 44% had a degree in clinical laboratory science. This highlights the close rela- tionship between nursing staff and patients and their colla- boration with other healthcare team members in promoting user well-being. Figure 1 shows the most common errors in blood culture sample collection reported by the respondents. The absence of protocols in the laboratory room (43.75%) was the most frequently cited error, followed by the lack of proper contai- ners for sample transportation (25%), too many staff mem- bers in the learning phase (18.75%), and 12.5% mentioned

J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 15 that orders were not made on time. Iqbal et al. (2023) sug- gested that the quality of the analytical phase and its results largely depended on the preanalytical phase, but it was not given the attention it deserved. Particularly in high-demand laboratories, students were not adequately trained during their internships. The economic cost of preanalytical errors was 10% of the total cost of obtaining and sending samples, and in public hospitals, these errors cost the country as a whole (Iqbal et al., 2023). Respondents reported a lack of preparation at the beginning of their clinical practice activi- ties and the absence of protocols. In Table 4, it is observed that 62.5% of the staff surveyed indicated that blood cultures are taken based on clinical sus- picion of sepsis, while 37.5% reported that they were taken prior to systemic antimicrobial therapy, which is corrobora- ted by the literature of Alados et al. (2014) in their Manual on Clinical Microbiology Procedures, where they stated that blood cultures remain the primary diagnostic method for determining the etiology of bacteremia. Its easy execution makes it accessible to any center, and it is the only method that allows the isolation of viable microorganisms necessary for determining their antibiotic sensitivity. Its usefulness is highly associated with its exclusive use in patients with a cli- nical presentation compatible with bacteremia. Performing it under other circumstances increases healthcare costs and does not provide clinically useful information. Table 4. Survey results on practices and protocol com- pliance in blood culture collection at the General Hospital of Portoviejo Indicator Frequency Percentage Reason for performing blood cultures Indicated before systemic antimicrobial therapy 6 37.5 Clinical suspicion of sepsis 10 62.5 Time of sample collection Near the fever peak 10 62.5 Far from the fever peak 6 37.5 Compliance with the blood culture collection protocol Yes 11 68.75 No 5 31.25 Strict compliance with biosafety standards Yes 10 62.5 No 6 37.5 Technique for performing blood culture Manual 10 62.5 Automatic 6 37.5 Compliance with training Yes 6 37.5 No 10 62.5 Delays hinder the practical value of diagnosis in obtai- ning results because it is not favorable in all patients, with its lowest performance being in patients on antibiotic treat- ment or those with fungal infections, slow-growing bacte- ria, or those requiring special growth conditions. Another limiting factor is the high proportion of contaminated blood Figure 1. Most common errors made in blood culture sample collection.

J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 16 cultures by microorganisms belonging to the skin microbio- ta; this process generates diagnostic errors and inadequate treatments and incurs high economic costs for the healthcare system. The sensitivity of blood cultures is related mainly to the sample volume, the timing of collection, and the absence of prior antibiotic treatment (Cohen et al., 2015). In Table 4, it is observed that 62.5% of the staff indicated that the samples were taken close to the fever peak, while 37.5% reported that the samples were taken far from the fe- ver peak and symptoms. This practice does not comply with the guidelines from the Murcia Health Clinical Practice Gui- de, which states that for blood cultures, the sample should be collected as soon as possible after a fever peak to avoid affecting the isolation of causative microorganisms. Howe- ver, a study (Hernández-Bou et al., 2016) found no signifi- cant differences in isolation rates if blood was drawn during afebrile intervals or simultaneously with the fever peak. It has been recommended that the optimal time for sample collection be as soon as possible after the appearance of cli- nical symptoms, although blood can be sampled at any time. The blood collection during or immediately after a fever peak is considered optimal, except in cases of endocarditis. The detection of bacteremia through blood cultures establi- shed that the presence of fever at the time of blood culture collection was neither sensitive nor specific for the presence of bacteremia. Hernández-Bou et al. (2016) evaluated the ti- ming of blood culture collection in relation to temperature elevation in more than 1,400 patients with bacteremia and fungemia. They found no correlation between the timing of sample collection and the likelihood of a positive blood cul- ture. Most of the healthcare staff (68.75%) followed the steps for properly collecting and delivering blood culture samples in chronological order, while 31.25% did not. According to Alados et al. (2014), the correct methodology for blood cul- ture extraction includes using gloves and a mask; cleaning the vials’ caps with chlorhexidine; selecting the blood co- llection site (avoiding blood extraction via catheter); disin- fecting the skin with chlorhexidine, letting the disinfectant act; performing the puncture without touching the patient’s skin with the hand; avoiding contact between the needle and cotton; extracting the necessary amount of blood (10 ml per bottle in adults and between 1 and 5 ml in children); ino- culating the anaerobic bottle first, followed by the aerobic one (without adding air), and other tubes if necessary; gently shaking the bottles, and urgent transport to the microbiology service or, if not possible, maintaining at room temperature. If done correctly, sensitivity and specificity are high, but in- correct execution can lead to erroneous results, so the proce- dure should not be performed unless optimal conditions are met (Cuervo et al., 2001). A total of 62.5% of the staff strictly followed laboratory safety rules, whereas 37.5% did not. Similar results were reported by Alados et al. (2014) in their microbiology ma- nual, where they emphasized that the blood inoculated in the vials may contain, in addition to bacteria or fungi, other via- ble microorganisms such as hepatitis or HIV viruses, which represent an infection risk for those handling the samples. Knowing and strictly adhering to universal prevention mea- sures for handling blood is crucial, particularly in preventing accidental needle sticks during blood extraction or proces- sing positive vials. The replacement of glass vials with plastic vials has helped reduce accidents caused by breakage. Some companies have recently developed blood collection and puncture systems that eliminate the risk of accidental needle sticks while pro- cessing positive vials. Cuervo et al. (2001) highlighted the importance of staff strictly following laboratory biosecurity rules to minimize these risks. One respondent indicated that blood cultures are taken by medical or nursing staff, not by laboratory personnel; however, laboratory technicians must have all the necessary knowledge to perform this procedure and its subsequent analysis, as they are the ones professionally trained to co- llect, process, and interpret biological samples. Techniques used for blood culture collection indicated that 62.5% were performed using the manual method, while 37.5% used the automatic method (Suk-Fong et al., 2018). A majority of the staff, 62.5%, reported not having recei- ved training on blood culture collection in the past two years, compared to 37.5% who had received it. Training on any topic within the healthcare team is fundamental for profes- sional growth and is recommended by all international stan- dards to ensure quality care for users. The research findings highlighted the need to update the knowledge of professio- nals involved in blood culture sample collection to avoid fal- se positives and contamination—factors that could compro- mise results, treatment, and, consequently, the patient’s life. Conclusions No specific procedure for blood culture collection was identified in the laboratory despite its importance in treating sepsis. The main errors detected include the absence of pro- tocols, the lack of suitable containers for sample transport,

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J. Adv. Educ. Sci. Humanit. (January - June 2024) 2(1): 11-18 18 Hepatology Communications, 3(1), 8-19. http://dx.doi. org/10.1002/hep4.1281 Tompkins, L.S., Tien, V., & Madison, A.N. (2023). Getting to zero: Impact of a device to reduce blood culture contamination and false-positive central-line-associat- ed bloodstream infections. Infection Control & Hos- pital Epidemiology, 44(9), 1386-1390. http://dx.doi. org/10.1017/ice.2022.284 Wilson, M.L, Mitchell, M., Morris, A.J., Murray, P.R., Re- imer, L.G., Barth, L., Towns, M., Weinstein, M.P., Well- stood, S.A., Dunne, W.M., Jerris, R.C., & Welch, D.F. (2007). M47-A Principles and Procedures for Blood Cultures; Approved Guideline. Clinical and Laboratory Standards Institute. https://clsi.org/standards/products/ microbiology/documents/m47/ Conflicts of interest The authors declare that they have no conflicts of interest. Author contributions Conceptualization: Carlos E. Mera, Jean P. Sanclemen- te. Research: Carlos E. Mera, Jean P. Sanclemente, Ivón Howland. Methodology: Ivón Howland. Supervision: Ivón Howland. Validation: Ivón Howland. Writing the original draft: Carlos E. Mera, Jean P. Sanclemente, Ivón Howland. Writing, review and editing: Carlos E. Mera, Jean P. San- clemente, Ivón Howland. Data availability statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Statement on the use of AI The authors acknowledge the use of generative AI and AI-assisted technologies to improve the readability and cla- rity of the article. Disclaimer/Editor’s note The statements, opinions, and data contained in all publi- cations are solely those of the individual authors and contri- butors and not of Journal of Advances in Education, Scien- ces and Humanities. Journal of Advances in Education, Sciences and Humani- ties and/or the editors disclaim any responsibility for any in- jury to people or property resulting from any ideas, methods, instructions, or products mentioned in the content.