Aim: The term candidemia simply indicates the presence of Candida species in the blood. Candida spp. are important pathogens in neonatal intensive care unit (NICU) patients, critically ill patients, and those with underlying immunocompromising conditions. Material and Method: In this retrospective study, 144 nosocomial candidemia episodes of 106 pediatric patients who had been admitted to pediatric units of Çukurova Univesity Hospital from 2010 January to 2013 December were evaluated. These 106 children’s demographic and clinical features, risk factors, blood and intravascular line cultures, antifungal resistance, treatment and clinical outcome were evaluated. Results: The most frequently isolated species were Candida parapsilosis (31.9%), C. albicans (29.2%). C. tropicalis (16.0%), C. kruseiandC. Famata (4.2%),C. Glabrata (3.5%). Risk factors of 106 pediatric patients with Candidemiae were investigated. The main risk factors for candidemia included the presence of intravascular (IV) lines (68.1%), multiple antibiotics therapy (79.1%), total parenteral nutrition (59.7%), nasogastric tube insertion (45.1%), mechanical ventilation (34.7%), admission to intensive care unit (32.6%), and neutropenia (22.9%). The overall mortality rate of candida species was 17.9%. Discussion: Regular surveillance of local Candida species, resistance profiles, and risk factors are important in order to identify patients at risk and to develop empirical treatment protocols to reduce the incidence and mortality of candidemia.

Keywords: Candida; Risk Factor; Mortality; Childhood

• Introduction

  During recent years, fungal infections are reported to constitute an increasing proportion among all nosocomial infections, only preceded by bacterial infections. Regarding invasive fungal infections, Candida species are the leading causative agents in hospitalized children. In the United States, Candidaspecies are reported as the third leading cause of healthcare-associated bloodstream infections [1,2].

  Although the most common agent in Candidemia is C. albicans, there has been an increasing incidence of non-albicans Candidaspecies in recent years [3,4]. Especially among child patients, C. parapsilosis and C. tropicalisconstitute more than half of the non-albicans Candidainfections [5,6]. According to studies, the most common risk factors for Candidemia are broad-spectrum antibiotic use, total parenteral nutrition (TPN), the presence of a central venous catheter, malignancy, chemotherapy, immunosuppressant use, neutropenia, and diabetes [7-9].

  Nosocomial fungal infections are an important cause of increased morbidity and mortality. Some causes of increased frequency of fungemia and fungemia-associated mortality are the increasing number of diagnostic and therapeutic interventions applied to patients as a result of the advances in modern medicine, widespread use of treatment regimes that suppress host defense mechanisms, and ever-growing utilization of broad-spectrum anti-bacterial drugs. Among all children with sepsis, fungal infections are the second leading cause of mortality (13%). Candidemiais often characterized by symptoms and signs of sepsis [2,3,10-12].

  In this study, we aimed to investigate the demographical and clinical features and treatment outcomes of Candidemiaepisodes that developed in patients hospitalized in pediatric wards of Çukurova University Hospital, to determine the predisposing risk factors for the purpose of taking necessary precautions, and to compare epidemiologic variations in nosocomial candidemia.

  Material and Methods

  In this study, we retrospectively examined 144 candidemiaepisodes in 106 patients who were treated as inpatients in intensive care units (pediatric, neonatal, cardiovascular surgery, pediatric surgery, neurosurgery), pediatric hematology and oncology wards, and other pediatric wards from January 2010 to December 2013. A data form was created; each patient’s demographical and clinical features, risk factors, blood and catheter culture results, antifungal resistance patterns, treatment, and clinical outcomes were obtained from patient files and were recorded on these data forms. In some patients, antifungal treatment was initiated empirically based on the patient’s age, the site of candidagrowth and underlying disease (especially febrile neutropenia); and it was adjusted according to antifungal sensitivity results. The study was approved by the institutional review board.

  Candidemiais defined as isolation of a Candidaspecies in at least one blood and/or catheter culture in the presence of systemic symptoms and signs. Catheter-associated Candidemia is regarded as >15 cfu candida growth in a catheter-tip culture. Utilization of one or more types of antibiotics for more than 14 days, neutropenia (absolute neutrophil count <1000/mm³), history of surgical operation within the last 30 days, TPN and catheter applications, chemotherapy and malignancy were accepted as risk factors. Deaths occurring within the first 30 days in the presence of clinical, microbiological and/or histological evidence were accepted as Candidemia-associated mortality.

  All the data were analyzed using SPSS version 20 (SPSS, Chicago, IL, USA) statistics package software. Student t-test and chi-square tests were used for comparison of the groups.

  p< 0.05 was accepted as the level of statistical significance.

  Results

  Table 1 shows the demographical and clinical features of 106 patients with Candidemiaincluded in this study. Mean age (± SD) was 43± 47 months (median age: 28 months). The male/female ratio was 54/52. Of these patients, 28.8% were admitted to intensive care units and 25.5% were admitted to pediatric hematology and oncology wards.

  Of the examined patients, 82 (77.3%) were younger than 60 months while 24 (22.7%) were older than 60 months. The general mortality rate was 17.9%. Of those patients who died, 68.4% (n: 13) were younger than 60 months, and 47.3% (n:9) were younger than 12 months.

  General mortality rate among cases with Candidemia was 17.9%. Mortality rate was higher among children younger than 60 months (68.4%), with C. albicansspecies (25%) and C.nonalbicansisolates (75%) (Table 7). Only one patient developed endophthalmitis (0.9%) as a complication.

  Table 2 summarizes risk factors for Candidemia. Main risk factors detected in 106 patients with Candidemiaexamined in our study were: intravascular catheter (68.1%), multiple antibiotherapy (79.1%), total parenteral nutrition (59.7%), nasogastric tube (45.1%), mechanical ventilation (34.7%), admission to intensive care unit (32.6%) and neutropenia (22.9%).

  Table 3 shows Candidaspecies isolated from blood and catheter cultures. The most frequently isolated Candidaspecies are C. parapsilosis(31.9%), C. albicans(29.2%), and C.tropicalis(16.0%).

  Table 4 shows the body fluids from which Candida species were isolated during Candidemia episodes. Candida species were isolated from blood culture in 94.4% and from catheter culture in 5.6% of Candidemia episodes.

  Table 5 shows the results of anti-fungal sensitivity test in isolated Candida species. Totally 7 of the 144 isolates (4.9%) were found to have antifungal resistance, and all of these isolates were NAC species. Amphotericin B resistance was detected in 2 (1.4%) isolates; fluconazole resistance was detected in 4 (2.8%) isolates; and voriconazole resistance was detected in 1 (0.7%) isolate. One isolated C.parapsilosisspecies had resistance against amphotericin B, while another isolated C.parapsilosisspecies had resistance against both amphotericin B and voriconazole.

  Table 6 shows sensitivity to fluconazole among Candida species, and the association between Candida species and mortality. All C. albicans isolates were sensitive to fluconazole, while 3.9% of Candida nonalbicans isolates were resistant against fluconazole. Of the 6 isolates of C. krusei, 4 (66.7%) had resistance against fluconazole.

  Treatment Outcomes of Patients

  Of all patients, 40.6% received fluconazole. According to the guideline on febrile neutropenic patients, 26.2% received amphotericin B, 15.3% received caspofungin, 9.6% received voriconazole, and 2.6% received amphotericin B + flucytosine. For those who had catheter infection, catheter was removed. When the treatment was initiated empirically in febrile neutropenic patients, the treatment was not changed if the antifungal sensitivity test results showed that the growing Candida species was sensitive to the administered drug.

  Table 7 shows risk factors responsible for the development of C. albicans andC.nonalbicansinfections. In comparison to C. albicans,the major risk factors in Candida nonalbicansinfections were as follows: mechanical ventilation, presence of the urinary catheter, nasogastric tube placement and admission to intensive care units (p < 0.01, p < 0.05). Among patients with Candidemiawho did not receive gastric acid suppression treatment, C. nonalbicansinfection was more frequent (%75.2) compared to C. albicans(%24.8) (p<0.05). C. albicansinfection was significantly more frequent compared to C.nonalbicans infection among patients treated in the intensive care unit (p<0.005).

  Discussion

  Candida-associated fungemia takes the lead among all nosocomial fungal infections.

  Studies related with Candidemia report the major risk factors for the development of these infections as broad-spectrum antibiotic use, total parenteral nutrition, central venous catheter, malignancy, chemotherapy, immunosuppressant drug use, neutropenia and diabetes [7,9]. According to our results, major risk factors for the development of all Candidainfections were found as multiple antibiotic use (79.1%), the presence of central venous catheter (68.1%), total parenteral nutrition (59.7%), in decreasing order of frequency; and our results seem to be consistent with previous reports. Moreover, in comparison with patients with C.albicans infections, mechanical ventilation (34.7%), the presence of nasogastric tube (45.1%), admission to intensive care unit (32.6%) and presence of urinary catheter (29.1%) were significantly more frequent among patients with Candida nonalbicansinfections (p<0.05). Candidanonalbicans infections were significantly more frequent in comparison to C.albicansinfection among patients who receive gastric acid suppression treatment (75.2% vs. 24.8%) (p<0.05).

  Considering admission rates to intensive care and neonatal intensive care units in our study group (31.2%+20.1%=51.3%), our results are predictable and consistent with the results of previous studies.

  Recent studies related with Candidemiain child patients show that the proportion of C.albicansinfection is decreasing, being replaced by Candida non-albicans infections [9,11,13,15-17]. In our study, the most frequently isolated Candida species during Candidemia episodes within the last 3 year was (% 29,2); and the proportion Candidemiadue to non-albicans Candida was 70.8% (Figure 1). Especially between the years 2011 and 2013, we detected an increase in the proportion of Candida non-albicans (62.5%-70.8%) infections in comparison to C.albicans infections (17.6%-37.5%), which was a finding that is consistent with previous reports (Figure 2).

  Studies report the most frequently isolated non-albicans Candidaspecies as C. tropicalis, C. parapsilosis,C. glabrata and C. krusei. While C. parapsilosis takes the lead among Candidemia causes in Europe, Canada, and Latin America countries; in the US, the most frequent cause is C. glabrata [14].In their study involving 102 pediatric patients, Çelebi et al. [15] detected C. albicans in 39.2%,C.parapsilosis in21.6%, C. tropicalis in 15.7%, andC.glabrata in 6.9%of patients.In our study, we detected C. albicans in 29.2%,C. parapsilosis in 31.9%, C tropicalis in 16%, andC glabrata in 3.2% of our patients, and our rates were similar to theirs (Figure 2, Table 3).

  Studies in pediatric age group indicate that fluconazole is effective and safe in treatment of Candidemia.Accordingly, 40.6% of the patients in our study group were treated with fluconazole only as the first line of the treatment [18,19]. Additionally, 26.2% of the patients which had febrile neutropenia were treated with amphotericin B, while 15.3% received caspofungin, 9.6% received voriconazole, and 2.6% who had neonatal meningitis were treated with amphotericin B + flucytosine. When the treatment was initiated empirically in febrile neutropenic patients, the treatment was not changed if the antifungal sensitivity test results showed that the growing Candida species was sensitive to the administered drug. Four patients (2.6%) who had ongoing Candidemiarefractory to treatment were treated with a combination of amphotericin B and fluconazole.

  Fluconazole is the most commonly used antifungal for the treatment of invasive Candidainfections, and resistance against fluconazole and other azoles is an important therapeutic problem. Studies show fluconazole resistance does not exist among C.albicans isolates but is present in high rates among C.nonalbicansstrains such as C.krusei (78.5%) and C.glabrata (63.16%). In our study, all of the C.albicans isolates were sensitive to fluconazole, while 2.8% of Candida non-albicansstrains showed resistance against fluconazole, and 66.7% of these resistant strains were C.krusei. Some authors relate the increasing rate of fluconazole resistance to the fact that it is the first line of a drug in the treatment of Candida infection [20].

  One study found Candidemia associated mortality rate as 42.4%; in that study, the majority of the mortal cases were infected with Candida non-albicans isolates and under 1 year of age [21]. Among infants and children, Candidemia- associated mortality rate varies in a wide range between 11% and 54% [17,22-24] . The variation in this rate depends on the underlying disease, age and Candida species. In our study, the general mortality rate among cases with Candidemiawas 17.9%. Mortality rate was 68.4% among children younger than 60 months of age, and 47.3% below 12 months of age. Mortality was higher among patients infected with Candida nonalbicans species (75%).

  As a conclusion, in order to control nosocomial Candidainfections and to reduce mortality associated with these infections, it is of vital importance that regular surveillance studies are carried out, predisposing risk factors are taken under control, and empirical treatment schemes are established after determining resistance profiles.

  Scientific Responsibility Statement

  The authors declare that they are responsible for the article’s scientific content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientific review of the contents and approval of the final version of the article.

  Animal and human rights statement

  All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.

  Funding: None

  Conflict of interest

  None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.

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Kocabas E, Cay U, Dayar GT, Cetin C, Seydaoglu G, Kibar F. Analysis of 144 pediatric nosocomial candidaemia episodes over a four-year period. Ann Clin Anal Med 2020;11(1):15-19

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Aim: Healthcare providers have an important role in notifying mothers about their children’s handicap, in other words, the bad news. The mothers’ acceptance of the newborns, adapting to the process and the family dynamics are thought to be affected by this first notification period, so it is assumed to be a very important period. There have been important speculations about notifications regarding the child’s physical and psychological well-being and about the parent’s unhappiness. It was found that there is insufficient literature about how and when the parents should be notified about their child’s handicap; in fact, it was noticed that this topic was not covered in our country at all. In this light, the aim of this study is to determine how mothers who have children with Down’s syndrome are notified, and how they prefer to be notified. Material and Method: In order to determine the mothers’ preferences for the notification, a comprehensive literature survey, and interviews that are a qualitative method have been used. The participants consisted of 21 mothers whose children attended two different private rehabilitation centers in Istanbul and Karabük. In analyzing the data, the “Interview Form” and the transcripts of the interview recordings were used. Consecutive coding from A1 to A21 was done to describe the participants. Results: The findings show that the mothers were not pleased with the first notifications and that they are critical of the current notification system. According to our results, various suggestions to doctors were done. Discussion: Informing mothers that they have a different child is an important task for doctors that will affect the relationship between thischild and the mother throughout their lives. For this reason, it is very important that doctors are knowledgeable and sensitive about the reporting process.

Keywords: Down Syndrome; Parents; Diagnose; Acceptance

• Introduction

  Down’s syndrome was the first chromosomal disease defined in the literature. It was defined by J. Langdon Down in 1866. In 1959, Lejeune, Gautier, and Turpin proved that the reason of the disease is the extra numbers of chromosomes. Resulting from the trisomy of the 21st chromosome, Down’s syndrome can occur in 1 out of 700 births and can be diagnosed during pregnancy [6]. These children may have phenotypic conditions as well as a deficiency in growth and cognition and also many health problems in different body systems [2]. From their birth onwards, they need to receive care and support to cope with these difficult conditions.

  All parents would like to have a completely healthy child. “To have a special child” is experienced differently in all families and for some, it can be more painful and hard. They want to employ positive methods to handle or their coping skills may fade. As a result, they can experience physical and emotional burnout syndrome, psychosomatic illnesses, social isolation, drug addiction, inappropriate anger problems, ignorance for the child’s illness and they might keep away from the child, wearing themselves and the children out [9]. For this reason, the first approach while giving information to the parents about the medical condition of the child and the statements used are very important for the process which follows.

  Informing mothers of disabled children can be seen as delivering bad news and the role of healthcare professionals is very important. After the diagnosis, healthcare professionals will be faced with various questions such as: “How to explain the diagnosis? What are some appropriate responses? How to deliver the news in a birth environment? How should the situation be discussed with the other parents informed about the birth?” It is very important that mothers are provided with the correct approach in the first process of learning about the syndrome that is thought to affect many areas such as acceptance of the newborn, adapting to the process and family dynamics. The timing of giving the news to the mother, choosing “the correct words” and up-to-date information are very crucial. According to researches, the positive approaches of healthcare professionals adopted towards the mother might determine the reaction of the mother to this unexpected situation. However, when there is a doubt about the disability of the baby or it needs confirmation, healthcare professionals feel disturbed about giving information or are unprepared. Doctors might refrain from giving information to the parents about the ultimate diagnosis. In the literature, it is reported that birth centers have made protocols on this subject, it is standing out that there is not enough study on how and when to give information to the parents about the physical or mental disabilities of the child and in our country, there is not any research on this topic [10].

  This study aims toexplain how the mothers learn about the Down’s Syndrome diagnosis, what they learn about the diagnosis and their choice of being informed; giving a draft to healthcare professionals on how to communicate and inform the parents about such a delicate subject and give information to parents both before and after birth.

  Accompanying a sensitive and appropriate treatment, the sincere and affectionate approach of healthcare professionals will help the family accept the fact and assist the parents of the disabled child in this lifelong journey.

  Material and Methods

  This research is structured by using qualitative research methods and techniques. Qualitative research is a process of making meaning by questioning social life and human problems with their own methods [3]. In the qualitative research process, the researcher presents the integrated research table, uses word analysis, detailed participant interview reports, and organizes the research in a natural setting. The qualitative research process is generally followed from part to entire /whole (inductive).

  In general terms, the qualitative researcher maintains the process by explaining concepts, meanings, and relationships, starting from observations, interviews and documents [5]. In this study, the semi-structured interview model was used as a qualitative research model.

  Population and Sampling:

  The population of the research was formed of 21 mothers having child with Down syndrome in two separate Special Education and Rehabilitation Centers training from different states which are Karabük and İstanbul. The average age of the mothers is 42.3 and their ages range from 28 to 63 years. The average age of children is 8.3, the oldest being 25 and the youngest being 1 year. There are six mothers who graduated from high schools, and six mothers from primary schools, five mothers from high schools, and three mothers from junior high schools. Lastly, one mother was not literate.

  Collection of Data:

  The data of the study were collected using the semi-structure. The semi-structured interview form was developed by researchers, taking into account similar studies in the literature. The form contains demographic information and 9 open-ended questions (Table 1.). The boundary of interview topics and questionnaires were defined and the interviews were recorded in this framework by recording with a voice recorder. Each interview lasted approximately 20 minutes, at least six minutes and maximum 73 minutes in accordance with the intended purpose and in the interactive environment. Mothers who agreed to participate in the interviews organized at the Special Education and Rehabilitation Centers were interviewed at the time of their child’s education at the institution. Interviews were organized in an empty room provided by the institution. Mothers who participated in the research were given a “consent form” to explain the purpose of study and to approve their participation.

  Analysis of Data

  In the analysis of the data, “Interview Form” and written records of the data were used. These were obtained by recording with a voice recorder. Sequential coding was performed from A1 to A21 for identifying the participants. The analysis process was modeled by Marshall and Rossman (1999) in five models: a) Organizing the data; b) Definition of themes and categories; c) Testing the emerging hypothesis with data; d) the search for alternative explanations of the data and e) writing the report. In this context, when creating themes, titles can be gathered under the same heading, and titles are arranged by making quotations from appropriate speeches.

  Results

  The Time of Learning the Down’s syndrome Diagnosis of the Children:

  When the answers of the mothers to the question about when they had learnt about the Down’s syndrome diagnosis of their children are investigated, it can be seen that they learnt it in time ranging from 3 days to 6 months after the birth (Table 2).

  Mothers expressed that they were relieved upon receiving the news. Two participants told that waiting for the final diagnosis was much more stressful.

  A3: “For three months, I had such a doubt which made me even worse. Isn’t it so? Uncertainty is really bad. I would constantly search for children with Down’s syndrome online. When my husband told me that our child has the syndrome, a huge burden was lifted off my shoulders. I calmed down, I settled down.”

  A5: “I lived with that suspicion for 6 months. Then, it took one more month before it became certain. For 6 months, I prayed for it not to happen. Waiting for 7 months was worse than learning about it.”

  A18 stated that learning about the syndrome before the birth was very important in order to be prepared for it: “It is an advantage to learn about the syndrome before birth. At least, one gets used to the idea and prepares herself for the possibility. I no longer pray for the syndrome not to occur, sure. If I had learnt about it after giving the birth, the shocking effect could have lasted longer. It could have been much more difficult to accept. Facing the fact all of a sudden while expecting a normal child would be harder.”

  A14 who said that she could have had an abortion: “To tell the truth, I would have had abortion thinking about the difficulties and distress that my child endured from his family life to school performance. I would not do it for myself, thinking about my ego or for feeling comfortable, but for my child so as not to make him endure such a difficulty”.

  Our participant A21 argues that not knowing about the syndrome increases anxiety and sadness. Learning about the disease makes the hard period of pregnancy more difficult: “I think that learning about the syndrome after giving birth contributed to my beautiful period of pregnancy. Luckily, I learnt it after the birth because I do not know what I would do if I learnt it before. I would not know how to cope with it, my pregnancy would be painful. Fortunately, I learnt it after the birth.”

  Nine mothers who learnt about the first suspicion of the child after the birth got the information from the pediatrician, two of got them from the gynecologist, two of them got the information from the explanation of both the pediatrician and the gynecologist; two mothers had a suspicion from their prior knowledge about the symptoms (Table 2).

  The evaluation of how and from whom the mothers learnt that their children had Down’s syndrome:

  Participant mothers explained their negative experiences with regard to the question: “Who gave you the informationthat your child has Down’s syndrome and how?”

  A16: “The Pediatrician told me: “Your child is retarded. He will not ask for any bread, water or do anything. He will eat only what you feed him or ask nothing.” This is how I learnt my child was disabled.”

  A11: “Our obstetrician told me the news right before he cut the umbilical cord. It was a vaginal delivery, he told me during delivery. It was awful. He told me that my child was a healthy Mongolian baby. I was devastated. I did not know what a Mongolian baby was. He told me that he would not be able to talk, walk or go to school like normal children. He told all this at the delivery room”.

  A19: “I no longer go to the genetics department. There was a woman whose name started with Z and she directly told me: “Why did you give birth?” When I left her room

  I was like “I gave birth to a monster, why did they give it to me, what am I supposed to do with it ’? When I went there for the second time, there was another professor. “You gave birth to this baby because of motherly instincts but he will suffer a lot. You did a wrong thing, why did you bring this child to this world” he said.

  A21: “The information given was very insufficient. It was this ambiguity that made me uncomfortable. It was the missing information. It was a wrong information. Maybe I was not given any information because I was a doctor. However, it is very incorrect. The person who is explaining should explain the facts with great care and detail as if the person who he/she is talking to is not a professional and knows about nothing”.

  The evaluation of opinions about how to deliver the news to the families

  When the answers provided by the mothers to the question “How would you prefer that this news is told to the families and when’’ were examined, it is seen that they provided a range of suggestions in parallel with their own experiences of distress (Table 4).

  Nine participants suggested that the initial information on Down syndrome should also include the positive aspects of the disease. One of these propositions was included in the participant’s statements as follows:

  A5: “They say it will be good, not bad. No need to talk about bad probability. He will be fine when I deal with him. Then I would be happier and I would not get exhausted. There is a choice of telling good news before telling bad. When doctors say bad things, you think your kid will not have a reason.”

  According to the distribution of the data, the second most frequent respondent is “the husband should be next to his wife (mother)” and as the reason for this:

  A14: “A person should not be alone when learning this news. I would very much like to share this burden with my husband. What mother and father need to do, two people who bring the child to the world, also father should do the truth consciously. The couples must be together when this news is given so that they can learn what to do.”

  Six participants expressed similarity with A7 that it is important to be informed about the development of their children and possible health problems:

  “What the Down syndrome is, what features does it have, deficiencies, improvements, developments should be explained, and it should be said that there may be some discomforts after giving birth. It should be said that they can come up to a certain degree with education. Physical therapy should be recommended. I mean they need to focus on education. If they have discomfort, they should be directed to the treatment.”

  The evaluation in terms of how mothers felt when they learnt about the syndrome

  Mothers were asked the following questions: “What was your first reaction after learning about the syndrome? What did you think? How did you feel?” and their answers are collected under five categories namely future anxiety, sorrow, acceptance, denial and despair. Table 5 consists of the distribution of the mothers’ first feelings.

  Mentioning problems about future anxiety, A8 has important expressions: “How can I look after my baby? Will I be powerful enough? Will I have any patience? What can I offer him? People are not informed about these issues. This child is special and first we thought about what we can offer him. I was sorry. It was my first child. I got married late. I was pregnant without any treatment. We were really happy because we didn’t think of another child because of my age. We hoped for the best.”

  Participant A21 says the first feelings changed during this process: “I wanted to get an eraser and erase it or change the time. I cursed my faith. When my baby came out of the incubator, all these feelings changed. I took my baby in my arms and we made a good connection from that moment onwards.”

  The evaluation in terms of factors, which have effect on the acceptance process of mothers

  Elevenparticipants gave answers to the question abouthow they got over the process of accepting the fact that their children have Down’s syndrome as they believed in faith. Other participants gave information about how they coped with and accepted the situation with a range of answers. The distribution of answers is given in Table 6.

  Because of their religious beliefs, they saw this as a “trial” or “grace” during the acceptance of the child.

  A11: “We thought that this is a grace from God. We always think about good things. We accepted and I recovered. I hugged her differently. I have two more children, but I can say that I have learned and experienced the feeling of motherhood with my daughter.

  A19: “I said one night: “My God, this human being is yours, and you gave the feeling of motherhood, and you gave me this child. You gave the health. If it is a punishment it’s my punishment if it is a reward, it’s my reward. If I deserved it, this came from you. But this person is very incapable. You lead me to an exit” I prayed.”

  A4: “It lasted no more than a week. What are you going to do? God has appreciated and gave. You can not put your child outside the door, or you cannot give your child to someone else, you have to look after your child.”

  Stating that she has received too much support to get through the acceptance process, A21 said she feels better when she sees that her child can do something. Unlike all participants, she asserted that the acceptance process precedes life long: ‘’The admission process is something that you have to face again when that child encounters everything that needs to be done. I accept it is not a completed process. You are constantly facing. When someone comes and asks you what problem does the child have, then you remember once again even if you forgot it.”

  The evaluation in terms of being informed about special education

  Mothers who learned about the importance of physical therapy and special education started physical education and the training process a couple of months after giving a birth with the guidance of a genetic doctor. Being different from the other candidates, A1 who learnt about special education via a radio channel expressed what she experienced 13 years ago: “When the baby was 3-4 years old, we used to hear every announcement made listening to the radios and leant about special education back then. The doctors did not mention any education when we visited the hospital for the vaccines. We never thought that education needed to start early. We thought it needed to start at the schooling age.”

  Discussion

  After the doubt disappears and diagnosis is accurate, families are informed by an expert. An Expert should consult the family in a suitable time and context. Both of parents should take part in this interview and one of the relatives can come next to them. This information should be given with pediatrist who follow up the child should be together with them. Parents should be welcomed with a positive and sincerely facial expression. Parents should be met with a positive and intimate facial expression. Parents should not be approached with pity as if doctors were giving bad news, or on the contrary, they should not have a paternalistic sense of air. It should be kept in mind that this interview is an informative meeting. Firstly, an expert should start with brief information about what is the Down syndrome and he should talk about the positive characteristics of these children. Afterwards, should be explained in a very brief way the medical and social difficulties life related to DS that will affect the child’s. It should be guided about the importance of the early start of special education, physical therapy and the process where to be applied. In this first meeting, the questions of the family must be answered clearly and briefly and then the family should be referred to a Medical Genetics or Child Genetics Specialist for further genetic counseling. Resource books list and the names of the institutions and associations to which they can apply should be given to family. Legal and social rights should be briefed. If the child is diagnosed during pregnancy, under no circumstances should not be given to directive about he termination or continuation of pregnancy to the family.

  “The doctor has a heavy rock in his hand, how can he deliver it so that the mother is not crushed under this rock. It is also possible to stay under this rock or holding this rock properly and also sculpting it.”

  Scientific Responsibility Statement

  The authors declare that they are responsible for the article’s scientific content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientific review of the contents and approval of the final version of the article.

  Animal and human rights statement

  All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.

  Funding: None

  Conflict of interest

  None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.

  References

  1. Abdelmoktader AM, Abd Elhamed KA. Egyptian mothers’ preferences regarding how physicians break bad news about their child’s disability. A structured verbal questionnaire. BMC Med Ethics.2012;13-14. DOI: 10.1186/1472-6939-13-14.

  2. Beckman PJ. Influence of selected child characreristics on stress in families of handicapped infants, Am J Intellect Dev Disabil.1983; 88 (2):150-6.

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  7. Shepard MP, Mahon MM. Chronic conditions and the family. In P. L. Jackson & J. A. Vessey (Eds.), Primary care of the child with a chronic condition . 3rd ed. New York: Mosby; 2000.p.50-65.

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  How best to deliver the news. Am J Med Genet A. 2009; 149A: 2361-67. DOI: 10.1002/ajmg.a.33082.

  9. Whaley LF, Wong DL. Nursing Care of Infants and Children, 5th ed. Mosby Company, Missouri.1995; 1017-22.

  10. Wright DB, Gronroos N, Burkhartsmeyer J., Johnson D. Delivering the Hard News Well: Your Child Has Mental Retardation. Southern California: Diagnostic Center. 2008.

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Başgül ŞS, Mutlu D, Alanay Y, Toker M. The process of how mothers with down’s syndrome children learn about the diagnosis: A qualitative research. Ann Clin Anal Med 2020;11(1):54-58

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Aim: Heroin is a semi-synthetic opioid, which is widely abused due to its euphoric effects. It is responsible for numerous deaths or diseases each year throughout the world. The goal of this work was to validate and establish a simple and reliable GC-FID method for quantitative analysis of heroin in seized drug samples in accordance with the predicted sample matrix. Material and Method: Detection parameters and chromatographic conditions were optimized in order to achieve an advanced method. Separation was accomplished on a HP-5 column (30 m-0.32 nm ID-0.25 μm) utilizing n-tetracosane as an internal standard at the concentration of 0.25 mg/mL in chloroform/methanol (1:1) mixture. Method validation was processed by means of specificity, linearity, accuracy, precision, range, quantitation limit and detection limit. Results: Method provided a great linearity with correlation coefficients (r2 =0.9994) for heroin. The limit of detection and limit of quantification values of GC-FID method for heroin analysis were 2.20 µg/mL and 7.33 µg/mL, respectively while the limit of linearity was 1000 µg/mL. Mean recovery value obtained from spike study was 99.89%, and relative error calculated after CRM analysis was equal to 1.80%, indicating that the method was accurate. Discussion: Inter-day stability of the instrument was demonstrated by use of the control chart. The method represented is comparatively simple, fast, precise, and pertinent for clandestine drug analysis in toxicological, pharmaceutical, and forensic laboratories.

Keywords: Drug Abuse; Clandestine Drug; Heroin; Toxicology; Forensic Chemistry; Validation; GC-FID

Introduction

Heroin is a semi-synthetic drug formulated by acetylation of morphine presenting as the main opiate in opium poppy tears. As an intravenous illicit drug, heroin addiction is a phenomenon involving all age groups in Europe, and it is responsible for many deaths or diseases each year throughout the world [1,2]. Although some individuals may use heroin in a controlled manner [3], most of those who try to use heroin become addictive to this substance [4]. Chemical structure of heroin, 6-monoacetylmorphine (6-MAM) and morphine, are shown in Figure 1.

Following oral administration, heroin is subjected to comprehensive pre-systemic biotransformation by deacetylation, resulting in a pharmacologically active drug for the systemic delivery of morphine [5]. Although injection of heroin bypasses of pre-systemic metabolism effect, it can pass the blood-brain barrier quickly by reason of the acetyl groups causing it further lipophilic than morphine [6,7]. Nevertheless, heroin is rapidly metabolized first to 6-MAM following systemic administration, and the half-life of heroin in humans is nearly 1.8 to 7.8 minutes [8]. Like other opioids, morphine also causes euphoric, analgesic, and anxiolytic effects that is liable for its addictive function. The µ-opioid receptor in the brain intermediates to these effects of heroin [9]. However, heroin itself shows a comparatively small affinity for the μ receptor [10].

Heroin is converted to morphine through the 6-MAM in the body within minutes. In cases, morphine is usually a dominant active metabolite and is excreted from the body by transformation 3- and 6-glucuronides through the urine and bile. The existence of 6-MAM in urine differentiates the use of heroin from morphine. Low amount of codeine can also be involved in the urine of abusers by reason of acetyl codeine in heroin [11].

Heroin arrives Europe through four major transportation ways. The two essential directions are the Balkan and southern routes. The Balkan route passes Turkey through Bulgaria, Greece or Romania as well as the central, southern and Western Europe. Furthermore, Syria and Iraq have also appeared as an outgrowth of the Balkan route. The southern route which involves transition directions from Pakistan and Iran through African countries or directly into Europe by air or sea has recently been used. Northern route and the southern Caucasus crossing the Black Sea are the remaining minor routes [12]. Based on the Turkey Drug Report (2018): toxicological examination was accessible for the entire approved drug-related deaths. More than half of expirations was associated with multi-substances involving opioids, particularly heroin, engaged in approximately one-third of the deaths. The statistic of opioid-linked deaths recorded in Tukey has been approximately steady from 2014 up to now. Moreover, 8179 heroin cases were reported in 2016, and 5585.1 kg of heroin were seized in Turkey.

In the last decades, many chromatographic assays were advanced to determine heroin concentration in seized drugs as well as biological samples for toxicological, clinical, pharmaceutical, and forensic purposes [13-17]. As an alternative to immunoassays, thin-layer chromatography (TLC) appears as a beneficial method by reason of it is one of the practical and the cheapest techniques. Yet, such screening methods can fail to detect low concentration of drugs. For this reason, mass spectroscopy (MS) connected to liquid chromatography (LC) and gas chromatography (GC) are good instrumental examples for quantification and confirmation [18,19].

The aim of this research essentially focuses on validation and development of a GC-FID system for quantitative determination of heroin in clandestine drug specimens in order to improve the criminal justice system by providing enhanced objective conclusions. There are numerous works on this topic. However, major significance and novelty of this paper fundamentally depends on the advanced chromatographic resolution and detection sensitivity throughout validation and optimization of the technique designed in accordance with the predicted sample matrix. Ultimately, the study showed a satisfactory separation of all analyte peaks within 13 minutes.

Material and Methods

Instrumentation

The analysis was performed using Agilent GC 6890N (Santa Clara, California, USA) equipped with a flame ionization detector (FID) and an automated liquid sampler. An Agilent 7683 Series Auto-Injector was utilized for the injection of samples. This instrumentation was utilized for validation and optimization of an analytical method based on the determination of heroin in illicit samples.

Standard Solutions and Reagents

Stock solutions of methanol, chloroform, and n-tetracosane powder were obtained from Merck (Darmstadt, Germany). Certified reference material (CRM) of heroin, and caffeine, codeine, morphine, and 6-MAM solutions were obtained from Lipomed Services to Health®, Switzerland. All the other solvents and chemicals used during laboratory work were of analytical reagent grade. Ultrapure water (Merck Millipore Direct-Q8, Germany) with a resistivity of 18MΩ.cm, was used to prepare the solutions during the experimental process.

Sample Preparation and Procedure

An Agilent Model 6890N gas chromatograph was utilized during analyses. One mL of the prepared solutions was placed into an autosampler vial for analysis, and separation was achieved on an HP-5 column (30 m, 0.32 mm ID, 0.25 μm) using an IS (tetracosan at the concentration of 0.25 mg/mL) in chloroform/methanol (1:1, v/v) mixture. Ultrahigh purity (99.999 percent) hydrogen was chosen as the carrier gas with a flow rate of 1.5 mL/minute. The flame ionization detector and the injection port were sustained at 280 °C. An Agilent 7683 Series Auto-Injector was used during injection of samples. In the splitless mode (20:1), 2 mL amounts of samples were injected. Then, isothermally programmed oven temperature was adjusted to 180 °C for 10.00 minutes, and nitrogen was utilized as the auxiliary make-up gas for the detector. Operating parameters of the GC-FID system for heroin analysis was given in Table 1.

All samples and calibration standard solutions were prepared by use of an appropriate amount of our working solution: chloroform/methanol (1:1, v/v) mixture containing the IS (tetracosane at the concentration of 250 μg/mL). In order to prepare the calibration standards at the concentrations of 0, 50, 100, 250, 500 and 1000 μg/mL, heroin powder standards were diluted in our working solution described above. The solution was kept at 4±1°C when not in use and warmed to room temperature before use.

Method Validation

As outlined in International Conference on Harmonization (ICH) guidelines, this GC-FID method for analysis of heroin content in illicit specimens was validated based on specificity, accuracy, precision, range, linearity, quantitation limit and detection limit.

Results

Calibration

All calibration standards were analyzed 5 times, and a calibration curve of area heroin/area tetracosane versus concentration of heroin standards was drawn (Figure 2). The correlation coefficient (r2) and equation of the calibration curve for heroin were respectively found to be r2=0.9994 and y=2.1106x-0.0003 where y stands for area heroin/area tetracosane, and x is the heroin concentration in μg/mL. The chromatogram of heroin standard at 600 μg/mL, obtained after analysis according to proposed GC-FID method, was illustrated in Figure 3.

Limit of Detection, Quantification, and Linearity

The limit of detection (LOD) and lowest limit of quantification (LOQ) were determined by means of the standard deviation of the response and the slope of the calibration curve, according to International Conference on Harmonization (ICH) guidelines, LOD=3.3σ/S, LOQ=10σ/S, where σ is the standard deviation of the response and S is the slope of the calibration curve. The LOD and LOQ values of GC-FID method for heroin analysis were 2.20 µg /mL and 7.33 µg /mL, respectively. Limit of linearity (LOL) is the concentration at which the calibration curve departs from the linearity. Dynamic range refers to concentration intervals from LOQ to LOL, which was found between 7.73 µg/mL and 1000 µg/mL, in this study.

Precision

The precision of the method was assessed by means of repeatability, intermediate precision, and reproducibility parameters. The reproducibility of the recommended method was characterized by analysis of six different samples at the same concentration of 700 μg/mL from the same certified reference solution of heroin. Caffeine concentration of these samples was adjusted as 300 µg/mL to make sure the matrix effect. The mean of measured heroin concentrations was found as 699.17±9.64 μg/mL with 1.38% relative standard deviation (RSD). The result of the reproducibility study was shown in Table 2. Repeatability was controlled by injecting six individual samples of heroin at 200 μg/mL concentration while the intermediate precision was assessed by two analysts. Mean heroin concentrations from Analyst-A and Analyst-B were calculated as 202.17±2.56 μg/mL and 201.33±2.50 μg/mL, respectively. Repeatability study was summarized in Table 3.

Control Chart

The control chart study provides observation of inter-day and intra-day differences in peak intensity. From this point of view, a convenient procedure for monitoring the inter-day stability of the instrument was verified by use of the control chart. A mixture solution containing heroin at 500 µg/mL and caffeine at 500 µg/mL concentration was analyzed by GC-FID method once a month during a year, and the mean concentration of heroin was found as 499.330±10.30 µg/mL. After that, warning limits were calculated from the following formula: Warning Limits = xmean±2σ. Lowest warning limit (LWL) and upper warning limit (UWL) were equal to 478.73 µg/mL and 519.93 µg/mL, respectively. Similarly, control limits were calculated from the formula: Control Limits = xmean± 3 σ. Lowest control limit (LCL) and upper control limit (UCL) were equal to 468.43 µg/mL and 530.23 µg/mL, respectively. The control chart study was demonstrated in Figure 4.

Discussion

Optimization

Several essential parameters were improved in order to accomplish the most excellent performance from this chromatographic study. The main principle was based on a choice of the convenient column, deciding the best oven temperature program, selection of a suitable internal standard (IS), the election of concentration range due to heroin concentration in illicit drug specimens, and building the linearity. From this perspective, preliminary investigations were performed for arrangement and decision of the chromatographic conditions for the GC-FID determination of heroin.

As the column shows a critical role in an improved chromatographic separation [20], the selection of the favorable capillary column was decided on the basis of four necessary items: column length, stationary phase, film thickness, and column I.D. [19]. According to Tony Taylor’s 2015 paper, temperature has an effect on retention and relative retention in GC. Once the temperature is varied, the selectivity of the separation is also changed [21]. In order to obtain the excellent chromatographic resolution and detection sensitivity, an outcome of the oven temperature rate was analyzed by examining not only various oven temperature programs but also ramp rates up to highest oven temperature of 300 oC. Hence, 280 oC was decided as the final temperature. Then, the gradient temperature program in the injection and the GC oven were examined. Subsequently, the improved program provided an adequate separation of entire analyte peaks within 13 minutes. Since utilizing of IS is suggested to avoid potential miscalculations following injection of different sample volumes in the chromatographic equipment [18], n-tetracosane was therefore chosen as IS. Selection of a favorable solvent is important to provide that both the IS and the target analyte are completely dissolved. Therefore, chloroform/methanol mixture (1:1, v/v) was chosen as a solvent.

Selectivity/ Specificity

Selectivity/Specificity of an analytical assay can be characterized as the detection ability of the desired component in the existence of alternative analytes which can be supposed to be present in a complex matrix such as impurities, degradation products, and diluents [19,21]. The components in the seized illicit drug are generally country dependent. For this reason, a method must either be validated or revised according to the predicted sample matrix [14]. To appraise the specificity of this chromatographic method, our working solution: chloroform/methanol (1:1, v/v) mixture and placebo solution containing the IS (n-tetracosane at the concentration of 0.25 mg/mL) without the heroin were injected into the GC-FID system. The specificity of the method was carried out in existence of our working solution and placebo, which has heroin free solution. A characteristic chromatogram of heroin standard solution is presented in Figure 3(A). As can be observed in Figure 3(B), there is no peak correlated to placebo or dilution solution was identified at the retention time of heroin. Figure 3(C) demonstrates the sample chromatogram in which heroin was mixed with compounds (caffeine, codeine, morphine, and 6-MAM) usually encountered in real cases. Satisfactory selectivity was verified for heroin and IS by investigating whether all these analytes were separated adequately from one another on a chromatogram.

Accuracy

Accuracy can be described as the proximity of a quantified magnitude to an approved certified value or known value [19]. The accuracy of the assay was evaluated by means of the percentage of recovery data from spike analysis. According to our experience, heroin samples seized in Turkey generally contain caffeine. Reference caffeine and heroin solution were therefore spiked in three different amounts. The mean of recovery was found as 99.89% for heroin (see Table 4). In addition, relative error (RE) and coefficient of variation (CV) were also used to assess the accuracy of the method (see Table 5).

Conclusion

Heroin addiction is still a global public health problem through new psychoactive substances such as synthetic cannabinoids have been widely used by abusers. Turkey is exposed to international illegal heroin trafficking as transit and/or destination country. Hence, a significant amount of heroin has been seized by law enforcement agencies, indicating the importance of forensic drug analysis. In this study, GC-FID method for heroin analysis in illicit drug samples was developed and validated for accuracy, precision, and linearity. The mean recoveries obtained from CRMs analysis were found as 99.89% with relative error equal to 1.8%, indicating the method was accurate. The method provided LOD and LOQ equal to 2.20 μg/mL and 7.73 μg/mL, respectively. The GC-FID method is relatively fast, simple, precise, and applicable for routine forensic and pharmaceutical analysis. This work will improve the criminal justice system by providing quantitative and objective conclusions while examiners are presenting forensic evidence in the court.

Acknowledgment

Dr. Bayram Yuksel would like to express his gratitude to TUBITAK-BIDEB-2219 Program for the financial support

Scientific Responsibility Statement

The authors declare that they are responsible for the article’s scientific content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientific review of the contents and approval of the final version of the article.

Animal and human rights statement

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.

Funding: None

Conflict of interest

None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.

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Yüksel B. Quantitative GC-FID analysis of heroin for seized drugs. Ann Clin Anal Med 2020;11(1):38-42

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