INTRODUCTION

In the case of active substances, liquid pharmaceutical forms are ideal for use in the pediatric population, as they are easily swallowed and allow greater control over the dose administered. Such adjustments are essential, since pediatric patients have pharmacokinetic and pharmacodynamic differences that vary rapidly throughout childhood, and are more exposed to drug-related adverse effects^1-4.

Although ideal, there is a notable absence of drugs in liquid pharmaceutical form in pediatric clinical practice, due to legal, ethical and economic issues, where this section of the population is generally not included in clinical trials for the development of new drugs, or due to the fact that some drugs have poor bioavailability or effectiveness when prepared in aqueous solution^5,6.

In this context, a common practice in many children's hospitals is the adaptation of pharmaceutical forms for administration to children, particularly inpatients. These adapted formulations can be obtained by different methods, from crushing a tablet or opening a capsule to use its contents. The resulting powder can be dissolved or suspended in various excipients to produce a liquid consistency medicine for oral use, or put back into smaller capsules or sachets^3,7.

The act of reformulating medicines into an oral extemporaneous preparation for prescription outside the approved indications (age range, dosage, presentation) or for use by a route of administration other than that originally developed is defined in science as unlicensed or off-label use⁸.

Although the overall burden of inefficient and unsafe pharmacotherapy in children has never been established, the high use of off-label medicines is worrying⁶. The prevalence of prescriptions with adapted pediatric medicines is estimated at 3.2% to 95%; where, 26% to 95% in neonates, 2.7% to 51.2% in outpatients and 9.0% to 79.0% in inpatients. Developing countries are the most severely affected, as people aged between 0 and 18 make up a large proportion of the population and are the most vulnerable to disease³.

It can be seen that adapted medicines are a solution not only for personalizing therapy in paediatric patients, but also for specific therapies when there are no commercial alternatives available. However, splitting the tablet, crushing and/or dissolving it to obtain a liquid pharmaceutical form offers different risks to the patient since there is not enough safety information on drug interactions, stability or efficacy caused by potential changes in bioavailability^3,9.

Environmental factors can affect the physicochemical and microbiological stability of these drugs, such as temperature, light, humidity, radiation, air, particle size, solvents, pH and the presence of contamination or the intentional mixing of different products, as well as the hygiene and sterility of the place where they will be handled¹⁰.

In the literature, most of the quality control work on pharmaceutical adaptations in hospitals focuses on analyzing the physicochemical stability of the formulations. However, it is important to emphasize the need to assess the load of 'non-compliant microorganisms' in these products, which can cause organoleptic changes and interfere in the drug's degradation process, leading to a reduction in its efficacy and safety. In addition, the high microbial load can cause serious infections in these populations¹².

Pharmaceutical professionals must consider microbial contamination and materials that pose a danger to patients, especially vulnerable groups such as pediatric patients. The addition of preservatives and good handling practice measures are crucial in controlling mold, inhibiting yeast growth and protecting against bacterial spread^13,14.

In view of this, the research justifies its raison d'être in the need to carry out microbiological control of these extemporaneous formulations in a hospital in order to contribute to improving the handling process, reducing the risk of contamination of patients or even accelerated degradation of the medicines produced and, finally, to generate sufficient data available on the microbiological control of these adaptations^11,15, since no studies on the subject were found in the city, although the use of adapted products has already been investigated in the city of Manaus^16,17.

Therefore, the aim of this study was to carry out microbiological control of adapted liquid pharmaceutical forms in a pediatric hospital, following the specifications of the Brazilian Pharmacopoeia for non-sterile products, using the most probable number for mesophilic microorganisms and testing for the pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella sp.

METHODS

Study design - A prospective study was carried out on the microbiological control of liquid pharmaceutical forms adapted in the pediatric hospital pharmacy, with collections from September 2017 to May 2018.

Survey and selection of adapted pharmaceutical forms – Initially, a survey was carried out of all the formulations adapted in the hospital pharmacy during the study period, and the total number of formulations produced was obtained. These formulations were then evaluated in terms of their composition and classified according to their pharmacological classes, determined using the ATC (Anatomical Therapeutic Chemical) methodology.¹⁸ After this process, exclusion criteria were applied to select which formulations would be evaluated microbiologically, including: being an antimicrobial agent, not being for oral use, a shelf life of less than 7 days and production in insufficient quantities for the study's analysis.

Together with the formulations selected for microbiological analysis, an aliquot of the excipients simple syrups and carboxymethylcellulose used in the preparation of the pharmaceutical adaptations were inserted, both of which have a shelf life of 180 days and are prepared in the hospital pharmacy itself.

After selecting the medicines and excipients used in the liquid formulations, the samples obtained for analysis were grouped according to the shelf life established by the site's standard operating procedure: Group 1 (7 to 20 days), Group 2 (30 days), Group 3 (35-48 days), Group 4 (60 days), Group 5 (90 days) and Group 6 (180 days).

For the microbiological analysis, 03 units were requested from the same batch of each selected sample, which were collected at “time zero”, i.e. on the day of handling. These were then transported in a refrigerated box to the laboratory and kept under refrigeration (4 °C) until the moment of analysis.

Microbiological analysis – Microbiological analyses were carried out immediately after preparing the formulations (time zero), halfway through the shelf life and when the shelf life was reached, using previously prepared culture media incubated in an oven at 32 ºC ± 2.5 ºC for 24 hours to assess sterility after preparation and sterilization in an autoclave. Tests were carried out for the total count of mesophilic microorganisms and for pathogens, in accordance with the criteria for non-sterile pharmaceutical products as specified in the Brazilian Pharmacopoeia 5th edition.¹⁹

Counting the total number of mesophilic microorganisms by most probable number

- Mesophiles were analyzed using the most probable number method, which consists of evaluating the growth of viable microorganisms within 5 days, in Casein-Soy broth (KASVI, São José dos Pinhais-PR) incubated in an oven at 32 ºC ± 2.5 ºC. The analysis was carried out by preparing dilutions, in triplicate, at a ratio of 1:10; 1:100 and 1:1000 of the formulations analyzed. Positive and negative tubes were recorded and compared according to the specifications in the Brazilian Pharmacopoeia, where the limit for total aerobic bacteria count (UFC/g or mL) is 200 UFC/g or mL of product. The result obtained is expressed as Most Probable Number per gram or milliliter of product, MPN per g or MPN per mL of product.

Testing for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus - The tests for E. coli, P. aeruginosa and S. aureus were initially carried out together, as the sample preparation and pre-incubation stages followed the same procedure. To prepare the sample, 1 mL of the formulation was inoculated into a tube containing 9 mL of Casein-Soy broth. The contents of this tube were transferred to a flask containing 90 mL of Casein-Soy broth (pre-incubation stage) and incubated for 18-24 hours at 32 ºC ± 2.5 ºC. After this period, the plates were sown on Cetrimide agar (HiMEDIA, Mumbai, Maharashtra, India) to test for Pseudomonas sp. and sown on Salted Mannitol agar (HiMEDIA, Mumbai, Maharashtra, India) to test for Staphylococcus sp. The plates were incubated in an oven for 18-24 hours at 32 ºC ± 2.5 ºC and colony growth was observed. For Escherichia coli, an aliquot of 1 mL of the pre-incubation medium was transferred to an erlenmeyer flask containing 100 mL of MacConkey broth (enrichment) (KASVI, São José dos Pinhais-PR), which was incubated for 18-24 hours at 43 ºC ± 1 ºC and then sown on a plate (subculture) containing MacConkey agar (HiMEDIA, Mumbai, Maharashtra, India), and incubated for 18-72 hours at 32 ºC ± 2.5 ºC.

The results obtained, following the limits determined by the Brazilian Pharmacopoeia, after the tests, should be negative, i.e. the absence of these pathogens, in the samples of the products analyzed.

Search for Salmonella sp. - For the sample preparation and pre-incubation period, 10 mL of sample was added to a flask containing 90 mL of Casein-Soy broth, which was then incubated for 18-24 hours at 32 ºC ± 2.5 ºC. After incubation, a 0.1 mL aliquot of the solution containing the sample was inoculated into a tube containing 10 mL of Selenite-Cystine broth (HiMEDIA, Mumbai, Maharashtra, India). It was incubated for 18-24 hours at 32 ºC ± 2.5 ºC. A loop of the Selenite-Cystine broth was removed using a bacteriological loop and sown on a plate containing Xylose Lysine Deoxycholate - XLD agar (Biokar Diagnost, Beauvais Cedex France), which was incubated for 18-72 hours at 32 ºC ± 2.5 ºC.

The results obtained, following the limits determined by the Brazilian Pharmacopoeia, after the tests, is the absence of this pathogen in the samples of the products analyzed.

Ethical aspects – This project was carried out without the need for approval by the Research Ethics Committee. However, a consent form was previously issued by the Health Unit's Board of Directors for the use of the formulations employed in the study.

RESULTS

Survey and selection of pharmaceutical adaptations– Data was collected from the register of formulations produced in 2017. During this period, 36 pharmaceutical adaptations were prepared. Half of them (50%) were antihypertensives, antibacterials, psycholeptics and diuretics, in the proportion of 11.1% each respectively, and antiepileptics (5.6%), according to the ATC (Anatomical Therapeutic Chemical) methodology¹⁸. The other half was distributed among other ATC classes. In view of this data, the exclusion criteria were applied, as shown in Table 1.

Among the exclusion criteria applied, 14 (41.6%) of the excluded adaptations did not meet the criterion of sufficient quantity for the study. As an example, we can mention the antiepileptic drug topiramate, which had its adaptation suspended because the tablets were out of date and would be taken for disposal. This was followed by the exclusion of 6 (16.6%) adaptations containing antimicrobial agents and 1 (3%) formulation because it was not administered orally. Adapted formulations with a shelf life of less than 7 days were not excluded.

As a result, 14 pharmaceutical adaptations were selected for the study, as shown in Table 2.

The composition of the pharmaceutical adaptations was then analyzed (Table 3). It was found that simple syrup is used as the vehicle and carboxymethylcellulose (CMC) as the viscosifying/dispersing agent. These have a shelf life of 180 days and are prepared in the hospital pharmacy itself, being incorporated into the adapted formulations according to production.

Table 3 shows that in half of the adaptations selected for the study, distilled water was added as a diluent for the drug in the syrup. The use of simple syrup in extemporaneous formulations is a positive factor both for the taste of pediatric patients, as it is able to mask the taste of some drugs, and to promote their greater stability, due to its hypertonic characteristic capable of inhibiting bacterial growth.²⁰

In addition, citric acid is an input used to acidify the medium, which can provide stability to the drug or promote its dissolution in the formulation, especially for the adaptation of captopril, being able to act as a stability promoter, as described by. No preservatives or flavor adjuvants are used.

Microbiological analysis - The total count of mesophilic microorganisms, after analysis of the times proposed methodologically, obtained the results that can be seen in Table 4, considering the microbial limits for non-sterile products in the total count of fungi/yeasts of 20 UFC/g or mL and for the total count of aerobic bacteria the limit of 200 UFC/g or mL of product.¹⁹

Based on the results shown in Table 4, together with the limits determined in the literature, of the 16 samples analyzed at time zero, 8 (50.0%) showed non-compliant results. Among these, clobazam, methadone, propranolol and dexamethasone did not comply with the limits stipulated for fungi/yeasts, while spironolactone, hydrochlorothiazide, methyldopa and carboxymethylcellulose did not meet the limits for aerobic bacteria.

In the half-life analysis of the formulations, 7 (43.75%) showed non-compliant results, with clobazam, spironolactone methadone, propranolol and captopril being outside the stipulated limits for fungi/yeasts and dexamethasone and methyldopa being outside the limits for aerobic bacteria.

In the analyses referring to the final deadline, 10 (62.5%) of the formulations did not comply with the established parameters. Spironolactone, methadone, propranolol and dexamethasone were found to be outside the permitted limit of CFU/g or mL of product and the samples of clobazam, sildenafil, hydrochlorothiazide, methyldopa and furosemide failed to meet the permitted limit for aerobic bacteria.

These data show that a large proportion of the formulations submitted for analysis, during the period of the deadline, do not comply with the microbiological limits, either in terms of aerobic bacteria or fungi/yeasts. Studies such as the one by Mugoyela (2010), show that the main contaminants of these non-sterile formulations are aerobic bacteria such as Bacillus spp and fungi such as Candida spp and Aspergillusspp.

In the results obtained from the captopril and hydrochlorothiazide formulations, variations were observed between their approval and disapproval, according to the limits stipulated by the pharmacopoeia, during the study period. Possible reasons for this include handling errors, environmental contamination during analysis or even a decline in the cell viability of the microorganism during storage.²¹

The pharmaceutical adaptations that maintained acceptable microbial limits throughout the study period were midazolam, nifedipine and potassium chloride 6%, representing 18.75% of the adapted drug samples. When analyzing the Standard Operating Procedure (SOP) for these adaptations, we found that the adaptation was carried out using sterile medication (ampoules), except in the case of nifedipine, which was made using slow-release tablets.

All the pharmaceutical adaptations complied with the legal requirement for the absence of the following pathogens: Escherichia coli, Salmonella sp., Pseudomonas aeruginosa and Staphylococcus aureus.

DISCUSSION

During the survey and classification of formulations that could be adapted in the pharmacy sector of the hospital where the study took place, it was found that the main pharmacological classes subjected to this process during the study period were: antihypertensives, antibacterials, psycholytics and diuretics. Other studies, such as the one by García-López (2020), using the same ATC classification, show that the main classes of off-label drugs are those for the central nervous system, gastrointestinal tract and respiratory system. The prescription profile is different to that of the hospital under study, except for psycholytics, which are classified as drugs that act on the central nervous system.

With regard to the composition of the formulations, it can be seen that no preservatives were used, which can largely contribute to the microbiological growth observed in the results obtained from the mesophilic microorganism count, where 13 (81.25%) samples showed a total number of mesophilic microorganisms above the permitted limits for fungi/yeasts or aerobic bacteria.¹⁷

Microbial contamination above the permitted limits can easily compromise the health of pediatric patients, because in some cases the presence of microorganisms alone is not harmful to health, but the toxins they produce can trigger symptoms such as diarrhea, acute gastroenteritis, stomach pain or even, in severe cases, death. ²³

Only 3 (18.75%) formulations were found to be within acceptable microbial limits during analysis: midazolam, nifedipine and potassium chloride 6%. These results show the need for training and adequate infrastructure for the manufacture of these products so that patient safety can be ensured. ^8,21

When it came to testing for pathogens, all the pharmaceutical adaptations were free of Escherichia coli, Salmonella sp., Pseudomonas aeruginosa and Staphylococcus aureus, thus within the limits recommended by the pharmacopoeia. It is therefore interesting to understand that in the case of testing for S. aureus, when not producing enterotoxins, and E. coli are important markers for assessing the hygiene of the hands of the handler and the workbench during the process.^24,25

Pseudomonas aeruginosa is described as an important human pathogen, it has the ability to contaminate non-sterile products due to its versatility to grow in unfavorable environments and its intrinsic and acquired resistance to antimicrobials, its absence in the analysis is a good marker in monitoring the quality of water and food, as well as the absence of Salmonella sp., being especially dangerous for children and immunocompromised, and can lead to fatal dehydration. ^26,27

The scarcity of commercial formulations suitable for the pediatric public, considering differences in physiological and metabolic response, makes it necessary to use off-label preparations. Therefore, in order to ensure their quality, rigorous safety assessment is necessary, considering the possibility of adverse reactions, toxicity and especially physicochemical and microbiological stability.

Therefore, effective microbiological quality control is essential to increase the safety of manipulated medicines, guiding pharmacists to improve their practices through appropriate procedures and access to crucial information on the stability, compatibility, bioavailability and safety of these products

Acknowledgments

This study was funded by the Amazonas State Research Foundation (FAPEAM) - Amazonas, Brazil. The samples were obtained in partnership with the Instituto de Saúde da Criança do Amazonas (ICAM) - Manaus, Amazonas, Brazil. The Federal University of Amazonas (UFAM) - Manaus, Amazonas, Brazil, for carrying out the project's analyses.

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