Reduction Of Bioburden Research Articles

Bacillus-based probiotic cleansers reduce the formation of dry biofilms on common hospital surfaces.

In the absence of liquid suspension, dry biofilms can form upon hard surfaces within a hospital environment, representing a healthcare-associated infection risk. Probiotic cleansers usinggenerally recognized as safe organisms, such as those of the Bacillus genus, represent a potential strategy for the reduction of dry biofilm bioburden. The mechanisms of action and efficacy of these cleaners are, however, poorly understood. To address this, a preventative dry biofilm assay was developed using steel, melamine, and ceramic surfaces to assess the ability of a commercially available Bacillus spp. based probiotic cleanser to reduce the surface bioburden of Escherichia coli and Staphylococcus aureus. Via this assay, phosphate-buffered saline controls were able to generate dry biofilms within 7 days of incubation, with the application of the probiotic cleanser able to prevent >97.7% of dry biofilm formation across both pathogen analogs and surface types. Further to this, surfaces treated with the probiotic mixture alone also showed a reduction in dry biofilm across both pathogen and surface types. Confocal laser scanning microscopy imaging indicated that the probiotic bacteria were able to germinate and colonize surfaces, likely forming a protective layer upon these hard surfaces.

Long-term experience with a collagen-elastin scaffold in combination with split-thickness skin grafts for the treatment of full-thickness soft tissue defects: improvements in outcome\u2014a retrospective cohort study and case report

PurposeThe management of severe soft tissue injuries to the extremities with full-thickness wounds poses a challenge to the patient and surgeon. Dermal substitutes are used increasingly in these defects. The aim of this study was to investigate the impact of the type of injury on the success rate of Matriderm® (MD)-augmented split-thickness skin grafting, as well as the role of negative pressure wound therapy (NPWT) in preconditioning of the wounds, with a special focus on the reduction of the bioburden.MethodsIn this study, 45 wounds (44 affecting lower extremities (97.7%)), resulting from different types of injuries: soft tissue (ST), soft tissue complications from closed fracture (F), and open fracture (OF) in 43 patients (age 55.0 ± 18.2 years, 46.7% female), were treated with the simultaneous application of MD and split-thickness skin grafting. The study was designed as a retrospective cohort study from March 2013 to March 2020. Patients were stratified into three groups: ST, F, and OF. Outcome variables were defined as the recurrence of treated wound defects, which required revision surgery, and the reduction of bioburden in terms of reduction of number of different bacterial strains. For statistical analysis, Student’s t-test, analysis of variance (ANOVA), Mann–Whitney U test, and Pearson’s chi-squared test were used.ResultsThere was no significant difference in the rate of recurrence in the different groups (F: 0%; OF: 11.1%; ST: 9.5%). The duration of VAC therapy significantly differed between the groups (F: 10.8 days; OF: 22.7 days; ST: 12.6 days (p < 0.05)). A clinically significant reduction of bioburden was achieved with NPWT (bacterial shift (mean (SD), F: − 2.25 (1.89); OF: − 1.9 (1.37); ST: − 2.6 (2.2)).ConclusionMD-augmented split-thickness skin grafting is an appropriate treatment option for full-thickness wounds with take rates of about 90%. The complexity of an injury significantly impacts the duration of the soft tissue treatment but does not have an influence on the take rate. NPWT leads to a relevant reduction of bioburden and is therefore an important part in the preconditioning of full-thickness wounds.

Nucleic acid peptide nanogels for the treatment of bacterial keratitis.

Cage-shaped nucleic acid nanocarriers are promising molecular scaffolds for the organization of polypeptides. However, there is an unmet need for facile loading strategies that truly emulate nature's host-guest systems to drive encapsulation of antimicrobial peptides (AMPs) without loss of biological activity. Herein, we develop DNA nanogels with rapid in situ loading of L12 peptide during the thermal annealing process. By leveraging the binding affinity of L12 to the polyanionic core, we successfully confine the AMPs within the DNA nanogel. We report that the thermostability of L12 in parallel with the high encapsulation efficiency, low toxicity and sustained drug release of the pre-loaded L12 nanogels can be translated into significant antimicrobial activity. Using an S. aureus model of infectious bacterial keratitis, we observe fast resolution of clinical symptoms and significant reduction of bacterial bioburden. Collectively, this study paves the way for the development of DNA nanocarriers for caging AMPs with immense significance to address the rise of resistance.

Cerium Nitrate Treatment Provides Eschar Stabilization through Reduction in Bioburden, DAMPs, and Inflammatory Cytokines in a Rat Scald Burn Model

In this study, we used a clinically relevant rat scald burn model to determine the treatment effects of cerium nitrate (CN) for stabilizing burn eschars through reduction of damage-associated molecular patterns (DAMPs), inflammatory cytokines, and bioburden. Forty-two male Sprague–Dawley rats were anesthetized before undergoing a scald burn at 99°C for 6 seconds to create a 10% full-thickness burn. The test groups included sham burn, burn with water bathing, and burn with CN bathing. End point parameters included circulating DAMPs, proinflammatory cytokines, tissue myeloperoxidase activity, and quantification of resident flora in burn skin. The high mobility group protein box 1 was found to be elevated in burn animals at postoperative days (POD) 1 and 7. CN significantly alleviated the increase (P < .05 at POD 1 and P < .01 at POD 7). CN also lessened the heightened levels of hyaluronan in burn animals (P < .05 at POD 7). Additionally, CN significantly reduced the burn-induced increases in interleukin-1β, growth-regulated oncogene/keratinocyte chemoattractant, and macrophage inflammatory protein-1α in burn wounds. The anti-inflammatory effect of CN was also demonstrated in its ability to mitigate the upregulated circulatory xanthine oxidase/dehydrogenase and increased tissue neutrophil infiltration in burn animals. Last, CN suppressed postburn proliferation of resident skin microbes, resulting in a significant 2-log reduction by POD 7. In conclusion, these results suggest that CN attenuates the burn-induced DAMPs, tissue inflammatory responses, and regrowth of resident skin flora, all of which collectively could improve the quality of burn eschar when applied at the point of injury in prolonged field care situations.

Evaluation of a new sink design incorporating ozonated water

Novel sink and U-trap designs have been developed to reduce contamination by users and diminish biofilm. Real-world experience with new sink designs and adjunctive measures such as ozone has been limited. To assess a new sink design for splashback and to evaluate the ozonated water feature for reduction of microbial bioburden. A portable sink unit was created that permitted the application of white absorbent paper to plexiglass shields beside and behind the sink. Participants, wearing painter coveralls and masks, spread 30 mL of tempera paint over their hands and washed for 20 s with neutral soap. Each participant repeated this five times sequentially, and cumulative results were recorded. Escherichia coli was exposed to ozonated water from the sink unit and to regular tap water and evaluated for microbial survival. Compared with a conventional sink, the SmartFLO3 sink had less environmental contamination within the sink, surrounding area and splashback on to the participant. Despite modifications to enhance ozone generation, readings of reactive oxygen species did not exceed 0.3 ppm, and no significant bactericidal effect was demonstrated. The SmartFLO3 sink reduces splashback and has the potential to reduce pathogen transmission from sinks. At the low levels of ozone generated in this study, no clear bacterial killing effect was observed compared with tap water alone.

Use of silicon nanoparticle surface coating in infection control: Experience in a tropical healthcare setting.

A nano-scale surface coating containing silicon nanoparticles (Bacterlon®) creates a hydrophobic surface which prevents the growth of bacteria. Study objective was to evaluate the performance of this silicon nano-coating in Sri Lankan healthcare setting. This prospective study was conducted from September 2015 to December 2015 in an Intensive Care Unit and a medical ward in Base Hospital Homagama and a bacteriology laboratory in Medical Research Institute, Colombo, Sri Lanka. Silicon nanoparticle coating was applied to 19 high touch surfaces from those three sites. During the follow-up period, these test sites and non-coated control sites were used for routine work and were cleaned routinely as per institute protocol. Swabbing was done for coated and non-coated sites once a week for 12weeksat unannounced times. Surfaces were categorized in to low (≤10CFU/cm2) and high (>10-99CFU/cm2) contamination by Aerobic Bacterial Count (ABC) in non-coated sites at any given time. In low and high contaminated surfaces, an improvement in the mean percentage bioburden reduction from 36.18% to 50.16% was observed from 4th week to 12th week with silicon nanoparticles and a significant reduction (p<0.05) was seen in ABC in each of the coated surface compared with their non-coated counterpart by the 12th week. The frequency of isolation of Acinetobacter spp. on coated surfaces had a significant reduction (p<0.01). Silicon nanoparticle coating demonstrates a significant reduction of the bacterial bioburden in low and high contaminated surfaces for 12 weeks in a tropical healthcare setting.

Effects and safety of atmospheric low‐temperature plasma on bacterial reduction in chronic wounds and wound size reduction: A systematic review and meta‐analysis

The use of atmospheric low‐temperature plasma (AP) on chronic wounds and its effect on microbial bioburden in open wounds has not been explored with a systematic review and meta‐analysis. PRISMA guidelines were followed and PubMed, Embase, CENTRAL, and CINAHL databases searched for randomised controlled trials (RCTs), which compared AP with no AP for the management of open, chronic wounds. The primary outcomes of reduction of bioburden or wound size were included. Meta‐analyses were performed; odds ratio (OR) and 95% confidence intervals (CIs) were extracted and pooled in a random effects model.Four RCTs investigated the effect of AP on chronic wound healing. Chronic wounds treated with AP did not show a significant improvement in healing (AP vs control: OR = 1.46; 95% CI = 0.89‐2.38; P = 0.13). Five further RCTs investigated the reduction of bioburden in wounds, but AP demonstrated no significant reduction of bioburden (AP vs control: OR = 0.85; 95% CI = 0.45‐1.62; P = 0.63). All nine RCTs recorded the presence of any severe adverse events (SAEs) in the 268 patients studied, with only one unrelated SAE identified in each group (AP vs control: OR = 1.00; 95% CI = 0.05‐19.96; P = 1.00). Use of AP in wound care is safe, but the retrieved evidence and meta‐analysis show that there is no clinical benefit of AP in chronic open wounds using currently available AP device settings.

Minocycline enhances the mesenchymal stromal/stem cell pro-healing phenotype in triple antimicrobial-loaded hydrogels.

Wound healing is a complex biological process that can be hindered by bacterial infection, excessive inflammation, and inadequate microvasculature. In this study, we develop a new formulation of poly(ethylene glycol) diacrylate and thiolated gelatin poly(ethylene glycol) crosslinked hydrogels loaded with minocycline, vancomycin, linezolid, and mesenchymal stromal/stem cells that induces a favorable wound healing phenotype in mesenchymal stromal/stem cells and prevents bacterial bioburden on the hydrogel. This combinatorial approach to biomaterial development has the potential to impact wound healing for contaminated full thickness cutaneous wounds.

Novel negative pressure wound therapy with instillation and the management of diabetic foot infections.

The use of negative pressure wound therapy with instillation (NPWTi) in complex or difficult-to-treat acute and chronic wounds has expanded rapidly since the introduction of commercially available NPWTi systems. We summarize the evidence related to NPWTi and particularly focus on the application of this technology in diabetic foot ulcers, diabetic foot infections and postoperative diabetic wounds. The benefits of negative pressure wound therapy (NPWT) are well documented in the treatment of complex acute and chronic wounds, including noninfected postoperative diabetic wounds and diabetic foot ulcers. Combining intermittent wound irrigation with NPWT may offer additional benefits compared to NPWT alone, including further reduction of wound bed bioburden, increased granulation tissue formation and provision of wound irrigation in a sealed environment, thus preventing potential cross-contamination events. Recently, available evidence suggests that adjunctive NPWTi may be superior to standard NPWT in the management of diabetic infections following surgical debridement and may promote granulation tissue formation in slow-to-heal wounds. Available evidence relating to the utilization of NPWTi in diabetic foot infections is promising but limited in quality, being derived mostly from case series or small retrospective or prospective studies. In order to confirm or refute the potential benefits of NPWTi in this patient cohort, well designed randomized controlled studies are required that compare NPWTi to NPWT or standard wound care methodologies.

One‐stage Versus Two‐stage Exchange

Liaison: Paul Lichstein MD, MSLeaders: Thorsten Gehrke MD (International), Adolph Lombardi MD, FACS (US), Carlo RomanoMD (International), Ian Stockley MB, ChB, MD, FRCS (International)Delegates: George Babis MD, Jerzy Bialecki MD, La´szlo´ Bucsi MD, Xu Cai MD, Li Cao MD, Briande Beaubien MD, Johannes Erhardt MD, Stuart Goodman MD, PhD, FRCSC, FACS, FBSE,William Jiranek MD, Peter Keogh, David Lewallen MD, MS, Paul Manner MD, WojciechMarczynski MD, J. Bohannon Mason MD, Kevin Mulhall MB, MCh, FRCSI, Wayne PaproskyMD, Preetesh Patel MD, Francisco Piccaluga MD, Gregory Polkowski MD, Luis Pulido MD, IanStockley MBBS, ChB, FRCS, Juan Suarez MD, Fritz Thorey MD, Rashid Tikhilov MD, JobDiego Velazquez MD, Heinz Winkler MD

A Prospective, Single-Center, Nonblinded, Comparative, Postmarket Clinical Evaluation of a Bovine-Derived Collagen With Ionic Silver Dressing versus a Carboxymethylcellulose and Ionic Silver Dressing for the Reduction of Bioburden in Variable-Etiology, Bilateral Lower-Extremity Wounds

There are numerous dressings designed to manage the overabundance of matrix metalloproteinases, while also addressing the excessive bioburden found in chronic wounds. The authors compared the efficacy of 2 such dressings: a sodium carboxymethylcellulose/1.2% ionic silver (CMC), which theoretically reduces bacteria by providing silver ions, versus a bovine native collagen (BDC)/ionic silver dressing, which also delivers silver ions in an aqueous environment. Both dressings theoretically modulate the wound bed; CMC through moist wound care and fibrin ingrowth and BDC through matrix metalloproteinase balancing. A prospective protocol was undertaken using patients as their own controls. Ten patients with bilateral venous stasis or diabetic foot ulcers were selected. One limb was randomized to treatment by either CMC or BDC, whereas the contralateral wound was treated with the other dressing. Biopsies for quantitative cultures were taken at weeks 1 and 4. Wound area was assessed at the weekly visits. The BDC wounds started with 1.0 × 10 (±1.2 × 10) bacteria, and the CMC wounds started with 1.4 × 10 (±1.3 × 10) bacteria. Over the 4-week period, the bacteria in the 3-ppm (parts per million) silver-treated wound increased 1.53 × 10, whereas in the 21-ppm silver-treated wound, the bacteria increased 1.42 × 10. The rates of closure for CMC-treated wounds was 0.79 ± 0.735 cm/wk and for BDC-treated wounds was 1.38 ± 1.44 cm/wk. Only 1 wound treated with either dressing exhibited a decrease in bacteria. Both CMC and BDC silver dressings appeared to have statistically similar efficacy regarding the rate of wound healing and little impact on the actual bioburden in chronic lower-extremity wounds. Interestingly, there was no correlation in the size of the wound and any effect on bioburden. Although the BDC dressing showed a higher absolute rate of wound closure, neither technology demonstrated a statistically significant difference in wound closure rate when corrected for initial wound size.

Topical antiseptics in wound care: time for reflection

Topical antiseptics in wound care: time for reflection

Wound dressings and other topical treatment modalities in bioburden control

Reduction of wound bioburden has traditionally been achieved by the use of topical antimicrobial agents, such as bactericidal antiseptics or antibiotics. This has worked well for many years, however, concerns about toxicity and resistance have prompted research into other mechanisms. There are various means of removing bacteria from the wound, without recourse to chemical agents; for example, larvae (maggots) ingest bacteria, together with the devitalised tissue of the wound, while the control of exudate restricts the availability of free water, impeding the growth of water-loving bacteria. The discovery that some materials selectively adsorb, or sequester, bacteria has led to the development of bacteriostatic dressing materials, which do not rely on antiseptics for their action. These are designed to physically remove microorganisms, such as bacteria, from the wound, thus reducing bioburden. However, this still demands validation as a clinically relevant mechanism. Until that time, it remains a fascinating theoretical concept. This is a chapter taken from the book Microbiology of Wounds, published by CRC Press in 2010.

Wound bed preparation of difficult wounds: an evolution of the principles of TIME

In the last few years, considerable progress has been made in the treatment of chronic ulcers, thanks to new therapy methods. Wound bed preparation is a modern approach for the removal of local barriers to healing by optimising debridement, reduction of bioburden and exudate management through the TIME principles, which have been introduced by the International Advisory Board on Wound Bed Preparation. However, this protocol does not evaluate the state of the repair process and therefore does not suggest the ideal therapeutic choice for each single patient. The revised TIME-H concept considers also the supposed healing time, H, and gives a score that correlates the wound condition with the incidental concomitance with medical pathologies related to the therapeutic measures, thus guiding the clinician towards a practical and systematic approach in the treatment. By applying this scheme to our situation, the average healing time was considerably reduced. The formulation of the new protocol TIME-H for a critical assessment of treatment scheme, which also includes the general conditions of the patient, represents a more rational and adequate approach for an accurate prognosis and therefore for a more suitable therapeutic choice in the treatment of difficult wounds.

An improved mixed dextran rejection test for characterization of UF membranes in an R&D environment

Ultrafiltration performance is often characterized using solutes of known molecular size that emulate protein molecules. At Millipore we currently use a mixed dextran rejection test for release of UF membranes. We have recently developed and validated a new mixed dextran test that shows improved precision and sensitivity. The improved precision results from changes in the test format, changes in and standardization of all operating conditions used, improvements in general lab practices that resulted in reduction of bioburden, as well as better management of the SEC calibration procedures. The %CV for R90 (MW corresponding to 90% rejection) has been reduced by about 50% compared to previous practices. The improved sensitivity results from the use of more sensitive detection equipment, better integration and processing algorithms and better understanding of chromatographic artifacts. Sensitivity has been improved by 10–100 x and it is now possible to robustly report R99.9 (MW corresponding to 99.9% rejection or sieving of 0.001 of a dextran molecule) for membranes that have that capability. The improved test is presently being used for membrane characterization within an R&D environment. The capability has provided a valuable guide to new membrane development projects. More specifically, the consequence of the precision improvement is the ability to quantify the effect of small changes in manufacturing parameters and thus establish the trends in the process. The consequence of the sensitivity improvement is that we have a finer lens to study the effect of various treatments on the membrane. Changes in membrane properties that are not reflected in R90 can now be detected. The improved test could be used as a tool to explore the correlation between dextran and protein sieving performance, based on the expectation that R99.9 might be a more sensitive parameter compared to R90. Other potential benefits include the ability to map and better understand UF membrane process capability. Ultimately, better membranes can only be characterized to their full potential if a better test is available.

Johne's Disease

Johne's disease (paratuberculosis) is a chronic, granulomatous infection of the intestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). There is no approved treatment, no known way to eliminate the infection once established, nor is there an effective vaccine for the disease. Johne's disease (JD) has emerged as an important disease of cattle due to the economic impact and the potential link to human Crohn's disease. The recently introduced herd Risk Assessment (RA) and Herd Management Plan (HMP) developed by the National Johne's Working Group (NJWG) have been incorporated into the national program standards by USDA, APHIS. Increased state and federal funding for Johne's disease has increased the focus on ways to control the condition. Methods to control the spread and to reduce within herd transmission of Johne's disease (JD) are being adopted by both dairy and beef herds in many states through implementation of the national Johne's disease program. Implementation of best management practices (BMP) following a herd risk assessment (RA) designed to reduce the risk of transmission of JD remains the focal point of the national effort to reduce the prevalence of JD in cattle herds today. Although a vaccine for JD is available in many states, its use is closely regulated by individual state veterinarians and usage by the profession remains less than 2,000 herds nationally. Test and culling of culture-positive cattle seems a low priority if the infected animals are profitable. Reduction of the herd bioburden of MAP seems a lower priority for many producers. Brumbaugh et al1 demonstrated a reduction in the number of colony forming units (cfu) of MAP from the livers of experimentally MAP-infected mice treated with monensin compared to non-treated controls. More recently he showed monensin reduced the severity of histological lesions in cattle with clinical signs of Johne's disease, including weight loss and diarrhea.2 Initial infection with MAP is generally considered to occur in the neonatal calves. Our laboratory at the University of Pennsylvania, New Bolton Center has successfully induced experimental MAP infection in neonatal calves via oral gavage of MAP on two consecutive days.4 The current experiment was designed to assess the efficacy of monensina to reduce pass-through fecal shedding and to reduce tissue bacterial load (bioburden) of MAP in calves.

Residual protein levels on reprocessed dental instruments

Reduction of the initial bioburden on instruments, prior to sterilization, is believed to reduce transmission risks of iatrogenic Creutzfeldt-Jakob disease. Endodontic files are used in the preparation of root canals and are likely to have close contact and become contaminated with neural material from branches of the maxillary and mandibular cranial nerves. This study examined methods used by 22 dental practices to clean endodontic files, and scored visible debris and residual protein levels adhering to 220 dental endodontic files that had been used, cleaned, autoclaved and were deemed ready for re-use. Visible debris was scored after examination under a dissecting light microscope. Residual protein was quantified using a fluorescent assay based on reaction of proteins with o-phthaldialdehyde/N-acetyl cysteine. There was wide variation in the methods used by practices to clean endodontic files. The cleaning process varied from a wipe with an alcohol-impregnated cloth to hand scrubbing and/or use of an ultrasonic bath. Surface debris was visually detected on 98% of files. Residual protein was detected on all the files examined (median amount: 5.4 microg; range: 0.5-63.2 microg). These results demonstrate that the cleaning of some instruments reprocessed routinely in primary care is incomplete, and such instruments cannot be excluded as a potential source of cross-infection.

Temperature–time issues in bioburden control for planetary protection

Heat energy, administered in the form of an elevated temperature heat soak over a specific interval of time, is a well-known method for inactivating organisms. Sterilization protocols, from commercial pasteurization to laboratory autoclaving, specify both temperature and time, as well as water activity, for treatments to achieve either acceptable reduction of bioburden or complete sterilization. In practical applications of planetary protection, whether to reduce spore load in forward or roundtrip contamination, or to exterminate putative organisms in returned samples from bodies suspected of possible life, avoidance of expensive or potentially damaging treatments of hardware (or samples) could be accomplished if reciprocal relationships between time duration and soak temperature could be established. Conservative rules can be developed from consideration of empirical test data, derived relationships, current standards and various theoretical or proven mechanisms for thermal damage to biological systems.

The use of topical antimicrobials in wound bioburden control

The control of wound bioburden is believed to be valuable in avoiding possible deterioration into infection; in reducing critical colonization; and in treating frank infection. While the immune response is the governing factor in the development of infection, the reduction of bioburden – if achieved to a sufficient degree and for sufficient duration – can enable host defences to regain control. Topical antimicrobial agents, carefully selected and correctly dosed, can help achieve these goals. This review examines the value of topical antiseptics and antibiotics in various formulations in controlling the bioburden of acute and, predominantly, chronic wounds. The currently held perception of antibiotic misuse is reinforced and reiterated. Topical antibiotics must be used with caution and only for specific, limited indications. Topical antiseptics have a much broader range of applications and are generally safer when used appropriately. Conditions and circumstances of use are provided for these agents.

Gamma irradiation for terminal sterilization of 17β-estradiol loaded poly-(d,l-lactide-co-glycolide) microparticles

17β-Estradiol-loaded microparticles using poly-(d,l-lactide-co-glycolide) polymer (PLG) were prepared by a modified spray-drying method and the effects of γ-irradiation on drug substance, polymer and microparticles were investigated. Irradiation doses ranging from 5.1 to 26.6 kGy were applied using a 60Co-radiation source. 17β-Estradiol drug substance showed excellent stability against γ-irradiation in the investigated dose range, whereas microencapsulated estradiol seems to be converted to conjugation products with PLG, and to a lesser extent to the degradation product 9,11-dehydroestradiol. The weight-average molecular weight of the PLG polymers decreased with increasing irradiation dose while polydispersity indices (Mw/Mn) remained nearly unchanged, compatible with a random chain scission mechanism in lactide/glycolide-copolymer degradation. In vitro drug release studies showed accelerated kinetics with increasing irradiation doses due to dose dependent polymer degradation. Microbiological process monitoring showed decreasing bioburden with increasing spraying time, which was successfully further reduced by applying irradiation sterilization. Microencapsulated test spore suspensions of Bacillus pumilus ATCC 27142, the official test specimen for the γ-sterilization process, revealed effective reduction of bioburden, confirming its published D10 value. In conclusion, our studies demonstrated efficacy of γ-irradiation as terminal sterilization method for poly-(d,l-lactide-co-glycolide) polymer-based drug delivery systems. The sterilization conditions need to be carefully adjusted for the final dosage form.