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Fecal Microbiota Transplantation in Irritable Bowel Syndrome: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.
Jamshidi, P, Farsi, Y, Nariman, Z, Hatamnejad, MR, Mohammadzadeh, B, Akbarialiabad, H, Nasiri, MJ, Sechi, LA
International journal of molecular sciences. 2023;24(19)
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Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder the cause of which is not yet fully elucidated. Probiotics, prebiotics and dietary changes have been shown to mitigate IBS symptoms whilst the results from studies of faecal microbiota transplants (FMT) have been inconsistent. The aim of this systematic review and meta-analysis of double-blind, randomised controlled trials (RCT) was to evaluate the efficacy and safety of FMT in IBS. 7 RCTs with a low risk of bias and no publication bias were included in the meta-analysis. Overall, no statistically significant effect was observed. A subgroup analysis by treatment modality showed that benefits were seen with lower GI administration of a single dose of multiple-donor FMT. Abdominal pain, nausea, diarrhoea and bloating were the most common adverse events, with no severe or critical adverse events reported. The authors call for larger and longer clinical trials to fill existing knowledge gaps.
Abstract
Irritable bowel syndrome (IBS) poses a significant challenge due to its poorly understood pathogenesis, substantial morbidity, and often inadequate treatment outcomes. The role of fecal microbiota transplantation (FMT) in managing IBS symptoms remains inconclusive. This systematic review and meta-analysis aimed to ascertain the effectiveness of FMT in relieving symptoms in IBS patients. A thorough search was executed on PubMed/Medline and Embase databases until 14 June 2023, including all studies on FMT use in IBS patients. We examined the efficiency of FMT in reducing patients' symptoms overall and in particular subgroups, classified by placebo preparation, FMT preparation, frequency, and route of administration. Among 1015 identified studies, seven met the inclusion criteria for the meta-analysis. The overall symptomatology of FMT-treated IBS patients did not significantly differ from the control group (Odds Ratio (OR) = 0.99, 95% Confidence Interval (CI) 0.39-2.5). Multiple doses of FMT compared with non-FMT placebo, or single-donor FMT therapy compared with autologous FMT placebo also showed no significant benefit (OR = 0.32, 95%CI (0.07-1.32), p = 0.11, and OR = 1.67, 95%CI (0.59-4.67), p = 0.32, respectively). However, a single dose of multiple-donor FMT administered via colonoscopy (lower gastrointestinal (GI) administration) significantly improved patient symptoms compared with autologous FMT placebo (OR = 2.54, 95%CI (1.20-5.37), p = 0.01, and OR = 2.2, 95%CI (1.20-4.03), p = 0.01, respectively). The studies included in the analysis showed a low risk of bias and no publication bias. In conclusion, lower GI administration of a single dose of multiple-donor FMT significantly alleviates patient complaints compared with the autologous FMT used as a placebo. The underlying mechanisms need to be better understood, and further experimental studies are desired to fill the current gaps.
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Disturbances of Vaginal Microbiome Composition in Human Papillomavirus Infection and Cervical Carcinogenesis: A Qualitative Systematic Review.
Wu, M, Li, H, Yu, H, Yan, Y, Wang, C, Teng, F, Fan, A, Xue, F
Frontiers in oncology. 2022;12:941741
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Cervical cancer remains the fourth most prevalent cancer in women worldwide. The infection of certain strains of human papillomavirus (HPV)) are thought to have an important causative role in the development of cervical cancer. But since a vast majority of HPV infections clear naturally within a few months, this indicates other factors at play determine the progression of the disease and its cancerous developments. Recent findings indicate that there may be a close link between disruptions of the vaginal microbiome and HPV infection, cervical lesions, cervical cancer and other gynaecological diseases. However, the evidence thus far is quite varied. Hence this systematic review sought to gather the current evidence and integrate it to create up-to-date knowledge. Included were the 22 studies relating to vaginal microbiota, and women with HPV-associated cervical diseases. The studies were conducted in various countries around the world and contained a mixture of case-controlled, cross-sectional and longitudinal studies. The authors acknowledge the challenges of summarising the findings due to differences in how the studies have been conducted. The results of the review showed that vaginal disturbances in HPV infections and related cervical diseases, seem to manifest in decreases in Lactobacilli, and increases in aerobic and anaerobic bacteria. Lactobacillus iners seemed to have either protective or pathogenic effects. They also noted that there are geographical and ethnic differences and patterns, which made the consolidation of results more challenging. For future research, the authors deemed the role of the Lactobacillus family of particular interest.
Abstract
BACKGROUND Emerging evidence has demonstrated a close association between perturbations in vaginal microbiota composition in women and human papillomavirus (HPV) infection, cervical lesions, and cervical cancer (Ca); however, these findings are highly heterogeneous and inconclusive. AIM: To perform a comprehensive systematic review of the global disturbance in the vaginal microbiota, specifically in women with HPV-associated cervical diseases, and to further conduct within- and across-disease comparisons. METHOD Twenty-two records were identified in a systematic literature search of PubMed, Web of Science, and Embase up to February 28, 2022. We extracted microbial changes at the community (alpha and beta diversity) and taxonomic (relative abundance) levels. Within- and across-disease findings on the relative abundance of taxonomic assignments were qualitatively synthesized. RESULTS Generally, significantly higher alpha diversity was observed for HPV infection, cervical lesions, and/or cancer patients than in controls, and significant differences within beta diversity were observed for the overall microbial composition across samples. In within-disease comparisons, the genera Gardnerella, Megasphaera, Prevotella, Peptostreptococcus, and Streptococcus showed the greatest abundances with HPV infection; Sneathia and Atopobium showed inconsistent abundance with HPV infection, and Staphylococcus was observed in Ca. Across diseases, we find increased levels of Streptococcus and varying levels of Gardnerella were shared across HPV infections, high-grade squamous intraepithelial lesions, and Ca, whereas Lactobacillus iners varied depending on the HPV-related disease subtype. CONCLUSIONS This systematic review reports that vaginal microbiome disturbances are correlated to the depletion of Lactobacillus, enrichment of anaerobes, and increased abundance of aerobic bacteria in HPV infection and related cervical diseases. Moreover, L. iners may exert either protective or pathogenic effects on different HPV-related diseases.
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Contribution of Lactobacillus iners to Vaginal Health and Diseases: A Systematic Review.
Zheng, N, Guo, R, Wang, J, Zhou, W, Ling, Z
Frontiers in cellular and infection microbiology. 2021;11:792787
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The vaginal microbiome is an important contributor to vaginal health. Here the dominance of Lactobacilli species, alongside their antimicrobial compounds play a critical role in contributing and protecting the vaginal milieu. Conversely the disruption or absence of Lactobacilli dominance is frequently associated with vaginal disease and infections. One of the Lactobacilli species prevalent in the vaginal microbiome is Lactobacillus iners. It was long overlooked in research due to it being so difficult to culture, and it was first identified in 1999 thanks to DNA sequencing. Researchers since discovered that the relationship between L. iners and vaginal health is far more complicated and ambiguous compared to the other Lactobacilli species. This systematic review explores the current knowledge of the characteristics of L. iners and its role in vaginal health and disease. The article discusses L. iners identification, genetic make up and differences to other Lactobacilli species and how they relate to vaginal health. The article also summarizes L. iners nutrient requirements and its role in diseases like dysbiosis, bacterial vaginosis, sexually transmitted infections and biofilm formation. Furthermore the authors look at the relation between L. iners and premature birthing, fertility and menstrual cycles. A final section in discusses the antimicrobial and immune sytem activating qualities of L. iners. In light of all these findings the authors describe L . iners as a very unique Lactobacilli due to its unusual characteristics. Whether L. iners is beneficial or pathogenic for the host remains controversial, as it can adapt to high and low pH environment and is seen in health and equally dysbiotic states of infection. Hence some describe it as a transitional species that colonizes the vagina after disturbances. It may be a risk factor for infections by contributing to the onset and maintenance of dysbiotic disturbances. Further studies are needed to clarify the role of L. iners and its role on vaginal health and whether it could serve as a biomarker for vaginal inflammation. This article is a useful summary about the characteristics and role of L. iners in vaginal health in disease.
Abstract
Lactobacillus iners, first described in 1999, is a prevalent bacterial species of the vaginal microbiome. As L. iners does not easily grow on de Man-Rogosa-Sharpe agar, but can grow anaerobically on blood agar, it has been initially overlooked by traditional culture methods. It was not until the wide application of molecular biology techniques that the function of L. iners in the vaginal microbiome was carefully explored. L. iners has the smallest genome among known Lactobacilli and it has many probiotic characteristics, but is partly different from other major vaginal Lactobacillus species, such as L. crispatus, in contributing to the maintenance of a healthy vaginal microbiome. It is not only commonly present in the healthy vagina but quite often recovered in high numbers in bacterial vaginosis (BV). Increasing evidence suggests that L. iners is a transitional species that colonizes after the vaginal environment is disturbed and offers overall less protection against vaginal dysbiosis and, subsequently, leads to BV, sexually transmitted infections, and adverse pregnancy outcomes. Accordingly, under certain conditions, L. iners is a genuine vaginal symbiont, but it also seems to be an opportunistic pathogen. Further studies are necessary to identify the exact role of this intriguing species in vaginal health and diseases.
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The Efficacy of Probiotics, Prebiotic Inulin-Type Fructans, and Synbiotics in Human Ulcerative Colitis: A Systematic Review and Meta-Analysis.
Astó, E, Méndez, I, Audivert, S, Farran-Codina, A, Espadaler, J
Nutrients. 2019;11(2)
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It is thought that ulcerative colitis (UC) may be caused by an excessive immune response to endogenous bacteria in genetically predisposed individuals, and therefore that manipulating of the gut flora may be of benefit. Microbial diversity and intestinal microbiota stability are lower in patients with inflammatory bowel disease (including UC), than in healthy people. This systematic review and metanalysis looked at clinical trials using probiotics, prebiotics and synbiotics (a combination of pro- and prebiotics) in UC. 18 papers were included, with a total of 1491 adult and 69 children. 16 of these were on probiotics, one on prebiotics and one on synbiotics. Outcomes considered in this systematic review were the effects on short chain fatty acids (SCFAs, metabolic end products of gut bacteria which have a beneficial effect on immune and gut health), inflammation levels, composition of faecal microbiota and UC remission. In trials on inactive UC patients, the faecal concentration of SCFAs did not differ significantly between the probiotic and placebo groups, whilst in trials with active UC patients, SCFAs significantly increased after probiotic supplementation. All studies reported a significant reduction in inflammation. Meta-analysis of studies which looked at induction/maintenance of remission by probiotics showed that this depends on a) the type of disease activity score used to assess remission, and b) the type of probiotics used, with bifidobacteria containing probiotics, VSL3 and Mutaflor showing benefits, but studies without bifidobacteria being no different from placebo. The authors conclude that bifidobacteria containing probiotics seem to be beneficial in terms of reaching remission in patients with UC, although there is insufficient information on necessary dose and duration of treatment. They note that there are only few studies on prebiotics and synbiotics and are calling for a standardisation of scales to assess remission.
Abstract
Studies of probiotics, fructan-type prebiotics, and synbiotics in patients with ulcerative colitis (UC) show significant heterogeneity in methodology and results. Here, we study the efficacy of such interventions and the reasons for the heterogeneity of their results. Eligible random controlled trials were collected from the PUBMED and SCOPUS databases. A total of 18 placebo-controlled and active treatment-controlled (i.e., mesalazine) studies were selected with a Jadad score ≥ 3, including 1491 patients with UC. Data for prebiotics and synbiotics were sparse and consequently these studies were excluded from the meta-analysis. The UC remission efficacy of probiotics was measured in terms of relative risk (RR) and odds ratio (OR). Significant effects were observed in patients with active UC whenever probiotics containing bifidobacteria were used, or when adopting the US Food and Drug Administration (FDA)-recommended scales (UC Disease Activity Index and Disease Activity Index). By the FDA recommended scales, the RR was 1.55 (CI95%: 1.13⁻2.15, p-value = 0.007, I² = 29%); for bifidobacteria-containing probiotics, the RR was 1.73 (CI95%: 1.23⁻2.43, p-value = 0.002, I² = 35%). No significant effects were observed on the maintenance of remission for placebo-controlled or mesalazine-controlled studies. We conclude that a validated scale is necessary to determine the state of patients with UC. However, probiotics containing bifidobacteria are promising for the treatment of active UC.
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The anxiolytic effect of probiotics: A systematic review and meta-analysis of the clinical and preclinical literature.
Reis, DJ, Ilardi, SS, Punt, SEW
PloS one. 2018;13(6):e0199041
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The microbiome-gut-brain axis in general and the possibility of altering the microbiome through administration of probiotics to support physical and mental health has received much attention in recent years. Here, a systematic review and meta-analysis were carried out to evaluate the clinical and preclinical evidence for the use of probiotics in anxiety. 22 preclinical (rodent) studies were included in the meta-analysis and showed an overall significant anxiolytic effect of probiotics in diseased, but not healthy, animals. Studies were heterogenous with regards to species and strains of probiotic used. Subgroup analysis showed that only Lactobacillus rhamnosus significantly reduced anxiety-like behaviour. 14 human studies were included in the meta-analysis and overall no anxiolytic effect was observed. Only three out of the 14 studies showed a positive effect (vs 12 out of the 22 animal studies), one of which used L. rhamnosus. Due to the small number of trials no subgroup analysis could be performed. Apart from the small number and heterogeneity of human studies, the authors discuss further possible reasons for the discrepancy between animal and human studies: • Dose: Dosages were typically 100 times higher (per kg) in animals than in humans. • Diseased vs healthy subjects: In animal studies, only those which investigated animals displaying anxiety related behaviour improved with probiotic administration. None of the human studies specifically recruited anxious individuals, eight of the studies included healthy subjects, the other six selected participants for other disorders, including four for irritable bowel syndrome. The authors conclude that more research into an anxiolytic effect of probiotics in humans is warranted, especially using L. rhamnosus, studying patients with anxiety, and using higher dosages and longer study duration.
Expert Review
Conflicts of interest:
None
Take Home Message:
- While preclinical animal studies suggest that probiotics may help reduce anxiety, such findings have not yet translated to clinical research in humans.
- Further investigation of probiotic treatment for clinically relevant anxiety is warranted, particularly with respect to the probiotic species L. rhamnosus.
Evidence Category:
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A: Meta-analyses, position-stands, randomized-controlled trials (RCTs)
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B: Systematic reviews including RCTs of limited number
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C: Non-randomized trials, observational studies, narrative reviews
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D: Case-reports, evidence-based clinical findings
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E: Opinion piece, other
Summary Review:
This review highlights how important it is for future studies to focus on clinically anxious patients and also to consider exploring the effects of differing doses of probiotics in this population.
Clinical practice applications:
Anxiety disorders affect as many as 3 in 10 people at some point during their lifetime. On that basis, it would be great to have a viable non-pharmaceutical option to help with some of the symptoms.
Considerations for future research:
If the results from the pre-clinical studies can be corroborated in human populations, this could have widespread clinical implications.
Abstract
BACKGROUND Probiotics have generated intensive research interest in recent years as a novel mode of treatment for physical and mental illness. Nevertheless, the anxiolytic potential of probiotics remains unclear. The present systematic review and meta-analysis aimed to evaluate the clinical and preclinical (animal model) evidence regarding the effect of probiotic administration on anxiety. METHODS The PubMed, PsycINFO, and Web of Science databases were reviewed for preclinical and clinical studies that met the defined inclusion and exclusion criteria. The effects of probiotics on anxiety-like behavior and symptoms of anxiety were analyzed by meta-analyses. Separate subgroup analyses were conducted on diseased versus healthy animals, specific preclinical probiotic species, and clinical versus healthy human samples. RESULTS Data were extracted from 22 preclinical studies (743 animals) and 14 clinical studies (1527 individuals). Overall, probiotics reduced anxiety-like behavior in animals (Hedges' g = -0.47, 95% CI -0.77 --0.16, p = 0.004). Subgroup analyses revealed a significant reduction only among diseased animals. Probiotic species-level analyses identified only Lactobacillus (L.) rhamnosus as an anxiolytic species, but these analyses were broadly under-powered. Probiotics did not significantly reduce symptoms of anxiety in humans (Hedges' g = -0.12, 95% CI -0.29-0.05, p = 0.151), and did not differentially affect clinical and healthy human samples. CONCLUSIONS While preclinical (animal) studies suggest that probiotics may help reduce anxiety, such findings have not yet translated to clinical research in humans, perhaps due to the dearth of extant research with clinically anxious populations. Further investigation of probiotic treatment for clinically relevant anxiety is warranted, particularly with respect to the probiotic species L. rhamnosus.
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Infectious complications following probiotic ingestion: a potentially underestimated problem? A systematic review of reports and case series.
Costa, RL, Moreira, J, Lorenzo, A, Lamas, CC
BMC complementary and alternative medicine. 2018;18(1):329
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This article is a systematic review of case reports and case series published between 1976 and 2018, evaluating serious infections in the context of probiotic intake. The authors found 67 articles, 60 case reports and 7 case series, corresponding to 93 patients. Whilst they believe that these cases represent only a small proportion of existing cases, they also point out that the proportion of cases of infectious complications is small when the total number of people who use probiotics is considered. Fungemia was the most commonly observed complication, with 37.6% of the cases, sepsis was identified in 31.2% patients; bacteraemia was responsible for 20.4% of cases, followed by endocarditis and abscess, with 4 and 3 cases, respectively. Of the fungal complications, 50.5% were due to Saccharomyces spp., Lactobacillus spp. were the etiologic agents in 26 and Bifidobacterium in 12 cases of infectious complications. Infectious complications were more likely in the elderly, children under 1 (especially premature babies), and patients with central venous access or receiving enteral or parenteral nutrition. Immunosuppressive medication was reported in 15% of patients and antibiotic use in 43%. All-cause mortality was 19.6%. The authors conclude that the use of probiotics cannot be considered risk free and should be carefully evaluated for high-risk groups of patients.
Abstract
BACKGROUND Little is studied about complications related to probiotic ingestion. This study proposes to present a synthesis and critical evaluation of the reports and series of cases on the infectious complications related to the ingestion of probiotics, which can raise awareness for the prescribing and use of probiotics for certain groups of patients. METHODS Systematic review of reports and series of cases researched in the PubMed, SciELO and Scopus databases published until August 2018. The references of the articles were investigated manually for the search of cross references. SPSS version 23.0 was used for descriptive statistics and univariate analysis. RESULTS We found 60 case reports and 7 case series, making up a total of 93 patients. Fungemia was the most common infectious complications with 35 (37.6%) cases. The genus Saccharomyces was the most frequent with 47 (50.6%) cases, followed by Lactobacillus, Bifidobacterium, Bacillus, Pedioccocus and Escherichia with 26 (27.9%), 12 (12.8%), 5 (5.4%), 2 (2.2%) and 1 (1.1%) case, respectively. Adults over 60 years of age, Clostridium difficile colitis, antibiotic use and Saccharomyces infections were associated with overall mortality. HIV infections, immunosuppressive drugs, solid organ transplantation, deep intravenous lines, enteral or parenteral nutrition were not associated with death. CONCLUSION The use of probiotics cannot be considered risk-free and should be carefully evaluated for some patient groups. TRIAL REGISTRATION CRD42016042289.
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Probiotics for the Treatment of Atopic Dermatitis in Children: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.
Huang, R, Ning, H, Shen, M, Li, J, Zhang, J, Chen, X
Frontiers in cellular and infection microbiology. 2017;7:392
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It is estimated that atopic dermatitis (AD) affects around 10-20% of children. This systematic review and meta-analysis evaluates the evidence for using probiotics for the treatment of AD in children. 13 randomised controlled trials (RCT) were included in the review, all but one were of a double-blind design. They included a total of 1070 children, 553 receiving probiotics and 517 controls. Overall, a beneficial effect of probiotics in AD was observed. Subgroup analysis showed a) positive results in children aged 1-18 years, but probiotics being ineffective in infants younger than 1 year; b) probiotics reduced AD in Asian, but not European studies, and c) certain strains proved beneficial (Lactobacillus salivarius, Lactobacillus fermentum and a multi-strain probiotic), whilst Lactobacillus rhamnosus GG and Lactobacillus plantarum showed no effect. The authors discuss that their meta-analysis is limited by the heterogeneity among the trials and inclusion of studies with small sample sizes. They conclude that probiotics may be of benefit in children with AD but that more adequately powered trials assessing specific probiotics and dosages are needed, to inform the best treatment protocols.
Abstract
Objective: Atopic dermatitis (AD) is a prevalent, burdensome, and psychologically important pediatric concern. Probiotics have been suggested as a treatment for AD. Some reports have explored this topic; however, the utility of probiotics for AD remains to be firmly established. Methods: To assess the effects of probiotics on AD in children, the PubMed/Medline, Cochrane Library Scopus, and OVID databases were searched for reports published in the English language. Results: Thirteen studies were identified. Significantly higher SCORAD values favoring probiotics over controls were observed (mean difference [MD], -3.07; 95% confidence interval [CI], -6.12 to -0.03; P < 0.001). The reported efficacy of probiotics in children < 1 year old was -1.03 (95%CI, -7.05 to 4.99) and that in children 1-18 years old was -4.50 (95%CI, -7.45 to -1.54; P < 0.001). Subgroup analyses showed that in Europe, SCORAD revealed no effect of probiotics, whereas significantly lower SCORAD values were reported in Asia (MD, -5.39; 95%CI, -8.91 to -1.87). Lactobacillus rhamnosus GG (MD, 3.29; 95%CI, -0.30 to 6.88; P = 0.07) and Lactobacillus plantarum (MD, -0.70; 95%CI, -2.30 to 0.90; P = 0.39) showed no significant effect on SCORAD values in children with AD. However, Lactobacillus fermentum (MD, -11.42; 95%CI, -13.81 to -9.04), Lactobacillus salivarius (MD, -7.21; 95%CI, -9.63 to -4.78), and a mixture of different strains (MD, -3.52; 95%CI, -5.61 to -1.44) showed significant effects on SCORAD values in children with AD. Conclusions: Our meta-analysis indicated that the research to date has not robustly shown that probiotics are beneficial for children with AD. However, caution is needed when generalizing our results, as the populations evaluated were heterogeneous. Randomized controlled trials with larger samples and greater power are necessary to identify the species, dose, and treatment duration of probiotics that are most efficacious for treating AD in children.
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Probiotics and oral health: A systematic review.
Seminario-Amez, M, López-López, J, Estrugo-Devesa, A, Ayuso-Montero, R, Jané-Salas, E
Medicina oral, patologia oral y cirugia bucal. 2017;22(3):e282-e288
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Oral cavity has its own microbiome. When the balance of the microbes living in the oral cavity is altered, e.g. due to poor oral hygiene, diet or the use of antimicrobial drugs, periodontal disease and dental caries can develop. Probiotics may provide a new approach for the prevention and management of these oral diseases. This review paper examined 12 published randomised clinical trials, 2 meta-analyses and 1 systematic review that assessed the effect of probiotics in the treatment and/or prevention of an infectious oral disease. Based on the reviewed studies, the authors concluded that the use of probiotics could be beneficial for the maintenance of oral health, due to its ability to decrease colony forming unit counts of oral pathogens. However, more clinical trials with longer-term follow-ups are needed to confirm the efficacy of probiotic therapy, identify specific probiotic strains and determine the correct dose, treatment time and delivery form required for each disease.
Abstract
BACKGROUND Probiotics are microorganisms, mainly bacteria, which benefit the host's health. Many studies support the role of probiotics as a contributor to gastrointestinal health, and nowadays many authors are trying to prove its influence in oral health maintenance. OBJECTIVES To review the published literature with the purpose of knowing the importance of using probiotics as a preventive and therapeutic method for oral infectious diseases management. MATERIAL AND METHODS An electronic search in PubMed database with the keywords "oral health AND probiotics AND dentistry" was conducted. The inclusion criteria were: randomized clinical trials (RCTs) that assess the action of any probiotic strain in the treatment and / or prevention of an infectious oral disease, RCTs that assess the action of any probiotic strain on counting colony forming units (CFU) of oral pathogens, systematic reviews and meta-analysis. The Jadad scale was used to assess the high quality of RCTs. RESULTS Fifteen articles were considered for this review. Of which, 12 were RCTs of good / high quality (Jadad scale), two meta-analysis and one systematic review. CONCLUSIONS The literature reviewed suggests probiotics usage could be beneficial for the maintenance of oral health, due to its ability to decrease the colony forming units (CFU) counts of the oral pathogens. However, randomized clinical trials with long-term follow-up periods are needed to confirm their efficacy in reducing the prevalence/incidence of oral infectious diseases. Furthermore, the recognition of specific strains with probiotic activity for each infectious oral disease is required, in order to determine exact dose, treatment time and ideal vehicles.