Impact and effectiveness of the 10-valent pneumococcal conjugate vaccine on invasive pneumococcal disease among children under 5 years of age in the Netherlands.

BACKGROUND: In 2011, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 10-valent vaccine (PCV10) in the Netherlands. We report on impact and effectiveness against invasive pneumococcal disease (IPD) in children aged under 5 years by switching from PCV7 to PCV10. METHOD: We included IPD cases between 2004 and 2019 in children aged < 5 years reported via the national surveillance system. To assess the impact of the PCV10 vaccination program we compared IPD incidence 6-8 years after PCV10 introduction (2017-2019) to the two years just before the switch to PCV10 (2009-2011). We estimated vaccine effectiveness (VE) using the indirect cohort method, comparing vaccination status (at least two vaccine doses) in IPD-cases caused by PCV10 serotypes (cases) to non-PCV10 IPD cases (controls), in children eligible for PCV10. RESULTS: The overall incidence decreased from 8.7 (n = 162) in 2009-2011 to 7.3 per 100.000 (n = 127) in 2017-2019 (Incidence rate ratio (IRR) 0.83, 95%CI: 0.66; 1.05). IPD caused by the additional serotypes included in PCV10 declined by 93% (IRR 0.07, 95%CI: 0.02; 0.23). Incidence of non-PCV10 IPD showed a non-significant increase (IRR 1.25, 95%CI: 0.96; 1.63). Among 231 IPD-cases eligible for PCV10, the overall VE was 91% (95%CI: 67; 97) and did not differ by sex or age at diagnosis. Effectiveness against non-PCV10 serotype 19A IPD was non-significant with an estimate of 28% (95%CI:-179; 81). CONCLUSION: PCV10 is highly effective in protecting against IPD in Dutch children under 5 years with limited serotype replacement after switching from PCV7 to PCV10. We found no evidence for significant cross-protection of PCV10 against 19A serotype IPD.

Predicted coverage by 4CMenB vaccine against invasive meningococcal disease cases in the Netherlands.

Neisseria meningitidis serogroup B is a major cause of invasive meningococcal disease in Europe. In the absence of a conjugate serogroup B vaccine, a subcapsular 4CMenB vaccine was developed. Data on 4CMenB vaccine efficacy is still limited. Recently, genomic MATS (Meningococcal Antigen Typing System) was developed as a tool to predict strain coverage, using vaccine antigens sequence data. We characterized all invasive meningococcal isolates received by the Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM) in two epidemiological years 2017-2019 using whole-genome sequencing and determined serogroup, clonal complex (cc) and estimated 4CMenB vaccine coverage by gMATS. Of 396 cases of invasive meningococcal disease, corresponding to an incidence of 1.22 cases/105 inhabitants, 180 (45%) were serogroup W, 155 (39%) serogroup B, 46 (12%) serogroup Y, 10 (3%) serogroup C, 2 non-groupable (0.5%) and 3 (0.7%) unknown. The incidence was the highest among 0-4 years olds (4 cases/105 inhabitants), and 57/72 (79%) of these cases were serogroup B. Serogroup W predominated among persons 45 years of age or older with 110/187 (59%) cases. Serogroup B isolates comprised 11 different clonal complexes, with 103/122 (84%) isolates belonging to 4 clonal complexes: cc32, cc41/44, cc269 and cc213. In contrast, serogroup W isolates were genetically similar with 95% belonging to cc11. Of 122 serogroup B isolates, 89 (73%; 95% CI: 64-80%) were estimated to be covered by 4CMenB and the degree of coverage varied largely by clonal complex and age. Among the 0-4 year olds, 25 of 43 (58%; 95% CI: 43-72%) MenB isolates were estimated to be covered. Since the coverage of the 4CMenB vaccine is dependent on circulating clonal complexes, our findings emphasize the need for surveillance of circulating meningococcal strains. In addition, estimation of age specific coverage is relevant to determine the right target age group for vaccination.

Intraspecific Diversity and Taxonomy of Emmonsia crescens.

Emmonsia crescens is known as an environmental pathogen causing adiaspiromycosis in small rodents. As the generic name Emmonsia is no longer available for this species, its taxonomic position is re-evaluated. The intraspecific variation of Emmonsia crescens was analyzed using molecular, morphological, and physiological data, and the relationship between frequency of adiaspiromycosis and body temperature of host animals was explored. A North American and a pan-global lineage could be discerned, each with subclusters at low genetic distance. European strains produced the classical type of very large adiaspores, while in the North American lineage adiaspores relatively small, resembling the broad-based budding cells of Blastomyces. Members of the closely related genus Emergomyces may exhibit large, broad-based in addition to small, narrow-based budding cells. We conclude that the morphology of the pathogenic phase in these fungi differs gradationally between species and even populations, and is therefore less suitable as a diagnostic criterion for generic delimitation. Two Emmonsia species are reclassified in Emergomyces.

Community-acquired pneumonia in patients with bacterial meningitis: a prospective nationwide cohort study.

OBJECTIVE: Pneumonia is considered a focus of infection in patients presenting with community-acquired bacterial meningitis but the impact on disease course is unclear. The aim was to study presenting characteristics, clinical course and outcome of meningitis patients with co-existing pneumonia on admission. METHODS: We evaluated adult patients with community-acquired bacterial meningitis with pneumonia on admission in a nationwide, prospective cohort performed from March 2006 to June 2017. We performed logistic regression analysis to identify clinical characteristics predictive of pneumonia on admission, and to quantify the effect of pneumonia on outcome. RESULTS: Pneumonia was diagnosed on admission in 315 of 1852 (17%) bacterial meningitis episodes and confirmed by chest X-ray in 256 of 308 (83%) episodes. Streptococcus pneumoniae was the causative organism in 256 of 315 episodes (81%). Pneumonia on admission was associated with advanced age (OR 1.03 per year increase, 95% CI 1.02-1.04, p < 0.001), alcoholism (OR 1.96, 95% CI 1.23-3.14, p 0.004), cancer (OR 1.54, 95% CI 1.12-2.13, p 0.008), absence of otitis or sinusitis (OR 0.44, 95% CI 0.32-0.59, p < 0.001) and S. pneumoniae (OR 2.14, 95% CI 1.55-2.95, p < 0.001) in the multivariate analysis. An unfavourable outcome defined as a score of 1-4 on the Glasgow Outcome Scale was observed in 172 (55%) episodes and 87 patients (28%) died. Pneumonia on admission was independently associated with unfavourable outcome and mortality in the multivariate analysis (OR 1.48, 95% CI 1.12-1.96; p 0.005). CONCLUSION: Pneumonia on admission in bacterial meningitis is a frequent coexisting infection and is independently associated with unfavourable outcome and mortality.

Extensive tinea capitis and corporis in a child caused by Trichophyton verrucosum.

A 3-year-old boy presented with multiple lesions of tinea corporis with dermatophytids, and subsequent inflammatory lesions with alopecia on the scalp. At the beginning, topical clobetasone butyrate was prescribed. The infection was diagnosed as dermatophytosis on the basis of positive direct microscopy and fungal culture. The etiological agent was isolated from all sampled sites and identified as Trichophyton verrucosum. Clonal nature of the infection was confirmed by random amplified polymorphic DNA (RAPD) analysis. The child lived in close vicinity of cattle. He was successfully treated with itraconazole.

Identification of chromoblastomycosis agents by PCR based reverse line blot (PCR-RLB) hybridization assay.

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

Variation in coagulation and fibrinolysis genes evaluated for their contribution to cerebrovascular complications in adults with bacterial meningitis in the Netherlands.

OBJECTIVE: To study whether genetic variation in coagulation and fibrinolysis genes contributes to cerebrovascular complications in bacterial meningitis. METHODS: We performed a nationwide prospective genetic association study in adult community-acquired bacterial meningitis patients. The exons and flanking regions of 16 candidate genes involved in coagulation and fibrinolysis pathways were sequenced. We analyzed whether genetic variation in these genes resulted in a higher risk of cerebrovascular complications, unfavorable outcome and differences in thrombocyte count on admission. RESULTS: From 2006 to 2011, a total of 1101 bacterial meningitis patients were identified of whom 622 supplied DNA for genotyping and passed genetic quality control steps. In 139 patients (22%) the episode of bacterial meningitis was complicated by cerebral infarction, and 188 (30%) had an unfavorable outcome. We identified the functional variant rs494860 in the protein Z (PROZ) gene as our strongest association with occurrence of cerebral infarction (odds ratio (OR) 0.49 (95% confidence interval 0.33-0.73), p = 5.2 × 10-4). After Bonferroni correction for multiple testing no genetic variant was significantly associated (p-value threshold 2.7 × 10-4). CONCLUSION: Our study suggests a functional genetic variation in the PROZ gene, rs494860, may be of importance in bacterial meningitis pathogenesis and cerebral infarction risk. Replication of this finding in other cohort studies populations is needed.

Indoor wet cells as a habitat for melanized fungi, opportunistic pathogens on humans and other vertebrates.

Indoor wet cells serve as an environmental reservoir for a wide diversity of melanized fungi. A total of 313 melanized fungi were isolated at five locations in Guangzhou, China. Internal transcribed spacer (rDNA ITS) sequencing showed a preponderance of 27 species belonging to 10 genera; 64.22% (n = 201) were known as human opportunists in the orders Chaetothyriales and Venturiales, potentially causing cutaneous and sometimes deep infections. Knufia epidermidis was the most frequently encountered species in bathrooms (n = 26), while in kitchens Ochroconis musae (n = 14), Phialophora oxyspora (n = 12) and P. europaea (n = 10) were prevalent. Since the majority of species isolated are common agents of cutaneous infections and are rarely encountered in the natural environment, it is hypothesized that indoor facilities explain the previously enigmatic sources of infection by these organisms.

Direct detection of Exophiala and Scedosporium species in sputa of patients with cystic fibrosis.

Detection of species of Exophiala and Scedosporium in the respiratory tracts of cystic fibrosis (CF) patients remains controversial because of highly variable results. The results of our study suggested a significantly higher prevalence and more complex colonization than previously estimated. Approximately 17% (27/162) of clinical sputum samples were found to be positive for Exophiala dermatitidis and 30% (49/162) were positive for Scedosporium apiospermum / S. boydii species complex determined by reverse line blot (RLB) hybridization. In contrast, only 14.2% (23/162) and 1.2% (2/162) of clinical sputa were positive for E. dermatitidis and S. apiospermum / S. boydii species complex when tested by culture, respectively. Molecular detection methods, such as loop-mediated isothermal amplification (LAMP) or reverse line blot (RLB) hybridization, have the potential to become powerful alternatives to selective culture, providing a more realistic understanding on the prevalence of E. dermatitidis and S. apiospermum / S. boydii species complex in the respiratory tract of CF patients.

Molecular Diagnostics of Arthroconidial Yeasts, Frequent Pulmonary Opportunists.

Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard ribosomal DNA (rDNA) identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). The interspecific similarity obtained using rDNA markers (the internal transcribed spacer [ITS] and large subunit regions) was in the range of 96 to 99%, whereas that obtained using protein-coding loci (Rbp2, Act, and Tef1α) was lower at 89.4 to 95.2%. Ultimately, Rbp2 was selected as the best marker for species distinction. On the basis of cloned ITS data, some strains proved to be misidentified in comparison with the identities obtained with phenotypic characters, protein sequences, and AFLP profiles, indicating that different copies of the ribosomal operon were present in a single species. Antifungal susceptibility testing revealed that voriconazole had the lowest MIC against M. capitatus, while amphotericin B had the lowest MIC against S. clavata Both species exhibited in vitro resistance to fluconazole and micafungin.

[Gastrointestinal symptoms with meningococcal infection. Emergence of Neisseria meningitidis serogroup W.] / Gastro-intestinale klachten bij meningokokkeninfectie.

BACKGROUND: Meningococcal disease usually presents as meningitis and/or septicaemia, but can also present as pneumonia or arthritis. Since 2000, a worldwide increase in meningococcal disease is reported which is caused by a new virulent clone of serogroup W (MenW:cc11). This subtype is more likely to give an atypical clinical presentation and results in high mortality rates. CASE DESCRIPTION: A 68-year-old woman with polymyalgia rheumatica, managed with prednisone, developed an acute gastrointestinal syndrome of nausea, diarrhoea, vomiting, fever and chills. She presented at the Emergency Department and was admitted to intensive care for septic shock. Blood cultures revealed MenW:cc11 infection. She received antibiotic treatment and left the hospital in good condition 8 days after admission. CONCLUSION: MenW:cc11 is associated with gastrointestinal symptoms and sepsis. Recognition of this atypical clinical presentation is important for a timely and adequate treatment and for antibiotic prophylaxis of family members and close contacts.

Diversity of opportunistic black fungi on babassu coconut shells, a rich source of esters and hydrocarbons.

The present study assessed the diversity of black yeast-like fungi present on babassu coconut shells, a substrate rich in lipids and several volatile organic compounds (VOCs) including aromatic hydrocarbons. Using different isolation methods, one-hundred-six isolates were obtained and were identified by ITS sequencing as members of the genera Exophiala, Cladophialophora, Veronaea, and Rhinocladiella. Two novel species were discovered. Eight strains were selected for assessing their ability to grow on toluene and phenyl acetate as the sole carbon and energy source. All strains tested were able to assimilate phenyl acetate, while two out of eight were able to use toluene. VOCs profiling in babassu samples was also investigated by GC-ToF MS, revealing that a complex mixture of VOCs was emitted, which included alkylbenzenes such as toluene. Assimilation of alkylbenzenes by the black yeasts might therefore be the result of evolutionary adaptation to symbiotic interactions with higher plants. The potential relationship between lipid/aromatic hydrocarbon metabolism and pathogenicity is also discussed.

Imported Talaromycosis in Oman in Advanced HIV: A Diagnostic Challenge Outside the Endemic Areas.

A 37-year-old male living in Oman was seen by his physician with complaints of cough, body aches with bilateral lower limb weakness and on and off fever. He was diagnosed with HIV infection and culture from blood and bone marrow grew Talaromyces marneffei. He had travelled to Malaysia on several occasions. Treatment with liposomal amphotericin B resulted in complete cure. This case is reported for its rarity and unusual presentation to alert clinicians and microbiologists to consider T. marneffei as an etiology in high risk individuals. Our case is the first recorded diagnosis of T. marneffei in Oman.

Meningococcal carriage in Dutch adolescents and young adults; a cross-sectional and longitudinal cohort study.

OBJECTIVES: Current information on rates and dynamics of meningococcal carriage is essential for public health policy. This study aimed to determine meningococcal carriage prevalence, its risk factors and duration in the Netherlands, where meningococcal C vaccine coverage is >90%. Several methods to identify serogroups of meningococcal carriage isolates among adolescent and young adults were compared. METHODS: Oropharyngeal swabs were collected from 1715 participants 13-23 years of age in 2013-2014; 300 were prospectively followed over 8 months. Cultured isolates were characterized by Ouchterlony, real-time (rt-) PCR or whole-genome sequencing (WGS). Direct swabs were assessed by rt-PCR. Questionnaires on environmental factors and behaviour were also obtained. RESULTS: A meningococcal isolate was identified in 270/1715 (16%) participants by culture. Of MenB isolates identified by whole genome sequencing, 37/72 (51%) were correctly serogrouped by Ouchterlony, 46/51 (90%) by rt-PCR of cultured isolates, and 39/51 (76%) by rt-PCR directly on swabs. A sharp increase in carriage was observed before the age of 15 years. The age-related association disappeared after correction for smoking, level of education, frequent attendance to crowded social venues, kissing in the previous week and alcohol consumption. Three participants carried the same strain identified at three consecutive visits in an 8-month period. In these isolates, progressively acquired mutations were observed. CONCLUSIONS: Whole genome sequencing of culture isolates was the most sensitive method for serogroup identification. Based upon results of this study and risk of meningococcal disease, an adolescent meningococcal vaccination might include children before the age of 15 years to confer individual protection and potentially to establish herd protection.

Nomenclatural notes on Nadsoniella and the human opportunist black yeast genus Exophiala.

The opportunistic black yeast are particularly known through the genus Exophiala, characterised by annellidic budding cells. However, this phenotype is polyphyletic within the order Chaetothyriales. Seventeen generic names are available in the family Herpotrichiellaceae, one of which is Exophiala. Future taxonomy will be based on molecular phylogeny; each multi-species clade may qualify for one of these names. This paper focuses on the genus Nadsoniella, which is the oldest valid name in the Herpotrichiellaceae. Despite its exophiala-like phenotype, the type species of Nadsoniella clusters in the jeanselmei-clade, competing with the sympodial genus Rhinocladiella. In contrast, Exophiala competes with morphologically pronounced genera Thysanorea and Veronaea. Replacing the current phenotypic system for phylogenetic nomenclature requires highly stable phylogenies, which currently are not available.

Microtensile Bond Strength of Composite Cement to Novel CAD/CAM Materials as a Function of Surface Treatment and Aging.

OBJECTIVES: To evaluate the effect of different surface treatments on the bond strength to a composite and a polymer-infiltrated ceramic CAD/CAM block after six-month artificial aging. METHODS AND MATERIALS: Two types of CAD/CAM blocks (Cerasmart, GC; Enamic, Vita Zahnfabrik) were cut in slabs of 4-mm thickness, divided into six groups, and subjected to the following surface treatments: group 1: no treatment; group 2: sandblasting (SB); group 3: SB + silane (Si); group 4: SB + Si + flowable composite (see below); group 5: 5% hydrofluoric acid etching (HF) + Si; and group 6: 37% phosphoric acid etching (H3PO4) + Si. Sections of the same group were luted together (n=3: 3 sandwich specimens/group) using a dual-cure self-adhesive cement for all groups, except for the sections of group 4 that were luted using a light-curing flowable composite. After three weeks of storage in 0.5% chloramine at 37°C, the sandwich specimens were sectioned in rectangular microspecimens and trimmed at the interface to a dumbbell shape (1.1-mm diameter). One half of the specimens was subjected to a microtensile bond strength (µTBS) test, and the other half was tested after six months of water storage (aging). Data were statistically analyzed with a linear mixed-effects model for the factors surface treatment, material type, and aging, together with their first-degree interactions (α=0.05). RESULTS: The lowest bond strengths were obtained in the absence of any surface treatment (group 1), while the highest µTBSs were obtained when the surface was roughened by either SB or HF, this in combination with chemical adhesion through Si. Loss in bond strength was observed after six-month aging when either surface roughening or silanization, or both, were omitted. CONCLUSIONS: Both the composite and polymer-infiltrated ceramic CAD/CAM blocks appeared equally bonding-receptive regardless of the surface treatment used. Creating a microretentive surface by either SB or HF, followed by chemical adhesion using Si, is mandatory to maintain the bond strength after six months.

Benzalkonium tolerance genes and outcome in Listeria monocytogenes meningitis.

OBJECTIVES: Listeria monocytogenes is a food-borne pathogen that can cause meningitis. The listerial genotype ST6 has been linked to increasing rates of unfavourable outcome over time. We investigated listerial genetic variation and the relation with clinical outcome in meningitis. METHODS: We sequenced 96 isolates from adults with listerial meningitis included in two prospective nationwide cohort studies by whole genome sequencing, and evaluated associations between bacterial genetic variation and clinical outcome. We validated these results by screening listerial genotypes of 445 cerebrospinal fluid and blood isolates from patients over a 30-year period from the Dutch national surveillance cohort. RESULTS: We identified a bacteriophage, phiLMST6 co-occurring with a novel plasmid, pLMST6, in ST6 isolates to be associated with unfavourable outcome in patients (p 2.83e-05). The plasmid carries a benzalkonium chloride tolerance gene, emrC, conferring decreased susceptibility to disinfectants used in the food-processing industry. Isolates harbouring emrC were growth inhibited at higher levels of benzalkonium chloride (median 60 mg/L versus 15 mg/L; p <0.001), and had higher MICs for amoxicillin and gentamicin compared with isolates without emrC (both p <0.001). Transformation of pLMST6 into naive strains led to benzalkonium chloride tolerance and higher MICs for gentamicin. CONCLUSIONS: These results show that a novel plasmid, carrying the efflux transporter emrC, is associated with increased incidence of ST6 listerial meningitis in the Netherlands. Suggesting increased disease severity, our findings warrant consideration of disinfectants used in the food-processing industry that select for resistance mechanisms and may, inadvertently, lead to increased risk of poor disease outcome.

Sex-based differences in adults with community-acquired bacterial meningitis: a prospective cohort study.

OBJECTIVES: To investigate sex-based differences in clinical features, causative pathogens, outcome and treatment of adult community-acquired meningitis. METHODS: From January 2006 to July 2014, we prospectively investigated sex-based differences in clinical features, causative pathogens, outcome and treatment of adult community-acquired meningitis in a nationwide cohort study in the Netherlands. Sex was analysed along with known predictors of unfavourable outcome using logistic regression. RESULTS: We evaluated 1412 episodes of meningitis, 707 (50%) in men. Men more often presented with a history of remote head injury (41/667 (6%) versus 14/658 (2%) women, p 0.0002) or alcoholism (61/652 (9%) versus 21/660 (3%) women, p <0.0001). Neck stiffness was less common in men (453/651 (70%) versus 524/671 (78%) women, p 0.0004). Despite greater illness severity, women were less likely to receive treatment in an intensive care unit (odds ratio (OR) 0.72, 95% CI 0.58-0.89, p 0.003) or mechanical ventilation (OR 0.67, 95% CI 0.54-0.85, p 0.001). Women exhibited higher serum inflammatory parameters than men (median C-reactive protein 211 versus 171, p 0.0001; median erythrocyte sedimentation rate 48 versus 33, p <0.0001). Corticosteroids improved prognosis in both sexes, but absolute risk reduction was higher in women (20% versus 15%, p 0.001), although we found no significant interaction between sex and dexamethasone (p 0.38). In the multivariable analysis, male sex was an independent predictor of unfavourable outcome (OR 1.34, 95% CI 1.03-1.75, p 0.03) and death (OR 1.47, 95% CI 1.04-2.07, p 0.03). CONCLUSIONS: Our findings show sex-based differences in adults with community-acquired bacterial meningitis. Male sex is an independent risk factor for adverse outcome. It is possible that sex-based differences in immune reaction could determine a distinct response to corticosteroids.

Monoclonal antibodies for the treatment of Ebola virus disease.

INTRODUCTION: To date, the management of patients with suspected or confirmed Ebolavirus disease (EVD) depends on quarantine, symptomatic management and supportive care, as there are no approved vaccines or treatments available for human use. However, accelerated by the recent large outbreak in West Africa, significant progress has been made towards vaccine development but also towards specific treatment with convalescent plasma and monoclonal antibodies. Areas covered: We describe recent developments in monoclonal antibody treatment for EVD, encompassing mAb114 and the MB-003, ZMAb, ZMapp™ and MIL-77E cocktails. Expert opinion: Preventive measures, are, and will remain essential to curb EVD outbreaks; even more so with vaccine development progressing. However, research for treatment options must not be neglected. Small-scale animal and individual human case studies show that monoclonal antibodies (mAbs) can be effective for EVD treatment; thus justifying exploration in clinical trials. Potential limitations are that high doses may be needed to yield clinical efficacy; epitope mutations might reduce efficacy; and constant evolution of (outbreak-specific) mAb mixtures might be required. Interim advice based on the clinical experience to date is that treatment of patients with mAbs is sensible, provided those could be made available in the necessary amounts in time.