"Such an institution represents the circle of life" - bringing an inpatient hospice into an academic setting: a pre-implementation exploratory study.

BACKGROUND: To combine the benefits of hospice and palliative care, the integration of both seems self-evident. Aim of this study was to explore clinical staff's and volunteers' expectations and concerns of the first university hospice in Germany planning for implementation. METHODS: Staff and volunteers of the Department of Palliative Medicine of the University Hospital in Cologne received questionnaires and were interviewed following three themes of interest: opportunities, challenges, general criteria. Questionnaire results were analyzed descriptively using mean ± SD and percentages, open-ended questions and interviews were analysed using content analysis. RESULTS: A total of 28/100 questionnaires was returned (n = 17 clinical staff, n = 11 volunteers) and 18 interviews conducted. The majority of both clinical staff and volunteers estimated the need for a university inpatient hospice as rather to very high (64.7% and 81.8%, respectively). Our findings revealed that most clinical staff and volunteers anticipated improvements with the intended university inpatient hospice, although their expectations were divided between both hope and concern while adhering to legal and general requirements, which they feared might oppose such a project. Participants expressed concern about leadership and staffing plans, albeit most pronounced among clinical staff. Nursing staff repeatedly articulated concerns about being interchanged between the palliative care ward and the intended inpatient hospice while they had explicitly chosen to work in palliative medicine. CONCLUSIONS: The overall high level of anticipated progress and excitement is very encouraging. Albeit serious concerns were mentioned, our results indicate that all participants believe in a positive impact and highlight the need of developing a solid concept. In order to implement such a hospice within a university setting, it is important to consider multilevel contextual factors such as system-level factors (funding, external and internal regulations), organization-level factors (leadership, staff motivation), and patient-level factors (adaptability to patients' needs). Our findings illustrate the importance of understanding the context of practice before implementation. Our pre-implementation study helps identify critical views from staff members and volunteers that may hinder or advance the implementation. TRIAL REGISTRATION: The study was registered at the German Clinical Trials Register (#DRKS00021258) on April 17th 2020.

Communication about the desire to die: Development and evaluation of a first needs-oriented training concept - A pilot study.

OBJECTIVE: Patients' desire to die (DD) is rarely discussed in palliative care (PC) due to health professionals' (HPs) feeling of uncertainty. The aim of the study was to develop and evaluate a training to increase HPs' self-confidence in responding professionally to patient's DD and to assess the feasibility of this approach. METHODS: The training course was developed via focus groups and relevant literature and refined with an advisory board. An evaluation design was developed to evaluate training outcomes and to examine feasibility. To assess self-confidence, knowledge, skills, and attitudes: (1) standardized surveys were applied at T1 (before training), T2 (directly after), and T3 (3 months later), and were analyzed by descriptive and non-parametric statistics; and (2) participants' open feedback was summarized by content. RESULTS: A two-day multi-disciplinary training was developed to improve self-confidence via diverse teaching methods. Twenty-four HPs from general and specialized PC were participated. Via self-rating on Likert scales at three time points, improvements were seen at T1, T2, and partly remained at T3, especially in the overall item of self-confidence in communicating with patients about their DD (means: 4.3. at T1, 5.7 at T2, and 5.9 at T3; on a 7-point scale with 1 = lowest value and 7 = highest value). Fewer improvements were found in skills (using different approaches) and attitudes (feeling less helplessness). Open feedback revealed a high appreciation for the training, especially the composition of participants, the role-play, and the overall increase of awareness of the topic. SIGNIFICANCE OF RESULTS: The developed training on addressing DD meets a need and was perceived by the participants to be of added value. Future research should measure training effects with a validated instrument, including more participants, diverse participant groups, and a control group. Effects on patients should be assessed.

Mobile Palliative Care Consultation Service (PCCS): Overview of Hospice and Palliative Care Evaluation (HOPE) Data on In-Patients With End-Stage Cancer, Multiple Sclerosis, and Noncancer, Nonneurological Disease From 4 PCCS Centers in Germany in 2013.

CONTEXT: During the last decade, numerous in-patient Palliative Care Consultation Service (PCCS) units were established throughout Germany. OBJECTIVE: To provide an epidemiological overview on a whole year cohort of palliative patients in terms of demography, complaints, and therapy on admission to PCCS and the impact of PCCS treatment, and identify differences and similarities in different palliative patient subgroups. METHODS: Chi-square, analysis of variance (ANOVA), Kruskal-Wallis followed by Games-Howell analysis of HOspice and Palliative care Evaluation (HOPE 2013) data on 4 PCCS centers and in total 919 patients, with solid tumors (237), metastatic cancer (397), leukemia and lymphoma (99), neurological (109, mostly multiple sclerosis [MS]), and noncancer, nonneurological disease (NCNND, 77). RESULTS: A mostly uniform block of 3 cancer subgroups in terms of demographics, admission complaints, and initial pharmacological treatment diverged from the neurologic/MS disease subgroup. The "intermediate," NCNND subgroup coalesced with the cancer or the neurologic/MS subgroups in part of the demographics, complaint, and drug parameters. Tetraparesis, requirement for nursing, and help with daily living were more, and pain, dyspnea, weakness, appetite loss, and fatigue were less frequent in neurologic patients compared with the cancer subgroups. Neurologic patients also showed more common use of coanalgetics and antidepressives, less opiates and nonopiate analgetics, corticosteroids, and antiemetics and antacids. NCNND patients had a particularly high rate of disorientation (48%) and death during PCCS (39%). In the 3 cancer subgroups, dyspnea, weakness, appetite loss, and anxiolytic use were less frequent in solid tumor patients. Palliative Care Consultation Service treatment was associated with reduction in symptom severity independent of subgroup entity. All listed differences were significant at P < .05 level. CONCLUSION: Despite divergence in demographics, symptoms, and medication, the data underline general usefulness of PCCS care in all end-stage patients and not only the cancer subgroups. Nevertheless, the strong differences revealed in the current study also underscore the need for a carefully tuned, disease-specific therapeutic approach to these subgroups of palliative patients.

Methicillin-resistant Staphylococcus aureus (MRSA) management in palliative care units and hospices in Germany: a nationwide survey on patient isolation policies and quality of life.

BACKGROUND: For palliative care settings, little is known about the benefits of specific methicillin-resistant Staphylococcus aureus containment regimens and the burdens patient isolation imposes on affected patients, their families, and professional caregivers. AIM: To explore the current practice of MRSA management and its impact on inpatients' quality of life as perceived by professional caregivers. DESIGN: Survey of inpatient palliative care institutions using 23-item questionnaires (infrastructural data: six items, management process: 14, clinical significance: three). SETTING/PARTICIPANTS: All palliative care units (179) and hospices (181) listed in Germany's directory of palliative care services. The χ(2) test was used to test for differences; significance level: p ≤ 0.05. RESULTS: 229 of 360 questionnaires were returned. More than 90% of the responding institutions employed specific MRSA protocols. Lack of resources was a more important issue for palliative care units than for hospices regarding availability of single rooms (p = 0.002) and staffing (p = 0.004). Compared to hospices, palliative care units more frequently isolated MRSA patients (p = 0.000), actively treated colonization (p = 0.026), assessed the efficacy of eradication (p = 0.000), provided information on MRSA management to patients (p = 0.014) and relatives (p = 0.001), more often restricted patients' activities (p = 0.000), and reported a negative impact on quality of life (p = 0.000). CONCLUSIONS: Rigorously applied MRSA protocols impose significant burdens at the end of life. Research on clinical outcomes including quality of life may identify interventions of questionable benefit. The issue of handling MRSA should be studied as a model for the management of other highly complex conditions and special needs such as patient isolation.

Identifying patients suitable for palliative care--a descriptive analysis of enquiries using a Case Management Process Model approach.

BACKGROUND: In Germany, case management in a palliative care unit was first implemented in 2005 at the Department of Palliative Medicine at the University Hospital Cologne. One of the purposes of this case management is to deal with enquiries from patients and their relatives as well as medical professionals. Using the Case Management Process Model of the Case Management Society of America as a reference, this study analysed (a) how this case management was used by different enquiring groups and (b) how patients were identified for case management and for palliative care services. The first thousand enquiries were analysed considering patient variables, properties of the enquiring persons and the content of the consultations. RESULTS: Most enquiries to the case management were made by telephone. The majority of requests regarded patients with oncological disease (84.3 %). The largest enquiring group was composed of patients and relatives (40.8 %), followed by internal professionals of the hospital (36.1 %). Most of the enquiring persons asked for a patient's admission to the palliative care ward (46.4 %). The second most frequent request was for consultation and advice (30.9 %), followed by requests for the palliative home care service (13.3 %). Frequent reasons for actual admissions were the need for the treatment of pain, the presence of symptoms and the need for nursing care. More than half of the enquiries concerning admission to the palliative care ward were followed by an admission. CONCLUSIONS: Case management has been made public among the relevant target groups. Case management as described by the Case Management Process Model helps to identify patients likely to benefit from case management and palliative care services. In addition, with the help of case management palliative patients may be allocated to particular health care services.

Workshop on Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test.

The Paul-Ehrlich-Institut (PEI), the Nederlands Vaccin Instituut (NVI) and the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised the international scientific workshop "Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test" at the PEI in Langen (Germany) on 09-10 June 2011. Twenty-seven experts (regulators, representatives from national control laboratories, vaccine manufacturers and academia) from 7 countries participated in this workshop. The meeting was triggered by the lack of satisfaction with the current safety testing for acellular pertussis vaccines, the "Histamine Sensitisation Test" (HIST) in mice, and the growing attention for the alternatives under development. The workshop objectives were: a) to review the current status of available alternative methods, b) to discuss the sensitivity that an alternative test needs, c) to plan experiments that allow for comparison of the alternative tests. The results of the workshop are summarised in this meeting report.

Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.

Monocyte activation test (MAT) reliably detects pyrogens in parenteral formulations of human serum albumin.

Disadvantages of the regulatory pyrogen test to assure safety of the end-product Human Serum Albumin (HSA) for parenteral use call for the implementation of an alternative test. In the current study, 16 HSA batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test, conventional and endotoxin-specific LAL assay and monocyte activation test (MAT). It was found that all HSA batches were contaminated with (1,3)-beta-glucans, which interfere with the conventional LAL. Endotoxin-specific LAL was not suitable to test HSA due to unacceptable endotoxin recovery. Experiments combining polymyxin B and MAT demonstrated that pyrogenic batches were mainly contaminated with endotoxins. However, endotoxin-specific LAL failed to detect one of them. The contaminating (1,3)-beta-glucans enhanced the MAT/IL-6 response to endotoxin, but not that of MAT/IL-1beta. The endotoxin equivalent concentrations obtained using the IL-6 readout were usually higher than those using IL-1beta, probably owing to the direct induction of IL-6 release from monocytes by (1,3)-beta-glucans. The MAT correlates with the rabbit pyrogen test, providing a higher safety level for pyrogenicity testing of HSA and probably other therapeutic proteins.

Laboratory Evaluation of the Effectiveness of Pathogen Reduction Procedures for Bacteria.

SUMMARY: Bacterial contamination remains a leading factor for transfusion-associated serious morbidity and mortality. Pathogen reduction procedures offer a pro-active approach to prevent bacterial contamination of cellular blood components and especially of platelet concentrates. In the past, the laboratory evaluation of the effectiveness of the pathogen reduction procedures to minimise the bacterial load of blood components has been primarily based on log reduction assays similar to the assessment of antiviral activities. Bacteria strains with the ability to multiply in the blood components are seeded in highest possible cell numbers, the pathogen reduction procedure is applied, and the post-treatment number of bacteria is measured. The effectiveness of the procedure is characterised by calculating the log reduction of the post- to pre-treatment bacteria titres. More recently, protocols have been developed for experiments starting with a low bacteria load and monitoring the sterility of the blood component during the entire storage period of the blood component. Results for 3 different pathogen reduction technologies in these experimental models are compared and critical determinants for the results are addressed. The heterogeneity of results observed for different strains suggests that the introduction of international transfusion-relevant bacterial reference strains may facilitate the validity of findings in pathogen reduction experiments.

Palliative care needs of chronically ill nursing home residents in Germany: focusing on living, not dying.

OBJECTIVES: To explore the palliative care needs of nursing home residents in Germany who had not yet entered the dying phase. METHODS: Semi-structured interviews were conducted with a sample of nine residents suffering from chronic disease or frailty. The interviews were audio-recorded, transcribed, and analysed using a grounded theory approach. FINDINGS: The residents described multidimensional needs, which were categorized as 'being recognized as a person', 'having a choice and being in control', 'being connected to family and the world outside', 'being spiritually connected', and 'physical comfort'. They emphasized their desire to control everyday matters. Physical impairment was a problem, especially when independence was threatened, e.g. by immobility or a reliance on pain killers. CONCLUSION: The desire for self-determination is key when designing and evaluating primary and palliative care programmes for nursing homes. Early integration of palliative care can improve the quality of life of chronically ill residents.

Lipopolysaccharide-induced experimental immune activation does not impair memory functions in humans.

Systemic immune activation occurring together with release of peripheral cytokines can affect behavior and the functioning of the central nervous system (CNS). However, it remains unknown whether and to what extent cognitive functions like memory and attention are affected during transient immune activation. We employed a human endotoxemia model and standardized neuropsychological tests to assess the cognitive effects of an experimental inflammation in two groups of 12 healthy young men before and after intravenous injection of lipopolysaccharide (LPS, Escherichia coli, 0.4 ng/kg) or physiological saline. Endotoxin administration caused a profound transient physiological response with elevations in body temperature, number of circulating neutrophils, and increases in plasma cytokine levels [interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α], and concentrations of norepinephrine, ACTH and cortisol. However, these changes in immune and neuroendocrine parameters were not associated with alterations of memory performance, selective attention or executive functions.

[Aspects of outpatient palliative care and assessment of the nursing workload. Survey among care givers in Germany]. / Versorgungssituation von Palliativpatienten und Einschätzung der Arbeitsbelastung der Pflegenden. Befragung unter ambulant tätigen Pflegenden in Deutschland.

INTRODUCTION: The aim of this study is to explore aspects of the health care situation of outpatient palliative patients in Germany as well as effort and workload of care from the viewpoint of involved care givers. Additionally the future development with regard to the cooperation with other health care providers is assessed. METHODS: A detailed questionnaire was developed and sent to 188 outpatient care givers, all members of the German Association for Palliative Medicine, in January 2009. All data was analyzed via SPSS version 16. RESULTS: 69 questionnaires were included into statistical analyses. Care givers estimate the effort of care of palliative patients as very high. 28 per cent of working time is spent on administration. Responders consider general and quantitative workload to be the highest. Most care givers assess the SAPV-directive of the Federal Joint Committee as well as the future health care situation of palliative patients and cooperation with other health care providers as good. DISCUSSION: Further studies should focus on the collection of longitudinal patient data for a more comprehensive insight.

Strategies of bacteria screening in cellular blood components.

Since the impressive reduction of transfusion-transmitted virus infections, bacterial infections by blood transfusion represent the most important infection risk. Platelet concentrates are the current focus of attention, as they are stored under temperature conditions which allow growth of contaminating bacteria up to 10(10) and more microbes per platelet bag. This paper does not consider the pathogen reduction methods but will assess suitable screening methods. Beside conventional microbiological approaches or surrogate markers, several efficient methods able to detect bacterial contamination in platelets are available on the market. They need to be divided into two different methodological principles: the cultivation methods and rapid methods. Cultivation or incubation methods require some time for signal production as they depend on growth of microbes. Thus, they have to be combined with early sampling, i.e., the sample to be examined has to be drawn from the blood component 1 day after donation. Their advantage is the relatively uncomplicated implementation into the logistics of blood banks. Because of the initially very low count of bacteria after donation, a certain small sampling error in application of that strategy remains. Rapid methods are able to produce the diagnosis within a short time. Therefore, they allow postponing of sample drawing, ideally up to the time immediately before transfusion. However, this procedure causes logistic complications. On the other hand, late sampling combined with a rapid method will prevent the transfusion of highly contaminated platelet concentrates leading to acute septic shock up to the death of the patient. Considering the sum of different aspects including the supply of patients, the potential improvement of microbial safety of platelet concentrates is comparable in both strategies.

Methods for the detection of bacterial contamination in blood products.

Culture-based and molecular assays have been developed for the screening of platelet concentrates and other blood components for bacterial contaminations. In this review, the principles of the assays are outlined. The focus of this review is the assessment of the analytical qualities of the methods. Spiking studies by adding defined levels of a wide range of bacteria to the complex biological matrix provide the first basis to evaluate and compare the qualities of methods for bacterial detection. The sensitivity acceptable for reliable screening for bacteria critically depends on the timing of either early sampling (within a period of up to 24 h after preparation of the blood component) or late sampling (a few hours before issuing the blood component). Large screening studies are essential to confirm both adequate sensitivity and specificity of the testing. In the ideal setting, these studies are prospectively planned and include systematic surveillance of adverse events in response to the administration of the screened products. The findings from sterility testing (predominantly with automated systems for detection of bacteria based on CO(2) generation) of more than 550,000 platelet concentrates in 13 studies are summarised. The limitations of the early sampling and the "negative-to-date" strategy to issue platelet concentrates are addressed. A few reported cases of probable transmission of bacteria by platelet transfusion despite negative screening tests emphasise the need to further develop optimised methods for testing of bacteria blood components.

Bacteria detection by flow cytometry.

Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.

Microbial safety of cell based medicinal products--what can we learn from cellular blood components?

Today, sterility of established parenteral drugs including biologicals, such as plasma derived products, is practically guaranteed. Bacterially contaminated products are extremely rare exceptions owing to the efficiency of the manufacturing processes in the pharmaceutical industry. In contrast, the manufacturing processes of cell based medicinal products or tissue preparations show much less defined conditions. The sterility of source materials cannot be guaranteed in many cases. As a rule, these source materials cannot be sterilised, as it holds true for the final products. Furthermore, the established methods for sterility testing are not applicable for cell preparations. Sterility of a restricted sample does not guarantee sterility of the whole preparation. Thus, small amounts of residual bacteria in the product can be overlooked and can grow up to enormous numbers during storage and shipping of cell based medicinal products. Considering these problems, there are some parallels in the warranty of microbial safety of cellular blood components. Therefore, the experiences collected in transfusion medicine in the past decade can be successfully used in the production of cell based medicinal products. Comparable to the situation regarding cellular blood components, there is a need for new principles in rapid bacteria detection.

Safety testing of cell-based medicinal products: opportunities for the monocyte activation test for pyrogens.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

Fever in the test tube--towards a human(e) pyrogen test.

The human whole blood IL-1 test exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after incubation. In order to understand any differences between the pyrogenic activity in this test and the existing live rabbit test (species differences versus aberrant response of the particular blood sample), the rabbit whole blood test was developed. This approach could also help to avoid the use of putatively infectious human blood for pyrogen testing in vitro.

International validation of pyrogen tests based on cryopreserved human primary blood cells.

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.