Re-Appraisal of Estrogen Receptor Negative/Progesterone Receptor Positive (ER-/PR+) Breast Cancer Phenotype: True Subtype or Technical Artefact?

Expression of the ER and PR receptors is routinely quantified in breast cancer as a predictive marker of response to hormonal therapy. Accurate determination of ER and PR status is critical to the optimal selection of patients for targeted therapy. The existence of an ER-/PR+ subtype is controversial, with debate centred on whether this represents a true phenotype or a technical artefact on immunohistochemistry (IHC). The aim of this study was to investigate the true incidence and clinico-pathological features of ER-/PR+ breast cancers in a tertiary referral symptomatic breast unit. Clinico-pathological data were collected on invasive breast cancers diagnosed between 1995 and 2005. IHC for ER and PR receptors was repeated on all cases which were ER-/PR+, with the same paraffin block used for the initial diagnostic testing. Concordance between the diagnostic and repeat IHC was determined using validated testing. Complete data, including ER and PR status were available for 697 patients diagnosed during the study period. On diagnostic IHC, the immunophenotype of the breast tumours was: ER+/PR+ in 396 (57%), ER-/PR- in 157 (23%), ER+/PR- in 88 (12%) and ER-/PR+ in 56 (8.6%) patients. On repeat IHC of 48/56 ER-/PR+ tumours 45.8% were ER+/PR+, 6% were ER+/PR- and 43.7% were ER-/PR- None of the cases were confirmed to be ER-/PR+. The ER-/PR+ phenotypic breast cancer is likely to be the result of technical artefact. Prompt reassessment of patients originally assigned to this subtype who re-present with symptoms should be considered to ensure appropriate clinical management.

Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy.

Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Management of landfill leachate: The legacy of European Union Directives.

Landfill leachate is the product of water that has percolated through waste deposits and contains various pollutants, which necessitate effective treatment before it can be released into the environment. In the last 30years, there have been significant changes in landfill management practices in response to European Union (EU) Directives, which have led to changes in leachate composition, volumes produced and treatability. In this study, historic landfill data, combined with leachate characterisation data, were used to determine the impacts of EU Directives on landfill leachate management, composition and treatability. Inhibitory compounds including ammonium (NH4-N), cyanide, chromium, nickel and zinc, were present in young leachate at levels that may inhibit ammonium oxidising bacteria, while arsenic, copper and silver were present in young and intermediate age leachate at concentrations above inhibitory thresholds. In addition, the results of this study show that while young landfills produce less than 50% of total leachate by volume in the Republic of Ireland, they account for 70% of total annual leachate chemical oxygen demand (COD) load and approximately 80% of total 5-day biochemical oxygen demand (BOD5) and NH4-N loads. These results show that there has been a decrease in the volume of leachate produced per tonne of waste landfilled since enactment of the Landfill Directive, with a trend towards increased leachate strength (particularly COD and BOD5) during the initial five years of landfill operation. These changes may be attributed to changes in landfill management practices following the implementation of the Landfill Directive. However, this study did not demonstrate the impact of decreasing inputs of biodegradable municipal waste on leachate composition. Increasingly stringent wastewater treatment plant (WWTP) emission limit values represent a significant threat to the sustainability of co-treatment of leachate with municipal wastewater. In addition, the seasonal variation in leachate production poses a risk to effective co-treatment in municipal WWTPs, as periods of high leachate production coincide with periods of maximum hydraulic loading in WWTPs.

Frontal and frontoparietal injury differentially affect the ipsilateral corticospinal projection from the nonlesioned hemisphere in monkey (Macaca mulatta).

Upper extremity hemiplegia is a common consequence of unilateral cortical stroke. Understanding the role of the unaffected cerebral hemisphere in the motor recovery process has been encouraged, in part, by the presence of ipsilateral corticospinal projections (iCSP). We examined the neuroplastic response of the iCSP from the contralesional primary motor cortex (cM1) hand/arm area to spinal levels C5-T1 after spontaneous long-term recovery from isolated frontal lobe injury and isolated frontoparietal injury. High-resolution tract tracing, stereological, and behavioral methodologies were applied. Recovery from frontal motor injury resulted in enhanced numbers of terminal labeled boutons in the iCSP from cM1 compared with controls. Increases occurred in lamina VIII and the adjacent ventral sectors of lamina VII, which are involved in axial/proximal limb sensorimotor processing. Larger frontal lobe lesions were associated with greater numbers of terminal boutons than smaller frontal lobe lesions. In contrast, frontoparietal injury blocked this response; total bouton number was similar to controls, demonstrating that disruption of somatosensory input to one hemisphere has a suppressive effect on the iCSP from the nonlesioned hemisphere. However, compared with controls, elevated bouton numbers occurred in lamina VIII, at the expense of lamina VII bouton labeling. Lamina IX boutons were also elevated in two frontoparietal lesion cases with extensive cortical injury. Because laminae VIII and IX collectively harbor axial, proximal, and distal motoneurons, therapeutic intervention targeting the ipsilateral corticospinal linkage from cM1 may promote proximal, and possibly distal, upper-limb motor recovery following frontal and frontoparietal injury.

CT angiography-guided single-stage release of adjacent webspaces in non-Apert syndactyly.

UNLABELLED: We describe the CT angiography protocol and surgical technique utilized at our institution for single-stage release of adjacent web-spaces in non-Apert syndactyly. In a series of seven consecutive hands we analyse syndactyly anatomy, CT angiographic findings, operative details, and complications. Outcomes were assessed with a functional activity evaluation, range of motion, and a parental visual analogue scale. Seven affected hands in four patients underwent single-stage release of adjacent webspaces. In all cases, the CT angiogram correctly predicted the presence of at least one artery supplying each digit. There were no cases of digital ischemia or loss. Angiographically guided, single-stage release of adjacent webspaces is technically feasible and benefits patients by reducing the number of surgical stages and allowing complete release to be achieved at an earlier age compared with the standard multi-stage approach. LEVEL OF EVIDENCE: IV.

Vulnerability of the medial frontal corticospinal projection accompanies combined lateral frontal and parietal cortex injury in rhesus monkey.

Concurrent damage to the lateral frontal and parietal cortex is common following middle cerebral artery infarction, leading to upper extremity paresis, paresthesia, and sensory loss. Motor recovery is often poor, and the mechanisms that support or impede this process are unclear. Since the medial wall of the cerebral hemisphere is commonly spared following stroke, we investigated the spontaneous long-term (6 and 12 month) effects of lateral frontoparietal injury (F2P2 lesion) on the terminal distribution of the corticospinal projection (CSP) from intact, ipsilesional supplementary motor cortex (M2) at spinal levels C5 to T1. Isolated injury to the frontoparietal arm/hand region resulted in a significant loss of contralateral corticospinal boutons from M2 compared with controls. Specifically, reductions occurred in the medial and lateral parts of lamina VII and the dorsal quadrants of lamina IX. There were no statistical differences in the ipsilateral CSP. Contrary to isolated lateral frontal motor injury (F2 lesion), which results in substantial increases in contralateral M2 labeling in laminae VII and IX (McNeal et al. [2010] J. Comp. Neurol. 518:586-621), the added effect of adjacent parietal cortex injury to the frontal motor lesion (F2P2 lesion) not only impedes a favorable compensatory neuroplastic response but results in a substantial loss of M2 CSP terminals. This dramatic reversal of the CSP response suggests a critical trophic role for cortical somatosensory influence on spared ipsilesional frontal corticospinal projections, and that restoration of a favorable compensatory response will require therapeutic intervention.

Using video ratings to assess multitasking performance in a naturalistic paradigm.

BACKGROUND: Multitasking measures, in which a series of tasks must be completed within a naturalistic setting not fully under the experimenter's control, have been shown to be more sensitive than traditional measures in detecting organisational problems in people with difficulties in executive functioning. There are a number of drawbacks to such tasks however. They can take considerable time to administer and are demanding in terms of examiners noting and recording all relevant aspects of performance. This potentially leaves them more open to subtle bias. One method that could offset these limitations is to video record performance. OBJECTIVES: The practicality and outcome of using video ratings to accurately score performance off-line is investigated here. METHODS: Nineteen participants completed a Multiple Errands Task (MET) while wearing a body-worn camera. Their performance was scored "live" and by an independent rater who had only access to video footage of the task. RESULTS: Significant relationships were seen on all variables of the MET between the live and video ratings. The inter-rater reliability of the measure appears strong. CONCLUSION: We provide initial support for the use of a video rater when assessing performance on an MET.

Intensive working memory training: a single case experimental design in a patient following hypoxic brain damage.

BACKGROUND: Recent reports suggest that intensive, progressive training on working memory tasks can lead to generalized cognitive gains. CASE STUDY: A patient, following hypoxic brain damage, showed significant difficulties in working memory and time-perception. This study examined the impact and specificity of any benefits resulting from automated working memory training (AWMT) in comparison with the effects of an equivalent programme that emphasized automated novel problem-solving (APST) which served as an active control. Following initial assessment, the patient trained for 4 weeks (20 days), 20-30 minutes a day on the APST tasks before repeating key outcome measures. He then trained for an identical period on AWMT. RESULTS: There were no cognitive gains apparent following APST. Furthermore, there were no disproportionate gains on digit span following AWMT. AWMT was, however, associated with improvement in time-perception that had previously been resistant to rehabilitation. In line with previous reports, AWMT was also followed by gains on a measure of planning. CONCLUSION: The results provide encouraging evidence that AWMT may have generalized benefits in the context of impaired WM capacity following brain injury.

The potential of algae blooms to produce renewable gaseous fuel.

Ulva lactuca (commonly known as sea letuce) is a green sea weed which dominates Green Tides or algae blooms. Green Tides are caused by excess nitrogen from agriculture and sewage outfalls resulting in eutrophication in shallow estuaries. Samples of U. lactuca were taken from the Argideen estuary in West Cork on two consecutive years. In year 1 a combination of three different processes/pretreatments were carried out on the Ulva. These include washing, wilting and drying. Biomethane potential (BMP) assays were carried out on the samples. Fresh Ulva has a biomethane yield of 183LCH4/kgVS. For dried, washed and macerated Ulva a BMP of 250LCH4/kgVS was achieved. The resource from the estuary in West Cork was shown to be sufficient to provide fuel to 264 cars on a year round basis. Mono-digestion of Ulva may be problematic; the C:N ratio is low and the sulphur content is high. In year 2 co-digestion trials with dairy slurry were carried out. These indicate a potential increase in biomethane output by 17% as compared to mono-digestion of Ulva and slurry.

Solitary fibrous tumours of the pleura: report of two cases and literature review.

BACKGROUND: Solitary fibrous tumours of the pleura (SFTPs) are rare pleural mesenchymal neoplasms with distinct clinicopathological and immunohistochemical features, accounting for less than 5 % of all neoplasms involving the pleura. METHODS: We present two cases of SFTP with a review of the current literature. RESULTS: Clinical presentation varies according to size and intrathoracic localisation. The molecular pathology of SFTPs is largely unknown. Complete surgical resection is recommended with long-term clinic and radiographic follow-up due to its malignant potential. CONCLUSIONS: This is a summary of available literature outlining current clinical practice in the diagnosis, management, and treatment of SFTPs in Ireland.

Recurrent dedifferentiated paratesticular liposarcoma with synchronous renal cell carcinoma and prostate cancer.

Paratesticular liposarcoma is a very rare cause of scrotal mass. It is thought that they arise from spermatic cord lipomas most commonly. While well differentiated tumors tend to share many histological similarities with dedifferentiated tumors, the latter has a much more aggressive phenotype. We present an unusual case of a 69-year-old male with synchronous prostate adenocarcinoma and unilateral renal cell carcinoma who was found to have a dedifferentiated paratesticular liposarcoma. Treatment was with radical resection, preserving the testis, followed by radiotherapy. Unusually recurrence did not occur until 4 years following initial treatment. This case demonstrates the high propensity of dedifferentiated liposarcoma to recur locally and examines the most frequently employed management strategies.

A role for MRP8 in in stent restenosis in diabetes.

OBJECTIVE: The most common cause of death in diabetes mellitus is cardiovascular disease. Patients frequently undergo vascular intervention such as stenting. The occurrence of in stent restenosis (ISR) has been reduced by the use of drug eluting stents in non-diabetic patients but the incidence of restenosis and stent thrombosis remains higher in diabetic patients. We investigated the pathogenesis of in stent restenosis in an animal model of type 2 diabetes mellitus. METHODS AND RESULTS: Stents were placed in Zucker Fatty rat (ZFR) and wild type rat carotid arteries, and tissues were harvested 14 days post surgery for morphometric analysis. Unstented carotid arteries from both groups were harvested for microarray analysis. In vitro apoptosis, proliferation and migration assays were performed on rat and human aortic endothelial cells (EC). ZFRs developed an exaggerated intimal response to stent placement compared to wild type controls 14 days post stent placement. MRP8 and MRP14 were up-regulated in unstented ZFR carotid arteries in comparison to controls. Expression of MRP8/14 was also elevated in EC exposed to high glucose conditions. EC function was impaired by high glucose concentrations, and this effect could be mimicked by MRP8 over-expression. MRP8 knockdown by shRNA significantly restored EC function after exposure to high glucose concentrations. MRP8 expression in glucose exposed cells was also inhibited using pharmacological blockade of glucose-induced pathways. CONCLUSIONS: EC dysfunction caused by elevated glucose levels could be mimicked by MRP8/14 over-expression and reversed/prevented by MRP8 knockdown. Thus, MRP8/14 likely plays a role in exaggerated ISR in diabetes mellitus, and MRP8 inhibition may be useful in improving outcome after stent placement in diabetes mellitus.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model.

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

Demographics, nature and treatment of orthopaedic trauma injuries occurring in an agricultural context in the West of Ireland.

BACKGROUND: Farming is a major industry in the West of Ireland. This prospective study examined the age profile, nature and treatment of orthopaedic injuries occurring in agricultural surroundings presenting at the Orthopaedic Unit of Merlin Park Hospital, Galway. METHODS: The cohort consisted of 70 direct or indirect farm employees presenting with orthopaedic injuries caused in an accident occurring on a farm. RESULTS: There were significantly (P < 0.01) more male than female farmers (73 vs. 27%) with a mean age of 63 years (ranging from 13 to 87 years). Average number of out-patient visits related to the injury was 3.6. Injuries involving livestock resulted in the majority of accidents (P < 0.05) with a significant rise in the number of visits with increasing age (P < 0.01) with older patients having more severe injuries. Soft tissue injuries and fractures were commonly encountered. Complex fractures were the slowest injury to heal requiring eight subsequent out-patient visits.

Asymptomatic primary fallopian tube cancer: an unusual cause of axillary lymphadenopathy.

Primary Fallopian tube malignancy is considered a rare disease and is often mistaken histologically and clinically for ovarian cancer. The etiology is poorly understood, and it typically presents at an advanced disease stage, as symptoms are often absent in the initial period. As a result, primary fallopian tube cancer is generally associated with a poor prognosis. We present the case of a 45-year-old female who presents with a 5-day history of left axillary swelling and a normal breast examination. Mammogram and biopsy of a lesion in the left breast revealed a fibroadenoma but no other abnormalities. Initial sampling of the axillary node was suspicious for a primary breast malignancy, but histology of the excised node refuted this. PET-CT showed an area of high uptake in the right pelvis, and a laparoscopy identified a tumor of the left fallopian tube which was subsequently excised and confirmed as a serous adenocarcinoma.

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.

BACKGROUND: Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. METHODS: Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. RESULTS: In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. CONCLUSIONS: AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.

Cardiovascular gene therapy: current status and therapeutic potential.

Gene therapy is emerging as a potential treatment option in patients suffering from a wide spectrum of cardiovascular diseases including coronary artery disease, peripheral vascular disease, vein graft failure and in-stent restenosis. Thus far preclinical studies have shown promise for a wide variety of genes, in particular the delivery of genes encoding growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) to treat ischaemic vascular disease both peripherally and in coronary artery disease. VEGF as well as other genes such as TIMPs have been used to target the development of neointimal hyperplasia to successfully prevent vein graft failure and in-stent restenosis in animal models. Subsequent phase I trials to examine safety of these therapies have been successful with low levels of serious adverse effects, and albeit in the absence of a placebo group some suggestion of efficacy. Phase 2 studies, which have incorporated a placebo group, have not confirmed this early promise of efficacy. In the next generation of clinical gene therapy trials for cardiovascular disease, many parameters will need to be adjusted in the search for an effective therapy, including the identification of a suitable vector, appropriate gene or genes and an effective vector delivery system for a specific disease target. Here we review the current status of cardiovascular gene therapy and the potential for this approach to become a viable treatment option.

Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury.

Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 x 10(9) PFU of adenoviral(Ad)eNOS, AdiNOS or Adbeta-galactosidase (Adbeta-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR. The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.07+/-0.044) and iNOS (0.087+/-0.086) transduced arteries compared with Adbeta-Gal (0.332+/-0.14) transduced arteries (n=7). In addition, AdeNOS treatment (4.21+/-3.12% de-endothelialized area) enhanced endothelial regeneration compared to Adbeta-Gal treatment (10.05+/-4.98), while treatment with AdiNOS (25.17+/-11.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n=7-8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains.

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (approximately 14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.