Haemorrhagic cystitis: a review of management strategies and emerging treatments.

Haemorrhagic cystitis (HC) is characterised by persistent haematuria and lower urinary tract symptoms following radiotherapy or chemotherapy. Its pathogenesis is poorly understood but thought to be related to acrolein toxicity following chemotherapy or fibrosis/vascular remodelling after radiotherapy. There is no standard of care for patients with HC, although existing strategies including fulguration, hyperbaric oxygen therapy, botulinum toxin A, and other intravesical therapies have demonstrated short-term efficacy in cohort studies. Novel agents including liposomal tacrolimus are promising targets for further research. This review summarises the incidence and pathogenesis of HC as well as current evidence supporting its different management strategies.

Low Energy Shock Wave Therapy Inhibits Inflammatory Molecules and Suppresses Prostatic Pain and Hypersensitivity in a Capsaicin Induced Prostatitis Model in Rats.

The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the eyes, hypolocomotion, and tactile allodynia, which effects were ameliorated by LESW treatment. LESW therapy (2Hz, energy flux density of 0.12 mJ/mm2) at 200 and 300 shocks significantly decreased capsaicin-induced inflammatory reactions, reflected by a reduction of tissue edema and inflammatory cells, COX-2 and TNF-α stained positive cells, however, the therapeutic effects were not observed at 100 shocks treated group. Capsaicin-induced IL-1ß, COX-2, IL-6, caspase-1, and NGF upregulation on day 3 and 7, while NALP1 and TNF-α upregulation was observed on day 7. LESW significantly suppressed the expression of IL-1ß, COX-2, caspase-1, NGF on day 3 and IL-1ß, TNF-α, COX-2, NALP1, caspase-1, NGF expression on day 7 in a dose-dependent fashion. LESW has no significant effect on IL-6 expression. Intraprostatic capsaicin injection activates inflammatory molecules and induces prostatic pain and hypersensitivity, which effects were suppressed by LESW. These findings might be the potential mechanisms of LESW therapy for nonbacterial prostatitis in humans.

Cancer survivorship issues with radiation and hemorrhagic cystitis in gynecological malignancies.

PURPOSE: Given that more cancers are being diagnosed earlier and that treatment of cancer is improving, health issues of cancer survivors are becoming more common and apparent. Pelvic radiation therapy for the treatment of gynecological cancers can lead to long-term collateral damage to the bladder, a condition termed radiation cystitis (RC). Late sequelae may take many years to develop and include incontinence and pain as well as hematuria. RC is a rare but potentially life-threatening condition for which there are few management and treatment options. METHODS: There are limited data in the literature regarding the effects of radiation on the bladder after gynecological cancer therapy and we hereby review the literature on cancer survivorship issues of pelvic radiation for gynecology literature. RESULTS: Treatment options are available for patients with radiation-induced hemorrhagic cystitis. However, most treatments are risky or only effective for a short timeframe and no therapy is currently available to reverse the disease progress. Furthermore, no standardized guidelines exist describing preferred management options. Common therapies include hyperbaric oxygen therapy, clot evacuation, fulguration, intravesical instillation of astringent agents, and surgery. Novel developing strategies include Botulinum Toxin injections and liposomal-tacrolimus instillations. These treatments and strategies are discussed. CONCLUSIONS: In this review, we will present current and advanced therapeutic strategies for RC to help cancer survivors deal with long-term bladder health issues.

New technology assessment and current and upcoming therapies for underactive bladder.

BACKGROUND AND AIMS: Stakeholders from around the world came together to address the unmet needs of underactive bladder (UAB) at the 3rd International Congress for Underactive Bladder. METHODS: The main recommendation from the regulatory working group is a need for a meeting of UAB stakeholders and regulatory agencies including the FDA to discuss guidance for regulatory trial design for devices, drugs, and/or biologics for UAB. RESULTS: The following issues to be discussed and agreed upon for UAB trials: 1) Appropriate inclusion and exclusion criteria. 2) Should residual urine volume be the primary outcome parameter and how often should it be measured? 3) Are there secondary measures that should have a place in UAB trials, such as change in the number of catheterizations, quality of life measures, etc.? 4) Use and format of bladder voiding and catheterization diary for trials. 5) Define role and technique of urodynamics in UAB trials. Are urodynamics required to monitor, and possibly exclude, individuals with high pressure voiding induced by bladder prokinetic therapies? 6) Development and use of UAB questionnaires. DISCUSSION AND CONCLUSION: The UAB regulatory working group recognizes the path forward should include engaging the FDA and other regulatory organizations that may harmonize and formalize guidance for regulatory trial designs for therapeutics for UAB.

Treatment of ulcerative compared to non-ulcerative interstitial cystitis with hyperbaric oxygen: a pilot study.

BACKGROUND: The etiology of interstitial cystitis (IC) is often idiopathic but can be due to Hunner's ulcers. Hyperbaric oxygen (HBO) is used to treat ulcerative disease of the superficial skin. We hypothesized that HBO can treat ulcerative IC (UIC) but would be less efficacious for non-ulcerative IC (NIC). METHODS: Patients with NIC and UIC enrolled in this study. Following informed consent, demographic information was collected. A visual analog pain scale and validated questionnaires were collected; each patient underwent cystoscopy prior to treatment. Each subject met with a hyperbaric specialist and after clearance underwent 30 treatments over 6 weeks. Adverse events were monitored. Patients repeated questionnaires, visual analog pain scale and global response assessment (GRA) immediately, 2 weeks, 3, 6 and 12 months after treatment. Patients also underwent cystoscopy 6 months after treatment. Differences before and after treatment were compared. RESULTS: Nine patients were recruited to this study. One was unable to participate, leaving two subjects with NIC and six with UIC. All patients completed HBO without adverse events. Three patients completed HBO but pursued other therapies 7, 8.5 and 11 months after treatment. On GRA, 83% of patients with UIC were improved. This treatment effect persisted, as 66% of UIC patients remained better at 6 months. In contrast, only one patient in the NIC group improved. Questionnaire scores improved in both groups. Pain scores improved by 2 points in the UIC group but worsened by 1.5 points in the NIC group. Two patients with ulcers resolved at 6-month cystoscopy. CONCLUSION: HBO appeared beneficial for both UIC and NIC. Data shows slightly better benefit in patients with UIC compared to NIC; both groups showed improvement. Given the small sample size, it is difficult to draw definitive conclusions from these data. Larger studies with randomization would be beneficial to show treatment effect.

Liposome Based Intravesical Therapy Targeting Nerve Growth Factor Ameliorates Bladder Hypersensitivity in Rats with Experimental Colitis.

PURPOSE: Pelvic organ cross sensitization is considered to contribute to overlapping symptoms in chronic pelvic pain syndrome. Nerve growth factor over expression in the bladder is reportedly involved in the symptom development of bladder pain syndrome/interstitial cystitis. We examined whether a reduction of over expressed nerve growth factor in the bladder by intravesical treatment with liposome and oligonucleotide conjugates would ameliorate bladder hypersensitivity in a rat colitis model. MATERIALS AND METHODS: Adult female rats were divided into 1) a control group, 2) a colitis-oligonucleotide group with intracolonic TNBS (2,4,6-trinitrobenzene sulfonic acid) enema and intravesical liposome-oligonucleotide treatments, 2) a colitis-saline group with intracolonic TNBS and intravesical saline treatments, 4) a sham oligonucleotide group with intravesical liposome-oligonucleotide treatment without colitis and 5) a sham-saline group with intravesical saline treatment without colitis. Liposomes conjugated with nerve growth factor antisense oligonucleotide or saline solution were instilled in the bladder and 24 hours later colitis was induced by TNBS enema. Effects of nerve growth factor antisense treatment were evaluated by pain behavior, cystometry, molecular analyses and immunohistochemistry 10 days after TNBS treatment. RESULTS: In colitis-oligonucleotide rats nerve growth factor antisense treatment ameliorated pain behavior and decreased a reduction in the intercontraction interval in response to acetic acid stimulation as well as nerve growth factor expression in the bladder mucosa. All were enhanced in colitis-saline rats compared to sham rats. CONCLUSIONS: Nerve growth factor over expression in the bladder mucosa and bladder hypersensitivity induced after colitis were decreased by intravesical application of liposome-oligonucleotide targeting nerve growth factor. This suggests that local antinerve growth factor therapy could be effective treatment of bladder symptoms in chronic pelvic pain syndrome.

Effect of Sacral Neuromodulation on Outcome Measures and Urine Chemokines in Interstitial Cystitis/Painful Bladder Syndrome Patients.

OBJECTIVES: Sacral neuromodulation (SNM) may improve interstitial cystitis/painful bladder syndrome (IC/BPS) symptoms of urinary frequency, urgency and perhaps even pain, but objective measures of improvement are lacking. We evaluated the potential for urinary chemokines to serve as measures of treatment response over time to SNM. METHODS: Women with IC/BPS undergoing SNM consented for this study. Three-day bladder/pain diaries were collected at baseline and validated Interstitial Cystitis Symptom Problem Index (ICSPI) scores and mid-stream urine specimens were collected at baseline and at 24 weeks after successful implant. Collected urine was screened for infection by dipstick and analyzed for chemokines by luminex xMAP analysis. RESULTS: At baseline (n = 16), urine levels of CXCL-1 positively correlated with pain score (r = 0.63, P = 0.009), urgency (r = 0.61, P = 0.01), ICSPI (r = 0.43, P = 0.09) and daily voids (r = 0.44, P = 0.08). ICSPI and pain scores also positively correlated with sIL-1ra (r = 0.50, P = 0.04) and monocyte chemotactic protein-1 (MCP-1) or CCL2 positively correlated with daily voids (r = 0.45, P = 0.07) only. At 24 weeks, the median ICSPI index fell from 28 to 15 (n = 7, P = 0.008). Urine levels of sIL-1ra (633.8 ± 188.2 vs. 149.9 ± 41.62 pg/mL) and MCP-1 (448.3 ± 11.6 vs. 176.9 ± 46.16 pg/mL) and CCL5 (20.78 ± 4.09 vs. 11.21 ± 4.12 pg/mL) were also significantly reduced at the follow-up relative to baseline values (P = 0.04). Multivariable analysis of data revealed that sIL-1ra and MCP-1 together explained the majority of variance in data. Levels of CXCL-1, CXCL-10, interleukin (IL)-8, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) were also reduced at 24 weeks, but differences were not significant. CONCLUSIONS: Concomitant decrease in urine levels of chemokines especially MCP-1 was associated with treatment response of SNM. These results support the role of chemokines as downstream effectors of neuromodulation response and could serve as potential non-invasive measures of treatment response. CLINICAL TRIAL REGISTRATION NUMBER: NCT01739946.

Advanced therapeutic directions to treat the underactive bladder.

Muscarinic agonists are the most commonly used agents for treating the underactive bladder (UAB). However, because of the absence of pharmacologic specificity for bladder-only effects and possibly as a result of degenerative and other post-synaptic changes involving detrusor smooth muscle cells, they are simply not effective and side effects are common. If safe and effective therapy for UAB is made available, then most experts agree that the potential market would exceed industry expectations, just as antimuscarinic agents for overactive bladder did in the late 1990 s. The pharmaceutical and biotechnology industries that have a pipeline to urology and women's health should consider UAB as a potential target condition. A rational approach to treating the pathology of UAB is presented with a discussion of potential targets that may allow the development of safe and effective agents for the treatment of UAB.

Nerve growth factor level in the prostatic fluid of patients with chronic prostatitis/chronic pelvic pain syndrome is correlated with symptom severity and response to treatment.

OBJECTIVE: • To explore whether levels of nerve growth factor (NGF) in expressed prostatic secretions (EPS) are correlated with symptom severity in chronic prostatitis (CP) and chronic pelvic pain syndrome (CPPS). PATIENTS AND METHODS: • All patients with CP/CPPS underwent a complete history and physical examination, and were scored according to the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). • Expressed prostatic secretion samples from 20 patients with CP/CPPS and from four asymptomatic control patients were collected and frozen, and NGF levels in EPS were measured by enzyme-linked immunosorbent assay. • Patients were asked to complete NIH-CPSI questionnaires at baseline and 8 weeks after treatment and patients with at least a 25% decrease in total NIH-CPSI score from the baseline values were classified as responders to treatment. RESULTS: • The mean (± sd) NGF levels in EPS of patients with CP/CPPS and asymptomatic control patients were 7409 (± 3788) pg/mL and 4174 (± 1349) pg/mL, respectively. The NGF level in patients with CP/CPPS correlated directly with pain severity (P= 0.014, r= 0.541). • There were no significant differences between NGF levels in EPS before and after treatment. However, successful treatment significantly decreased NGF levels in responders (P= 0.001). CONCLUSION: • Nerve growth factor might contribute to the pathophysiology of CP/CPPS as changes in NGF level in EPS occurred in proportion to pain severity. Therefore, these results suggest that NGF could be used as a new biomarker to evaluate the symptoms of CP/CPPS and the effects of treatment.

Cost analysis of interventions for antimuscarinic refractory patients with overactive bladder.

OBJECTIVES: To estimate average, initial, and cumulative procedure related costs from a US payer perspective extending up to 3 years for the overactive bladder (OAB) interventions: sacral neuromodulation (SNM), intra-detrusor botulinum toxin A (BoNTA), and augmentation cystoplasty (AC) for antimuscarinic refractory patients. METHODS: Costs (2007 US dollars) were calculated using Current Procedural Terminology (CPT) codes, Ambulatory Payment Classification (APC) codes; Diagnosis Related Group (DRG) payments, and Healthcare Common Procedure Coding System (HCPCS) Level II Codes extracted from the literature and from the SNM device manufacturer. CPT codes were converted to costs using the Center for Medicare and Medicaid Services (CMS) Relative Value Unit (RVU) fee schedule. Sensitivity analyses were performed to evaluate assumptions and uncertainty of results based on plausible variation in estimates of key cost drivers. RESULTS: The initial treatment cost was $22,226, $1,313, and $10,252 for SNM, intra-detrusor injection of BoNTA, and AC respectively. The first-year cost was $23,614, $2626, and $11,637 respectively. Three years after initiating treatment, the cumulative cost was $26,269, $7651, and $14,337 respectively. Sensitivity analyses revealed that SNM persisted as the most costly intervention in all scenarios. The 3-year cumulative cost range produced by the sensitivity analyses for SNM, BoNTA, and AC was $25,384-$27,357, $4586-$11,476, and $12,315-$16,830, respectively. CONCLUSIONS: All estimates of cost endpoints for SNM were greater than those for BoNTA and AC. These cost estimates, when combined with data on outcomes and risks, are important components of a robust health care technology assessment of antimuscarinic treatment failure options.

Induction of apoptosis in human bladder cancer cells by green tea catechins.

Cell culture and animal studies have demonstrated strong chemopreventative effects of green tea and its associated polyphenols in multiple cancers, though the exact mechanisms of action are not well understood. This in vitro study examined the antiproliferative/pro-apoptotic potential of green tea extract (GTE), polyphenon-60 (PP-60), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in both normal and malignant human bladder cells. Cell growth (proliferation/apoptosis) was measured in UROtsa (normal), SW780 (tumorigenic; low-grade), and TCCSUP (tumorigenic; high-grade) human bladder urothelial cells by cell proliferation (XTT) assay after treatment with 0-80 microg/mL of GTE, PP-60, ECG and EGCG for 72 h. Molecular signaling pathways of catechin-induced apoptosis were analyzed using Human signal transduction RT(2) Profiler PCR array (SuperArray). Compared to control-treated cells, treatment with catechin agents significantly suppressed cell growth in a dose-dependent fashion (P < 0.01), with strongest effects evoked by ECG and EGCG in UROtsa cells, ECG in low-grade RT4 and SW780 cells, and PP-60 and EGCG in high-grade TCCSUP and T24 cells. Microarray analysis indicated distinct differences in mRNA gene expression regarding growth signaling pathway activation induced by EGCG in normal/tumorigenic human bladder cell lines, providing a rationale for the putative therapeutic usage of green tea polyphenols against bladder disease.

Instillation of liposomes vs dimethyl sulphoxide or pentosan polysulphate for reducing bladder hyperactivity.

OBJECTIVE: To investigate the efficacy of intravesical liposomes against dimethyl sulphoxide (DMSO), and pentosan polysulphate (PPS) in reducing chemically induced bladder hyperactivity in rats. MATERIALS AND METHODS: Bladder reflex activity of female Sprague-Dawley rats was evaluated by continuous cystometry under urethane anaesthesia (1.0 g/kg). After obtaining a control cystometrogram (CMG) with normal saline (0.04 mL/min) for 2 h, bladder hyperactivity was then induced by 1 h infusion of protamine sulphate (10 mg/mL) followed by a 1-h infusion of KCl (500 mm). Six rats each were then infused with KCl-based preparations containing either 50% DMSO, PPS (6 mg/mL), or liposomes (2 mg/mL) for 2 h. The variables measured included the intercontraction interval (ICI), pressure threshold (PT) and baseline pressure (BP). RESULTS: Sequential infusion of protamine sulphate/KCl induced hyperactive bladder with no significant difference in ICI, PT or BP among groups before initiating treatment. ICI was significantly increased after infusion of PPS (58.1% increase) and liposomes (156.8% increase) but there was no increase with DMSO. PT was not significantly affected by liposome infusion but slightly increased with PPS (12.4% increase). There was a large and significant increase in PT and BP with DMSO (116.5% increase) and BP largely remained unchanged after instillation with liposomes or PPS. CONCLUSIONS: Intravesical liposomes and PPS have a beneficial effect in a bladder hyperactivity rat model, while acute instillation of DMSO does not. Intravesical liposomes were effective in doubling the ICI compared with PPS, and might be a new treatment option for bladder hyperactivity.

Antioxidant effects of green tea and its polyphenols on bladder cells.

Genitourinary tract inflammation/ailments affect the quality of life and health of a large segment of society. In recent years, studies have demonstrated strong antioxidant effects of green tea and its associated polyphenols in inflammatory states. This in vitro study examined the antioxidant capabilities (and putative mechanisms of action) of green tea extract (GTE), polyphenon-60 (PP-60, 60% pure polyphenols), (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in normal/malignant human bladder cells following catechin treatment+/-1 mM H2O2 (oxidative agent). Cell viability, apoptosis and reactive oxygen species (ROS) formation were evaluated. Our results showed that H2O2 exposure significantly reduced normal (UROtsa) and high-grade (TCCSUP, T24) bladder cancer (BlCa) cell viability compared with control-treated cells (p<0.001). No affect on low-grade RT4 and SW780 BlCa cell viability was observed with exposure to H2O2. Compared to H2O2-treated UROtsa, treatment with PP-60, ECG and EGCG in the presence of H2O2 significantly improved UROtsa viability (p<0.01), with strongest effects evoked by ECG. Additionally, though not as effective as in UROtsa cells, viability of both high-grade TCCSUP and T24 BlCa cells, in comparison to H2O2-treated cells, was significantly improved (p<0.01) by treatment with PP-60, ECG, and EGCG in the presence of H2O2. Overall, our findings demonstrate that urothelium cell death via H2O2-induced oxidative stress is mediated, in part, through superoxide (O2-.;), and potentially, direct H2O2 mechanisms, suggesting that green tea polyphenols can protect against oxidative stress/damage and bladder cell death.

Early capsaicin intervention for neurogenic bladder in a rat model of spinal cord injury.

We explored capsaicin pretreatment, prior to spinal trauma, as a method to prevent the development of neurogenic detrusor overactivity (NDO) and urethral-bladder dyssynergia reflex after spinal cord injury (SCI). In addition, the duration of effect of capsaicin therapy on NDO in a rat model of SCI was investigated. Two sets of experiments were performed on female Sprague Dawley rats transected at the T9-T10 spinal level. First, SCI rats received capsaicin (125 mg/kg s.q.) 3-4 days before and 4-5 days after SCI. Cystometrograms (CMG) was performed 4 weeks after injury. In the second set of experiments, serial CMG in the same SCI animal was performed after one time injection of capsaicin (125 mg/kg s.q.) 4 weeks after spinalization. There were no differences in intercontraction intervals, voiding efficiency, or voiding pressure between the capsaicin pretreated and control SCI rats. However, the number of uninhibited detrusor contractions decreased 4 weeks after injury. We found that a single dose of capsaicin suppressed uninhibited detrusor contractions for 34 days in the chronic SCI animals. Early therapy with capsaicin was able to prevent/reduce detrusor hyperreflexia in spinal cord injured animals 4 weeks after injury. Early vanilloid therapy may prevent development of urologic sequelae after SCI.

Effect of hyperforin-enriched extract on pro-ejaculatory effect of 8-hydroxy-2-(di-N-propylamino)tetralin in anesthetized rats.

OBJECTIVES: Premature ejaculation is the most common male sexual dysfunction and, yet, no approved effective therapies are currently available. We studied the in vivo effectiveness of hyperforin (HF), a concentrated extract of Hypericum perforatum in an experimental model for the expulsion phase of ejaculation in anesthetized rats. METHODS: The ejaculation model involved inducing rhythmic bulbospongiosus (BS) muscle contractions in male rats under urethane anesthesia (1.2 g/kg subcutaneously) by transiently raising the internal urethral pressure with saline infusion for 2 seconds at a rate of 116 microL/s. Electrodes in the BS muscles recorded the electrical activity during the contractions as a cluster of bursts on the electromyogram. Injection of the 5-hydroxytryptamine type 1A agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) (0.4 mg/kg subcutaneously) intensified the BS muscle contractions induced by increases in urethral pressure. RESULTS: Administration of 8-OH-DPAT strongly accelerated the ejaculation in the vehicle-treated rats and the amplitude of electrical discharges and the duration of electrical bursts accompanying the increases in urethral pressure were increased from baseline by 203.2% +/- 32.9% and 178.1% +/- 22.9%, respectively. The HF extract reduced the effects of 8-OH-DPAT on ejaculation at lower doses when tested in the dose range of 5 to 80 mg/kg. The reduction in the amplitude of bursts with HF extract remained unchanged after a midthoracic spinal transection, suggesting that the action of HF is either at the spinal ejaculation generator or directly on the neurons innervating the BS muscles. CONCLUSIONS: This is the first report of the effect of HF in a rat model of ejaculation. HF can be considered a novel new treatment of premature ejaculation.

Intraprostatic capsaicin injection as a novel model for nonbacterial prostatitis and effects of botulinum toxin A.

OBJECTIVES: An animal model for nonbacterial prostatitis in rats was developed with the use of intraprostatic injection of capsaicin, an agent thought to excite C-afferent fibers and cause neurogenic inflammation. The analgesic and anti-inflammatory properties of botulinum toxin type A (BoNT-A) was tested in this model. METHODS: Adult male Spraque-Dawley rats were injected with varying doses of capsaicin into the prostate. The nociceptive effects of capsaicin were evaluated for 30min by using a behavior approach; then the prostate was removed for histology and cyclooxygenase (COX) 2 protein concentration measurement. Evans blue (50mg/kg) was also injected intravenously to assess for plasma protein extravasation. A second set of animals were injected with up to 20U of BoNT-A into the prostates 1 wk prior to intraprostatic injection of 1000micromol/l capsaicin. RESULTS: Capsaicin dose dependently induced modifications in pain behavior: closing of the eyes, hypolocomotion, and inflammatory changes: increase of inflammatory cell accumulation, COX2 expression, and plasma extravasation at the acute stage, but completely recovered at 1 wk. BoNT-A pretreatment dose dependently reversed pain behavior and inflammation. BoNT-A 20U significantly decreased inflammatory cell accumulation, COX2 expression, and Evans blue extraction (82.1%, 83.0%, and 50.4%, respectively), and reduced pain behavior (66.7% for eye score and 46.5% for locomotion score). CONCLUSIONS: Intraprostatic capsaicin injection induced neurogenic prostatitis and prostatic pain, and may be a useful research model. BoNT-A produced anti-inflammatory and analgesic effects, and support clinical evaluation in prostatitis.

Green onions: potential mechanism for hepatitis A contamination.

The largest documented foodborne hepatitis A outbreak in U.S. history occurred in November 2003. The source of that outbreak was green onions from a farm in Mexico. Two biomarkers were used to determine ways in which hepatitis A virus (HAV) can contaminate onions. Fluorescent microspheres (1.0 to 10 microm) and HAV vaccine were placed on the soil and the surfaces of pot-grown onions and in the liquid medium of hydroponically cultivated onions. Reverse transcription PCR (RT-PCR) was used to identify HAV RNA. Microspheres were found on the outside and inside of the pot-grown onions for up to 60 days. RT-PCR revealed HAV RNA from the vaccine in well-washed green onions. In the hydroponically grown onions, microspheres were found throughout the onion after only 1 day. RT-PCR also revealed HAV RNA inside the hydroponically grown onions. Both biomarkers support the hypothesis that HAV can contaminate the inside of the growing onion and can be taken up intracellularly through the roots. Once inside, the particles are impossible to remove by cleaning.