Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Rabies control in rural Africa: evaluating strategies for effective domestic dog vaccination.

Effective vaccination campaigns need to reach a sufficient percentage of the population to eliminate disease and prevent future outbreaks, which for rabies is predicted to be 70%, at a cost that is economically and logistically sustainable. Domestic dog rabies has been increasing across most of sub-Saharan Africa indicating that dog vaccination programmes to date have been inadequate. We compare the effectiveness of a variety of dog vaccination strategies in terms of their cost and coverage in different community settings in rural Tanzania. Central-point (CP) vaccination was extremely effective in agro-pastoralist communities achieving a high coverage (>80%) at a low cost (US$5/dog) and inadequate (<20% coverage); combined approaches using CP and either house-to-house vaccination or trained community-based animal health workers were most effective with coverage exceeding 70%, although costs were still high (>US$6 and >US$4/dog, respectively). No single vaccination strategy is likely to be effective in all populations and therefore alternative approaches must be deployed under different settings. CP vaccination is cost-effective and efficient for the majority of dog populations in rural Tanzania and potentially elsewhere in sub-Saharan Africa, whereas a combination strategy is necessary in remote pastoralist communities. These results suggest that rabies control is logistically feasible across most of the developing world and that the annual costs of effective vaccination campaigns in Tanzania are likely to be affordable.

Molecular epidemiology identifies only a single rabies virus variant circulating in complex carnivore communities of the Serengeti.

Understanding the transmission dynamics of generalist pathogens that infect multiple host species is essential for their effective control. Only by identifying those host populations that are critical to the permanent maintenance of the pathogen, as opposed to populations in which outbreaks are the result of 'spillover' infections, can control measures be appropriately directed. Rabies virus is capable of infecting a wide range of host species, but in many ecosystems, particular variants circulate among only a limited range of potential host populations. The Serengeti ecosystem (in northwestern Tanzania) supports a complex community of wild carnivores that are threatened by generalist pathogens that also circulate in domestic dog populations surrounding the park boundaries. While the combined assemblage of host species appears capable of permanently maintaining rabies in the ecosystem, little is known about the patterns of circulation within and between these host populations. Here we use molecular phylogenetics to test whether distinct virus-host associations occur in this species-rich carnivore community. Our analysis identifies a single major variant belonging to the group of southern Africa canid-associated viruses (Africa 1b) to be circulating within this ecosystem, and no evidence for species-specific grouping. A statistical parsimony analysis of nucleoprotein and glycoprotein gene sequence data is consistent with both within- and between-species transmission events. While likely differential sampling effort between host species precludes a definitive inference, the results are most consistent with dogs comprising the reservoir of rabies and emphasize the importance of applying control efforts in dog populations.

Anti-hyperalgesic effect of octreotide in patients with irritable bowel syndrome.

BACKGROUND: Octreotide has been found to be beneficial in the treatment of chronic pain, although the mechanisms underlying its therapeutic effect are incompletely understood. AIMS: To assess the effect of octreotide on perceptual responses to rectal distension in irritable bowel syndrome patients and healthy controls at baseline and following the experimental induction of rectal hyperalgesia. METHODS: In study 1, rectal perception thresholds for discomfort were determined in seven irritable bowel syndrome patients and eight healthy controls on three separate days using a computer-controlled barostat. Subjects received saline, low-dose and high-dose octreotide in a random double-blind fashion. In study 2, perceptual responses to rectal distension were obtained in nine irritable bowel syndrome patients and seven controls before and after repetitive high-pressure mechanical sigmoid stimulation. RESULTS: Octreotide increased the discomfort thresholds in irritable bowel syndrome patients, but not in controls, without changing rectal compliance. Repetitive sigmoid stimulation resulted in decreased rectal discomfort thresholds in the patient group only. In irritable bowel syndrome patients, octreotide prevented the sensitizing effect of repetitive sigmoid stimulation on rectal discomfort thresholds. CONCLUSIONS: Octreotide effectively increased discomfort thresholds in irritable bowel syndrome patients, but not in controls, at baseline and during experimentally induced rectal hyperalgesia. These findings suggest that octreotide exerts primarily an anti-hyperalgesic rather than analgesic effect on visceral perception.

Alosetron controls bowel urgency and provides global symptom improvement in women with diarrhea-predominant irritable bowel syndrome.

OBJECTIVES: Bowel urgency is one of the most bothersome symptoms for nonconstipated IBS patients. The efficacy of alosetron in control of bowel urgency and Global Improvement of IBS symptoms were evaluated in a multicenter double-blind, randomized, placebo-controlled study. METHODS: Female IBS patients with lack of satisfactory control of bowel urgency were randomized 2:1 to alosetron 1 mg twice daily or placebo treatment groups. The primary endpoint was the proportion of days with satisfactory control of bowel urgency during the 12-wk treatment period and 2-wk follow-up period. Secondary endpoints included IBS Global Improvement (responder defined as patient-reported moderate or substantial improvement in IBS symptoms) and improvements in bowel function (stool frequency, consistency, and sensation of incomplete evacuation). RESULTS: A total of 801 women were randomized to the alosetron (n = 532) or placebo groups (n = 269). Physicians classified 98% of patients with diarrhea-predominant IBS. Patients treated with alosetron had a significantly greater proportion of days with satisfactory control of urgency compared to placebo for the treatment period (73% vs 57%, p < 0.001). A significantly greater number of patients treated with alosetron were IBS Global Improvement responders compared to placebo at week 12 (76% vs 44%, p < 0.001). IBS Global Improvement responders had more days with satisfactory control of urgency at week 12 (88% vs 48%) as well as firmer stools, fewer stools/day, and fewer days with incomplete evacuation compared with nonresponders. Alosetron-treated patients showed improvements in bowel functions compared to placebo-treated patients. Constipation was the most commonly reported adverse event.

Gastroenterological causes of pelvic pain.

Chronic pelvic pain is a common condition, which accounts for up to 10% of gynecological consultations and for over a third of diagnostic laparoscopies. In addition to gynecological etiologies for the pelvic pain, the physician must also consider gastroenterological, urological, and neurological disease as a possible basis for the pain. This article discusses the major gastroenterological causes of pelvic pain.

Iatrogenic Horner's syndrome in an experimental pig.

An adult domestic female pig (Sus scrofa) exhibited clinical signs of right-sided Horner's syndrome after experimental placement of a woven aortic stent followed by aortic catheterization. The clinical signs included a miotic pupil, ptosis of the upper eyelid, prolapse of the nictitating membrane, and enophthalmos. Necropsy revealed a large mass in the right midcervical region that encased or was in contact with the carotid artery, internal jugular vein, and vagus nerve. Closer evaluation of the mass revealed that it was a small piece of surgical suture material that was embedded within the lumen of the carotid artery. This extrinsic material served as a nidus for an inflammatory reaction involving the vagus nerve.

Development of constipation in nursing home residents.

PURPOSE: Constipation is a common complaint among geriatric patients and may result in significant morbidity, especially among nursing home residents. The prevalence of constipation increases with advancing age and may be a result of the aging process, but the exact cause is unknown. The aim of this study was to describe the prevalence of constipation and to determine risk factors for the development of constipation in a large population of nursing home residents. METHODS: The Minimum Data Set is an assessment instrument used in nearly all Medicare-certified and Medicaid-certified nursing facilities. Nursing home residents who were at least 65 years of age and who had assessments at baseline and at three months were included in the study (N = 21,012). Baseline risk factors were included in a multivariate logistic regression to determine their association with the development of constipation. To allow causal implications, nursing home residents with constipation at baseline were excluded. The variables examined included medications, mobility, comorbid illness, and nutrition. RESULTS: The mean age (+/- standard deviation) of nursing home residents was 83 +/- 8 years, and the population was 70 percent female and 83 percent white. At baseline, the prevalence of constipation was 12.5 percent (N = 2,627). By the three-month assessment, 7 percent (N = 1,291) of nursing home residents had developed constipation. The factors associated independently with the development of constipation were, in order of magnitude, race, decreased fluid intake, pneumonia, Parkinson's disease, and the presence of allergies. Congestive heart failure and the use of a feeding tube were two factors identified as having a protective effect. CONCLUSION: The variables associated with the development of constipation may be used to identify geriatric nursing home residents at risk and to prevent constipation and its potential complications. Further study is needed to demonstrate a causal relationship between the risk factors and the development of constipation.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

GERD progressing to diffuse esophageal spasm and then to achalasia.

The pathophysiology of achalasia is not completely understood. Several reports have suggested that esophageal motility disorders may progress from one type to another. We report a patient with symptoms and esophageal motility findings consistent with gastroesophageal reflux who subsequently developed a diffuse esophageal spasm and then achalasia. We believe this to be the first report showing such a progression in esophageal motility.

Inflammation of the gastro-oesophageal junction (carditis) in patients with symptomatic gastro-oesophageal reflux disease: a prospective study.

BACKGROUND: Recent data have suggested that cardia biopsy specimens may be more reflective of gastro-oesophageal reflux disease (GORD) than squamous biopsy specimens. AIMS: To assess the distribution, severity, and types of mucosal injury in GORD. PATIENTS: Thirty patients with symptomatic GORD with no or minimal erosions. METHODS: Biopsies were performed at the squamocolumnar junction (Z-line) and 1-2 cm below the Z-line. Injury to the columnar mucosa was scored for inflammatory cells, epithelial cell abnormalities, and for the presence of intestinal metaplasia and Helicobacter pylori. A carditis score above 2 was considered positive (maximum score = 9). RESULTS: Mean carditis scores and percentages of patients with a positive carditis score were higher in Z-line biopsy specimens containing both squamous and columnar mucosa than in those with just columnar mucosa or in specimens taken 1-2 cm below the Z-line. Carditis at the Z-line was focal in 49% of the specimens and was always present adjacent to the squamous epithelium. Goblet cells were present more frequently in the specimens immediately at the Z-line than in those 1-2 cm below the Z-line. H pylori was present in only four patients. The mean carditis scores of specimens 1-2 cm below the Z-line in these patients was significantly higher than in those patients without H pylori. CONCLUSIONS: Mucosal injury at the gastric cardia is highly localised to the region adjacent to the squamocolumnar junction in patients with GORD. Morphological studies of the cardia in GORD should focus on tissue samples that contain both squamous and columnar epithelium in order to obtain an accurate picture of the spectrum of injury.

Symptoms and visceral perception in patients with pain-predominant irritable bowel syndrome.

OBJECTIVE: Abdominal pain is thought to be a hallmark of the irritable bowel syndrome (IBS), although currently used symptom criteria do not differentiate between abdominal pain and discomfort. By focusing on viscerosensory symptoms, we sought to determine: 1) which type of symptoms are most commonly reported by IBS patients, and 2) whether patients who report pain as their most bothersome symptom differ in clinical, psychological, and physiological characteristics. METHODS: A total of 443 consecutive new patient referrals to a tertiary referral center for functional gastrointestinal disorders who met symptom criteria for IBS were given validated, psychometric, health status, and bowel symptom questionnaires containing specific questions regarding the patients' predominant viscerosensory gastrointestinal symptom. Of these patients, 155 (35%) also met criteria for functional dyspepsia. A representative subset of the total IBS patient population (n = 58) underwent evaluation of perceptual responses to controlled rectal distension before and after a noxious sigmoid conditioning stimulus. RESULTS: Viscerosensory symptoms clustered into four groups: 1) abdominal pain, 2) bloating-type discomfort, 3) sensation of incomplete rectal evacuation, and 4) extraabdominal (chest pain or pressure and nausea). A total of 66% of patients reported gas as one of their viscerosensory symptoms, whereas 60% reported abdominal pain as one of their symptoms. Only 29% rated abdominal pain as their most bothersome symptom, whereas bloating-type symptoms were listed by 60% as most bothersome. Although pain predominance did not correlate with the severity of gastrointestinal or psychological symptoms, there was a significant correlation with the development of rectal hypersensitivity in response to the sigmoid conditioning stimulus. CONCLUSIONS: In a tertiary referral population of IBS patients: 1) abdominal pain is reported by only one third of patients as their most bothersome viscerosensory symptoms; and 2) pain-predominance correlates with development of rectal hypersensitivity after a noxious sigmoid stimulus.

Approaches to the modulation of abdominal pain.

Despite their high prevalence and significant economic impact on the health care system, functional gastrointestinal disorders have evaded successful therapy. Conventional medical therapies are based on inadequate disease models, and the great majority of published treatment trials are flawed in their design, permitting no conclusions to be drawn about the true efficacy of any particular treatment. During the past several years, a new, comprehensive disease model based on alterations in brain-gut interactions has rapidly evolved. Even though the precise mechanisms and sites underlying these alterations remain incompletely understood, plausible targets for the development of effective pharmacological treatments are receptors on peripheral terminals of visceral afferent nerves (opioids and serotonin), ion channels and receptors on dorsal horn neurons within the spinal cord (opioids, glutamate, calcitonin gene-related peptide and neurokinin-1), and supraspinal targets in the brainstem within the limbic system and in the prefrontal cortex (serotonin, catecholamines, dopamine and acetylcholine). Regardless of the primary pathophysiology underlying functional gastrointestinal disorders (ie, central versus peripheral), different pharmacological strategies targeted at different sites in the periphery or within the central nervous system may become effective therapies in the future.

Involvement of stretch-sensitive calcium flux in mechanical transduction in visceral afferents.

The spinal and vagal visceral innervation to the gastrointestinal tract contains mechanosensitive afferents that are activated by contraction, distension of smooth muscle or movement in the receptive field. The mechanism by which free nerve endings detect changes in smooth muscle tension is not clear. The present study investigated the effects of mechanical stimulation on dorsal root ganglion neurons in vitro. Neurons were cultured using standard techniques and used in experiments after 24-72 h. Intracellular calcium [Ca2+]i was visualized using a video microscopic technique (Attoflour) in Fura-2 loaded neurons. DRG neurons innervating the stomach or colon were identified by the presence of a retrograde tracer, dextran-conjugated Texas Red, injected into the visceral wall 14-28 days previously. Increases in [Ca2+]i were measured in response to transient (0.5 s) mechanical stimulation of the cell soma using a flame polished probe. Approximately 25% of the whole population of DRG neurons (n = 199) were mechanosensitive, showing a transient rise in [Ca2+]i. In labeled afferents (n = 12), approximately 40% of neurons were mechanosensitive. The increase in [Ca2+]i in response to mechanical stimulation was reduced (100 microM) or abolished (250 microM) by superfusion with gadolinium or by removal of extracellular calcium. Cell somata of visceral spinal afferents show a stretch-sensitive calcium flux that may be involved in sensory transduction of mechanical stimuli that lead to autonomic and sensory reflexes.

Audio and visual stimulation reduces patient discomfort during screening flexible sigmoidoscopy.

OBJECTIVE: Despite current recommendations calling for regular screening flexible sigmoidoscopies over the age of 50, only a small percentage of the population have regular examinations. Improving patient tolerance of flexible sigmoidoscopies could therefore increase patient compliance with these recommended guidelines. The aim of this study was to determine whether audio and visual stimulation reduces discomfort during flexible sigmoidoscopy and whether the effects of the stimulation are secondary to distraction. METHODS: A total of 37 patients undergoing routine screening flexible sigmoidoscopy were randomized to receive no intervention, audio stimulation alone, or audio and visual stimulation. Patient discomfort ratings and affect states were measured prior to and immediately following flexible sigmoidoscopy using a visual analogue scale and the Stress Symptom Ratings (SSR) ratings. RESULTS: Patients receiving audio and visual intervention had lower abdominal discomfort ratings (7.1 +/- 1.4) than patients receiving audio stimulation (9.5 +/- 1.3) or no intervention (10.8 +/- 1.6) (p < 0.05). Patients receiving audio and visual intervention also had higher arousal (7.3 +/- 0.4) and attention (9.2 +/- 0.2) ratings than patients receiving no intervention (6.1 +/- 0.4 and 6.2 +/- 0.7, respectively) (p < 0.05). Anxiety and anger ratings, on the other hand, were significantly lower in patients receiving audio and visual intervention (2.5 +/- 0.4, 1.4 +/- 0.3, respectively) than patients receiving no intervention (4.4 +/- 0.6, 3.6 +/- 0.7). CONCLUSION: Audio and visual stimulation reduces abdominal discomfort associated with flexible sigmoidoscopy. This effect appears to be due to distraction.

The effects of preoperative irradiation on peripheral nerve regeneration.

The purpose of this study was to evaluate the effects of preoperative external cobalt60 beam irradiation on nerve regeneration. Ninety 250-g male Sprague-Dawley rats were studied. Peripheral nerve regeneration was measured by walking track analysis and histomorphology of the proximal, grafted, and distal nerve segments. Ninety animals were randomly assigned to one of five treatment groups, receiving a total fractionated dose of 30, 50, 70, and 90 Gy. Each animal received a 15-mm interposition nerve graft into the right posterior tibial nerve 6 weeks following completion of radiation therapy. The left leg served as a control. The remaining 10 animals received a nerve isograft subjected to a single dose of 30 Gy prior to placement (group 5). Walking track analysis was performed monthly through 8 months. At the conclusion of 120 and 240 days, sections of the proximal, grafted, and distal nerve were harvested, stained, and examined histomorphologically. Evaluation of the print length index demonstrated no statistical difference between our previously established nonirradiated controls, the irradiated groups, and the irradiated isograft group (group 5). The total number of axons per square millimeter was significantly decreased in the distal segment of all irradiated groups when compared with the controls. No statistical difference in number of axons per square millimeter was noted in the irradiated isograft group. Furthermore, no statistical difference was noted in the nerve fiber density between the control group, the preoperative irradiated groups, or the irradiated isograft group (group 5). Despite the reduction in myelinated regenerating fibers, no reduction in function was observed as measured by walking track analysis. Thus, immediate reconstruction of peripheral nerve defects in the face of preoperative irradiation may not be contraindicated.

Postirradiation sarcomas in Sprague-Dawley rats.

A series of radiation-induced neoplasms occurred in Sprague-Dawley rats 4-8 months after irradiation of a single hind leg with 60Co gamma rays. The rats were exposed to fractionated cumulative doses that ranged from 0 to 106 Gy. Osteosarcomas, malignant fibrous histiocytomas and fibrosarcomas developed in the radiation fields of a number of the rats in the higher-dose groups. Tumors did not develop throughout an 8-month observation period in rats that received doses of only 0 or 46 Gy. The most common postirradiation sarcomas in humans are osteosarcoma, malignant fibrous histiocytoma and fibrosarcoma. The Sprague-Dawley rat may serve as a good animal model in studying the development of sarcoma in humans after regional radiotherapy.

Brain-gut interactions: implications for newer therapy.

Despite their high prevalence and significant economic impact on the healthcare system, functional GI disorders have evaded successful therapy. Conventional medical therapies are based on inadequate disease models, and the great majority of published treatment trials are flawed in their design, thus not permitting to draw any conclusions about true efficacy of any particular treatment. The past several years have seen the rapid evolution of a new, comprehensive disease model, based on alterations in brain-gut interactions. Even though the precise mechanisms and sites underlying these alterations remain incompletely understood, plausible targets for the development of effective pharmacologic treatment are receptors on peripheral terminals of visceral afferent nerves (opioids, serotonin), ion channels and receptors on dorsal horn neurons within the spinal cord (opioids, glutamate, CGRP, NK-1) and supraspinal targets in the brainstem, within the limbic system and in the prefrontal cortex (serotonin, catecholamines, dopamine, acetylcholine). Regardless of the primary pathophysiology underlying functional GI disorders (i.e. central vs. peripheral), different pharmacological strategies targeted at different sites in the periphery or within the central nervous system may be effective.