Post-mortem chemical excitability of the iris should not be used for forensic death time diagnosis.

Post-mortem chemical excitability of the iris is one of the non-temperature-based methods in forensic diagnosis of the time since death. Although several authors reported on their findings, using different measurement methods, currently used time limits are based on a single dissertation which has recently been doubted to be applicable for forensic purpose. We investigated changes in pupil-iris ratio after application of acetylcholine (n = 79) or tropicamide (n = 58) and in controls at upper and lower time limits that are suggested in the current literature, using a digital photography-based measurement method with excellent reliability. We observed "positive," "negative," and "paradox" reactions in both intervention and control conditions at all investigated post-mortem time points, suggesting spontaneous changes in pupil size to be causative for the finding. According to our observations, post-mortem chemical excitability of the iris should not be used in forensic death time estimation, as results may cause false conclusions regarding the correct time point of death and might therefore be strongly misleading.

Effects of Post-Treatment Hydrogen Gas Inhalation on Uveitis Induced by Endotoxin in Rats.

BACKGROUND Molecular hydrogen (H2) has been widely reported to have benefiicial effects in diverse animal models and human disease through reduction of oxidative stress and inflammation. The aim of this study was to investigate whether hydrogen gas could ameliorate endotoxin-induced uveitis (EIU) in rats. MATERIAL AND METHODS Male Sprague-Dawley rats were divided into a normal group, a model group, a nitrogen-oxygen (N-O) group, and a hydrogen-oxygen (H-O) group. EIU was induced in rats of the latter 3 groups by injection of lipopolysaccharide (LPS). After that, rats in the N-O group inhaled a gas mixture of 67% N2 and 33% O2, while those in the H-O group inhaled a gas mixture of 67% H2 and 33% O2. All rats were graded according to the signs of uveitis after electroretinography (ERG) examination. Protein concentration in the aqueous humor (AqH) was measured. Furthermore, hematoxylin-eosin staining and immunostaining of anti-ionized calcium-binding adapter molecule 1 (Iba1) in the iris and ciliary body (ICB) were carried out. RESULTS No statistically significant differences existed in the graded score of uveitis and the b-wave peak time in the Dark-adapted 3.0 ERG among the model, N-O, and H-O groups (P>0.05), while rats of the H-O group showed a lower concentration of AqH protein than that of the model or N-O group (P<0.05). The number of the infiltrating cells in the ICB of rats from the H-O group was not significantly different from that of the model or N-O group (P>0.05), while the activation of microglia cells in the H-O group was somewhat reduced (P<0.05). CONCLUSIONS Post-treatment hydrogen gas inhalation did not ameliorate the clinical signs, or reduce the infiltrating cells of EIU. However, it inhibited the elevation of protein in the AqH and reduced the microglia activation.

Aqueous humor tyrosinase activity is indicative of iris melanocyte toxicity.

Antibiotics such as fluoroquinolones (FQLs) are commonly used to treat ocular infections but are also known to cause dermal melanocyte toxicity. The release of dispersed pigments from the iris into the aqueous humor has been considered a possible ocular side effect of the systemic administration of FQLs such as Moxifloxacin, and this condition is known as bilateral acute iris transillumination (BAIT). Bilateral acute depigmentation of iris (BADI) is a similar condition, with iris pigment released into the aqueous, but it has not been reported as a side effect of FQL. Iris pigments are synthesized by the melanogenic enzyme tyrosinase (TYR) and can be detected but not quantified by using slit-lamp biomicroscopy. The correlation between dispersed pigments in the aqueous and the extent of melanocyte toxicity due to topical antibiotics in vivo is not well studied. Here, we aimed to study the effect of topical FQLs on iris tissue, the pigment release in the aqueous humor and the development of clinically evident iris atrophic changes. We evaluated this process by measuring the activity of TYR in the aqueous humor of 82 healthy eyes undergoing cataract surgery following topical application of FQLs such as Moxifloxacin (27 eyes, preservative-free) or Ciprofloxacin (29 eyes, with preservative) or the application of non-FQL Tobramycin (26 eyes, with preservative) as a control. In addition, the patients were questioned and examined for ocular side effects in pre- and post-operative periods. Our data showed a significantly higher mean TYR activity in the aqueous humor of Ciprofloxacin-treated eyes compared to Moxifloxacin- (preservative free, p < 0.0001) or Tobramycin-treated eyes (p < 0.0001), which indicated that few quinolones under certain conditions are toxic to the iris melanocytes. However, the reduced TYR activity in the aqueous of Moxifloxacin-treated eyes was possibly due to the presence of a higher drug concentration, which inhibits TYR activity. Consistently, immunoblotting analysis of the aqueous humor from both Ciprofloxacin- and Moxifloxacin-treated eyes showed the presence of soluble TYR enzyme, thus reflecting its toxicity to iris melanocytes and corresponding to its activity in the aqueous humor. Intriguingly, none of these patients developed any clinically appreciable ocular side effects characteristic of BAIT or BADI. Overall, our results suggest that topical antibiotics cause different levels of iris melanocyte toxicity, releasing dispersed pigments into the aqueous humor, which can be measured through TYR enzyme activity. Hence, we conclude that topical FQLs may cause subclinical toxicity to the iris melanocytes but may not be the sole cause of the development of BAIT or BADI.

Intraoperative floppy iris syndrome and its association with various concurrent medications, bulbus length, patient age and gender.

PURPOSE: To evaluate the association between intraoperative floppy iris syndrome (IFIS) and concurrent medications containing selective alpha1A receptor antagonists as well as nonselective alpha1-adrenergic receptor antagonists, bulbus length, patient age and gender. METHODS: We performed a prospective data acquisition of IFIS occurrence and grading, and retrospective evaluation of concurrent medications, bulbus length, patient age and gender of all patients undergoing cataract surgery over a 6-month period. RESULTS: IFIS was observed in 119 of 947 cases (12.6 %). 31 of those 119 patients (26.1 %) had a concurrent medication with a drug that is associated with a higher risk of causing IFIS. Tamsulosin was the drug most commonly associated with IFIS (n = 11), followed by a combination of drugs (n = 7), doxazosin (n = 4), quetiapine (n = 4), finasterid (n = 2), prothipendyl (n = 2), and mianserin (n = 1). Bulbus length and age did not show any significant association with occurrence or grade of IFIS. Gender distribution among IFIS cases was 57.1 % males (n = 68) and 42.9 % (n = 51) females. CONCLUSIONS: The occurrence of IFIS has to be expected with a variety of concurrent medications. The number of IFIS cases and the percentage of females in this series are higher compared to previous reports. The observations might be due to a rising awareness of surgeons or to an increasing number of causative medications on the market.

Retrospective Evaluation of Topical Bimatoprost and Iris Pigmentation Change.

BACKGROUND: Topical bimatoprost is a topical prostaglandin analog originally used to treat glaucoma and more recently used to cosmetically induce hypertrichosis of the eyelashes. Iris pigmentation change has been noted in the treatment of glaucoma but has not been assessed with the cosmetic periorbital application of bimatoprost. OBJECTIVE: To evaluate for iris pigmentation change with the long-term cosmetic use of topical bimatoprost. MATERIALS AND METHODS: A retrospective chart review in a cosmetic dermatology practice of women (N = 50) who consistently purchased topical bimatoprost over an average of 4.59 years was compared with that of age-matched non-bimatoprost patients (N = 50). A blinded evaluator assessed each patient for iris pigmentary change. RESULTS: No iris pigmentation change was noted with the cutaneous application of bimatoprost. CONCLUSION: The cutaneous application of bimatoprost appears to be safe with minimal risk for iris pigmentation change.

A 5-Minute Interval between Two Dilating Eye Drops Increases Their Effect.

PURPOSE: Patients are usually advised to wait 5 minutes between eye drops. This delay supposedly allows the first drop not to be washed out by the second one, thereby increasing the combined effect. However, in the only experimental study conducted in humans on the concurrent administration of two different eye drops, the authors concluded that a 10-minute time interval between eye drops did not increase their combined effect. Our study was designed to address this puzzling observation. METHODS: Using digital photographs shot in photopic conditions in 40 eyes of 20 healthy volunteers, we compared relative pupil surface (i.e., pupil to iris surface area ratios) before and after the administration of one drop of 10% phenylephrine and one drop of 0.5% tropicamide either immediately or after a 5-minute time interval. RESULTS: Waiting 5 minutes yielded a 5.6% relative pupil surface gain (observer 1: P = .003, observer 2: P = .005) indicating an additional combined effect with a 5-minute time interval. CONCLUSIONS: These results show a detectable additive effect that is probably the result of methodological refinements including the challenging of the mydriasis by photopic conditions and the use of pupil and iris surface areas, which may show differences that would be undetectable in terms of diameter.

Prostaglandin E2-Glyceryl Ester: In Vivo Evidence for a Distinct Pharmacological Identity from Intraocular Pressure Studies.

Prostaglandin E2 (PGE2)-2-glyceryl ester is a cyclo-oxygenase 2 product of the endocannabinoid 2-arachidonyl glycerol. It is claimed as pharmacologically novel, but this is complicated by rapid and irreversible isomerization to the 1(3) ester. For ocular studies, enzymatic hydrolysis of the ester moiety creates an additional complication. PG-glyceryl esters were stabilized to isomerization and hydrolysis by replacing the noncarbonyl O with NH, to form the serinolamide and propanediolamide as stable analogs of PG-2-glyceryl and PG-2-1(3) glyceryl esters, respectively. Intraocular pressure was measured in conscious dogs and conscious laser-induced ocular hypertensive monkeys. Pharmacological studies involved stable transfectants for each of the human recombinant prostanoid receptors and the isolated feline iris for prostamide activity. PGE2-serinolamide and PGE2- propanediolamide were essentially inactive at all receptors except the EP3 receptor (EC50, ∼500 nM). This obliged elucidation of EP3 receptor involvement in the intraocular pressure response to these PGE2-glycyerl ester analogs. Since the EP3 receptor agonists sulprostone and GR 63799 did not lower monkey intraocular pressure, a role for EP3 receptors in mediating the effects of PGE2-serinolamide and PGE2-propanediolamide is not indicated. PGE2-glyceryl ester (0.01% and 0.1%) substantially lowered intraocular pressure in monkeys. PGE2-propanediolamide was more efficacious than PGE2-serinolamide in lowering intraocular pressure in monkey eyes, but both appeared equieffective in dog eyes. PGE2-serinolamide dose-dependently (0.01- 0.1%) lowered intraocular pressure in both species, but PGF2 α-serinolamide was inactive. In conclusion, stable PGE2-glyceryl ester analogs lowered intraocular pressure. These findings are consistent with the presence of a PGE2-glyceryl ester-specific recognition site in the eye.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.

[Moxiflaxin and iris transillumination]. / Moxifloxacine et transillumination irienne : cas clinique.

Bilateral Acute Iris Transillumination (BAIT) is a new clinical entity characterized by acute onset of pigment dispersion in the anterior chamber and angle, depigmentation of the iris stroma and permanent iris transillumination, masquerading as uveitis. An association with oral moxifloxacin is reported in some articles. We describe one case of bilateral acute iris transillumination, following the use of systemic moxifloxacin.

La Transillumination Irienne Bilatérale Aiguë (TIBA) est une nouvelle entité clinique, caractérisée par une dispersion pigmentaire aiguë en chambre antérieure et dans l'angle iridocornéen, une dépigmentation du stroma irien et une transillumination irienne définitive, mimant une uvéite antérieure. Une association avec un traitement systémique par moxifloxacine est relatée dans plusieurs articles. Nous rapportons un cas de transillumination irienne bilatérale aiguë ayant suivi la prise systémique de moxifloxacine.

The effects of tamsulosin and alfuzosin on iris morphology: an ultrasound biomicroscopic comparison.

CONTEXT: It is well known that Alpha-1 adrenergic receptor antagonists affect the receptors in the prostate and also iris dilator muscle, leading to loss of iris muscle tone. OBJECTIVE: To compare morphological alterations of iris secondary to tamsulosin and alfuzosin use. PARTICIPANTS: Patients included in the study were grouped as follows: 16 patients treated with tamsulosin (Group 1), 14 patients treated with alfuzosin (Group 2) and 18 untreated controls (Group 3). MATERIALS AND METHODS: All patients underwent ultrasound biomicroscopic and pupillometric examination. Iris thickness was measured at the dilator muscle region (DMR; measured at half of the distance between the scleral spur and the pupillary margin) and sphincter muscle region (SMR; Standardized at 0.75 mm from the pupillary margin). DMR/SMR was also calculated for each patient. Differences among groups were analysed. Main outcome measures were DMR, SMR, DMR/SMR and pupillary diameter. RESULTS: Mean duration of treatments were 2.4 ± 0.96 years (1-4) and 2.3 ± 1.01 years (1-4) in Groups 1 and 2. Pupillary diameters were reduced in Groups 1-2 compared to Group 3 (p < 0.001, p < 0.001). The SMR was similar in Groups 1 and 2 (p: 0.114). These values were not significantly different from that of Group 3 (p: 0.196, p: 0.209). However, thickness in the DMR in Groups 1-2 were significantly lower than that of controls (Group 3) whereas there was no significant difference between Groups 1 and 2 (p: 0.041, p: 0.039 and 0.986, respectively). Mean DMR/SMR ratios were significantly lower in Groups 1-2 than that of Group 3 (p: 0.040 and p: 0.040, respectively). CONCLUSIONS: In patients using these medications, the iris seems to be thinner at the dilator muscle region, but preserving the sphincter muscle region.

The effect of topical 1% cyclopentolate on IOLMaster biometry.

PURPOSE: To investigate the effects of topical cyclopentolate on ocular biometry parameters in healthy young adults. METHODS: Ocular biometry measurements were performed twice with 45-minute intervals by use of the IOLMaster. Effects of topical application of 1% cyclopentolate were examined in 25 eyes from 25 young adults (group 1) before and after its application. As a control (cyclopentolate-free, group 2), 30 eyes from 30 age- and sex-matched healthy subjects were evaluated within the same period. RESULTS: There was no difference between the groups with respect to mean (±SD) ages (group 1: 23 [±3.19] years [range, 18 to 28 years] vs. group 2: 22.6 [±2.7] years [range, 18 to 28 years], p = 0.616). In group 1, the anterior chamber depth and horizontal iris width (white-to-white distance) measurements between the two sessions were significantly different, whereas the axial length was not. In group 2, none of these parameters were different between the two sessions. CONCLUSIONS: Topical application of 1% cyclopentolate caused an increase in the anterior chamber depth and white-to-white distance values without any significant effect on axial length measurement. It is necessary to consider these effects on measurements taken with an IOLMaster in young adults in whom 1% cyclopentolate has been applied.

Bilateral diffuse iris atrophy after the use of oral clarithromycin.

PURPOSE: To report a newly recognised advert event of clarithromycin. METHODS: Retrospective, case report. RESULTS: A 30-year old female referred to our centre with bilateral diffuse iris atrophy after the use of oral clarithromycin for a pulmonary infection. CONCLUSIONS: Bilateral acute depigmentation of the iris is a new clinical entity. Iris transillumination and sphincter paralysis are recognised adverse effects of oral antibiotic therapy, but to our knowledge no other publication has referred to uveitis-like syndrome due to oral administration of clarithromycin.

Postmortem sympathomimetic iris excitability.

BACKGROUND: A reliable estimation of time since death can be important for the law enforcement authorities. The compound method encompassing supravital reactions such as the chemical excitability of the iris can be used to further narrow intervals estimated by temperature-based methods. Postmortem iris excitability was mostly assessed by parasympatholytic or parasympathomimetic substances. Little is known regarding sympathomimetic agents. The present study aims to describe the postmortem iris excitability using the sympathomimetic drug phenylephrine. METHODS: Cadavers were included after body donors gave written informed consent during lifetime. Exclusion criteria were known eye disease, or a postmortem interval exceeding 26 hours. A pupillometer with a minimum measurement range of 0.5 mm was used to determine the horizontal pupil diameter before and 20 minutes after the application of phenylephrine. Increase in pupil diameter was labeled as positive reaction, unchanged pupil diameter was labeled as negative reaction, and decrease in pupil diameter was labeled as paradox reaction. RESULTS: 30 eyes from 16 cadavers (median age = 80.0; 9 males, 7 females) were examined. Initial pupil size was in median 3.5 mm (interquartile range [IQR]: 3.0-4.5 mm) and progressed to 4.0 mm (IQR: 3.5-5.0 mm) 20 minutes after drug instillation. The achieved pupil diameter difference comprised in median 0.5 mm (IQR: 0.0-1.0 mm). A positive reaction was observed in 21 cases. Negative reactions were observed in 5 cases and paradox reactions in 4 cases. Overall, there was a statistically significant difference in diameter between the initial and the reactive pupil (P = 0.0002). CONCLUSION: Although relatively rarely used, sympathomimetic drugs seem to be eligible for chemical postmortem iris excitability. Currently, assessment of postmortem iris excitability usually only involves parasympatholytic and parasympathomimetic agents. The findings of the present study give a hint that the application of a third agent with a sympathomimetic mechanism of action could provide additional information. Further studies assessing such a triple approach in the compound method in comparison with the current gold standard for estimation of time since death are mandatory to ensure reliable results.

Transient oblong anisocoria and orbital surgery.

We present a rare case of transient oblong (segmental) anisocoria occurring at the time of limited orbital surgery. Observation of this previously undescribed phenomenon prompted us to review the relevant anatomy and physiology of the iris and the pharmacokinetics of lidocaine as it pertains to surgery in the region of the eyelids and the orbit.

Risk factors for intraoperative floppy iris syndrome: a meta-analysis.

PURPOSE: To evaluate risk factors (hypertension, diabetes mellitus, and current tamsulosin, alfuzosin, terazosin, or doxazosin use) for intraoperative floppy iris syndrome (IFIS) in patients undergoing phacoemulsification cataract surgery. DESIGN: Systematic review and meta-analysis of the literature. PARTICIPANTS: Seventeen eligible studies (17 588 eyes) examining the association between IFIS and risk factors. METHODS: Pertinent publications were identified through a systematic search of PubMed. All references of relevant reviews and eligible articles were also screened. Language restrictions were not used, and data were extracted from each eligible study by 2 investigators working independently. For medications, 2 separate analyses were performed: an analysis using a dichotomous criterion (use/non-use of the examined agent) and an alternative analysis performing comparisons with patients not receiving any α(1)-blocker. The fixed-effects model (Mantel-Haenszel method) or the random-effects (DerSimonian Laird) model was appropriately used to calculate the pooled odds ratio (OR). Publication bias was appropriately assessed. MAIN OUTCOME MEASURES: Pooled OR for the incidence of IFIS. RESULTS: The pooled OR for IFIS after tamsulosin use was approximately 40-fold greater (or 16.5 at the alternative analysis) than that after alfuzosin use, that is, the second α(1)-blocker in order of effect size. Alfuzosin and terazosin were also associated with IFIS with comparable ORs; the effect of doxazosin reached formal statistical significance at the alternative analysis. Intraoperative floppy iris syndrome was positively associated with hypertension (pooled OR = 2.2, 95% confidence interval [CI], 1.2-4.2, fixed effects) but not with diabetes mellitus (pooled OR = 1.3, 95% CI, 0.7-2.2, fixed effects). CONCLUSIONS: This meta-analysis has highlighted a hierarchy concerning the role of α(1)-blockers in IFIS, indicating an extremely sizeable effect size of tamsulosin; this may entail important physiologic implications. Alfuzosin, terazosin, and doxazosin presented with comparable effect sizes. Hypertension, but not diabetes mellitus, emerged as a risk factor for IFIS.

BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.

Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. Understanding how this process is regulated might provide clues about why lens regeneration does not occur in higher vertebrates. The genes six-3 and pax-6 are known to induce ectopic lenses during embryogenesis. Here we tested these genes, as well as members of the bone morphogenetic protein (BMP) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. We show that the lens can be regenerated from the ventral iris when the BMP pathway is inhibited and when the iris is transfected with six-3 and treated with retinoic acid. In intact irises, six-3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3 and retinoic acid or by BMP inhibitors. These data suggest that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex than the dorsal expression of lens regeneration-specific genes.

Intravitreal triamcinolone acetonide induced changes in the anterior segment in a pig model of branch retinal vein occlusion.

BACKGROUND: Triamcinolone acetonide (TA) has applications for the treatment of a large range of intraocular vascular diseases. The present study in pigs was performed to investigate histopathological and histochemical changes in the levels of myocilin deposition in the anterior segment in a model of branch retinal vein occlusion (BRVO) after vitreal administration of TA. METHODS: After ophthalmoscopic examination, intraocular pressure (IOP) measurement and fundus photography, a BRVO was created photothrombotically in each eye of six pigs, using argon green photocoagulation. The left eye was then injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined, animals sacrificed, and eyes enucleated and processed in paraffin and epoxy resin. Immunofluorescence cytochemistry on paraffin sections was performed to localise the distribution of myocilin in the anterior segment and histology by light and transmission electron microscopy on epoxy resin sections on TA-treated and untreated eyes. RESULTS: Histology revealed pathological changes in the TA-treated eye, including swollen mitochondria, layered long endoplasmic reticulum, pleomorphic nuclei, dense fibrillar extracelluar deposits and aggregates of unusual cell inclusions. Myocilin levels were significantly higher in the TA-treated eyes in the trabecular meshwork (p = 0.001), ciliary process (p = 0.011) and iris (p = 0.030) than in the untreated eyes. CONCLUSIONS: This study suggests that increased myocilin synthesis and related ultrastructural changes in the anterior segment after treatment with intravitreal TA in a porcine model of retinal oedema in BRVO may contribute to IOP elevation.

Early diagnosis of Horner syndrome using topical apraclonidine.

The diagnosis of Horner syndrome (HS) using apraclonidine eye drops is an alternative to the use of topical cocaine drops. A number of reports have described the efficacy of apraclonidine testing, but there is some debate over its sensitivity in the acute setting. We describe a patient with HS secondary to carotid dissection who had a positive response to apraclonidine 3 hours after the onset of symptoms. The case is made for a larger study of apraclonidine use to determine its true sensitivity and specificity, identify confounding factors, and redefine the criteria for positive testing.