Can donor cornea transmit microsporidial infection?
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Adult↗
Biomedical subjects
U Gopinathan
Publications and source records attributed to U Gopinathan.
The value of corneal transplantation in reducing blindness.
PURPOSE: To analyse the role of keratoplasty in reducing world blindness due to corneal diseases. METHODS: Review of published literature. We collected and analysed articles published in the English language literature related to the prevalence and causes of blindness in different parts of the world, causes of corneal blindness, and outcome of corneal transplantation for various corneal diseases. RESULTS: A total of 80% of the world's blind live in developing countries. Retinal diseases are the most important causes of blindness (40-54%) in established economy nations while cataract (44-60%) and corneal diseases (8-25%) are the most common causes of blindness in countries with less developed economies. Keratitis during childhood, trauma, and keratitis during adulthood resulting in a vascularized corneal scar and adherent leucoma are the most frequent causes of corneal blindness in developing countries. Corneal diseases are responsible for 20% of childhood blindness. Nearly 80% of all corneal blindness is avoidable. The outcome of keratoplasty for vascularized corneal scar and adherent leucoma is unsatisfactory, necessitating repeat surgery in a high proportion of these cases. Other barriers for keratoplasty in these nations are suboptimal eye banking, lack of trained human resources, and infrastructure. CONCLUSIONS: Since the developing world carries most of the load of corneal blindness and the major causes of corneal blindness are corneal scar and active keratitis, development of corneal transplantation services need a comprehensive approach encompassing medical standards in eye banking, training of cornea specialists and eye banking personnel and exposure of ophthalmologists to care of corneal transplants for better follow-up care. However, concerted efforts should be made to develop and implement prevention strategies since most corneal blindness is preventable.
Blindness↗
Polymerase chain reaction based detection of fungi in infected corneas.
AIMS: To evaluate a polymerase chain reaction (PCR) based assay to detect fungi in scrapings from infected corneas. METHODS: A PCR assay was developed to amplify a portion of the fungal 18S ribosome gene. Corneal scrapings from 30 patients with presumed infectious keratitis were evaluated using this assay, as well as by standard microbiological techniques, and the results were compared. Conjunctival swabs from each patient's healthy, fellow eye were also evaluated by PCR. RESULTS: PCR and fungal culture results matched (were both positive or both negative for fungi) in 22 (74%) of 30 scrapings from infected corneas. Three (10%) of 30 samples were PCR positive but fungal culture negative; two of these appeared clinically to represent fungal infections, and the third was clinically indeterminate. Four (13%) scrapings were positive by PCR but also by bacterial and not fungal culture. One specimen (3%) was PCR negative but fungal culture positive. Of the conjunctival swabs from each patient's healthy fellow eye, five (17%) of 30 were positive by PCR, and the opposite, infected eye of all five of these harboured a fungal infection. CONCLUSIONS: PCR is promising as a means to diagnose fungal keratitis and offers some advantages over culture methods, including rapid analysis and the ability to analyse specimens far from where they are collected.
Candida↗
Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits.
Mycotic keratitis, being frequently refractive to most of the currently available antifungal therapy, continues to pose a therapeutic challenge to the clinician. In keratitis of infectious etiology stromal dissolution may be brought about by a combination of agent and host factors. An understanding of the source and nature of corneal tissue damage is essential for evolving more effective therapeutic modalities in the treatment of fungal keratitis. In the present study, we have characterized the extracellular proteases produced in vitro by corneal fungal pathogens namely the Aspergillus flavus and Fusarium solani when collagen was provided as the sole nitrogen source. In addition, fungal infected rabbit corneas were investigated for proteolytic activities and nature of inflammatory reaction. Gelatin zymography detected protease bands with molecular mass ranging from 100 to 200 kDa in the culture extracts of A. flavus, and a single major band of molecular mass approximately 200 kDa in the culture extracts of F. solani. A basal proteolytic activity of mass 65 kDa was visualized in all uninfected and infected rabbit corneal extracts. Infected corneas in addition revealed the presence of additional proteolytic species of mass 92 and 200 kDa. The enzyme inhibitory profile suggested that fungal cultures in vitro contained predominantly serine protease activity and to a lesser extent metalloprotease activity. However, fungal infected corneal homogenates showed the presence of metalloproteinase activity alone, the enzymatic activities entirely being sensitive to ethylene diamine tetra acetate (EDTA), a metalloprotease inhibitor. Interestingly, the serine proteolytic activity detected in fungal cultures in vitro was not present in the fungal infected corneas in vivo. However, the possible role of fungal serine proteases in the activation of corneal matrix metalloproteinases (MMPs) cannot be ruled out. Based on the criteria of molecular mass, proteolytic activity in the presence of calcium at neutral pH, and sensitivity to inhibition by a metalloprotease inhibitor, the 65 and 92 kDa gelatinases were identified as MMP 2 and MMP 9, respectively. The expression of 92 and 200 kDa gelatinases correlated positively with the amount of polymorphonuclear cells present in the infected tissues. Activated resident corneal cells or inflammatory cells may largely contribute to the increased proteolytic activities in fungal infected corneas resulting in tissue matrix degradation in fungal keratitis.
Animals↗
Aspergillus fumigatus keratitis with wreath pattern infiltrates.
PURPOSE: To report a case of Aspergillus fumigatus keratitis with clinical features simulating Nocardia keratitis and to highlight the utility of microbiologic investigation in the successful management of infectious keratitis. METHOD: Case report. RESULTS: A 62-year-old man presented with complaints of pain, redness, and watering of 10 days' duration in his right eye. Direct microscopic observation of smears of corneal scrapings revealed a fungal etiology. The patient was treated with 5% natamycin eye drops and 1% atropine sulphate eye drops and was advised to visit the hospital for observation. During his visit to the hospital on day 10 after medication, the eye demonstrated a wreath pattern corneal infiltrate that simulated Nocardia keratitis. The fungus grown from culture of corneal scraping was identified as A. fumigatus. CONCLUSION: This report highlights the significance of subjecting corneal scrapings from suspected cases of infectious keratitis to microbiologic evaluation and emphasizes the fact that a complete microbiologic work-up helps in establishing a definitive etiologic diagnosis and initiating specific antimicrobial therapy.
Antifungal Agents↗
Aspergillus flavus keratitis after laser in situ keratomileusis.
PURPOSE: To report a case of fungal keratitis caused by Aspergillus flavus after laser in situ keratomileusis surgery. METHODS: Case report. A 24-year-old woman developed pain, redness, decreased vision, and an infiltrate posterior to the corneal flap in her right eye 3 days after laser in situ keratomileusis. On referral, approximately 3 weeks after laser in situ keratomileusis, examination of the right eye revealed light perception vision, a large full-thickness corneal infiltrate, and hypopyon. Corneal scrapings were taken for direct microscopic examination and culture. RESULTS: Corneal scraping revealed the presence of fungal filaments in smears and in culture. The fungus was identified as A. flavus. Intensive topical natamycin and systemic ketoconazole therapy was initiated. Despite intensive medical treatment, the infiltrate progressed and the patient was subjected to a therapeutic penetrating keratoplasty. There was no recurrence of infection after surgery. At last follow-up 4 months after surgery, the eye was quiet with graft edema. CONCLUSION: Fungal keratitis is a rare complication of laser in situ keratomileusis surgery. In a case that does not respond to medical treatment, early surgical intervention must be considered.
Adult↗
Keratomycosis: clinical and microbiologic experience with dematiaceous fungi.
OBJECTIVE: To assess the significance of dematiaceous fungi in the causation of keratomycosis. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Eighty-eight cases of dematiaceous fungal keratitis seen at the L. V. Prasad Eye Institute, Hyderabad, India from January 1991 through December 1996. INTERVENTION: Only culture-proven cases were analyzed. MAIN OUTCOME MEASURES: Predisposing factors, clinical characteristics, microbiology, treatment methods, and outcome. RESULTS: Of 557 cases of fungal keratitis seen during the study period, dematiaceous fungi were the etiologic agents in 88 (15.7%), after Fusarium in 210 (37.6%) and Aspergillus species in 170 cases (30.4%), respectively. Trauma was the most common predisposing factor (47.7%). Fifty-three eyes (61.3%) had the classical clinical picture of yellow-white, dry raised infiltrate with feathery hyphate edges at initial examination. The characteristic macroscopic pigmentation was seen in only 24 eyes (27.27%). Septate branching fungal filaments were identified in 78 smears (88.63%) on light microscopy, of which 5 (5.7%) also showed the presence of bacteria. Curvularia species dominated the spectrum (22.7%). Treatment was started in 48 eyes with topical antifungal agents, whereas 37 received both oral and topical antifungal agents. Outcome data were available for 68 cases. Forty-nine (72%) responded to medical therapy, whereas 13 eyes required therapeutic penetrating keratoplasty and 6 eyes had to be eviscerated. CONCLUSIONS: This is the largest series of keratitis caused by dematiaceous fungi reported to date. It clearly brings out the clinical importance of this group of corneal infections.
Adolescent↗
Infectious keratitis in climatic droplet keratopathy.
PURPOSE: To report the ulcer characteristics, microbiologic data, and outcome of infectious keratitis associated with climatic droplet keratopathy (CDK), suggesting that CDK is a predisposing factor for infectious keratitis. METHODS: Medical records of 32 patients (34 eyes) in whom infectious keratitis was seen in association with CDK were retrospectively reviewed. Data were collected regarding the nature of CDK lesions, infiltrate characteristics, organisms isolated, and outcome. RESULTS: CDK was peripheral in 16 (47.1%) eyes, central in 8 (23.5%), and diffuse in 10 (29.4%). The CDK lesions were nodular and elevated in all eyes. The infiltrate was adjacent to the CDK lesions in 28 eyes (82.4%). The infiltrate size (widest dimension) ranged 2-6 mm in 18 eyes (52.9%) and was >6 mm in 7 (20.5%). The infiltrate was full thickness in 15 eyes (44.1%) and was involving up to the middle third of the corneal stroma in another 18 (52.8%). Hypopyon was seen in all. Bacteria were commonly isolated. Staphylococcus epidermidis (six eyes) and Streptococcus pneumoniae (five eyes) were the common bacteria isolated. Resolution with medical treatment was seen in 20 (58.8%) eyes. CONCLUSIONS: CDK is a predisposing factor for infectious keratitis. Treatment should be considered for advanced and nodular lesions, even if they are peripheral, to prevent infectious keratitis.
Adult↗
Corneal ulceration in the elderly in Hyderabad, south India.
AIMS: To report demographic, microbiological, therapeutic, anatomical, and visual results of corneal ulceration in the elderly patients seen at a tertiary eye care centre in south India. METHODS: 102 consecutive cases of microbial keratitis in patients 65 years and older were studied. Inclusion criteria were: (i) presence of corneal stromal infiltrate upon slit lamp examination; and (ii) microbiological evaluation of corneal scrapings for suspected microbial keratitis. RESULTS: The principal predisposing factors identified in this study were ocular disease (38.2%), previous ocular surgery in the same eye (29.4%), trauma (17.6%), and severe systemic disease (16.7%). Contact lens wear was associated with only two cases (2.0%). 99 organisms were isolated in cultures of corneal scrapings from 74 (72.5%) of the 102 cases. Staphylococcus epidermidis (31.1%), filamentous fungi (25.7%), and Streptococcus pneumoniae (13.5%) were the most common isolates. 12 eyes (11.8%) required surgery, 15 (14.7%) eventually required evisceration, and nine (9.6%) of the 94 followed patients achieved an unaided vision of 20/60 or better at last follow up. CONCLUSIONS: This work represents the largest recent single centre study on (non-viral) microbial keratitis in the elderly, its management, and outcomes of therapy. While the predisposing factors differ from those of general population, the spectrum of microbes responsible for keratitis in the elderly appears to reflect the local microbial flora rather than a predilection for elderly patients. Delay in diagnosis and systemic conditions associated with advancing age probably contribute to poorer outcome from therapeutic measures.
Acanthamoeba Keratitis↗
Microbiologic spectrum and susceptibility of isolates: part I. Postoperative endophthalmitis. Endophthalmitis Research Group.
PURPOSE: To present the microbial spectrum and susceptibilities of isolates in postoperative endophthalmitis. METHOD: Isolates from 206 eyes of 206 patients who underwent vitrectomy for postoperative endophthalmitis were examined. RESULTS: One-hundred twelve (54.4%) of 206 vitreous samples were culture positive and 14 (12.5%) of 112 culture-positive cases were polymicrobial, yielding a total of 126 isolates. Isolates included 59 (46.8%) gram-positive cocci, eight (6.3%) gram-positive bacilli, 33 (26.2%) gram-negative organisms, five (4.0%) Actino-mycetes-related organisms, and 21 (16.7%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This is the largest, single-center, prospective series on microbial susceptibilities in postoperative endophthalmitis. We report a high prevalence of gram-negative species and fungi, suggesting that empiric therapy should include coverage for gram-negative pathogens and for fungal pathogens in appropriate settings.
Anti-Bacterial Agents↗
Microbiologic spectrum and susceptibility of isolates: part II. Posttraumatic endophthalmitis. Endophthalmitis Research Group.
PURPOSE: To present the microbial spectrum and susceptibilities of isolates in posttraumatic endophthalmitis. METHOD: Isolates from 182 eyes of 182 patients who underwent vitrectomy for posttraumatic endophthalmitis were examined. RESULTS: One hundred thirteen (62.1%) of 182 vitreous samples were culture-positive, and 23 (20.4%) of 113 culture-positive cases were polymicrobial, including three (2.7%) trimicrobial cases, yielding a total of 139 isolates. Isolates included 63 (45.3%) gram-positive cocci, 24 (17.3%) gram-positive bacilli, 25 (18.0%) gram-negative organisms, seven (5.0%) Actinomycetes-related organisms, and 20 (14.4%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This study represents a large series on microbial spectrum and susceptibilities in posttraumatic endophthalmitis. We report a high prevalence of gram-positive bacilli species and polymicrobial infections containing gram-negative species, underscoring the importance of broad-spectrum, combination antibiotics in the empiric treatment of posttraumatic endophthalmitis.
Anti-Bacterial Agents↗
Role of intravitreal dexamethasone in exogenous fungal endophthalmitis.
PURPOSE: To determine the effects of intravitreal dexamethasone in patients with exogenous fungal endophthalmitis. METHODS: Twenty cases of culture-proven exogenous fungal endophthalmitis following cataract surgery (11/20) and trauma (9/20) were retrospectively analysed for pre- and postoperative visual acuity, anterior chamber and vitreous inflammation and media clarity. All patients were managed with pars plana vitrectomy with intravitreal amphotericin B and oral ketoconazole with (steroid plus group) or without (steroid minus group) intravitreal dexamethasone. Results were analysed by Fisher's exact test. RESULTS: Following vitrectomy 9 of 20 patients (45%) achieved a visual acuity better than counting fingers at 3 m. No statistically significant difference was observed in anatomical and visual outcome between the steroid plus and steroid minus groups, though the number of patients with favourable visual outcome was greater in the steroid plus group. Rate of clearance of inflammation was better in the steroid plus group (40 +/- 15.5 vs 55 +/- 8.6 days). All patients (6/20) with pre-operative vision better than counting fingers showed good anatomical and visual outcome in both groups. CONCLUSIONS: The results of our retrospective study suggest that steroids may be beneficial in promoting faster clearance of inflammation in fungal endophthalmitis. Sensitivity of the fungi to antifungals, dose and timing of steroid and institution of effective antifungal medication prior to the use of steroids are the essential factors which need to be examined further in a prospective manner.
Administration, Topical↗
Microbial keratitis in children.
OBJECTIVE: Microbial keratitis is a major cause of corneal blindness worldwide. This problem is particularly relevant to children, because most of their visual life is ahead of them, and they are uniquely at risk for irreversible ocular deficits, such as those resulting from amblyopia. The objective of this study was to determine the etiologic agents and predisposing factors in childhood infectious keratitis and to examine the outcome of treatment in terms of structure and visual acuity. DESIGN: The study design was a retrospective cases series. PARTICIPANTS: The authors studied 113 eyes in 107 children 16 years of age and younger who were treated for (nonviral) microbial keratitis at the LV Prasad Eye Institute in Hyderabad, India, during the 4.5-year period between February 1, 1991, and June 30, 1995. INTERVENTION: The patients who met the following criteria were included in the study: (1) corneal stromal infiltrate was present on slit-lamp examination; and (2) a corneal scraping was taken at the time of examination for suspected microbial keratitis. MAIN OUTCOME MEASURES: Etiologic micro-organisms, predisposing factors, treatment method, structural treatment outcome, and visual acuity treatment outcome of the infectious keratitis episode were measured. RESULTS: The principal predisposing factors identified in this study were trauma (21.2%), ocular disease (17.7%), systemic disease (15.9%), and prior penetrating keratoplasty in the same eye (8.8%). Vitamin A deficiency was an important factor within the category of severe systemic disease, and contact lens wear was not involved in any of the cases. A total of 85 organisms were isolated in cultures of corneal scrapings from 64 (56.6%) of the 113 cases. Staphylococcus species (43.7%), Streptococcus pneumoniae (18.8%), and fungi (17.2%) were the most common isolates. Eighteen eyes (15.9%) required surgery, and 28 (36.4%) of the 77 patients on whom visual acuity was assessed at last follow-up achieved an unaided visual acuity of 20/60 or better at last follow-up. CONCLUSION: This work represents the largest recent study on childhood (nonviral) microbial keratitis, its management, and treatment outcomes. In this study, amblyopia is highlighted as a potentially significant sequela of childhood microbial keratitis. Identification of the appropriate predisposing factors, etiologic microbial organisms, and treatment outcome from this study may aid in early recognition and treatment of microbial keratitis in children.
Adolescent↗
Antimicrobial effect of ciprofloxacin, povidone-iodine, and gentamicin in the decontamination of human donor globes.
PURPOSE: Clinical research addressing the issue of donor globe decontamination is yet to establish convincing data for the optimal choice of an antimicrobial agent. METHODS: In a donor-globe decontamination study, the antimicrobial effectiveness of a fluoroquinolone antibiotic (ciprofloxacin, 0.3%) was evaluated for the first time and compared with povidone-iodine (P-I, 5%) and gentamicin (0.3%). RESULTS: Ciprofloxacin and gentamicin were found to be less effective than P-I (p < 0.05) in converting culture-positive donor globes to culture negative. In eliminating coagulase-negative staphylococci that predominated the bacterial spectrum, again P-I scored better than ciprofloxacin (p = 0.003) and gentamicin (p = 0.006). Overall, P-I performed better than the other two in the 3-min decontamination procedure. Decontamination was carried out with the same agent for 15 min to assess the effect of duration of decontamination on the antimicrobial activity of P-I. With time, there was no significant increase in the antimicrobial efficacy of the agent except for Corynebacterium species. CONCLUSION: P-I continues to be the preferred agent for decontaminating donor globes. Whereas a contact of 3-min duration between P-I and donor globe remains satisfactory in decontamination procedures, corneal tolerance of this procedure needs investigation.
Anti-Bacterial Agents↗
Early diagnosis of mycotic keratitis: predictive value of potassium hydroxide preparation.
Potassium hydroxide (KOH) preparation is an underutilized modality in the diagnosis of mycotic keratitis. We have earlier shown its utility in the diagnosis of Nocardia and Acanthamoeba keratitis. The aim of this study was (i) to evaluate the sensitivity, specificity and predictive value of KOH preparation, and (ii) to compare its efficacy with other methods of corneal scraping examination, for the diagnosis of mycotic keratitis. The study was conducted in two phases. In phase I, randomized corneal scrapings were examined by KOH, Gram's stain, and lactophenol cotton blue (LPCB) in 91 infectious keratitis subjects. In phase II, 53 corneal scrapings were stained with KOH and calcofluor white (CFW), and viewed with bright field (KOH) and fluorescence (CFW) microscopy. The KOH and CFW readings were recorded by an observer masked to the clinical findings and culture results. Nineteen scrapings were examined by two masked observers. In 22 culture positive fungal keratitis patients in phase I, the sensitivity of KOH, Gram's stain, and LPCB methods was 100%, 86.4%, and 77.3%, respectively. In phase II, the specificities of KOH and CFW were identical (83.8%), while the sensitivities were 81.2% and 93.7%, respectively (p = 0.59), in 16 culture positive mycotic keratitis patients. There was no significant difference between the negative and positive predictive values of KOH and CFW. Furthermore, no significant interobserver variability was found in the specificity and sensitivity. The KOH method compares well with other microscopy methods in the diagnosis of keratomycosis and has a definite place in the armamentarium of diagnostic techniques.
Benzenesulfonates↗
Influence of intraocular lens haptic material on bacterial isolates from anterior chamber aspirate.
PURPOSE: To determine whether intraocular lens (IOL) type affects the bacterial count in anterior chamber aspirates obtained immediately after cataract surgery. SETTING: L.V. Prasad Eye Institute, Hyderabad, India. METHODS: This in vivo study evaluated two groups of eyes that had uneventful cataract extraction and implantation of one of two types of IOLs: all poly(methyl methacrylate) (PMMA) (n = 73) or polypropylene haptic (n = 83). Anterior chamber fluid aspirates (0.1 mL) were obtained with a 30 gauge cannula at the end of surgery and inoculated onto chocolate agar and in thioglycolate broth. Microbiology evaluation was performed using standard methods. RESULTS: Seven eyes (9.5%) with all-PMMA IOLs and 21 (25.3%) with polypropylene haptic IOLs were positive for bacterial isolates (P = .0322; chi-square test). Mean colony count (+/-SD) was 11.43 +/- 3.78 and 13.16 +/- 4.78 colony-forming units/ milliliter in the PMMA and polypropylene haptic IOL groups, respectively. No eye developed endophthalmitis. CONCLUSION: Polypropylene haptic IOLs were associated with a significant increase in bacteria in the anterior chamber.
Aged↗
Microbial contamination of hydrogel contact lenses.
Bacterial contamination of contact lenses (CLs) may contribute to CL-related corneal infection and inflammation. This study reports CL biota over time during daily and extended wear. Microbial contamination of a 58% water, ionic hydrogel CL and a 38% water, non-ionic hydrogel CL was evaluated in an Australian and an Indian population. Fifty wearers were repeatedly sampled over 18 months. Overnight CL use did not alter the frequency of positive cultures, nor the spectrum of organisms compared with daily CL wear. There were no differences in type and frequency of CL contamination between the CL types. Positive cultures were more frequently recovered from the Indian population compared with the Australian population. Streptococcus spp. and Propionibacterium spp. were more frequently isolated from the Australian population. Fungi and Bacillus spp. were more frequently isolated from the Indian population. Normal CL biota alone cannot explain the increased rate of infection and inflammation in extended wear.
Bacillus↗
Sensitivity and predictability of vitreous cytology, biopsy, and membrane filter culture in endophthalmitis.
PURPOSE: Forty-seven consecutive patients with endophthalmitis were prospectively studied to: 1) compare the undiluted vitreous biopsy culture and membrane filter culture of vitrectomy cassette fluid; 2) determine the sensitivity and specificity of initial smear examinations in relation to final culture; and 3) characterize vitreous cytology in culture positive and negative endophthalmitis. METHODS: Examinations included smears (Gram stains, Diff Quik [Bacto Laboratories Pvt. Ltd., Liverpool, Australia] and unstained) and aerobic/anaerobic culture of undiluted vitreous biopsy specimens and diluted vitreous collected in the cassette. The cassette fluid was passed through 5-microns polycarbonate filter for cytology and a 0.22-microns polyvinylidene difluoride millipore filter for culture. RESULTS: By either method culture results were positive in 27 (57.4%) patients. Culture positive of the vitreous biopsy alone was 44.6% and cassette fluid alone was 49% (P > 0.05). In the initial smear examination the sensitivity and specificity of the Gram stain were 66.6% and 84.2%, respectively. comparatively, Diff Quik provided lower sensitivity and specificity at 40.7% and 80.9% respectively. Examination of wet film of vitreous samples did not provide any useful information. Although polymorphs were seen in large numbers in all cultures that yielded positive results on examination, they also were seen in 80% of the sterile samples. There was no significant difference in the quantity of macrophages observed in infected and noninfected samples. CONCLUSIONS: Initial smear examination and cytology have limited roles in the diagnosis of infectious endophthalmitis. Obtaining cultures of both an undiluted vitreous biopsy sample and the vitrectomy cassette fluid has a significant advantage compared with culture of only one sample.
Adolescent↗