Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses.

Objective: The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK). Design: This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK. Subjects: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India. Methods: The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction for the diagnosis of FK. Main Outcome Measures: The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods. Results: The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across 4 common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% confidence interval [CI], 86.62%-97.25%) and a specificity of 89.47% (95% CI, 66.86%-98.70%) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35-124 minutes). Conclusions: The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Acanthamoeba keratitis - A review.

This is a comprehensive review after a thorough literature search in PubMed-indexed journals, incorporating current information on the pathophysiology, clinical features, diagnosis, medical and surgical therapy, as well as outcomes of Acanthamoeba keratitis (AK). AK is a significant cause of ocular morbidity, and early diagnosis with timely institution of appropriate therapy is the key to obtaining good outcomes. The varied presentations result in frequent misdiagnosis, and co-infections can increase the morbidity of the disease. The first line of therapy continues to be biguanides and diamidines, with surgery as a last resort.

Bacterial orbital cellulitis - A review.

Infections of orbit and periorbita are frequent, leading to significant morbidity. Orbital cellulitis is more common in children and young adults. At any age, infection from the neighboring ethmoid sinuses is a likely cause and is thought to result from anatomical characteristics like thin medial wall, lack of lymphatics, orbital foramina, and septic thrombophlebitis of the valveless veins between the two. Other causes are trauma, orbital foreign bodies, preexisting dental infections, dental procedures, maxillofacial surgeries, Open Reduction and Internal Fixation (ORIF), and retinal buckling procedures. The septum is a natural barrier to the passage of microorganisms. Orbital infections are caused by Gram-positive, Gram-negative organisms and anaerobes in adults and in children, usually by Staphylococcus aureus or Streptococcus species. Individuals older than 15 years of age are more likely to harbor polymicrobial infections. Signs include diffuse lid edema with or without erythema, chemosis, proptosis, and ophthalmoplegia. It is an ocular emergency requiring admission, intravenous antibiotics, and sometimes surgical intervention. Computed tomography (CT) and magnetic resonance imaging (MRI) are the main modalities to identify the extent, route of spread from adjacent structures, and poor response to intravenous antibiotics and to confirm the presence of complications. If orbital cellulitis is secondary to sinus infection, drainage of pus and establishment of ventilation to the sinus are imperative. Loss of vision can occur due to orbital abscess, cavernous sinus thrombosis, optic neuritis, central retinal artery occlusion, and exposure keratopathy, and possible systemic sequelae include meningitis, intracranial abscess, osteomyelitis, and death. The article was written by authors after a thorough literature search in the PubMed-indexed journals.

A 5-Year Review of Coinfections in Acanthamoeba keratitis From South India.

PURPOSE: To ascertain the frequency of coinfections in Acanthamoeba keratitis, the nature of copathogens involved, and to analyze the implications in the context of current research on amoebic interactions. METHODS: A retrospective case review from a Tertiary Care Eye Hospital in South India. Smear and culture data for coinfections in Acanthamoeba corneal ulcers were collected from records over a 5-year period. The significance and relevance of our findings in the light of current research on Acanthamoeba interactions were analyzed. RESULTS: Eighty-five cases of culture-positive Acanthamoeba keratitis were identified over a 5-year period (43 of them being coinfections). Fusarium was most commonly identified species, followed by Aspergillus and the dematiaceous fungi. Pseudomonas spp was the commonest bacterial isolate. CONCLUSION: Coinfections with Acanthamoeba are common at our centre, and account for 50% of Acanthamoeba keratitis. The diverse nature of the organisms involved in coinfections suggest that such amoebic interactions with other organisms are probably more widespread than recognized. To the best of our knowledge, this is the first documentation from a long-term study of pathogen diversity in Acanthamoeba coinfections. It is possible that Acanthamoeba itself may be virulence enhanced and secondary to the co-organism, breaching the ocular surface defenses in an already compromised cornea. However, observations from the existing literature on Acanthamoeba interactions with bacteria and certain fungi are based mainly on nonocular or nonclinical isolates. It would be illuminating if such studies are performed on Acanthamoeba and coinfectors from corneal ulcers-to ascertain whether interactions are endosymbiotic or virulence enhanced through amoebic passage.

Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum.

Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.

Rapid Office-Based Diagnosis of Demodex Using an Innovative Smartphone-Aided Intraocular Lens Tool.

ABSTRACT: Demodex is an important pathogen causing eyelid and eyelash diseases. This article describes a quick, efficient, cost-effective office-based imaging of demodex with the help of an intraocular lens (IOL) and smartphone-aided innovated tool. Eyelid photography of a 56-year-old man with suspected demodex infestation was obtained using a 20-diopter (D) IOL over the smartphone camera called as Anterior Segment Photography using IOL (ASPI). A video or photograph of the epilated eyelash was taken using four 30-D IOLs attached to the smartphone camera to form an optical system called IOLSCOPE, which clearly detected the demodex parasite. The importance of ASPI and IOLSCOPE for the rapid office-based diagnosis of demodex pathogen in peripheral health centers devoid of slitlamp and microscopes has been emphasized here.

A case of stromal microsporidiosis: medical strategies employed for successful resolution.

We describe the diagnosis and successful management of a case of stromal microsporidiosis, an important emerging ocular disease. Stromal microsporidiosis is recalcitrant and very often requires therapeutic keratoplasty for effective eradication. We successfully managed a steroid-treated case diagnosed only after 9 months, with a combination of polyhexamethyl biguanide 0.04%, chlorhexidine 0.04% and fluconazole 0.3% eye drops supplemented with tablet albendazole. However, complete resolution was achieved only after epithelial debridement. Toxicity due to the drugs was not noted. Diagnostic delays, steroid use and inappropriate therapy are commonly observed in stromal microsporidiosis. In spite of these potential disadvantages, our case responded well with complete eradication of the infection. The disease being fairly indolent and slowly progressive, medical therapy should be continued, in the absence of progression or other complications. Epithelial debridement may facilitate healing.

Office-Based Rapid Diagnosis and Management of External Ophthalmomyiasis Via Smartphone-Aided Innovative Tools in an Under-Resourced Setting.

PURPOSE: To evaluate the use of smartphone-based innovative tools named Anterior segment photography with intraocular lens (ASPI) and smartphone based intraocular lens microscope (IOLSCOPE) to demonstrate, treat, and diagnose the patients of ophthalmomyiasis caused by Oestrus ovis. METHODS: A retrospective interventional case series of six patients infested with O. ovis presenting in a rural health center with symptoms of burning, itching, watering, and foreign body sensation diagnosed with ophthalmomyiasis. Diagnosis and treatment were performed using an innovative imaging technique that is, an optical system formed by intraocular lens (IOL) and smartphone. RESULTS: The smartphone-based ASPI clearly detected and removed the moving larvae from the ocular surface, followed by microbiological diagnosis of O.ovis larvae with the help of an IOLSCOPE. CONCLUSION: The importance of ASPI and IOLSCOPE for the management of ophthalmomyiasis in peripheral health centers devoid of slitlamp and microscopes has been emphasized here.

Voriconazole in the successful management of a case of Acanthamoeba-Cladosporium keratitis.

PURPOSE: Acanthamoeba and fungal infections can be recalcitrant to therapy - more so when the deeper layers of the corneas are involved. We describe the diagnosis and successful management strategies employed in a case of deep keratitis due to co-infection with Acanthamoeba and Cladosporium sp. OBSERVATIONS: Once the diagnosis of co-infection with both Acanthamoeba and Cladosporium was made, treatment was initiated with a combination of PHMB, chlorhexidine, natamycin, and voriconazole; to which the response was favorable. Signs of relapse with spread of the infection to the deeper plane and the presence of endothelial exudates were noted at 5 weeks. This was attributed to poor compliance. Though the response to re-initiation of therapy under direct supervision was once again favorable; it was only after the introduction of intrastromal voriconazole repeated at timely intervals that rapid and complete resolution was obtained. CONCLUSIONS: Severe keratitis due to fungi or Acanthamoeba very often requires surgical intervention. Complete resolution with medical therapy was obtained only after the introduction of intrastromal voriconazole; thereby avoiding a therapeutic keratoplasty. The addition of voriconzole both topically and particularly intrastromally facilitated faster resolution as well as restricted the duration of therapy with more toxic drugs such as phmb and chlorhexidine.

Cluster of Post-Operative Endophthalmitis Caused by Acanthamoeba T10 Genotype - A First Report.

PURPOSE: To report a cluster of postoperative Acanthamoeba endophthalmitis after routine cataract surgeries. METHODS: A brief summary of sentinel events leading to the referral of 4 patients of postoperative endophthalmitis to our hospital is followed by clinical descriptions and the various diagnostic approaches and interventions used. Genotyping and phylogenetic analysis are also discussed. RESULTS: Four cases of postoperative cluster endophthalmitis, presumed to be bacterial and treated as such, were referred to our hospital. The presence of an atypical ring infiltrate in the first case facilitated the diagnosis of Acanthamoeba endophthalmitis. All patients had vitritis, corneal involvement, and scleral inflammation. Multiple diagnostic methods, such as corneal scrapings, confocal microscopy, aqueous and vitreous taps, scleral abscess drainage, histopathological studies, polymerase chain reaction, and genotyping and phylogenetic analyses of isolated Acanthamoeba, were used to confirm the diagnosis of endophthalmitis and to establish the extent of ocular involvement. Various medical and therapeutic interventions used to control the infections were also documented. The isolated Acanthamoeba were confirmed as belonging to the T10 genotype, an environmentally and clinically rare variety. CONCLUSIONS: This is the first report of a cluster of postoperative T10 genotype Acanthamoeba endophthalmitis, occurring after routine cataract surgery in immunocompetent individuals. Contrary to current perceptions, a rapidly evolving infection can occur with Acanthamoeba.

Conidiobolus, a hitherto unidentified pathogen in microbial keratitis.

Fungal infections are a significantly increasing cause of ocular and systemic morbidity; the vast majority of cases being ascribed to a handful of species. Fungal keratitis, unlike systemic infections, usually occur in immunocompetent individuals. Rarely, systemic infections can be associated with ocular involvement (e.g., Candida, Mucor, Pythium), or a fungus that predominantly causes systemic disease can affect the eye. One such fungus is Conidiobolus which is known to cause muco-cutaneous infections. We report the identification and successful treatment of a case of Conidiobolus corneal ulcer in an immunocompetent individual, who had no co-existing muco-cutaneous disease. Identification of this particular fungus and awareness of its potential to cause systemic disease is especially relevant, given its potential for chronic indolent infection of the subcutaneous tissues. To the best of our knowledge, this is the first reported case of a Conidiobolus corneal ulcer.

Fulminant Acanthamoeba Endophthalmitis After Cataract Surgery-A Case Report.

PURPOSE: To report a case of Acanthamoeba endophthalmitis after an uneventful cataract surgery. METHODS: Description, management, and outcomes of a biopsy-proven case of Acanthamoeba endophthalmitis. RESULTS: Two days after a routine cataract surgery elsewhere, the patient presented with acute endophthalmitis diagnosed as a bacterial infection based on timing and severity. When conventional methods of management failed, the patient was referred to us. Only the presence of an atypical ring infiltrate suggested Acanthamoeba as a probable cause. Subsequent diagnostic evaluations confirmed the initial suspicion. Management with topical antiamoebics and intracameral and intravitreal voriconazole was attempted. Systemic voriconazole and metronidazole were also administered. However, because of relentless disease progression, the eye had to be eviscerated. The cornea, aqueous, vitreous, and sclera were positive by culture and/or polymerase chain reaction and histopathology. CONCLUSIONS: Acanthamoeba usually causes a chronic smoldering keratitis and, very rarely, scleritis. This report, which is the first of its kind, emphasizes the fact that fulminant endophthalmitis with associated scleritis can occur after ocular surgery in an immunocompetent individual, with no significant ophthalmic history.

A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial pathogens.

PURPOSE: Ocular bacterial pathogenesis is a serious sight threatening infection due to several bacterial species like Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa which are predominant. It is necessary to expedite diagnosis of pathogens for early treatment. Hence, a SYBR Green based multiplex Real-Time PCR assay coupled with melting curve analysis has been developed for rapid detection and differentiation of Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa in a single reaction. METHODS: The assay was designed for simultaneous detection and differentiation of pathogens based on their distinct melting curve. The analytical specificity, sensitivity and reproducibility of the assay were examined using various reference strains. Clinical validation was carried out with 100 ocular samples collected from patients suffering from ocular infections. RESULT: Each reaction tested for the targets individually generated three non overlapping melting curves with well alienated peaks corresponding to each gene. Among 100 ocular samples tested, 40 samples diagnosed with positive results in RT-PCR. Thus assay showed 100% specificity with high sensitivity and reproducibility. CONCLUSION: The developed assay consistently established as a rapid and accurate diagnosis of ocular bacterial pathogens compared to the conventional laboratory techniques. Such precise method would aid greatly in clinical management of devastating ocular infections.

The Acanthamoeba-Fungal Keratitis Study.

PURPOSE: To ascertain the incidence of Acanthamoeba keratitis and the coexistence of Acanthamoeba and fungi in microbial keratitis. DESIGN: Prospective cross-sectional study. METHODS: Patients presenting with stromal keratitis were additionally tested for Acanthamoeba irrespective of the clinical diagnosis. Culture positivity was the gold standard. RESULTS: Of the 401 cases included in the study, 40 were positive for Acanthamoeba (10%); of these 40, 16 were positive for both Acanthamoeba and fungi (4.5% of the study group was Acanthamoeba and fungal keratitis positive); 5 were positive for Acanthamoeba and bacteria; and 2 had triple infection with Acanthamoeba, fungi, and bacteria. Ring infiltrates and stromal edema are frequently associated with Acanthamoeba keratitis, as well as in Acanthamoeba coinfections. Ring infiltrates in particular were more frequently seen in the Acanthamoeba and fungal keratitis group (8/16) and they were often yellowish with hyphate edges (vs ring infiltrates only, which are seen in the patients with Acanthamoeba alone). Only 2 patients were contact lens wearers: however, they presented with history of trauma. CONCLUSIONS: Acanthamoeba coinfections are much more frequent and are not restricted to contact lens users. Anticipating coinfections is necessary for establishing a diagnosis as well as for appropriate and timely therapeutic interventions.

Pythium insidiosum and Acanthamoeba keratitis in a contact lens user.

The oomycete Pythium and the protozoan Acanthamoeba can cause fulminant and recalcitrant keratitis, respectively. These infections are not only sight-threatening but can also threaten the structural integrity of the eye. A high index of suspicion is required to identify Pythium keratitis given its uncommon occurrence. Acanthamoeba keratitis is most commonly associated with contact lens wear. However, its coexistence with Pythium has not been reported. We present the successful management of a case of contact lens-related keratitis, coinfected with Pythium and Acanthamoeba.

Diagnostic Evaluation of Co-Occurrence of Acanthamoeba and Fungi in Keratitis: A Preliminary Report.

PURPOSE: To test the hypothesis that the coexistence of Acanthamoeba with other forms of microbial keratitis, especially fungal keratitis (FK), is more prevalent than suspected. METHODS: A prospective diagnostic study whereby patients presenting with stromal keratitis were additionally tested for Acanthamoeba, irrespective of the initial diagnosis. In addition to the routine workup with Gram stain, KOH mount, and cultures on blood agar and potato dextrose agar, nonnutrient agar was included. Confocal microscopy was performed where feasible. Samples for polymerase chain reaction studies were also obtained. We present the preliminary report of the first 100 culture-positive cases. The primary outcome measured was the number of coexistent Acanthamoeba and FK. The secondary outcomes were the total number of Acanthamoeba cases detected and the correlation between clinical diagnosis and microbiological observations. RESULTS: Of the first 100 cases, 22 were culture positive for Acanthamoeba, of which 9 were associated with concurrent FK, 5 with bacterial keratitis, and 8 in isolation. However, only 2 cases were diagnosed clinically as Acanthamoeba, whereas 5 were Acanthamoeba suspects. An additional 4 cases of fungal/Acanthamoeba coexistence in keratitis were revealed purely by confocal microscopy. CONCLUSIONS: Acanthamoeba can coexist with other forms of microbial keratitis. The frequency of infection coexistent or otherwise is higher than reported, and the possibility of coinfection must be considered especially in unresponsive cases. Including nonnutrient agar and confocal microscopy in all cases of keratitis would perhaps translate into better treatment strategies and outcomes.

Synthesis of carboxymethyl chitosan and coating on wound dressing gauze for wound healing.

Wound healing is a long and complex process. To improve wound healing, the wound dressing cotton gauze can be functionalized by imparting moisture holding and antibacterial ability. Moisture is an important factor for wound healing and the absence of microbial intervention can accelerate wound healing process. Direct alkylation method was used to synthesis carboxymethylated chitosan with water solubility, biocompatibility and antibacterial activity. Calcium alginate was used along with modified chitosan as moisture gaining polymeric agent. Pad-dry-cure method was employed to coat both the polymers on cotton gauze surface, which was weaved using 40s Ne cotton yarn. After coating, the cotton was analysed for its polymer add-on percentage, antibacterial action against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 10229. The persistence analysis of antibacterial activity ensures the polymer withstanding ability on cotton gauze surface. SEM detection of polymers with cotton threads confirms their presence. Wound healing action of the polymer coated cotton gauze was determined using albino rats as animal model.

Bispecific enzyme-linked signal-enhanced immunoassay with subattomole sensitivity.

A bispecific enzyme-linked signal-enhanced immunoassay (BiELSIA) was developed with markedly increased sensitivity. Antimyosin, the detection antibody, was linked to the signal probespecific antibody. Probes consisted of diethylenetriamine pentaacetic acids attached to polylysine modified with up to seven or eight horseradish peroxidase (HRP) units. Each bispecific antibody bound two polymer probes, providing twice the signal. Using BiELSIA in a competitive inhibition immunoassay format with an average of 1.5, 3, 4.5, 6, and 7.5 HRP units per polymer-probe, the sensitivity of standard enzyme-linked immunosorbent assay (10(13) mole) was increased to 10(15), 10(18), 10(19), 10(20), and 10(-21) mol (< or = 1,000 molecules), respectively. BiELSIA detected cardiac myosin heavy chain fragments in sera of patients obtained at the time of emergency department admission for acute myocardial infarction, but not in normal sera. This technology should be applicable for detection of cancer, human immunodeficiency virus, prion, and other antigens that are present in concentrations too low for detection by current immunoassays.