Corneal Scrapings Research Articles

Ochrobactrum anthropi Keratitis with Focal Descemet's Membrane Detachment and Intracorneal Hypopyon.

Purpose. To describe a unique case of O. anthropi keratitis associated with a rare manifestation of Descemet's membrane detachment and intracorneal hypopyon and to discuss challenges in diagnosis and management. Methods. Best-corrected visual acuity was measured with Snellen letters. Corneal scrapings were performed and aerobic, viral, herpetic, acid-fast bacilli, Acanthamoeba, and fungal stains and cultures were obtained. Following evisceration, tissue was evaluated for histologic features and again stained for bacteria, mycobacteria, Acanthamoeba, fungi, and viral particles. Results. Initial presentation to our institute was notable for a corneal ulcer, focal Descemet's membrane detachment, and intracorneal hypopyon. Speciation of initial corneal scrapes revealed Ochrobactrum anthropi and initial management included fortified tobramycin. Despite medical therapy, the patient developed a corneal perforation and required subsequent evisceration. Conclusion. O. anthropi is an emerging ocular pathogen that has not been previously reported in cases of keratitis. As this pathogen becomes increasingly recognized as a source of ocular infections, it is important to identify and treat aggressively to avoid vision-threatening disease.

A Case of Successful Treatment Using Topical Colistin in Multidrug-resistantPseudomonas aeruginosaBacterial Ulcer

Purpose: To report a successful case of corneal ulcer caused by multidrug-resistant Pseudomonas aeruginosa using topical colistin in an immobilized patient who was hospitalized for a long period. Case summary: An immobilized 58-year-old female who was admitted for a long stay due to cerebral aneurysm hemorrhage presented with left ocular discharge and hyperemia, and was referred to our clinic. The patient was treated at a local clinic with topical antibiotics, but showed no improvement. At initial visit, she had difficulty communicating with the medical team and taking ophthalmic examination, and she had severe chemosis and corneal infiltration, corneal opacity, and hypopyon on her left eye with a portable slit lamp. Gram staining, bacterial and fungal cultures, and an antibiotic sensitivity test were performed from a corneal scrape. The cultures revealed growth of Pseudomonas aeruginosa, which is resistant to all antibiotics except colistin. As soon as we were aware of the results of the antibiotic sensitivity test, she was treated with topical colistin 0.19% every 1 hour on her left eye, starting immediately. After 28 days of treatment, the infection was resolved except for the remaining corneal opacity. She had a persisted stable corneal lesion at 1-year-follow up after colistin treatment, which indicated no recurrence. Conclusions: Due to gait disturbance, the patient almost missed appropriate ophthalmic examination or treatment. However, as we started immediate topical colistin treatment, we report a successful therapy of corneal ulcer induced by multidrug-resistant Pseudomonas aeruginosa without severe complications, such as perforation. J Korean Ophthalmol Soc 2016;57(8):1307-1311

Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis

Background Techniques used to diagnose Acanthamoeba keratitis (AK), a sight-threatening corneal infection, are either insensitive or time-consuming. Early and accurate diagnosis and appropriate therapy are the key to a good prognosis.Objective The objective of this study is to shed light on the efficacy of PCR in the diagnosis of AK in comparison with other diagnostic techniques.Patients and methods Corneal swabs and scrapings from 95 cases suspected to have AK were examined by microscopy, culture on non-nutrient agar seeded with the avian fecal Escherichia coli AFEC49, and by PCR targeting the 18S rRNA gene.Results The highest number of positive swabs and scrapings was detected by the PCR technique (5.26 and 27.37%, respectively), followed by culture (1.05 and 13.68%, respectively), and the lowest was detected by direct microscopy (0 and 7.37%, respectively). Corneal scrapings showed more positive cases than corneal swabs with statistically significant differences irrespective of the technique used. PCR results were significant versus culture (P<0.05) and direct microscopy (P<0.001). Out of the 26 positive cases, 20 were contact lens wearers, 13 were swimming pools users, five had a history of eye trauma, and five had undergone previous eye operation.Conclusion PCR proved to be a better technique for the detection of Acanthamoeba infection compared with direct microscopy and culture techniques. Scrape specimens are more efficient than swabs in detecting Acanthamoeba by different diagnostic techniques. The important risk factors of AK were contact lens wearers, use of swimming pools, a history of previous eye operation as well as eye trauma.

Two Cases of Keratomycosis Caused by Fusarium Solani: Therapeutic Management

Backgrounds: To report 2 cases of multidrug-resistant fungal keratitis caused by Fusarium solani. The patients underwent a different conservative and surgical therapy including in both of them a keratoplasty à chaud. Methods and findings: A 21-years-old male and a 26- years-old female patient, who both used soft contact lenses developed corneal infiltrates. In the first case the diagnosis was delayed because multiple microbiological examinations of corneal scrapings were initially negative for fungi. A combined empirical therapy against Acanthamoeba and bacteria resulted in no improvement of the clinical findings. The patient underwent a keratoplasty à chaud and the histopathologic analysis of the corneal button yielded Fusarium solani multiresistant to all antifungal agents. In the second case, Fusarium solani was detected in the initial microbiological examination of the corneal scrapings. Although the fungus was multiresistant to all antifungal agents, an aggressive multiple topical antifungal therapy as well as lyposomal Amphotericin B i.v was applied successfully after the keratoplasty à chaud combined with application of Polyvidon-Iodine on the infected cornea and cryocoagulation circular at limbus. Results: Although a therapy with topical Amphotericin B and systemic lyposomal Amphotericin B was applied by the first case the infection progressed to an endophthalmitis. Unfortunately an enucleation was unavoidable. The multiple conservative antifungal therapy in the second case was successful, and the corneal transplant remained free from new infiltrations. Conclusion: We consider the fast diagnosis of multidrug resistance of great importance. Secondly, a penetrating keratoplasty early on the course of the disease increases the chances of a successful outcome.

Microbial Profile of Corneal Ulcers in a Tertiary Care Hospital in South India.

Purpose:To identify the prevalence and microbial profile of infectious keratitis in a tertiary eye care hospital, and to test for the in vitro antimicrobial resistance of the bacterial isolates.Methods:A total of 312 patients presenting to a tertiary eye care hospital with infected corneal ulcer were enrolled in this study. Their socio-demographic data and risk factors were recorded. Corneal scrapings collected from the edge of the ulcer were processed for direct gram stain and KOH mount. Culture was recovered on blood agar, chocolate agar, MacConkey agar and Sabouraud's dextrose (SDA) agar in multiple C shaped streaks. After overnight incubation, bacterial culture was followed by standard biochemical tests and antimicrobial sensitivity according to the clinical and laboratory standards institute (CLSI) guidelines. Inoculated SDA was inspected daily for up to 10 days and the growth was identified by its colony morphology, pigment production and lacto-phenol cotton blue mount examination.Results:Of 312 patients, a microbial etiology was established in 117 cases (37.5%). Of these, 72 (61.5%) were male. The age range of 41-60 years was the most affected group. Of 117 positive cases, 52 (44.5%) were bacterial, 58 (49.5%) were fungal and 7 (6%) patients showed mixed bacterial and fungal infection. The most common isolated fungus was Fusarium which was detected in 36 (31%) cases, followed by Aspergillus spp in 13 (11%) subjects. Staphylococcus aureus was the most common isolated bacteria. All Gram positive cocci were susceptible to vancomycin followed by gatifloxacin, whereas all Gram negative bacilli were susceptible to gatifloxacin.Conclusion:Routine microbiological examination of patients with corneal ulcer is necessary in order to analyze and compare the changing trends of the etiology and their susceptibility patterns.

Comparative study of impression smear with conventional mechanical corneal scrapping by potassium hydroxide (10%KOH) in diagnosis of fungal keratitis

Background: Diagnosis of fungal keratitis remains a challenge to an ophthalmologist due to variety of presentation of patients. If a prompt, aggressive and early treatment is not started disease turns indolent resulting in sloughing of cornea, serious loss of vision, possible perforation and loss eye. Methods: This observational study was conducted in upgraded department of ophthalmology, BRD Medical College, Gorakhpur, India among 50 patients of suspected fungal keratitis. Results: Fungal keratitis is more common in young males of rural area. Most of fungal corneal (>5 mm) with presence of hypopyon, pigmentation and satellite lesion as consistent features. KOH smear with impression smear technique was positive in 36 (72.0%), out of total 50 cases. KOH smear with conventional mechanical scrapings was positive in 35 (70.0%), out of total 50 cases. Sensitivity of impression smear technique as compared to conventional scraping was 100.0%. Sensitivity of mechanical scraping technique as compared to impression smear technique was 92.11%. Conclusions: Impression cytology is a recent diagnostic tool in fungal keratitis. It has various advantages over mechanical scraping like no expensive equipments like slit lamp and operation microscope required. Impression smear KOH mount is comparative to conventional mechanical corneal scraping KOH examination in making and tentative diagnosis of fungal keratitis and can be accurately replied upon for initiation antifungal therapy.

Microbiological evaluation of mycotic keratitis in north Maharashtra, India: A prospective study

Objective: Corneal blindness is a major public health problem worldwide and mycotic keratitis is one of the predominant causes. Infection is difficult to treat and can lead to severe visual impairment or blindness. It is worldwide in distribution but is more common in tropics and subtropical region. Trauma is the major predisposing factor, followed by ocular or systemic defects, prior application of corticosteroids, and prolonged use of antibiotic eye drops. The purpose of this study was to document the clinical and epidemiological features and laboratory diagnosis of fungal corneal ulcer. Methods: Patients who presented with clinically suspected corneal ulcer to ophthalmology department registered for the study. Data were collected through history and slit lamp examination. Corneal scrapping was performed. A portion of each scrapping was examined by direct microscopy. Another portion was inoculated directly on to solid culture media. Results: This study included 40 subjects with corneal ulcer based on clinical suspicion, of whom 14 cases were diagnosed with mycotic keratitis in the laboratory. Among these 14 cases, culture showed fungal growth only in 12 cases where the remaining cases were positive only by Potassium hydroxide (KOH) preparation. Males were more commonly affected and were mostly in the age group of 21 to 50 years. Aspergillus species and Fusarium species were the major isolates. Conclusion: Rapid diagnosis and early institution of anti-fungal therapy is necessary to prevent ocular morbidity and blindness. The direct microscopy method by KOH is rapid, inexpensive and reliable method and culture helps in definite diagnosis and identification. J Microbiol Infect Dis 2015;5(3): 99-102 Key words: Mycotic keratitis, potassium hydroxide, Aspergillus species, Fusarium species.

Corynebacterium spp as causative agents of microbial keratitis

PurposeTo report the clinical and microbiological profile of keratitis caused by Corynebacterium spp.MethodsThe medical and microbiology records of 22 patients, who had presented at the L V Prasad Eye Institute,...

Etiological Analysis of Fungal Keratitis and Rapid Identification of Predominant Fungal Pathogens.

Fungal keratitis is a worldwide-distributed refractory and potentially blinding ocular infection caused by various fungi. It is necessary to investigate the etiological and epidemiological characteristics of this disease and establish a rapid and specific pathogenic identification method. Here, we isolated and identified fungal pathogens of 275 patients with presumed fungal keratitis from Jilin Province, China, and conducted statistical analyses of epidemiological information. The positive rate of fungal culture was 72.0 %. Fusarium sp. was the most common genus among 210 fungal isolates. The predominant species were Fusarium solani, Aspergillus fumigatus, and Candida glabrata, which accounted for over 50 % of the isolated organisms. Corneal trauma and previous use of drugs were the most important predisposing factors. In addition, a multiplex polymerase chain reaction (PCR) was designed with species-specific primers of the three species that could identify them with amplicons of approximately 330 bp from F. solani, 275 bp from A. fumigatus, and 230 bp from C. glabrata. Additionally, PCR with fungal universal primers and multiplex PCR were performed using DNA prepared by an improved DNA extraction method from corneal scrapings. With this method, fungal pathogens from corneal scrapings could be specifically and rapidly identified within 8 h. The culture-independent rapid identification of corneal scrapings may have great significance for the early diagnosis and treatment of fungal keratitis.

Epidemiology and etiological diagnosis of infective keratitis in eastern region of Nepal

Objective: This study aimed to determine the epidemiological characteristics and risk factors predisposing to corneal ulceration in patients presenting to Biratnagar Eye Hospital (Nepal). Methods: All patients presenting to Biratnagar Eye Hospital between January 1 and December 31, 2011 with corneal ulceration were retrospectively reviewed. Sociodemographic data and information pertaining to risk factors were recorded, all patients were examined and corneal scrapping and cultures were carried out. Results: Over one year period 1644 patients with corneal ulcer were evaluated, out of which 76.6% of patients were in the age range of 30 to 69 years and 65% of patients had presenting visual acuity &lt; 3/60. Ocular trauma was the most common cause of keratitis accounting for 60.3% of corneal ulcer and majority of the patients (40%) presented after 2 weeks of symptoms. Among corneal scraping positive cases 1150 (70%) showed fungus, 73 (4.4%) showed bacteria and 20 (1.2%) showed both bacterial and fungus. Conclusion: Corneal ulcer continues to be one of the leading causes of preventable blindness in this region. Lack of awareness about gravity of this disease, financial constraints and geographic barriers remain the major reasons for delay in seeking proper medical help.

Epidemiology and etiological diagnosis of infective keratitis in eastern region of Nepal.

Objective : This study aimed to determine the epidemiological characteristics and risk factors predisposing to corneal ulceration in patients presenting to Biratnagar Eye Hospital (Nepal). Methods : All patients presenting to Biratnagar Eye Hospital between January 1 and December 31, 2011 with corneal ulceration were retrospectively reviewed. Sociodemographic data and information pertaining to risk factors were recorded, all patients were examined and corneal scrapping and cultures were carried out. Results : Over one year period 1644 patients with corneal ulcer were evaluated, out of which 76.6% of patients were in the age range of 30 to 69 years and 65% of patients had presenting visual acuity < 3/60. Ocular trauma was the most common cause of keratitis accounting for 60.3% of corneal ulcer and majority of the patients (40%) presented after 2 weeks of symptoms. Among corneal scraping positive cases 1150 (70%) showed fungus, 73 (4.4%) showed bacteria and 20 (1.2%) showed both bacterial and fungus. Conclusion: Corneal ulcer continues to be one of the leading causes of preventable blindness in this region. Lack of awareness about gravity of this disease, financial constraints and geographic barriers remain the major reasons for delay in seeking proper medical help.

A study of microsporidiosis in corneal scrapings of keratitis patients referring to Farabi Eye Hospital, Tehran, Iran in 2013-14.

Background and Purpose:Microsporidiosis is one of the emerging and opportunistic infections, which causing various clinical symptoms in humans. The prevalence of this infection varies, depending on the infected organ, diagnostic methods, and geographical conditions. In the present study, we aimed to investigate microsporidial keratitis in patients referring to Farabi Eye Hospital Tehran, Iran in 2013-14.Materials and Methods:Two scraping samples were collected from 91 keratitis patients, five cases had prior history of receiving immune suppressive drugs. One of the two collected samples from each participant was used for Vero cell culture and the other was used for the preparation of Giemsa and Gram staining slides. After 30 days, the cells were scrapped and used for DNA extraction; afterwards, nested polymerase chain reaction (PCR) detection method was applied. Primer pairs of small-subunit ribosomal RNA gene were designed by CLC Genomics workbench software to amplify all major microsporidian pathogens, as well as E. bieneusi , which was used as the positive control in this study. Results:The nested PCR showed negative results regarding the presence of microsporidia in the samples. Similarly, Giemsa and Gram staining slides did not detect any spores.Conclusion:The prevalence of human microsporidiosis ranges between 0% and 50%, worldwide. Based on all the negative samples in the present study, we can conclude that the prevalence of this infection among Iranian patients falls in the lower quartile. By gathering further evidence, researchers can take a step forward in this area and open new doors for the assessment of AIDS patients and users of immunosuppressive drugs.

A rare case of Arthrographis kalrae keratomycosis in a non-contact lens wearer

&#x0D; Purpose: Keratomycoses secondary to Arthrographis kalrae are rarely reported. Previous cases involved patients with contact lens wear. Herein we report a rare case of severe fungal keratitis due to Arthrographis kalrae in a non-contact lens wearer.Methods: Case report.Results: A 52-year-old male presented with a left paracentral corneal ulcer with stromal infiltrates following contact with a foreign body while riding a motorbike. Initial treatment with intensive topical antibiotic drops failed. Antifungal therapy with hourly topical amphotericin B 0.15% and fluconazole 0.2%, as well as oral fluconazole was initiated. His ulcer progressed rapidly despite aggressive treatment. This led to a small, peripheral corneal perforation which was treated with corneal gluing and bandage contact lens. He also developed secondary glaucoma. Microbiological cultures of his corneal scrapings revealed Arthrographis kalrae. A single dose of subconjunctival amphotericin B 0.015% o.1mL was then given. At two months after presentation, he eventually underwent therapeutic penetrating keratoplasty (PK) for non-resolving keratomycosis. Following PK, he was maintained on topical amphotericin B 0.15%, fluconazole 0.2% and ciprofloxacin 0.3% four-hourly, together with fusidic acid ointment 2% twice daily. The graft failed three months post-transplant, albeit with no evidence of residual or recurrent infection.Conclusion: Arthrographis kalrae-related keratitis may occur in non-contact lens wearer. Management remains clinically challenging because this organism causes severe, rapidly progressive keratitis.&#x0D;

Trends in Microbiological and Antibiotic Sensitivity Patterns in Infectious Keratitis: 10-Year Experience in Mexico City.

To report the distribution and trends in microbiological and antibiotic sensitivity patterns of infectious keratitis in a 10-year period at a reference center in Mexico City. In this retrospective observational case series, samples were obtained from corneas with a diagnosis of infectious keratitis from January 2002 to December 2011 at the Institute of Ophthalmology "Conde de Valenciana" in Mexico City. Results of cultures, stains, and specific sensitivity/resistance antibiograms for each microorganism were analyzed. A total of 1638 consecutive corneal scrapings were analyzed. Pathogen was recovered in 616 samples (38%), with bacterial keratitis accounting for 544 of the positive cultures (88%). A nonsignificant increasing trend in gram-negative isolates (P = 0.11) was observed. The most commonly isolated pathogen was Staphylococcus epidermidis, and the most common gram-negative isolated species was Pseudomonas aeruginosa. Methicillin-resistant Staphylococcus aureus (MRSA) was present in 45% of the S. aureus isolates; meanwhile, 53.7% coagulase-negative Staphylococcus isolates were methicillin resistant (MRCNS). Pseudomonas aeruginosa resistance to ceftazidime increased from 15% in the first period to 74% for the last 5 years of the study (P = 0.01). The overall sensitivity for vancomycin of MRSA was 87.5%, whereas 99.6% of the MRCNS were sensitive. There was a nonsignificant increase in the recovered gram-positive and gram-negative microorganisms over time. We observed an increased resistance to methicillin in almost half of the MRSA and MRCNS isolates.

Clinico-epidemiological profile of patients with microbial keratitis in Kashmir valley

Purpose: This study was undertaken to provide information on the clinico-epidemiological profile of patients clinically suspected with bacterial and fungal keratitis, who were admitted in the Ophthalmology unit of a tertiary center during a 2 year period. Methods: This is a prospective study of all the patients who were admitted in the eye ward of Government Medical College Srinagar (India) between September 2010 and September 2012. 100 patients of bacterial and fungal corneal keratitis without any history of preexisting ocular or systemic disease were included in the study. All patients were assessed, admitted and completely evaluated in hospital and managed accordingly. Corneal ulcer scrapings were taken at presentation, at the Department of Microbiology. Results: Maximum numbers of patients among males (61.90%) were agricultural workers and among females, 81.08% were housewives. It was seen that majority of patients (79%) presented within 1st week of onset of symptoms and among them growth was seen in 64.6%. Growth was seen in 32cases (72.72%) out of 44 cases who gave a history of trauma whereas growth was seen in 36 cases (64.28%) who did not give a clear history of trauma. Conclusion: Bacterial and fungal keratitis in Kashmir is more prevalent in rural areas, with men being more at risk of developing the disease. There is a seasonal variation in the incidence of the disease; higher cases being reported during the harvesting season. Trauma with organic or vegetable matter is a common preceding factor. Microbiological studies should preferably be carried out in all cases.

Clinico-epidemiological profile of patients with microbial keratitis in Kashmir valley

Purpose: This study was undertaken to provide information on the clinico-epidemiological profile of patients clinically suspected with bacterial and fungal keratitis, who were admitted in the Ophthalmology unit of a tertiary center during a 2 year period. Methods: This is a prospective study of all the patients who were admitted in the eye ward of Government Medical College Srinagar (India) between September 2010 and September 2012. 100 patients of bacterial and fungal corneal keratitis without any history of preexisting ocular or systemic disease were included in the study. All patients were assessed, admitted and completely evaluated in hospital and managed accordingly. Corneal ulcer scrapings were taken at presentation, at the Department of Microbiology. Results: Maximum numbers of patients among males (61.90%) were agricultural workers and among females, 81.08% were housewives. It was seen that majority of patients (79%) presented within 1st week of onset of symptoms and among them growth was seen in 64.6%. Growth was seen in 32cases (72.72%) out of 44 cases who gave a history of trauma whereas growth was seen in 36 cases (64.28%) who did not give a clear history of trauma. Conclusion: Bacterial and fungal keratitis in Kashmir is more prevalent in rural areas, with men being more at risk of developing the disease. There is a seasonal variation in the incidence of the disease; higher cases being reported during the harvesting season. Trauma with organic or vegetable matter is a common preceding factor. Microbiological studies should preferably be carried out in all cases.

Corneal inlay implantation complicated by infectious keratitis

Background/aimsTo report five cases of infectious keratitis following corneal inlay implantation for the surgical correction of presbyopia.MethodsThis was a retrospective, observational case series. Five eyes of five patients were identified...

The role of domestic tap water on Acanthamoeba keratitis in non-contact lens wearers and validation of laboratory methods.

Acanthamoeba is increasingly recognized as an important cause of keratitis in non-contact lens wearers while contact lens wear is the leading risk factor for Acanthamoeba keratitis (AK). It is unlikely that the Acanthamoeba colonization is a feature which is effective only in patient's homes with infectious keratitis since the organism has been isolated from domestic tap water. Two hundred and thirty-one (231) corneal scrapings were taken from infectious keratitis cases, and four contact lens solutions and domestic tap waters were taken from 22 out of 44 AK-diagnosed patient's homes. Microscopic examination, culture, PCR, real-time PCR and DNA sequencing analyses were used for AK-diagnosed samples. The real-time PCR was the most sensitive (100 %) one among the methods used in diagnosis of AK. The 44 (19.0 %) out of 231 corneal scrapings, 4/4 (100 %) contact lens solution and 11/22 (50 %) of domestic tap water samples were found to be positive by real-time PCR for Acanthamoeba. A. griffini (T3), A. castellanii (T4) and A. jacobsi (T15) genotypes were obtained from corneal scrapings, contact lens solutions and domestic tap water samples taken from the patient's homes diagnosed with AK. The isolation of Acanthamoeba containing 6/22 (27.3 %) A. griffini (T3), 14/22 (63.6 %) A. castellanii (T4) and 2/22 (9.1 %) A. jacobsi (T15) from the domestic tap water outlets of 22 of 44 (50 %) of patient's homes revealed that is a significant source of these organisms. A. griffini (T3) and A. jacobsi (T15) genotypes have not been determined from AK cases in Turkey previously. Thus, we conclude that Acanthamoeba keratitis is associated with exposition of patients who has ocular trauma or ocular surface disease to domestic tap water in endemic or potentially endemic countries.

Application of real-time PCR in pathogenic detection of necrotizing herpes stromal keratitis

Background Herpes stromal keratitis (HSK) is a common infectious ocular surface disease, with a higher recurrent rate, especially in necrotizing HSK.The diagnosis of HSK primarily depends on signs and symptoms, and specific laboratory diagnostic is lack. Objective This study was to clarify the expression of herpes simplex virus (HSV) in the corneal epithelium scrapings and tears of necrotizing HSK patients. Methods Thirty eyes of 30 patients with necrotizing HSK were enrolled in Nanjing Hospital of Nanjing Medical Hospital from September 2012 to September 2013 under the patient's informed consent.The eyes were examined by slit lamp microscope and scored.HSK patients received local and systemic therapy for 8 weeks and then an oral maintenance dose for 6 months.Corneal epithelial scrapings and tears samples were collected for HSV DNA detection by real-time PCR before and the 1st, 2nd, 4th, 6th and 8th week after therapy respectively.The difference of HSV positive rate was compared between corneal epithelium scrapings and tears samples using Chi-square test.Multilevel mixed effective model was employed to evaluate HSV concentration change in the samples at various time points in the HSV-positive patients of initial visit.The correlation between HSV concentration and clinical score was analyzed by Spearman rank correlation. Results HSV-positive rate was 46.4% (13/30) in the corneal epithelial scrapings and 13.3% (4/30) in the tear samples, showing a significant difference between them (P=0.006). HSV-positive rate was significantly lower in the corneal epithelial scrapings 1 week, 2 weeks and 4 weeks after treatment than before (P=0.001, 0.003, 0.004), and no HSV was detected 6 weeks and 8 weeks after treatment.No significant change in HSV-positive rate in tear samples in 1 week and 2 weeks after treatment in comparison with before treatment (P=1.000, 0.583), and no HSV was detected after 4 weeks following treatment.The HSV concentration was 2 460(2 165-636 500)/ml in initial 13 HSV-positive eyes of corneal epithelial scrapings and 0(0-1 150)/ml in initial 13 HSV-positive eyes of tear samples.Multilevel mixed effective model determined that HSV concentration was significantly lower in corneal epithelial scraping than that in tear (P=0.005), and HSV concentration was reduced with the lapse of time (P=0.001), with the faster rate of decline in the corneal epithelial scrapings (P=0.049). A positive correlation was found between initial HSV concentration and clinical scores (rs=0.844, P=0.000). Conclusions Real-time PCR appears to be a powerful molecular tool for the detection of HSV in the HSK, especially in corneal epithelial scrapings of lesion.The initial positive outcome of viral DNA in corneal epithelial scrapings predicts a severe clinical procedure. Key words: Herpes simplex/diagnosis; Corneal stroma; Keratitis; Necrosis; DNA, viral/analysis; Epithelium/virology; Tears/virology; Real-time PCR

Pythium insidiosum keratitis: clinical profile and role of DNA sequencing and zoospore formation in diagnosis.

To report the molecular and microbiological diagnosis and clinical profile of 13 patients with Pythium insidiosum keratitis. Phase 1 of the study consisted of DNA sequencing of the ITS region of the rDNA of 162 stocked morphologically unidentified nonconsecutive fungal isolates from corneal scraping of patients with keratitis (2010-2012). Blast and phylogenetic analyses of the sequences showed 9 to be P. insidiosum. A retrospective review of archived photographs of colony and direct microscopy of corneal scrapings and clinical records of the cases were performed. Phase 2 began in 2014, in which a simple method of zoospore formation was used for fungal colonies resembling those of P. insidiosum followed by DNA sequencing. The prevalence of P. insidiosum among unidentified fungal isolates from keratitis was 9/162 (5.5%) in phase 1. In phase 2, 4/102 cases (3.9%) of fungal keratitis were identified as P. insidiosum (January-February, 2014). Phylogenetic analysis of all 13 fungal isolates confirmed the identification of P. insidiosum. Corneal infiltrates exhibited hyphate edges, tentacle-like extensions, and dot-like infiltrates surrounding the main infiltrate. Response to topical 5% natamycin eye drops with or without oral antifungals was poor (penetrating keratoplasty: 9 and evisceration: 2) with a mean follow-up period of 82 days. P. insidiosum keratitis needs to be considered in the differential diagnosis of severe fungal keratitis. It can be identified using the zoospore formation method and confirmed by ITS DNA sequencing. Lack of response to currently used antifungal drugs calls for evaluation of newer drugs for medical therapy and consideration for early penetrating keratoplasty.