Clinical Features and Treatment Outcomes of Carbapenem-Resistant Pseudomonas aeruginosa Keratitis.

Importance: Evaluation of the microbiological diagnostic profile of multidrug-resistant Pseudomonas aeruginosa keratitis and potential management with rose bengal-photodynamic antimicrobial therapy (RB-PDAT) is important. Objective: To document the disease progression of carbapenemase-resistant P aeruginosa keratitis after an artificial tear contamination outbreak. Design, Setting, and Participants: This retrospective observation case series included 9 patients 40 years or older who presented at Bascom Palmer Eye Institute and had positive test results for multidrug-resistant P aeruginosa keratitis between January 1, 2022, and October 31, 2023. Main Outcomes and Measures: Evaluation of type III secretion phenotype, carbapenemase-resistance genes blaGES and blaVIM susceptibility to antibiotics, and in vitro and in vivo outcomes of RB-PDAT against multidrug-resistant P aeruginosa keratitis. Results: Among the 9 patients included in the analysis (5 women and 4 men; mean [SD] age, 73.4 [14.0] years), all samples tested positive for exoU and carbapenemase-resistant blaVIM and blaGES genes. Additionally, isolates were resistant to carbapenems as indicated by minimum inhibitory concentration testing. In vitro efficacy of RB-PDAT indicated its potential application for treating recalcitrant cases. These cases highlight the rapid progression and challenging management of multidrug-resistant P aeruginosa. Two patients were treated with RB-PDAT as an adjuvant to antibiotic therapy and had improved visual outcomes. Conclusions and Relevance: This case series highlights the concerning progression in resistance and virulence of P aeruginosa and emphasizes the need to explore alternative therapies like RB-PDAT that have broad coverage and no known antibiotic resistance. The findings support further investigation into the potential effects of RB-PDAT for other multidrug-resistant microbes.

Nocardia keratitis: amikacin nonsusceptibility, risk factors, and treatment outcomes.

PURPOSE: To report the increasing trends in Nocardia keratitis species diversity and in vitro antibiotic susceptibility, to demonstrate contact lens wear as a risk factor, and to report visual acuity outcomes after treatment. METHODS: A retrospective clinical case series was performed at a single academic referral center which identified 26 patients with culture-confirmed Nocardia keratitis between 2014 and 2021. A combination of conventional microbiology and molecular techniques were used to identify isolates. Antibiotic susceptibilities were determined using both commercial and in-house laboratory methods. Microbiology and electronic medical records were used to characterize patients' clinical profiles. RESULTS: Patients' median age was 32.5 years with a 2:1 male to female ratio. Eighty-four percent (n = 21/25) of patients were diagnosed within two weeks of symptom onset. Nocardia amikacinitolerans (n = 11/26) was the most recovered Nocardia isolate among study patients. Sixty-four percent (n = 16/25) of all isolates, including all 11 N. amikacinitolerans isolates, were resistant to amikacin. All isolates were susceptible to trimethoprim sulfamethoxazole. Contact lens wear was the leading identified risk factor (n = 23/26) in this population. Median time to resolution was 44 days (n = 23, range: 3-190 days). Seventy-one percent of patients (n = 15/21) had a final visual acuity of 20/40 or better. CONCLUSION: Amikacin resistant Nocardia isolates were the majority in the current study. Trimethoprim sulfamethoxazole may be the preferred alternative antibiotic treatment based on in vitro susceptibilities. Contact lens wear was the major risk factor for Nocardia keratitis in South Florida. Overall visual acuity treatment outcomes of patients were favorable.

Presence of SARS-CoV-2 Viral RNA in Aqueous Humor of Asymptomatic Individuals.

PURPOSE: The purpose of this study was to determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detectable in the aqueous of asymptomatic individuals presenting for ophthalmic surgery. DESIGN: Prospective cross-sectional study. METHODS: Setting and participants: all patients undergoing anterior segment surgery at an ambulatory surgical center (ASC) belonging to a tertiary academic center in South Florida during a 102-day period between June and September 2020 received nasal swab testing for SARS-CoV-2 and underwent a relevant review of symptoms prior to surgery, with negative results required for both in order to proceed with surgery. Main outcomes and measurements: a small sample of aqueous humor (approximately 0.2 cc) was acquired at the beginning of anterior segment surgery from all participants. Aqueous humor was analyzed for SARS-CoV-2 viral ribonucleic acid (RNA) using real-time reverse transcriptase polymerase chain reaction. Demographic information was acquired from participants for secondary analyses. RESULTS: A total of 70 samples were acquired. Of those, 39 samples were excluded due to insufficient material or inconclusive results. Of 31 samples that were successfully analyzed, 6 (19.4%) demonstrated detectable SARS-CoV-2 RNA. None of the 6 individuals (0%) with detectable viral RNA in aqueous humor reported symptoms during the year, compared to 2 of 25 individuals (8%) with negative samples (P = 1). Positive samples were distributed throughout the study period, including both the first and the last days of enrollment. CONCLUSIONS: The presence of SARS-CoV-2 viral RNA in aqueous despite negative nasal swab testing confirmed its presence beyond the blood-ocular barrier in asymptomatic individuals and raises the possibility that the virus may persist in immunoprivileged spaces despite an absence of symptoms.

In vitro Susceptibilities of Methicillin-Susceptible and Resistant Staphylococci to Traditional Antibiotics Compared to a Novel Fluoroquinolone.

BACKGROUND: To assess the in-vitro efficacy of delafloxacin, a new fourth generation fluoroquinolone, against Staphylococcus vitreous isolates from patients with clinically diagnosed endophthalmitis. This is the first investigation of delafloxacin in ocular tissues. METHODS: Intravitreal isolates of culture-proven S. aureus and S. epidermidis were identified between 2014 and 2018. Minimum inhibitor concentrations (MIC) were determined using ETEST strips. The antibiotic susceptibilities were tested against a panel of drugs including glycopeptides such as vancomycin, as well as traditional and newer fluoroquinolones (levofloxacin, moxifloxacin, and delafloxacin). RESULTS: Of 45 total isolates identified between 2014 and 2018, 13% (6) were methicillin-resistant S. aureus (MRSA), 9% (4) were methicillin-sensitive S. aureus (MSSA), 53% (24) were methicillin-resistant S. epidermidis (MRSE), and 24% (11) were methicillin-sensitive S. epidermidis (MSSE). Among the fluoroquinolones, resistance rates were 61% for levofloxacin, 50% for moxifloxacin, and 12% for delafloxacin. Inter-class comparisons between delafloxacin and the two other fluoroquinolones demonstrated higher Gram-positive susceptibility to delafloxacin (p < 0.01). MIC90 values were lowest for delafloxacin (1.0 µg/mL) compared to levofloxacin (8.0 µg/mL) and moxifloxacin (8.0 µg/mL). Vancomycin was 100% effective against all isolates with MIC90 value of 0.75 µg/mL. CONCLUSION: Compared to levofloxacin and moxifloxacin, the newer fluoroquinolone delafloxacin demonstrated the lowest MICs values and lowest rates of resistance for Gram-positive in-vitro S. epidermidis and S. aureus vitreous isolates.

Rose bengal photodynamic antimicrobial therapy to inhibit Pseudomonas aeruginosa keratitis isolates.

To evaluate the in vitro efficacy of rose bengal and riboflavin photodynamic antimicrobial therapy for inhibition the growth of four Pseudomonas aeruginosa (P. aeruginosa) isolates. Four different clinical P. aeruginosa isolates were collected from patients with confirmed keratitis. Each strain was mixed with either sterile water, 0.1% riboflavin solution, or 0.1% rose bengal solution to yield a final bacteria concentration of 1.5 × 107 CFU/mL. Aliquots from each suspension were plated onto nutrient agar in triplicate. Plates were separated into two groups: (1) no irradiation and (2) 5.4 J/cm2 of radiant exposure with custom-made LED irradiation sources. Separate irradiation sources were used for each photosensitizer. The riboflavin groups used a UV-A light source (375 nm) and rose bengal groups used a green light source (525 nm). Plates were photographed at 72 h and custom software measured bacterial growth inhibition. Growth inhibition to riboflavin and rose bengal PDAT showed strain-dependent variability. All four strains of P. aeruginosa showed greatest growth inhibition (89-99%) in the green irradiated-rose bengal group. The UV-A-irradiated riboflavin showed inhibition of 24-44%. UV-A irradiation only showed minimal inhibition (7-14%). There was little inhibitory effect in the non-irradiated photosensitizer groups. Rose bengal PDAT had the greatest inhibitory effect on all four P. aeruginosa isolates. In the UV-A-irradiated riboflavin group, there was moderate inhibition within the irradiation zone; however, there was no inhibition in the non-irradiated groups. These results suggest that rose bengal PDAT may be an effective alternative treatment for Pseudomonas aeruginosa infections.

Molecular epidemiology and resistance profiles among healthcare- and community-associated Staphylococcus aureus keratitis isolates.

PURPOSE: To characterize the molecular, epidemiological, and resistance profiles of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) keratitis isolates. PATIENTS AND METHODS: We used a combination of standard microbiological techniques and DNA microarray analysis to characterize the molecular and antibiotic resistance profiles of 75 Staphylococcus aureus keratitis isolates collected over an 11-year period (2006-2016). RESULTS: Two major USA clonal complexes (CC), CC5 (n=30, 40%) and CC8 (n=28, 37.3%), accounted for 77.3% of the collected S. aureus isolates. USA100, traditionally healthcare associated (n=18/47, 38.3%), and USA300, traditionally community associated (n=12/47, 25.5%), were the dominant MRSA strains. Four (22.2%) of the USA100 MRSA isolates were recovered from patients with no prior healthcare exposure. Eleven (91.7%) of the USA300 isolates were recovered from patients with documented healthcare risk factors. MSSA isolates were polyclonal (n=13). Ninety-three percent of MSSA infections were of healthcare origin. Thirty-seven of 61 (60.6%) healthcare- and 11 of 14 (78.6%) community-associated strains were resistant to three or more antibiotic classes. Sixty-eight percent (n=51) of isolates harbored three of more resistance determinants (genes). The Panton-Valentine Leucocidin gene was detected in 11 (14.7%) of the study isolates. The majority (72.7%) of the strains were members of the USA300 MRSA clone. CONCLUSION: Clonal complexes CC5 and CC8 were the most frequent clones detected among both the MSSA and the MRSA keratitis isolates. USA100 and USA300 clones were the dominant MRSA genotypes. The USA300 MRSA clone has become a leading cause of healthcare-associated keratitis in South Florida. The USA100 MRSA clone has emerged as an increasing cause of community-associated corneal infections in our outpatient population. This shifting epidemiology coupled with the increasing prevalence of multidrug resistance among both MSSA and MRSA keratitis is a cause of concern.

Multiplex Polymerase Chain Reaction Assay for Screening of Mycotoxin Genes From Ocular Isolates of Fusarium species.

PURPOSE: To identify mycotoxin genes among clinical ocular isolates of Fusarium species and to correlate these with clinical outcomes of Fusarium keratitis. METHODS: Fifty-four clinical isolates of Fusarium were retrieved from the Bascom Palmer Eye Institute Ocular Microbiology Laboratory data bank. Multiplex polymerase chain reactions were run to confirm the identification of Fusarium species [internal transcribed spacer sequence, translation elongation factor 1-α (TEF) and ß-tubulin] and to detect the presence of genes encoding production of fumonisin B mycotoxins (FUM1 and FUM8) and trichothecene mycotoxins (deoxynivalenol and nivalenol). The presence or absence of mycotoxins was compared with patient outcomes. RESULTS: Forty-three (79%) of the 54 isolates were confirmed as Fusarium species, by an internal transcribed spacer sequence in 3 (5.6%) and by TEF in 43 (79.6%) of the 54 isolates. Fumonisin biosynthetic gene 1 (FUM1) was detected in 57.4% (n = 31/54) of the Fusarium isolates. No FUM8, deoxynivalenol genes, and nivalenol genes were detected among these in the clinical isolates group. Initial best-corrected visual acuity ranged from 20/25 to 20/80 in the FUM1 gene-negative group and from 20/20 to light perception in the FUM1 gene-positive group. There was no difference in the time to cure between both groups. The presence of FUM1 genes in 5 fungal isolates seemed to be associated with progression to penetrating keratoplasty in the 5 patients from whom the fungi were isolated. Fusarium solani was recovered from all patients requiring penetrating keratoplasty. CONCLUSIONS: Fumonisin B biosynthetic gene 1 may be common among clinical Fusarium isolates and contribute to worse initial visual acuity and high-risk progression to penetrating keratoplasty.

A novel rat contact lens model for Fusarium keratitis.

PURPOSE: The aim of this study was to develop and characterize a new contact lens-associated fungal keratitis rat model and to assess the ability of non-invasive spectral-domain optical coherence tomography (SD-OCT) to detect pathological changes in vivo in fungal keratitis. METHODS: We used SD-OCT to image and measure the cornea of Sprague Dawley rats. Fusarium infection was initiated in the rat eye by fitting Fusarium solani-soaked contact lenses on the experimental eye, while the control animals received contact lenses soaked in sterile saline. The fungal infection was monitored with periodic slit-lamp examination and in vivo SD-OCT imaging of the rat eye, and confirmed by histology, counting of viable fungi in the infected rat cornea, and PCR with specific primers for Fusarium sp. RESULTS: We imaged and measured the rat cornea with SD-OCT. Custom-made contact lenses were developed based on the OCT measurements. Incubation of contact lenses in a F. solani suspension resulted in biofilm formation. We induced contact lens-associated Fusarium keratitis by fitting the rat eyes for 4 h with the Fusarium-contaminated contact lenses. The SD-OCT images of the cornea correlated well with the slit-lamp and histopathological results and clearly defined clinical signs of infection, namely, increased corneal thickening, loss of epithelial continuity, hyper-reflective areas representing infiltrates, and endothelial plaques characteristic of fungal infection. Moreover, in three cases, SD-OCT detected the infection without any clear findings on slit-lamp examination. Infection was confirmed with histological fungal staining, PCR, and microbiological culture positivity. CONCLUSIONS: We developed a highly reproducible rat contact lens model and successfully induced contact lens-associated Fusarium keratitis in this model. The clinical presentation of contact lens-associated Fusarium keratitis in the rat model is similar to the human condition. SD-OCT is a valuable tool that non-invasively revealed characteristic signs of the fungal infection and could provide sensitive, objective monitoring in fungal keratitis.

Uso del análisis de restricción en el control de calidad de cepa del biolarvicida cubano BACTIVEC / Use of the restriction analysis in the quality control of the strain of Cuban biolarvicide BACTIVEC

Se mostró la caracterización genómica de la cepa utilizada en la producción del biolarvicida Bactivec. Se compararon los patrones de digestión de la cepa de referencia 266 de Bacillus thuringiensis de la colección del Instituto Pushkin del antiguo Leningrado y 2 cepas aisladas de 2 lotes del biolarvicida. Las digestiones del ADN cromosomal se realizaron con las enzimas HindIII y EcoRI. En todos los casos se observó el mismo patrón de restricción, lo que muestra la estabilidad genética del ingrediente activo del biolarvicida Bactivec(AU)

Uso del análisis de restricción en el control de calidad de cepa del biolarvicida cubano BACTIVEC / Use of the restriction analysis in the quality control of the strain of Cuban biolarvicide BACTIVEC

Se mostró la caracterización genómica de la cepa utilizada en la producción del biolarvicida Bactivec. Se compararon los patrones de digestión de la cepa de referencia 266 de Bacillus thuringiensis de la colección del Instituto Pushkin del antiguo Leningrado y 2 cepas aisladas de 2 lotes del biolarvicida. Las digestiones del ADN cromosomal se realizaron con las enzimas HindIII y EcoRI. En todos los casos se observó el mismo patrón de restricción, lo que muestra la estabilidad genética del ingrediente activo del biolarvicida Bactivec

[Use of the restriction analysis in the quality control of the strain of Cuban biolarvicide BACTIVEC]. / Uso del análisis de restricción en el control de calidad de cepa del biolarvicida cubano BACTIVEC.

The genomic characterization of the strain used in manufacturing biolarvicide BACTIVET was shown in this paper. Digestion patterns of Bacillus thuringiensis reference strain 266 from the collection of Pushkin Institute located in former Leningrad and of two strains isolated from 2 batches of biolarvicide were compared. Chromosomal DNA digestion was performed by enzymes Hindlll and EcoRI. The same restriction pattern was observed in all cases, which is indicative of genetic stability of the active ingredient of biolarvicide BACTIVET.

Eficacia antiamebiana del metronidazol demostrada en un estudio realizado en la provincia de Cienfuegos / Effectiveness antiamebiana of the metronidazol demonstrated in a study carried out in the county of Cienfuegos

Se realizó una serie de 3 estudios en la provincia de Cienfuegos para demostrar que la amebiasis intestinal en Cuba podría ser un problema de salud sobredimensionado. Con los 2 primeros, se demostraron 2 componentes de esta sobredimensión: el sobrediagnóstico microscópico y el desconocimiento de la presencia de Entamoeba dispar, especie no patógena, en mucho de los casos en que la observación microscópica fue correcta. Aquí se reportó el estudio que demuestra el tercer componente: la creencia errónea de que existe resistencia de Entamoeba histolytica al metronidazol. Para ello, a 35 individuos con infección por una o ambas especies del complejo E. histolytica / E. dispar se les administró metronidazol a la dosis de 250 mg 3 veces al día, durante 10 dias. A muestras de heces colectadas inmediatamente después del tratamiento se les aplicó la prueba ENZYMEBA, para detectar la presencia de una o ambas especies del complejo E. histolytica / E. dispar, y un procedimiento de reacción en cadena de la polimerasa múltiple, para precisar la especie presente. Los resultados de la aplicación de estos ensayos permitieron comprobar la desaparición de la infección por E. histolytica en todos los casos y, por lo tanto, concluir que en aquella provincia, como posiblemente en el resto del país, el metronidazol continúa siendo una droga eficaz en el tratamiento de la amebiasis intestinal(AU)

Eficacia antiamebiana del metronidazol demostrada en un estudio realizado en la provincia de Cienfuegos / Antiamebic effect of metronidazole proved in a study conducted in Cienfuegos province

Se realizó una serie de 3 estudios en la provincia de Cienfuegos para demostrar que la amebiasis intestinal en Cuba podría ser un problema de salud sobredimensionado. Con los 2 primeros, se demostraron 2 componentes de esta sobredimensión: el sobrediagnóstico microscópico y el desconocimiento de la presencia de Entamoeba dispar, especie no patógena, en mucho de los casos en que la observación microscópica fue correcta. Aquí se reportó el estudio que demuestra el tercer componente: la creencia errónea de que existe resistencia de Entamoeba histolytica al metronidazol. Para ello, a 35 individuos con infección por una o ambas especies del complejo E. histolytica / E. dispar se les administró metronidazol a la dosis de 250 mg 3 veces al día, durante 10 d. A muestras de heces colectadas inmediatamente después del tratamiento se les aplicó la prueba ENZYMEBA, para detectar la presencia de una o ambas especies del complejo E. histolytica / E. dispar, y un procedimiento de reacción en cadena de la polimerasa múltiple, para precisar la especie presente. Los resultados de la aplicación de estos ensayos permitieron comprobar la desaparición de la infección por E. histolytica en todos los casos y, por lo tanto, concluir que en aquella provincia, como posiblemente en el resto del país, el metronidazol continúa siendo una droga eficaz en el tratamiento de la amebiasis intestinal

Aplicación de la técnica de hibridación en colonias para la identificación de Vibrio cholerae O1 toxigénico / Application of the hybridization technique on colonies to identify toxigenic Vibrio cholerae 01

Por medio de la reacción en cadena de la polimerasa (RPC), se obtuvo una sonda para el gen que codifica la subunidad B de la toxina colérica (CTxB) que portaba una cepa de referencia de Vibrio cholerae 01. La comprobación del producto amplificado se realizó por la técnica de hibridación en colonias. El producto amplificado hibridó con el gen que codifica la subunidad B de la toxina colérica aislada de Perú y Ecuador, representante de la presente epidemia en América Latina y no lo hizo con las cepas filogenéticamente relacionadas(AU)

Aplicación de la técnica de hibridación en colonias para la identificación de Vibrio cholerae 01 toxigénico

Por medio de la reacción en cadena de la polimerasa (RPC), se obtuvo una sonda para el gen que codifica la subunidad B de la toxina colérica (CTxB) que portaba una cepa de referencia de Vibrio cholerae 01. La comprobación del producto amplificado se realizó por la técnica de hibridación en colonias. El producto amplificado hibridó con el gen que codifica para la subunidad B de la toxina colérica aislada de Perú y Ecuador, representante de la presente epidemia en América Latina y no lo hizo con las cepas filogenéticamente relacionadas.

By means of the polymerase chain reaction (PCR) it was obtained a probe for the gen that codifies the subunit B of cholerae toxin (CTxB), which carried a Vibrio cholerae 01 reference strain. The checking of the amplified product was performed by using the hybridization techniques in colonies. This product hybridized with the gen that codifies for the subunit B of cholerae toxin isolated from Peru and Ecuador, representing the present epidemics in Latin America, but it did not so with the phylogenetically related strains.

Ensayo de la prueba cutánea con el antígeno soluble del Mycobacterium leprae en un grupo de riesco

Se realizó un estudio mediante pruebas cutáneas con antígeno soluble de M. leprae (ASMI y derivado purificado de porteínas (PPD) en 192 convivientes de pacientes de lepra multibacilar, con el objetivo de evaluar la utilidad del método para identificación de indivíduos presumiblemente infectados com M. leprae. Todos ellos residían en el municipio Guantánamo, que es el área de más alta prevalencia de lepra en Cuba. El tamaño medio de la reacción fue 5'03 mm. mientras que en los grupos de control utilizado fue 0 mm. en pacientes lepromatosos, 18'62 mm. en pacientes tuberculoide, 2'75 mm. en pacientes tuberculosis pulmonar y 1'72 en individuos sanos. El criterio de positividad para ambas pruebas fue de 6 mm. Se observó que la prueba cutánea con ASMI puede der útil para al conducción de estudios epidemiológicos ya que, entre los convivientes positivos, el tamaño de la reacción fue significativamente mayor que el observado con el PPD.