K. Lily Therese

Detection of Herpes simplex virus (HSV) genome using polymerase chain reaction (PCR) in clinical samples Comparison of PCR with standard laboratory methods for the detection of HSV

BACKGROUND Diagnosis of Herpes simplex virus (HSV) infections is achieved by detecting the antigen and isolating the virus from the specimen, which requires 7-28 days. With the recently introduced molecular biological technique of polymerase chain reaction (PCR), the diagnosis of HSV infections has been made more rapid and specific. OBJECTIVE We evaluated PCR in comparison with the standard laboratory methods on different types of clinical specimens referred to in our laboratory. STUDY DESIGN A total of 54 specimens, from 54 patients, were investigated. Antigen detection on direct smears was carried out using fluorescent antibody test (FAT) and virus isolation was performed using conventional tube culture method. PCR was carried out with the DNA extracted from various specimens using primers, which coded for the DNA polymerase gene giving a 179 base pair (bp) product. RESULTS The primers were specific for HSV-1 and HSV-2, and the sensitivity of the primers was found to be 0.5 and 0.2 fg in the detection of HSV-1 and HSV-2 DNA, respectively. Of the 50 specimens (excluding 4 archival formalin fixed tissue specimens, which were not subjected to virological methods of detection) HSV was detected by virological methods and PCR in nine specimens, and by PCR alone in 15 additional specimens, thus increasing the analytical sensitivity significantly by 30% (McNemar test: P = 0.0001). The positivity of PCR in all nine virologically positive specimens and the 4 archival specimens obtained from proven lesions of HSV infections further confirmed the specificity of the PCR. CONCLUSION PCR, in our study, was found to be a rapid, specific and highly sensitive method for the detection of HSV in clinical specimens.

The association of rubella virus in congenital cataract — a hospital-based study in India

BACKGROUND The association of rubella virus (RV) with congenital cataract has been well established. Since the data on association of RV with congenital cataract in India are scanty, a study was done based on virus isolation from lens aspirates in patients undergoing therapeutic lensectomy and serology. OBJECTIVE To determine the incidence of the association of rubella virus with congenital cataract. STUDY DESIGN The lens aspirates collected during the 9-year period (from 1990 to 1998), from 70 children up to 12 years of age with congenital cataract were processed for the isolation of rubella virus by conventional viral isolation culture method using BHK-21 and Vero cell lines. Identification of the virus was confirmed by immunofluorescence using human anti-rubella virus specific hyperimmune serum. Serum samples were collected from 55 out of these 70 children and the presence of antibodies to RV was detected by ELISA test. RESULTS RV was isolated from lens aspirates in seven (10%) out of the 70 children with congenital cataract. Of the 55 sera tested, 22 had both anti-rubella IgM and IgG antibodies, in 13 only anti-RV IgG antibodies, in seven only IgM antibodies and the rest of the 13 samples did not have detectable levels of rubella antibodies. Among the children who had IgM antibodies, 12 (24.5%) were below the age of 6 months. CONCLUSION It can be concluded based on virus isolation that 10% of patients with congenital cataract were due to rubella infection and the detection of 24.5% anti-RV IgM antibodies in children below 6 months old shows the possible association of rubella virus with congenital cataract.

Diagnostic Value of Specific Local Antibody Production and Nucleic Acid Amplification Technique-Nested Polymerase Chain Reaction (nPCR) in Clinically Suspected Ocular Toxoplasmosis

Objective: The study was to evaluate the diagnostic efficacy of nested polymerase chain reaction (nPCR) using primers targeting B1 gene of Toxoplasma gondii (T. gondii) with Witmer Desmonts coefficient (WDC) technique in intraocular fluids of clinically suspected toxoplasma retino choroiditis (TRC) patients. Materials and Methods: Two hundred and seventy eight specimens from 189 patients (25 TRC patients and 164 controls) consisting of 189 serum samples and 89 intraocular fluids were included in the study. The clinical specimens were categorized into TRC patients (typical TRC lesion-group I & atypical TRC lesion-group II) and controls (voluntary blood donors-group III, patients undergoing uncomplicated cataract surgery-group IV, ocular inflammation of nontoxoplasma origin-group V). Detection of anti T. gondii IgG and IgM antibodies in serum samples and intraocular fluids were performed and WDC was calculated by the standard method. The standardized nPCR was applied on the 89 intraocular fluids. Results: Clinical diagnosis of TRC based on fundus examination was considered to be the “gold standard.” Anti T. gondii IgG/IgM antibodies were detected in serum by ELISA in 95.6% of 25 clinically suspected TRC patients (gp I and II), 28% of gp III, 40.4% of gp IV, and in 58.3% of gpV. Witmer Desmonts coefficient was positive in 72.7% (16/22) and nPCR in 59.1% (13/22) of TRC patients (gp I and II). Both WDC and nPCR were negative in all the controls. The difference in sensitivity of WDC and nPCR was not statistically significant (p = 0.5247). Conclusions: Though both WDC and nPCR were reliable diagnostic techniques for ocular toxoplasmosis, nPCR is more acceptable because of the amount of specimen(s) required, rapidity, cost effectiveness, and direct evidence of T. gondii DNA in the intraocular fluids.

Acute Postoperative Bacillus cereus Endophthalmitis Mimicking Toxic Anterior Segment Syndrome

OBJECTIVE To study the clinicomicrobiologic characteristics and treatment outcomes in eyes with acute postoperative endophthalmitis (APE) owing to Bacillus cereus from a tertiary eye-care center. DESIGN Retrospective, interventional case series. PARTICIPANTS Case records of all eyes with culture-proven APE attributable to B cereus from January 2000 to May 2011 were identified from a computerized database and evaluated. METHODS Clinical features at time of presentation, microbiological characteristics, and treatment measures were recorded. A thorough literature search using PubMed and the Cochrane Library databases was done to identify all cases of APE owing to Bacillus species reported to date and clinical characteristics of these eyes was compared with our series. MAIN OUTCOME MEASURES Structural (globe salvage) and functional (visual rehabilitation) outcomes at last follow-up visit. RESULTS We found 6 sporadic cases that experienced APE during the study period. All eyes had a fulminant onset within the first 24 hours of cataract surgery with extremely high intraocular pressure (IOP) and corneal edema similar to toxic anterior segment syndrome (TASS). However, these eyes progressed rapidly to develop corneal infiltrates, scleral and uveal tissue necrosis with hyphema, brownish exudates in anterior chamber and necrotizing retinitis within hours despite immediate initiation of intravitreal pharmacotherapy and vitrectomy. All eyes demonstrated gram-positive bacilli from the aqueous and B cereus was isolated, which was sensitive to conventional antibiotics except penicillin. Two eyes required therapeutic keratoplasty, combined with a scleral patch graft in 1 eye, 1 eye was eviscerated after 48 hours of onset of symptoms, and 2 eyes experienced phthisical changes within 10 days of onset. CONCLUSIONS We found that APE owing to B cereus has an onset within 12 to 24 hours of intraocular surgery and simulates TASS in the first few hours. The clinical course is marked by rapidly worsening necrotizing infection, leading to very poor outcomes despite early institution of appropriate therapy. One must closely observe every case of TASS that presents with intense pain and extremely high IOP and rule out APE owing to B cereus with microbiologic testing. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Identification of bacteria in culture negative and polymerase chain reaction (PCR) positive intraocular specimen from patients with infectious endopthalmitis

A novel Denaturing High-Performance Liquid Chromatography (dHPLC)-based technique allows rapid high-resolution analysis of PCR products. We show the application of this PCR/dHPLC approach for direct detection and identification of bacterium from the Eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endopthalmitis and to differentially identify the culture negative cases and initiate appropriate therapy. The aim of this study is to identify culture negative PCR positive cases by the application of PCR based DNA sequencing. A total of 116 intraocular specimens were subjected for the study. Sixty-nine different bacteria were identified using dHPLC based DNA sequencing of which predominant ones were Gram-positive bacteria and cannot be cultured by conventional methods. Forty eight different bacteria detected in this study is being reported for the first time in infectious endopthalmitis.

Detection of Mycobacterium tuberculosis by polymerase chain reaction in a case of orbital tuberculosis

Orbital tuberculosis is quite uncommon. We report a case of orbital tuberculosis in a 3-year-old child from Bangladesh who presented with swelling and discharging sinus in the lower part of the orbit. Histopathology revealed a granulomatous inflammation with caseation necrosis. Polymerase chain reaction (PCR) showed amplification of the Mycobacterium tuberculosis genome. The patient responded to a course of antituberculous treatment. Mycobacterium tuberculosis should be considered in the differential diagnosis of inflammatory orbital disease in the Indian subcontinent where tuberculosis is prevalent.

Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1)

Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins—namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis.

Detection of Escherichia fergusonii by PCR-based DNA sequencing in a case of delayed-onset chronic endophthalmitis after cataract surgery.

UNLABELLED We report a case of chronic low-grade endophthalmitis after cataract surgery presenting with recurrent episodes of severe anterior chamber reactions and hypopyon uveitis caused by Escherichia fergusonii, which was isolated from vitreous aspirate by polymerase chain reaction-based DNA sequencing. Polymerase chain reaction has emerged as an essential, powerful, and rapid laboratory diagnostic technique and a useful adjunct to the conventional gold standard. FINANCIAL DISCLOSURE No author has a financial or proprietary interest in any material or method mentioned.

Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes

Pseudomonas aeruginosa (P. aeruginosa) is a menacing opportunistic, nosocomial pathogen; become a growing concern as conventional antimicrobial therapy is now futile against it. Multi‐drug resistant P. aeruginosa (MDRPA) has distinctive resistance mechanisms such as production of β‐lactamases, repression of porin genes and over‐expression of efflux pumps. The focus of this study is to standardize and application of multiplex PCR (mPCR) to detect the presence of betalactamase genes encoding blaTem, blaOXA, blaCTX‐M‐15, blaVim, blaGes, blaVeb, blaDIM, AmpC and Efflux pump genes encoding Mex A,B‐oprM, Mex C,D‐oprJ, Mex X,Y‐oprN, oprD, nfxB, MexR. A total of 200 clinical isolates of P. aeruginosa were tested for the presence of the above mentioned genes genotypically through mPCR and characterized by phenotypic methods for ESBL and MBL production. Out of 200 isolates, 163 (81.5%) nfxB regulator gene, 102 (51%) MexA, 96 (48%) MexC, 93 (46.5%) MexB, 86 (43%) MexD, 81 (40.5%) OprM, 74 (37%) OprJ, 72 (36%) OprD and MexR, 53 (26.5%) Mex X and OprN, 49 (24.5%) MexY gene. Betalactamase genes 145 (72.5%) blaTem, 67 (33.5%) blaOXA, 35 (17.5%) blaVim, 25(12.50%), 23 (11.50%) blaVeb, 21 (11.5%) blaGes, 14 (7%) Ctx‐m and 10 (5%) AmpC and 5 (2.5%) blaDim‐1 gene were tested positive by mPCR. Phenotypically 38 (19%) and 29 (14.5%) out of 200 tested positive for ESBL and MBL production. Application of this mPCR on clinical specimens is fast, accurate, specific and low‐cost reliable tool for the screening, where culture negative Eubacterial PCR positive cases for an early molecular detection of drug resistance mechanism assisting the clinician to treat the disease with appropriate antibiotic selection.

Endogenous endophthalmitis due to Roseomonas mucosa presenting as a subretinal abscess

BackgroundEndogenous bacterial endophthalmitis is an infrequently reported entity. Although Roseomonas mucosa has been reported to cause systemic infections in immunosuppressed individuals, ocular infection due to Roseomonas has been rarely reported in literature previously.FindingsA 74-year-old diabetic was diagnosed to have Klebsiella urinary tract infection and septicemia following which he developed ocular pain and redness. Further investigation revealed endophthalmitis with subretinal abscess and retinal detachment. The patient underwent pars plana vitrectomy with drainage of the abscess and silicone oil tamponade. The subretinal aspirate was found to contain R. mucosa confirmed on culture and PCR.ConclusionMicrobiological evaluation of the subretinal purulent material revealed pink-colored colonies. Nested PCR was positive for detection of the eubacterial genome as well as for detection of the Mycobacterium tuberculosis genome (Ref)-targeting MPB64 gene. PCR examination of the subretinal pus sample ruled out M. tuberculosis and confirmed R. mucosa. The occurrence of Roseomonas endogenous endophthalmitis presenting as a subretinal abscess has not yet been reported in English literature so far to the best of our knowledge.