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Biomedical subjects

H N Madhavan

Publications and source records attributed to H N Madhavan.

At least 19 recordsLinked to original sources
PubMed2007

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens.

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.

Cytomegalovirus↗
PubMed2006

Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus.

PURPOSE: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. MATERIALS AND METHODS: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. RESULTS: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. CONCLUSION: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.

Aqueous Humor↗
PubMed2006

Evaluation of three polymerase chain reaction tests targeting morphological transforming region II, UL-83 gene and glycoprotein O gene for the detection of human cytomegalovirus genome in clinical specimens of immunocompromised patients in Chennai, India.

BACKGROUND: Human Cytomegalovirus (HCMV) continues to be an important cause of morbidity and occasional mortality in immunocompromised patients. Polymerase chain reaction (PCR) is the most sensitive and commonly used method for the assessment of HCMV infection in the immunocompromised patients at risk from severe associated clinical manifestations. However, there is little consistency in the qualitative PCR used for different regions of HCMV genome. Therefore, the performance of three Qualitative PCR tests to detect HCMV genome in clinical specimens from immunocompromised patients was evaluated. With pp65 antigenemia assay as the "gold standard", nested PCR for morphological transforming region II (mtr II) and glycoprotein O (gO) gene and uniplex PCR for UL 83 gene were applied on 92 consecutive clinical specimens obtained from 74 immunocompromised patients with clinically suspected HCMV disease. Virus isolation was attempted on 12 clinical specimens from six pp65 antigenemia positive patients. Based on the pp 65 antigenemia results as "gold standard", the sensitivity, specificity, positive predictive value and negative predictive value for each PCR was calculated. RESULTS: The PCR targeting mtr II region showed a higher sensitivity (100%) and negative predictive value (100%) than the other two PCRs in detecting HCMV DNA from clinical specimens obtained from different immunocompromised patient population of Chennai region, India. CONCLUSION: The results suggests that the optimal method of detection of HCMV DNA could be achieved by PCR using primer sequences targeting mtr II region of genome of HCMV in Chennai region, India.

Antigens, Viral↗
PubMed2006

Failure to genotype Human Cytomegalovirus by PCR-RFLP method due to sequence variation within the primer binding site.

Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) is one of the rapid methods for genotyping Human Cytomegalovirus (HCMV). When genotyping clinical samples by a sensitive nested PCR-based RFLP method for the glycoprotein B (gB) gene of HCMV, it was found that some of the clinical specimens did not give an amplification signal. Analysis of the prototype sequences of the different genotypes showed base pair mismatches over the primer binding site. An alternative assay is suggested for genotyping of HCMV.

Binding Sites↗
PubMed2006

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 non-toxoplasma 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 Desmont's 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.

Animals↗
PubMed2006

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates.

PURPOSE: To standardize in-vitro antifungal susceptibility testing by agar dilution method to find out the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole and ketoconazole on ocular fungal isolates. METHODS: A total of 180 ocular fungal isolates (130 filamentous fungi and 50 yeasts) were included. The antifungal drugs such as amphotericin B (0.0625-8 microg/mL), fluconazole (0.2-819.6 microg/mL) and ketoconazole (0.025-6.4 microg/mL) were incorporated in doubling dilutions in the yeast nitrogen base medium. The MIC was determined as the lowest concentration of the antifungal drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug-free control plates. RESULTS: All 50 ocular isolates of yeast were susceptible to amphotericin B, while two (4%) and five (10%) strains were resistant to fluconazole and ketoconazole respectively. Of the 130 filamentous fungi tested, six (4.6%) were resistant to amphotericin B, 49 (37.7%) and 10 (7.6%) were resistant to fluconazole and ketoconazole respectively. Percentile 50 (MIC 50) and Percentile 90 (MIC 90) for all the three antifungal agents were calculated. Aspergillus niger, Aspergillus terreus and Candida krusei were found to be resistant to fluconazole and ketoconazole. CONCLUSION: This technique was found to be reliable, cost effective and easy to perform with consistent results.

Amphotericin B↗
PubMed2005

Chronic microsporidial stromal keratitis in an immunocompetent, non-contact lens wearer.

An 82-year-old healthy man with unilateral chronic stromal keratitis, initially diagnosed to have viral keratitis and refractory to medical therapy, showed numerous oval, microsporidial organisms, measuring 4-5 m in length in the corneal biopsy. Penetrating keratoplasty, followed by treatment with systemic albendazole and topical propamidine isethionate resulted in resolution of the infection. Electron microscopy of the keratoplasty specimen demonstrated sporoblasts with diplokaryotic nuclei and multiple coils of the filament. The light and electron microscopic features were consistent with microsporidial keratitis.

Aged↗
PubMed2005

Application of PCR-based restriction fragment length polymorphism for the identification of mycobacterial isolates.

BACKGROUND AND OBJECTIVE: Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level. METHODS: The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests. RESULTS: PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum. INTERPRETATION AND CONCLUSION: PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.

DNA Primers↗
PubMed2005

Application of nested polymerase chain reaction (nPCR) using MPB 64 gene primers to detect Mycobacterium tuberculosis DNA in clinical specimens from extrapulmonary tuberculosis patients.

BACKGROUND & OBJECTIVE: The conventional culture technique for diagnosis of extrapulmonary tuberculosis is time consuming. In order to find a sensitive and rapid technique nested polymerase chain reaction (nPCR) targeting the conserved MPB 64 gene of Mycobacterium tuberculosis was evaluated for detection of M. tuberculosis DNA directly from clinical specimens of extrapulmonary origin. METHODS: A total of 400 clinical specimens from clinically suspected cases of extrapulmonary tuberculosis and 30 control specimens of nontuberculous aetiology were processed by smear and culture and by nPCR technique for detection of M. tuberculosis. The specimens were divided into 3 groups, (group I--280 specimens [104 peritoneal fluid (PF), 120 cerebrospinal fluid (CSF), 44 lymph node biopsies 3 pericardial fluid and 9 other biopsy specimens], group II--120 aqueous humour (AH) from idiopathic granulomatous uveitis cases, and group III--30 control specimens (10 CSF and 20AH). RESULTS: The conventional culture was positive only in 16 of 400 specimens. The overall positivity of nPCR was 35.2 per cent (141/400). Among the 280 specimens from extrapulmonary lesions (group I), 15 were bacteriologically positive, while 115 of 265 bacteriologically negative specimens (43.4%) were positive by nPCR. All the 30 control specimens were negative by nPCR. INTERPRETATION & CONCLUSION: The nPCR using MPB64 gene primers might be a rapid and reliable diagnostic technique for detection of M. tuberculosis genome in clinically suspected extrapulmonary tuberculosis specimens, as compared to the conventional techniques.

Aqueous Humor↗
PubMed2005

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

PURPOSE: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. METHODS: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. RESULTS: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. CONCLUSIONS: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

DNA, Viral↗
PubMed2004

Efficacy and safety of 1% rimexolone versus 1% prednisolone acetate in the treatment of anterior uveitis--a randomized triple masked study.

PURPOSE: To evaluate the efficacy and safety of 1% rimexolone versus 1% prednisolone acetate ophthalmic suspension in the treatment of anterior uveitis. METHODS: A randomised triple masked, parallel comparison of rimexolone and prednisolone acetate ophthalmic suspensions was carried out on 78 patients with acute, chronic and recurrent anterior uveitis. Treatment regimen included instillation of one or two drops of drug one hourly through the waking hours during the first week, two hourly in the second week, four times a day in the third week, two times a day for the first 4 days and once a day for the 3 days in the last week. The patient was clinically evaluated on the 3-4th, 7-10th, 14th, 21st and 28th days. The patient was also reviewed on the 30th day. Anterior chamber cells and flare reactions were compared for evaluating the efficacy of the drugs. RESULT: Rimexolone is as effective as prednisolone acetate ophthalmic suspension in the treatment of anterior uveitis. The largest difference found was 0.1 in the flare reaction (statistically insignificant; p = 0.3) and 0.2 score units (statistically significant; p = 0.01) in the cells. Overall, comparison of the drugs shows no clinical significance in the treatment of anterior uveitis by either drug. Difference in intraocular pressure (IOP) was also statistically insignificant (p > 0.05). However, three patients in the prednisolone acetate group and 1 patient from the rimexolone group showed a rise in IOP. CONCLUSION: Rimexolone 1% ophthalmic suspension is as effective as and safer than prednisolone acetate 1% ophthalmic suspension in the treatment of anterior uveitis.

Acute Disease↗
PubMed2004

Current perspectives of herpesviral retinitis and choroiditis.

Vision-threatening viral retinitis are primarily caused by members of the herpesvirus family. The biology and molecular characterization of herpesviruses, clinical presentations of retinopathies, pathology and pathogenesis including the host responses, epidemiology and the laboratory methods of aetiological diagnosis of these diseases are described. Clinical syndromes are acute retinal necrosis (ARN), progressive outer retinal necrosis (PORN), cytomegalovirus (CMV) retinitis, multifocal choroiditis and serpiginous choroiditis besides other viral retinopathies. Herpes simplex virus (HSV) retinitis is more common in immunocompetent persons while varicella zoster virus (VZV) affects both immunocompetent and immunosuppressed patients equally. CMV retinitis is most common among patients with AIDS. The currently employed laboratory methods of antigen detection, virus isolation and antibody detection by enzyme linked immuno-sorbent assay (ELISA) have low sensitivity. Polymerase chain reaction (PCR) has increased the value of diagnosis due to its high clinical sensitivity and absolute specificity in detection of herpesviruses in intraocular specimens.

Choroiditis↗
PubMed2004

Polymerase chain reaction to detect Chlamydia trachomatis and adenovirus in the nasopharyngeal aspirates from paediatric patients with lower respiratory infections.

Polymerase chain reaction (PCR) was evaluated to detect Adenoviruses and Chlamydia trachomatis on nasopharyngeal aspirates (NPA) obtained 4-5 days after the onset of lower respiratory tract illness in children. Forty-five nasopharyngeal aspirates (NPA) from 45 children with lower respiratory tract infections were processed for the detection of C. trachomatis and Adenovirus by Fluorescent antibody test (FAT), culture and PCR for the cryptic plasmid of C. trachomatis and the gene coding for hexon of Adenoviruses. Seven (13.3%) and 4 (6.6%) of the 45 specimens were positive for C. trachomatis and adenovirus by PCR respectively, which included one specimen each positive for these agents. Cultures were negative for both the organisms. PCRs showed a statistically significant (McNemar test--p= 0.004) higher sensitivity. PCR test is necessary to detect C. trachomatis and adenovirus in nasopharyngeal aspirates obtained 4-5 days after the onset of illness.

Adenovirus Infections, Human↗
PubMed2003

Phenotypic and genotypic methods for the detection of herpes simplex virus serotypes.

Typing of herpes simplex virus (HSV) into its serotypes plays a major role in epidemiology and management of reactivation. To develop and evaluate a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was employed using Hae III and Taq I against neutralization test, allele-specific PCR and DNA sequencing for the detection of HSV serotypes. Neutralization test, allele-specific PCR, DNA sequencing and PCR-based RFLP were applied simultaneously to 2 standard strains (HSV-1 and HSV-2) and 23 clinical isolates. PCR-based RFLP was applied further to 20 culture negative PCR positive clinical specimens. The 179 bp product of the clinical isolates and specimens amplified using the type-common primers of HSV was subjected to DNA sequencing and PCR-based RFLP. Allele-specific PCR was absolutely specific and highly sensitive. All the typing methods differentiated concordantly 23 clinical isolates into 12 HSV-1 and 11 HSV-2. DNA sequencing did not reveal any nucleotide variations within the serotypes among the isolates sequenced. PCR-based RFLP typed a further 20 culture negative clinical specimens into 15 HSV-1 and 5 HSV-2. PCR-based RFLP was a reliable, less laborious and cost-effective molecular biological tool for the determination of HSV serotypes both for the clinical isolates and culture negative specimens.

Alleles↗