Balaji Nandagopal

SRI NARAYANI HOSPITAL & RESEARCH CENTER, Velluru, Tamil Nādu, India

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Publications (16)21.06 Total impact

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    ABSTRACT: Salmonella typhi, Mycobacterium tuberculosis, and Burkholderia pseudomallei are among the most important monocyte-tropic bacterial agents causing pyrexia of unknown origin (PUO), with a significant number of endemic infections in both South and Southeast Asian regions. These infections pose a major risk to travelers to these regions as well. We developed and evaluated a multiplex nested polymerase chain reaction (PCR) for the simultaneous detection of the three pathogens in 305 patients' buffy coat samples. The assay for S. typhi and B. pseudomallei was able to detect down to 1 colony forming unit/5 μL PCR input and M. tuberculosis was detected down to 20 genome copies/5 μL PCR input. S. typhi was detected in 10 (3.3 %) individuals, B. pseudomallei in 10 individuals (3.3 %), and M. tuberculosis in 18 individuals (5.9 %). Co-infections of M. tuberculosis and B. pseudomallei were detected in three individuals and S. typhi and B. pseudomallei in two individuals. This protocol is efficient for PUO diagnosis especially in Asian countries.
    Molecular diagnosis & therapy. 01/2014;
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    ABSTRACT: BACKGROUND: Typhoid fever is endemic in India, and a seasonal increase of cases is observed annually. In spite of effective therapies and the availability of vaccines, morbidity is widespread owing to the circulation of multiple genetic variants, frequent migration of asymptomatic carriers, unhygienic food practices and the emergence of multidrug resistance and thus continues to be a major public health problem in developing countries, particularly in India. Classical methods of strain typing such as pulsed-field gel electrophoresis, ribotyping, random amplification of polymorphic DNA and amplified fragment length polymorphism are either laborious and technically complicated or less discriminatory. METHODS: We investigated the molecular diversity of Indian strains of Salmonella enterica serovar Typhi (S. Typhi) isolated from humans from different parts of India to establish the molecular epidemiology of the organism using the variable number tandem repeat (VNTR)-PCR analysis. The electrophoretic band pattern was analysed using the GelCompar II software program. RESULTS: Of the 94 strains tested for three VNTRs loci, 75 VNTR genotypes were obtained. Of the three VNTRs tested in this study, VNTR1 was amplified in all the strains except one and found to be predominant. VNTR2 was amplified only in 57 strains with a Simpson diversity index of 0.93 indicating the high variability of this region within the strains. VNTR3 was amplified in 90 strains. The discriminatory power of this typing tool has been greatly enhanced by this VNTR2 region as the other two regions could not discriminate strains significantly. In our study, about 55 % of the strains amplified all three VNTR regions and 39 % of the strains lacked the VNTR2 region. Among the three VNTR regions tested, the majority of the strains produced similar banding pattern for any two regions grouped into a cluster. The strains grouped as a genotype were from the same geographical location. Strains collected from each geographical region were also highly heterogeneous. CONCLUSION: Such analysis is important to identify the genetic clones of the pathogen associated with sporadic infections and disease outbreak to identify the common source and implement public health measures.
    Molecular Diagnosis & Therapy 04/2013; · 2.59 Impact Factor
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    ABSTRACT: BACKGROUND: Acute respiratory tract infections (ARTIs) are one of the major causes of morbidity and mortality among young children in developing countries. Information on the incidence of human metapneumovirus (hMPV) and human bocavirus (HBoV) infections in developing countries, especially among rural children, is very limited. OBJECTIVES: This study was conducted to identify whether these viruses were associated with ARTI among children ≤5 years of age in rural and peri-urban populations in South India. METHODS: The study was cross-sectional with prospective sample collection. Oropharyngeal swabs were collected from children ≤5 years of age presenting with ARTI. None of the children in this study were known to have any immunosuppressive conditions. The two viruses, hMPV and HBoV, were identified using semi-nested polymerase chain reaction (PCR) assays and one-step PCR assays, respectively. The lower limits of detection of hMPV and HBoV were 6.69 × 10(5) plasmid copies and 5.77 × 10(3) plasmid copies, respectively, per 5 μL PCR reaction input. RESULTS: The frequency of hMPV infection in children was higher than that of HBoV infection. The different frequencies of hMPV in patients in various age groups with upper and lower respiratory tract infections were compared, and the variance was found to be insignificant. In the 38 children who were hMPV positive, the majority (73.7 %) were from rural communities. The overall hMPV-positive rate was higher in the rural population than in the peri-urban population, but the difference was statistically insignificant. The youngest age at which hMPV-positive status was recorded was 5 months. CONCLUSION: This study demonstrated that hMPV was associated with a significant number (i.e. >10 %) of ARTIs in children in South India, whereas a relatively smaller number of HBoV infections was observed.
    Molecular Diagnosis & Therapy 04/2013; · 2.59 Impact Factor
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    ABSTRACT: BACKGROUND: Human metapneumovirus (hMPV), which has a global distribution, is an important cause of acute respiratory tract infections, especially in children and immunocompromised patients. METHODS: We investigated the genetic variability of partial nucleoprotein (N) gene sequences of hMPV strains identified among young children in South India. The sequences of the N gene were compared with previously reported sequences available in the GenBank repository. RESULTS: The results showed that strains are localized in a geographically circumscribed area (topotype). The results also demonstrates that viruses from the same genetic lineage can circulate concurrently within a given location during a given season. The close clustering of the majority of our hMPV isolates indicates that the N gene sequences in the virus population are relatively homogeneous, and suggests temporal rather than geographic variations in the evolutionary pattern. In our study, the majority of the strains belonged to genetic lineage B2 (71.1 %), followed by A2b (18.4 %), A2a (7.9 %), and B1 (2.6 %), demonstrating the presence of 4 of the 5 known genotypes of hMPV. Global alignment of the nucleotide sequences showed that the strains are closely related to sequences from Canada, The Netherlands, and Australasia. Differences at the nucleotide level and the amino acid level were identified. The results provide evidence for the diversity of the N gene of hMPV in samples collected from South India compared with global strains. When investigated for selective pressure, the sequences showed 1 positively selected site and 19 negatively selected sites. CONCLUSION: These data should prove useful in further investigations of the evolutionary dynamics of hMPV infection.
    Molecular Diagnosis & Therapy 04/2013; · 2.59 Impact Factor
  • S Sankar, H Narayanan, S Kuppanan, B Nandagopal
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    ABSTRACT: Extended-spectrum β-lactamase (ESBL) producers were reported only from hospital settings previously, but, nowadays, its common presence in community settings is evident from reports. Our primary aim was to assess the frequency of ESBL-producing Gram-negative bacteria (GNB) and their antibiogram pattern among the clinical isolates received during an 8-month time period. The clinical isolates which belonged to the family Enterobacteriaceae from the clinical specimens were included in the study. These clinical isolates were tested for ESBL production using the double-disk synergy test. In total, 301 patients were included in this study, of which 146 (48.50 %) were found to harbor strains of ESBL producers. The acquisition of ESBL in relation to age, sex, inhabitancy, inpatients, and outpatients was also analyzed. In our study, 50.29 % of inpatients and 45.86 % of outpatients were found to harbor ESBL producers. The difference between the two groups was not statistically significant. We found five meropenem-resistant ESBL-producing strains among the 146 ESBL producers. Rural inhabitants were found to contain more ESBLs when compared to peri-urban inhabitants. The study showed a high frequency of ESBLs in both community-acquired and hospital-acquired infections. The frequency of ESBLs was higher among isolates from patients who were from rural populations than those from peri-urban populations. The data on ESBL frequency suggests the need for a rational antibiotic use which would reduce the spread of ESBL-producing members of the family Enterobacteriaceae.
    Infection 04/2012; 40(4):425-9. · 2.44 Impact Factor
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    ABSTRACT: Melioidosis and Brucellosis are important endemic infections among people in India, especially in rural settings. Conventional detection techniques have several limitations. Only a few studies exist on the prevalence of Melioidosis and Brucellosis in rural area especially in India. We sought to evaluate detection of Burkholderia pseudomallei and Brucella spp. among patients presenting febrile illness. Previously described polymerase chain reaction (PCR) assays for both pathogens were evaluated with Deoxyribonucleic acid extracts of buffy coat samples collected from 301 patients recruited prospectively. Data was not amenable to statistical analysis. The PCR showed specific amplification and no non-specific amplification with heterologous Gram-negative bacilli. The lower limit of detection of the assay for B. pseudomallei was determined to be 1 colony-forming unit /mL and for Brucella it was 1.95 × 10(3) plasmids per microliter. Blood culture in automated blood culture system was negative for all the samples. This prospective study carried out in southern India for the first time. PCR for Brucella was positive in 1% of the patient samples whereas 0.3% was positive for B. pseudomallei. The finding of Brucella and Burkholderia infections in our populations leads us to suggest that tests for Brucella and B. pseudomallei should also form part of a diagnostic platform for patients with Pyrexia of unknown origin in tropical developing countries.
    Journal of global infectious diseases 01/2012; 4(1):31-7.
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    ABSTRACT: The IS6110 belongs to the family of insertion sequences (IS) of the IS3 category. This insertion sequence was reported to be specific for Mycobacterium tuberculosis complex and hence is extensively exploited for laboratory detection of the agent of tuberculosis and for epidemiological investigations based on polymerase chain reaction. IS6110 is 1361-bp long and within this sequence different regions have been utilized as targets in the identification of M. tuberculosis by PCR. However, the results are not always consistent, specific and sensitive. In recent years, a few clinical investigations raised concerns over IS6110 specificity and sensitivity in the diagnosis of tuberculosis due to false-positive (homology with other target DNA besides M. tuberculosis) or false negative (due to absence of copies of IS6110) results with IS6110 specific primers. To unravel the variations in IS6110 sequences, an insilico analysis of IS6110 sequence of different strains of M. tuberculosis was carried out. Our results of comparative analysis of IS6110 insertion sequences of M. tuberculosis complex suggests that, IS6110 insertion sequences harbored variations in its sequence, which is evident from the phylogenetic analysis. Importantly, IS6110 sequence has divergence within the copies of same strain and formed different clusters. A list of IS6110 specific primers used in various clinical investigation of tuberculosis was obtained from the literature and their performance scrutinized. Our study emphasizes the need to develop PCR assays (multiplex format) targeting more than one region of the genome of M. tuberculosis.
    Bioinformation 06/2011; 6(7):283-5. · 0.50 Impact Factor
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    ABSTRACT: Infectious diseases are a major global public health problem. Multiple agents are now recognized to cause indistinguishable illnesses. The term 'syndrome' applies to such situations, for which early and rapid diagnosis of the infecting agent would enable prompt and appropriate therapy. Public health physicians would also get timely information on the specific etiology of the infectious syndrome, facilitating intervention at the community level in the face of outbreaks or epidemics. A variety of molecular techniques have been evaluated for rapid diagnosis of infectious syndromes. These techniques include real-time multiplex PCR, DNA microarray, loop-mediated isothermal amplification, and other similar assays. This review surveys such state-of-the-art technologies.
    Molecular Diagnosis & Therapy 06/2011; 15(3):145-58. · 2.59 Impact Factor
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    ABSTRACT: Tuberculosis is an under-recognized yet catastrophic health problem, particularly in developing countries. The HIV pandemic has served to increase the number of susceptible individuals, and multidrug-resistance and poor socioeconomic conditions also augment the prevalence and the consequences of the disease. To control the disease and its spread, it is vital that tuberculosis diagnostics are accurate and rapid. Whereas microscopy and culture have several limitations (low sensitivity is a problem for the former, while the latter has a delayed turnaround time), PCR-based techniques targeting regions of the Mycobacterium tuberculosis genome such as IS6110 have proved to be useful. The purpose of this review is to assess the use of PCR-RFLP, nested PCR and real-time PCR protocols and the choice of target regions for the detection of M. tuberculosis. Real-time PCR for the detection of M. tuberculosis target genes in clinical specimens has contributed to improving diagnosis and epidemiologic surveillance in the past decade. However, targeting one genome sequence such as IS6110 may not by itself be sufficiently sensitive to reach 100% diagnosis, especially in the case of pulmonary tuberculosis. Additional testing for target genome sequences such as hsp65 seems encouraging. An interesting approach would be a multiplex real-time PCR targeting both IS6110 and hsp65 to achieve comprehensive and specific molecular diagnosis. This technology needs development and adequate field testing before it becomes the acceptable gold standard for diagnosis.
    Molecular Diagnosis & Therapy 01/2011; 15(1):1-11. · 2.59 Impact Factor
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    ABSTRACT: Indian medicinal plants are now recognized to have great potential for preparing clinically useful drugs that could even be used by allopathic physicians. Traditionally, practitioners of Indian medicine have used plant products in powder, syrup or lotion forms, without identification, quantification and dose regulation, unlike their allopathic counterparts. The present review explores the immense potential of the demonstrated effect of Indian medicinal plants on microbes, viruses and parasites. In the present context, with the available talent in the country like pharmaceutical chemists, microbiologists, biotechnologists and interested allopathic physicians, significant national effort towards identification of an "active principle" of Indian medicinal plants to treat human and animal infections should be a priority.
    Indian journal of medical microbiology 01/2011; 29(2):93-101.
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    ABSTRACT: With 1.8 million new cases each year, India carries 20% of the global burden of tuberculosis, a situation that is now further exacerbated with the emergence of drug resistance. The current diagnostic technique suggested by the Government of India's Revised National Tuberculosis Control Programme is Ziehl-Neelsen staining of a sputum smear. This technique is known to be inadequate. The aim of this study was to evaluate nested PCR (nPCR) in the detection of pulmonary tuberculosis in sputum samples in comparison with conventional smear findings, in an effort to improve detection rates from those obtained by the smear-alone approach. Patients attending a tertiary-care hospital (situated in a rural area of Vellore district) with clinical suspicion of pulmonary tuberculosis were prospectively recruited from mid-April 2009 to mid-December 2009 and investigated. The sputum samples were stained by Ziehl-Neelsen staining for smear examination. DNA extracted from concentrated sputum was tested by nPCR, targeting the IS6110 sequence in the Mycobacterium tuberculosis genome. Among 84 patients tested (median age 45.5 years), 80.95% were from the rural community and 19.05% were from the peri-urban community. Seventeen patients (20.24%; mid-p 95% CI 31.5, 52.4) tested positive by the smear examination and 35 (41.67%; mid-p 95% CI 12.7, 29.8) tested positive by nPCR. The difference in detection rates was statistically significant (chi(2) = 9.02; p = 0.002). The kappa coefficient between smear findings and nPCR findings was 0.47, which was a statistically significant agreement (Z = 4.91; p < 0.0001). This report describes the molecular detection of M. tuberculosis in patients' sputum samples tested by the nPCR format, using IS6110 as a target sequence. A high prevalence of pulmonary tuberculosis was identified by the nPCR assay, which was shown to have a significantly higher detection rate than conventional smear staining.
    Molecular Diagnosis & Therapy 08/2010; 14(4):223-7. · 2.59 Impact Factor
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    ABSTRACT: Fever is one of the most common illnesses in all age groups in India. Typhoid fever is a continuing problem in developing countries such as India, which has poor sanitation facilities. The diagnosis of typhoid fever is still made by conventional culture-based isolation and identification. Serologic diagnostic tests, though widely used, have many deficiencies. Our objective was to investigate a molecular nested polymerase chain reaction (nPCR) technique to detect Salmonella typhi among patients with febrile illness in rural and peri-urban communities in Vellore district (Tamil Nadu, India). nPCR targeting the flagellin gene (fliC) was carried out using HotStar Taq DNA polymerase on DNA extracted from the buffy coat fraction of blood samples. Blood culture was done in a completely automated blood culture system, BacT/Alert(R), on prospectively collected blood samples. Relevant clinicopathologic data were obtained. nPCR was found to have a lower limit of detection of 0.01 colony-forming units per milliliter. The prevalence of typhoid fever as estimated by nPCR was 4.7% in pyrexia of unknown origin (PUO) in the rural/peri-urban communities of Vellore district. The prevalence of S. typhi as estimated by blood culture was 2.0%, which was lower than the nPCR estimation. nPCR had sensitivity and specificity of 100% and 97.3%, respectively. The observed agreement between blood culture and nPCR was 0.973 and the Kappa coefficient was 0.59 (p < 0.0001). The frequency of typhoid fever as detected by nPCR was 4.35% among rural patients and 5.32% among peri-urban individuals. nPCR on DNA extracts of buffy-coat samples using HotStar Taq was found to be a valuable and specific technique for diagnosis of typhoid fever.
    Molecular Diagnosis & Therapy 04/2010; 14(2):107-12. · 2.59 Impact Factor
  • Kavitha Balaji, Sathish Sankar, Balaji Nandagopal
    Indian Journal of Community Medicine 04/2010; 35(2):362-4.
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    ABSTRACT: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.
    Indian journal of medical microbiology 01/2010; 28(3):227-32.
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    ABSTRACT: We investigated the faecal carriage of extended spectrum β-lactamases (ESBL) producing Escherichia coli in different groups of human subjects and in the environment. A total of 363 E. coli strains were isolated from stool samples of patients (n = 77), healthy subjects (n = 170) and from different environmental samples (n = 116). A total of 124 ESBL producing E. coli strains were isolated in this study. The frequency of ESBL producing E. coli was found to be highest (60.3%) among the strains isolated from patients, followed by healthy individuals (38%) and the environment (10.5%). The environment was observed to have a very low number of ESBL producing E. coli.
    Indian journal of medical microbiology 32(2):172-4.
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    ABSTRACT: Tuberculosis remains an important health problem all over the world, especially in resource poor settings like India. The Ziehl-Neelsen (ZN) staining of sputum smear is still the method of choice in the diagnosis of tuberculosis in spite of its low sensitivity and specificity. This paper evaluates comparison of two different polymerase chain reaction (PCR) assays with sputum smear findings to detect Mycobacterium tuberculosis. A total of 191 sputum samples were collected from 84 patients attending a tertiary care hospital, who were suspected of having pulmonary tuberculosis, were examined by PCR targeting two different genomic regions, namely, TRC 4 by non-nested format and IS6110 insertion element by nested format in comparison to ZN staining of sputum smears. Among the patients tested, 20.24% (Mid-p 95%CI: 31.5-52.4) were smear positive, 7.14% (Mid-p 95%CI: 2.94-14.26) were positive by TRC 4 PCR and 41.67% (Mid-p 95%CI: 12.7-29.8) were positive by IS6110 nested PCR (nPCR). The median age of overall positive cases was 42 years. Among the nPCR positives, the median for age of rural and peri-urban community was 46 and 32 years, respectively. The kappa coefficient between smear findings and TRC4 PCR findings was 0.27 and an agreement of 0.83 was observed (Z = 2.99; one-tailed P = 0.001). TRC 4 PCR picked two unique positives that were negative by smear and IS6110 nPCR. The non-nested TRC 4 PCR showed inability for accurate detection of M. tuberculosis in sputum samples. The study concluded that the nPCR targeting IS6110 is superior and more sensitive than TRC 4 PCR.
    Indian journal of medical microbiology 28(4):303-7.