Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

Kalghatgi, AT; Praharaj, Ashok Kumar; Sahni, A.K.; Pradhan, D.; Kumaravelu, S; Prasad, PL; Nagendra, A

doi:10.1016/s0377-1237(08)80141-4

Cited by 9 publications

(5 citation statements)

References 20 publications

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“…This is probably due to similar circulating strains spreading over the South-East Asia [28]. For Alu1 , Kalghatgi et al performed an experiment to differentiate K. pneumoniae from other pathogenic bacteria using some restriction enzymes where 60% of K. pneumoniae isolates were digested by Alu1 and showed band at 476 bp, 220 bp and 65 bp [29]. In this study, 63.4% of isolates were sensitive to Alu1 and displayed similar banding pattern like earlier study (476 bp and 220 bp) [29].…”

Section: Discussionmentioning

confidence: 99%

“…PCR alone or sometimes in combination with RFLP has been extensively used for precise detection and analysis of pathogens for many years [8]. Traditional culture based technologies are time consuming, labor intensive, and sometimes frequent use of antibiotics may affect culture positive isolates thus difficult to interpret data correctly [9].…”

Section: Introductionmentioning

confidence: 99%

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Molecular detection and PCR-RFLP analysis using Pst 1 and Alu 1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh

Mahmudunnabi

Majlish

Momtaz

et al. 2018

Journal of Genetic Engineering and Biotechnology

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Klebsiella pneumoniae is the second leading causative agent of UTI. In this study, a rapid combined polymerase chain reaction and restriction fragment length polymorphism analysis was developed to identify K. pneumoniae in women, infected with urinary tract infection in the Sylhet city of Bangladesh. Analysis of 11 isolates from women at the age range of 20–55 from three different hospitals were done firstly by amplification with K. pneumoniae specific ITS primers. All of the 11 collected isolates were amplified in PCR and showed the expected 136 bp products. Then, restriction fragment length polymorphism analysis of 11 isolates were conducted after PCR amplification by 16s rRNA universal primers, followed by subsequent digestion and incubation with two restriction enzymes, Pst1 and Alu1. Seven out of 11 isolates were digested by Pst1 restriction enzymes, six isolates digested by Alu1, and while others were negative for both enzymes. Data results reveal that, women at age between 25 and 50 were digested by both enzymes. A woman aged over than 50 was negative while bellow 20 was digested by only Pst1. The results could pave the tactic for further research in the detection of K. pneumoniae from UTI infected women.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular detection and PCR-RFLP analysis using Pst 1 and Alu 1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh

Mahmudunnabi

Majlish

Momtaz

et al. 2018

Journal of Genetic Engineering and Biotechnology

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“…Body fluids, especially the CSF from child, present a diagnostic challenge due to the small sample volume and paucity of slow growing or uncultivable organisms those results in lower test sensitivity [23, 24]. This study was carried out with the objectives to recommend a rapid screening system of identifying common encephalitis pathogens in CSF by means of advanced fragment analysis (AFA).…”

Section: Discussionmentioning

confidence: 99%

Detection of varicella-zoster virus from cerebrospinal fluid using advanced fragment analysis in a child with encephalitis: a case report

Song

Jiang

et al. 2019

BMC Infect Dis

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Background Varicella zoster virus (VZV) encephalitis is an infectious inflammatory disease of brain that can cause irreversible mental damage without timely treatment. In fact, many viruses can cause encephalitis, and the viral loads in cerebrospinal fluid (CSF) in the early stage of the disease are usually too low to be detected. Here we report a case of VZV encephalitis diagnosed by advanced fragment analysis (AFA), which could potentially to contribute to early diagnosis of VZV central nervous system (CNS) infections with a small volume of CSF samples. Case presentation A 10-year-old boy was admitted to the hospital with obvious neurological symptoms of headache, dizziness and vomiting for one day. Physical examination showed left facial paralysis. Complete blood count (CBC) test only showed an unspecific inflammation, and the culture of cerebrospinal fluid and microscopic staining examination were all negative. AFA was performed to screen the common 18 encephalitis related pathogens in CSF. Obvious VZV DNA fragments were observed by capillary electrophoresis at 160 nt, suggesting the existence of VZV CNS infection in children. The results were consistence with real-time quantitative PCR and concomitant symptoms in the acute stage of the disease. Conclusions We report a case of acute VZV encephalitis in a child without obvious skin manifestations, which was rapidly diagnosed by AFA. Overall, we would recommend the use of AFA analysis as the rapid screening system for the identification and differentiation of encephalitis pathogens in children.

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“…For PCR analysis, a eubacterial broad-range and M. tuberculosis PCR assay based on a previously published primer pair [18,19] was adapted to a D-PCR protocol. The protocol took advantage of competitive DNA amplification due to which when M. tuberculosis was present, amplification of smaller and repetitive units of IS6110 region was favoured in spite of presence of 16SrDNA sequence.…”

Section: Discussionmentioning

confidence: 99%

“…Identification of non-TB bacterial organisms was done by using a broad range primers [18] Ul-5 0 -CCA GCA GCC GCG GTA ATA CG-3 0 , and U2-5 0 -ATC GG(C/T) TAC CTT GTT ACG ACT TC-3 0 . Identification of M. tuberculosis was done using a specific pair of primers designed to amplify an insertion sequence IS6110 in the M. tuberculosis complex and the expected band size was about 123-bp.…”

Section: Primersmentioning

confidence: 99%

Rapid Diagnosis and Simultaneous Identification of Tuberculous and Bacterial Meningitis by a Newly Developed Duplex Polymerase Chain Reaction

et al. 2015

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The present study describes the development and evaluation of a duplex polymerase chain reaction (D-PCR) for diagnosis and simultaneous identification of tuberculous meningitis (TBM) and bacterial meningitis (BM) in a single reaction. A D-PCR with primers amplifying portions of the Mycobacterium tuberculosis IS6110 and the eubacteria 16SrDNA sequence in a same reaction mix was developed and tested on DNA extracted from 150 clinical CSF samples from different categories (TBM = 39, BM = 26, control infectious and non-infectious category = 85). The results indicate a clear differentiation between bands for eubacteria and M. tuberculosis with an analytical sensitivity of 10 3 cfu/ml for eubacteria and 10 2 cfu/ml for M. tuberculosis. When evaluated in clinical samples, D-PCR overall diagnosed 100 % confirmed TBM and 100 % confirmed BM cases with overall specificity of 96.5 %. D-PCR can be an effective tool for diagnosis and simultaneous identification of TBM or BM in a single PCR reaction. It saves time, cost, labour and sample amount and help in administration of appropriate antimicrobial therapy.The proposed diagnostic assay would be helpful in correct and rapid management of TBM and BM patients.

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Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

Cited by 9 publications

References 20 publications

Molecular detection and PCR-RFLP analysis using Pst 1 and Alu 1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh

Molecular detection and PCR-RFLP analysis using Pst 1 and Alu 1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh

Detection of varicella-zoster virus from cerebrospinal fluid using advanced fragment analysis in a child with encephalitis: a case report

Rapid Diagnosis and Simultaneous Identification of Tuberculous and Bacterial Meningitis by a Newly Developed Duplex Polymerase Chain Reaction

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