Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

Nirmala, Lini; N, Shankernarayan; Dharmalingam, Kuppamuthu

doi:10.1099/jmm.0.007252-0

Cited by 30 publications

(26 citation statements)

References 32 publications

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“…Fortunately, in recent years, several molecular techniques based on PCR have become available that are helpful in the diagnosis of leprosy based on detection of M. leprae DNA in clinical samples. 10,14,18,19 In this study, we developed and validated a real-time PCR assay and showed that this test could rapidly detect M. leprae DNA from formalin-fixed paraffin-embedded specimens.…”

Section: Discussionmentioning

confidence: 98%

Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

Wen¹,

Xing²,

Yuan³

et al. 2014

The American Society of Tropical Medicine and Hygiene

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Abstract. The TaqMan real-time polymerase chain reaction (PCR) assay was evaluated systematically with respect to the standard curve, linear range, and used for detecting Mycobacterium leprae DNA in paraffin-embedded skin biopsy specimens from 60 confirmed leprosy patients and three healthy individuals and 29 other dermatoses and bacterial DNA from 21 different species. The test was further evaluated with 51 paucibacillary (PB) patients. The results showed that the test had good sensitivity (8 fg) and good specificity with no cross-reactivity with 21 other bacterial species and the control specimens, except one with Xanthomatosis. The real-time PCR detection rate for the 51 PB specimens was 74.5% (38 of 51). We conclude that the real-time PCR test is a useful adjunct test for diagnosing early stage or PB leprosy cases.

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Section: Discussionmentioning

confidence: 98%

Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

Wen¹,

Xing²,

Yuan³

et al. 2014

The American Society of Tropical Medicine and Hygiene

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“…However, correlation between disease severity and qPCR quantification of bacterial infections in clinical samples from animals and humans has been reported for Borrelia burgdorferi [15], Mycoplasma genitalium [16], Brucella spp. [17], Helicobacter pylori [18], Mycobacterium leprae [19,20], Mycoplasma gallisepticum [21], Streptococcus pneumoniae [22], Brucella melitensis [23], Haemophilus influenzae [24] and Legionella penumophila [25]. Further, clinical cut-off levels for bacterial load has been established for Streptococcus pneumoniae [22,26], Haemophilus influenzae [24], Mycobacterium tuberculosis [27], Gardnella vaginalis and Atopobium vaginae [28].…”

Section: Discussionmentioning

confidence: 99%

Association between faecal load of lawsonia intracellularis and pathological findings of proliferative enteropathy in pigs with diarrhoea

et al. 2012

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BackgroundThe study was designed to investigate correlation between histological findings of Lawsonia intracellularis in porcine cases of diarrhoea and the quantitative detection of Lawsonia intracellularis in faeces. A total of 156 pigs (10 to 70 days post weaning) with diarrhoea were randomly selected from 20 herds: The pigs were subjected to necropsy, histopathology, immunohistochemistry and faecal quantification of Lawsonia intracellularis by real time PCR.ResultsThe median Lawsonia intracellularis excretion was significantly higher in pigs with gross lesions of proliferative enteropathy (median excretion: 5.92 log10 bacteria/g faeces) compared to pigs without gross lesions of proliferative enteropathy (median excretion: <3.3 log10 bacteria/g faeces) (P<0.001). Spearman’s correlation coefficient between the measureable PE lesions and L. intracellularis excretion was 0.50 (P<0.001). A significantly increasing trend in Lawsonia intracellularis excretion level for increasing proliferative enteropathy histopathology and immunohistochemistry scores was demonstrated (P<0.001; P<0.001). Spearman’s correlation coefficient between the histopathology scores and L. intracellularis excretion was 0.67 (P<0.001). Spearman’s correlation coefficient between the IHC scores and L. intracellularis excretion was 0.77 (P<0.001).ConclusionsThe histological and quantitative PCR detection of Lawsonia intracellularis were correlated in pigs with diarrhoea. Overall the results suggest that clinically important levels for Lawsonia intracellularis excretion in faeces may be established. Such clinical threshold levels may be used in practice to confirm a diagnosis of Lawsonia intracellularis associated diarrhoea.

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“…A recent and similar approach to monitor the effectiveness of chemotherapy using hsp18 as the gene target was developed by Lini and coworkers [75]. The copy number of bacterial DNA and hsp18 mRNA was estimated from 47 leprosy patients during treatment using paraffin-embedded biopsy samples.…”

Section: Pcr For Diagnosis Of Difficult Casesmentioning

confidence: 99%

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

Talhari²,

et al. 2014

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In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.

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Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

Cited by 30 publications

References 32 publications

Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

Association between faecal load of lawsonia intracellularis and pathological findings of proliferative enteropathy in pigs with diarrhoea

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

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