Enumeration of Mycobacterium leprae Using Real-Time PCR

Truman, Richard W.; Andrews, Preston K.; Robbins, Naoko; Adams, Linda B.; Krahenbuhl, James L.; Gillis, Thomas P.

doi:10.1371/journal.pntd.0000328

Cited by 117 publications

(117 citation statements)

References 21 publications

Supporting

Mentioning

105

Contrasting

Unclassified

Order By: Relevance

“…The sodA mRNA transcript, encoding superoxide dismutase A (ML0072c) of M. leprae, was selected as the mRNA target for this assay because of the gene transcripts tested, including hsp18 (ML1795), gyrA (ML0006), and rpoB (ML1891c); the sodA transcript levels remained stable for at least 48 h after harvesting from the MFP tissues but rapidly degraded after the death of M. leprae cells (data not shown). Also, since real-time RLEP DNA-based PCR was previously characterized to be a rapid and objective molecular enumeration tool for detecting and quantifying bacterial numbers in an M. leprae preparation (29), it was chosen to serve as a normalizer for these assays.…”

Section: Discussionmentioning

confidence: 99%

“…The levels of M. leprae sodA mRNA and 16S rRNA in M. leprae from axenic medium, from cultured M⌽, or in skin biopsy specimens were determined using real-time RT-PCR. These levels were normalized for bacterial numbers using a previously characterized, DNA-based, real-time PCR assay for RLEP (29). Primers and probes were designed using Primer Express 2.0 software (Applied Biosystems) ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Determination of Mycobacterium leprae Viability by Use of Real-Time PCR

et al. 2009

View full text Add to dashboard Cite

Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (M⌽), or within immune-activated M⌽. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.Leprosy is a chronic infectious disease of skin and peripheral nerves and is of special concern because it can progress to peripheral neuropathy and permanent progressive deformity. Despite a marked reduction in the prevalence of leprosy since the implementation of multidrug therapy (MDT), the detection rate for new cases has not shown a substantial decline (2). One explanation is that standard immunological and histological approaches for disease assessment are less effective in the diagnosis of early leprosy, and therefore, disease and transmission can progress. In addition, in vitro Mycobacterium leprae viability assays such as those based on radiorespirometry (RR) and the Live/Dead BacLight fluorescent bacterial viability assay require large quantities of bacteria, 10 7 and 10 6 bacteria, respectively, for reliable detection and are therefore not applicable for direct detection in clinical specimens (14, 28). The bacterial index (BI) is a long-established method for monitoring the patients' responses to chemotherapy by giving an estimation of the number of acid-fast bacilli present in skin smears of lesions and other specific sites of the skin of leprosy patients. The BI range is 1 to 6, where 1 is the least amount of bacilli detectable and 6 is the most. However, the BI drops very slowly during t...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Molecular Determination of Mycobacterium leprae Viability by Use of Real-Time PCR

et al. 2009

View full text Add to dashboard Cite

show abstract

“…DNA was not amplified by nested PCR of any of 17 other Mycobacterium species or 4 other bacterial species, and there was also no cross-reaction with DNA from 18 cases of TB. Truman and others 14 developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. The assay was specific and able to detect 10 fg purified M. leprae DNA or approximately 300 bacteria in infected tissues.…”

Section: Discussionmentioning

confidence: 99%

Whole-Blood Nested-PCR Amplification of M. leprae-Specific DNA for Early Diagnosis of Leprosy

Wen¹,

Xing²,

Yuan³

et al. 2013

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.25% (6/96) of HHC, but it was not detected in 18 TB or 35 normal controls. The sensitivities of the anti-bovine serum albumin (ND-O-BSA) immunoglobulin M (IgM) and antifusion protein of ML0405-ML2331 IgG for MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) had lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these promising findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases.

show abstract

“…Enumeration of M. leprae cells in mouse footpads. The number of M. leprae cells in footpad homogenates was assessed by DNA amplification, using real-time PCR with previously described primers and probes (62). Suspensions of nude mouse-derived M. leprae were used as a standard to establish relative numbers of M. leprae in tissues.…”

Section: Methodsmentioning

confidence: 99%

Vaccination with the ML0276 Antigen Reduces Local Inflammation but Not Bacterial Burden during Experimental M ycobacterium leprae Infection

et al. 2009

Self Cite

View full text Add to dashboard Cite

Leprosy elimination has been a goal of the WHO for the past 15 years. Widespread BCG vaccination and multidrug therapy have dramatically reduced worldwide leprosy prevalence, but new case detection rates have remained relatively constant. These data suggest that additional control strategies, such as a subunit vaccine, are required to block transmission and to improve leprosy control. We recently identified several Mycobacterium leprae antigens that stimulate gamma interferon (IFN-␥) secretion upon incubation with blood from paucibacillary leprosy patients, a group who limit M. leprae growth and dissemination. In this study, we demonstrate that M. leprae-specific mouse T-cell lines recognize several of these antigens, with the ML0276 protein stimulating the most IFN-␥ secretion. We then examined if the ML0276 protein could be used in a subunit vaccine to provide protection against experimental M. leprae infection. Our data demonstrate that combining ML0276 with either a Toll-like receptor 4 (TLR4) (EM005), TLR7 (imiquimod), or TLR9 (CpG DNA) agonist during immunization induces Th1 responses that limit local inflammation upon experimental M. leprae infection. Our data indicate that only the ML0276/EM005 regimen is able to elicit a response that is transferable to recipient mice. Despite the potent Th1 response induced by this regimen, it could not provide protection in terms of limiting bacterial growth. We conclude that EM005 is the most potent adjuvant for stimulating a Th1 response and indicate that while a subunit vaccine containing the ML0276 protein may be useful for the prevention of immune pathology during leprosy, it will not control bacterial burden and is therefore unlikely to interrupt disease transmission.Leprosy, which is caused by infection with Mycobacterium leprae, can manifest across a wide spectrum of disease symptoms. Through the use of clinical, histopathological, and immunological diagnoses, five forms of leprosy have been characterized: lepromatous (LL), borderline lepromatous (BL), mid-borderline, borderline tuberculoid (BT), and tuberculoid (TT) leprosy (53, 56). Multibacillary (MB) patients, encompassing the BB, BL, and LL forms, are characterized as having multiple skin lesions largely devoid of functional lymphocytes. At the extreme MB pole, LL patients demonstrate high titers of anti-M. leprae antibodies but an absence of specific cellmediated immunity (53). In the absence of a strong cellular immune response, LL patients do not control bacterial replication and have high bacterial indices (BI). It is still unclear why these patients do not mount effective cell-mediated immunity, but factors such as ineffective initial antigen presentation or priming of Treg cells may contribute. In marked contrast, paucibacillary (PB) leprosy patients, encompassing the BT and TT forms, are characterized as having one or few skin lesions and granulomatous dermatopathology with a low or absent BI. At the extreme PB pole, TT patients demonstrate specific cell-mediated immunity against M. leprae and have...

show abstract

Enumeration of Mycobacterium leprae Using Real-Time PCR

Cited by 117 publications

References 21 publications

Molecular Determination of Mycobacterium leprae Viability by Use of Real-Time PCR

Molecular Determination of Mycobacterium leprae Viability by Use of Real-Time PCR

Whole-Blood Nested-PCR Amplification of M. leprae-Specific DNA for Early Diagnosis of Leprosy

Vaccination with the ML0276 Antigen Reduces Local Inflammation but Not Bacterial Burden during Experimental M ycobacterium leprae Infection

Contact Info

Product

Resources

About