ABSTRACT.
Pure neuritic leprosy (PNL) often remains underdiagnosed due to the lack of simple, reliable diagnostic tools to detect Mycobacterium leprae. This study aimed to investigate the utility of multiplex polymerase chain reaction (MPCR) in easily accessible and less invasive biopsy sites, including skin biopsy samples and nasal swabs (NSs), to detect M. leprae. A total of 30 (N = 30) clinically suspected and untreated patients with PNL were recruited. Nasal swabs and skin biopsy samples from the innervation territory of an “enlarged nerve” were collected. DNA was extracted and subjected to MPCR (targeting leprae-specific repetitive element [RLEP], 16S rRNA, and SodA genes) and RLEP-PCR (individual gene PCR). The PCR products were analyzed by 3% agarose gel electrophoresis. In 30 patients with clinically suspected PNL, 60% (N = 18) of skin biopsy samples and 53% (N = 16) of NSs were found positive for M. leprae DNA by MPCR, whereas only 23.3% (N = 7) of skin biopsy samples and 10% (N = 3) of NSs were found positive by RLEP-PCR. MPCR demonstrated a greater positivity rate than did RLEP-PCR for detection of M. leprae. Serologic positivity for anti-natural disaccharide-octyl conjugated with bovine serum albumin (ND-O-BSA) antibodies was 80% (16/20), including 35% (7/20) of PNL patients for which the skin MPCR was negative. Both serologic positivity and skin MPCR positivity were observed in 65% of patients (N = 20). Multiplex polymerase chain reaction is a useful tool for detection for M. leprae in skin biopsy samples and NSs in clinically suspected cases of PNL, with the added advantages of being less invasive and technically easier than nerve biopsy.
INTRODUCTION
Pure neuritic leprosy (PNL) is a distinct subset of leprosy frequently seen in India. Pure neuritic leprosy is difficult to diagnose because of the absence of skin lesions and acid-fast bacilli (AFB) in slit skin smears (SSS).1 The criteria for diagnosis, proposed by Jardim et al.,2 are not routinely practiced by clinicians in developing countries as they involve invasive peripheral nerve biopsy for histopathological examination and demonstration of Mycobacterium leprae DNA in the specimen. However, detection of M. leprae is difficult, and histological findings may be nonspecific as the nerve involvement is patchy.3 Furthermore, nerve biopsy is a skill-based invasive procedure possible only at specialized centers. It is also limited by sampling errors, low sensitivity, and sometimes permanent nerve deficit. Thus, diagnosis of PNL has remained subjective because of overdependence on clinical expertise and a lack of simple and reliable diagnostic methods.
In the present era, molecular techniques are being applied to diagnose diseases with higher diagnostic positivity and efficacy. Multiplex polymerase chain reaction (MPCR), an alternative form of PCR, employs more than one gene specific to target DNA and thereby increases the efficacy of conventional PCR. Its diagnostic utility has already been investigated for detecting M. leprae in leprosy cases belonging to different disease spectra, including indeterminate, paucibacillary (PB), and multibacillary (MB) leprosy, as well as in household contacts and also for a variety of clinical samples (SSS, nerve biopsy samples, blood, nasal swabs [NSs], and saliva).4–8
The present study aimed to investigate the diagnostic utility of MPCR in patients with clinically suspected PNL by using three different gene targets, namely, the leprae-specific repetitive element (RLEP), 16S rRNA, and SodA genes, to identify M. leprae in their skin biopsy samples and NSs.
MATERIALS AND METHODS
This was a prospective, observational study conducted in 30 clinically suspected and untreated patients with PNL over a period of 2 years. Untreated patients with PNL with complaints of hypoesthesia/anesthesia, dysesthesia with/without loss of muscle strength in hands and feet (with or without motor impairment or loss of function), and thickening of innervating nerves but showing no skin lesions suggestive of leprosy were included.1 Patients with other diseases known to cause peripheral neuropathy, such as alcoholism, diabetes, HIV infection, thyroid disorders, metabolic disturbances, or systemic vasculitis, as well as patients with limited cognitive ability who were unable to respond adequately to sensory testing were excluded. In addition to a detailed history and clinical examination (both cutaneous and neurological), SSS testing was performed in all these patients as a part of the routine diagnostic workup. Patients with AFB demonstrated in SSS were excluded from the study. Nerve conduction studies of main peripheral nerve trunks were performed in all patients to establish the pattern of neuropathy. All patients diagnosed with PNL were started on the 12-month WHO multibacillary multidrug therapy (WHO MB-MDT) regimen.9 Patients having acute neuritis or silent nerve function impairment of less than 6 months duration were treated with oral prednisolone per standard guidelines.
Sample collection.
Punch skin biopsy (5 mm) from the area of sensory loss supplied by the clinically involved peripheral nerve trunk was performed in all patients at the time of enrollment. The biopsy sample was divided into two halves: one was collected in 70% ethanol for PCR, and the other half was formalin fixed and paraffin embedded using standard techniques for histopathologic examination (HPE). Genomic DNA was extracted from the skin biopsy samples by using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) per the manufacturer’s instructions.
Nasal swabs were collected from the surface of the turbinate bone of the nasal septum of both nostrils with sterilized wet cotton swabs which had been soaked earlier in sterilized normal saline (0.9% [wt/vol] NaCl).10 The swab cotton was stored in microcentrifuge tubes at −20°C until further processing. Swabs from both nostrils of an individual were lysed together in one tube and processed for DNA extraction by a method described by Jadhav et al.11 Serum for IgM anti-ND-O-BSA ELISA and the skin biopsy sample for histopathological examination were collected from only 20 patients. Figure 1A describes the investigations done and samples collected from patients with PNL for this study.
Figure 1.
(A) Flow chart depicting methodology. (B) Clinical photographs showing WHO grade 2 deformities in hands and feet of pure neuritic leprosy (PNL) patients. (C) Results of multiplex polymerase chain reaction (MPCR) and IgM anti-ND-O-BSA serology in PNL patients (N = 20); the dashed horizontal line represents the cutoff value (0.66) for IgM anti-ND-O-BSA antibodies; each dot above this line represents one PNL patient, each showing serological positivity for IgM anti-ND-O-BSA antibodies per comparison.
MPCR and RLEP-PCR for detection of M. leprae DNA.
Multiplex PCR and RLEP-PCR were done for all patients on both skin biopsy samples and NSs. Multiplex PCR using three genes, i.e., the RLEP, 16S rRNA and SodA genes, and RLEP-PCR were done as per methods/protocols described earlier.5,11 Each batch of samples was set up with one positive control (i.e., genomic DNA from M. leprae strain NHDP-63 [kindly provided by the Biodefense and Emerging Infections Research Resources Repository; https://www.beiresources.org/]) and one negative control (i.e., molecular biology-grade water) as shown in Supplemental Figure 1. PCR products were confirmed using 4% agarose for MPCR and 2% agarose for RLEP-PCR by gel electrophoresis. The representative images of agarose gel electrophoresis of MPCR and RLEP-PCR are given in Supplemental Figure 1A and B.
IgM anti-ND-O-BSA ELISA.
Serology for IgM anti-ND-O-BSA was done for a subset of patients (20/30) by using the protocol described by Sinha et al. with minor modification.12 Briefly, microplates were coated with 1 µg/mL of natural disaccharide-octyl conjugated with bovine serum albumin (ND-O-BSA) in carbonate buffer for 18 hours at 37°C. Blocking was done with 3% bovine serum albumin (BSA)–phosphate-buffered saline. Diluted serum (1:100) was added and incubated at 37°C for 2 hours. Peroxidase-conjugated anti-human IgM (1:2,000) was added to each well, followed by substrate solution (orthophenylene diamine plus H2O2). The reaction was stopped with 3 M H2SO4. Absorbance was recorded at 492 nm using a Multiskan Fc microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA). A cutoff value of 0.66 has already been previously established in our laboratory. This cutoff value was calculated by the addition of a mean optical density (OD) value obtained from healthy controls with 2 SD (mean OD of healthy individuals plus 2SD) using sera from 30 healthy subjects. Optical densities greater than this cutoff value were considered positive and those lower than this value were considered negative for IgM anti-ND-O-BSA antibodies.
STATISTICAL ANALYSES
The collected data were entered into a database and analyzed using Microsoft Excel and the statistical software IBM SPSS Statistics 22.0 (SPSS, Chicago, IL). Descriptive statistics such as mean, median, and SD were calculated for continuous variables such as age and duration of disease. Frequencies and percentages were calculated for categorical variables such as gender, presenting complaints, pattern of neurological involvement, disability grading, and MPCR positivity on skin biopsy samples and NSs. Logistic regression analysis was done for correlations between skin MPCR positivity and number of nerves involved.
RESULTS
Demographic details and clinical characteristics (N = 30).
Overall, 30 patients with suspected and untreated PNL were recruited. The mean age of patients was 39.7 (±15.45) years, ranging between 18 and 65 years (Table 1). The male-to-female ratio was 2.7:1 (73.3% and 26.6%, respectively). The mean duration of illness was 26.6 months (range, 3 months to 15 years). A history of leprosy contact was found in three cases (10%). Grade 2 disability (24 [80%]) was noted to be more common than grade 1 disability (6 [20%]) (Figure 1B). Fourteen of the 30 patients (46.6%) presented with sensory complaints in the hands and feet with negative sensory symptoms (hypoesthesia). Motor weakness was the presenting complaint in 8 (26.6%) patients, while trophic ulceration was the main symptom in 9 (30%) patients. The pattern of neurological involvement showed mononeuropathy in 7 (23.3%), mononeuropathy multiplex in 9 (30%), and polyneuropathy in 14 (46.6%) patients. Nerve thickening on manual palpation was seen as involvement of the ulnar nerve in 21 (70%), the common peroneal nerve in 20 (66.6%), the median nerve in 10 (33.3%), the radial cutaneous nerve in 10 (33.3%), the sural nerve in 8 (26.6%), the posterior tibial nerve in 2 (6.6%), and other nerves in 3 (10%) patients (Supplemental Figure 2).
Table 1.
Basic clinicodemographic characteristics of the study participants (N = 30)
| S. No. | Parameter | n | % | |
|---|---|---|---|---|
| 1 | Age at presentation (mean) | 39.77 years | – | – |
| 2 | Male:female ratio | 2.75:1 | – | – |
| 3 | Duration of illness (mean) | 3 months–15 years (26.66 months) | – | – |
| 4 | Epidemiological nexus (any leprosy contact) | 3 | 10.0 | |
| 5 | WHO disability grade | Grade 1 | 6 | 20.0 |
| Grade 2* | 24 | 80.0 | ||
| 6 | Chief complaints | Sensory symptoms | ||
| Hypoesthesia | 14 | 46.6 | ||
| Numbness | 6 | 20.0 | ||
| Paresthesia (tingling) | 3 | 10.0 | ||
| Motor symptoms | ||||
| Weakness | 5 | 16.6 | ||
| Muscle wasting (atrophy) | 3 | 10.0 | ||
| Trophic injuries/ulcers | 9 | 30.0 | ||
| 7 | Disabilities | Hands | 16 | 53.3 |
| Feet | 21 | 70.0 | ||
| Eyes | 0 | 0.0 | ||
| 8 | Neurological examination | Impairment in sensory examination only (thermal, pain, and tactile sensations) | 24 | 80.0 |
| Impairment in both sensory and motor examination | 6 | 20.0 | ||
| Nerve thickening | 30 | 100.0 | ||
| 9 | Pattern of neurological involvement | Mononeuropathy | 7 | 23.3 |
| Mononeuropathy multiplex | 9 | 30.0 | ||
| Polyneuropathy | 14 | 46.6 |
Grade 2 disabilities include visible deformity or damage of hands or feet (including trophic ulcers). S. No. = serial number.
Nerve conduction studies revealed five patterns of nerve involvement in terms of type and number of nerves involved (Supplemental Table 1). Asymmetrical sensory motor axonal mononeuropathy was noted in 9 patients (30%), asymmetrical sensory motor axonal polyneuropathy was noted in 12 patients (40%), symmetrical sensory motor axonal polyneuropathy was noted in 6 patients (20%), symmetrical sensorimotor axonal and demyelinating neuropathy was noted in 2 patients (6.7%), and normal study was seen in 1 patient (3.3%).
MPCR and RLEP-PCR in skin biopsy samples and NSs (N = 30).
By RLEP-PCR, the diagnostic positivity for M. leprae DNA detection was 23.3% (n = 7/30) for skin biopsy samples and 10% (n = 3/30) for NS samples. To test the efficiency of MPCR over that of RLEP-PCR, MPCR was performed with the same clinical samples. Using MPCR, the rate of M. leprae DNA detection was increased to 60% (n = 18/30) for skin biopsy samples and to 53% (n = 16/30) for NS samples (Supplemental Figure 3). No correlation was found between skin MPCR positivity and number of nerves involved (P value = 0.519) on logistic regression analysis.
Level of IgM against ND-O-BSA in PNL cases (N = 20).
Serologic positivity for anti-ND-O-BSA antibodies was found in 16 out of 20 PNL patients whose sera were tested, including 7 patients who were skin MPCR negative for M. leprae DNA.
Comparison of MPCR results with IgM anti-ND-O-BSA levels (N = 20).
A comparison of MPCR results with IgM anti-ND-O-BSA levels could be made in only 20 patients, for whom results of both IgM anti-ND-O-BSA serology and MPCR were available. IgM anti-ND-O-BSA serology showed a higher (80%; 16/20) positivity rate than skin MPCR did (65%; 13/20), as shown in Figure 1C.
Histopathologic examination of skin biopsy samples (N = 20).
Skin biopsy samples taken from apparently healthy looking skin in the area of sensory loss for HPE were available in 20/30 cases only. All cases showed normal epidermis and mild perivascular lymphocytic infiltrate in the upper dermis. There was no evidence of granuloma, foam cell infiltrate, or any perineural infiltrate. Fite stain for M. leprae bacilli was negative in all cases.
Comparison of MPCR results with HPE of skin biopsy samples (N = 20).
Multiplex PCR analysis of skin biopsy samples was positive in 60% (12/20) of PNL cases despite the absence of diagnostic histopathological features of leprosy and AFB on Fite staining.
DISCUSSION
Timely diagnosis of PNL is essential to prevent irreversible nerve damage and minimize disability in affected individuals. Pure neuritic leprosy primarily targets peripheral nerves, leading to sensory and motor deficits. Prompt identification and treatment can help halt the progression of nerve damage, alleviate symptoms, and improve the overall quality of life for patients. Assessing dermato-neurological changes in suspected PNL patients is complicated because of the subjectivity and variations in both inter- and intraindividual assessments. This challenge is especially exacerbated in PNL cases where nerve enlargement is equivocal and there are no observable skin lesions.
The present study investigated the utility of MPCR for diagnosis of PNL by detecting M. leprae in the DNA extracted from skin biopsy samples and NSs. Skin biopsy is the easiest tool at disposal, and performing an additional investigation of MPCR increases the pickup rate of diagnosis manifold as elucidated in our study.
A few studies have explored the potential of fine needle aspiration cytology (FNAC) as a less invasive alternative to nerve biopsy, which, however, needs complementary diagnostic methods, such as conventional acid-fast staining and MPCR, to enhance the accuracy of diagnosis in PNL patients.13–16 Reja et al. have shown improved rates of M. leprae detection by using MPCR in comparison to conventional acid-fast staining (84.6% versus 38.4%) in nerve tissue FNAC aspirates.16
There are challenges in diagnosing PNL cases when cytological/histopathological evidence of M. leprae in nerve biopsy samples is lacking because of patchy nerve involvement. Limited studies evaluating the significance of PCR-based detection of M. leprae in nerve samples have demonstrated a variable but considerable increase in detection rate (with high sensitivity and specificity) of AFB by PCR in comparison with Fite staining on histopathological specimens from patients with PNL.17–22
We observed a higher rate of positivity with MPCR than with RLEP-PCR for detection of M. leprae, i.e., 60% positivity with MPCR versus 23.3% with RLEP-PCR in skin biopsy samples from PNL cases. Jardim et al. performed PCR on material extracted from nerves and observed positive results in 87% of clinically suspected PNL cases.2 Pitta et al. detected M. leprae DNA via the PCR-based method in 36.5% (15/41) of nerve biopsy samples from PNL patients.23 Santos et al. demonstrated that quantitative PCR (qPCR) of nerve biopsies was positive in 60.8% (17/28) of PNL cases (SSS negative on bacilloscopy) whereas qPCR of the superjacent skin area was positive in only 10.7% (3/28) of cases.17 The results of our study employing MPCR in skin biopsy samples and the pickup rate of PCR-based M. leprae detection on nerve samples as described in various studies showed a strong concordance.
We observed a positivity rate of 53% with MPCR for M. leprae detection in comparison to 10% with RLEP-PCR in NS samples from PNL cases. An increased positivity for the presence of M. leprae DNA in NS samples using MPCR has been observed in household contacts of both PB and MB leprosy cases.4,5 Nasal swab detection of M. leprae offers additional benefits over conventional strategies of performing biopsies by being completely noninvasive. The expertise of a nerve biopsy and facilities like the use of Semmes-Weinstein filaments are not usually available at the primary health care level, especially in countries where leprosy is endemic such as India and Brazil. Tools like NSs, on the other hand, can be effectively implemented at the level of basic health care in such areas.
Santos et al.17 reported anti-PGL-1 serologic positivity in 52.9% (37/70) of PNL cases, which was similar to that observed by Pitta et al.23 (54.2%, 19/33). Jardim et al.24 observed a positive anti-PGL-1 antibody response in 21% (14/67) of PNL cases. We observed anti-ND-O-BSA serologic positivity in 80% (16/20) of PNL cases, which indicates that all these patients had widespread disease albeit not yet manifested in the skin. Positive serology at times helps in recognizing this uncommon clinical form of leprosy (PNL), including in oligo/asymptomatic patients.17 Seven patients with clinical and electrophysiological findings suggestive of PNL, and for whom skin MPCR was negative, demonstrated positive anti-ND-O-BSA ELISA results. Four patients for whom serology was negative were diagnosed with PNL based on positive skin MPCR results for M. leprae DNA. A combined evaluation of IgM ND-O-BSA serology and skin MPCR resulted in a 100% positivity rate of detecting PNL cases when clinically suspected, thereby almost obviating the need of invasive neural biopsy in PNL.
Leprosy-specific histological alterations covering the whole spectrum of disease (including indeterminate, borderline tuberculoid and borderline lepromatous) may or may not be found in biopsy samples taken from visually normal-appearing skin in PNL patients.1,25–29 In our cohort, histologic examination of skin biopsy samples from apparently normal-looking skin in the innervation territory of an affected nerve (in 20 PNL cases where skin biopsy samples were available for HPE) did not show leprosy-specific histology or AFB on Fite’s staining, though mild nonspecific mononuclear cell infiltrates around the blood vessels in the papillary dermis were noted in all of them, suggesting an inflammatory response to M. leprae. In 60% (12/20) of these PNL patients, MPCR demonstrated the presence of M. leprae despite negative Fite staining/leprosy histopathology findings. In the remaining eight cases, both HPE and MPCR were negative.
Since nerve biopsy is a gold standard investigation for PNL, it should be done in all such cases whenever possible for confirmation of diagnosis and histological characterization.30,31 In our study, none of the patients underwent nerve biopsy, so we do not have AFB positivity status/histological typing of involved peripheral nerve trunks available. Our observation of 80% serologic positivity (representing a higher relative risk of the disease) in this cohort indicates that PNL patients are closer to the MB pole of leprosy (with a predominant humoral response), reiterating the need for 12-month MB-MDT in PNL patients.17 In the absence of a nerve biopsy-based demonstration of M. leprae (either by PCR or microscopy) and histological subtyping as in our study, a holistic approach including clinical evaluation, electrophysiological evaluation, and laboratory-based anti-ND-O-BSA serology as well as histologic examination and molecular MPCR detection of M. leprae in skin biopsy samples can be of great help in diagnosing PNL patients.
Limitations.
Negative controls as well as healthy controls were not recruited, so we cannot comment upon sensitivity, specificity, or positive predictive value of MPCR. Records of nerve ultrasonography and nerve biopsy histopathology (since nerve biopsy was not done) were not available for a better correlation of findings. The small sample size remains another limitation.
CONCLUSION
Multiplex PCR is a useful tool for M. leprae detection in skin and NSs, reducing the need for invasive nerve biopsy in PNL cases. The heightened diagnostic sensitivity of MPCR over RLEP and the ease of obtaining samples such as skin biopsy samples and NSs make its application more easily widespread, especially in countries where leprosy is endemic, like India. It should complement clinical and serological assessments, suggesting its potential in PNL screening/detection and management, which will also help in preventing deformities and disabilities.
Supplemental Materials
ACKNOWLEDGMENTS
The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.
Note: Supplemental material appears at www.ajtmh.org.
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