Diagnosis of Leprosy: Current Updates and Future Directions

Procedure

The smear is taken while squeezing the skin to diminish the bleeding and incision (about 5 mm long and 3 mm deep) is given to collect tissue fluid. Precaution is to be taken to avoid blood in the collected material as it dilutes the number of bacilli in the smear. Thin smear of the material is prepared and heat fixed by passing the slide 3–4 times through the flame of a Bunsen burner. Carbol fuchsin (lipid soluble and penetrates the waxy cell wall) is poured over smear and underside of the slide is gently heated by passing a flame until fumes appear (without boiling). Overheating should be avoided and the stain is allowed to stand for 5 min. Smears are rinsed with water until no colour appears in the effluent. Sulphuric acid (5%) or acid-alcohol (1%) is poured over the slide and kept for one minute and this step is repeated until the slide appears light pink in colour (15–20 s). Smear is washed well with clean water, and then, the smears are covered with methylene blue or malachite green stain for 1–2 min. The stain is washed with clean water and slide is examined under microscope, using the ×100 oil immersion objective.

Interpretation of smears

The solid staining bacilli, representing living bacilli, appear as uniformly stained rods, whereas dead bacilli appear irregularly stained (fragmented bacilli) or as granules (granular bacilli). The density of bacilli (both living and dead) in smears is known as the bacteriological index (BI) and is expressed by Ridley’s logarithmic scale based on the number of bacilli seen in an average microscopic field using an oil-immersion objective [Figure 2]. Histoid lesions typically show longer appearing bacilli with tapered ends, which are not usually clumped as globi.

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Figure 2:

Leprosy smears (Modified Ziehl -Neelsen staining). (a) BI 4+: 10–100 bacilli in a single oil immersion field, (b) BI 5+: 10–100 bacilli in a single oil immersion field, (c) BI 6+: globi (> 1000 bacilli) in a single oil immersion field, (d) Mycobacterium leprae highlighted on fluorescent staining, showing solid, fragmented, and granular bacilli (HiMedia^® K021-1KT).

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It requires about 10⁴ bacilli/g of tissue for reliable detection by Z-N staining.^[2,3] Thus, acid–fast bacilli (AFB) are absent in a typical tuberculoid (TT) lesion and either absent or scanty in borderline tuberculoid (BT) lesions. BI does not fall during first 12 months in lepromatous patients under treatment, as both dead and living bacilli are counted; however, it gradually declines at a rate of about 0.62 log/year thereafter and disappears over the next 5–10 years.

The percentage of solid staining bacilli is expressed as morphological index (MI), which is calculated after examining preferably 200 singly lying red staining elements. MI is a crude measure of viability of bacilli and is helpful in determining the activity of the disease.^[4] Further, MI can be used to monitor response to treatment and an increase in MI on treatment suggests drug resistance or defaulter status. MI of lepromatous patients will be between 25% and 75% before initiation of multi-drug therapy (MDT) and there is a steady fall in MI to zero in 4–6 months of dapsone monotherapy, whereas it is considerably faster with MDT. However, there exists significant differences in MI depending on the different sites examines, with smears from nasal mucosa of lepromatous leprosy (LL) patients often revealing higher MI compared to those from skin and ear lobes.^[5] Further, the higher MI may persist for longer in nasal mucosa and normal bacilli may reappear here but not elsewhere.

There are different guidelines regarding the number of skin smear sites and it has been changing over the years. Latest WHO recommendation is to prepare smears from a minimum of three sites (one ear lobe and two active lesions).^[6] Two smears have to be taken from diametrically opposite active edge of the lesion when a single lesion is present. ILEP recommends use of two sites for initial smear (ear lobe and edge of most active area of an active looking lesion).^[7]

The diagnostic specificity of SSS is 100% when a proper staining procedure is followed; however, recent studies have shown that SSS has a 5-year average sensitivity of 31.4%.^[8,9] A study from India reported a SSS positivity of 100% in LL and histoid leprosy, 86.4% in borderline lepromatous (BL), 38.8% in BT and none in TT, indeterminate and pure neuritic leprosy (PNL).^[2] The overall sensitivity of SSS was 59.8% in multibacillary (MB) and 1.8% in paucibacillary (PB) leprosy.^[2]

Indeterminate leprosy

AFBs may be seen occasionally in normal nerve, arrector pylori muscle, hair follicles, subepidermal zone and/or perivascular infiltrates in the early stage, whereas lymphocyte infiltration or Schwann cell proliferation characterise the late stage. Lymphocyte infiltration usually involves perineural sheath with preservation of nerve parenchyma, but the nerve fibre could be completely replaced by lymphocytes occasionally.^[13] Proliferation of Schwann cells results in loss of wavy pattern of nerves and loss of longitudinal orientation of individual Schwann cell nuclei resulting in ‘baton’-shape of nuclei in normal nerves.

Histoid leprosy

Histopathology reveals hypercellular granuloma, predominantly composed of spindle-shaped cells. Pseudocapsule is seen as the centrifugal growth of these cells compresses the fibrous tissue. Solid staining bacilli are arranged in parallel stalks within the cells, referred to as histoid habitus. Islands of epithelioid cells without any organism inside may be seen in few histoid lesions, known as epithelioid contaminants.

Staining of AFBs on tissue

Different staining methods have been used for demonstration of AFBs in tissue samples. The density of the bacilli required to identify a single bacillus in the section by Fite-Faraco (FF) method is about 1000 per cubic millimetre of the tissue.^[15] Fluorescent method has been found to be more sensitive than modified FF and ZN methods in detecting lepra bacilli in tissue sections especially when BI is less than three.^[16] Sensitivity of fluorescent stain for indeterminate and BT leprosy was found to be 100%, and thus, this is most reliable in the categorisation of PB and MB leprosy (Figure 2).^[16]

Molecular diagnosis of leprosy

While smear examination and histopathology enable precise diagnosis of the disease and its spectrum in most instances, additional diagnostic methods may be required in the following scenarios:

1. Early leprosy

2. Indeterminate leprosy

3. Paucibacillary leprosy

4. PNL

5. Early reactions

6. Asymptomatic contacts.

The definitive identification of M. leprae is possible through the extraction of nucleic acid, amplification and identification of M. leprae DNA in clinical specimens using polymerase chain reaction (PCR) [Flow chart 1]. Samples on which PCR can be performed include skin biopsy, skin smears, nerves, urine, oral or nasal swabs, blood and ocular lesions.^[17] RLEP is the most sensitive genes for detecting M. leprae and RLEP and 16SrRNA are most commonly used.^[18,19] Common genes employed in PCR assays to diagnose leprosy are listed in [Table 3].^[20-29] PCR has facilitated the direct quantification of the bacterial DNA content in clinical samples, thereby increasing the reliability of the results.^[20]

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Flow chart 1:

PCR procedure in leprosy. PCR: Polymerase chain reaction.

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Quantitative PCR (qPCR) is emerging as the most suitable method for diagnosis of leprosy, particularly PB leprosy.^[4] It is also useful in the identification of drug resistant bacilli, and this, in fact, has become an important use in leprosy in current times. Notably, it helps in differentiation of relapse from reaction by way of viability assays. Viability of bacilli can be detected by extraction of RNA from clinical samples, followed by complementary DNA synthesis and amplification by real time PCR. However, there are certain drawbacks as it has many targets and data on Good Manufacturing Practices products used for diagnostic purposes are lacking.

PCR sensitivity ranges from 30% to 83% in patients with a negative BI or with tuberculoid leprosy, while the sensitivity is 87–100% in those with a positive BI.^[18,30] Recently, PCR has been developed by multiplexing of two or three genes specific for M. leprae to increase the sensitivity for diagnosing early leprosy cases/household contacts/patients with vague or no symptoms.^[25,26]

Recent advance in molecular diagnosis of leprosy includes duplex-droplet digital PCR with greater sensitivity of detecting M. leprae DNA in PB patients compared with qPCR (79.5% vs. 36.4%), while both assays had a 100% sensitivity in MB patients.^[31] Loop-mediated isothermal amplification is developed as a field friendly, cost-effective diagnostic tool and utilise RLEP and 16S rRNA gene targets to detect M. leprae.^[32-34]

Drug resistance testing of M. leprae can be done using mouse foot pad inoculation as M. leprae is uncultivable in artificial media. However, it is time taking, cumbersome and expensive method. PCR followed by Sanger sequencing can be used for detection of drug resistant strains of M. leprae by targeting RpoB gene for rifampicin, FolP gene for dapsone and GyrA gene for ofloxacin resistance [Figure 4].

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Figure 4:

Chromatogram showing point mutations in rpoB, folP, and gyrA gene followed by BLAST result.

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Serology

Phenolic glycolipid 1 (PGL-1), a cell wall species-specific glycolipid, is the most widely used antigen for serological assays in leprosy.^[35] The synthetic sugars – natural trisaccharide (NT) and natural disaccharide (ND) were synthesised and conjugated with either bovine serum albumin (BSA) or human serum albumin (HSA) using either octyl (O) or phenyl (P) linker arms (ND-O-BSA/HSA or NT-O-BSA/NT-P-BSA) as these showed higher affinity for IgM antibody than PGL-1. PGL-1 ELISA has 90–95% positivity for diagnosis of BL/LL cases and 25–60% positivity for diagnosis of TT/BT cases.^[36] These antigens are also utilised in M. leprae dipstick assay and particle agglutination assay.^[37] An immunochromatographic strip test is a quick output lateral flow assay for the detection of antibodies in field conditions and it takes only 10 min to perform.^[38]

Recombinant 35kD protein can diagnose 40% of PB cases.^[39] Leprosy Infectious Disease Research Institute Diagnostic 1 (LID-1) was found to be positive in 95% of MB cases and 20–40% of PB cases.^[39] Antibody level against PGL-1, LID-1 and NDO-LID (synthetically conjugated LID-1 and ND-OBSA) was declined significantly after 6 month and 12 month of MDT treatment and, thus, can be used for monitoring treatment response. The major membrane proteins-I and II are the other antigens which have been used for serological diagnosis.^[37,40-42] However, they seem to add a little value in the diagnosis.^[43]

There are many studies that stress about high risk of developing leprosy among household contacts with positive anti-PGL-I titres. However, the role of positive anti-PGL-1 titres in the detection of preclinical leprosy among household contacts of leprosy patients may not be applicable in endemic areas as many individuals with seropositivity will never develop leprosy. In fact, it has been shown that more than half of the individuals with antibodies against PGL-1 will never develop leprosy.^[44,45]

Studies have been conducted to analyse the utility of serological tests in the prediction of reactions in leprosy patients. A recent study found higher anti-LID-1 levels in patients with type 2 reaction (T2R) at diagnosis compared to type 1 reaction (P = 0.008) and non-reactional patients (P = 0.020). The author concluded that high and persistent anti-LID-1 antibody levels in MB leprosy might be a useful tool to predict susceptibility of patients to develop T2R.^[45] Similar findings were later reported by Devides et al. using anti-PGL-1 and anti-NDO-LID-1 levels.^[46]

Cytology in leprosy

Singh et al. have suggested cytology criteria for subclassification of leprosy.^[47] Samples for cytology may be from skin lesions, nerve or lymph nodes. Cytology demonstrates cohesive epithelioid cell granulomas with lymphocytes, not infiltrating the granuloma, in tuberculoid leprosy.^[47] The cohesion between the cells of the granulomas declines, with concurrent increase in infiltration of lymphocytes within them, as the disease downgrades toward the lepromatous pole.

Important use of cytology lies in diagnosis of pure neural leprosy where tissue for histopathology is difficult to obtain, as even motor and mixed nerves can be safely sampled for cytology.^[4] Further, the aspirate can be used for PCR detection of M. leprae as well, enhancing diagnostic yield in PB spectrum.

Nerve biopsy

Nerve biopsy is considered as gold standard for the diagnosis of PNL (as skin lesions are absent). A thickened sensory nerve which lacks motor fibres such as a supraorbital branch of the 5^th cranial nerve, a supraclavicular nerve, the great auricular nerve in the neck, the radial nerve at the wrist, a cutaneous nerve of forearm or thigh, the sural nerve behind the lateral malleolus or a superficial peroneal nerve on the dorsum of the foot are considered suitable for nerve biopsy [Figure 5]. We mostly perform sural nerve biopsy at our centre due to ease of isolation, ability to obtain sufficient sample for biopsy (considering the bulk of the nerve) and as sensory loss in area of distribution of sural nerve is less concerning to patients than sensory loss following biopsy of radial cutaneous nerve.

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Figure 5:

(a and b) Marking and dissection of sural nerve in nerve biopsy (Image: Khurana A. Diagnosis of leprosy. In: Sardana K, Khurana A, editors. Jopling’s Handbook of Leprosy. 7^th ed. New Delhi: CBS publishers; 2023).

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Nerve conduction studies

NCS in nerves affected by leprosy may show reduced amplitude of sensory nerve action potentials and compound muscle action potential or CMAP suggestive of axonal damage, and decreased nerve conduction velocity and increased latency due to demyelination.^[53] A prospective study found that 100% MB and 50% PB leprosy cases showed abnormalities on NCS at the time of diagnosis of leprosy.^[54]

Studies demonstrate early detection of nerve dysfunction by NCS, before the appearance of typical symptoms and signs of nerve function impairment, thus helping in detecting subclinical neural involvement, although the therapeutic implication of this is yet undetermined.^[55] Interestingly, a recent study comparing combination of nerve palpation with Semmes-Weinstein monofilament testing and voluntary muscle testing showed comparable efficacy to NCS in detecting nerve damage.^[56]

High-frequency ultrasonography

High-frequency ultrasonography (15–20 MHz) helps in better identification of nerves and gives details about features such as exact site and size of nerve thickness, morphological variations in nerve trunk such as texture, pattern of fascicles and vascularity.^[55] Loss and destruction of fascicular pattern is the most specific feature for neural impairment in leprosy.^[56] It is particularly important in the diagnosis of PNL and is most useful for the assessment of nerves that are inaccessible for clinical palpation, such as the median nerve at the wrist; however, higher sensitivity and specificity have been reported for ulnar and common fibular nerves. Reactions in PNL shows increased vascularity and oedema of nerve trunk, suggestive of neuritis.

The cross-sectional area (CSA) of peripheral nerves is an important tool in the detection of large areas of nerve damage in leprosy. Frade et al. have suggested the measurement of the asymmetry index in the evaluation of leprosy and have demonstrated that this index is highly sensitive and specific for the differentiation between the nerves of healthy individuals and the nerves of patients with leprosy.^[56] The ROC analysis of CSAs showed the best specificity and sensitivity at the pre-tunnel (PT point) of the ulnar and common fibular nerves, respectively. Leprous neuropathy shows an increased CSA and the pattern of asymmetry (ΔCSA>2.5 mm² with an RR of 13) with high sensitivity and specificity for its early diagnosis.^[57]

Some older test, not in common use in current practice, are detailed in [Table 4].^[4]