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Role of histopathological, serological and molecular findings for the early diagnosis of treatment failure in leprosy

BMC Infectious Diseases volume 24, Article number: 1085 (2024) Cite this article

Summary

Background

Treatment failure (TF) in leprosy following multidrug therapy (MDT) presents a significant challenge. The current World Health Organization (WHO) fixed-duration MDT regimen, based on lesion count, might not be adequate. Leprosy lacks clear-cut objective cure criteria, and the predictive value of post-MDT histopathological findings remains uncertain. This study aims to identify predictive factors for TF among leprosy patients who have completed the WHO-recommended MDT.

Methods

An analysis was conducted on 80 individuals from a national leprosy reference center, comprising 40 TF cases (with a mean relapse at 13.0 months) and 40 controls (with a mean of 113.1 months without disease signs). Various epidemiological and clinical-laboratory parameters were assessed post-MDT.

Results

In skin samples, the presence of foamy granuloma (OR = 7.36; 95%CI2.20-24.60; p = 0.0012) and histological bacillary index (hBI) ≥ 1+ (OR = 1.55; 95%CI1. 22-1.99; p = 0.0004) were significantly associated with TF, with odds ratios of 7.36 and 1.55, respectively. Individuals who experienced TF had a mean hBI of 3.02+ (SD ± 2.02), while the control group exhibited a mean hBI of 1.8+ (SD ± 1.88). An hBI ≥ 3 + showed a sensitivity of 73% and a specificity of 78% for TF detection (AUC: 0.75; p = 0.0001). Other histopathological features like epithelioid granulomas, and skin changes did not show significant associations (p > 0.05). Additionally, higher anti-phenolic glycolipid-I (anti-PGL-I) ELISA index (EI) levels were linked to a 1.4-fold increased likelihood for TF (OR = 1.4; 95%CI1.13-1.74; p = 0.0019). A mean EI of 4.48 (SD ± 2.80) was observed, with an EI ≥ 3.95 showing a sensitivity of 79% and a specificity of 59% for TF detection (AUC: 0.74; p = 0.0001). Moreover, the presence of Mycobacterium leprae (M. leprae) DNA in real-time polymerase chain reaction (qPCR) was associated with a 3.43-fold higher likelihood of TF. Multivariate regression analysis indicated that concurrent presentation of neural/perineural lymphocytic infiltrate, foamy granuloma, hBI ≥ 1+, and EI ≥ 1 markedly increased the likelihood of TF by up to 95.41%.

Conclusion

Persistence of nerve-selective lymphocytic infiltrate, foamy granulomas, and bacilli in skin biopsies, and elevated EI post-MDT, may serve as predictive factors for identifying individuals at higher probability of TF.

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Introduction

150 years after the discovery of M. leprae, leprosy remains a concern for the medical community and public health in developing countries [1,2,3]. These acid-fast bacilli extend beyond Schwann cells and macrophages, parasitizing various other cell types throughout the body tissues and organs [4].

According to the Ridley and Jopling (R&J) classification, leprosy exhibits a wide immunopathological spectrum, ranging from a robust cellular immune response (Th1 pattern) in tuberculoid leprosy (TT) to a Th2 response dominance with an increased humoral pathogenesis in lepromatous leprosy (LL) [3, 5] [3]. In contrast, the WHO operational classification oversimplifies this to paucibacillary (PB) and multibacillary (MB) leprosy [6,7,8]. Therefore, patients are managed with the same three-drug MDT - comprising rifampicin, dapsone, and clofazimine - with a fixed-duration treatment of six months for PB and 12 months for MB leprosy [9].

The scientific community has engaged in discussions and raised concerns regarding the utilization of the WHO classification for the treatment selection, as it may lead to the inappropriate assignment of PB regimens to MB leprosy patients [10,11,12]. Thus, adopting the R&J spectral classification could potentially facilitate selecting more appropriate treatments, ultimately leading to lower therapeutic insufficiency, TF, and leprosy relapse rates [13,14,15,16].

[16]TF is a scenario where patients do not demonstrate clinical improvement despite receiving appropriate and well-indicated MDT, and may exhibit high levels of EI after completing therapy and within five years [16,17,18]. Insufficient data on TF and associated numbers exist due to the lack of standardized defining criteria for such cases. In the literature, the TF rate ranges from 4.72 to 31.68% within the initial first five years following MDT [19,20,21].

In cases of TF, clinicians should conduct a molecular investigation to assess resistance to the MDT drugs [22,23,24,25]. Drug resistance rates related to mutated M. leprae strains range from 8.0–43.24% [26,27,28]. However, the drug resistance rate may be underestimated [29]. The swift initiation of treatment, prompt case detection, and effective monitoring of MDT resistance are fundamental components of the global leprosy control strategy [30].

Clinically, the resolution of skin lesions and the reduction or negative outcomes of BI, EI, and qPCR are crucial in assessing the efficacy of MDT and distinguishing between TF and leprosy reaction during and after treatment [31,32,33]. In these instances, a comprehensive assessment combining clinical observations with laboratory results is vital for the timely identification of TF cases.

There has been limited focus on identifying predictive clinical and laboratory factors with minimal attention given to the significance of post-MDT skin histopathological findings in relation to TF. This study aimed to pinpoint histopathological, serological, and molecular predictive indicators upon MDT completion following the initial treatment of patients who were later diagnosed with TF, within five years post-treatment release.

Materials and methods

Study design and selection of cases

In a case-control study conducted at the National Reference Center for Leprosy/Dermatological Health (CREDESH), Minas Gerais, Brazil, between 2008 and 2020, 567 leprosy cases were initially selected from a total of 1,869 reported by CREDESH. Data analysis was based on secondary data obtained from medical records, specific software, and national databases like National Reference Center and the Notifiable Diseases Information System (SINAN) of the Brazilian Ministry of Health (MH).

This study included adults (age ≥ 18 years) treated with a fixed MDT regimen of six, 12, or 24 doses, diagnosed and managed by experienced leprologists at CREDESH. According to clinical, histopathological, bacillary, serological, and molecular characterization, patients were classified into the R&J spectrum [5]: borderline-tuberculoid (BT), borderline-borderline (BB), borderline-lepromatous (BL) or lepromatous leprosy (LL) and, for treatment purposes classified as PB or MB leprosy according to the WHO operational classification [7].

The sample was divided into two groups for comparison: the treatment failure group consisting of 92 cases identified as TF cases between 2013 and 2020; and the control group consisting of 475 new cases of leprosy between 2008 and 2013, with no reentries reported for at least 60 months post-treatment and followed up until 2020. Patients who completed the MDT and had results from bacillary exams, IgM anti-PGL-I serology, and qPCR for M. leprae DNA at the completion of treatment were included. Cases with other infectious comorbidities such as tuberculosis, HIV/AIDS, and viral hepatitis were excluded, as were cases with insufficient or unavailable formalin-fixed paraffin-embedded blocks for new histological sections. Both groups were paired, resulting in 40 patients in each group (Fig. 1).

Fig. 1
figure 1

Selection and pairing of individuals from the treatment failure group and the control group

Ridley and Jopling (R&J) classification. BT: borderline-tuberculoid leprosy; BB: borderline-borderline leprosy; BL: borderline-lepromatous leprosy; LL: lepromatous leprosy. World Health Organization (WHO) standard operational classification. PB: paucibacillary leprosy; MB: multibacillary leprosy. Anti-PGL-I serology (IgM), positive if ELISA index (EI) ≥ 1. qPCR: real-time polymerase chain reaction

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Definition and diagnostic criteria of treatment failure in leprosy

The definition of the MH for TF was adopted, encompassing all cases of leprosy that do not present signs of clinical response during regular and appropriately indicated treatment of six or 12 MDT doses or cases of MB patients who receive up to 24 MDT doses, and demonstrate signs of clinical activity and/or the presence of well-defined solid bacilli in the dermal smear, and/or histopathological examination of the skin and maintenance of high levels of anti-PGL-I (IgM) [16]. In this study, the diagnosis of TF was established by experienced leprologists from CREDESH based on clinical and laboratory findings, ruling out leprosy reactions, therapeutic insufficiency, and leprosy relapse.

Study variables

Epidemiological and clinical variables. The data points included sex, age, R&J clinical form, WHO operational classification, number of MDT doses, time (in months) between treatment release and TF for the TF group, and time (in months) elapsed since treatment release for the control group.

Laboratory variables. Intradermal smear bacillary index. The smear bacillary index (sBI) was calculated by obtaining samples from a minimum of six skin sites, including earlobes, elbows, and knees. The smears were then stained using the cold Ziehl-Neelsen technique following the protocol specified by the MH [34].

Histopathological analysis. Skin samples were fixed in 10% buffered formalin, embedded in paraffin, and then 3 μm histological sections were prepared. These sections were stained with hematoxylin-eosin (H&E) and Fite-Faraco (FF) staining techniques [35] for assessing the histopathological variables (Table 1). To determine the bacillary load [35], the logarithmic hBI was calculated according to the Ridley and Hilson scale [36]. The analysis of blinded slides from both the TF and the control groups was conducted by four observers using a multi-head optical microscope (Nikon Eclipse 80i, Nikon Corporation, Japan). Three of the four observers were pathologists with experience in R&J classification.

Table 1 Histopathological characteristics assessed in the skin sample at the completion of multidrug therapy for leprosy: definitions
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[4, 35, 36]IgM anti-PGL-I serology. IgM anti-PGL-I serum antibodies were detected using an ELISA against native PGL-I purified from the M. leprae cell wall. The reagents utilized, including M. leprae PGL-I and monoclonal anti-M. leprae PGL-I, were sourced as donations from BEI Resources, NIAID, NIH (NR-19342 and NR-19370, respectively). The titer of anti-PGL-I antibodies in the dilution of 1:10,000 was quantified. The optical density (OD) was obtained at 492 nm. The antibody titers were expressed as the EI according to the following formula: EI = ODsample/ODcut−off, with EI values equal to or greater than 1.0 considered positive [37].

DNA extraction and quantitative polymerase chain reaction. DNA was extracted from skin biopsies preserved by freezing in liquid nitrogen. The DNA extraction was conducted with NucleoSpin® Tissue/Macherey-Nagel® kit. In summary, cellular lysis was accomplished with a lysis buffer and Proteinase K. Subsequent wash steps removed impurities, resulting in DNA elution in an alkaline buffer. Following this, quantification assays were performed to evaluate the quantity and quality of the obtained DNA. The extracted DNA was the used to amplify the specific genomic region of the bacillus (RLEP3) through a qPCR system (ABI 7300, Applied Biosystems, Foster City, CA, United States) [38, 39].

Statistical analyses

Data normality was assessed with the Shapiro-Wilk test. Continuous variables were described with mean, median, maximum, minimum, and standard deviation values, while dichotomous variables were presented as percentages. The Binomial Test examined associations between TF group and control group regarding histopathological characteristics [40].

Univariate and multivariate logistic regression analyses estimated odds ratios to predict TF, considering clinical, histopathological, and laboratory features. The logit function from logistic regression was utilized to calculate the probability of TF based on multiple independent variables in a multivariate model [41]. This model included lymphocytic neural/perineural infiltrate, hBI ≥ 3+, and EI ≥ 3.95 to estimate the probability of TF. Although hBI and EI are continuous variables, they were transformed into binary variables using cutoff points (hBI ≥ 3 + and EI ≥ 3.95) to determine the final probability. ROC curves were generated for hBI variables in skin biopsy and anti-PGL-I serology to compare their diagnostic performance for TF detection. All analyses used a significance level of 5% (p < 0.05) and were conducted using SPSS version 22.0 (IBM, Armonk, NY, USA).

Results

The mean time interval between treatment completion and TF was 13.0 months (Min. 1, Max. 42 months), while the mean duration of disease-free period in the control group was 113.1 months (Min. 83, Max. 158 months) (See additional file 1). From 2013 to 2020, CREDESH diagnosed 92 cases as TF out of 1,385 reported leprosy cases, resulting in a TF rate of 6.7% (92/1,385).

Analysis using the G Test (likelihood ratio) to assess associations between categorical variables and groups revealed no statistically significant differences in epidemiological and clinical variables (p > 0.05) (See additional file 2). Regarding the number of MDT doses, the majority in the TF group received 24 doses, while in the control group, doses were evenly distributed among 6, 12, and 24 doses.

The mean hBI in the skin sample was significantly higher in the TF group compared to the control group (3.02 + vs. 1.8+, p = 0.004). Using the ROC curve analysis, a cutoff value of hBI ≥ 3+ demonstrated good sensitivity (73%) and specificity (78%) for discriminating TF (AUC: 0.75; cutoff of ≥ 3+; p = 0.0001) (Fig. 2) (See additional file 3).

Fig. 2
figure 2

Sensitivity, specificity, and area under curve for the skin histological bacillary index (hBI) and IgM anti-PGL-I ELISA index (EI)

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The mean EI for anti-PGL-I was also significantly higher in the TF group compared to the control group (4.48 vs. 2.44, p = 0.0024), with The EI demonstrating good sensitivity (79%) and specificity (59%) for TF detection (AUC: 0.74; cutoff of ≥ 3.95; p = 0.0001).

Regarding the histopathological characteristics of the skin biopsies at the end of MDT (Table 2), an association was observed between TF group and the features such as foamy granuloma (p = 0.0003), skin hBI (p = 0.0111), and neural/perineural lymphocytic infiltrate (p = 0.0402) (Figs. 3, 4 and 5). However, for other characteristics analyzed, there were no significant differences in the proportions of these categories between the two groups (p > 0.05).

Fig. 3
figure 3

Foamy granulomas. In a BT leprosy case treated with 12 doses of MDT. Treatment failure was observed after 3 months of follow-up. (A) Foamy granuloma throughout the dermis and subcutaneous fat (H&E, 4x). (B) Collection of foamy macrophages (H&E, 40x). (C) Numerous granular and fragmented bacilli (Fite-Faraco, 40x)

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Fig. 4
figure 4

Histological bacillary index. In a LL leprosy case treated with 24 doses of MDT. Treatment failure was observed after only 2 months of follow-up post-MDT. At the treatment release, there were numerous granular and fragmented bacilli (Fite-Faraco, 40x). In detail, solid bacillus (arrow) (Fite-Faraco, original 100x)

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Fig. 5
figure 5

Neural/perineural lymphocytic infiltrate. In a BL leprosy case treated with 24 doses of MDT. Treatment failure was observed after 14 months of follow-up since treatment completion. (A) Mild perineural lymphocytic infiltrate, (B) Lymphocytic delamination of the perineurium, and (C) Hyaline fibrosis of the nerve (H&E, 100x)

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Table 2 Histopathological characteristics in skin samples biopsied at the completion of multidrug therapy
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The data were submitted to logistic regression analysis, the results of which are displayed in Table 3.

Table 3 The statistical significance of the analyzed variables at the completion of multidrug therapy, assessed by univariate and multivariate logistic regression
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In the univariate logistic regression analysis, significant associations were found with the LL initial clinical form (OR = 3.12; 95%CI1.25-7.78; p = 0.0150), indicating a 3.12-fold higher odds for TF. Histopathologically, significant associations were observed (p < 0.05) for the presence of foamy granuloma and hBI. Presence of foamy granuloma at the end of treatment resulted in 7.4-fold greater odds (OR = 7.36; 95% CI 2.20–24.60; p = 0.0012) for TF. Similarly, hBI ≥ 1 + increased the odds by 1.6-fold (OR = 1.55; 95%CI 1.22–1.99; p = 0.0004) for TF, with odds rising by 55% per log, reaching up to 330% for hBI = 6+.

Laboratory findings showed a significant association with IgM anti-PGL-I serology. According to the analysis, having an EI ≥ 1 at the end of treatment increased the odds of TF occurrence were 1.4-fold (OR = 1.40; 95%CI 1.13–1.74; p = 0.0019). It is worth noting that these odds increase by 40% for each unit increase in EI. Also, for qPCR results of skin biopsy being positive for the presence of M. leprae DNA, the odds of TF were 3.4-fold higher (OR = 3.43; 95% CI 1.21–9.69; p = 0. 0201).

In the multivariate regression analysis, the combined presence of neural/perineural lymphocytic infiltrate (OR = 5.24; 95%CI 1.37–19.98; p = 0.0153), hBI (OR = 1.55; 95%CI 1 0.16-2.07; p = 0.0033) and EI (OR = 1.26; 95%CI 1.00-1.58; p = 0.0467) significantly predicted TF. A statistical model incorporating the presence of lymphocytic neural/perineural infiltrate, hBI ≥ 3+, and EI ≥ 3.95 indicated a high probability (up to 95.41%) for TF (See additional file 4).

Overall, the study identified several key predictive factors for TF in leprosy patients post-MDT, emphasizing the importance of histological, serological, and molecular markers in identifying individual at higher probability of TF.

Discussion

In the current case-control study, following the initial selection of 567 individuals, 80 participants were allocated into two paired groups: 40 participants in the TF group and 40 individuals in control group. This final sample was achieved through the application of stringent research inclusion criteria and precise pairing between groups, ensuring the exclusion of any statistical differences between the samples (p > 0.05). All patients were evaluated at the time of treatment completion of a fixed-duration MDT regimen consisting of 6, 12, or 24 doses of rifampicin, dapsone, and clofazimine. Given the limited literature on the clinicopathological and bacteriological evaluation of patients showing persistent signs of disease activity in skin lesions post- MDT [42], this study aimed to assess the predictive value of cutaneous histopathological and serological findings for TF in leprosy.

TF can be defined as the presence or worsening of skin lesions and failure to decrease the BI in MB leprosy [17, 18]. Patients with TF do not exhibit the expected improvements in terms of lesion disappearance and bacilli elimination following therapy completion, and may even experience disease progression throughout MDT [21]. TF in this study encompasses cases where (a) leprosy patients show no signs of clinical improvement despite receiving regular and appropriately indicated 6 or 12 doses of MDT [16, 43]; (b) MB patients treated with up to 24 doses of MDT who display signs of clinical activity or the presence of solid bacilli post-treatment, along with elevated anti-PGL-I (IgM) levels [16]. Different studies establish various criteria for TF, ranging from persistent active lesions or the appearance of new ones after 12 months of MDT, to a 2-log increase in BI post-MDT [44, 45]. Another clinical aspect in diagnosis TF is the emergence of new lesions and downgrading of the patient. In cases of TF, patients may exhibit these characteristics, contrasting with leprosy relapse where old lesions reappear without significant downgrading of the patient [46]. Despite the distinction between TF and relapse, there remains a lack of a precise and universally accepted criterion for identifying TF in leprosy, often attributed to factors such as poor treatment adherence or the development of drug resistance [22, 47] [22].

In this 12-year follow-up period study, an average time to TF of 13.0 months, and a TF rate of 6.7% (92/1,385) were identified. A previous study over six years reported a compatible TF rate of 4.7% (50/1,059) [20]. Symptoms recurring within 9 months post-treatment completion is often indicative of TF rather than leprosy relapse, which commonly occurs later after MDT [48]. Therefore, TF should be recognized as an early event, often shortly treatment completion, with a majority of cases manifesting within the initial 3 years following the conclusion of MDT [21].

All cases in the TF group (40/40) were initially classified as MB before commencing treatment with MDT. The WHO categorizes leprosy into PB and MB based on factors such as the number of skin lesions, neural involvement, and BI results [9]. There exists a notable yet significant risk of undertreatment in cases designated as PB but harboring a high bacillary load [49]. In a study involving 264 untreated leprosy patients, it was found between 38 and 51% of individuals (100–134/264) classified as PB met the criteria for MB, suggesting a risk of undertreatment as per WHO guidelines [10]. In another group of PB patients with up to 5 skin lesions and with bacterio-histopathological confirmation, approximately 24.2% (15/62) were identified to have the MB form of leprosy [50]. Therefore, the correlation of clinical, histopathological, and bacteriological findings could be more useful in diagnosing leprosy than considering just a single parameter in isolation [51].

Most patients in the TF group (34/40) received 24 doses of MDT, indicating individuals who did not show improvement during MDT or surpassed the criteria for therapeutic insufficiency. According to the WHO, a shorter regimen (six doses) of MDT poses a potential increase in the risk of TF [9]. In cases where the treatment duration is insufficient to eliminate the bacilli, the ongoing inflammatory response results in persistent clinical activity for 12 to 18 months, potentially resulting in TF [52]. A study that evaluated 25 MB leprosy patients treated with MDT, including 11 BL and 14 LL patients, revealed the persistence of M. leprae in tissues even after 2 years of treatment [53]. Patients classified as MB are at a heightened risk of recurrence following 2 years of MDT [54].

In this study, individuals diagnosed with LL at the onset of treatment had 3.12 times higher odds of experiencing TF. Relapse and TF primarily affect BL or LL patients with high initial BI [8]. A study involving 189 MB patients treated with four different drug regimens and monitored for up to 12 years post- treatment reported relapse rates of up to 3% in BL and LL patients with a high bacterial load following MDT [55]. There remains a lack of a precise definition of a definitive cure of leprosy, beyond simply the fixed-duration therapy [47]. The therapeutic response of patients receiving MDT can be evaluated based on three key parameters: (1) resolution of skin lesions; (2) reduction in BI, and (3) recurrence rate [31].

Resolution of skin lesions

For effective treatment, an accurate diagnosis of leprosy is crucial, necessitating precise histopathological characteristics, BI, and clinical correlations [56]. Histopathological examination of skin lesions is considered the gold standard for diagnosing leprosy, enabling classification of leprosy and its reactions, estimation of BI, monitoring treatment and disease activity, as well as distinguishing TF from leprosy reaction [46, 51, 57]. Even after completing of MDT, some TT and BT patients may exhibit active and persistent skin lesions, potentially due to drug-resistant bacilli or an autoimmune phenomenon triggered and sustained by persistent bacillary antigens [42]. Recommendations suggest PB patients should be monitored for at least 3 years, while MB patients should be followed for 9 years to detect most leprosy recurrences [21]. At CREDESH, patients typically undergo annual are follow-ups for 10 or 20 years depending on clinical and laboratory status post-MDT [20].

The study data indicated that the presence of foamy granuloma resulted in a 7.36-fold higher odds for TF. This could be attributed to various factors in the host-pathogen interaction, such as high bacillary load, persistent bacilli, clinical form of leprosy, and duration of MDT.

The key question about foamy macrophages is whether they can conceal live bacilli. M. leprae rather than just recognizing antigen from the dead ones. M. leprae alters lipid metabolism to enhance its survival and dissemination while promoting host tolerance [58]. Studies indicate that foamy macrophages create an anti-inflammatory milieu in LL skin lesions, hindering bactericidal responses and aiding M. leprae evasion, thus increasing skin lesions [59,60,61,62]. Consequently, the foamy phenotype likely results from more than just the breakdown of dead M. leprae [63]. Identifying foamy granulomas in post-treatment skin biopsies may potentially indicate TF.

Decrease in the bacillary index

The BI indicates the concentration of bacilli in smears or tissues, including both live and deceased bacilli [51]. In most PB cases, the hBI is generally higher compared to sBI [4, 56, 64]. Conversely, there is typically a strong correlation between hBI and sBI in MB cases [51, 56].

Findings indicated that an hBI ≥ 1 + in the skin biopsies post- MDT increased the odds of TF by 1.55-fold per log, potentially reaching up to 9.3-fold in cases where hBI is 6+. A study of 46 cases of TF and relapse identified that 20.8% of cases exhibited a BI of ≥ 4+ [65]. Accurate patient classification through BI and biopsy is crucial, as BI enables effective monitoring, particularly when TF or leprosy reactions are suspected [66]. However, some authors suggest that a mildly positive sBI at treatment completion, especially in patients originally diagnosed with a negative smear, may not indicate TF if clinical improvement is evident [67].

After initiating treatment, BI typically decreases by approximately 1 log per year, presenting a more reliable measure of bacillary clearance compared to smear negativity, which largely reflects the initial classification [18, 68]. Patients with high initial BI are at greater risk of recurrence post-MDT compared to those with negative or low BI [21]. However, the rate of decline or a negative BI does not definitely predict the success or failure [8]. This research suggests using the downward trend or absence of bacilli in hBI as an objective criterion for declaring a leprosy cure.

Distinguishing between TF and reactions, predominantly within the initial 3 to 5 years post-treatment, can be challenging. Here, hBI is useful for diagnostic clarification, particularly in patients with granulomatous reactivation at the conclusion of MDT [4, 69]. In cases of recurrence, a Type 1 reaction may be perceived as a partially effective immune reconstitution (upgrading reaction) or a hypersensitivity reaction (downgrading) [70]. The study demonstrated moderate sensitivity (73%) and specificity (78%) in discriminating TF using an hBI cutoff ≥ 3 + in skin biopsies post-MDT. Additional data comparing hBI, sBI, and EI in the TF group is provided, highlighting results from both initial diagnosis and at the completion of MDT (See additional file 5).

Persistent bacilli are organisms that remain permanently or partially dormant, capable of surviving despite adequate chemotherapy. They are present in approximately 10% of MB patients, with a likelihood possibly higher in cases with high BI [18, 21, 70]. These dormant bacilli reside in immunologically privileged sites such as dermal nerves, smooth muscle cells, lymph nodes, iris, bone marrow, and liver, making them less accessible to medications [71].

The post-inflammatory reparative process can lead to perineural and endoneural fibrosis of the skin nerves, creating fibrotic areas that serve as sanctuaries for M. leprae, which may revert to normal division rates after MDT [47, 57]. Therefore, routine skin biopsies and dermal smears should be conducted at least once every six months during treatment and for 5 years after achieving BI negativity [21]. Regular monitoring in this way can help in detect any potential disease resurgence.

Decrease in the ELISA index

After MDT for leprosy, distinguishing between leprosy activity, reactions, therapeutic insufficiency, TF, relapse, and reinfection becomes challenging. In addition to histopathological findings, serological assessments play a valuable role in differential diagnosis [21]. When treating MB patients, repeated ELISA tests can serve as an adjunct tool to assess therapy effectiveness, with elevated antibody potentially indicating disease recurrence [32, 72, 73]. In a study involving LL patients, anti-PGL-I was detected in 94.1% (64/68) of new cases with a BI ranging from 3.2 to 5.8 + and in 78.8% (26/33) of patients with relapsed leprosy with a sBI ranging from 3.0 to 5.3+ [74]. Similarly, other researchers observed anti-PGL-I in 93.1% (27/29) of patients with an hBI of 4.0 + or higher and 68.2% (15/22) of those patients with hBI ranging from 3.0 to 3.9+, but none of the 37 patients with hBI less 1.9 + exhibited detectable anti-PGL-I levels [75].

A study involving 115 cases of leprosy reported a substantial agreement of 82% (94/115) between serology and sBI [32]. Elevated IgM anti-PGL-I levels in MB patients compared to PB cases have been observed in various studies, reinforcing the correlation with bacillary load [76,77,78]. The presence of serological results with an EI value ≥ 1 at the conclusion of MDT indicated 1.40-fold higher odds per each unit increase, suggesting an increased likelihood of TF, with an EI cutoff value ≥ 3.9 showing moderate sensitivity (79%) and specificity (59%) for discriminating the TF group.

The onset of positivity or elevation in EI levels post-MDT tends to precede the detection of bacilli in the skin, making it an early indicator of disease recurrence [32, 79]. The gradual decrease in IgM anti-PGL-I antibodies following treatment suggests its utility in monitoring treatment effectiveness [74]. Seronegative conversion of anti-PGL-I is considered valuable for assessing bacterial load elimination and determining the treatment effectiveness [80]. The study recommends utilizing EI values to define leprosy cure based on a decline or negativity in serological anti-PGL-I levels.

Real-time polymerase chain reaction of skin biopsy

Leprosy, known as “the great imitator”, shares clinical and histopathological similarity with various skin conditions like pityriasis alba, pityriasis versicolor, vitiligo, tinea circinata, psoriasis, and others [4, 11, 33, 35]. In the diagnostic process, especially for PB cases, and in evaluating the recurrence, the detection of M. leprae DNA by PCR serves as a pivotal role [21, 81,82,83,84]. In this study, a positive result in skin biopsy qPCR at the conclusion of MDT was associated with 3.43 times greater odds for TF. A study examining undetermined leprosy cases post-4 to 6 years of MDT without active disease sings discovered M. leprae DNA positivity in 54.5% (18/38) of cases, suggesting the presence of bacilli much longer than anticipated, albeit reinfection could not be ruled out [85]. Positive qPCR in individuals suspected of having leprosy may indicate subclinical infection, potentially making them carriers and transmitters of M. leprae [86].

An RT-PCR assay conducted on samples from 24 PB patients, all with sBI = 0, revealed positive results in 21% (5/24), 25% (6/24), and 96% (23/24) for intradermal smear examinations of the earlobe and cutaneous lesions and skin biopsy, respectively, enhancing the diagnostic capacity of the histopathological examination by up to 17% [33]. Skin biopsies qPCR is reported to have a sensitivity of 50% and a specificity of 94% for diagnosis leprosy [87].

There is a lack of available data addressing the likelihood of TF in leprosy based on histopathological findings in skin samples post-MDT. The data presented in our study suggests, for the first time, that the combined presence of neural/perineural lymphocytic infiltrate in skin biopsy and hBI ≥ 3 + and EI ≥ 3.95 at the end of MDT results in a 95.41% probability of TF.

Conclusion

This research has made a significant contribution by shedding light on the predictive value of histopathological findings in post-MDT skin biopsy for TF, offering essential insights for monitoring leprosy cases managed with WHO fixed-duration regimens. Unlike relapse, TF should be viewed as an early event occurring closer to the conclusion of MDT, with the LL patients facing a higher likelihood of TF.

Key factors that elevate TF odds in patients who have under gone regular MDT include the presence of foamy granulomas and hBI ≥ 1 + at the conclusion of treatment. Furthermore, seropositivity for IgM anti-PGL-I (IE ≥ 1) and detection of M. leprae DNA via qPCR at treatment conclusion serve as predictive factors for TF. When combined with parameters such as neural/perineural lymphocytic infiltrate, foamy granulomas, hBI ≥ 3+, and EI ≥ 9.5, the likelihood of TF post-MDT is significantly heightened. This offers a promising avenue for future research with larger sample sizes.

There remains a crucial need to establish universal criteria for leprosy cure, therapeutic insufficiency, TF and leprosy relapse that are globally applicable, based on objective clinical and laboratory findings. The R&J classification and skin biopsy are deemed vital in this endeavor. It is essential to uphold active surveillance, particularly for patients at a heightened likelihood of TF post-MDT, to ensure timely intervention and proper management.

Limitations of the study

One potential limitation of this research is the relatively small sample size. However, it is important to note that this sample reflects the incidence of FT over seven years at a national reference center for leprosy. Future studies with larger sample may help validate the conclusions stated herein.

A major limitation is the impossibility to obtain pre-treatment skin biopsies and compare with those post-MDT.

Another limitation concerning qPCR analysis is the challenge of interpreting results quantitatively due to the diverse units which values were expressed over the 12-year study period. Standardizing units could enhance the interpretability and comparability of qPCR results in future research efforts.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

ABI:

Applied Biosystems

AUC:

Area Under the Curve

BB:

Borderline-Borderline

BEI:

Biodefense and Emerging Infections Research Resources Repository

BI:

Bacillary Index

BL:

Borderline-Lepromatous

BT:

Borderline-Tuberculoid

CI:

Confidence Interval

CREDESH:

National Reference Center for Leprosy/Dermatological Health

DNA:

Deoxyribonucleic Acid

ELISA:

Enzyme-Linked Immunosorbent Assay

EI:

ELISA Index

FF:

Fite-Faraco

H&E:

Hematoxylin-Eosin

HIV/AIDS:

Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome

hBI:

Histological Bacillary Index

IgM:

Immunoglobulin M

LL:

Lepromatous-Lepromatous

MB:

Multibacillary

MDT:

Multidrug Therapy

M. leprae:

Mycobacterium leprae

MH:

Ministry of Health

NIAID:

National Institute of Allergy and Infectious Diseases

NIH:

National Institutes of Health

NR:

National Institutes of Health Biodefense and Emerging Infections Research Resources Repository

OD:

Optical Density

OR:

Odds Ratio

PB:

Paucibacillary

PCR:

Polymerase Chain Reaction

PGL-I:

Phenolic-Glycolipid-I

qPCR:

Quantitative Polymerase Chain Reaction

ROC:

Receiver Operating Characteristic

RT-PCR:

Reverse Transcription Polymerase Chain Reaction

R&J classification:

Ridley and Jopling classification

SD:

Standard Deviation

SINAN:

Notifiable Diseases Information System

SPSS:

Statistical Package for Social Sciences

TF:

Treatment Failure

WHO:

World Health Organization

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Funding

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001. This work was supported by the National Council for Scientific and Technological Development (CNPq), the State Funding Agency of Minas Gerais (FAPEMIG), and the National Health Fund – Brazilian Ministry of Health [TED 123/2020].

Author information

Authors and Affiliations

  1. Pathology Unit, Hospital of Clinics, Federal University of Uberlândia, Brazilian Company for Hospital Services (HC-UFU/EBSERH), Uberlândia, MG, Brazil

    Bruno de Carvalho Dornelas, João Pablo Ferraz de Abreu, Juliana Salomão Daud & Deiriene Rodrigues de Oliveira Campos

  2. Post-Graduation Program in Health Science, School of Medicine, Federal University of Uberlândia, Uberlândia, MG, Brazil

    Bruno de Carvalho Dornelas, Diogo Fernandes dos Santos & Isabela Maria Bernardes Goulart

  3. School of Medicine, Federal University of Uberlândia, Uberlândia, MG, Brazil

    Willian Vargas Tenório da Costa & Felipe dos Anjos Rodrigues Campos

  4. National Reference Center for Leprosy/Dermatological Health (CREDESH), HC- UFU/EBSERH, Uberlândia, MG, Brazil

    Douglas Eulálio Antunes, Diogo Fernandes dos Santos & Isabela Maria Bernardes Goulart

  5. School of Mathematics, Federal University of Uberlândia, Uberlândia, MG, Brazil

    Lúcio Borges de Araújo

  6. Lauro de Souza Lima Institute (ILSL), Bauru, SP, Brazil

    Cleverson Teixeira Soares

  7. Unidade de Anatomia Patológica. Hospital de Clínicas de Uberlândia, Av. Pará, 1720 - Umuarama, Uberlândia, MG, 38405-320, Brazil

    Bruno de Carvalho Dornelas

Authors
  1. Bruno de Carvalho Dornelas
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  2. Willian Vargas Tenório da Costa
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  3. João Pablo Ferraz de Abreu
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  4. Juliana Salomão Daud
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  5. Felipe dos Anjos Rodrigues Campos
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  6. Deiriene Rodrigues de Oliveira Campos
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  7. Douglas Eulálio Antunes
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  8. Lúcio Borges de Araújo
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  9. Diogo Fernandes dos Santos
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  10. Cleverson Teixeira Soares
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  11. Isabela Maria Bernardes Goulart
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Contributions

BCD, DFS, and IMBG developed the conceptualization. BCD, WVTC, and IMBG collected the data. FAR and DROC performed the histological techniques. BCD, WVTC, JPFA, and JSD analyzed the histological sections. BCD, LBA, DEA, and IMBG evaluated the data. BCD, DEA, DFS, IMBG assisted with study design and methodology. BCD and IMBG wrote the manuscript. IMBG and CTS reviewed the manuscript. IMBG obtained resources and funds. BCD, WVTC, FAR, JPFA, JSD, DFS, and IMBG carried out the investigation. IMBG administered and supervised the project. BCD, WVTC, FAR, DROC, JPFA, JSD, LBA, DEA, and IMBG validated the work.

Corresponding author

Correspondence to Bruno de Carvalho Dornelas.

Ethics declarations

Ethics approval and consent to participate

The research followed the principles of the Declaration of Helsinki. Informed consent was waived and the research was approved by the Research Ethics Committee of the Federal University of Uberlândia (CAAE: 42878620.0.0000.5152) on 16/04/2021. The data were obtained from medical records, specific software, and SINAN, and were blinded for analysis. The informed consent waiver was also requested due to the difficulty of obtaining written consent from patients in a long-term retrospective study.

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Not applicable.

Competing interests

The authors declare no competing interests.

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de Carvalho Dornelas, B., da Costa, W.V.T., de Abreu, J.P.F. et al. Role of histopathological, serological and molecular findings for the early diagnosis of treatment failure in leprosy. BMC Infect Dis 24, 1085 (2024). https://doi.org/10.1186/s12879-024-09937-2

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Keywords

BMC Infectious Diseases

ISSN: 1471-2334

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