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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 2  |  Page : 155-161

Clinical, histopathological, and molecular characterization of leprosy in an endemic area of the colombian caribbean


1 Department of Internal Medicine, University of Cartagena, Cartagena de Indias, Colombia
2 Department of Molecular Research Unit Group (UNIMOL), Laboratory of Tropical Medicine, Faculty of Medicine, University of Cartagena, Cartagena de Indias, Colombia
3 Department of Dermatology, Faculty of Medicine, University of Cartagena, Cartagena de Indias, Colombia
4 Department of Molecular Research Unit Group (UNIMOL), Faculty of Medicine, University of Cartagena, Cartagena de Indias, Colombia

Date of Submission24-Feb-2021
Date of Acceptance06-Apr-2021
Date of Web Publication14-Jun-2021

Correspondence Address:
Maria Carolina Fragozo-Ramos
Cra. 50 #24120, Zaragocilla Campus, Faculty of Medicine, University of Cartagena, Cartagena de Indias-130001
Colombia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_43_21

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  Abstract 


Background: Mycobacterium leprae was considered the only causal agent of leprosy until Mycobacterium lepromatosis was identified' which it has been suggested has greater pathogenicity and is linked to diffuse lepromatous leprosy (DLL) and Lucio's phenomenon (LPh). Our objective is to identify Mycobacterium spp. in an endemic area of leprosy in Colombia. Methods: The study included cases with a diagnosis of leprosy by clinical and histopathological analysis. DNA extraction and two specific rounds of semi-nested polymerase chain reaction (PCR) were performed in paraffin biopsies skin to identify M. leprae and M. lepromatosis. Demographic, clinical, and histopathological data were extracted and tabulated for analysis. Results: Forty-one cases of leprosy were analyzed. The most frequent clinical diagnosis was lepromatous leprosy (36.6%); there was one case with DLL and two with LPh. The most common histopathological finding was tuberculoid leprosy (36.59%); three cases had negative histopathology. M. lepromatosis was not detected; all cases corresponded to M. leprae including cases with negative histopathology' DLL, and LPh. Conclusion: In this study, M. leprae was the causative agent of leprosy, encompassing even its most severe phenotypic forms. It is appropriate to consider PCR as an indispensable tool for the diagnosis of leprosy and to continue to carry out the active search for M. lepromatosis.

Keywords: Clinical diagnosis, histopathological diagnosis, leprosy, Mycobacterium leprae, Mycobacterium lepromatosis


How to cite this article:
Fragozo-Ramos MC, Cano-Pérez E, Sierra-Merlano RM, Camacho-Chaljub F, Gómez-Camargo D. Clinical, histopathological, and molecular characterization of leprosy in an endemic area of the colombian caribbean. Int J Mycobacteriol 2021;10:155-61

How to cite this URL:
Fragozo-Ramos MC, Cano-Pérez E, Sierra-Merlano RM, Camacho-Chaljub F, Gómez-Camargo D. Clinical, histopathological, and molecular characterization of leprosy in an endemic area of the colombian caribbean. Int J Mycobacteriol [serial online] 2021 [cited 2022 Nov 28];10:155-61. Available from: https://www.ijmyco.org/text.asp?2021/10/2/155/318378




  Introduction Top


Leprosy also known as Hansen's disease is a chronic mycobacteriosis that mainly affects the skin and the peripheral nervous system resulting in neuropathy, deformity, and long-term disability.[1] Leprosy in Colombia was considered eliminated as a public health problem (defined as a prevalence of <1/10,000 inhabitants). However, the transmission of leprosy has not been interrupted, and according to the World Health Organization (WHO) for 2018, there were 208,641 new cases and 184,238 prevalent cases in the world. 5.3% of the cases present Grade 2 disability at the time of diagnosis, evidencing the late detection of the disease.[2] In Colombia, 381 new cases of leprosy were reported in 2019 (national incidence rate of 0.76/100,000 inhabitants), of which 61.9% were multibacillary forms. The departments with the highest incidence rate were Cesar (3.15), Huila (2.31), Norte de Santander (2.28), Tolima (2.20), Arauca (1.76), Bolívar (1.65), Barranquilla (1.38), and Cartagena (1.34).[3]

Leprosy has a heterogeneous clinical behavior, the immune and genetic response of the host determines its phenotypic manifestation, and the spectrum ranges from indeterminate leprosy (IL) and the paucibacillary tuberculoid pole until borderline forms and lepromatous pole with high bacillary load.[1],[4],[5] Some cases of leprosy have acute immune reactions with skin and systemic symptoms; these acute reactions are classified as Type 1 or reversal reactions,[6] Type 2 corresponding to erythema nodosum leprosum,[7] and Type 3 named Lucio's phenomenon (LPh) or necrotizing erythema.[8]

Mycobacterium leprae was considered the only causal agent of leprosy. However, in 2008, a new species of Mycobacterium were identified, described as Mycobacterium lepromatosis due it was isolated in Mexican natives with diffuse lepromatous leprosy (DLL), which is a subtype of severe multibacillary leprosy associated with LPh.[9] It has been suggested that M. lepromatosis has higher virulence and a lower reproducibility index concerning M. leprae. These differences could explain its identification in phenotypic forms of the disease with a high bacillary load.[7] The M. lepromatosis species has been documented mainly in Mexico and less frequently in countries such as Singapore[10] and Myanmar,[11] suggesting an intercontinental distribution of the species. The methods for identifying the causative species of leprosy using polymerase chain reaction (PCR) are a valuable tool to better understand the epidemiology of the disease, its transmission routes, and better diagnosis of leprosy cases.[12]

The present study aims to identify the etiological agents of leprosy (M. leprae and M. lepromatosis) by PCR assays in Cartagena and Bolívar, an endemic area of leprosy in Colombia, and to describe the clinicopathological forms of the disease.


  Methods Top


Study design and population

This was a descriptive ambispective study. Leprosy is a low prevalence disease; therefore, the sampling was carried out in a nonprobabilistic way for convenience. All cases with leprosy diagnosis confirmed for clinical or laboratory, with a skin biopsy, complete clinical and pathological information, and signed informed consent, were included in the study. The included cases were obtained from January 2015 to February 2020. This study was approved by the Ethics Committee of the University of Cartagena, Colombia (FM-CB-CERTI. 01-2020).

Collection and preparation of skin biopsies

Skin biopsies were collected in three reference pathology centers of Cartagena. The tissues were fixed in formaldehyde and embedded in paraffin according to accepted protocols. For the molecular study, the tissue blocks were recovered, individually wrapped, and transported to the laboratory.

Fine cuts of the paraffined tissues were made with the help of a scalpel; the blade was changed for each sample to avoid cross-contamination. Sections from each tissue were placed in labeled 1.5-ml microcentrifuge tubes. Deparaffinization was performed using the xylene protocol.[13] Briefly, 2 or 3 xylene washes were performed with incubating for 10 min at room temperature and shaking carefully to remove the paraffin. The pellet was rehydrated in an ethanol cycle at decreasing concentrations (absolute ethanol, 70%, and 50% ethanol). Each change was preceded by vortex homogenization and centrifugation. Finally, after removing the ethanol supernatant from the last wash, the pellet was air-dried for 5 min.

DNA extraction and differentiation of Mycobacterium by polymerase chain reaction

For the differentiation of the Mycobacterium species, DNA was previously extracted from samples using PureLink™ Genomic DNA Kits (Invitrogen®, Carlsbad, CA, USA) following the manufacturing protocol. The specific detection of M. leprae and M. lepromatosis was performed using two semi-nested PCRs to increase the sensitivity and specificity of the detection. The first round comprised one reaction mix with primers AFBFO (5′-GCGTGCTTAACACATGCAAGTC-3′) and MLER4 (5′-CCACAAGACATGCGCCTTGAAG-3′) that amplify a 171-bp region of the 16S rRNA gene common to all known Mycobacterium species. A second round was carried out from the amplification products of the first PCR; this second round comprised two mixes of independent reactions, one with the primers MLER4 and LPMF2 (5'-GTCTCTTAATACTTAAACCTATTAA-3′) to identify M. lepromatosis (142 bp) and another with the MLER4 and LERF2 (5'-CTAAAAAATCTTTTTTAGAGATAC-3′) primers to identify M. leprae (135 bp).[14] Each reaction mix was adjusted to a final volume of 25 μl and amplified under the following cycling conditions: activation of the enzyme at 95°C for 2 min; 35 cycles of denaturation (95°C for 20 s), primer annealing (58°C for 20 s for the first round of PCR or 48°C for 20 s for the second round of PCR), and extension (72°C for 40 s); and finally, the final extension of 5 min.[14] The PCR products were analyzed by electrophoresis on agarose gels using a 100 bp DNA ladder (Promega, Madison, WI, USA).

Statistical analysis

The database with the clinical and histopathological information was registered in Excel and later analyzed in the statistical software SPSS version 21.0 (IBM Corp, Armonk, NY, USA). The categorical variables were analyzed with absolute frequencies (count) and relative frequencies (%); the variables' numerical type was evaluated using the Shapiro–Wilk statistic to evaluate the assumption of normality. The variables were represented through the mean and the standard deviation (SD) or median with percentiles and quartiles according to the nature of their distribution. Fisher's exact test or Chi-square test was used as appropriate to examine differences between variables in different subgroups. Statistical significance was defined as P < 0.05.


  Results Top


Sociodemographic and clinicopathological characteristics

In this study, we collected 64 cases of leprosy, and 41 cases were included in the final analysis. The most frequent reason for exclusion was not having the histopathological sample [Figure 1]. The mean age was 50.5 years (SD: 16.4), the female sex was the most frequent with 54%, the cases came from Cartagena in 85%, and 73% were at the poverty level [Table 1]. The distribution of leprosy cases according to locality and communal unit of government shows in [Figure 2].
Table 1: Sociodemographic variables in patients with leprosy

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Figure 1: Flow diagram illustrating the number of participants included in the study for the identification of Mycobacterium leprae and Mycobacterium lepromatosis by polymerase chain reaction

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Figure 2: Distribution of leprosy cases according to locality and CUG. LOC: Locality, MB: Multibacillary, PB: Paucibacillary, CUG: Communal unit of government

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Of the 41 cases included in the analysis, 61% of the cases were multibacillary leprosy according to the WHO classification, 24 had lymph smear and bacillary index (BI) with a mean 0.6 and standard deviation 1.3 (SD 1.3) (range 0-6.4). The BI was paucibacillary (<0.1) in 39% and multibacillary (≥0.1) in 19% and 41% did not have a BI. One LLD case and two cases with LPh were presented. The time of evolution of the symptoms, the clinical classification of leprosy, and other clinical variables of interest are described in [Table 2]. Regarding the histopathology, 36% were LT, IL 29%, and lepromatous leprosy (LL) 19%; three cases were negative for leprosy.
Table 2: Time of evolution of symptoms, clinical classification of leprosy, and other clinical variables of interest

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When analyzing the distribution of variables between different groups, there was a statistically significant difference between the variables “grade of disability” and “type of case” (P < 0.001); we did not find significant statistical differences between the classification of the case and clinical diagnosis and the variables: health insurance scheme, location, the maximum grade of disability, and lepra reactions (P > 0.05).

Molecular differentiation of Mycobacterium and comparison of clinicopathological variables

The biopsies studied were stored between 2015 and 2020 with a median storage time of 2.4 years. The molecular identification of the leprosy species was obtained in 83% of the cases. All the cases corresponded to M. leprae including the cases with LLD, LPh, and negative histopathology; none of the studied samples corresponded to M. lepromatosis.

Of the 34 samples with positive PCR for M. leprae, the most frequent clinical diagnosis was LL in 41.2% and LT in 17.6%; in the samples without Mycobacterium detection, the most common clinical subtype was LT [Table 3]. Regarding the type of histopathological diagnosis, IL was present in 35% of the cases, followed by LT with 26%. In the cases with negative PCR, 6 were LT, and one LL [Table 3].
Table 3: Clinical and histopathological diagnosis of leprosy according to the molecular identification of Mycobacterium spp.

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  Discussion Top


Colombia reached the leprosy elimination goal proposed by the WHO in 1997, less than one in 10,000 inhabitants. Even when it is considered in the postelimination stage, an average of 400 new cases of leprosy are still diagnosed per year.[15] This is the first study, as far as is known, that examines the presence of M. leprae and M. lepromatosis in a Colombian population with leprosy. Furthermore, this research is essential due to Cartagena, Bolívar is considered an endemic area of leprosy where there were two hospitals for lepers: the Real Hospital de San Lázaro of Cartagena de Indias (1760–1810) and Lazareto de Caño de Loro (1870–1930).[16]

In this study, we observed LL being the most frequent clinical diagnosis and tuberculoid leprosy as the most common histopathological presentation. A significant difference in statistics was found between the highest degree of disability and type in terms of recurrences, and it is worth noting the importance of the molecular study in leprosy as there were three cases with histopathology that did not favor the disease. In all cases, M. leprae was identified, including cases with negative histopathology and even the most severe phenotypic forms of expression: LPh and DLL that have been associated with M. lepromatosis, which was not detected in this study.

Our results are similar to those reported by Vidal et al.[17], who found a high detection rate of multibacillary cases and patients with Grade 2 disability at the time of diagnosis. This finding suggests a late diagnosis of leprosy. Leprosy transmission is dynamic and complex with factors that depend on the host's social, economic, family, and immunogenetic environment;[18],[19] leprosy has historically been associated with poverty and social exclusion.[17],[20] Cartagena is the third city with the highest monetary poverty in Colombia, and a significant number of cases studied were grouped by areas with high population density and high social risk, extreme poverty, and a deficit of essential utilities such as water and sewerage.[21]

Another study by Serrano-Coll et al.[22] on cohabitants of a leprosy case described one of the main determinants of M. leprae infection as having low socioeconomic status and living in a vulnerable population. Cabral-Miranda et al.[23] show similar results in Brazil and point to unequal distribution of wealth, high population density, and overcrowding as risk factors that perpetuate the transmission of leprosy. In general, leprosy continues to be a deeply neglected disease linked to stigma and urban poverty. People affected with leprosy often feel fear, anxiety, and sorrow, as a result of stigma and discrimination.[24]

In the analyzed sample, it is evident that five of seven relapsed patients presented numbness of the hands or feet and visible deformity in contrast to seven of 34 new cases (P < 0.001). Prabu et al.[25] had similar findings in a retrospective cohort study where they identified 58 relapses, 31% (n = 18) with deformity. These data suggest that although the burden of relapses is low, the presence of physical deformity in this group is more frequent. The high bacillary load and the presence of leprosy reactions are also related to physical disability.[26]

Regarding the typification of species, Han et al. in 120 cases with clinical-pathological criteria of leprosy from 12 Mexican states identified the mycobacterial species in 72.5%, 55 cases for M. lepromatosis, 18 cases with M. leprae, and 14 cases with both. M. lepromatosis was isolated from all patients with DLL, and it was therefore concluded that this species specifically caused DLL.[14] In contrast, our data show that DLL and LPh were caused by M. leprae. Other authors have also identified M. leprae in cases of LPh and DLL.[27],[28],[29] This finding could suggest a host component, rather than the etiologic agent, as the leading risk factor for this reaction; however, the total number of cases is small in our study.

Phylogenetically, M. lepromatosis and M. leprae come from a common ancestor and must have separated more than 13.9 million years ago.[30] A study by Han et al.,[9] in which the etiological agents of leprosy were analyzed in four countries: Brazil (n = 52), Malaysia (n = 31), Myanmar (n = 9), and Uganda (n = 4), reported a higher frequency of M. leprae, in addition, they detected M. lepromatosis in various clinical forms of leprosy, which suggests that the phenotypic expression of DLL by M. lepromatosis is mainly endemic in Mexico. Furthermore, the presence of this species in Myanmar[9] and Singapore[8] favors the hypothesis of the Asian origin of the disease. It is striking that a study carried out in the province of Shandong, China, that involved 85 cases of leprosy for the genetic analysis of Mycobacterium spp., did not manage to identify M. lepromatosis in any of the samples studied, as it did in our population.[31]

In Colombia, molecular typing of M. leprae using variable number tandem repeat has been used to differentiate the strains. Thus, two groups have been found that show different origins of leprosy that reached Colombia. In the Andean region, there is an isolated mycobacterial group that coincides with European strains. In Cartagena, Bolívar, however, another type of M. leprae is found that corresponds to the Caribbean region where leprosy arrived with African slaves during the colonization period.[32] These findings could also explain differences in the geographical distribution of the species that cause leprosy and coincide with previously described models based on single-nucleotide polymorphisms.[33]

The diagnosis of leprosy continues to be predominantly clinical, however, the variability in its phenotypic forms leads to erroneous and late diagnoses, especially in paucibacillary forms of the disease. Conventional diagnostic tests such as smear microscopy, histopathology, and serological tests lack sensitivity, especially in the early stages of the disease or in asymptomatic individuals.[19],[34],[35] A study evaluated the clinical and histopathological correlation in 75 patients with clinically diagnosed leprosy; the results showed that the histopathology favored the diagnosis of leprosy in 72 of the cases. Similar to our findings, three cases did not show histopathological evidence of the disease and were diagnosed as dermatitis superficial perivascular. The correlation between the clinical and histopathological diagnoses was observed in 45.33% of the study participants.[36] In general, the degree of agreement between the clinical and histopathological diagnoses is very heterogeneous and varies between the different studies from 32.5% to 80.4%.[37],[38]

Given the limitations associated with routine diagnostic methods, PCR has emerged as an incredibly valuable tool for accurate diagnosis, especially in settings of clinical uncertainty or inconsistent clinical-pathological findings. A meta-analysis carried out by Gurung et al. showed a quantitative PCR sensitivity of 78.5% (95% confidence interval [CI]: 61.9–89.2) and a specificity of 89.3% (95% CI: 61.4–97.8). Conventional PCR showed a sensitivity of 75.3% (95% CI: 67.9–81.5) and specificity of 94.5% (95% CI: 91.4–96.5), and as observed in our population, up to 70% of the patients with leprosy may be smear negative and much smaller proportion histopathologies do not favor the diagnosis or are inconclusive.[39] Girma et al. evaluated the performance of PCR as an alternative diagnostic method versus standard histopathological diagnostic methods. In cases with negative histopathology (n = 24), an endpoint PCR test was performed, achieving confirmation of leprosy in 10/24 cases. PCR achieved better performance compared to pathology, even improving its sensitivity if enzymatic lysis was combined with mechanical tissue lysis (96.8%, P < 0.05).[40]

Previous studies support our findings and indicate the potential value of the primary performance of PCR to support clinical diagnosis without the requirement of pathology or other staining tests. However, the difficulty of carrying it out in first-level centers, the cost of the test, and the need for technical and laboratory infrastructure make its use a complementary method to establish the diagnosis of leprosy in histologically doubtful cases and especially in early stages or paucibacillary forms of the disease.[39],[41] Consequently, efforts should be made to include PCR as a diagnostic option under challenging cases with primers that allow the identification of both etiological species.

The strength of this study is to have included newly diagnosed individuals, which allowed us to describe relevant clinical information. Among the limitations, we find the poor condition and scarcity of tissue present in some samples, which possibly limited the identification of mycobacterium in cases with negative PCR. The use of molecular methods in fresh tissue samples or paraffin biopsies is suggested for future studies to guarantee an adequate quantity of the biological specimen.


  Conclusion Top


PCR assays performed to identify the causative agent of leprosy (M. lepromatosis and M. leprae) allowed a better diagnosis of the disease. In this series of cases of Cartagena and Bolivar (Colombia), M. leprae was the only species identified and was related to the most severe phenotypic forms such as LPh and DLL, so host-related factors should be considered as the main risk factors. Sociodemographic determinants must be evaluated as factors that favor and perpetuate lepromatous infection. Finally, PCR should be considered as an indispensable tool for the diagnosis of leprosy, the use of molecular assays for the identification of both species should be emphasized and actively search for M. lepromatosis in cases with negative PCR for M. leprae.

Ethical clearance

Within the framework of legal regulations in Colombia for research, this study was considered a research study with greater than minimal risk, based on article 11 of the ethical aspects of research in humans, resolution number 8430 of 1993. This study was approved by the ethics committee of the University of Cartagena (FM-CB- CERTI. 01-2020).

Acknowledgments

The authors would like to thank the Administrative Department of Health (DADIS) for their support in the identification and characterization of the cases and the personnel of the pathology centers for their collaboration in the collection of paraffin tissues.

Financial support and sponsorship

This work was supported by the UNIMOL Research group, University of Cartagena, with its own resources.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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