Blood test for leprosy: Laboratory Diagnostics | Hansen’s Disease (Leprosy)

29.07.197316.09.2021 by alexxlab

Содержание

Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae, the Causative Agent of Leprosy

J Clin Microbiol. 2014 Feb; 52(2): 613–619.

,^a,^b,^b,^b,^b,^c,^c and ^a

Malcolm S. Duthie

^aInfectious Disease Research Institute, Seattle, Washington, USA

Marivic F. Balagon

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Armi Maghanoy

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Florenda M. Orcullo

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Marjorie Cang

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Ronaldo Ferreira Dias

^cOrangeLife, Rio de Janeiro, Brazil

Marco Collovati

^cOrangeLife, Rio de Janeiro, Brazil

Steven G.

Reed

^aInfectious Disease Research Institute, Seattle, Washington, USA

G. A. Land, Editor

^aInfectious Disease Research Institute, Seattle, Washington, USA

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

^cOrangeLife, Rio de Janeiro, Brazil

Corresponding author.

Received 2013 Aug 2; Revisions requested 2013 Sep 3; Accepted 2013 Dec 5.

Abstract

Leprosy remains an important health problem in a number of regions. Early detection of infection, followed by effective treatment, is critical to reduce disease progression. New sensitive and specific tools for early detection of infection will be a critical component of an effective leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms, but few clinicians are able to confidently identify these. Simple tests to facilitate referral to leprosy experts are not widely available, and the correct diagnosis of leprosy is often delayed. In this report, we evaluate the performance of a new leprosy serological test (NDO-LID). As expected, the test readily detected clinically confirmed samples from patients with multibacillary (MB) leprosy, and the rate of positive results declined with bacterial burden. NDO-LID detected larger proportions of MB and paucibacillary (PB) leprosy than the alternative, the Standard Diagnostics leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%, respectively), while also demonstrating improved specificity (97.4% versus 90.4%). Coupled with a new cell phone-based test reader platform (Smart Reader), the NDO-LID test provided consistent, objective test interpretation that could facilitate wider use in nonspecialized settings. In addition, results obtained from sera at the time of diagnosis, versus at the end of treatment, indicated that the quantifiable nature of this system can also be used to monitor treatment efficacy. Taken together, these data indicate that the NDO-LID/Smart Reader system can assist in the diagnosis and monitoring of MB leprosy and can detect a significant number of earlier-stage infections.

INTRODUCTION

Despite advances toward the elimination of leprosy over the last 2 decades, new case detection rates have stabilized over the last decade and leprosy remains an important health problem in many regions (1). Like the number of registered cases, however, the number of clinicians who can reliably diagnose leprosy has waned, and delays in determining the correct diagnosis are common (2, 3). Efforts to develop and improve surveillance and referral systems appear necessary to achieve the early detection required to ensure that prompt and appropriate treatment can be provided to limit disabilities. The development of simple and practical tools to facilitate diagnosis would appear prudent.

Currently, the diagnosis of leprosy is dependent on the appearance and recognition of clinical signs and symptoms. When skin-smear or pathology services are available, leprologists utilize the Ridley-Jopling scale to characterize five forms of the disease (4, 5). In practice, however, most field programs lack such services, and the clinical system suggested by WHO to classify individual patients and to select their treatment regimen is used (6). The WHO system uses skin lesions, bacterial positivity by skin smear, and the number of involved nerves to group leprosy patients into one of two simplified categories: multibacillary (MB) leprosy (patients are typically smear positive, have involvement of more than one nerve, or have more than 5 lesions) and paucibacillary (PB) leprosy (patients are smear negative, have no nerve involvement or involvement of one nerve, and have a maximum of 5 lesions). In many settings, particularly the poorest rural settings where access to experienced leprologists is limited or entirely absent, primary care providers cannot identify the signs of leprosy and patients are commonly misdiagnosed and mistreated (7,–9). With the delays in diagnosis of MB leprosy, transmission of Mycobacterium leprae from infected individuals to their contacts continues, and in many cases, irreversible nerve damage occurs before those infected are registered as patients (10,–12). Simple tools that can facilitate leprosy diagnosis could help to address this deficit.

While PB leprosy patients have absent bacterial indices (BI; a measure of the number of acid-fast bacilli in the dermis expressed in a logarithmic scale) and generally have low or absent anti-M. leprae antibody responses, MB leprosy patients do not control bacterial replication and demonstrate titers of anti-M. leprae antibodies that correlate with their bacterial burdens (4). Phenolic glycolipid I (PGL-I) is well recognized as a target of the antibody response of leprosy patients, with the magnitude of the response strongly correlating with BI (13). To date, leprosy rapid diagnostic tests have comprised only PGL-I mimetics as the antigenic target (ML Flow test and Standard Diagnostics leprosy test [hereinafter called SD Leprosy], containing tri- or disaccharides NTP and NDO, respectively) (14,–17). Using laboratory-based assays, our group and others have identified numerous native and recombinant proteins recognized by antibodies in MB leprosy patient serum (18,–25). One example is the chimeric fusion protein leprosy IDRI diagnostic 1 (LID-1), an antigen specifically recognized by sera from leprosy patients from geographically and ethnically diverse populations, with a direct correlation between seroreactivity and BI (18, 26,–28). Complementary detection of antibodies against PGL-I or the components of LID-1 could lead to improved sensitivity within tests.

In conjunction with OrangeLife, Rio de Janeiro, we have created simple immunochromatographic lateral flow tests with the capacity to detect PGL-I- and LID-1-specific antibodies. In this report, we assess the performance of this new rapid diagnostic test against that of a previously available test using newly acquired and archived serum samples from clinically confirmed leprosy patients in Cebu, Philippines.

MATERIALS AND METHODS

Study site and participants.

Blood samples were collected following local ethics committee approval from Cebu Skin Clinic attendees after they signed informed consent forms. Patients were fully characterized on the Ridley-Jopling scale by slit skin smear and biopsy (4). Healthy household contacts (HHCs) of MB leprosy patients were enrolled as individuals at elevated risk of developing leprosy (29). Individuals presenting with other skin conditions/diseases were also enrolled as endemic controls (ECs). Serum samples from a total of 208 newly diagnosed MB leprosy patients, 62 newly diagnosed PB leprosy patients, 51 healthy household contacts of MB leprosy patients, and 63 endemic controls were evaluated in distinct panels (, and ). Sera were prepared by centrifugation. Following diagnosis, each leprosy patient was provided with a standard multidrug therapy (MDT) regimen as recommended by WHO (for MB leprosy patients, rifampin [600 mg once a month], dapsone [100 mg daily], and clofazimine [300 mg once a month and 50 mg daily] for 12 months; for PB leprosy patients, rifampin [600 mg once a month] and dapsone [100 mg daily] for 6 months).

TABLE 1

Initial evaluation panel to determine specificity and sensitivity of rapid diagnostic tests by subjective interpretation

Sample type	Study A			Study B
	No. of samples	% positive results^{^a}		No. of samples	% positive results^{^a}
	No. of samples	NDO-LID	SD Leprosy	No. of samples	NDO-LID	SD Leprosy
MB^{^b}	48	93. 8	77.1	40	85.0	77.5
PB	19	52.6	0.0	13	30.8	8.3
HHC				11	0.0	0.0
EC	12	0.0	25.0	26	3.8	0.0

TABLE 2

Specificity and sensitivity of rapid diagnostic tests following development with a secondary serum panel

Sample type	BI^{^a}	No. of samples	% positive results^{^b}
Sample type	BI^{^a}	No. of samples	NDO-LID	SD Leprosy
MB	High	40	100.0	97.5
	Medium	40	85.0	90.0
	Low	40	65.0	65.0
PB		30	20. 0	10.0
HHC		40	2.5	12.0
EC		25	0.0	12.5

TABLE 3

Cumulative performance of rapid diagnostic tests following subjective interpretation

Sample type	No. of samples	No. (%) of positive results^{^a}
Sample type	No. of samples	NDO-LID	SD Leprosy
MB	208	181 (87. 0)	170 (81.7)
PB	62	20 (32.3)	4 (6.5)
HHC	51	2 (3.9)	5 (9.8)
EC	63	1 (1.6)	6 (9.5)
Leprosy	270	201 (74.4)	174 (64.4)
Not leprosy	114	3 (2.6)	11 (9.6)

Rapid diagnostic tests.

Sera were tested either within 2 h of collection or after thawing following storage at −20°C for up to 6 years. Two rapid diagnostic tests were evaluated: the SD Leprosy test was purchased from Standard Diagnostics (Yongin, South Korea), and NDO-LID was fabricated by OrangeLife (Rio de Janeiro, Brazil). Each is a simple immunochromatographic (lateral flow) test with the purpose of detecting circulating antibodies. The SD Leprosy test detects IgM antibodies to M. leprae-specific PGL-I through the use of NDO-bovine serum albumin (NDO-BSA; a synthetic mimetic of PGL-I conjugated to BSA), while NDO-LID detects IgM antibodies to PGL-I and IgG antibodies specific to LID-1 (the synthetic mimetic conjugated to the recombinant fusion protein product of the M. leprae genes ML0405 and ML2331) (18). Evaluations with each rapid diagnostic test involved the addition of undiluted serum (10 μl) and running buffer (2 to 3 drops; ∼100 μl) to a sample well, followed by readings of line development in the detection window after 10 min. Validation of the results required the visualization of a colored control line. A positive result was defined by the staining of both the control line and the test line; faint or no staining was considered a negative result. Visual readings were performed by a minimum of two independent readers.

Objective measurement of NDO-LID.

NDO-LID tests have been adapted such that they can be read electronically by a Smart Reader, an Android-based smartphone rapid test reader platform mechanically attached to the existing camera unit (). This reader collected test images and objectively quantified the signal intensities of the control and test lines in each NDO-LID test. The calculation of Smart Reader cutoff values was based on the receiver operating curve, taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual readings, the Smart Reader cutoff was calculated as 9.99. For data analysis, the cutoff for positive results by the Smart Reader was therefore considered 10. 0. Assuming this cutoff, the sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87% (95% confidence interval [CI], 79.2 to 92.7%) and the specificity was 96.1% (95% CI, 91.7 to 98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, 0.01; P < 0.0001) (30).

Representative images and subjective scoring system for NDO-LID. Tests were developed, and examples of each scoring group that was subjectively assigned are shown. The NDO-LID tests have been adapted such that they can be read electronically by a Smart Reader application, an Android-based cell phone rapid test reader platform mechanically attached to the existing camera unit.

Statistical analysis.

Statistical significance was assessed using an unpaired t test for comparison between two groups. Results were considered statistically significant when P values of <0.05 were obtained.

RESULTS

Comparison of two rapid diagnostic tests for leprosy.

We analyzed sera using both the SD Leprosy test (based on the detection of IgM antibodies against the PGL-I mimetic NDO antigen) and the NDO-LID rapid diagnostic test (based on the detection of IgM antibodies against NDO and IgG antibodies against the LID-1 protein) to permit direct comparisons between these tests. In an initial study, subjective interpretation indicated that when developed with sera from MB leprosy patients, NDO-LID tests produced a significantly stronger band than that observed with SD Leprosy tests (). This was true for both fresh and frozen samples (P values, 0.011 and 0.003, respectively). Sensitivity for MB leprosy patient samples in this initial study was found to be 93.8% (45 of 48 samples) with NDO-LID versus 77.1% (37 of 48) for SD Leprosy (). While previous storage did not affect performance in the NDO-LID tests, the signal intensity of SD Leprosy tests was, surprisingly, lower when freshly prepared sera were added (). These results were verified against another panel of sera (, P value of 0. 02).

Improved performance of NDO-LID over SD Leprosy. In an initial study (A), stored (from MB leprosy patients only, n = 38) or fresh (MB leprosy patients, n = 10; PB leprosy patients, n = 19; and EC, n = 12) sera were tested by either the SD Leprosy or the NDO-LID rapid diagnostic test. The strength of the test band was then subjectively interpreted on a scale of 0 to 4 (negative to strong positive). In a follow-on study (B), fresh sera (MB leprosy patients, n = 40; PB leprosy patients, n = 13; HHC, n = 26; and EC, n = 11) were evaluated in the same manner, with the exception that the scoring scale had a maximum value of 3. Results from one interpreter are shown, and they were verified/corroborated by the additional interpreter. Strength of the NDO-LID test and control bands was then objectively measured using the Smart Reader (panel C indicates the objective measurement of the tests that were subjectively assessed in panel A, and panel D depicts the objective measurement of the tests subjectively assessed in panel B). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 between the indicated groups. Results in panels A and B are shown as means and standard errors of the means (SEM) for each group; horizontal bars in panels C and D indicate means.

PB leprosy patients have low or absent antibody responses and are not well recognized in rapid diagnostic tests containing only PGL-I mimetics (14, 16). In agreement, when NDO-LID and SD Leprosy rapid diagnostic tests were developed with PB leprosy patient sera, only a subset of PB leprosy patient samples could be distinguished. A stronger signal was, however, observed with the NDO-LID tests than with the SD Leprosy tests, such that a greater proportion of PB leprosy patient samples were positive (, P value of <0.0001) (, 52.6% positive by NDO-LID versus 0.0% by SD Leprosy). Despite returning stronger results with patient samples, the NDO-LID tests were less likely to be positive than SD Leprosy tests when developed with sera from control individuals ( and , 0. 0% positive by NDO-LID versus 25.0% by SD Leprosy). An independent follow-up study using only fresh sera confirmed that the NDO-LID test had a greater band intensity when developed with leprosy patient sera () and that a greater proportion of patients could be discriminated (). Together, these data indicate that the NDO-LID test provides a greater differential of positive and negative results than the SD Leprosy test, with improved discrimination of leprosy patients from healthy individuals.

Objective and quantitative evaluation by NDO-LID.

Visual interpretation of results is highly subjective and represents an important limitation when performed by personnel lacking expertise, limiting their scope of use. Results from any diagnostic test would ideally also be blinded from the clinical evaluation, but this is difficult to achieve in rural settings with limited resources. To address this deficit, a simple test reader (Smart Reader) can be used to permit objective scrutiny of data following the subjective evaluation of each NDO-LID. While readings on the control line were relatively consistent, a wide range of values were obtained when tests developed with sera from patients with MB leprosy were analyzed (). The Smart Reader identified an additional 1 and 5 samples as positive, respectively, increasing sensitivity to 95.8% in study A (46 of 48 samples) and 97.5% in study B (39 of 40). When signal intensities were compared, there was a highly significant correlation of readings (Spearman r, 0.967) (). Repetitive Smart Reader quantification of the same test, and repeat testing of the same sample, yielded highly consistent results (data not shown). In addition, although the manufacturer does not provide guidelines for reevaluation, only a minor but consistent drop in signal intensity was measured when values were reevaluated approximately 1 month later (Spearman r, 0.980) (). Thus, when coupled with a Smart Reader, NDO-LID tests provide rapid, consistent, robust, and objective quantification of seroreactivity.

NDO-LID/Smart Reader provide a robust system for evaluation. (A) NDO-LID tests were developed, and subjectively assigned values were plotted versus objective Smart Reader measurements to determine correlation. (B) Smart Reader values obtained 10 min after test development (initial value) are plotted versus values obtained by reading the same tests 1 month later. The solid line represents the best-fit linear regression, and the Spearman r value is shown.

Correlation of rapid diagnostic test results with BI.

We then evaluated rapid diagnostic test performance across sera from patients with MB leprosy identified to have either high (>4.0), medium (2.0 to 3.9), or low (<2.0) BI at the time of clinical diagnosis. As expected, test bands were most intense for the high-BI patients and diminished as BI decreased (). While both rapid diagnostic tests performed well in detecting MB leprosy patients, the signal intensity was significantly greater in NDO-LID tests than in SD Leprosy tests. NDO-LID tests detected 20% of the PB leprosy serum samples in this testing round versus 10% detected by SD Leprosy tests. More importantly, while the SD Leprosy tests returned positive results for similar proportions of healthy household contacts and endemic controls (12.0 and 12.5%, respectively), the NDO-LID tests improved specificity (2.5% healthy household contacts were identified as being positive, and there were no positive results against endemic controls) (). The subjective NDO-LID results were corroborated by Smart Reader (). Thus, the NDO-LID test can readily detect MB leprosy patients, and compared to the SD Leprosy test, it permits improved discrimination of PB leprosy patients from the general population.

Measurement of patient response to treatment by NDO-LID. Archived sera were selected based on patient BI at time of collection and then evaluated by NDO-LID and SD Leprosy (MB leprosy patients with high BI, n = 40, medium BI, n = 40, and low BI, n = 40; PB leprosy patients, n = 30; HHC, n = 30; and EC, n = 25). (A) The strength of the test band in each rapid diagnostic test was subjectively interpreted on a scale of 0 to 3 (negative to strong positive). Results are shown as means and SEM for each group. (B) The objective NDO-LID/Smart Reader of sera collected from patients at either time of diagnosis or end of MDT are shown. The values for “at diagnosis” samples were generated from the tests depicted in panel A, while the same number of “after treatment” samples. Horizontal bars indicate means. *, P < 0.05; **, P < 0.01 between indicated groups.

Monitoring treatment by NDO-LID and Smart Reader.

Smart Reader measurements could confer additional utility beyond initial detection and patient classification. To evaluate if the rapid diagnostic test/Smart Reader combination was sensitive enough to monitor treatment, we contrasted results generated using sera collected from patients at the time of diagnosis against sera collected at MDT completion. Overall, there was a reduced signal intensity in the after-treatment samples, with the decline most obvious in sera from patients that had the highest BI at intake (, P value of 0. 003). In these high-BI MB leprosy patients, the mean reading of 46.3 at diagnosis declined to 27.7 after treatment, while in medium-BI MB leprosy patients, the decline was from 27.2 to 20.7, and in low-BI MB leprosy patients, it was from 10.2 to 6.1. These data suggest the utility of continued testing during, and even after, MDT.

DISCUSSION

Leprosy control programs are currently structured around the treatment of cases as they are reported. Case numbers are now relatively low in most regions, however, such that fewer clinicians have experience with the disease and only a limited number can confidently recognize the early signs of leprosy. Diagnosis is therefore commonly delayed, and the appearance of leprosy-associated disabilities may become more frequent (2, 3). By lessening the reliance on the clinical exam and the recognition of symptoms, simple tools like the rapid diagnostic tests evaluated here could address this shortcoming. Tests could greatly aid general practitioners in their evaluation of suspected cases. This would appear particularly prudent in regions where a large proportion of patients present as MB leprosy cases, such as the Philippines (31). Importantly, the ability to objectively read and quantify NDO-LID test results using a Smart Reader eliminates the need for prior training/experience in interpreting test results. This also provides an objective threshold and generates results that are consistent regardless of the many variables that could adversely affect test interpretation (different users, days, times of day, locations, etc.). This is of particular importance considering the possibility of individual bias in subjective reading of rapid tests in field conditions. The quantitative information could also be used to monitor individuals suspected of being infected with M. leprae over time or to expedite an informed referral to a leprosy expert.

The uptake of previous rapid tests for leprosy was restricted due to the relatively high proportion of seropositive results in the general population in regions of endemicity, despite the fact that the individuals with these results did not display clinical symptoms (15). In any test, the threshold for a positive result is obviously critical to establish test performance, balancing sensitivity against specificity. Our previous results, obtained in the laboratory setting by enzyme-linked immunosorbent assays (ELISA), indicate that LID-1 provides an improved discrimination of leprosy patient samples from those of control subjects (18). This would appear to continue through to the NDO-LID that contains a combination of NDO and LID-1. Our data not only demonstrate the utility of the NDO-LID but also strongly indicate an improved performance, in terms of both sensitivity and specificity, over that of the SD Leprosy rapid diagnostic test.

Consistent with WHO recommendations, Cebu Skin Clinic staff clinically examine contacts of MB leprosy patients at 6-month intervals over the course of 2 years following index case reporting (6). While this system facilitates the earlier recognition of leprosy among these contacts, it is labor-intensive and time-consuming, especially given Cebu’s size. Additional practical and economic considerations (presence during visits, ensuring that work is not impacted, etc.) necessitate vigorous and sustained efforts to ensure that as many individuals can be observed as possible. Because the NDO-LID/Smart Reader is simple and rapid (10 to 20 tests can be conducted by one individual in 30 min), their integration could simplify and enhance this type of monitoring. The duration of any household visit could be markedly reduced, and evaluations could potentially be made at a much greater frequency than that of clinical exams. Any marked increase in test values could trigger a full clinical exam along with regularly scheduled visits. In this regard, strong results in LID-1 laboratory-based ELISA have already been used to draw attention to individuals who have subsequently developed clinical symptoms (24, 27). The levels detected in ELISA that have triggered such attention are readily detected in the NDO-LID test (30). In addition, the robustness/stability of developed tests suggest that if tests perform equally well when using whole blood, they could be sent in advance to patients so that each of their household members could use them at a convenient time proximate to a surveillance visit. The long-term preservation of signal in the tests also suggests that they could even be returned to a central facility for quantitation. By simplifying the referral system, enhancing surveillance programs, or a combination of both, the use of an objective and quantifiable rapid diagnostic test could provide earlier detection and, through prompt treatment, a further reduction in leprosy-associated disabilities.

By lab-based ELISA, we previously identified reductions in patient antigen-specific antibody responses during treatment (24, 32). These subtle changes can be captured by the NDO-LID/Smart Reader combination. In another study, we identified patients who were mistakenly undertreated or who had poor compliance with treatment (28). We hypothesize that, in parallel with clinical examinations, thorough quantification of serological antibody responses by Smart Reader will allow us to capture nonresponse to treatment. Given that truncated treatment regimens are being proposed (33,–36), projecting how a patient will respond to treatment without the need for invasive skin slits or biopsies would be an important and practical tool in trial design. Expanding evaluations into the treatment phase could ultimately provide objective guidelines for clinicians to identify high-risk groups requiring additional monitoring, permitting streamlining and prioritization within currently stretched control programs.

In summary, the highly quantifiable nature of the NDO-LID test/Smart Reader platform appears to have utility for detection and monitoring of MB leprosy. We believe it could enhance surveillance, facilitate referrals, and be an important tool in trials of new interventions and treatments.

ACKNOWLEDGMENTS

We are extremely grateful to the patients and their contacts for generous participation and thank the field and laboratory staff of Cebu Skin Clinic and Leonard Wood Memorial Center for Leprosy Research for their excellent clinical and technical assistance.

This work was supported by the American Leprosy Missions and the Renaissance Health Service Corporation.

Marco Collovati is the owner of OrangeLife, the company producing and marketing the NDO-LID rapid test. Ronaldo Ferreira Dias is an employee of OrangeLife.

Footnotes

Published ahead of print 11 December 2013

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Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae, the Causative Agent of Leprosy

J Clin Microbiol. 2014 Feb; 52(2): 613–619.

,^a,^b,^b,^b,^b,^c,^c and ^a

Malcolm S. Duthie

^aInfectious Disease Research Institute, Seattle, Washington, USA

Marivic F. Balagon

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Armi Maghanoy

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Florenda M. Orcullo

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Marjorie Cang

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Ronaldo Ferreira Dias

^cOrangeLife, Rio de Janeiro, Brazil

Marco Collovati

^cOrangeLife, Rio de Janeiro, Brazil

Steven G. Reed

^aInfectious Disease Research Institute, Seattle, Washington, USA

G. A. Land, Editor

^aInfectious Disease Research Institute, Seattle, Washington, USA

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

^cOrangeLife, Rio de Janeiro, Brazil

Corresponding author.

Received 2013 Aug 2; Revisions requested 2013 Sep 3; Accepted 2013 Dec 5.

Abstract

INTRODUCTION

MATERIALS AND METHODS

Study site and participants.

TABLE 1

Initial evaluation panel to determine specificity and sensitivity of rapid diagnostic tests by subjective interpretation

Sample type	Study A			Study B
	No. of samples	% positive results^{^a}		No. of samples	% positive results^{^a}
	No. of samples	NDO-LID	SD Leprosy	No. of samples	NDO-LID	SD Leprosy
MB^{^b}	48	93.8	77.1	40	85.0	77.5
PB	19	52.6	0.0	13	30.8	8.3
HHC				11	0.0	0.0
EC	12	0.0	25.0	26	3.8	0.0

TABLE 2

Specificity and sensitivity of rapid diagnostic tests following development with a secondary serum panel

Sample type	BI^{^a}	No. of samples	% positive results^{^b}
Sample type	BI^{^a}	No. of samples	NDO-LID	SD Leprosy
MB	High	40	100.0	97.5
	Medium	40	85.0	90.0
	Low	40	65.0	65.0
PB		30	20.0	10.0
HHC		40	2.5	12.0
EC		25	0.0	12.5

TABLE 3

Cumulative performance of rapid diagnostic tests following subjective interpretation

Sample type	No. of samples	No. (%) of positive results^{^a}
Sample type	No. of samples	NDO-LID	SD Leprosy
MB	208	181 (87.0)	170 (81.7)
PB	62	20 (32.3)	4 (6.5)
HHC	51	2 (3.9)	5 (9.8)
EC	63	1 (1.6)	6 (9.5)
Leprosy	270	201 (74.4)	174 (64.4)
Not leprosy	114	3 (2.6)	11 (9.6)

Rapid diagnostic tests.

Objective measurement of NDO-LID.

NDO-LID tests have been adapted such that they can be read electronically by a Smart Reader, an Android-based smartphone rapid test reader platform mechanically attached to the existing camera unit (). This reader collected test images and objectively quantified the signal intensities of the control and test lines in each NDO-LID test. The calculation of Smart Reader cutoff values was based on the receiver operating curve, taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual readings, the Smart Reader cutoff was calculated as 9.99. For data analysis, the cutoff for positive results by the Smart Reader was therefore considered 10.0. Assuming this cutoff, the sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87% (95% confidence interval [CI], 79.2 to 92.7%) and the specificity was 96.1% (95% CI, 91.7 to 98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, 0.01; P < 0.0001) (30).

Statistical analysis.

Statistical significance was assessed using an unpaired t test for comparison between two groups. Results were considered statistically significant when P values of <0.05 were obtained.

RESULTS

Comparison of two rapid diagnostic tests for leprosy.

PB leprosy patients have low or absent antibody responses and are not well recognized in rapid diagnostic tests containing only PGL-I mimetics (14, 16). In agreement, when NDO-LID and SD Leprosy rapid diagnostic tests were developed with PB leprosy patient sera, only a subset of PB leprosy patient samples could be distinguished. A stronger signal was, however, observed with the NDO-LID tests than with the SD Leprosy tests, such that a greater proportion of PB leprosy patient samples were positive (, P value of <0.0001) (, 52.6% positive by NDO-LID versus 0.0% by SD Leprosy). Despite returning stronger results with patient samples, the NDO-LID tests were less likely to be positive than SD Leprosy tests when developed with sera from control individuals ( and , 0.0% positive by NDO-LID versus 25.0% by SD Leprosy). An independent follow-up study using only fresh sera confirmed that the NDO-LID test had a greater band intensity when developed with leprosy patient sera () and that a greater proportion of patients could be discriminated (). Together, these data indicate that the NDO-LID test provides a greater differential of positive and negative results than the SD Leprosy test, with improved discrimination of leprosy patients from healthy individuals.

Objective and quantitative evaluation by NDO-LID.

Correlation of rapid diagnostic test results with BI.

Monitoring treatment by NDO-LID and Smart Reader.

Smart Reader measurements could confer additional utility beyond initial detection and patient classification. To evaluate if the rapid diagnostic test/Smart Reader combination was sensitive enough to monitor treatment, we contrasted results generated using sera collected from patients at the time of diagnosis against sera collected at MDT completion. Overall, there was a reduced signal intensity in the after-treatment samples, with the decline most obvious in sera from patients that had the highest BI at intake (, P value of 0.003). In these high-BI MB leprosy patients, the mean reading of 46.3 at diagnosis declined to 27.7 after treatment, while in medium-BI MB leprosy patients, the decline was from 27.2 to 20.7, and in low-BI MB leprosy patients, it was from 10.2 to 6.1. These data suggest the utility of continued testing during, and even after, MDT.

DISCUSSION

ACKNOWLEDGMENTS

This work was supported by the American Leprosy Missions and the Renaissance Health Service Corporation.

Marco Collovati is the owner of OrangeLife, the company producing and marketing the NDO-LID rapid test. Ronaldo Ferreira Dias is an employee of OrangeLife.

Footnotes

Published ahead of print 11 December 2013

REFERENCES

Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae, the Causative Agent of Leprosy

J Clin Microbiol. 2014 Feb; 52(2): 613–619.

,^a,^b,^b,^b,^b,^c,^c and ^a

Malcolm S. Duthie

^aInfectious Disease Research Institute, Seattle, Washington, USA

Marivic F. Balagon

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Armi Maghanoy

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Florenda M. Orcullo

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Marjorie Cang

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Ronaldo Ferreira Dias

^cOrangeLife, Rio de Janeiro, Brazil

Marco Collovati

^cOrangeLife, Rio de Janeiro, Brazil

Steven G. Reed

^aInfectious Disease Research Institute, Seattle, Washington, USA

G. A. Land, Editor

^aInfectious Disease Research Institute, Seattle, Washington, USA

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

^cOrangeLife, Rio de Janeiro, Brazil

Corresponding author.

Received 2013 Aug 2; Revisions requested 2013 Sep 3; Accepted 2013 Dec 5.

Abstract

INTRODUCTION

MATERIALS AND METHODS

Study site and participants.

TABLE 1

Initial evaluation panel to determine specificity and sensitivity of rapid diagnostic tests by subjective interpretation

Sample type	Study A			Study B
	No. of samples	% positive results^{^a}		No. of samples	% positive results^{^a}
	No. of samples	NDO-LID	SD Leprosy	No. of samples	NDO-LID	SD Leprosy
MB^{^b}	48	93.8	77.1	40	85.0	77.5
PB	19	52.6	0.0	13	30.8	8.3
HHC				11	0.0	0.0
EC	12	0.0	25.0	26	3.8	0.0

TABLE 2

Specificity and sensitivity of rapid diagnostic tests following development with a secondary serum panel

Sample type	BI^{^a}	No. of samples	% positive results^{^b}
Sample type	BI^{^a}	No. of samples	NDO-LID	SD Leprosy
MB	High	40	100.0	97.5
	Medium	40	85.0	90.0
	Low	40	65.0	65.0
PB		30	20.0	10.0
HHC		40	2.5	12.0
EC		25	0.0	12.5

TABLE 3

Cumulative performance of rapid diagnostic tests following subjective interpretation

Sample type	No. of samples	No. (%) of positive results^{^a}
Sample type	No. of samples	NDO-LID	SD Leprosy
MB	208	181 (87.0)	170 (81.7)
PB	62	20 (32.3)	4 (6.5)
HHC	51	2 (3.9)	5 (9.8)
EC	63	1 (1.6)	6 (9.5)
Leprosy	270	201 (74.4)	174 (64.4)
Not leprosy	114	3 (2.6)	11 (9.6)

Rapid diagnostic tests.

Objective measurement of NDO-LID.

NDO-LID tests have been adapted such that they can be read electronically by a Smart Reader, an Android-based smartphone rapid test reader platform mechanically attached to the existing camera unit (). This reader collected test images and objectively quantified the signal intensities of the control and test lines in each NDO-LID test. The calculation of Smart Reader cutoff values was based on the receiver operating curve, taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual readings, the Smart Reader cutoff was calculated as 9.99. For data analysis, the cutoff for positive results by the Smart Reader was therefore considered 10.0. Assuming this cutoff, the sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87% (95% confidence interval [CI], 79.2 to 92.7%) and the specificity was 96.1% (95% CI, 91.7 to 98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, 0.01; P < 0.0001) (30).

Statistical analysis.

Statistical significance was assessed using an unpaired t test for comparison between two groups. Results were considered statistically significant when P values of <0.05 were obtained.

RESULTS

Comparison of two rapid diagnostic tests for leprosy.

PB leprosy patients have low or absent antibody responses and are not well recognized in rapid diagnostic tests containing only PGL-I mimetics (14, 16). In agreement, when NDO-LID and SD Leprosy rapid diagnostic tests were developed with PB leprosy patient sera, only a subset of PB leprosy patient samples could be distinguished. A stronger signal was, however, observed with the NDO-LID tests than with the SD Leprosy tests, such that a greater proportion of PB leprosy patient samples were positive (, P value of <0.0001) (, 52.6% positive by NDO-LID versus 0.0% by SD Leprosy). Despite returning stronger results with patient samples, the NDO-LID tests were less likely to be positive than SD Leprosy tests when developed with sera from control individuals ( and , 0.0% positive by NDO-LID versus 25.0% by SD Leprosy). An independent follow-up study using only fresh sera confirmed that the NDO-LID test had a greater band intensity when developed with leprosy patient sera () and that a greater proportion of patients could be discriminated (). Together, these data indicate that the NDO-LID test provides a greater differential of positive and negative results than the SD Leprosy test, with improved discrimination of leprosy patients from healthy individuals.

Objective and quantitative evaluation by NDO-LID.

Correlation of rapid diagnostic test results with BI.

Monitoring treatment by NDO-LID and Smart Reader.

Smart Reader measurements could confer additional utility beyond initial detection and patient classification. To evaluate if the rapid diagnostic test/Smart Reader combination was sensitive enough to monitor treatment, we contrasted results generated using sera collected from patients at the time of diagnosis against sera collected at MDT completion. Overall, there was a reduced signal intensity in the after-treatment samples, with the decline most obvious in sera from patients that had the highest BI at intake (, P value of 0.003). In these high-BI MB leprosy patients, the mean reading of 46.3 at diagnosis declined to 27.7 after treatment, while in medium-BI MB leprosy patients, the decline was from 27.2 to 20.7, and in low-BI MB leprosy patients, it was from 10.2 to 6.1. These data suggest the utility of continued testing during, and even after, MDT.

DISCUSSION

ACKNOWLEDGMENTS

This work was supported by the American Leprosy Missions and the Renaissance Health Service Corporation.

Marco Collovati is the owner of OrangeLife, the company producing and marketing the NDO-LID rapid test. Ronaldo Ferreira Dias is an employee of OrangeLife.

Footnotes

Published ahead of print 11 December 2013

REFERENCES

Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae, the Causative Agent of Leprosy

J Clin Microbiol. 2014 Feb; 52(2): 613–619.

,^a,^b,^b,^b,^b,^c,^c and ^a

Malcolm S. Duthie

^aInfectious Disease Research Institute, Seattle, Washington, USA

Marivic F. Balagon

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Armi Maghanoy

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Florenda M. Orcullo

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Marjorie Cang

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

Ronaldo Ferreira Dias

^cOrangeLife, Rio de Janeiro, Brazil

Marco Collovati

^cOrangeLife, Rio de Janeiro, Brazil

Steven G. Reed

^aInfectious Disease Research Institute, Seattle, Washington, USA

G. A. Land, Editor

^aInfectious Disease Research Institute, Seattle, Washington, USA

^bLeonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines

^cOrangeLife, Rio de Janeiro, Brazil

Corresponding author.

Received 2013 Aug 2; Revisions requested 2013 Sep 3; Accepted 2013 Dec 5.

Abstract

INTRODUCTION

MATERIALS AND METHODS

Study site and participants.

TABLE 1

Initial evaluation panel to determine specificity and sensitivity of rapid diagnostic tests by subjective interpretation

Sample type	Study A			Study B
	No. of samples	% positive results^{^a}		No. of samples	% positive results^{^a}
	No. of samples	NDO-LID	SD Leprosy	No. of samples	NDO-LID	SD Leprosy
MB^{^b}	48	93.8	77.1	40	85.0	77.5
PB	19	52.6	0.0	13	30.8	8.3
HHC				11	0.0	0.0
EC	12	0.0	25.0	26	3.8	0.0

TABLE 2

Specificity and sensitivity of rapid diagnostic tests following development with a secondary serum panel

Sample type	BI^{^a}	No. of samples	% positive results^{^b}
Sample type	BI^{^a}	No. of samples	NDO-LID	SD Leprosy
MB	High	40	100.0	97.5
	Medium	40	85.0	90.0
	Low	40	65.0	65.0
PB		30	20.0	10.0
HHC		40	2.5	12.0
EC		25	0.0	12.5

TABLE 3

Cumulative performance of rapid diagnostic tests following subjective interpretation

Sample type	No. of samples	No. (%) of positive results^{^a}
Sample type	No. of samples	NDO-LID	SD Leprosy
MB	208	181 (87.0)	170 (81.7)
PB	62	20 (32.3)	4 (6.5)
HHC	51	2 (3.9)	5 (9.8)
EC	63	1 (1.6)	6 (9.5)
Leprosy	270	201 (74.4)	174 (64.4)
Not leprosy	114	3 (2.6)	11 (9.6)

Rapid diagnostic tests.

Objective measurement of NDO-LID.

NDO-LID tests have been adapted such that they can be read electronically by a Smart Reader, an Android-based smartphone rapid test reader platform mechanically attached to the existing camera unit (). This reader collected test images and objectively quantified the signal intensities of the control and test lines in each NDO-LID test. The calculation of Smart Reader cutoff values was based on the receiver operating curve, taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual readings, the Smart Reader cutoff was calculated as 9.99. For data analysis, the cutoff for positive results by the Smart Reader was therefore considered 10.0. Assuming this cutoff, the sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87% (95% confidence interval [CI], 79.2 to 92.7%) and the specificity was 96.1% (95% CI, 91.7 to 98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, 0.01; P < 0.0001) (30).

Statistical analysis.

Statistical significance was assessed using an unpaired t test for comparison between two groups. Results were considered statistically significant when P values of <0.05 were obtained.

RESULTS

Comparison of two rapid diagnostic tests for leprosy.

PB leprosy patients have low or absent antibody responses and are not well recognized in rapid diagnostic tests containing only PGL-I mimetics (14, 16). In agreement, when NDO-LID and SD Leprosy rapid diagnostic tests were developed with PB leprosy patient sera, only a subset of PB leprosy patient samples could be distinguished. A stronger signal was, however, observed with the NDO-LID tests than with the SD Leprosy tests, such that a greater proportion of PB leprosy patient samples were positive (, P value of <0.0001) (, 52.6% positive by NDO-LID versus 0.0% by SD Leprosy). Despite returning stronger results with patient samples, the NDO-LID tests were less likely to be positive than SD Leprosy tests when developed with sera from control individuals ( and , 0.0% positive by NDO-LID versus 25.0% by SD Leprosy). An independent follow-up study using only fresh sera confirmed that the NDO-LID test had a greater band intensity when developed with leprosy patient sera () and that a greater proportion of patients could be discriminated (). Together, these data indicate that the NDO-LID test provides a greater differential of positive and negative results than the SD Leprosy test, with improved discrimination of leprosy patients from healthy individuals.

Objective and quantitative evaluation by NDO-LID.

Correlation of rapid diagnostic test results with BI.

Monitoring treatment by NDO-LID and Smart Reader.

Smart Reader measurements could confer additional utility beyond initial detection and patient classification. To evaluate if the rapid diagnostic test/Smart Reader combination was sensitive enough to monitor treatment, we contrasted results generated using sera collected from patients at the time of diagnosis against sera collected at MDT completion. Overall, there was a reduced signal intensity in the after-treatment samples, with the decline most obvious in sera from patients that had the highest BI at intake (, P value of 0.003). In these high-BI MB leprosy patients, the mean reading of 46.3 at diagnosis declined to 27.7 after treatment, while in medium-BI MB leprosy patients, the decline was from 27.2 to 20.7, and in low-BI MB leprosy patients, it was from 10.2 to 6.1. These data suggest the utility of continued testing during, and even after, MDT.

DISCUSSION

ACKNOWLEDGMENTS

This work was supported by the American Leprosy Missions and the Renaissance Health Service Corporation.

Marco Collovati is the owner of OrangeLife, the company producing and marketing the NDO-LID rapid test. Ronaldo Ferreira Dias is an employee of OrangeLife.

Footnotes

Published ahead of print 11 December 2013

REFERENCES

Fast New Test Could Find Leprosy Before Damage Is Lasting

For centuries, some observant doctors have noticed early signs: the numb skin patches, missing eyebrows, drooping earlobes, bulging neck nerves, the flat “lion face” caused by nasal cartilage dissolving.

Since nothing could be done for them before the age of antibiotics, victims lost the use of their hands and had to beg. Some also went blind as the blinking muscles degenerated and their eyes dried out. In the Middle Ages, some towns banned lepers, while others required them to ring bells to warn of their approach. Religious charities created “leper colonies.”

And they still exist, even in the United States. A few elderly residents have chosen to stay on in Carville, La., and Kalaupapa, Hawaii, despite having been cured. Several thousand live at one in northeast Brazil, said John S. Spencer, a leprosy researcher at Colorado State University who has worked there. “People say things like ‘People outside won’t understand what’s wrong with my face,’ ” he said.

Nowadays, he said, most patients are cured before their faces are severely disfigured. Still, he said, he had read a survey in which health experts asked Brazilians whether they would rather have the human immunodeficiency virus or leprosy. Most chose H.I.V. — even though leprosy does not kill, can be cured, and does not make a victim risky to have sex with. “The stigma is that strong,” he said.

A new test was crucial because trained microscope diagnosticians are rare in the rural areas where the disease persists. It is simple: one drop of blood goes into a well on a plastic test strip followed by three drops of solution.

It took a long time to develop, Dr. Spencer said, because researchers needed a steady supply of the bacterium, and no way to grow it in a laboratory has ever been found.

It grows vigorously in one animal: the armadillo, a fact discovered only in the 1970s at a federal laboratory in Baton Rouge, La. But armadillos come with their own complications. After a year of harboring the slow-growing bacteria, they must be killed for their livers and spleens — and armadillos do not breed in captivity.

“Luckily,” Dr Duthie said, in Louisiana and Texas, “they’re everywhere, and they’re easy to catch.”

However, armadillo hunting is not risk-free. Some Southerners hunt them for food and their armored skins, and some wild armadillos harbor strains of leprosy bacteria. Two years ago, federal researchers estimated that about a third of the human cases discovered in the United States each year are caught from armadillos — which have the honor of being one of the state mammals of Texas.

To wipe out leprosy, we have to find it

By Tiffany O’Callaghan

“Early detection based on clinical symptoms is often too late”

(Image: IDRI)

With a new rapid-result blood test and a vaccine in the works, leprosy eradication may soon be a reality, says immunologist Malcolm Duthie, who created the test

Most people think of leprosy as a problem of the past. How common is it today?
There are about 250,000 new cases reported each year. But that’s probably about 5 to 6-fold lower than actually occur. In one study in Bangladesh, for example, they detected a rate sixfold higher than what was reported.

Why is leprosy so under-reported?
It is very easily misdiagnosed. In the mid-1980s there were about 12 million cases globally. Then the World Health Organization led a drive to reduce cases to less than 1 per 10,000 people by 2000. Since then levels have plateaued. But an unfortunate consequence of that success is there are now fewer clinicians who can diagnose leprosy – and the front line is clinical recognition.

How is a leprosy infection confirmed?
You need to collect lymph fluid, or take a biopsy and look for evidence of Mycobacterium leprae. But none of this is rapid, and it requires significant expertise. People are commonly treated for fungal infections or other skin conditions. It is often a last resort, after multiple wrong diagnoses, that they end up at leprosy reference centres. That delay is critical; the longer the infection goes on, the greater the chance that person is going to have lasting nerve damage.

Your team has developed a new blood test for leprosy. How early can it detect infection?
In a lab-based study, we were able to identify most cases about 9 to 12 months in advance of clinical symptoms. That is probably conservative.

How easy is it to use the test?
It is like a home pregnancy test, but with blood. You take a finger prick, collect a drop of blood, add it to a window in the test, and it causes a colour change. It doesn’t have to be done by a specialist.

The test was developed as an offshoot of your leprosy vaccine programme. How close are you to a vaccine?
Our timeline, if everything goes to plan, is to have a phase 1 clinical trial at the end of this year or the start of next year. Actual implementation of the vaccine is probably several years down the track.

Do you think we can eradicate leprosy?
Yes, there is the potential. About 65 per cent of cases are reported in India. The one significant hurdle is that, with a population of 1.2 billion and a disease that officially affects about 150,000, there’s probably not going to be widespread implementation of the vaccine. That is why we want our blood test, so we can target regions to implement the vaccine. The WHO mantra is early detection, early treatment. Well, early detection based on clinical symptoms is often too late.

So would the strategy be like that used to eradicate smallpox – by targeting hotspots?
Exactly. An infection with a clinical onset of seven years is not going to advance as rapidly as smallpox, but that’s the model. Identify the hotspot, treat the hotspot, knock the disease down in a particular region and then keep an eye on that region.

Profile

Malcolm Duthie is a senior scientist at the Infectious Disease Research Institute in Seattle. He led a team that developed a blood test for leprosy, which can provide results in 10 minutes

This article appeared in print under the headline “One minute with… Malcolm Duthie”

Preparation and Examination of Skin Smears

Printer-Friendly Preparation and Examination of Skin Smears (PDF- 225 KB)

Printer-Friendly Skin Smear/Biopsy Chart (PDF-56 KB)

The skin smear is a valuable, cost-effective tool in the routine management of the patient with Hansen’s disease. The smear is a means of estimating the number of acid-fast bacteria present, reported as the Bacterial Index (BI), and is important in determining the type and severity of disease as well as assessing the response to treatment.

General

Initial skin smears are usually taken from 6 “routine sites” (both earlobes, elbows, and knees) as well as several typical lesions from the patient. Repeat smears are obtained from 3 to 4 of the most active sites previously tested to evaluate progress.
The time interval between repeat smears is determined by the physician, but in general, annual smears are adequate for monitoring response to treatment and during the following-up period to detect any evidence of relapse.
All microscopic slides on which skin smears are made should be precleaned in 70% alcohol, acetone, or alcohol-acetone to remove amorphous debris. The slides are wiped dry with a clean hand towel. Blades that are used in smear taking are likewise cleaned.
Slides should be air-dried and NEVER heat fixed.
They may be sent in protective mailers to:

National Hansen’s Disease Program
Attention: Clinical Laboratory – Skin Smears
1770 Physicians Park Drive
Baton Rouge, La. 70816
Phone: (225) 756-3735

Procedure for Obtaining Smears

Universal precautions should be observed in obtaining skin smears.
The skin is cleansed with 70% alcohol and air-dried or wiped dry with cotton. (Zephiran tends to make the skin too slippery and is not recommended.)
A fold of skin is made relatively avascular by pinching or mild clamping. If the skin cannot be grasped by pinching, it can be compressed. A surgeon’s glove may aid in grasping.
Local anesthesia is generally unnecessary. (If there is not adequate decrease in sensation, obtain local anesthesia with 1% Xylocaine or Ethyl Chloride spray.) The compression of the skin by pinching aids in the anesthesia.
An incision 3-5 mm long and 2-3 mm deep is made with a alcohol cleansed, single-edged razor blade. A scalpel with a #15 Bard-Parker blade may also be used. Mild pressure to maintain relative avascularity is continuously applied to the area until an adequate smear has been obtained.
A small amount of blood does not interfere with the reading, but large amounts should be avoided and can usually be controlled by the amount of pressure of the pinch. If excessive bleeding occurs, it can be wiped away with a cotton swab.
After the incision is made, and before the blade is withdrawn, the inner surface of the wound is scraped with the blade held at a right angle to the incision. Upon scraping, tissue fluid and dermal tissue are obtained.
The material is transferred to the cleaned microscope slide. A moderately thick smear, with a visible uniform opacity is made. The smear is made in a circular manner on the slide, no larger than a pencil eraser (5-7 mm) , beginning peripherally and ending in the center, leaving a central “button” (2-4 mm) which can be easily focused upon with the microscope. Slides should be properly labeled as shown below in the sample diagram for 3 routine sites.
A Band-Aid is generally sufficient to protect the smear site.
A single technician takes all smears to insure more uniform and consistent results.
The smears are then sent to the National Hansen’s Disease Programs for reading.
A chart to diagram sites of the skin smears is linked from the top of this page.

Staining of Skin Smears

Dry the slide with smear at room temperature. DO NOT HEAT FIX .
Place slides on a staining rack and flood with 10% formalin for 15 minutes for fixation.
Gently rinse well with tap water. All formalin must be removed to prevent the formation of precipitates.
Flood slides with Ziehl-Neelsen carbol-fuchsin for twenty minutes. The carbol-fuchsin must be filtered before each use. Filtering can be accomplished by placing pre-cut filter paper strips on the slide prior to the addition of stain and left in place for the full twenty minutes.
After removing and discarding filter paper strips, gently rinse slides well with tap water to remove excess stain.
Decolorize with 2% acid alcohol for 1 minute. This is best accomplished by placing slide into a two-slide plastic slide mailer filled with acid alcohol. Occasional up and down movement of the slide in the acid alcohol should remove all excess carbol fuchsin.
Gently rinse slides thoroughly with tap water.
Counterstain with alkaline methylene blue for 30 seconds to 1 minute.
Gently rinse well with tap water and air dry.

NOTE: Positive and negative control slides must be used each day for quality control purposes.

Z-N Carbol Fuchsin Stain:

Basic fuchsin ————————— 1.0 gm.
Phenol crystals (melted)—————-5.0 mls.
95% ethanol ————————— 10.0 mls.
Water, to make ———————- 100.0 mls.

Dissolve stain in alcohol, and then add phenol/water mixture. Let stand overnight before use. Store in dark brown bottle. Stable for 1 year.

Acid alcohol:

Conc. HCl —————————— 2.0 mls.
95% ethanol ————————– 98.0 mls.

Alkaline Methylene Blue:

KOH (10%) ————————– 0.10 mls.
Methylene blue ———————- 0.35 gms.
95% ethanol —————————30.0 mls.
Water to make ———————–100.0 mls.

Dissolve the stain in the alcohol, then add the KOH and water mixture and allow to sit overnight. Filter before use.

Microscopic Examination of Skin Smears

The stained smears are examined with a quality microscope using the oil immersion objective (x100) to determine the total number of bacilli. The same individual should read all smears for the purpose of consistency. The smear will have similar numbers of bacilli throughout. However, four separate quadrants of the smear are examined and averaged to establish the Bacterial Index.

Reporting the Bacterial Index

The results are reported on a 0 to 6+ semi-logarithmic scale using a descriptive phrase or numerical code. This is an indicator of the total bacillary load of the patient. It falls about 1 point per year during effective treatment as dead bacilli undergo lysis and are absorbed.

Very Numerous	( +6 )	over 1000 bacilli per oil immersion field.
Numerous	( +5 )	100 to 1000 bacilli per oil immersion field.
Moderate	( +4 )	10 to 100 bacilli per oil immersion field.
Few	( +3 )	1 to 10 bacilli per oil immersion field.
Very few	( +2 )	1 to 10 bacilli per 10 fields.
Rare	( +1 )	1 to 10 bacilli per 100 fields.
None found	( NF )	No AFB seen on entire site.

Revised by Clinical Lab 11/06/2008

90,000 Lepra

Introduction

Lepra is a chronic infectious disease caused by Mycobacterium leprae, an acid-fast bacillus bacillus. The disease mainly affects the skin, peripheral nerves, the mucous membrane of the upper respiratory tract and the eyes. Leprosy is curable, and early treatment can prevent disability.

A Brief History – Disease and Treatment

Lepra has been known for a long time and is mentioned in written sources of ancient civilizations.Throughout history, people with leprosy have often been ostracized by their communities and families.

In the past, leprosy has been treated in many ways, but the first real breakthrough in treatment came in the 1940s when dapsone, a drug that stops disease, was developed. But the treatment was long-term, even lifelong, which made it difficult for patients. In the 1960s. M. leprae began to develop resistance to dapsone, the only antileprotic drug known in the world at that time.In the early 1960s, rifampicin and clofazimine were discovered and included in a regimen later called combination drug therapy (KLT).

In 1981, WHO recommended KLT. The currently recommended course of KLT includes the following drugs: dapsone, rifampicin, and clofazimine. The course of treatment lasts six months in the case of oligobacillary leprosy and 12 months in its multibacillary form. KLT kills the pathogenic microorganism and cures the patient.

Since 1995, WHO has provided CRT free of charge to all leprosy patients worldwide. The free KLT was originally funded by the Nippon Foundation, and since 2000, the free KLT has been delivered under an agreement with Novartis, which recently pledged to extend it to at least 2020.

The target of eliminating leprosy as a public health problem (i.e. reducing its prevalence to less than 1 case in 10,000 people) was achieved globally in 2000.Over the past 20 years, more than 16 million patients have been treated with KLT.

WHO activities

In 2016, to accelerate efforts to control leprosy, WHO launched the Global Leprosy Strategy 2016–2020: Accelerating Action for Global Leprosy Elimination. The main focus of this Strategy is on children and on the prevention of disability.

At the heart of the Global Leprosy Strategy 2016-2020 there are three main components:

Component I: Strengthen government ownership, coordination and partnerships

Main events

Ensure political commitment and adequate resources for leprosy control programs.
Promote universal health coverage, with a focus on children, women and disadvantaged groups, including migrants and displaced persons.
Strengthen partnerships with government and non-government actors and promote intersectoral collaboration and partnerships at the international level and within countries.
Promote and conduct basic and operational research on all aspects of leprosy and maximize the evidence base for policy, strategy and intervention development.
Strengthen surveillance and health information systems for program monitoring and evaluation (including geographic information systems).

Component II: Stop Leprosy and Its Complications

Main events

Raise patient and community awareness of leprosy.
Facilitate early case detection by proactively searching (eg campaigning) in areas of high endemicity and by monitoring people who have contact with patients.
Ensure prompt initiation and adherence to treatment, including work to improve treatment regimens.
Improve prevention and management of disabilities.
Strengthen antimicrobial resistance surveillance, including the laboratory network.
Promote innovative approaches to training, referral and continuing education in leprosy, such as e-health.
Promote activities for the prevention of infections and diseases.

Component III: End discrimination and promote social inclusion

Main events

Promote social inclusion by addressing all forms of discrimination and stigma.
Empower people affected by leprosy and build their capacity to actively participate in leprosy treatment services.
Engage communities in activities to improve leprosy treatment services.
Facilitate the building of coalitions among people affected by leprosy and facilitate the alignment of these coalitions and / or their members with other organizations at the community level.
Facilitate access to social and financial support services, such as facilitating income generation, for people affected by leprosy and their families.
Support community-based rehabilitation of people with leprosy-related disabilities.
Work to eliminate discriminatory laws and promote policies for the social inclusion of people affected by leprosy.

Objectives of the Global Leprosy Strategy

90,028 90,029 no new child patients with disabilities;

90,029 disability of the second group occurs in less than one in a million people;

No country has legislation allowing discrimination on the basis of leprosy.

In August 2016WHO has published Operational Guidelines to help adapt and implement the Global Leprosy Strategy 2016–2020. This guide provides guidance to managers of national leprosy programs (or similar) on how to adapt and implement the Global Leprosy Strategy to address the epidemiological burden in their countries.

In March 2017, the WHO Global Leprosy Control Program published the Guidelines for Monitoring and Evaluating the Implementation of the Global Leprosy Control Strategy.Under the leadership of the Global Leprosy Program, efforts are being made to expand the leprosy drug-resistance surveillance network, which is one of the key activities of the Global Leprosy Strategy. A guide to the surveillance of antimicrobial resistance in leprosy has also been published.

Russian scientists want to oblige migrants to be tested for leprosy

https: // ria.ru / 201

/ 1550081735.html

Russian scientists want to oblige migrants to be tested for leprosy

Russian scientists want to oblige migrants to be tested for leprosy – RIA Novosti, 29.01.2019

Russian scientists want to oblige migrants to be tested for leprosy

Scientists The Research Institute for the Study of Leprosy sent a proposal to the Ministry of Health of Russia to use a new test system for early diagnosis … RIA Novosti, 29.01.2019

2019-01-29T15: 52

society

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ROSTOV-ON-DON, January 29 – RIA Novosti, Yulia Nasulina. Scientists of the Research Institute for the Study of Leprosy sent a proposal to the Russian Ministry of Health to use a new test system for early diagnosis of leprosy as a mandatory examination of migrants, the director of the institute, Doctor of Medical Sciences Viktor Duiko told RIA Novosti.According to him, in 2018, the institute received a patent for the invention of a test system for the early diagnosis of leprosy “A method for identifying the DNA of mycobacterium leprosy using a polymerase chain reaction.” “This is a method for diagnosing leprosy, which makes it possible to detect a disease by blood at a stage when there are no clinical manifestations yet,” Duiko said, noting that early diagnosis is very important for subsequent treatment with a good outcome. “We proposed to include this test system in the complex mandatory screening of migrants, which is very important with the development of tourism, uncontrolled migration processes and an increase in cases of imports of leprosy from endemic countries, “said the source.He added that now migrants arriving in Russia are being examined by narrow specialists, including to exclude leprosy. The specialist added that if a decision is made to introduce a test system, this method can become an auxiliary in diagnosing the disease. Lepra, or leprosy is a chronic infectious disease caused by mycobacteria. It is characterized by lesions of the skin, mucous membranes, peripheral nervous system, internal organs. The Research Institute for the Study of Leprosy is based in Astrakhan and is the only government agency in the country engaged in the diagnosis and treatment of this disease.According to the institute, the Astrakhan region today remains the most significant and active leprosy focus in the Russian Federation, 60% of all registered patients live there. In 2017-2018, six new cases of leprosy and one relapse were registered among the residents of the region.

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society

ROSTOV-ON-DON, January 29 – RIA Novosti, Yulia Nasulina .Scientists of the Research Institute for the Study of Leprosy sent a proposal to the Russian Ministry of Health to use a new test system for early diagnosis of leprosy as a mandatory examination of migrants, the director of the institute, Doctor of Medical Sciences Viktor Duiko told RIA Novosti.

According to him, in 2018 the institute received a patent for the invention of a test system for the early diagnosis of leprosy “A method for identifying the DNA of mycobacterium leprosy using a polymerase chain reaction.” “This is a method for diagnosing leprosy, which allows the blood to identify the disease at a stage when there are no clinical manifestations yet,” Duiko said, noting that early diagnosis is very important for subsequent treatment with a good outcome.

January 16, 2019, 14:24

The Ombudsman asked to remind migrants about the length of stay in Russia

endemic countries, “- said the interlocutor of the agency.

He added that now migrants arriving in Russia are being examined by narrow specialists, including to exclude leprosy.

“The examination is carried out by classical methods (visual examination of the skin, bacterioscopic examinations). The test system is a modern high-tech method related to preclinical research,” Duiko said.

The specialist added that if a decision is made to introduce a test system, this method can become auxiliary in the diagnosis of the disease.

Leprosy, or leprosy, is a chronic infectious disease caused by mycobacteria.It is characterized by lesions of the skin, mucous membranes, peripheral nervous system, internal organs. The Research Institute for the Study of Leprosy is based in Astrakhan and is the only government agency in the country engaged in the diagnosis and treatment of this disease.

According to the Institute, the Astrakhan region today remains the most significant and active leprosy focus in the Russian Federation, 60% of all registered patients live there. In 2017-2018, six new cases of leprosy and one relapse were registered among the residents of the region.

Why one of the most dangerous infections was never eradicated

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Why one of the most dangerous infections was never eradicated

Why one of the most dangerous infections was never exterminated – RIA Novosti, 10.06.2019

Why one of the most dangerous infections was never eradicated

In the world, less than one case of leprosy is detected per ten thousand people per year. Having declared the disease harmless, WHO, however, believes that it is too early to rejoice.In the last … RIA Novosti, 10.06.2019

2019-06-10T08: 00

Science

Great Britain

Paul Gauguin

dna

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MOSCOW, June 10 – RIA Novosti, Tatyana Pichugina.In the world, there is less than one case of leprosy per ten thousand people a year. Having declared the disease harmless, WHO, however, believes that it is too early to rejoice. Recently, the number of new cases has been growing, mainly in India, Brazil, Indonesia. This means that the pathogen lives in some hidden foci and not all of its pathways are known. It penetrates into the cells of the skin and mucous membranes and develops very slowly there.The bacillus divides every two weeks – a record duration among pathogens. For comparison: a tubercle bacillus takes only 20 hours. The incubation period takes years. Symptoms can appear even after two decades: spots on the skin, growths. In advanced cases, the microbe affects the peripheral nerves, which causes the fingers to curl up, and sensitivity is lost. The disease greatly disfigures a person. Lepers used to be driven out, resettled on reservations. In Japan, the law on the compulsory isolation of patients with leprosy was in force until 1996.In India, lepers cannot work or be in public places. The causative agent of leprosy was discovered at the end of the 19th century, but a cure for it appeared only in 1940 – the antibiotic dapsone. Twenty years later, since the mycobacterium developed resistant strains, two more drugs were developed. In the 1980s, there were 11 million lepers in the world. WHO set a goal to eradicate the disease, treatment was provided free of charge. In 2000, it was announced that leprosy was no longer a public danger. In India, for example, most programs to combat leprosy have been phased out since 2005.Now there are almost four hundred thousand patients on the planet, 74 percent are in India, Brazil and Indonesia. Recently, however, doctors have identified more and more new cases. According to the WHO, in 2015 210,758 people fell ill with leprosy, in 2017 – already 211,009. New foci foundThe causative agent of leprosy cannot be cultivated in an artificial environment, it lives only inside living cells. This made it difficult to study until, in the 1970s, scientists discovered leprosy-infected nine-banded and six-banded armadillos, shell-covered small mammals native to South America and the southern United States.For a long time, battleships remained the only laboratory models for studying leprosy. Later they learned to grow it in the cells of the pads of mouse paws, but this is very laborious. In 2014, it was established that the microbe survives in acanthamoeba cysts – unicellular ones that are found in fresh water, moist soils. There, the bacterium remains active for eight months. This alarmed the scientific community, since another route of infection was discovered – through water or soil. In 2016, scientists, taking samples of warty growths from common squirrels from the UK and Ireland, isolated Mycobacterium leprae and another related species of the microbe, Mycobacterium lepromatosis.It was later discovered in humans. The microbe can also live in the ground. Its traces were identified by DNA in soil samples from Bangladesh, Suriname, from the British Isles of Browns and Arran, where sick squirrels are found. All of this will help shed light on how leprosy is transmitted. It is believed that the main route is airborne, through sputum, sneezing, blowing your nose. They become infected with prolonged numerous contacts with patients. Most often – family members, blood relatives. However, the increase in the number of new cases indicates that there are probably other ways of transmission of the pathogen, not only from person to person.Genetics Reveals Mysteries Scientists view wild armadillos as a natural source of leprosy. There are known cases of infection from them in the southern states of the United States. The danger of squirrels in the British Isles is out of the question. Also, pathogens have been identified in primates in Africa and Southeast Asia. How did the strains that cause leprosy in humans come about? The answer to this question is provided by the study of DNA from ancient burials. In 2018, an international team of scientists led by German geneticist Johannes Krause published the results of a complete sequencing of the genomes of the causative agent of leprosy isolated from human remains in different European countries.It was possible to trace the evolution of this pathogen over one and a half millennia, starting from the fourth century BC. Krause and colleagues identify ten genomes belonging to different branches of the causative agent of leprosy, which indicates a large genetic diversity. The strains circulating in squirrels in the British Isles and armadillos from the southern United States are very similar to those that afflicted humans in late medieval Europe, and are still active in various regions of the world. Maybe the disease was transmitted to animals from people in the Middle Ages and the era of the Great Discoveries? This is not yet clear.There is also no unequivocal understanding of how leprosy is spreading now, what role does genetic predisposition play (it is known that this infection is quite selective), how quickly strains resistant to existing antibiotics will develop. There are still no tests to detect contamination from a blood sample, and vaccines are under development. With this in mind, WHO has launched a new program to combat leprosy until 2020.

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Great Britain, Paul Gauguin, DNA

MOSCOW, June 10 – RIA Novosti, Tatyana Pichugina. There is less than one case of leprosy in every ten thousand people worldwide. Having declared the disease harmless, WHO, however, believes that it is too early to rejoice. Recently, the number of new cases has been growing, mainly in India, Brazil, Indonesia.This means that the pathogen lives in some hidden foci and not all of its pathways are known.

The infection raises its head

Leprosy is caused by the mycobacterium Mycobacterium leprae, akin to the tubercle bacillus. It penetrates into the cells of the skin and mucous membranes and develops very slowly there. The bacillus divides every two weeks – a record duration among pathogens. For comparison: a tubercle bacillus takes only 20 hours.

The incubation period takes years.Symptoms can appear even after two decades: spots on the skin, growths. In advanced cases, the microbe affects the peripheral nerves, which causes the fingers to curl up, and sensitivity is lost.

Disease severely disfigures a person. Lepers used to be driven out, resettled on reservations. In Japan, the law on the compulsory isolation of patients with leprosy was in force until 1996. In India, lepers cannot work or be in public places.

The causative agent of leprosy was discovered at the end of the 19th century, but a cure for it appeared only in 1940 – the antibiotic dapsone.Twenty years later, since the mycobacterium developed resistant strains, two more drugs were developed.

In the 1980s, there were 11 million lepers in the world. WHO set a goal to eradicate the disease, treatment was provided free of charge. In 2000, it was announced that leprosy was no longer a public danger. In India, for example, most programs to combat leprosy have been phased out since 2005.

Now there are almost four hundred thousand sick people on the planet, 74 percent are in India, Brazil and Indonesia.Recently, however, doctors have identified more and more new cases. According to the WHO, in 2015 210,758 people fell ill with leprosy, in 2017 – already 211,009.

New foci found

The causative agent of leprosy cannot be cultivated in an artificial environment, it lives only inside living cells. This made it difficult to study until, in the 1970s, scientists discovered leprosy-infected nine-banded and six-banded armadillos, shell-covered small mammals native to South America and the southern United States.For a long time, battleships remained the only laboratory models for studying leprosy. Later they learned to grow it in the cells of the pads of mouse paws, but this is very laborious. In 2014, it was established that the microbe survives in acanthamoeba cysts – unicellular ones that are found in fresh water, moist soils. There, the bacterium remains active for eight months. This alarmed the scientific community, since another route of infection was discovered – through water or soil. In 2016, scientists, taking samples of warty growths from common squirrels from the UK and Ireland, isolated Mycobacterium leprae and another related species of the microbe, Mycobacterium lepromatosis.It was later discovered in humans. The microbe can also live in the ground. Its traces were identified by DNA in soil samples from Bangladesh, Suriname, from the British Isles of Browns and Arran, where sick squirrels are found.

All of this will help shed light on how leprosy is transmitted. It is believed that the main route is airborne, through sputum, sneezing, blowing your nose. They become infected with prolonged numerous contacts with patients. Most often – family members, blood relatives. However, the increase in the number of new cases indicates that there are probably other ways of transmission of the pathogen, not only from person to person.

Genetics Reveals Secrets

Scientists view wild armadillos as a natural source of leprosy. There are known cases of infection from them in the southern states of the United States. The danger of squirrels in the British Isles is out of the question. Also, pathogens have been identified in primates in Africa and Southeast Asia.

How did the strains that cause leprosy in humans come about? The answer to this question is provided by the study of DNA from ancient burials. In 2018, an international team of scientists led by German geneticist Johannes Krause published the results of a complete sequencing of the genomes of the causative agent of leprosy isolated from human remains in different European countries.It was possible to trace the evolution of this pathogen over one and a half millennia, starting from the fourth century BC.

Krause and colleagues identify ten genomes belonging to different branches of the causative agent of leprosy, which indicates a large genetic diversity. The strains circulating in squirrels in the British Isles and armadillos from the southern United States are very similar to those that afflicted humans in late medieval Europe, and are still active in various regions of the world.

Maybe the disease was transmitted to animals from people in the Middle Ages and the era of the Great Discoveries? This is not yet clear.

There is also no unequivocal understanding of how leprosy is spreading now, what role does genetic predisposition play (it is known that this infection is quite selective), how quickly strains resistant to existing antibiotics will develop. There are still no tests to detect contamination from a blood sample, and vaccines are under development.

With this in mind, WHO launched a new program to combat leprosy until 2020.

Identification of the genetic material of Mycobacterium leprae using various test systems by real-time PCR

Target

To compare the effectiveness of single-copy and multicopy markers of the Mycobacterium leprae genome, used to identify the presence of a pathogen in the human body, by real-time PCR.

Description

A method for diagnosing leprosy, based on the use of real-time PCR, allows detecting the presence of a DNA pathogen in the early stages of the disease, which increases the effectiveness of therapy and prevents serious consequences for patients.

Tasks

1. Study of the basic rules of work in the clinical diagnostic laboratory, including work with pathogens.

2. Mastering the laboratory method of PCR in real time.

3. Isolation of DNA from clinical samples of skin scarification of patients, characterization of DNA preparations.

4. Analysis of the presence of Mycobacterium leprae in clinical samples using diagnostic kits using amplification of the following regions: RLEP (repeating element of the leprosy genome), sodA gene (encodes the enzyme superoxide dismutase), rpoB gene (encodes β-subunit of bacterial RNA polymerase), mntH gene (encodes a manganese transporter).

5. Evaluation of the efficiency of amplification of various regions of the genome of Mycobacterium leprae by real-time PCR.

Lepra is a chronic infectious disease with a long incubation period caused by the pathogenic acid-fast bacterium Mycobacterium leprae. The basic laboratory method for diagnosing leprosy is the detection in preparations of Mycobacterium leprae using light microscopy after staining according to Ziehl-Nielsen. Bacterioscopic examination is not always informative, as it requires a high titer of the microorganism. Diagnosis of the disease is also based on clinical signs and histopathological presentation, since Mycobacterium leprae is uncultivated.

Currently, leprosy is considered curable, but even in the Middle Ages, the disease covered entire continents. Now on the territory of our country there are isolated cases, however, due to the presence of a large number of patients in other countries (India, Brazil, Malaysia) and active migration of the population, as well as the severe consequences of late diagnosis of the disease, the use of modern means of early diagnosis of leprosy is relevant. Left untreated, leprosy can cause progressive and persistent damage to the skin, nerves, limbs, and eyes.Early treatment can prevent disability.

The genome of Mycobacterium leprae has been completely deciphered. The uniqueness of the genome lies in the presence of repeating sequences of four families: RLEP, REPLEP, LEPREP, and LEPRPT. The use of various markers of the Mycobacterium leprae genome for early diagnosis of the presence of a pathogen in the human body is shown. Genome research is carried out by real-time polymerase chain reaction (PCR), the modern version of which is the use of a hydrolyzing sample.

When detecting DNA of Mycobacterium leprae, amplification of the region of repeated elements of RLEP is used (up to 29 repeats in the bacterial chromosome, the absence of other species of the genus Mycobacterium in the genome). The use of the family of repeating elements with the highest copy number RLEP for the detection of microorganisms by PCR showed better results in comparison with genes traditionally used for identification of bacterial genomes (rpoB, SodA, and 16S rRNA).Clinical diagnostic practice in Russia has also shown that test systems based on different regions of the Mycobacterium leprae genome exhibit unequal efficiency.

The analysis was carried out on clinical samples in the form of scrapings of skin, nasal mucus, skin biopsy obtained from patients with a diagnosis of leprosy, lepromatous type, multibacillary form, which are monitored by the Sergiev Posad branch of the State Research Center for Healthcare of the Russian Federation. The primary processing of the clinical samples was carried out by the staff of the State Scientific Center for Contemporary Arts.

Genetic material from M. leprae was found in three samples out of eight analyzed. It was shown that the concentration of M. leprae in the samples is different, which can be judged by changes in the value of the threshold cycle during real-time PCR. The absence of a relationship between the concentration of total DNA (a mixture of human and M. leprae DNA) and the detection of pathogen DNA was shown.

The shape of the curves on the graphs of the fluorescence signal accumulation obtained during the experiment indicates the correct course of the reaction and the possibility of analyzing the results.Comparison of the cycle threshold (Ct) values of single-copy genes indicates almost equal amplification efficiency when using systems for SodA and MntH genes, regardless of the concentration of mycobacterium leprosy DNA in the sample. The amplification rate with the rpoB gene system was the lowest, even when compared with the MntH system, which has the same threshold fluorescence level. The absence of detection in one of the M. leprae DNA samples by the system for the single-copy SodA gene was shown, which probably indicates the unstable operation of such a detection system at low concentrations of mycobacteria in the sample.

Equipment and equipment used in the work

• Set “NK Sample”

• Automatic homogenizer “Tissue Lyser II”

• Spectrophotometer NanoVue 2000

• DNA amplifier StepOne 5c

• Sets of probes and primers for the analysis of the repetitive region of the leprosy genome RLEP and single-copy genes SodA and MntH

• Leprosy std

set

Results

1.Comparison of test systems for the detection of Mycobacterium leprae DNA in clinical samples of patients diagnosed with leprosy showed the highest efficiency of the detection test system based on the use of a repeating element of the Mycobacterium leprae RLEP genome.

2. The mntH system showed the best efficiency among the test systems based on the analysis of regions of single-copy genes – manganese transporter mntH, superoxide dismutase enzyme sodA, β-subunit of bacterial RNA polymerase rpoB.

3. The lowest intensity is shown for a commercial kit from Genesig (UK) using a region of the rpoB gene.

The obtained patterns are shown for different degrees of M. leprae DNA concentrations in clinical samples of patients.

Prospects for using the results of work

The obtained results are part of the work on the creation of a diagnostic kit for the determination of the genetic material of leprosy in clinical practice.

Collaboration with the institution on job creation

Federal State Budgetary Institution “State Scientific Center for Dermatovenereology and Cosmetology” of the Ministry of Health of Russia.

Dissenting opinion

“The opportunity to take part in real research work arose due to the creation of conditions in our school for the implementation of an independent project. The school’s management did everything possible to get me allowed to work at the scientific center of the Russian Ministry of Health.From the proposed topics, I chose research in the field of microbiology of leprosy, because I wanted to learn more about this disease and work on modern equipment. It was difficult to study literature in the chosen direction, especially English, although it turned out to be more informative. This period of theoretical training became the most difficult. I was able to do this with the assistance of a leader. A separate stage was the preparation of a speech at the conference, the selection of submitted abstracts and, of course, the presentation of the report was very exciting.I wanted to tell everyone about the importance and interesting aspects of the work, to make the report bright and understandable, memorable and original. Receiving a II degree diploma was a pleasant surprise, since I spoke publicly for the first time and just wanted to share my research with the guys. “

90,000 A Modern View of Leprosy | # 05/18

Leprosy (leprosy, Hansen’s disease) is a chronic infectious disease from the group of mycobacteriosis, characterized by a long incubation period and a recurrent course.The disease is systemic in nature and affects the derivatives of the ectoderm – the skin, mucous membranes and the peripheral nervous system. Currently, despite the use of an effective antibiotic regimen and the elimination of the threat of the leprosy epidemic, new cases of the disease continue to be detected every year around the world, so the prospect of complete elimination of the disease is questioned [1-3].

The prevalence of leprosy in the world has been steadily declining from year to year. According to the WHO, the number of new cases detected each year worldwide has decreased from 763,000 in 2001.up to 249,000 in 2008 [1]. In 2013, 215 656 new cases of the disease were detected, in 2014 – 213 899, in 2015 – 211 973 [2, 3].

Global statistics show that 96% (203,600 people) of new cases of leprosy were detected in 22 states (such as India, Brazil, Angola, Congo, Sudan, Ethiopia, etc.). Other countries account for the remaining 4% [3].

Brazil is one of the six countries in the world with the highest prevalence of leprosy, with more than 30,000 new cases of the disease diagnosed each year.In 2014, the prevalence of leprosy in Brazil was 1.27 cases per 10,000 inhabitants. The level of prevalence of dermatosis across the country is uneven: in addition to the regions endemic for leprosy, there are also regions in which there is a low level of prevalence of leprosy [4].

Population migration plays a significant role in the spread of the disease. In Europe, most cases of leprosy occur in refugees from other countries. Thus, of the 168 cases of leprosy reported in 2013in Spain, 40 (24.6%) patients were found among the indigenous inhabitants of Spain, 128 (76.2%) – among migrants living in the country, mainly from Brazil, Paraguay and Bolivia [5]. In Italy, the number of patients with leprosy among the indigenous population in the period from 1990 to 2009 was 12 people, among migrants – 159 patients [6]. In France in 2009 and 2010 identified 39 new cases of the disease, of which 7 (18%) were observed in patients of French origin [7]. In Denmark, in the period from 1980 to 2010, 15 cases of the disease were detected, of which 87% were migrants from South and Southeast Asia [8].

In Russia, the Astrakhan region is an endemic region for leprosy. Over the past decades, patients with leprosy have been identified in other regions of the Russian Federation: in Siberia, the North Caucasus and the Far East [9, 10]. However, it should be noted that, thanks to the introduction of a whole range of antileprosy measures into practice, the incidence of leprosy in Russia is persistent and sporadic. In 2015, 240 patients were registered, including 135 in the Astrakhan region [10].

The causative agents of leprosy are Mycobacterium leprae (M. leprae) and Mycobacterium lepromatosis (M. lepromatosis). M. leprae was first discovered by the Norwegian physician Gerhard Hansen in 1873 [11]. This microorganism belongs to the Mycobacteriaceae family and is an acid- and alcohol-resistant bacterium, which is a gram-positive straight or curved rod 1–7 µm long and 0.2–0.5 µm in diameter [12]. M. leprae can be viable for a long time at low temperatures and drying.This microorganism is characterized by extremely slow growth, which is often not typical for bacteria (one division lasts about 12 days) [12]. The causative agent of the disease is an obligate intracellular parasite. M. leprae is able to persist for a long time in human macrophages, which is provided by the interaction of various mechanisms (antigenic variability, etc.). That is why patients discharged from leper colony for outpatient treatment with persistent forms of leprosy can be a source of infection [9].

In 2008, the second causative agent of leprosy was discovered – M. lepromatosis , which, unlike M. leprae, is a non-acid-resistant bacterium and mainly causes a severe diffuse lepromatous type of leprosy [13, 14].

Leprosy is distinguished from other infectious diseases by a long incubation period, which varies from 2–3 months to 50 years (averaging 4–6 years) [11].

The airborne route of transmission of infectious agents is generally recognized, but other routes of infection are not excluded – through the bites of blood-sucking insects and damaged skin.Leprosy is a low-contagious disease. Infection with mycobacterium leprosy occurs as a result of long-term close communication with a patient who is not receiving treatment, due to sensitization that increases with repeated contacts, a decrease in the body’s resistance (as a result of malnutrition, heavy physical exertion, frequent colds, alcoholism and other intoxication) and immunogenetic susceptibility [ eleven].

Susceptibility to leprosy is influenced by a variety of gene sets, including the human leukocyte antigen (HLA) system.Changes in candidate genes involved in the host’s response to an infectious agent are currently being studied. Genomic scan studies have identified binding peaks for leprosy at chromosome regions 6p21, 17q22, 20p13, and 10p13 [15, 17].

Resistance to infection with M. leprae is provided, on the one hand, by the low virulence of M. leprae , on the other hand, by the individual characteristics of innate immunity. An important role in maintaining innate immunity is played by the integrity of the epithelium, glandular secretions and surface immunoglobulin A (IgA).In addition, NK cells, cytotoxic T lymphocytes and activated macrophages can destroy mycobacteria, regardless of the activation of adaptive immunity. Upon infection, the regulation of inflammatory cytokines and chemokines leads to the proliferation of either type 1 T-helpers (Th2) or type 2 T-helpers (Th3), which contributes to the activation of the cellular or humoral immunity, which determines the clinical form of the disease [16 , 17].

Cellular immunity is ineffective in preventing the development of the disease in individuals with tuberculoid leprosy.Humoral immunity in individuals with lepromatous disease, which is responsible for the production of IgM against PGL-1 (phenolic glycolipid-1), does not provide protection and does not prevent the dissemination of bacteria [17].

Studies of in situ T-lymphocyte phenotype using immunohistochemical methods with monoclonal antibodies demonstrate the predominance of T-helpers (CD4 +) in tuberculoid leprosy, with a CD4 / CD8 ratio of 2: 1 (the same ratio was found in the blood).The ratio of memory cells / intact T cells is 1: 1 in blood and 14: 1 in lesions. This means that CD4 + cells in tuberculoid lesions express the phenotype of memory T cells (CD45R0 +). In lepromatous lesions, a population of TCD8 + lymphocytes with a CD4 / CD8 ratio of 0.6: 1 predominates, regardless of the blood ratio, half of the CD4 + cells belong to the subclass of T-intact cells. Most CD8 + cells belong to the CD28 phenotype, which indicates that they are T-suppressor cells, while T-cytotoxic cells (CD28 +) predominate in tuberculoid lesions [16, 17].It has been noted that CD4 + cells (memory T cell phenotype) bind to macrophages in the center of the tuberculoid granuloma, and CD8 + cells are the surrounding cuff. In lepromatous granulomas, CD8 + cells (T-suppressor phenotype) are mixed with macrophages and CD4 + cells. Different types of cytokines are produced by the CD4 + and CD8 + subclasses. CD4 + cell clones from TB patients produce high levels of interferon gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) [17].These clones (TCD4 + cells, Th2 pattern) enhance cell-mediated immunity and reduce proliferation of M. leprae . Clones of CD8 + cells from patients with leprosy produce high levels of suppressor cytokines of macrophage activity, IL-4, IL-5, and IL-10, as well as low levels of IFN-γ [17]. Given the structure of the secretion of cytokines by T-suppressor cells, in particular IL-4, these cell clones were named TCD8 + cells, the Th3 pattern. They promote the stimulation of B-lymphocytes, which increase the humoral immune response, and induce the production of antibodies that provide a person’s susceptibility to the development of the disease [16, 17].

The high level of TNF-α in the blood serum in patients with tuberculoid leprosy indicates the participation of this cytokine in the destruction of M. leprae and the formation of granulomas. TNF-α is involved in immune defense through the activation of macrophages, but the overproduction of TNF-α and its interaction with IFN-γ contribute to tissue damage and the formation of erythema nodosum leprosy (ENL) [17].

In the lepromatous form of leprosy, there is an increased level of transforming growth factor beta (TGF-β), which is absent in the tuberculoid form and manifests itself in small amounts in the borderline form of leprosy.TGF-β suppresses the activation of macrophages, which inhibits the production of TNF-α and IFN-γ, contributing to the persistence of infection [17].

About the mechanisms of transmission of leprosy, it is known that mycobacteria penetrate into endothelial cells and settle in Schwann cells of skin nerves, to which they have tropism, where a long-term period of their adaptation and reproduction takes place. It remains unknown how colonization of Schwann cells by mycobacterium leprosy leads to the spread of infection to other tissues [18].Neural tropism of M. leprae is due to its binding to the G region on the bridge of the laminin alpha-2 molecule, and alpha-dystroglycan serves as a receptor for M. leprae on Schwann cells [18].

Masaki et al. (2013), in the study in vitro and in vivo using mice, the interaction of M. leprae with Schwann cells was determined. Research has shown that M. leprae alters the differentiation of Schwann cells to progenitor cells [19].Cellular rearrangement leads to a decrease in the regulation of the Sox10 Schwann cell line [19]. Thus, M. leprae promote the spread of the infectious process through two mechanisms: direct differentiation of Schwann cells into mesenchymal tissues and the formation of granuloma-like structures that are secreted by bacteriological macrophages [19]. The study expands the understanding of the plasticity of mature cells and demonstrates the properties of M. leprae , leading to the rearrangement of adult cells into stem cells [18].The spread of infection by differentiation of Schwann cells is possible when they are infected with a large number of M. leprae . The methodology used in mice lacking T cells simplifies the inflammatory microenvironment into predominantly macrophages [19]. This work describes a promising in vitro model to explain the pathogenesis of M. leprae , but detailed studies are needed before extrapolating conclusions to the course of the infectious process in the human body [18].

There are two classifications of leprosy: the Madrid classification, adopted in 1953, as well as its subsequent modification, proposed by D. S. Ridley and V. Jopling in 1973 [11].

According to the Madrid classification, there are two polar types of leprosy: tuberculoid and lepromatous and two intermediate types: undifferentiated and borderline (dimorphic) [11].

In the Ridley-Jopling classification, three types of leprosy are distinguished – undifferentiated (I – Indeterminate), tuberculoid (Tuberculoidtype – TT) and lepromatous (Lepromatoustype – LL).Lepromatous and tuberculoid types are polar. In addition, subpolar and borderline groups of the disease are distinguished. The Ridley – Jopling classification has not found wide application due to its complexity; therefore, in practice, the lepromatous and tuberculoid types of leprosy are distinguished, as well as the borderline type, which can later be transformed into one of the first two forms [20].

The clinical manifestations of the disease are determined by the type of leprosy process. The tuberculoid form of leprosy is more benign than the lepromatous form.With the tuberculoid type, mainly the skin and the peripheral nervous system are affected; internal organs are affected less often. The defeat of the skin is characterized by sharply delineated depigmented spots, reminiscent of the manifestations of vitiligo, or asymmetric bright reddish-cyanotic spots with a pale center and flat polygonal purple nodules along the periphery. The nodules often merge with the formation of a slightly raised ridge about 2–3 cm wide (“curb elements”).As the plaque grows, its central part becomes depigmented and atrophic. The size of the plaques can vary from one to tens of centimeters in diameter. In some cases, with a tuberculoid form, sarcoid-like tubercles up to one centimeter in diameter, reddish-brown in color, with a tendency to grouping are formed on the skin. The lesion of the skin appendages is characterized by hair loss and impaired sweating in the area of the affected areas [11]. The tuberculoid type of leprosy is characterized by early damage to the peripheral nervous system with the formation of sensitivity disorders (pain, temperature and tactile).Polyneuritis in the tuberculoid form is characterized by a more favorable course than polyneuritis in the lepromatous form [11].

Cases of a rare form of tuberculoid leprosy with changes in peripheral nerves of the “tennis racket” type, clinically manifested as a thickening of the branch of the nerve emerging from the tuberculoid granuloma, are described. It occurs as a result of damage to the cutaneous nerves caused by the formation of granulomas, and leads to local pain or sensory impairment [21, 22].

The lepromatous type of leprosy is a more severe form of the disease, in which there is damage to the skin, nervous system, mucous membranes and internal organs. A characteristic sign of skin lesions is the appearance of numerous symmetrically located small reddish spots with a purple tint [11]. Over time, their color changes to brownish or copper. Local sensitivity in the area of rashes in the early stages of the disease is not disturbed [23]. Gradually, massive infiltration forms on the extensor surface of the limbs and face.Infiltrates localized on the face in the area of the forehead, eyebrows, nose and cheeks lead to impaired facial expressions and disfigurement of facial features (the so-called “lion’s face”, facies leonina is formed). Lesions may be accompanied by hair loss, hypo- or anhidrosis [8]. The process also involves subcutaneous adipose tissue with the formation of nodes – leprosy, representing brownish tubercles ranging in size from 2 mm to 2 cm, of a dense consistency, with a shiny surface. The tubercles tend to ulcerate and are most often localized on the face, in the area of the auricle lobes, on the skin of the limbs, buttocks and back [11].

With the lepromatous type of leprosy, both the peripheral and central nervous systems are affected with the development of neurotic disorders, less often psychoses and lesions such as neuritis and polyneuritis. In most cases, the radial, peroneal and large ear nerves are affected: they thicken and become palpable. Subsequently, motor and trophic disorders develop, as well as sensitivity disorders. Patients are characterized by neuralgia, hyperesthesia, paresthesia, inadequate or delayed response to irritation, analgesia.Against the background of trophic disorders, the process of mutation of the hands and feet develops. The defeat of internal organs is characterized by nonspecific changes in the liver, lungs, spleen and dysfunction of some endocrine glands [11].

A rare form of the lepromatous type of leprosy, manifested by warty keratosis, has been described [23]. To date, only 25 cases of this form of the disease have been reported [24].

With an undifferentiated form of leprosy, specific rashes are absent.This form is characterized by the appearance of a small number of pale spots of various sizes with indistinct boundaries, as well as damage to the peripheral nervous system by the type of polyneuritis. As a rule, it is extremely rare to identify the pathogen in this category of persons [11].

With the development of exacerbations of the leprosy process, the form of the disease can change. Despite the many clinical manifestations of leprosy, the final diagnosis is not always an easy task, since there are no pathognomonic clinical signs of the disease [25].

When diagnosing leprosy, data from an epidemiological history (stay in an endemic region, contacts with patients with leprosy), an objective examination (pay attention to the nature and duration of the existence of rashes, the presence of signs of violations of peripheral innervation) are taken into account.

The generally accepted laboratory method for diagnosing leprosy is bacterioscopic examination. Scrapings for research are taken from lesions on the skin and nasal mucosa by light scraping.The smear is placed on a glass slide and stained according to Ziehl-Nielsen. Punctate from the femoral or inguinal lymph nodes is also examined. However, bacterioscopic examination has a very low sensitivity, especially in patients with intermediate or tuberculoid lesions [26].

Lepromine reaction (Mitsuda test) is an indicator of the host’s ability to maintain cellular immunity to M. leprae . Mitsuda’s test is not always reliable, since 10% of healthy people with leprosy may have a negative reaction.The use of the lepromine test is also limited by the technical difficulties associated with obtaining lepromine and its intradermal administration [11].

For the serological diagnosis of leprosy, the complement binding test and the indirect hemagglutination test are used. However, it is impossible to unambiguously interpret the results obtained in the course of serological diagnostics, due to the presence of antigenic persistence, and in the absence of clinical manifestations of the disease, so-called “trace” antibodies can be detected [9, 27].

The most sensitive diagnostic method for determining M. leprae is polymerase chain reaction (PCR). PCR is currently considered the most promising of direct diagnostic techniques and is used to diagnose any type of leprosy [10, 18]. The advantage of PCR is non-invasiveness and ease of obtaining clinical material, which makes it possible to screen a large number of samples when examining patients in regions highly endemic for this disease.The use of PCR allows improving the diagnosis of leprosy and detecting the disease at an early stage [26].

Currently used traditional diagnostic methods, such as lepromine test and bacterioscopic examination, do not always allow to confirm the diagnosis of leprosy in the early stages of the disease. At the same time, the effectiveness of therapeutic and preventive measures is determined by the possibility of early diagnosis of the disease. New cases of leprosy are constantly being recorded in the world, therefore, the development and implementation of new, more accurate, diagnostic methods that would become available for widespread use and make it possible to diagnose leprosy with a high degree of reliability at the early stages of the development of the disease remains an urgent issue.

Treatment of the disease was standardized by WHO in 1981 [28]. Combination drug therapy involves the use of three main drugs: dapsone, rifampicin, and clofazimine [28].

Dapsone is a bacteriostatic drug that acts as a competitive inhibitor of the enzymes dihydrofolate synthetase and dihydrofolate reductase, which are key enzymes in the pathways of folate biosynthesis in mycobacterium leprosy [28].

Rifampicin – has M.leprae bactericidal action. It is a selective inhibitor of DNA-dependent RNA polymerase and blocks RNA synthesis [29].

Clofazimine is a reddish fat-soluble crystalline dye with bacteriostatic and anti-inflammatory properties. The mechanism of the antibacterial action of clofazimine is not well understood. It is probably associated with blocking the matrix function of DNA, an increase in the phagocytic activity of macrophages and the synthesis of lysosomal enzymes [28]. Clofazimine and rifampicin are effective against dapsone-resistant microorganisms.

In 1997, the WHO established the duration of the course of treatment: 6 months for multibacillary forms of leprosy and 12 months for oligobacillary forms. Dapsone is prescribed in a dosage of 100 mg for adults once a day, rifampicin in a dosage of 600 mg once a month, clofazimine in a dosage of 300 mg once a month. Lower doses of drugs are used to treat children [28]. If it is impossible to use one or two of the above drugs, there are treatment regimens using fluoroquinolones, which have also been shown to be effective against M.leprae [30].

Identification of sources of infection and implementation of the prescribed course of combination drug therapy are considered the main principles of the strategy for the subsequent reduction of the prevalence of leprosy [3]. However, combined drug therapy does not exclude the possibility of developing resistance to antileprosy drugs. Due to the duration of therapy, patients often do not adhere to the treatment regimen. Of the three drugs included in the combination drug therapy regimens, only two are registered in the Russian Federation – dapsone and rifampicin, which complicates the use of standardized therapy regimens [28].The development of domestic analogues of drugs, the improvement of existing methods of therapy and the search for new treatment regimens that would reduce the duration of therapy and thereby increase the adherence of patients to treatment, as well as reduce resistance to antileprotic drugs, seem relevant.

Timely diagnosis of leprosy, prevention of the spread of leprosy by refugees and internally displaced persons, especially from highly endemic states (Bangladesh, the Philippine Islands, India, Angola, Brazil, Sri Lanka, etc.), are a serious and urgent problem for world health, one of the priority tasks in exercising control over the health of foreign citizens and stateless persons entering the country. In the Russian Federation, in accordance with the existing procedure established at the legislative level, in order to control the spread of diseases among the population, non-residents of the Russian Federation must undergo a medical examination in medical organizations. However, this examination procedure is complicated by the lack of a comprehensive methodological platform.

Thus, despite the decrease in the prevalence of leprosy, it seems relevant to strengthen control over the mandatory examination for leprosy of foreign citizens arriving in the country, the development of domestic analogues of drugs and the search for new treatment regimens for patients with leprosy.

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Medeiros M. Z., Filho G. H., Takita L. C. et al. Verrucous lepromatous leprosy: a rare form of presentation – report on two cases // An.Bras. Dermatol. 2014. Vol. 89, No. 3. P. 481–484.
Ruiz-Fuentes J. L., Diaz A., Entenza A. E. et al. Comparison of four DNA extraction methods for detection of Mycobacterium leprae from Ziehl-Neelsen-stained microscopic slides // Int. J. Mycobacteriol. 2015. Vol. 4, No. 4. P. 284–289.
Turankar R. P., Pandey S., Lavania M. et al. Comparative evaluation of PCR amplification of RLEP, 16 S rRNA, rpoT and Sod A gene targets for detection of M.leprae DNA from clinical and environmental samples // Int. J. Mycobacteriol. 2015. Vol. 4, no. 1. P. 54–59.
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A.A. Kubanov *, Doctor of Medical Sciences, Professor, Corresponding Member of the Russian Academy of Sciences
T.V. Abramova **, Candidate of Medical Sciences
E.K. Murakhovskaya * ^{, 1}, Candidate of Medical Sciences
V.A.Lasachko *

* FGBOU DPO RMANPO MH RF, Moscow
** FGBU GNTSDK MH RF, Moscow

¹ Contact information: [email protected]

Modern view of leprosy / A. A. Kubanov, T. V. Abramova, E. K. Murakhovskaya, V. A. Lasachko
For citation: Attending physician No. 5/2018; Page numbers in the issue: 48-52
Tags: mycobacteriosis, skin diseases, lesions of the ectoderm, antibiotic therapy

90,000 Leproux was treated with death (from the history of the fight against leprosy) | Gorelova L.E.

MMA named after I.M. Sechenov

P rokaza, known to mankind since ancient times, is shrouded in a fog of legends and fears. Its homeland is considered to be the southeast of the Asian continent (Japan. China, India), from where Babylonian captives brought it to the north of Africa. In turn, the Phoenician sailors contracted leprosy from the ancient Egyptians and spread it throughout Europe. In Greece, leprosy was called the “Phoenician disease” or leprosy.

It is known from the ancient Egyptian papyri that leprosy was widespread in Egypt.The doctors of Pharaoh Meneptekh, the son of Ramses II, were apparently among the first who, studying this disease, expressed the idea of isolating lepers (for millennia, there was a struggle between scientists about the causes of leprosy infection).

Holoav the Leper (sculpture found in Alexandria, Egypt)

This strange disease is also mentioned in the Old Testament: “When a person develops a swelling on the skin, lichen poured a white spot that resembles a leprosy ulcer, he must be brought to the high priest Aaron or one of his sons… The high priest will examine the wound. If the hair on it has turned white and it deepens under the skin of the body, this is a leprosy ulcer; the priest who performed the inspection must declare the person’s body “unclean.” Sometimes the patient was closed for seven days to make sure that “the ulcer did not change or spread over the skin.”

Many biblical stories contain recommendations. For example, a leper covered with sores had to wear torn clothes, walk with his head uncovered, cover his head and shout: “Unclean, unclean, unclean.”In the Old Testament, it was also recommended to destroy the homes of lepers so that no one could live in them anymore, and burn their clothes and personal items.

In all ancient manuscripts, the idea of the transmission of this formidable disease through contacts runs like a red thread, so that the isolation of the sick was declared the only salvation. Therefore, many legends were associated with the hermitage of lepers. Ancient Egyptian books – “Vedas” also tell us about this: “If a leper man marries a leper woman, then they will have a child with leprosy.”And again a logical conclusion follows: the leper must retire into the jungle and live there as a hermit, feeding on fruits and tree roots. Only in solitude could one hope for a favorable outcome. A remedy was also indicated – the fruits of the kaloo tree. Currently, this remedy is identified with sholmogrovy oil – the most valuable medicine. Sholmogrovaya oil, used in Indian and Chinese medicine as early as the 10th century, appeared in European medicine only in the middle of the 19th century. Its analogue – hydrocarp oil for many centuries was the only cure for leprosy: it prevented the destruction of the nasal bone, the falling off of the ears, fingers and toes.

However, these oils were toxic and caused severe kidney damage. It was necessary to look for more effective means of combating leprosy. We learn from ancient Byzantine books that Constantine the Great, who founded the future capital of the Byzantine Empire in the 4th century and established Christianity as the state religion, suffered from leprosy. The pagan doctors suggested that he bathe in the blood of newborn children every morning to heal him. Constantine rejected this “method”. According to legend, St.Sylvester and as a token of gratitude healed the emperor. After that, Constantine converted to Christianity. Where is the myth, and where is the reality – it is difficult to say.

However, even if we leave aside the historical inaccuracies of Indian and Byzantine mythology, one cannot but recall such a legal document as the laws of Manu (rules of conduct for the population of Ancient India). This code prohibited those with leprosy, as well as the sons and daughters of lepers, from marrying healthy people.

However, the requirement to isolate the sick (as the ancient Egyptians, Jews, Chinese believed, according to sources) acquires the status of state measures only in the 6th century., when in France lepers were obliged to live in special houses – leper colony. On the basis of the decree of 503 throughout the entire period of the Middle Ages, “rules” were drawn up for the behavior of the leper and his relatives. Here is one of them. “As soon as the disease was discovered, the person was taken to a religious tribunal, which … condemned him to death.” What does this mean? The unfortunate man was taken to the church, where everything was prepared for the funeral. They put the patient in a coffin, served a funeral service, took him to the cemetery, lowered him into the grave and threw several shovels of earth on him with the words: “You are not alive, you are dead for all of us.”

After this, the patient was dragged out of the grave and taken to the leper colony. Forever and ever. He never returned home to his family again. He was dead to everyone. If he left the leper colony for a while, he had to wear a gray cloak with a hood, and carry a bell around his neck, notifying those around him of the approach of this “living dead”.

The history of the military monastic order, which competed for many decades with other orders – the Teutonic, Templar, is also connected with the history of the leper colony.It was the monks of this order of St. Lazarus, established in the XII century, began to care for patients with leprosy in leper colony. Hence the name “infirmary” (shelter of St. Lazarus) came from. How much courage one had to have in those distant times (and even now) to decide to work in leper colony! The work of doctors, paramedics, nurses, nurses and in modern leper colony is a real feat. And it is necessary. According to British, American and other leprologists, by the beginning of the 20th century on the globe. there were up to 15 million patients.According to the World Health Organization (WHO), by the middle of the 20th century, there were 12 million patients with leprosy. And they all need medical attention …

Care of the lepers (old engraving)

The unknown reason causing this disease made these people outcasts in society (the expression “run like from lepers” is still preserved). In fact, for many centuries, the fight against leprosy has been a history of the moral and physical destruction of the sick.Lepers were buried alive in the ground, burned at bonfires, thrown into gorges, drowned in rivers. So, in 1321 in the French province of Languedoc, 600 people were burned in one day, half of whom were sick with leprosy. The rest were only suspected of this terrible disease.

The causative agents of leprosy – bacteria were discovered only at the end of the 19th century. By the name of the scientists who first described this microbe and developed a method of staining it, the pathogens were called Hansen-Neisser bacteria. Until now, scientists continue to study the question of the cultivation of leprosy bacteria on artificial media.Unfortunately, leprosy is a sad fate only for the human body. Except for people, no one gets sick with leprosy in nature. Typical experimental leprosy cannot be induced on animals (rare experiments are carried out on chimpanzees).

Therefore, the experiments of doctors … on themselves became a tragic page in the history of medicine. This is what D.K. Danielsen, a Norwegian leprologist, who infected himself with pieces of diseased skin, transplanted them under his skin, injected himself with the blood of lepers.He had many followers. But not a single experiment yielded results. Some people became infected with leprosy even after short-term contact with sick people, others, inoculating themselves with this terrible infection, remained healthy.

Russian scientists made a great contribution to the study of the nature of leprosy and the fight against it. Among them, V.I. Kedrovsky (1865-1937). As head of the leprosy department of the Tropical Institute, he conducted unique research on the study of the causative agent of the disease and the fight against it.At the end of the XIX – beginning of the XX century, leprosy was registered in Russia in 59 provinces, the disease at one time threatened St. Petersburg.

The secret of leprosy has not been solved until now. Lepra is still waiting for his explorer-winner.

I. Gundarov: “The disease of Yushchenko and Tymoshenko threatens the world with a pandemic of leprosy”

To avoid this, a well-known doctor proposes to hold an international consultation.

KM.RU again addresses the topic of the illness of the ex-Prime Minister of Ukraine Yulia Tymoshenko.The disease is no less mysterious than the one that struck her colleague Viktor Yushchenko on the eve of the Orange Revolution.

Recall that in the opinion of the famous Russian doctor, Doctor of Medical Sciences, Professor, Academician of the Russian Academy of Natural Sciences Igor Gundarov, in both cases we are talking about a serious illness – leprosy (leprosy). “With a 99% probability, Tymoshenko can be suspected of leprosy (leprosy),” – , he said in a recent interview with our website.

Today we again turned to Igor Alekseevich with a request to comment on the development of the situation around Yulia Tymoshenko.

KM.RU: Igor Alekseevich, in 2004 you diagnosed Viktor Yushchenko with leprosy, contrary to the official version of dioxin poisoning. Although it was supported by such medical centers as the Austrian clinic “Rudolfinnerhaus”, the Geneva clinic of dermatology at the University Hospital, American experts from the Critical Incident Analysis Group, the Dutch laboratories of Bio Detection Systems, Rikit, the German laboratory Eurofins, the Kiev Institute of Dermatology and Cosmetology, the Ministry of Health of Ukraine.In this diagnostic battle, the winner was a Moscow professor. And now there is again a diagnostic confrontation on the topic of leprosy, but with Yulia Timoshenko in the leading role and with opponents represented by German doctors from the Charite clinic plus colleagues from Canada. Are you still claiming that the former prime minister contracted leprosy from the former president?

I.G. : I have never stated directly that Yushchenko and Tymoshenko have leprosy. This requires a special examination.I only argued that the available signs are enough to suspect a dangerous disease with a 99.9% probability. Any competent infectious disease specialist will confirm this. And in leprology, there is a rule: if there is a reasonable suspicion, the patient is subject to unconditional isolation with a mandatory examination by a dermatovenerologist. According to the letter of the Ministry of Health and Social Development of the Russian Federation of 13.09.2005, “a conclusion on the absence of leprosy should be issued … by a dermatovenerologist.”

KM.RU: What instrumental examinations are required to exclude the diagnosis of leprosy? First Deputy Prosecutor General of Ukraine Kuzmin said that a blood test could help establish what Tymoshenko is sick with, a blood test will reveal what diseases she suffers from. But the patient refuses to donate blood. What will the blood test show?

I.G .: I think it won’t show anything. With leprosy, there may be nothing in the blood. At the initial stage, it is required to do a scraping from the nasal mucosa and a biopsy from the affected skin.In leprosy, microscopy will reveal Hansen’s mycobacteria plus the characteristic clinical symptoms.

KM.RU: Did Tymoshenko have such examinations?

I.G .: No. It is difficult to point out the mistakes of colleagues, but I must say about this, since the situation is epidemically dangerous. It is known that for any skin disease, if the disease does not respond to conventional treatment for a long time, it is necessary to think about leprosy and conduct a special study. Similar recommendations are given for long-term neuropathies, the cause of which is not clear.Sometimes the only manifestations of leprosy are neuritis. After all, leprosy is primarily a neuroinfection. Inflammation of the nerves leads to their thickening, which causes compression and scarring, accompanied by severe pain that does not respond to anti-pain therapy. The debilitating pains are so intense that they lead the patient to despair. In this case, leprosy neuritis often precedes skin symptoms. There is even a type of leprosy that affects only the peripheral nervous system.

Therefore, the frivolous approach of foreign colleagues to the selection of the sent specialists is surprising.Tymoshenko thinks that “abroad will help her,” and among the visiting doctors there was no infectious disease specialist and dermatologist, which are obligatory in this case.

KM.RU: Recently, at a press conference in Berlin, doctors from the Charite clinic showed journalists MRI images of Yulia Timoshenko’s spine with a hernia. It turns out that there is a hernia?

I.G. : And where is the evidence that these are pictures of Tymoshenko? The case has taken on a political turn, so now nobody can take their word for it.Everything must be absolutely reliable. The falsifications with Yushchenko’s blood samples are still fresh in the memory, when respected laboratories found dioxin concentrations exceeding the norm from 1000 to 20,000 times. To exclude falsification, all analyzes should be: 1) taken in the presence of an unbiased commission, 2) copied, 3) sealed and anonymously sent to three certified laboratories, 4) answers from laboratories should be opened in a sealed form in the presence of the same commission. Leprosy tests should likewise be sent to certified bacteriological laboratories.

KM.RU : Does this mean that Tymoshenko was prescribed the wrong treatment?

I.G. : The commission’s report correctly noted that the manifestation of Tymoshenko’s disease is “compression of a nerve with neurological consequences in the form of partial paralysis … pain and vertebral posture disorders”, “concern about one’s life.” And the essence of the treatment is the blockade of pain, which requires a special clinic. In this regard, they complain that the Kharkiv hospital “does not have a pain clinic, which plays a central role in cases of primary blockages due to pain and additional secondary ones… pain caused by impaired posture and immobilization. ”

But this is all – symptomatic treatment, the fight against the consequences, and the cause remains hidden. Colleagues do not suggest that a specific process can be the cause of the pain syndrome, which requires etiological treatment to eliminate. Therefore, the basic therapy was not carried out by an infectious disease specialist, but by the neuropathologist Lutz Harms, who, as he himself said, performed procedures and psychotherapy. Psychotherapy for leprosy is needed, but this is not the main thing.He has now been replaced by physiotherapist Anette Reisgauer, who will continue to follow up on the recommendations. And what can a physiotherapist do to treat leprosy exacerbation? She even believes that she will be able to treat Tymoshenko without tests, without knowing the diagnosis. This was directly stated at the briefing in response to the question whether treatment is possible without appropriate tests: “Yes, treatment is possible in the main.”

In exactly the same way, they basically treated Yushchenko, not knowing what disease, based on the recommendations of the Swiss professor Jean Sora.Here are the questions of the journalist of “Ukrainian Truth” and the answers of Olga Bogomolets, the attending physician of the President of Ukraine: “But before you start treatment, you must know what you are treating a patient for?” – “Yes”. – “What did you treat Viktor Yushchenko for?” – “In December, I treated Viktor Andreevich from those manifestations on the skin that I observed.”

KM.RU: Who selects specialists sent to Ukraine?

I.G. : Amazing – the officials! A BBC correspondent asked Canadian physician Peter Kuytan, “How were the doctors selected for the job?”He replied: “The Department of Foreign Affairs approached me, they asked me to take part in this mission. Nobody else asked me. ”

KM.RU : What explains such gross blunders of titled specialists?

IG: I see two explanations. I will describe one thing with quotes from the former director of the Astrakhan Research Institute for the Study of Leprosy Anatoly Yushchenko (a fatal coincidence of names). “First of all, it should be recognized that, despite the fact that patients with leprosy can be found in any region … the ideas about this infection among the majority of doctors, even dermatovenerologists, often do not correspond to the current state of leprology… This leads to the fact that when meeting a patient with leprosy, the doctor often does not even have a suspicion of this disease, and the patient is being treated for other ailments for years … infectious diseases, and had to enter into the third millennium, accompanied by an increase in the incidence of tuberculosis, syphilis, malaria … With some delay, the same will happen with leprosy. The causative agent of leprosy, like other pathogens, develops drug resistance, mutants may appear, the reproduction of which today’s powerful combination therapy will not stop.On the other hand, unfavorable environmental and socio-economic conditions reduce the individual and population immunity of the population ”.

The second is the intervention of politicians in professional medical issues, violating the laws of hygiene and public health by driving human rights to the point of absurdity. The world has moved from the extreme of an authoritarian dictatorship to the opposite extreme of a liberal dictatorship. They decided that the leaders of the state could not get sick with leprosy, and on this basis they began to act as if it did not exist in reality.The laws of epidemiology are being canceled, you can safely meet with the broad masses of the population. Of course, politicians have the personal right to keep their illness secret when meeting with other politicians, but this does not mean that their colleagues are deprived of the same right to information about their health safety. Is it permissible to ensure the preservation of the rights of one person at the expense of violating the rights of another? Could it be a private matter that threatens the safety of many? Is it fair for the well-being of one VIP to endanger other VIPs and their families? However, doctors are prohibited from interfering with the “private life” of any patient, especially the government level.Compulsory examination is not allowed even if there is a reasonable suspicion of syphilis, leprosy, plague, and dangerous schizophrenia.

In this regard, let me turn to my colleagues – doctors of the world via the Internet. So far, the situation with Yushchenko’s disease, and now that of Tymoshenko’s, is the mistake of the century, but it can become the crime of the century if we do not throw off the yoke of political hypnosis. To prevent the outbreak of a pandemic of the medieval infection, I propose to hold an international consultation to develop a resolution addressed to the World Health Organization.And the KM.RU portal should be made a coordinating platform, if its management agrees to it.