Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

The number of new cases of leprosy reported worldwide has remained essentially unchanged for the last decade despite continued global use of free multidrug therapy (MDT) provided to any diagnosed leprosy patient. In order to more effectively interrupt the chain of transmission, new strategies will be required to detect those with latent disease who contribute to furthering transmission. To improve the ability to diagnose leprosy earlier in asymptomatic infected individuals, we examined the combined use of two well-known biomarkers of M. leprae infection, namely the presence of M. leprae DNA by PCR from earlobe slit skin smears (SSS) and positive antibody titers to the M. leprae-specific antigen, Phenolic Glycolipid I (anti-PGL-I) from leprosy patients and household contacts living in seven hyperendemic cities in the northern state of Pará, Brazilian Amazon. Combining both tests increased sensitivity, specificity and accuracy over either test alone. A total of 466 individuals were evaluated, including 87 newly diagnosed leprosy patients, 52 post-treated patients, 296 household contacts and 31 healthy endemic controls. The highest frequency of double positives (PGL-I+/RLEP+) were detected in the new case group (40/87, 46%) with lower numbers for treated (12/52, 23.1%), household contacts (46/296, 15.5%) and healthy endemic controls (0/31, 0%). The frequencies in these groups were reversed for double negatives (PGL-I-/RLEP-) for new cases (6/87, 6.9%), treated leprosy cases (15/52, 28.8%) and the highest in household contacts (108/296, 36.5%) and healthy endemic controls (24/31, 77.4%). The data strongly suggest that household contacts that are double positive have latent disease, are likely contributing to shedding and transmission of disease to their close contacts and are at the highest risk of progressing to clinical disease. Proposed strategies to reduce leprosy transmission in highly endemic areas may include chemoprophylactic treatment of this group of individuals to stop the spread of bacilli to eventually lower new case detection rates in these areas.

Introduction

Leprosy, caused by the human pathogen Mycobacterium leprae (M. leprae), causes a slowly developing granulomatous disease that affects mainly skin and peripheral nerves, resulting in disfiguring skin lesions and progressive nerve damage with subsequent muscle weakness, bone and tissue resorption, with disfigurement and disability causing stigma and social isolation. Over 200,000 new cases of leprosy have been diagnosed annually in the world during the last 10 years. It is still a public health problem in some endemic areas, particularly in India, Brazil and Indonesia, where 79% of all cases were reported in 2019 [1]. Moreover, independent data allow us to conclude that the real prevalence is much higher than those reported numbers [2, 3]. The global hidden prevalence is estimated at 3 million cases, but can be six times higher than the registered prevalence in some areas [4]. Recent mathematical models predict that elimination of leprosy as a public health risk by 2061 would require over 40 years in the three regions with the highest prevalence in Brazil (North, Northeast and Midwest), primarily due to the long delay in detection of cases [5]. Currently, Brazil is still the only country in the world that has not met the WHO goal of <1 new case per 10,000 population, with 27,863 new cases detected in 2019, around 1.3/10,000 nationally based on Brazil’s National Notifiable Diseases Information System (SINAN) [6].

There is no laboratory test capable of detecting all clinical forms of leprosy. Thus, the diagnosis is essentially based on clinical examination of skin and peripheral nerves, ideally by trained dermatologists or leprosy clinicians. However, diagnosing leprosy in Brazil is often made by health care personnel in the basic health units, who may lack training in diagnosing leprosy. The knowledge and skills of leprosy diagnosis, treatment and management by general health workers are unsatisfactory, resulting in delayed detection, leading to an increase in physical disabilities, socioeconomic impairment and continued M. leprae transmission [7, 8]. Therefore, the development of a more sensitive diagnostic test suitable for early-stage leprosy and for paucibacillary or asymptomatic disease is considered a research priority [9, 10].

The diagnosis of leprosy is based mainly on physical examination to detect clinical signs and symptoms (hypopigmented or scaly skin lesions with loss of sensation; pain or swelling of nerves; weakness of muscles or loss of function). The five Ridley-Jopling forms used to categorize the disease spectrum are polar tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and polar lepromatous (LL), with an increasing bacterial load in the lepromatous forms [11]. Indeterminate leprosy is an early stage of the disease with ill-defined skin lesions while pure neural leprosy (PNL) occurs when nerves are enlarged without any detectable skin lesions. The form of the disease is used for classifying patients into paucibacillary (PB) or multibacillary (MB) categories that determines the length of treatment with multidrug therapy (MDT) for 6 or 12 months, respectively. In hyperendemic areas in Pará, Brazil, around 70% of individuals diagnosed are classified as MB. Various tests have been developed to assess anti-PGL-I antibody positivity, a known biomarker of M. leprae infection, including the standard ELISA assay [12] and lateral flow rapid tests that incorporate synthetic PGL-I (ND-O-BSA) or novel protein glycoconjugates, like NDO-LID [13, 14]. There is an excellent correlation between the bacillary load (BI) and the anti-PGL-I titer, showing a progressive increase in the titer in lepromatous forms (BB, BL and LL) while the antibody titer is low to negative in tuberculoid forms (TT, BT). The molecular detection of M. leprae DNA in earlobe slit skin smears (SSS) or blood [15], skin lesions, nasal swabs or biopsies using standard PCR [16] or quantitative PCR (qPCR) [17, 18] has also been found to be very useful to detect asymptomatic carriers or to diagnose difficult cases. These confirmatory tests are currently being used to aid in the diagnosis of leprosy patients and are among the strategies that have been recommended for implementation for better leprosy control and patient management [19].

In the current cross-sectional study, we have combined the use of the standard ELISA assay to measure the anti-PGL-I titer in serum with the detection of M. leprae DNA by PCR amplification of the M. leprae-specific repetitive sequence, RLEP, in earlobe SSS samples in a cross-section of leprosy patients, healthy household contacts and healthy endemic controls from seven hyperendemic municipalities in different regions in the northern state of Pará in the Brazilian Amazon.

Methods

Ethics statement

This study conforms to the Declaration of Helsinki and the research protocols were approved by the institutional review boards at the Federal University of Pará (UFPA) (IRB protocol CAAE 26765414.0.0000.0018) and Colorado State University (IRB protocols 15-6340H, 18-8369H and 20-8369H). All individuals who agreed to participate read and signed a written informed consent document. In the case of minors, consent was obtained from a parent or guardian of the child. All data were anonymized.

Study area

Pará state is in northern Brazil, occupying an area of 1,253,164 km2, being the second largest state in Brazil. To include a broad representation of individuals from cities of various sizes, we selected seven municipalities that came from the six mesoregions in this state. The Lower Amazonas region was represented by samples from the city of Santarém (302,667 inhabitants). In the southwest, samples were collected from Senador José Porfírio (11,839 inhabitants). The town of Breves (101,891 inhabitants) represents the Marajó mesoregion. The southeast was represented by the city of Redenção (83,997 inhabitants), while the northeast was represented by the city of Acará (55,513 inhabitants). The metropolitan region of the capital of Belém is represented by the district of Mosqueiro (approximately 27,000 inhabitants), Castanhal (198,294 inhabitants) and the capital city itself (2.3M inhabitants).

Sampling design and methods

Leprosy is a compulsory notifiable disease in Brazil and patients detected through either clinic-based passive diagnosis or active surveillance have their clinical data and addresses registered in the national notifiable diseases information system (SINAN). At each site visited a random sampling of subjects from the seven cities surveyed was performed using available data for locating treated patients. We also relied on the local community health agents working with the basic healthcare units to assist us in locating households where new cases of leprosy were suspected. These households were visited by our team where new leprosy patients were diagnosed and their household contacts were assessed. All individuals received a free dermatologic exam performed by experienced leprosy clinicians, and the sample of blood and SSS was taken from each person by a trained phlebotomist. Blood was processed by centrifugation at each site to yield serum that was frozen and transported to the laboratory for analysis by ELISA. Earlobe SSS samples were placed in microcentrifuge tubes containing 70% ethanol and transported to the laboratory for DNA extraction and analysis by PCR. The diagnosis of leprosy by experienced leprosy clinicians was performed using internationally accepted clinical criteria based on the presence of skin lesions with sensory loss and/or nerve damage associated with nerve swelling and pain, muscle weakness or disability. Individuals diagnosed with leprosy received free MDT treatment from their local basic health unit. A total of 466 individuals from different cities in Pará agreed to provide blood and earlobe SSS for the purpose of assessing the anti-PGL-I titer and M. leprae DNA positivity by PCR, respectively. The individuals surveyed included 87 newly diagnosed leprosy patients, 52 former patients who had completed their MDT treatment, 296 household contacts (individuals living in a household with at least one confirmed diagnosed case of leprosy) and 31 healthy endemic control (HEC) subjects with no known exposure to a leprosy patient, mainly undergraduate and graduate students attending the Federal University of Pará, Belém, Pará.

Assessment of anti-PGL-I titer by ELISA

An indirect ELISA was used to measure the anti-PGL-I IgM titer of each of the serum samples tested at a 1:300 dilution using a protocol previously reported [20]. The cut-off for positivity was established at an optical density (O.D.) of 0.295 based on the average plus three times the standard deviation of healthy subjects from a hyperendemic area as reported. The O.D. for each well was read at 490 nm using an ELISA plate reader.

DNA extraction and RLEP amplification

Total DNA extraction of earlobe SSS samples using the Qiagen Blood & Tissue DNA kit (Qiagen, Germantown, MD) was performed according to the manufacturers’ protocol with minor modifications. Amplification of the M. leprae repetitive RLEP sequence (up to 37 copies are found within the genome) was achieved using Qiagen Multiplex PCR Master Mix (Qiagen) according to a previously published protocol [21] using the primer pairs LP1 (5’-TGCATGTCATGGCCTTGAGG -3’) and LP2 (5’-CACCGATACCAGCGGCAGAA-3’) described to amplify a 129-base pair fragment found in the genome [22].

Statistical analysis

The Mann-Whitney U test was used to compare the titers of anti-PGL-I IgM between groups, Student’s t-test (unpaired and nonparametric) to evaluate IgM anti-PGL-I titers, and Fisher’s exact test was used to compare the proportion of new cases detected among seropositive and seronegative individuals and to calculate the correlation between anti-PGL-I IgM and RLEP amplification. Sensitivity, accuracy, and specificity were determined by exact method of Clopper and Pearson. All analyses were performed using GraphPad Prism version 6.0.

Results

A total of 466 individuals chosen from seven different municipalities were divided into four groups: 87 newly diagnosed leprosy patients, 52 treated patients who had completed MDT (averaging 4 years after completing treatment), 296 HHC who did not have any clinical signs of disease and 31 HEC (Table 1). Children under the age of 15 years old were included. Of the total number evaluated, 92/466 (19.7%) were children; 38/87 (43.7%) in the new case group; 3/52 (5.8%) in the treated group; 51/296 (17.2%) in the HHC group; and 0/31 (0%) in the HEC group.

Table 1

Operational classification and number of subjects per group and municipality.

Characteristics of newly diagnosed leprosy patients, treated leprosy patients, healthy household contacts (HHC) and healthy endemic controls (HEC) from the seven cities surveyed.

		New leprosy cases			Treated leprosy patients				HHC	HEC
City	PB	%	MB	%	PB	%	MB	%
Acará	4	50.0	4	50.0	1	10.0	9	90.0	66	-
Breves	1	20.0	4	80.0	1	100.0	-	-	18	-
Castanhal	-	-	-	-	1	20.0	4	80.0	26	-
Belém/Mosqueiro	6	14.6	35	85.4	-	-	-	-	74	31
Redenção	1	25.0	3	75.0	2	20.0	8	80.0	24	-
Santarém	4	16.7	20	83.3	6	37.5	10	62.5	52	-
Senador José Porfirio	-	-	5	100.0	3	30.0	7	70.0	36	-
Total	16	18.4	71	81.6	14	26.9	38	73.1	296	31

We first examined the anti-PGL-I titer in serum samples from all individuals. As shown in the percent positivity was 55.2% for new cases, 50% in treated individuals, 51.7% in HHC and 22.6% in HEC. There was no statistical difference in the overall percentage of anti-PGL-I positivity between the patient and HHC groups, while positivity in HEC was significantly lower than all other groups. The O.D. for each individual was plotted for the three groups to determine the range and the median O.D. for each group (). Although the median O.D. was somewhat higher in the treated group, it was not significantly higher than the median O.D. for new cases or household contacts. The median for the HEC group was the lowest, with only seven individuals slightly exceeding the cut-off. Within the new case group, detection of a positive anti-PGL-I titer was 68.8% (42/61) in MB cases and 37.5% (6/16) in PB cases. When cases were subdivided according to Ridley-Jopling classification for the different forms across the disease spectrum, anti-PGL-I was positive in 57% (4/7) for the indeterminate form, 20% (1/5) for TT, 70.8% (34/48) for BT, 31.6% (6/19) for BB and 50% for BL and LL (2/4), respectively. In addition, four cases of primary neural form (PNL) were diagnosed, and 25% (1/4) were positive.

Frequency of anti-PGL-I positivity in new cases, treated cases, HHC and HEC.

A) Positivity versus negativity in anti-PGL-I titers with a similar percentage of positives observed in newly diagnosed cases, treated cases, and household contacts, while those in the HEC group were negative or weakly above the cut-off. B) Anti-PGL-I optical density (O.D.) for all individuals was plotted for each group with the median O.D. indicated by the solid horizontal line. The significant p value differences between groups are shown.

RLEP amplification was performed on DNA prepared from earlobe SSS to assess the presence of M. leprae DNA (). In the new case group, 73/87 individuals (83.9%) were RLEP positive; in the treated group (median 4 years after finishing treatment), 23/52 (44.2%) were positive; in the HHC group, 81/296 (27.4%) were positive, while in the HEC group, none were positive (0/31). When individuals in each group were divided into RLEP positive or negative and examined for anti-PGL-I titer, there were no statistical differences between the median O.D. values for RLEP positive versus negative individuals within the patient or HHC groups (). Within the new case group, detection of RLEP was 87.3% (62/71) in MB cases and 68.8% (11/16) in PB cases. When cases were subdivided according to Ridley-Jopling classification for the different forms across the disease spectrum, RLEP amplification was positive in 57.1% (3/7) for the indeterminate form, 80% (4/5) for TT, 87.5% (42/48) for BT, 84.2% (16/19) for BB and 100% for BL and LL (4/4). In addition, four cases of primary neural form (PNL) were diagnosed, and all were positive (100%, 4/4).

Analysis of RLEP and PGL-I titer for new cases, treated cases, HHC and HEC.

A) RLEP positivity within each of the four groups examined based on PCR of earlobe SSS. New cases were 83.9% positive, treated cases were 44.2% positive, HHC were 27.4% positive whereas no HEC were positive (0%). B) anti-PGL-I titer was plotted for each individual based on being RLEP positive or negative within each group. Solid line indicates the median O.D. for each group. There was no significant difference between the median anti-PGL-I titer when positive and negative RLEP groups were compared between any of the two patient and HHC groups.

Finally, we examined both RLEP and anti-PGL-I results within the four groups of subjects. The results showed the highest frequency of double positives (PGL-I+/RLEP+) in the new case group (40/87, 46%) with lower numbers for treated (12/52, 23.1%), HHC (46/296, 15.5%) and none for HEC (0/31, 0%). The frequencies in the groups were reversed for double negatives (PGL-I-/RLEP-) for new cases (6/87, 6.9%), treated cases (15/52, 28.8%) and the highest in HHC (108/296, 36.5%) and HEC (24/31, 77.4%) (). We did not detect any differences in sensitivity of anti-PGL-I or RLEP PCR detection based on either the geographic origin of the samples, nor were there differences based on whether the samples were from male or female subjects.

IgM anti-PGL-I serology showed 55% sensitivity, 51% specificity and 52% accuracy, while detection of RLEP DNA in SSS was higher, with sensitivity of 84%, specificity of 75% and accuracy of 77%. Combining both laboratory tests increased sensitivity to 88%, specificity to 77%, and accuracy to 79%.

The socioeconomic data collected from all of the individuals studied shown in Table 3 revealed that more than 20% of new cases and 34% of treated cases and HHC declared they had suffered food deprivation at least once during the year, while food insecurity did not exist in the HEC group. The financial situation of many of the families was quite precarious, showing that 63.2% of new cases, 57.7% of treated cases and 58.1% of HHC had ≤ one Brazilian minimum salary per month (approximately $200 USD), while none of the HEC fell into this category. A source of good clean drinking water (filtered or mineral water versus untreated, strained or chlorinated water) was lacking for 51.7% of new cases, 59.6% of treated cases, and 65.5% of HHC, while none of the HEC were without clean drinking water. Low (only elementary schooling) or no education was recorded among 88.5% of new cases, 73.1% of treated cases and 78.7% of HHC, while 100% of the HEC were university educated. Finally, there was a much higher density in living conditions in households of new cases, treated cases and HHC averaging between 5.0 to 5.5 individuals, while HEC households had on average only 3 individuals living together.

Table 3

Demographic information of the four groups studied (new leprosy cases, treated patients, HHC and HEC) including household density (average number of people living in the house), type of water used for drinking and cooking, income level, education level, receiving governmental support, median age and range, ratio of number of males to females, incidence of food deprivation and living in an urban versus a rural area.

	Number of people per house	Type of water used for drinking/cooking	Salary	Education (highest grade)	Receive governmental support (%)	Median age (range)	Sex (ratio M:F)	Food deprivation (%)	Living in urban area (%)
New cases (n = 87)	5.5	Not treated: 4Strained water: 39Chlorinated: 2Filtered: 36Mineral water: 6	≤ 1 minimum salary: 55Up to two minimum salary: 21Up to three minimum salary: 5Greater than 3MS: 6	No Education: 66Elementary School: 11High school: 9University education: 1	60/87 (69%)	25 (5–81)	Female: 52Male: 35(0.6: 1)	18/87 (20.7%)	81/87 (93.1)
Treated (n = 52)	5	Not treated: 3Strained water: 21Chlorinated: 7Filtered: 12Mineral water: 5	≤ 1 minimum salary: 30Up to two minimum salary: 13Up to three minimum salary: 4Greater than 3MS: 3	No Education: 30Elementary School: 8High school: 8University education: 6	35/52 (67.3%)	45 (12–87)	Female: 22Male: 30(1: 0.58)	18/52 (34.6%)	42/52 (80.8%)
HHC (n = 296)	5	Not treated: 15Strained water: 147Chlorinated: 32Filtered: 70Mineral water: 32	≤ 1 minimum salary: 172Up to two minimum salary: 81Up to three minimum salary: 27Greater than 3MS: 16	No Education: 106Elementary School: 127High school: 52University education: 11	193/296 (65.2%)	31 (6–79)	Female: 160Male: 136(0.46: 1)	102/296 (34.4%)	241/296 (81.4%)
HEC (n = 31)	3	Not treated: 0Strained water: 0Chlorinated: 0Filtered: 2Mineral water: 29	≤ 1 minimum salary: 0Up to two minimum salary: 1Up to three minimum salary: 2Greater than 3MS: 28	No Education: 0Elementary School: 0High school: 0University education: 31	0/31 (0%)	33 (19–62)	Female: 20Male: 11(0.35: 1)	0/31 (0%)	31/31 (100%)

Discussion

Bacilloscopy is the gold standard laboratory test to detect acid fast M. leprae in the skin of diagnosed leprosy patients, and is important to establish the bacillary index (BI, a logarithmic scale of the number of acid fast bacilli detected in the skin, where 0 is none detected and 6+ is the highest) for determining the treatment regimen (MB or PB, one year or 6 months MDT treatment, respectively). However, this test, although highly specific, has a low sensitivity (only 44%) [23], is labor intensive, requiring experienced lab personnel and may require taking a skin punch biopsy, which is somewhat invasive. Thus, it is performed only in presumed leprosy cases, and is negative in the majority of PB or primary neural forms. Biopsies are not performed on household contacts since, absent any lesions, almost all would be negative. For this reason, we have been using less invasive methods, namely taking samples of blood and earlobe SSS to assess anti-PGL-I positivity by ELISA and the presence of M. leprae DNA by PCR, respectively. Each of these biomarkers of infection pose an independent risk for an individual to progress to disease. People who are positive for anti-PGL-I have about a 6-fold higher risk of progressing to disease [24], and we have previously established that by following 10 seropositive individuals, there is a >90% chance that one of these individuals will progress to disease within a two-year timeframe [25]. Similarly, confirming molecular amplification of M. leprae DNA in nasal or oral mucosa [26, 27] or skin biopsies or smears can be used as a biomarker of infection, indicating colonization in the skin. After infection of the nasal mucosa by M. leprae, one of the secondary sites of infection is the earlobe due to its proximity to the nose and is a preferred site because of its relative coolness. Demonstration of M. leprae infection of the earlobe by identifying acid fast bacilli in SSS or by detecting a positive PCR for bacterial DNA can be used as confirmatory evidence in the diagnosis of a patient with other clinical signs of disease. Studies have shown that the use of the repetitive element RLEP as the target in detecting M. leprae DNA is more sensitive than single copy genes such as rpoT, sodA and 16S rRNA [28, 29], so for this reason we used primers to detect the RLEP sequence.

Although it has been previously established that being anti-PGL-I positive puts an individual at higher risk for eventually coming down with leprosy [30-32], our previous studies in cities in the state of Pará have shown that seropositivity among residents living in hyperendemic areas is generally quite high, usually 40–60%, reflecting the very high levels of M. leprae circulating in the general population [2, 20, 21, 25, 33, 34]. Despite these high rates of positivity, there is an overall genetic resistance towards developing leprosy, with over 90% of people having a natural immunity [35]. Even in this study, anti-PGL-I positivity in newly diagnosed leprosy patients was only slightly higher than in HHC, 55.2% versus 51.7%, respectively. The main reason for this is that the majority of newly diagnosed leprosy patients were classified as BT (48/87, 55.2%), known for having low or no anti-PGL-I antibody titer. For this reason, we wanted to pair an additional biomarker of M. leprae infection, namely RLEP PCR of earlobe SSS, to determine if the two biomarkers together could be more informative as far as identifying those with latent disease. For example, we discovered that some of the households we examined in the town of Acará, just two hours from the capital of Belém, had extremely high percentages of anti-PGL-I and RLEP PCR positivity. In one household with 12 people living together with a newly diagnosed index case, 92% (11/12) were found to be anti-PGL-I positive, 75% (9/12) had evidence of M. leprae colonization of their earlobes by RLEP PCR, and 75% were positive for both biomarkers. Only one individual from this family was negative for both biomarkers (anti-PGL-I-/RLEP-). At the time of visiting this household, six other blood related family members were clinically diagnosed, supporting published reports that HHC who have a genetic relatedness with an untreated MB index case have the highest risk of progressing to disease [36, 37]. These very high percentages for anti-PGL-I and RLEP PCR double positivity were seen in several other large extended households in the same neighborhood, indicating high rates of transmission and infection in this area.

These high levels of double positives in large families in many households in hyperendemic settings led us to conduct this current survey in multiple cities to determine if these results were generalizable in different areas of Pará state. Although there were no differences between the three groups (new patients, treated patients and HHC) as far as the percent of anti-PGL-I positivity, there were large differences in the rates of RLEP positivity in these groups. Newly diagnosed cases were overwhelmingly positive by PCR of SSS (83.9%), indicating earlobe colonization, while just under half of individuals who were treated were positive (44.2%) indicating a large reduction in the bacterial burden following treatment in this group. HHC showed the lowest rates of RLEP PCR positivity at only 27.4% even though slightly more than half of this group were anti-PGL-I positive (51.7%). The lack of RLEP positivity in HEC can be explained by the fact that most of these individuals (almost all are university students) come from a higher socioeconomic background and have no known contact with a leprosy patient. When these two biomarkers of infection were paired together, it revealed more compelling information. Almost half of newly diagnosed patients were double positive (anti-PGL-I+/RLEP PCR+, 46%), while a minority in this group were double negative (anti-PGL-I-/RLEP PCR-, 6.9%). Double negatives were higher in the treated group (28.8%), likely indicating treatment efficacy. In contrast, HHC had slightly more double negatives overall (36.5%) and much fewer double positives than either of the patient groups (15.5%). Despite this lower number, HHC who are double positive were found to have a much higher risk of progressing towards disease (OR = 19) since they most closely resemble the high rate of double positives found in newly diagnosed patients. For this reason, longitudinal long-term follow-up of these individuals would be critical to understanding their proclivity to progress towards disease over those who are double negative.

The four different possible combinations of ELISA/PCR results can be cautiously interpreted in several ways. We propose that those individuals without clinical signs and symptoms of leprosy who are PGL-I+/RLEP+ have latent leprosy infection, allowing permissive growth to allow infection of M. leprae in the earlobe and spread to other sites in the skin and induce an antibody response. These individuals most resemble newly diagnosed patients, the majority of whom are double positive, and thus are at the greatest risk of progressing to disease and spreading it to others. Individuals who are PGL-I+/RLEP- are infected but their functional cell mediated immune response has limited bacterial infection in the earlobe, which can evolve to a cure or can progress to paucibacillary disease. PGL-I-/RLEP+ individuals are also infected but the bacillary load has not increased to the point that induces an anti-PGL-I response. These individuals could either control the bacilli or progress to disease if the cell mediated response allows permissive growth and spread. Individuals who are double negative, PGL-I-/RLEP-, may not have been exposed to enough of a bacterial load to infect them or were more resistant to infection. These results could change over time depending on continued exposure to an untreated index case or other factors that can degrade a robust cell mediated immune response (co-infections, poor nutritional status).

Our demographic data confirm the close relationship between leprosy and several socioeconomic indicators. Human development index (HDI) scores that take into account three main metrics including life expectancy at birth, level of education and per capita income show a good correlation between lower HDI and higher new case detection rates throughout Brazil. Meta-analysis of secondary data has shown that poor socioeconomic conditions are associated with an increased risk of acquiring leprosy, with food deprivation and low socioeconomic levels being the most critical [38-41]. Food deprivation has also been indicated as an important factor for the development of clinical leprosy as the insufficient intake of macronutrients/micronutrients impairs the immune system and decreases host protection, leading to increased frequency and severity of infections [42, 43]. Related to this is the poor quality of drinking water that can be contaminated by disease causing bacteria, amoeba and helminths, co-infections that can cause a shift to a Th2 cytokine profile that does not protect against intracellular infections like M. leprae. Amoebal cysts can also carry M. leprae that can be ingested by drinking contaminated water [44]. The deficiencies observed in the patient and household contact groups with regard to low income levels, reliance on government aid programs, low educational levels, food insecurity, lack of clean drinking water, overcrowding conditions, lack of primary health care coverage and living in a leprosy hyperendemic environment all contribute to a higher risk of succumbing to leprosy.

Over the last few years, the principal stakeholders, including the WHO, involved in promulgating strategies aimed at reducing the global burden of leprosy, particularly in hot spots or high to hyperendemic regions, agree that early diagnosis, contact tracing, and treatment of all patients should be part of the overall strategy. The reported number of new leprosy cases worldwide has been above 200,000 for the last five years, with cases in children averaging around 8%, indicating continued leprosy transmission, and rates of grade 2 disability hovering around 6%, indicating serious delays in diagnosis [45]. However, there are many poor areas of the world that do not report statistics on leprosy [46] and low levels of contact tracing and follow-up could theoretically lead to large numbers of unreported cases in the coming years [47]. It has been suggested that chemoprophylaxis involving the use of single dose rifampicin (SDR) treatment of contacts of leprosy cases might be one way to reduce the number of cases in high endemic areas. In recent developments, there are large-scale clinical trials underway coordinated by national programs that are examining the efficacy of the use of Leprosy Post-Exposure Prophylaxis (LPEP), either SDR or other multidrug short course therapy regimens in multi-country locations to evaluate the potential of accelerating the reduction of transmission in high and hyperendemic areas [48]. Our results indicate that up to 25% of the contacts in highly endemic areas are already infected by M. leprae with no clinical disease. This may be one of the reasons why SDR has not be effective to control leprosy in hyperendemic areas. Instead of SDR, the use of MDT or other short course drug combinations currently being tested may be necessary to treat those infected contacts with latent infectious disease [49].

Considering all of the available data, it might be possible to target HHC that are double positive (anti-PGL-I+/RLEP PCR+), as these individuals have latent leprosy infection, are probably shedding bacilli and contributing to infecting their household contacts and are therefore most likely to progress to disease. Combining both of these tests increased the sensitivity and specificity over either test alone and may provide added benefit to detecting those with latent leprosy. Prophylactic treatment of this high-risk group and their HHC would likely be an effective strategy to end transmission within all of the contacts of these households. There are strong hopes that the use of these kinds of aggressive strategies will ultimately break the lines of transmission and successfully remove leprosy as a major health concern.

Limitations of the study

The diagnosis of leprosy is still based on the detection of classic signs and symptoms of skin lesions with loss of sensation, nerve damage with loss of function, swelling or pain and visible deformity as detected by well-trained clinicians or health care personnel since M. leprae cannot be cultivated. Despite the use of adjunct laboratory tests to detect acid fast bacilli in skin smears or M. leprae DNA by PCR, these tests are not always available in resource constrained settings. The anti-PGL-I assay, although easy to perform, only indicates prior infection by the bacillus and a positive test by itself does not trigger the administration of MDT since the majority of positive individuals will not progress to clinical disease. Although we have shown that the majority of newly diagnosed leprosy cases with clinical symptoms are positive for both anti-PGL-I and M. leprae DNA by PCR, whether household contacts who are also double positive have latent disease and are at the highest risk of succumbing to disease can only be determined in future long term longitudinal follow-up studies.

Frequency of anti-PGL-I positivity in new cases, post-treated cases, HHC and HEC.

A) Positivity versus negativity in anti-PGL-I titers with a similar percentage of positives observed in newly diagnosed cases, post-treated cases, and household contacts, while those in the HEC group were negative or weakly above the cut-off. B) Anti-PGL-I optical density (O.D.) for all individuals were plotted for each group with the median O.D. indicated by the solid horizontal line. The significant p value differences between groups are shown.

(DOC)

Click here for additional data file.

Analysis of RLEP and PGL-I titer for new cases, post-treated cases, HHC and HEC.

A) RLEP positivity within each of the four groups examined based on PCR of earlobe SSS. New cases were 83.9% positive, treated cases were 44.2% positive, HHC were 27.4% positive whereas no HEC were positive (0%). B) anti-PGL-I titer was plotted for each individual based on being RLEP positive or negative within each group. Solid line indicates the median O.D. for each group. There was no significant difference between the median anti-PGL-I titer when positive and negative RLEP groups were compared between any of the two patient and HHC groups.

(DOC)

Click here for additional data file.

(DOC)

Click here for additional data file.

(DOCX)

Click here for additional data file.

Demographic information of the four groups studied (new leprosy cases, treated patients, HHC and HEC) including household density (average number of people living in the house), type of water used for drinking and cooking, income level, education level, receiving governmental support, median age and range, ratio of number of males to females, incidence of food deprivation and living in an urban area.

(DOCX)

Click here for additional data file.

11 Sep 2020

PONE-D-20-19814

RLEP and anti-PGL-I double positivity in leprosy patient household contacts represents an important source of transmission in hyperendemic cities in Pará, Brazil.

PLOS ONE

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Reviewer #1: Dear authors,

please make your manuscript shorter and focus mainly on your finding

line 412"Prophylactic treatment of this high risk group and their HHC would likely be an effective strategy.." what did you mean from prophylactic treatment? MDT or SDR?

Reviewer #2: The design and research of the paper are highly operable. It has certain value for the diagnosis and prevention of leprosy. The disadvantage is that the sensitivity of the test is low, and the leprosy patients cannot be completely screened.

Reviewer #3: Comments to the authors

The overall recommendation to the authors is they should well acquit themselves with the STROBE guidelines and re-write the paper (I’m assuming the study design is cross-sectional). The paper has an overall message they are trying to communicate but it is not supported by the methods, results and the discussion.

Abstract

The abstract vaguely reports what the problem is, what is known about the problem and what the authors did to solve the problem.

However on introducing the bio-markers they should have ideally been written in full. Then abbreviations could be used thereafter (line 40 and 41).

Introduction

The introduction overall fairy tries to highlight what the problem is, what the gaps are and what was done about it. However:

(Line 118) The Dr. Marcelo Candia Reference Unit in Sanitary Dermatology (UREMC), I don’t understand the relevance of this clinic. Does it offer the standard testing; is it the Center of Excellence in Para? More clarity maybe needed for the reader.

Line 144-Line 149 Are actually Methods and Materials in the introduction section. The study design has been highlighted as well as the study description. I think this should be moved to the methods section.

Line 149-line 153 this is actually the discussion section in the introduction. This part should be moved to the discussion section.

Methods

Overall, the methods are poorly presented and hence the study cannot be reproduced. We don’t know what study design was used. A prospective study design was mooted in the introduction but it is not anywhere else. I advise the authors to look up the STROBE guidelines by Von Elm et al.

This the study is about probable diagnostic tests, the authors should tell us if the tests were done in series or in parallel since the results change depending on what is done.

There are some result tables within the methods section.

Results

I still advise the authors to have a look at the STROBE guidelines. However, it is important to add the socio-demographics of the study subjects unless this is a secondary analysis. The socio-demographics will help us with generalization and also perspective.

The author should use the statistical methods mentioned in the results section to find out if they are significant. Currently all we have are the proportions. We are not sure if they are purely by chance.

Discussion

Overall the discussion is about the bio-markers and leprosy in general. Very little effort is made to discuss the results. Their significance and the recommendations if any, the overall recommendation is hinted all over the paper with no possible justification given from the results.

Reviewer #4: 1) This is a good study trying to address the issue of transmission in leprosy

2) In fact this investigation is trying to look at possible sources of infection in the community and their role in transmission of the disease

3) In Table 2 for all parameters the author should give number of MB and PB cases in each group

4) In HCC it will be interesting to know the type of Index cases whether they are PB or MB

5) If skin smear BI available in Index case was there a correlation between high BI positivity and HCC positive

6) Is there a correlation between smear positivity (If available) and positivity in the two tests performed?

7) In HCC was there any other household which had many members who were positive for any of the two tests and the author should mention about number of families which were positive.

8) In HCC compare the positivity in different age groups and was there any correlation with higher age group?

9) The author needs to mention about the age groups of subjects and among the cases positive for any test in all groups, was there any child case?

10 There is no legend for the Tables mentioned

11) How many HHC who were doubly positive were followed up if followed up and what was the outcome of that follow up?

Reviewer #5: Leprosy is still a public health problem in some countries. In Brazil, the highest prevalence of the disease is observed in North, Northeast and Midwest regions. One important fact regarding the elimination of leprosy is the absence of a gold standard test for diagnosis that results in a significant number of hidden cases. Prophylactic treatment of contacts from multibacillary patients has been evaluated as an effective strategy to control the disease, but the identification of contacts with subclinical infection is important to determine the targets of chemoprophylactic strategy. Here, Silva and colleagues described that contacts that are positive for both RLEP and PGL-1 have latent disease and are at highest risk of progressing to clinical disease. This paper is interesting. However, I have some concerns:

1. Although the experimental design seems correct, authors need to describe the methodology for analyzing the data. How did they calculate sensitivity, specificity and accuracy? Please include in the methodology section.

2. The "Discussion" is rather long and sometimes confusing. It contains common information that is not directly related to the specific topic of this study.

3. Several studies have reported that PGL-1 is a marker of exposure, but not necessarily of infection. It is not clear in the discussion what is the hypothesis for the different profiles observed in the contacts. For example, PGL-1+RLEP+ are the latent patients. But, what is the hypothesis for PGL-1-RLEP+? Please discuss it.

**********

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Reviewer #1: Yes: Azin Ayatollahi, MD

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Reviewer #3: Yes: Michael Kakinda

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Reviewer #5: Yes: Roberta Olmo Pinheiro

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Submitted filename: Comments to the authors.docx

Click here for additional data file.

10 Nov 2020

We thank the reviewers very much for their questions and comments which were very helpful. We have endeavored to answer all of the questions and make the changes and additional clarifying paragraphs to the manuscript based on these suggestions wherever possible, keeping in mind that the reviewers also asked us to shorten the manuscript where possible. Our responses are in blue after each reviewer comment. We hope that the revised version of this manuscript is improved from the original.

PONE-D-20-19814

RLEP and anti-PGL-I double positivity in leprosy patient household contacts represents an important source of transmission in hyperendemic cities in Pará, Brazil.

Revised title: Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

PLOS ONE

Please submit your revised manuscript by Nov 4, 2020 11:59PM. Please upload your review as an attachment if it exceeds 20,000 characters

Reviewer #1:

Dear authors,

Please make your manuscript shorter and focus mainly on your findings

Thank you for these suggestions, we will endeavor to make changes wherever possible to achieve this.

line 412 "Prophylactic treatment of this high risk group and their HHC would likely be an effective strategy." what did you mean from prophylactic treatment? MDT or SDR?

This is a very important question that we can address in this response, but we will not debate this in our manuscript because it would be too long and is tangential to our study. Studies to examine the efficacy of the broad use of SDR under the LPEP (Leprosy Post Exposure Prophylaxis) trials have been ongoing since 2015, having been implemented alongside national leprosy programs in India, Indonesia, Myanmar, Nepal, Sri Lanka, Tanzania, Brazil and Cambodia. To date, they have not published any data on the efficacy of widespread use of SDR. There has been much debate among leprosy clinicians and academics as to whether SDR is a cost-effective intervention and there are concerns that a single dose of rifampin is not sufficient to protect against developing multibacillary (MB) disease.1 Others are concerned about SDR potentially increasing drug resistance. There may also be ethical problems in telling people that they will be protected since results of the large Chemoprophylaxis of Leprosy (COLEP) trial in Bangladesh found that SDR only protects some people from some types of leprosy (not MB disease) and only for up to 2 years.1 We include a new paragraph in the Discussion to address this.

1. Lockwood DNJ, Krishnamurthy P, Kumar B, Penna G. 2018. Single-dose rifampicin chemoprophylaxis protects those who need it least and is not a cost-effective intervention. PLoS Negl Trop Dis 12: e0006403. doi.org/10.1371/journal.pntd.0006403

Reviewer #2:

The design and research of the paper are highly operable. It has certain value for the diagnosis and prevention of leprosy. The disadvantage is that the sensitivity of the test is low, and the leprosy patients cannot be completely screened.

Thank you for these observations. We agree that the sensitivity of the anti-PGL-I ELISA is low in the newly diagnosed patient group (55%), but the majority of these patients are categorized as BT (42/87, 48.3%), and since patients in this category have a low BI, anti-PGL-I responses are generally weak or negative compared to MB patients. Those in the HHC group had a positivity rate slightly less than the new patient group. This is the main reason why we decided to couple anti-PGL-I with RLEP PCR results from earlobe skin smears, as the RLEP positivity in the new patient group was 83.9% (73/87). By detecting the number of double positives in HHC (15.5%) and showing that double positivity represents the largest percentage in newly diagnosed leprosy patients (46%), it suggests to us that this group of HHC has the highest risk of disease progression. Our findings are similar to results in other articles published in the literature, showed a sensitivity of the serological test of 55% in a hyper-endemic population, and 84% for conventional PCR, while the association of both tests reached 88% sensitivity.

The detection sensitivity presented by conventional PCR (88%) to a similar test for tuberculosis approved by the FDA (TB Amplicor-Roche), which presents 79 to 91% sensitivity1, was superior to staining methods, which shows around 59% sensitivity.2 We recognize that greater sensitivity is the goal to be achieved, however other detection tools, such as qPCR and ddPCR, are being tested by us to achieve that goal.

1. Yang S and Rothman RE. 2004. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 4: 337-48.

2. Girma S, Avanzi C, Bobosha K, Desta K, Idriss MH, Busso P, et al. 2018. Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia. PLoS Negl Trop Dis 12(9): e0006706. doi.org/10.1371/journal.pntd.0006706

Reviewer #3:

The overall recommendation to the authors is they should well acquit themselves with the STROBE guidelines and re-write the paper (I’m assuming the study design is cross-sectional). The paper has an overall message they are trying to communicate but it is not supported by the methods, results and the discussion.

Thank you for your suggestions. We will adhere to the STROBE guidelines where possible.

Abstract

The abstract vaguely reports what the problem is, what is known about the problem and what the authors did to solve the problem.

Thank you for your suggestion. Lines 37-45 in the Abstract straightforwardly lay out what the problem is, what is known about the problem and what we did to solve the problem. “In order to more effectively interrupt the chain of transmission, new strategies will be required to detect those with subclinical disease who contribute to spreading disease. To improve the ability to diagnose leprosy earlier in asymptomatic infected individuals, we examined the combined use of two well-known biomarkers of M. leprae infection, namely the presence of M. leprae DNA by PCR from earlobe slit skin smears (SSS) and positive serum antibody to the M. leprae-specific Phenolic Glycolipid I antigen (anti-PGL-I) from leprosy patients and household contacts living in seven hyperendemic cities in the northern state of Para, Brazilian Amazon.”

However on introducing the bio-markers they should have ideally been written in full. Then abbreviations could be used thereafter (line 40 and 41).

Thank you for your suggestion. The term for earlobe slit skin smears (SSS) is defined in line 40, while the presence of testing M. leprae DNA by PCR, these abbreviations should not have to be further explained. The term for the antibody to the M. leprae-specific antigen, Phenolic Glycolipid I (PGL-I) is now first defined in line 43.

Introduction

The introduction overall fairy tries to highlight what the problem is, what the gaps are and what was done about it. However: (Line 118) The Dr. Marcelo Candia Reference Unit in Sanitary Dermatology (UREMC), I don’t understand the relevance of this clinic. Does it offer the standard testing; is it the Center of Excellence in Para? More clarity maybe needed for the reader.

Thank you for your suggestion. The Dr. Marcelo Candia Reference Unit in Sanitary Dermatology (UREMC) is the Pará state reference laboratory for the diagnosis and treatment of Hansen's disease and offers specialized multidisciplinary clinical care to its users. UREMC was responsible for 19.9% (489/2,548) of the total leprosy notifications in Pará in 2019. People with suspected signs of leprosy from all over the state are referred to UREMC, some who have been misdiagnosed for years. Several leprosy dermatologists, including Dr. Salgado, have been examining leprosy patients and diagnosing them based on clinical signs and symptoms for over 21 years. Follow-up laboratory tests can include bacilloscopy of skin lesions to determine the BI and/or presence of acid fast bacilli as well as determining the anti-PGL-I titer, both which can be confirmatory in the diagnosis and are used to determine the length of the treatment regimen, MDT-PB or MDT-MB. We have modified the text to reflect the importance of this leprosy reference laboratory in the care and treatment of leprosy patients.

Line 144-Line 149 Are actually Methods and Materials in the introduction section. The study design has been highlighted as well as the study description. I think this should be moved to the methods section.

According to the STROBE checklist for cross-sectional studies (von Elm et al. Lancet, 2007), in the Introduction item #2 for Background/rationale states “Explain the scientific background and rationale for the investigation being reported.” These sentences are critically important to establish the rationale for investigating these two particular biomarkers in leprosy patients, healthy household contacts and healthy endemic control subjects that we examined.

Line 149-line 153 this is actually the discussion section in the introduction. This part should be moved to the discussion section.

Again, according to the STROBE checklist for cross-sectional studies, in the Introduction item #3 for Objectives states “State specific objectives, including any prespecified hypothesis”. These sentences are critically important so that the specific objectives are made clear and a hypothesis about the potential risks for individuals who are double positive for both biomarkers is also stated clearly.

Methods

Overall, the methods are poorly presented and hence the study cannot be reproduced. We don’t know what study design was used. A prospective study design was mooted in the introduction but it is not anywhere else. I advise the authors to look up the STROBE guidelines by Von Elm et al.

This the study is about probable diagnostic tests, the authors should tell us if the tests were done in series or in parallel since the results change depending on what is done.

There are some result tables within the methods section.

Thank you for your suggestion. According to the STROBE checklist for cross-sectional in series studies, we have included the following items in the Materials and Methods section and made some selective modifications in the text on the Study Design, Setting, Participants, Variables, Data measurements, Study size, Quantitative variables and Statistical methods. We have now added a new paragraph “Sampling design and methods”, lines 180-202, to further clarify how patients and household contacts were identified and sampled. As far as reproducibility, the ELISA anti-PGL-I assay and RLEP PCR described have been in use by us for over 12 years and both of these tests have been used by leprosy investigators all over the world, they are well-established and easily reproducible in well equipped laboratories. Based on item #10 for Study size the STROBE checklist states “Explain how the study size was arrived at”, Table 1 shows the number of individuals in each group (new leprosy patients, treated leprosy patients, healthy household contacts and healthy endemic controls) and from what cities they came from to answer this question. We have moved Table 1 to the Results section.

Results

I still advise the authors to have a look at the STROBE guidelines. However, it is important to add the socio-demographics of the study subjects unless this is a secondary analysis. The socio-demographics will help us with generalization and also perspective.

The author should use the statistical methods mentioned in the results section to find out if they are significant. Currently all we have are the proportions. We are not sure if they are purely by chance.

Thank you for your suggestion. Sociodemographic aspects are extremely relevant for understanding the epidemiology of leprosy, historically recognized as a disease of poverty1 and sociodemographic data have previously been explored by other groups for meta-analysis studies2. Although this data was not the primary focus of this study, we did collect it from all of the study participants. In previous studies by us, we highlighted potential risk factors that likely lead to higher rates of leprosy in the Amazon region including living in a very high or hyperendemic area for leprosy, poverty, lack of clean water and sanitation in the house, high household density with more than 2 people sleeping per bedroom, poor nutritional status and lack of health care availability.3,4 Certainly the individuals in the leprosy patient, treated patient and healthy household contact groups are more disadvantaged and likely have multiple risk factors among the above, while those in the healthy endemic group who are mostly university students are better off and live in higher socioeconomic areas of the capital of Belem. We have added Table 3 (see below) that includes this sociodemographic information in the Results and a paragraph in the Discussion.

1. Nery JS, Ramond A, Pescarini JM, Alves A, Strina A, Ichihara MY, Penna MLF, Smeeth L, Rodrigues LC, Barreto ML, Brickley EB, Penna GO. 2019. Socioeconomic determinants of leprosy new case detection in the 100 Million Brazilian Cohort: a population-based linkage study. Lancet Glob Health 7: e1226-1236.

2. Pescarini JM, Strina A, Nery JS, Skalinski LM, Andrade KVF, Penna MLF, et al. 2018. Socioeconomic risk markers of leprosy in high-burden countries: A systematic review and meta-analysis. PLoS Negl Trop Dis 12(7): e0006622. doi.org/10.1371/journal.pntd.0006622

3. Barreto, J. G., D. Bisanzio, L.deS. Guimarães, J. S. Spencer, G. M. Vazquez-Prokopec, U. Kitron, and C. G. Salgado. 2014. Spatial analysis spotlighting early childhood leprosy transmission in a hyperendemic municipality of the Brazilian Amazon region. PloS Negl. Trop. Dis. 8(2):e2665. doi: 10.1371/journal.pntd.0002665.

4. Barreto JG, Bisanzio D, Frade MAC, Moraes TMP, Gobbo AR, Guimarães L de S, da Silva MB, Vazquez-Prokopec GM, Spencer JS, Kitron U, Salgado CG. 2015. Spatial epidemiology and serologic cohorts increase the early detection of leprosy. BMC Inf Dis 15: 527 doi 10.1186/s12879-015-1254-8.

As for the application of statistical tests:

Lines 257-258: The statistical differences between groups were calculated by Student’s t-test

Lines 280-282: When individuals in each group were divided into RLEP positive or negative and examined for anti-PGL-I titer, there were no statistical differences between the median O.D. values for RLEP positive versus negative individuals within the patient or HHC groups (Figure 2B).

Discussion

Overall the discussion is about the bio-markers and leprosy in general. Very little effort is made to discuss the results. Their significance and the recommendations if any, the overall recommendation is hinted all over the paper with no possible justification given from the results.

We have expanded the discussion of the results.

Reviewer #4:

1) This is a good study trying to address the issue of transmission in leprosy.

Thank you for this comment. We feel that there are many unknowns about leprosy transmission that unfortunately cannot be answered because M. leprae is not cultivatable.

2) In fact this investigation is trying to look at possible sources of infection in the community and their role in transmission of the disease.

Thank you for this comment. In hyperendemic areas as in all of the cities that we visited in this state, the percentage of people diagnosed by an experienced leprosy dermatologist based on clinical signs and symptoms ranged from 3.4% to 22.3%. These are truly staggering numbers, hundreds of time higher than the nationally reported new case detection rate (NCDR) reported yearly by the Brazil Ministry of Health database (SINAN), which usually relies on passive detection rates (currently around 1.3 new cases per 10,000 population). We have published these results in numerous articles over the years highlighting that active surveillance by us in cities in the state of Pará generally shows a new case detection rate of around 4% in children and 8% in household contacts in this region.

3) In Table 2 for all parameters the author should give number of MB and PB cases in each group

We have revised Table 2 that shows the breakdown of MB and PB cases for the new case and treated groups for each test combination (double positive, single positive and double negative). MB and PB case categories do not apply for the HHC and HEC groups.

PGL+/ RLEP+ PB MB PGL-/RLEP+ PB MB PGL+/RLEP- PB MB PGL-/RLEP- PB MB

n % n % n % n % n % n % n % n % n % n % n % n %

New cases (n = 87) 40 46.0 4 10 36 90 33 37.9 7 21.2 26 78.8 8 9.2 2 25 6 75 6 6.9 3 50 3 50

Treated

(n = 52) 12 23.1 4 33.3 8 66.7 11 21.2 4 36.4 7 63.6 14 26.9 2 14.3 12 85.7 15 28.8 5 33.3 10 66.7

HHC

(n = 296) 46 15.5 35 11.8 107 36.1 108 36.5

HEC

(n = 31) 0 0% 0 0% 7 22.6 24 77.4

Table 3. Demographic information of the four groups studied (new leprosy cases, treated patients, healthy household contacts and healthy endemic controls. Highlights of findings: There are overall slightly more females than males except in the treated group, but the ratio does not seem to be overly skewed. Some observations: it is apparent that all of the HEC have a clean source of drinking water (2 filtered, 29 bottled mineral water) while less than half of the people in the other three groups have access to this, possibly leading to more exposure to water borne pathogens such as amoeba and bacteria causing diarrheal disease. Income for HEC is also skewed to 100% having two minimum salaries or more whereas more than half of people in the other three groups have one minimum salary or less, suggesting conditions of poverty. Education levels for HEC are all at university level, while the other three groups have a majority with no education or only primary level schooling. Food deprivation is between 20% (one in 5) to 34% (one in three) for three groups, while HEC do not experience this problem of hunger at all. The number of people per household is around 3 for HEC while at least 5 for the other three groups, indicating overcrowded conditions.

Number of people per house Type of water used

for drinking/cooking Salary Education (highest grade) Receive governmental support

(%) Median age (range) Sex

(ratio M:F) Food deprivation

(%) Living in urban area

(%)

New cases

(n = 87) 5.5 Not treated: 4

Strained water: 39

Chlorinated: 2

Filtered: 36

Mineral water: 6 ≤ 1 minimum salary: 55

Up to two minimum salary: 21

Up to three minimum salary: 5

Greater than 3MS: 6 No Education: 66

Elementary School: 11

High school: 9

University education: 1 60/87

(69%) 25

(5-81) Female: 52

Male: 35

(0.6 : 1) 18/87

(20.7%) 81/87

(93.1)

Treated

(n = 52) 5 Not treated: 3

Strained water: 21

Chlorinated: 7

Filtered: 12

Mineral water: 5 ≤ 1 minimum salary: 30

Up to two minimum salary: 13

Up to three minimum salary: 4

Greater than 3MS: 3 No Education: 30

Elementary School: 8

High school: 8

University education: 6 35/52

(67.3%) 45

(12-87) Female: 22

Male: 30

(1 : 0.58) 18/52

(34.6%) 42/52

(80.8%)

HHC

(n = 296) 5 Not treated: 15

Strained water: 147

Chlorinated: 32

Filtered: 70

Mineral water: 32 ≤ 1 minimum salary: 172

Up to two minimum salary: 81

Up to three minimum salary: 27

Greater than 3MS: 16 No Education: 106

Elementary School: 127

High school: 52

University education: 11 193/296

(65.2%) 31

(6-79) Female: 160

Male: 136

(0.46 : 1) 102/296

(34.4%) 241/296

(81.4%)

HEC

(n = 31) 3 Not treated: 0

Strained water: 0

Chlorinated:

Filtered: 2

Mineral water: 29 ≤ 1 minimum salary: 0

Up to two minimum salary: 1

Up to three minimum salary: 2

Greater than 3MS: 28 No Education: 0

Elementary School: 0

High school: 0

University education: 31 0/31

(0%) 33

(19-62)

Female: 20

Male: 11

(0.35 : 1)

0/31

(0%) 31/31

(100%)

4) In HHC it will be interesting to know the type of Index cases whether they are PB or MB

This data was not collected.

5) If skin smear BI available in Index case was there a correlation between high BI positivity and HHC positive

Since most of the work was done in the field, the BI of index cases was not assessed. However, we always do a bacilloscopy on leprosy cases attended at the UREMC reference center, resulting in a high correlation between our clinical definition and the BI.

6) Is there a correlation between smear positivity (If available) and positivity in the two tests performed?

We think there is since double positivity is highest in the new case group (46%) while it is markedly reduced in the treated patient group (23.1%) showing the treatment efficacy of MDT.

7) In HHC was there any other household which had many members who were positive for any of the two tests and the author should mention about number of families which were positive.

The size of each household visited varied considerably, from 3 to 12 and sampling at each house relied on who was at home at the time we visited with some household members away or at work. We cited one family as an example where out of twelve individuals with one index case, six other blood related individuals in this household were diagnosed that day and double positivity in this particular family was 75%. Although this was an extreme finding, there were other families where more than one person was diagnosed in the household that showed high rates of being double positive.

8) In HHC compare the positivity in different age groups and was there any correlation with higher age group?

We now have a new table that shows some of the variables for each of the groups (age, sex, income, household density, availability of clean water, etc.) (Table 3).

9) The author needs to mention about the age groups of subjects and among the cases positive for any test in all groups, was there any child case?

The total population evaluated is made up of 466 individuals, of which 92/466 (19.7%) were under 15 yo. Of the 87 new cases, 38/87 (43.7%) were in children under 15 years old. We have added this to the Results section.

10) There is no legend for the Tables mentioned

All tables have a legend that appears just above the tables.

Lines 237-240: Table 1. Operational classification and number of subjects per group and municipality. Characteristics of newly diagnosed leprosy patients, treated leprosy patients, healthy household contacts (HHC) and healthy endemic controls (HEC) from the seven cities surveyed.

Lines 294-297: Table 2. Correlation of RLEP and anti-PGL-I titer within each group. Double positive (PGL-I+/RLEP+), single positive (PGL-I+/RLEP- or PGL-I-/RLEP+) and double negative (PGL-I-/RLEP-) were calculated for each of the four groups.

11) How many HHC who were doubly positive were followed up if followed up and what was the outcome of that follow up?

We intend to do this in the future as we are getting funding for a project for long-term follow-up of contacts who were clinically healthy, but with positive laboratory results (ELISA and/or PCR). The main limiting factor is the lack of resources available for follow-up visits, which is certainly desirable in studies involving neglected chronic diseases like leprosy. Resources for continued leprosy surveillance projects are needed especially in hyperendemic areas, such as the state of Pará, with a territorial area of 1,248,000 km², and the municipalities selected in this project represent each one of the state's macro-regions. Follow-up visits to several of these cities are not possible logistically. For example, Senador José Porfirio is 516 miles away from the capital, Belém, and can only be reached by driving over 18 hours on the Trans-Amazon Highway. Another city, Breves, can only be reached by taking an overnight boat trip by river. In some cities visited we have provided the diagnosis and positivity data to the local health municipality leprosy control coordinators to perform follow-up with these families. Cities that are more easily visited like Acará, Belém and the surrounding metropolitan area (Mosqueiro) and others will be followed up in a future study. We have already published one study and a 2-year follow-up in the city of Castanhal where we found that in a follow-up of 10 individuals who came from anti-PGL-I positive households there would be a 90% chance of diagnosing one new case within 2 years and that individuals in anti-PGL-I positive households had a 2.7-fold higher risk of progressing to disease than those from negative households.1,2

1. Barreto JG, Bisanzio D, Guimarães L de S, Spencer JS, Vazquez-Prokopec GM, Kitron U, Salgado CG. 2014. Spatial analysis spotlighting early childhood leprosy transmission in a hyperendemic municipality of the Brazilian Amazon region. PLoS Negl Trop Dis 8(2): e2665. doi: 10.1371/journal.pntd.0002665.

2. Barreto JG, Bisanzio D, Frade MAC, Moraes TMP, Gobbo AR, Guimarães L de S, da Silva MB, Vazquez-Prokopec GM, Spencer JS, Kitron U, Salgado CG. 2015. Spatial epidemiology and serologic cohorts increase the early detection of leprosy. BMC Inf Dis 15: 527 doi 10.1186/s12879-015-1254-8.

Reviewer #5:

Leprosy is still a public health problem in some countries. In Brazil, the highest prevalence of the disease is observed in North, Northeast and Midwest regions. One important fact regarding the elimination of leprosy is the absence of a gold standard test for diagnosis that results in a significant number of hidden cases. Prophylactic treatment of contacts from multibacillary patients has been evaluated as an effective strategy to control the disease, but the identification of contacts with subclinical infection is important to determine the targets of chemoprophylactic strategy. Here, Silva and colleagues described that contacts that are positive for both RLEP and PGL-1 have latent disease and are at highest risk of progressing to clinical disease. This paper is interesting. However, I have some concerns:

1. Although the experimental design seems correct, authors need to describe the methodology for analyzing the data. How did they calculate sensitivity, specificity and accuracy? Please include in the methodology section.

Thank you for this question. Accuracy is the test ability to identify correctly subjects with leprosy (true positive) and healthy endemic controls (true negative). Sensitivity is the test ability to identify correctly subjects with leprosy (true positive). Specificity is the test ability to identify correctly subjects without leprosy, named as healthy endemic controls (true negative). All concepts described above were based on well-established terms of statistics and contextualized to leprosy disease1,2. Sensitivity, accuracy, and specificity were determined by the exact method of Clopper and Pearson3 using the GraphPad prism 6 (GraphPad Software, California, USA).

1. Gurung P, Gomes CM, Vernal S, Leeflang MMG. 2019. Diagnostic accuracy of tests for leprosy: a systematic review and meta-analysis. Clin Microbiol Infect 25: 1315-1327.

2. Vogels CBF, Brito AF, Wyllie AL et al. 2020. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat Microbiol doi.org/10.1038/s41564-020-0761-6

3. Clopper C and Pearson ES. 1934. The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26: 404-413. doi:10.1093/biomet/26.4.404

2. The "Discussion" is rather long and sometimes confusing. It contains common information that is not directly related to the specific topic of this study.

We will try our best to limit the Discussion directly to our results although we were asked to include additional data in Table 3 with discussion.

3. Several studies have reported that PGL-I is a marker of exposure, but not necessarily of infection. It is not clear in the discussion what is the hypothesis for the different profiles observed in the contacts. For example, PGL-I+/RLEP+ are the latent patients. But what is the hypothesis for PGL-I-/RLEP+? Please discuss it.

These are excellent questions. We would argue that in order to induce a positive anti-PGL-I response there must be an active infection. There is consensus and support in the literature that “the presence of circulating anti-PGL-I among healthy contacts was considered to indicate a subclinical infection”.1 However, not everyone who is infected has a positive antibody response, e.g. only around 20-40% of diagnosed PB patients have a positive titer because their bacillary load is low. Studies indicate that having a positive anti-PGL-I titer increases the risk of succumbing to leprosy by around 6-fold.2 Our hypothesis is that those individuals who are PGL-I-/RLEP+ are also subclinical, they just do not have a sufficient bacillary load to induce an anti-PGL-I response. We are attempting to use quantitative RT-PCR to correlate the bacillary load in earlobe SSS with the anti-PGL-I response. We have added a paragraph into the Discussion to clarify this:

The four different possible combinations of ELISA/PCR results can be cautiously interpreted in several ways. We propose that those individuals without clinical signs and symptoms of leprosy who are PGL-I+/RLEP+ have latent leprosy infection, allowing permissive growth to allow infection of M. leprae in the earlobe and spread to other sites in the skin and an antibody response. These individuals most resemble newly diagnosed patients, the majority of whom are double positive, and thus are at the greatest risk of progressing to disease. Individuals who are PGL-I+/RLEP- are infected but their functional cell mediated immune response has limited bacterial infection in the earlobe, which can evolve to a cure or can progress to paucibacillary disease. PGL-I-/RLEP+ individuals are also infected but the bacillary load has not increased to the point that induces an anti-PGL-I response. These individuals could either control the bacilli or progress to disease if the cell mediated response allows permissive growth and spread. Individuals who are double negative, PGL-I-/RLEP-, may not have been exposed to enough of a bacterial load to infect them or were more resistant to infection. These results could change over time depending on continued exposure to an untreated index case or other factors influencing a robust cell mediated immune response (co-infections, poor nutritional status).

1. Araújo S, Lobato J, Reis E de M, Souza DOB, Goncalves MA, Costa AV, Goulart LR, Goulart IMB. 2012. Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission. Mem Inst Oswaldo Cruz 107 (Suppl I): 55-59.

2. Goulart IMB, Souza DOB, Marques CR, Pimenta VL, Goncalves MA, Goulart LR. 2008. Risk and protective factors for leprosy development determined by epridemiological surveillance of household contacts. Clin Vacc Immunol 15: 101-105.

Submitted filename: Response to reviewers.pdf

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22 Jan 2021

PONE-D-20-19814R1

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies

PLOS ONE

Dear Dr. John S. Spencer, Greetings.

Thank you for submitting your revised manuscript "Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies" to the PLOS ONE for consideration. This is an interesting manuscript in an important area, so the editors sought external views to add to our own. However, the reviewers' comments and recommendations were mixed. The primary concerns are regarding the presentation and feeble language. The quality of the English used throughout your manuscript does not currently meet our minimum requirements, as there are substantial incorrect sentence constructions and grammatical errors throughout, obscuring the message the authors want to convey. After discussing the paper further, the editors felt that the manuscript need significant changes, mostly the sentences rephrasing. We recommend consulting native speakers.

By considering the lengthy review process and considerable improvement in the revised manuscript, we would like to give the authors a final chance to fix the issues. If you can address the points raised by editors and reviewers, we would encourage you to submit a revised manuscript. Once we have received your revised manuscript, a decision will be made, which we expect by 22 February 2021.

If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Dr. Supram Hosuru Subramanya, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

The quality of the English used throughout your manuscript is not acceptable for publication. Many sentences are of 4 to 5 lines; sometimes, it's impossible to understand what authors want to convey. I suggest to break the long sentence and make it concise and clear. The first sentence of the abstract itself confusing. I feel like reading google translated texts. Also, please check the word "Positivity" in the title is suitable over there????.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: Overall, PONE-D-20-19814R1 is better written compared to PONE-D-20-1981. Some of my comments were addressed in the review however; I still have a few more comments.

Introduction

The introduction does clearly explain the problem, but it could be summarized. The flow is good till line 109, however after that what was done should have been summarized.

I understand the importance of UREMC in confirmation of Leprosy disease however, it is just a referral site for the region and the authors should state likewise. However, if the authors must include it in the paper, then they should shift it to the methods section, and then give details about it and what role it plays including the staff who they are and what they do.

Line 145-to Line 154 is actually Methods and Materials and the results in the introduction section. Despite the authors attempt to highlight that they are giving a rationale for the investigation. They actually stated the study design and what they found. If they were tempting to give the hypothesis, there is need for re-wording

“We show that the majority of newly 151 diagnosed leprosy cases with clinical symptoms are positive for both of these 152 biomarkers of infection suggesting the possibility that household contacts who are also 153 double positive may have latent disease and should be carefully monitored through 154 follow-up examinations.”

Methods

The methods and materials have improved however the study cannot be reproduced.

It may be important to state the sampling methods used to get the evaluation areas. The authors do not that it was divided into Rural and Urban. But there is no other mention of any sampling technique and why it was chosen.

It may be important to find out how the authors arrived at 466 individuals with 87 newly infected, 52 former patients, 296 household contacts and 31 healthy endemic controls. Was this also random or it was the available data?

There is completely no mention of how the data was collected or stored prior to being analyzed

Results

Line 255 to 256, in the results section we expect to get the results got from the use of the t-test not what statistical test was used.

Discussion

This may seem like a contradiction on my last review, but I think there is need summarize the key finding of the study and discuss only those..

Reviewer #4: 1) Line 268 _Within the 268 new case group, detection of RLEP was 87.3% (62/71) in MB cases and 68.8% (11/16) in PB cases. When cases were subdivided according to Ridley-Jopling classification for the different forms across the disease spectrum, RLEP amplification was positive in 57.1% (3/7) for the indeterminate form, 80% (4/5) for TT, 87.5% (42/48) for BT, 84.2% (16/19) for BB and 100% for BL and LL (4/4). In addition, four cases of primary neural form (PNL) were diagnosed, and all were positive (100%, 4/4).

Similar results can be documented for anti-PGL-I.

2.) comment no 9) The author needs to mention about the age groups of subjects and among the cases positive for any test in all groups, was there any child case?

The total population evaluated is made up of 466 individuals, of which 92/466 (19.7%) were under 15 yo. Of the 87 new cases, 38/87 (43.7%) were in children under 15 years old. We have added this to the Results section

However, we did not find the above results mentioned in the revised manuscript. Also, findings on the number of children in new, treated and HHC group, will give information about the transmission.

3) Line 337 - For this reason, we have been using

338 less invasive methods, namely taking samples of blood and earlobe SSS.

(In response to our comment no 5) If skin smear BI available in Index case was there a correlation between high BI positivity and HHC positive

Since most of the work was done in the field, the BI of index cases was not assessed.

However, we always do a bacilloscopy on leprosy cases attended at the UREMC reference center, resulting in a high correlation between our clinical definition and the BI. )

We suggest that to obtain the BI, SSS from all earlobe, forehead and active lesion can be taken on slide, heat fixed, stained by ZNCF and quantified under oil immersion. There is no need of taking invasive punch biopsy.

Reviewer #5: Authors have adequately addressed my comments raised in a previous round of review and the manuscript is now acceptable for publication.

**********

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Reviewer #1: Yes: Azin Ayatollahi, MD

Reviewer #3: Yes: Michael Kakinda

Reviewer #4: No

Reviewer #5: Yes: Roberta Olmo Pinheiro

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Submitted filename: PONE-D-20-1981 R1 Review.docx

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25 Feb 2021

The file "Response to Reviewers" has been attached addressing all questions.

Submitted filename: Response to Reviewers.doc

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19 Mar 2021

PONE-D-20-19814R2

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies

PLOS ONE

Dear Dr. John S. Spencer,

Thank you for submitting your revised manuscript " Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies" to PLOS ONE. Peer review of your manuscript is now completed. Based on these reports and my own assessment as Editor, I am pleased to inform you that it is potentially acceptable for publication in PLOS ONE once you have carried out some essential revisions suggested by a reviewer. Currently, reviewer-3 has raised few genuine concerns which need to be addressed. If you can't address all the issues, I recommend discussing those points in the discussion part of the manuscript or state as a limitation of the study.

Some manuscripts require many rounds of revisions, so this is a standard but necessary stage of the editorial process. Therefore, I invite you to revise your paper, considering the points raised during the review process.

Please go over your manuscript text and ensure that it is written concisely and clearly. At the same time, we ask you to make sure your manuscript complies with our format requirements detailed on the journal website.

Please submit your revised manuscript by 03 April 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Supram Hosuru Subramanya, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: Overall, PONE-D-20-19814R2 is better written compared to PONE-D-20-1981 R1. Some of my comments were addressed in the review however; I still have a few more comments.

Introduction

The introduction does clearly explain the problem, but it could be summarized. The flow is good till line 109, however after that what was done should have been summarized.

Line 145-to Line 154 is actually Methods and Materials and the results in the introduction section. Despite the authors attempt to highlight that they are giving a rationale for the investigation. They actually stated the study design and what they found. If they were tempting to give the hypothesis, there is need for re-wording

Methods

The methods and materials have improved however the study cannot be reproduced.

It may be important to state the sampling methods used to get the evaluation areas. The authors do not that it was divided into Rural and Urban. But there is no other mention of any sampling technique and why it was chosen.

It may be important to find out how the authors arrived at 466 individuals with 87 newly infected, 52 former patients, 296 household contacts and 31 healthy endemic controls. Was this also random or it was the available data?

There is completely no mention of how the data was collected or stored prior to being analyzed

Results

Line 255 to 256, in the results section we expect to get the results got from the use of the t-test not what statistical test was used.

Discussion

This may seem like a contradiction on my last review, but I think there is need summarize the key finding of the study and discuss only those.

Reviewer #4: It is observed that the comments made earlier in the review has been addressed satisfactorily. The comments made have been responded to and have been also incorporated in the manuscript. As the comments have been addressed and incorporated in the manuscript the issues related have been clear and it therefore helps to relate and convey the objectives and purpose of the study and the results arising out of the study which are very important and have epidemiological implications.

Reviewer #5: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Azin Ayatollahi, MD

Reviewer #3: Yes: Michael Kakinda

Reviewer #4: No

Reviewer #5: Yes: Roberta Olmo Pinheiro

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-20-1981 R2 Review.docx

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1 Apr 2021

We have responded to all comments from Reviewer #3 in Response to Reviewers with notations in the Revised Manuscript with Track Changes as necessary.

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19 Apr 2021

PONE-D-20-19814R3

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies

PLOS ONE

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27 Apr 2021

We have added a paragraph "Limitations of the Study" after the Discussion as requested by the editor.

Submitted filename: Response to Reviewers.doc

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30 Apr 2021

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies

PONE-D-20-19814R4

Dear Dr. John S. Spencer,

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Supram Hosuru Subramanya, Ph.D.

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PLOS ONE

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Reviewers' comments:

5 May 2021

PONE-D-20-19814R4

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

Dear Dr. Spencer:

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PLOS ONE

Table 2

Correlation of RLEP and anti-PGL-I titer within each group.

	PGL+/RLEP+		PB		MB		PGL-/RLEP+		PB		MB		PGL+/RLEP-		PB		MB		PGL-/RLEP-		PB		MB
	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
New cases (n = 87)	40	46.0	4	10	36	90	33	37.9	7	21.2	26	78.8	8	9.2	2	25	6	75	6	6.9	3	50	3	50
Treated (n = 52)	12	23.1	4	33.3	8	66.7	11	21.2	4	36.4	7	63.6	14	26.9	2	14.3	12	85.7	15	28.8	5	33.3	10	66.7
HHC (n = 296)	46	15.5					35	11.8					107	36.1					108	36.5
HEC (n = 31)	0	0%					0	0%					7	22.6					24	77.4

Double positive (PGL-I+/RLEP+), single positive (PGL-I+/RLEP- or PGL-I-/RLEP+) and double negative (PGL-I-/RLEP-) were calculated for each of the four groups. The numbers of PB and MB cases are shown for the new case and treated case groups.