Abstract
Leprosy, also known as Hansen’s disease, is a chronic infectious disease caused by the intracellular pathogen Mycobacterium leprae. This mutilating and highly stigmatized disease is mainly characterized by dermatoneurological signs and symptoms. The upper respiratory tract is considered the main portal for entry and exit of M. leprae. Nevertheless, there is still a lack in full understanding of the transmission pathways of M. leprae. Disease control is therefore a serious challenge, leading to continuing endemicity of leprosy in some regions of the world, including the Union of the Comoros. This is an island group located in the Indian Ocean, between Madagascar and Mozambique. Despite decades of a solid leprosy control program, the Comoros islands of Anjouan and Mohéli continue to be hyperendemic for leprosy, while there is no direct evidence of endemicity on the third island, Grande Comore. The high proportion (>38%) of disease in children indicates that recent transmission is a major driver of the persistent endemicity, and that present control measures are not sufficient. The low proportion (2.4% on average in last 10 years) of grade II disabilities in newly diagnosed cases indicates that case detection is early. The unexplained high endemicity in the Comoros requires new insights in the transmission of the disease and the islands provide a unique geographical and epidemiological situation, studying the broader scope of transmission. This thesis will approach the issue from two different sides, investigating the possibility of involvement of ticks in the transmission of leprosy from a zoonotic reservoir, as well as assessing the quality of DNA extracts of historical skin biopsy samples as a source of information in transmission research.
Ticks play an important role in the transmission of various infectious diseases, due to their low host specificity and worldwide distribution. Moreover, transovarial transmission of M. leprae in artificially-fed adult female ticks has already been demonstrated by da Silva Ferreira et al. (2018), together with the ability of infected tick larvae to inoculate viable M. leprae bacilli during blood-feeding on a rabbit and the ability of M. leprae to grow within cells of an embryo-derived tick cell line (1). In 2010, wild ticks (Rhipicephalus microplus, Amblyomma variegatum, and Rhipicephalus appendiculatus) were collected from animals (cattle and goats) across the three islands of the Comoros. According to Yssouf et al. (2011), the tick population differs over the three islands (2). This master dissertation will determine, in collaboration with the research group of Dr. Yssouf, whether M. leprae DNA is present in a selection of the collected ticks. If we would confirm the presence of M. leprae DNA in ticks from the islands of Anjouan and Mohéli, and the absence of M. leprae DNA in the ticks from Grande Comore, this would suggest that ticks are involved in the transmission of leprosy in the Comoros.
As the long incubation period of the disease complicates research on leprosy transmission mechanisms, historical skin biopsies collected on the Comoros in the early 2000s and stored at the Institute of Tropical Medicine (ITM) in paraffin, could be a unique addition to the transmission study that is currently being conducted by the ITM in the Comoros, bridging the incubation gap to identify chains of transmission. The study of the ITM will extract and examine mycobacterial DNA out of sampled skin biopsies by targeted next generation sequencing techniques. However, extracting good quality DNA from paraffin embedded tissue is a challenge. This thesis will validate a technique, which, starting from paraffin embedded tissue, extracts DNA of good quality to perform the targeted NGS, used in the ongoing molecular epidemiological study.
1. State of the art literature
1.1 Leprosy in the world
Leprosy, caused by Mycobacterium leprae, has been known for centuries and is still endemic in various parts of the world, with in 2017, 210,671 new cases reported from 150 different countries. The number of new cases has been stabilizing since 2006, but the number of grade II disabilities (G2D) is going down. The South East Asian region (mainly India and Indonesia), together with Brazil account for 80.2% of all the new cases and are seen as the highest burden countries. In 2017, about 9% of all newly detected cases globally were children (16,979 new child cases), indicating continued disease transmission (3,4).
1.2 The disease
Mycobacterium leprae is an intracellular, slow growing bacillus (dividing every 12-14 days), which is grown in mouse footpads or the nine-banded armadillo, since cultivation in artificial media is not possible. This mycobacterium multiplies best at temperatures around 30°C (5) and therefore mainly infects Schwann cells in peripheral nerves and macrophages in the skin, but also eyes and (nasal) mucosa, leading to a progressive chronic disease with a spectrum of clinical symptoms in a small minority of those who are exposed or infected. These clinical manifestations can appear after a long incubation period of 6 months up till 20 years and are mainly depending on the cellular immune response of the host, rather than on the infectiousness and the multiplication speed of the bacteria. Therefore the susceptibility to develop leprosy is mainly determined by the host immune response, but other factors, like genetics and nutrition, have been suggested to play a role as well. The proportion of the human population that shows this susceptibility is fairly low, only 3-5% (6). A classification of the spectrum was made by Ridley and Jopling in 1966, with tuberculoid (TT) leprosy on one end of the spectrum, in which the disease is limited by a strong cellular response (CMI), a T helper 1 (Th1) type response, and lepromatous (LL) leprosy on the other end, which is characterized by a strong humoral response (T helper 2 type (Th2)), with the production of inefficient antibodies, and a weak cellular response (7). In the centre of the spectrum the unstable borderline forms of the disease are situated, divided into borderline tuberculoid (BT), borderline borderline (BB) and borderline lepromatous (BL). An additional indeterminate class is described, to classify relatively nonspecific perineural infiltrates without sufficient acid-fast bacilli to classify them. Classification of a case to a specific class entirely relies on the clinical representation of the patient. The clinical signs can range from the milder symptoms in TT leprosy, being single or few hypopigmented or erythematous skin lesions, with clear, often elevated borders, sensory loss and possible nerve thickening; to the severe LL form showing multiple smaller, symmetric lesions with indefinite borders, mostly no loss of sensation and no nerve thickening. In LL leprosy the skin lesions can also be plaques or nodules (lepromas) and can cause leonine facies and eyelash loss (madarosis). In this advanced stage other disabilities can develop as well. The borderline forms present symptoms intermediate between the polar forms. The WHO handles a simplified classifications method based on the bacterial index (BI), dividing cases in paucibacillar (PB), when a negative slit-skin smear is seen or only up to five skin lesions are present and multibacillar (MB), with a positive slit-skin smear (SSS) or more than five skin lesions (8–10). During the course of the disease or the recovery phase, two types of reactions can occur, which result from shifts in the balance between host immune response and the pathogen. These reactions are able to cause an improvement (moving towards the tuberculoid side) or worsening (moving towards the lepromatous side) of the clinical picture, which can result in neurological disabilities and morbidity (10–12).
1.3 Diagnosis
Because of the possible sequelae and physical disabilities associated with leprosy, an early diagnosis is important. Diagnosis of leprosy is based on the presence of at least one of three cardinal signs, being definite sensory loss of a hypopigmented or erythematous skin patch, enlarged or thickened peripheral nerves with loss of sensation and/or muscle weakness in the ones supplied by that nerve, and/or a positive result in a slit-skin smear. Recently, also the treatment is included in defining leprosy cases, as a case is seen as a person having at least one of the cardinal signs and has not completed the full course of chemotherapy, this way completing treatment is considered equal to cure (3,4,12).
1.4 Treatment and prevention
Since 1982 multidrug therapy (MDT) for leprosy is available, more specifically a 3-drug regimen consisting of rifampicin, dapsone and clofazimine (8). This MDT is used for all leprosy patients, during a period specific for the form present: in paucibacillary patients the treatment is given for 6 months, whereas multibacillary patients are treated for 12 months (13–15). In cases of rifampicin resistance, it is recommended to treat those patients daily with at least two second-line drugs (clarithromycin, minocycline or a quinolone) and clofazimine for 6 months and subsequently a daily clofazimine administration together with one of the second-line drugs for an additional 18 months (16,17). Regarding the prevention of leprosy, there is a need for innovative approaches to tackle the stagnant new case detection rate (NCDR). Despite the fact that several studies have shown that Post Exposure Prophylaxis (PEP) with single dose rifampicine (SDR) reduced the incidence over a two-year follow-up period, there is no consensus on who to target with PEP. The WHO argues for further evaluation on PEP administration strategies (18–22). Immunoprophylaxis by a repeated dose of the Bacillus Calmette-Guérin (BCG) vaccine for contacts of leprosy patients is also suggested by some papers, as it might have a protective effect (23–26).
1.5 Detection of M. leprae
The detection of M. leprae in different samples can be done with a few specific methods, since in vitro isolation is not possible. The method that is most frequently used in the confirmation of leprosy diagnosis with laboratory tests remains a biopsy of a skin lesion and a histopathological examination, detecting acid-fast bacilli within the nerves (17). For this purpose a Fite stain is recommended, since M. leprae is only weakly acid-fast and therefore the standard acid-fast Ziehl-Neelsen staining might be negative, unless performed with a short decolouration (27). Also slit skin smears can be used, which requires a shallow incision into the dermis on six standard sites on the body (both ears, knees and elbows), from which dermal fluid is collected and smeared onto a microscopic slide. Subsequently, the bacillary index is measured, examining using microscopy after staining. This method might reduce the variation seen in biopsies and can be done in resource limited settings. On the other hand, the sampling is not standardized and no tissue response, as can be seen in biopsies, can be detected. Immunological tests are not (yet) helpful in diagnosis or management of leprosy, due to the spectral variability in the immune response in leprosy. Because of this wide variation most tests only show a positive response in either lepromatous (antibody detection) or tuberculoid leprosy (CMI detection by mononuclease cell culture) (10). Various nucleic acid amplification techniques like PCR can be useful for the identification of the pathogen, but as a diagnostic confirmation, it usually appears not to be more sensitive than histological examinations using Fite staining and is prone to false positive results, due to possible environmental contaminations etc. However, in suspected early leprosy and paucibacillary patients, which are difficult to detect clinically or histologically, PCR detection could be useful, provided that good practice and control of the PCR conditions are applied (28,29). To counter the problems of conventional PCR, semi-quantitative PCR (qPCR) and reverse transcriptase-based PCR (RT-PCR) systems were developed. Different targets genes were studied, including various M. leprae proteins and the M. leprae-specific repetitive element (RLEP), with the latter recently demonstrated to be a highly specific target for M. leprae detection by Braet et al. (2018) (30). Martinez et al. (2011) showed that RLEP qPCR is a sensitive assay that could be used as a diagnostic test for detecting a M. leprae infection before the clinical symptoms show (31). Several different clinical samples can be tested for M. leprae DNA, like skin, hair bulbs, blood, saliva etc., but also environmental or vector samples can be examined by the use of nucleic acid amplification (29). Therefore in this study, RLEP qPCR will be used to examine the presence of M. leprae DNA in tick samples and in historical skin biopsy samples, as this is a highly specific and sensitive assay.
1.6 Route of transmission
The understanding of the transmission mechanisms of leprosy is still incomplete. M. leprae has lost its own energy metabolism due to its reductive evolution, wherefore it can only grow intracellularly in vivo. Therefore, in laboratory settings, due to its inability to be cultured in vitro, passage through mouse footpad is required. This, together with the long incubation time of leprosy, makes it difficult to study the transmission of the disease. At the moment it is known that leprosy is mainly transmitted in close contact between a multibacillary infected patient and a susceptible person (32). However, frequently newly diagnosed patient cannot be linked to an index case among their contacts. Although paucibacillary and sub-clinical multibacillary patients might also be involved in transmission, the multibacillary patients are considered the main reservoir of leprosy and can shed the bacilli via nasal secretions and droplets or, to lesser extent, via the skin. The main route of entry is considered to be the nasal mucosa, although entry via the oral cavity or the skin are not ruled out (4,32,33).
1.6.1 Non-human reservoirs
As human-to-human transmission alone cannot explain the presence of leprosy in areas without reported new cases and in patients with no previous contact with a leprosy patient, other reservoirs have been (and are being) studied. To date the only known non-human reservoir is the nine-banded armadillo in the southern states of the USA and Brazil (4,32–34). Other animals have been suggested as possible reservoirs, like the red squirrels in the UK and some non-human primates (32–34). Water, soil and also amoebas might form an environmental source for leprosy (4,32–35) and the possibility of a role for insects cannot be ruled out either (32,33,36). Different studies have suggested a possible role for various insects and arthropods, like mosquitos, flies, bedbugs and recently also the kissing bug, as a carrier (32,36–40), but the actual transmission itself and the subsequent development of the disease has not been proven.
1.6.2 Ticks as possible vectors/reservoirs
Ticks are small arachnids, belonging to the Parasitoformes. All ticks are obligate hematophagous ectoparasites and most of them (about 90% of all tick species) are host specific and do not feed on humans, domestic animals or livestock. Nevertheless, the remaining 10% do feed on animals that are close to humans and occasionally on humans themselves, as an accidental host. These ticks are of medical and economic concern, since many play an important role in the transmission of various infectious diseases (e.g. Borrelia burgdorferi, Rickettsia rickettsia, Francisella tularensis etc.) (41). Due to their low host specificity and worldwide distribution, ticks can be an interesting target in the research for the mechanisms behind leprosy transmission (42). Moreover, some potential non-human reservoirs of leprosy, described above, like the nine-banded armadillo and the red squirrel, are frequently infested by ticks (43–47). In addition, da Silva Ferreira et al. (2018) already demonstrated the occurrence of transovarial transmission of M. leprae in artificially-fed adult female ticks, the ability of infected tick larvae to inoculate viable M. leprae bacilli during blood-feeding on a rabbit and the ability of M. leprae to grow within cells of an embryo-derived tick cell line (1).
1.7 Leprosy in the Comoros
In 2000, Leprosy has been eliminated as a public health problem globally, as the prevalence reduced to under one per 10,000 population. However, for individual countries this target is not reached yet, with the Comoros having a 5-10 times higher incidence than the WHO elimination rate (15). The Union of the Comoros, located in the Indian Ocean between Mozambique and Madagascar, consists of three islands (Grande Comore, Anjouan and Mohéli) and has approximately 800,000 inhabitants. With 429 new cases of leprosy in 2017, resulting in a prevalence of 4.58 per 10,000 population, the Comoros islands are hyperendemic for leprosy, despite a solid leprosy control program with a highly effective treatment, including active case finding and follow-up on therapeutic outcome. Anjouan and Mohéli account for the vast majority of the cases (369 and 58 resp.), while on the largest island Grande Comore only two leprosy cases were detected in 2017. Also the number of child cases is very high compared to the WHO AFRO region (over 38% compared to 10%). The grade II disabilities rate at time of diagnosis, on the other hand, is much lower than the rate in the region (2% compared to 14% in the region), indicating early case detection (48,49).
The island of Anjouan has served as a leprosarium in the past and still shows a remarkably high prevalence of leprosy, with more than 9 per 10,000 population and the highest percentage of child cases in the world (approximately 40%). Aside from the possibility that the present population is more genetically susceptible, also poverty, malnutrition and the density of the population, might favour transmission of the disease. The Comoros’ health ministry is applying a solid leprosy control program since 1980, with the National Program to control Tuberculosis and Leprosy (NTLP) (49). Yet the islands remain hyperendemic. Therefore, further upscaling of the active case finding efforts and an even more systematic organisation will be needed, using modern technologies and considering prophylactic treatment of contacts. An epidemiological study by Hasker et al. (2017) could not demonstrate clear patterns in the clustering in time and space, indicating again the lack of knowledge about the leprosy transmission (49). Discoveries in the transmission pathways could lead to very useful insights in the identification of the inhabitants at higher risk of developing the disease (48–50). Due to its specific geographical and epidemiological situation, the Comoros could be an interesting area to study the transmission and the presence of M. leprae in ticks. Yssouf et al. (2011) already studied the tick populations in animals (cattle and goats) on the three islands, in light of the severe East Coast Fever epidemic on the Comoros islands of 2004 (2). In 2010, three hard tick species were collected from the animals, namely Amblyomma variegatum, Rhipicephalus microplus and Rhipicephalus appendiculatus, showing a different distribution over the three islands, with R. appendiculatus only present on Grande Comore. The one host tick R. microplus, also called the cattle tick, was the most common species found on the three islands and is a vector in bovine babeiosis, anaplasmosis and spirochaetosis in cattle (51). R. appendiculatus (The brown ear tick) and A. variegatum (The tropical bont tick) are both three host ticks, transmitting respectively East Coast Fever in cattle, and heartwater, bovine ehrlichiosis and benign bovine theileriosis (51). Moreover, different Amblyomma spp. were shown to infest armadillo species (43,46,47). Therefore, in collaboration with the research group of Dr. Yssouf, 133 ticks from Anjouan, 129 ticks from Mohéli and a selection of the ticks collected on Grande Comore, will be examined for the presence of M. leprae DNA. The ones from the non-endemic Grand Comore will be investigated as control ticks. The presence of M. leprae DNA in ticks from the islands of Anjouan and Mohéli, and the absence of M. leprae DNA in the ticks from Grande Comore, could suggest that ticks are involved in the transmission of leprosy in the Comoros.
Additionally, the Institute of Tropical Medicine (ITM) is currently conducting a study in the Comoros in which skin biopsies of leprosy patients are collected, for extracting and examining the mycobacterial DNA residing in the samples. With the help of targeted next generation sequencing, genotypes are generated and transmission chains can be identified. The ITM first collaborated with the Comoros (National Leprosy Control Programme of the Comoros) on leprosy research in the 1980’s with Dr. Stefaan Pattyn (50). During the early 2000s, under his leadership, skin biopsies were collected from leprosy patients and stored in paraffin. As the long incubation period of leprosy impedes transmission studies, this unique historical collection of skin biopsies could be a source for bridging this period. However, extracting good quality DNA from paraffin embedded tissue remains a challenge. Formalin fixation causes the formation of crosslinks between nucleic acids and proteins, which might impede the extraction of DNA and RNA. Also fragmentation of the DNA strands and chemical modifications are common (52). This thesis will therefore validate a technique to extract DNA from paraffin embedded tissue that is of good quality to perform targeted NGS.
The subjects handled in this thesis fit into the framework of the WHO Global Leprosy Strategy 2016-2020, which aims amongst others to keep on reducing the burden before 2020 and especially to reduce the number of children with leprosy-affected deformities (53). Therefore, more insight in the transmission chains of the disease is necessary, to identify unsuspected routes of transmission and to enable the set-up of interventions to stop transmission. This would also enable to define risk-populations, which could aid in the application of control programs by authorities and for example assigning target populations for prophylactic treatment. This thesis approaches the transmission question from two different sides, investigating the potential of ticks as a M. leprae reservoir in transmission and evaluating the quality of DNA extracts from historical samples holding great promise in bridging the incubation period and thereby possibly revealing the source of transmission of patients in the present time that happened several years ago.
2. Hypothesis and objectives
There is still insufficient understanding of how transmission of leprosy works. Therefore, this thesis will contribute to filling the knowledge gap with two different aims.
2.1 First aim: Possible role of ticks in leprosy transmission on the Comoros
As armadillos are the only known non-human reservoir, the transmission in the Comoros is presumably human-to-human. However, the persistent high NCDR despite a solid leprosy control program urges to investigate other potential reservoirs of M. leprae. Since ticks are involved in the transmission of several infectious diseases, we hypothesize that ticks play a role in leprosy transmission and that, as leprosy is only hyperendemic on two of the Comoros islands, M. leprae DNA would only be present in ticks collected on Anjouan and Mohéli. To investigate this hypothesis two objectives were set:
- Validate the DNA extraction method for ticks, in order to be able to accurately examine the presence of M. leprae DNA in the sampled ticks.
- Determine the presence of M. leprae DNA in ticks collected on the three islands of the Comoros with the RLEP qPCR assay in order to assess the hypothesized difference in between the islands.
2.2 Second aim: M. leprae DNA extraction from FFPE preserved leprotic skin biopsies of the Comoros
Formalin-fixed paraffin-embedded (FFPE) historical samples could be of great use in future transmission studies, if the M. leprae DNA that can be extracted from them is of a good quality. Therefore in this thesis the DNA extraction methods using the Maxwell 16 FFPE PLUS Tissue LEV DNA purification Kit (Promega, USA) will be optimized, validated and the quality of the obtained M. leprae DNA will be assessed. Therefore, the following objectives:
- Validate the DNA extraction from FFPE skin biopsies.
- Detect M. leprae DNA in FFPE preserved skin biopsies.
3. Material and method
3.1 First aim: Possible role of ticks in leprosy transmission
3.1.1 Sample population
In the frame of a cattle surveillance program by CRVOI (Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Océan Indien) ticks were collected by the research group of Dr. Yssouf, at randomly selected sampling sites on the three islands of the Union of the Comoros (2). This was done during the rainy season, from January to February 2010. Multiple farms on the three islands of the Comoros were randomly chosen to sample five animals (cows or goats) per farm. Based on a rapid visual screening, highly tick-infested animals were sampled. When more than 150 ticks were present on one animal, sequential collection of up to 20 ticks was done from feet, legs, anus, scrotum, udder, neck, ears and eyes. In case no highly infested animals were present on the farm, five animals with a lower infestation were screened and all ticks were sampled. On farms with less than five animals, animals from neighbouring farms were examined as well.
From the resulting sample collection of Yssouf et al. (2011), 133 ticks from Anjouan, 129 ticks from Mohéli, and 562 ticks collected on Grande Comore were sent over to the ITM with the collaboration documented in a material transfer agreement (2). All available ticks of Anjouan and Mohéli and a selection of 262 ticks of Grand Comore will be examined for the presence of M. leprae DNA. Three different tick species were identified in those samples, Amblyomma variegatum, Rhipicephalus microplus and R. appendiculatus, with the latter only appearing among the samples of Grande Comore.
3.1.2 Power analysis
Given a sample size of 262 ticks per group, being Anjouan and Mohéli combined, and Grand Comore, a 3% difference in the proportion of ticks positive for M. leprae DNA between both groups can be identified with a power of 0.80 (ClinCalc, USA).
3.1.3 Morphological classification of ticks
Classification of the sampled ticks will be done morphologically based on the guide of Walker et al. (2003) (51). The stages of the ticks will be defined first, based on number of legs, size and colour. Presence of a scutum (female) or conscutum (male) will be used to determine the sex of the ticks. Decision on the species will be based on following characteristics: overall body shape, scutum shape and colour, colouration on the legs, length and shape of the mouthparts, festoons and the posterior grooves. The classification of the tick species will be compared to the information provided by Yssouf et al. and in case of doubt, their expertise will be invoked.
3.1.4 DNA extraction
Half of every tick will be used. After submerging the ticks in 500µl phosphate buffered saline (PBS) in a gentleMACSTM M tube, they will be dissociated using the RNA-2 program on a gentleMACSTM Dissociator (Miltenyi Biotec, Germany). A tick suspension will be obtained. After treating with an in-house lysis buffer, a DNA extraction procedure will be done using the Maxwell 16 FFPE PLUS Tissue LEV DNA purification Kit (Promega, USA), according to the manufacturers’ protocol.
3.1.5 Molecular detection of M. leprae DNA in ticks
For the molecular detection of M. leprae DNA in the tick samples, an RLEP qPCR assay will be used. Targeting 35 of the 37 RLEP copies present in the genome, this method shows to be highly sensitive and specific (31). In this study, another platform will be used compared to the one used in Martinez et al. (2011), namely the StepOnePlusTM qPCR cycler (Applied Biosystems, USA). An internal positive control (IPC) that permits identification of inhibiting factors present in the prepared specimen will be included in this qPCR assay. The RLEP and inhibition control-specific oligonucleotides are labelled with a reporter dye (FAM- and Yakima Yellow®-fluorescent (YY) respectively) and a quencher dye. The amplification of RLEP DNA and internal positive control DNA are measured independently at different wavelengths, respectively FAM and VIC channel, permitting independent identification of RLEP DNA and inhibition control DNA. To allow for quantification of the DNA present in the samples, a serial dilution series of the reference M. leprae strain NHDP will be added. Also positive and negative DNA controls/extraction controls will be included during the whole process, to enable ruling out contamination.
3.1.6 Data analysis
As thresholds and baselines for Cq are automatically set, the StepOne software V2.3 will generate Cq values. The signals obtained for the included controls will be checked (see Table 1) before interpreting the sample results, since particular conditions must be met. In case one of the controls shows a discordant result, appropriate action will be taken. An RLEP-signal (FAM-signal) of Cq value < 40.00, obtained for a sample, demonstrates the presence of M. leprae DNA. Accordingly, if a Cq value ≥ 40.00 is detected or the FAM-signal is undetermined and the YY-signal (IPC) is present, the sample does not contain DNA of M. leprae. An undetermined FAM-signal or one with Cq value ≥ 40.00 and an absent YY-signal can indicate the presence of inhibiting factors or the failure of the assay steps. In this case the DNA extraction and/or qPCR must be repeated. The proportions of tick samples with amplification for M. leprae DNA will be calculated for the endemic islands (Anjouan and Mohéli) and the non-endemic island of the Comoros (Grand Comore).
Controls Result RLEP (FAM) Result IPC (VIC)
Negative DNA control – +
Negative extraction control – +
Positive extraction control + +
Positive M. leprae DNA control + +
Table 1: Internal quality controls for DNA extraction and amplification
3.1.7 Statistical analysis
R version 3.3.2 for Windows (The R foundation, Vienna, Austria) will be used to perform a Chi-squared test. A significance level of 0.05 will be applied.
3.1.8 Limitations of the study
The stadia of the sampled ticks (larvae, nymph or adult) remain to be determined in the study. The stadia that mainly parasitize humans are the larvae and nymphs (42). Therefore, if those stadia are underrepresented in the sample population or all ticks that come out positive for M. leprae DNA are solely adult ticks, the inference regarding the role of ticks in the transmission of leprosy to humans is less strong. It would however not exclude ticks as a vector in leprosy transmission, since it was already demonstrated that transovarial transmission of M. leprae is possible (1). Also, in light of leprosy transmission, other ticks feeding on other non-human hosts might be a more interesting target to investigate. In our proposed study ticks have been sampled from cattle and goats of different farms on the three islands. However, in Brazil, which is hyperendemic for leprosy, cattle (Nelore, Bos indicus) do not seem to be involved in leprosy transmission, as they do not present characteristic lesions. Nevertheless, ticks in this study could have been collected from a different breed of cattle, which might have a different susceptibility to leprosy. As the suggested non-human leprosy reservoirs in the Americas and UK are mostly small animals, a tick that feeds on smaller animals as well as on humans, like R. sanguineus, could be a more interesting target to study (42,51). Hence, if the sampled ticks of this study come out negative for M. leprae DNA, a prospective study in collaboration with the entomologist of the Comoros will be initiated to collect and examine R. sanguineus ticks.
3.2 Second aim: M. leprae DNA extraction from FFPE preserved leprotic skin biopsies of the Comoros
3.2.1 Sample population and rationale
Skin biopsy samples taken from leprosy patients on the Comoros between 2003 and 2006 were fixed with formalin and preserved in paraffin. Based on the available histological data and clinical data about these patients, 20 FFPE skin biopsies from multibacillary patients, presumably highly positive for M. leprae, will be selected to validate the extraction technique. Once validated, the remainder of the samples will have DNA extracted for RLEP qPCR analysis. Ultimately, beyond the scope of this thesis, this will allow to extend molecular epidemiological analyses to these historic samples, to identify chains of leprosy transmission.
3.2.1 DNA extraction
The skin biopsies will be submerged in 500µl phosphate buffered saline (PBS) and tissue dissociation will be done using the RNA-2 program on a gentleMACSTM Dissociator (Miltenyi Biotec, Germany). The obtained tissue suspension will be treated with an in-house lysis buffer and a DNA extraction procedure will be done using the Maxwell 16 FFPE PLUS Tissue LEV DNA purification Kit (Promega, USA), according to the manufacturers’ protocol.
3.2.2 Quality control and molecular detection of M. leprae DNA in skin biopsies
To assess the quality of the DNA extracted from 20 sampled biopsies, the Infinium FFPE QC Kit (Illumina, USA) will be used. Therefore, the DNA concentration of the samples will be determined using the Qbit assay and will be diluted to 1ng/µl. Hereafter, the Infinium HD FFPE QC assay will be executed as prescribed by the manufacturer, using the StepOnePlusTM qPCR cycler (Applied Biosystems, USA). The kit provides a quality control template (QCT) and a no template control (NTC) will be included.
The same RLEP qPCR assay as used in the M. leprae DNA detection in the ticks will be used on those skin biopsy suspensions that are shown to be of sufficiently high quality and quantity.
After validation, the remainder of the biopsies will be processed following the same approach.
3.2.3 Data analysis
As thresholds and baselines for Cq are automatically set, the StepOne software V2.3 will generate Cq values. For the quality control assay, the signal for NTC will be checked first. No or very inefficient amplification should be seen. After removing any replicates presenting a divergent Cq value (with more than half a unit), an average Cq value will be determined for both the FFPE samples and the QCT sample. Then, the difference between the average QCT Cq value and the average sample Cq value (Delta Cq) is calculated for each sample. Good quality samples are considered the ones with a Delta Cq value below five.
Data analysis of the RLEP qPCR assay will be done as described above in 3.1.6, resulting in a classification as M. leprae DNA positive or negative for every sample. The ultimate goal is to retrospectively extend ongoing molecular epidemiological analyses of M. leprae on the Comoros with the historical collection, to allow for the identification of chains of transmission of this very slow infectious agent.
4. Potential alternative research strategies
As mentioned above, other ticks like R. sanguineus could be interesting targets to investigate regarding the role of ticks in leprosy transmission. However, also the absence of M. leprae DNA in the studied ticks can be informative. It might indicate that on the Comoros, cattle and goats are not involved in the transmission of M. leprae and/or that the three sampled tick species are not suited to carry the mycobacteria. Nevertheless, it does not exclude that ticks are playing a role in the transmission of leprosy, as absence of evidence is not the evidence of absence (54). Therefore, future research on ticks as a vector in leprosy will be informed by the outcome of the proposed analysis, regardless of whether we obtain a positive or negative finding. This will be done in co-operation with entomologists and researchers involved in leprosy projects on the Comoros islands and Brazil. Furthermore, also other non-human reservoirs and/or vectors will be included in future studies to clarify the unexplained leprosy transmission pathways.
Regarding the secondary aim, different alternative methods are available for the sample preparation and DNA extraction. The alternative most often used for sample preparation of the FFPE tissue samples is xylene deparaffinization and ethanol rehydration (55). Also a heating method, melting away the paraffin wax can be used, as well as a direct digestion of the sample (56) or deparaffinization using mineral oil (57,58). For digestion proteinase K is being used. A few alternative commercial kits for the DNA purification are available (e.g. QIAamp DNA FFPE Tissue kit (Qiagen)) (59). Also an in-house procedure can be done using phenol and chloroform (60).
Conclusion
To conclude, this project could contribute to the understanding of leprosy transmission. Positive results in each of the two parts could lead to a better insight in the populations, which are at highest risk for leprosy on the Comoros, but also in the rest of the world. Based on that, more targeted prevention measurements could be taken, contributing to the control and in the long run, elimination of the mutilating disease.
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