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      First Isolation of Mycobacterium ulcerans from an Aquatic Environment: The End of a 60-Year Search?

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      PLoS Neglected Tropical Diseases
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          Abstract

          In a landmark paper in this issue of PLoS Neglected Tropical Diseases, Portaels et al. [1] describe the first isolation in pure culture of Mycobacterium ulcerans from an aquatic environment, ending a quest that began over 60 years ago when MacCallum and his Australian colleagues identified M. ulcerans as the causative agent of the ulcerative skin disease that later became known as Buruli ulcer [2]. This is a major achievement and will serve as the definitive reference point for scientists intent on revealing the ecology, environmental reservoir, and precise mode of transmission of M. ulcerans. Buruli ulcer is a terrible, disfiguring disease of skin and soft tissue that may leave sufferers permanently disabled (Figure 1). Those most affected are children living in rural West and Central Africa, but the disease is known in more than 30 countries worldwide, and people of all ages and races are susceptible. In some highly endemic regions, Buruli ulcer is now more common than the two most notorious mycobacterial diseases, leprosy and tuberculosis (TB) [3]. 10.1371/journal.pntd.0000216.g001 Figure 1 River Offin in the Amansie West District of Ghana showing a typical environmental setting for a region of Buruli ulcer endemicity, and M. ulcerans infection of the elbow of a female patient from the same district. Recently, the combination of the potent antimycobacterial drugs rifampicin and streptomycin has been shown to be able to kill the causative agent, M. ulcerans, in early nodular Buruli ulcer [4], and a new WHO protocol has been implemented in several endemic countries [5]. The new protocol is proving very effective, greatly reducing costs and often avoiding the need for surgery [6]. More research is required if we are to understand and control this potentially devastating disease. Exactly how M. ulcerans is introduced into the skin of humans remains unknown, but unlike TB or leprosy, the infection is acquired directly or indirectly from the environment and not from contact with other patients. The highly focal epidemiology and association with swamps and slow-flowing water are hallmarks of Buruli ulcer (Figure 1), and these observations have led to 50 years of failed attempts by many research groups to try to cultivate M. ulcerans from a variety of environmental sources that has included bats, sand flies, rodents, fish, molluscs, vegetation, water, and soil [7]–[11]. And while isolation of M. ulcerans from clinical specimens is relatively straightforward, efforts to isolate the bacterium from the environment have been confounded by its slow growth, its predicted paucity (based on PCR) in the environment, and the complex, competing microbial flora in these sample types. The discovery of the M. ulcerans−specific insertion sequence IS2404 in 1997 [12] and the subsequent development of molecular diagnostics for M. ulcerans was the catalyst for renewed efforts to find its environmental sources. The detection of IS2404 in carnivorous water bugs indicated that aquatic insects (among other things) might harbour the bacterium [13]. Laboratory-based feeding studies with M. ulcerans in Naucouris sp. have shown that the bacterium can colonize the salivary glands of these insects and also be transferred by biting a mammalian (mouse) host to cause disease [14]. These observations led Portaels et al. to try and culture M. ulcerans directly from homogenates of five aquatic insects captured from a Buruli ulcer–endemic region of Benin. In a complicated and patience-testing process spanning more than 2 years and involving classical mycobacterial culture techniques and monitoring of cultures by IS2404 PCR, followed by three rounds of blind passaging through mice and then further culture, the team were finally able to obtain a single M. ulcerans isolate from a water strider (Gerris sp.). Phenotypic analysis showed this isolate produced the same polyketide toxin as clinical isolates and was fully virulent for mice. Most importantly, molecular characterization confirmed it was M. ulcerans and not another recently reported M. ulcerans–like mycobacterium [15]. Molecular studies also revealed a novel single nucleotide polymorphism in the 16S rRNA gene of this strain, proving that the result was not a laboratory artefact caused by contamination. The same mutation has since been found in clinical M. ulcerans isolates recovered from patients in the same region of Benin, suggesting a common origin for environmental and patient isolates. Portaels and colleagues were the first to link insects with M. ulcerans [13], and in Australia, two recent papers have taken her observation one step further by demonstrating M. ulcerans DNA in mosquitoes and establishing that mosquito exposure increases the odds of Buruli ulcer in humans [16],[17]. However, whether insects transmit M. ulcerans to humans or just mark its presence in the environment has yet to be definitively established. The research presented in this paper represents considerable technical prowess, and is the result of knowledge accrued over many decades of M. ulcerans research. It does not end the quest to establish the environmental source of M. ulcerans but provides encouragement (or perhaps discouragement depending on your natural disposition) and an opportunity for future studies to also attempt M. ulcerans isolation from the environment. It will only be through the analysis of many different M. ulcerans isolates from the environment, in the context of solid epidemiological and ecological data, that the lingering and critical questions surrounding the reservoirs and modes of transmission of M. ulcerans will finally be resolved. Much work remains to be done.

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          Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer, Southeastern Australia

          Buruli ulcer (BU), also known as Bairnsdale ulcer ( 1 ), Daintree ulcer ( 2 ), and Mossman ulcer in Australia, is an emerging disease of skin and soft tissue with potential to cause scarring and disability ( 3 ). It is caused by Mycobacterium ulcerans ( 4 ), an environmental pathogen that produces a destructive polyketide toxin, mycolactone ( 5 ); the genes for the production of this toxin are encoded on newly described plasmid pMUM001 ( 6 ). BU occurs in >30 countries worldwide, but it affects mainly children in sub-Saharan Africa, where it is now more common than tuberculosis and leprosy in some regions ( 7 ). This disease occurs in people of all ages and races who live in or visit BU-endemic areas, but the precise mode of transmission remains unknown. Analysis of the recently sequenced M. ulcerans genome has shown that in addition to pMUM001, there are unusually high copy numbers of 2 independent insertion sequences (IS2404 and IS2606) and a high incidence of pseudogene formation ( 8 ). These data suggest that M. ulcerans is unlikely to be free-living in the environment but is instead undergoing adaptation to a specific ecologic niche in which the products of some ancestral genes are no longer essential. One such niche may be in aquatic insects because M. ulcerans has recently been reported to colonize the salivary glands of carnivorous water bugs (Naucoridae) under laboratory conditions ( 9 ), and mycolactone production appears to be necessary for this colonization ( 10 ). Studies from disease-endemic areas in Africa have reported that farming activities near rivers ( 11 ) and swimming in rivers or marshes ( 12 ) may be risk factors for BU; bites from contaminated water bugs may transmit the infection. In temperate southeastern Australia, outbreaks of M. ulcerans infection occur in localized areas, but few patients report direct contact with environmental water other than the ocean, which led to the proposal that aerosols from contaminated water may cause human infections ( 13 ). However, these low-lying disease-endemic areas also harbor large populations of mosquitoes, and some patients have reported that BU first appeared at the site of what may have been a mosquito bite (Figure 1). These observations, and knowledge from field studies in Africa implicating insects as either a reservoir or mode of transmission, led us to capture and screen mosquitoes during our investigation of a large outbreak of BU in humans in a small coastal town in southeastern Australia (Point Lonsdale), ≈60 km south of Melbourne (Figure 2). Figure 1 Ear of an 18-month-old child with culture- and PCR-confirmed Buruli ulcer who briefly visited St. Leonards, Australia, in 2001 (Figure 2). The initial lesion resembled a mosquito bite or that of another insect. Figure 2 Map of central coastal Victoria, Australia, showing towns and places referred to in the text or in associated references. Methods Outbreak Investigation M. ulcerans infection has become increasingly common in the southern Australian state of Victoria since the early 1990s ( 14 , 15 ) and characteristically causes localized outbreaks ( 16 ). In 1995, a research group at the Royal Children’s Hospital in Melbourne developed an IS2404 PCR to improve speed and accuracy of diagnosis of BU ( 17 ). This method has now become the initial diagnostic method of choice in Australia and elsewhere ( 18 ). All PCR- and culture-positive cases of M. ulcerans infection in Victoria have been unofficially reported to the Victorian Department of Human Services (DHS) since the 1990s, and investigators from DHS began to routinely contact and interview all new reported case-patients in 2000. All new cases of M. ulcerans infection were made legally reportable in Victoria in January 2004 ( 19 ). Case Definition For this study, a case of BU was defined as a patient with a suggestive clinical lesion from which M. ulcerans was identified by PCR or culture from a swab or tissue biopsy specimen from January 2002 through April 2007; the patient must have been either a resident of, or a visitor to, Point Lonsdale or Queenscliff (adjacent coastal towns on the Bellarine Peninsula) who did not report a recent history of contact with another known BU-endemic area. Australian Bureau of Statistics data derived from the 2001 Australian Census for Point Lonsdale/Queenscliff (postcode 3225) were used to obtain the resident population numbers and age distribution in the outbreak area ( 20 ). Mosquito Trapping A total of 8–13 overnight mosquito traps were placed at Point Lonsdale on 22 occasions from December 2004 through January 2007. Adult mosquito sampling was conducted with CO2-baited miniature light traps ( 21 ). Traps were 2-L, cylindrical, insulated containers designed to hold CO2 pellets that continuously produce CO2, which then diffuses through holes in the bottom of the container. A small electric light and fan at the base of the container deflected attracted mosquitoes into a holding container. The traps were set before dusk and then retrieved several hours after dawn the next morning. The catches were transported to Primary Industries Research in Attwood, Victoria, where they were counted, sorted, and pooled by sex and species. Mosquito species were identified by using the key of Russell ( 22 ). All captured mosquitoes were tested except in February–March 2005 and again in October 2005 when recent rains led to large spikes in mosquito numbers. Screening of Mosquitoes by PCR DNA was extracted from pools of 55 years of age than in those 1 pool was positive; otherwise uncorrected. Thirty-five IS2404-positive pools did not contain IS2606 and KR. However, the cycle threshold (Ct) values for IS2404 were lower for those pools that did have IS2606 and KR, which suggested that failure to detect KR and IS2606 in some pools was caused by low DNA concentration, rather than lack of specificity for M. ulcerans. This finding is consistent with known differences in copy number per cell of targets used for PCR screening and confirmation ( 23 ). A total of 124 pools of mosquitoes that were negative for IS2404 by PCR were screened with probes for KR and IS2606. None were positive, which indicated that these 2 loci are consistently linked to IS2404 and do not occur independently. The MLE (bias corrected) for all mosquitoes over the entire testing period at Point Lonsdale was 4.3 M. ulcerans PCR-positive mosquitoes/1,000 tested (95% confidence interval [CI] 3.2–5.6). However, mosquito numbers varied widely between trappings, as did proportions of positive pools. On 1 occasion, only 269 mosquitoes were trapped, but 6 of the pools were positive (December 2005; MLE 22.4, 95% CI 10.3–50.3). Most PCR-positive pools had relatively high Ct values for IS2404 PCR, which indicated low numbers of contaminating M. ulcerans cells. With reference to spiking experiments under laboratory conditions, ≈10–100 M. ulcerans were likely to have been present per contaminated mosquito ( 23 ). Mosquito Numbers, Proportion PCR Positive, and Reporting of BU Trapping was conducted at Point Lonsdale between December 2004 and January 2007. Mosquito numbers varied during the period, and traps were not set when local reports suggested low mosquito numbers (Appendix Figure). There appeared to be a qualitative relationship between PCR-positive mosquitoes in spring and summer (September–February) and reporting of new cases of human disease in autumn and winter (March–August). The exposure-to-reporting interval is typically longer than the actual incubation period because patients do not always seek medical assistance immediately and doctors do not always diagnose BU when a patient is first seen ( 28 ). Mosquitoes Caught at Other Locations in Victoria To test that the observed association between M. ulcerans and mosquitoes only occurs in outbreak areas, we tested 3,385 mosquitoes from several inhabited areas with lower BU endemicity than Point Lonsdale. From October 2005 through January 2007, a total of 2,119 mosquitoes (89% Ae. camptorhynchus) were trapped in townships on the Bellarine Peninsula where 30 cases of BU have been reported in the past 5 years; 3 pools of Ae. camptorhynchus were positive by IS2404 PCR. In January and June 2006, a total of 795 mosquitoes (82% Ae. camptorhynchus) were trapped in the Bass Coast Shire, which includes Phillip Island, a region that has previously been endemic for M. ulcerans ( 14 ) but has only reported 2 cases in the past 5 years. One pool of Ae. notoscriptus was positive for IS2404. From February through April 2006, 471 mosquitoes were captured from inhabited areas in northern and central Victoria where no human cases of M. ulcerans have been reported. Ten different species were trapped, including 226 Ae. camptorhynchus (48%), but all pools were negative for IS2404. When analyzed together, an association was observed between degree of endemicity and probability of trapping mosquitoes that are positive by PCR for M. ulcerans (Table 2), but this association did not show statistical significance (p = 0.07). Table 2 Relationship between cases of Buruli ulcer, mosquitoes tested, and maximum likelihood estimate (MLE) per 1,000 mosquitoes trapped in Victoria, Australia, and tested by PCR for insertion sequence IS2404 of Mycobacterium ulcerans* Region No. cases past 5 y No. mosquitoes tested (% Aedes camptorhynchus)† No. pools positive MLE (95% CI) Point Lonsdale 79 11,504 (91.8) 48 4.2 (3.08–5.53) Bellarine Peninsula (excluding Point Lonsdale) 30 2,119 (88.7) 3 1.42 (0.37–3.85) Bass coast Shire including Phillip Island 2 795 (82.1) 1 1.25 (0.07–6.03) Central and northern Victoria (Mildura, Swan Hill, Moira, Shepparton) 0 471 (48.0) 0 0 (0–7.34) Total 111 14,889 (89.4) 52 3.57 (2.70–4.64) *MLE bias was corrected when >1 pool was positive, otherwise uncorrected. CI, confidence interval
†p value = 0.07 (χ2: 4 × 2 table; pools positive/no. tested). Discussion To our knowledge, the outbreak of BU in Point Lonsdale is the largest ever recorded in Australia and has now caused more than twice as many cases as the well-described outbreak at Phillip Island a decade earlier ( 16 , 29 ). A striking feature of both outbreaks is their intensely localized nature. We identified 79 cases that were epidemiologically linked to Point Lonsdale and the western edges of Queenscliff, but the town of Queenscliff, only 4 km to the east along the same beach, has so far remained disease free. The cumulative attack rate for both towns is estimated to be 1.2% of the resident population, but it could be up to twice as high if only the population of Point Lonsdale, where all transmission appears to have occurred, were considered. Although Queenscliff remains unaffected, the nearby towns of Barwon Heads and Ocean Grove, ≈12 km west of Point Lonsdale, began reporting their first cases in 2005. The first case at Point Lonsdale was reported in January 2002. In 2004, the outbreak increased in intensity and began to involve visitors as well as residents, which suggested that environmental contamination with M. ulcerans has steadily increased over 5 years. Among local residents, we found a higher attack rate in the elderly, with 3.7% of residents of Point Lonsdale/Queenscliff >75 years of age with BU. The reasons for this age distribution are not known, but increasing risk with age could be caused by an age-related immune defect or an unrecognized behavioral factor. Among visitors, there was a pronounced bimodal age distribution, which probably represents a skewing of the exposed population (e.g., young children going to stay with their retired grandparents over the summer while their parents stayed at work) but may also reflect increased susceptibility in young persons. This bimodal pattern, which included increased incidence in young persons and the elderly, has also been reported in Africa ( 30 ). During our investigations at Point Lonsdale, we focused initially on several marshy areas and obtained positive PCR results for plant material from 2 small ornamental lakes and soil from storm water drains ( 23 ). However, case-patients did not report direct contact with these lakes or drains (these sources of water are not used for swimming or wading). Thus, how people were exposed is not clear. In an outbreak in Phillip Island, many cases were clustered around a newly formed wetland and a golf course irrigation system, and we proposed transmission from these sites by aerosol ( 16 , 29 ). However, this hypothesis may not be supported by our new evidence, which suggests that M. ulcerans may not be free-living in the environment but may have adapted to specific niches within aquatic environments, including salivary glands of some insects. Thus, we investigated whether M. ulcerans could be detected in mosquitoes, which had been reported in higher than usual numbers at Point Lonsdale. We also investigated behavior in a case-control study (the subject of a separate report), which found that being bitten by mosquitoes increased the odds of having BU ( 31 ). A total of 14,889 mosquitoes obtained over a 25-month period (11,504 from Point Lonsdale) were tested for M. ulcerans by using a highly sensitive and specific real-time PCR ( 23 ). We used PCR because direct culture of M. ulcerans from the environment is extremely difficult and was only achieved when IS2404 PCR screening of environmental samples accurately directed researchers to specific microenvironments that include water insects and aquatic plants ( 32 ). Although IS2404, IS2606, and the mycolactone-producing virulence plasmid have been detected in mycobacteria other than M. ulcerans ( 33 – 35 ), identification of these targets in expected relative proportions and the VNTR locus 9 sequence identical to that of the outbreak strain in a subset of mosquito pools with sufficiently high DNA concentrations confirms that we identified the outbreak strain ( 23 ). We also demonstrated that over a 2-year cycle at Point Lonsdale absolute numbers of mosquitoes and PCR-positive mosquitoes increased in spring and summer followed by a cluster of new human cases in autumn and winter. This pattern is consistent with recent point estimates that suggest the incubation period for BU in Australia is 3–7 months (2 cases) ( 36 ) and 1–4 months (3 cases) ( 28 ), and that an additional 1–6 weeks may elapse before cases are diagnosed and reported ( 28 ). The predominant species trapped was Ae. camptorhynchus; however, identification of M. ulcerans in 4 other species suggests that M. ulcerans contamination of mosquitoes is not species specific. Ae. camptorhynchus is a salt marsh species, an aggressive biter, and a major pest in coastal areas of southeastern Australia that has been linked to transmission of Ross River virus. The mosquito appears in large numbers after rain as minimum temperatures begin to increase, with a lag time of ≈1 month ( 37 ). Of the other species from which at least 1 PCR-positive pool was identified, An. annulipes and Cq. linealis are fresh water species ( 38 ). Ae. notoscriptus is a peridomestic species that breeds in containers (e.g., in roof gutters) ( 39 ), can transmit dog hookworm, and has a limited flight range (e.g., <200 m) ( 40 ). In contrast, Cx. australicus may have a flight range of many kilometers ( 41 ). A limited number of other biting or aquatic insects were also tested and none were positive for M. ulcerans. However, larger numbers must be screened before it can be concluded that they do not transmit M. ulcerans. Our results do not demonstrate viability or transmissibility of M. ulcerans at the time mosquitoes were captured, and the method we used does not answer questions about location of M. ulcerans within the insect. Because M. ulcerans is an environmental pathogen, PCR-positive mosquitoes may only be indicators of its presence in the environment and not linked to transmission. The Ct values obtained for mosquito pools suggest that only 10–100 organisms were present per positive pool, which is more consistent with organisms being acquired on outer surfaces of mosquitoes when resting or feeding in storm water drains ( 23 ), rather than mosquitoes being a true productive reservoir and vector. However, if some bacterial cells were present on the proboscis, they could have been injected beneath the keratin layer during feeding. Although the inoculum size required to cause a human infection is unknown, the long incubation period suggests a low initial inoculum. Our findings do not demonstrate that mosquitoes are responsible for transmission, but this possibility should be investigated. Studies are underway to artificially infect mosquito larvae with M. ulcerans and initiate infection in a mouse model, as has been conducted with naucorids ( 9 ). Although our findings may not apply to the situation in Africa, the close genetic relationship of Australian isolates of M. ulcerans with strains from humans with BU in Africa ( 35 ) should encourage similar search on M. ulcerans in mosquitoes from the primary BU-endemic regions of West Africa. We have shown that a small proportion of mosquitoes of 5 species captured in a BU-endemic area during an intense human outbreak of BU can carry M. ulcerans; PCR-positive mosquitoes are likely present at times of peak transmission and mosquitoes captured in areas with few human cases appear less likely to be positive for M. ulcerans. We hypothesize that transmission by mosquitoes offers a partial explanation for the outbreak at Point Lonsdale and possibly at other sites in southeastern Australia. Supplementary Material Appendix Figure Relationship between reporting of cases of Buruli ulcer (BU) and mosquitoes tested from Point Lonsdale, Australia, December 2004-January 2007. Increased mosquito activity in spring and summer (September-February) appears to be followed by a wave of new reports in autumn and winter (March-August). A) No. mosquitoes tested by month at Point Lonsdale (traps were not set when local reports suggested low mosquito activity). B) Proportion of tested mosquitoes positive by PCR for Mycobacterium ulcerans by month. C) No. of new cases of BU epidemiologically linked to Point Lonsdale by month.
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            Aquatic insects as a vector for Mycobacterium ulcerans.

            Mycobacterium ulcerans is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking M. ulcerans to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of M. ulcerans, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that M. ulcerans may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition, M. ulcerans appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated M. ulcerans from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of M. ulcerans.
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              Efficacy of the combination rifampin-streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans.

              Mycobacterium ulcerans disease is common in some humid tropical areas, particularly in parts of West Africa, and current management is by surgical excision of skin lesions ranging from early nodules to extensive ulcers (Buruli ulcer). Antibiotic therapy would be more accessible to patients in areas of Buruli ulcer endemicity. We report a study of the efficacy of antibiotics in converting early lesions (nodules and plaques) from culture positive to culture negative. Lesions were excised either immediately or after treatment with rifampin orally at 10 mg/kg of body weight and streptomycin intramuscularly at 15 mg/kg of body weight daily for 2, 4, 8, or 12 weeks and examined by quantitative bacterial culture, PCR, and histopathology for M. ulcerans. Lesions were measured during treatment. Five lesions excised without antibiotic treatment and five lesions treated with antibiotics for 2 weeks were culture positive, whereas three lesions treated for 4 weeks, five treated for 8 weeks, and three treated for 12 weeks were culture negative. No lesions became enlarged during antibiotic treatment, and most became smaller. Treatment with rifampin and streptomycin for 4 weeks or more inhibited growth of M. ulcerans in human tissue, and it provides a basis for proceeding to a trial of antibiotic therapy as an alternative to surgery for early M. ulcerans disease.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                March 2008
                26 March 2008
                : 2
                : 3
                : e216
                Affiliations
                [1 ]Department of Microbiology, Monash University, Clayton, Victoria, Australia
                [2 ]Department of Infectious Diseases, Austin Hospital, Heidelberg, Australia
                Institut Pasteur, France
                Author notes
                Article
                08-PNTD-EC-0043R1
                10.1371/journal.pntd.0000216
                2267217
                18365037
                e23e7403-0462-42e7-a95f-9ce52c84dc49
                Stinear, Johnson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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                Pages: 2
                Categories
                Expert Commentary
                Public Health and Epidemiology/Epidemiology
                Public Health and Epidemiology
                Infectious Diseases/Neglected Tropical Diseases
                Infectious Diseases/Bacterial Infections

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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