a family cluster was defined as multiple family members independently given a diagnosis of m. ulcerans disease who were living at the same residence at the time of diagnosis . data was collected by using epi info 6 ( centers for disease control and prevention , atlanta , ga , usa ) and analyzed by using stata 12 ( statacorp llc , college station , tx , usa ) . to determine the genetic relatedness of isolates derived from family clusters , we performed whole - genome sequencing and single - nucleotide polymorphism ( snp ) analysis for 6 isolates derived from 3 family cluster pairs ( tables 1 , 2 ) . we sequenced dna as 300-bp paired - end reads by using an miseq sequencer ( illumina , inc . resulting reads were mapped against the m. ulcerans agy99 genome ( 9 ) , including plasmid pmum001 ( 10 ) , by using bowtie2 ( 11 ) . raw sequence reads for the 6 isolates have been deposited in the national center for biotechnology information ( bethesda , md , usa ) sequence read archive under bioproject accession no . we also performed whole - genome snp analysis for 6 additional unrelated previously sequenced human m. ulcerans isolates ( sequence read archive accession no . * bh , barwon heads ; nt , not tested ; ptl , point lonsdale ; que , queenscliff ; who , world health organization . * a total of 4,918 core single - nucleotide polymorphisms were identified for all 6 isolates compared with the african agy99 reference genome . a total of 324 patients with m. ulcerans disease from the bellarine peninsula , victoria , australia , were managed in the barwon health observational cohort during january 1 , 1998april 12 , 2016 . median age was 57 years ( iqr 3474 years ) , and 164 patients ( 50.6% ) were men . for the whole cohort , a combined time of 1,968.5 years had elapsed from diagnosis of the initial m. ulcerans lesions until the time of study analysis ( april 12 , 2016 ) . the median duration elapsed from initial diagnosis until study analysis was 4.7 years ( iqr 2.89.7 years ) . twenty - one ( 6.5% ) patients were part of a family cluster ( table 1 ) , 9 genetically related and 12 related by marriage . we found that significantly fewer family clusters were diagnosed during the first half of the study period ( 0 of 92 cases during 19982007 ) than in the second half ( 21 of 232 cases during 20082016 ) ( p<0.01 ) . the median time between diagnoses of m. ulcerans lesions in an additional family member , after the initial family member was given a diagnosis , was 2.8 months ( iqr 1.120.6 months ) . the rate of new diagnosis of an m. ulcerans lesion in another family member was 5.69/1,000 person - years ( 95% ci 3.1510.29/1,000 person - years ) . we determined the cumulative proportion of patients given a diagnosis who had an affected family ( figure 1 ) . cumulative proportion of patients with a family member affected by mycobacterium ulcerans disease , barwon health cohort , bellarine peninsula , victoria , australia , 19982016 . core snps based on common variable nucleotide positions were identified for the 6 examined family isolates by whole - genome sequencing . a total of 4,918 core snps ascribed to the african agy99 reference genome were identified according to strict filtering criteria . only 8 snps were specific to > 1 of the 6 isolates ( table 2 ) . of the 8 snps that differed among the isolates , only 3 were nonsynonymous substitutions . pairwise comparisons of family cluster isolates showed that isolates from the 4a/4b pair were genetically identical . in contrast , isolates from the 2a/2b and 1a/1b pairs contained several isolate - specific snps ( table 2 ; figure 2 ) . snp analysis of unrelated m. ulcerans isolates from the same disease - endemic area showed that 3 of the 6 isolates were also genetically identical ( figure 2 ) , which demonstrated that unrelated isolates can share a common genotype . thus , family cluster isolates were not any more closely genetically related than 6 random isolates from the same geographic region . median joining network of 12 snps of 12 mycobacterium ulcerans isolates from patients with mycobacterium ulcerans disease , barwon health cohort , bellarine peninsula , victoria , australia , 19982016 . the size of each node is proportional to the number of genetically identical isolates with identical genotypes . our examination of family clusters of m. ulcerans disease provides useful insights into the environmental reservoir and mode of transmission of this organism . first , the median time to diagnosis between family members was short ( 2.8 months ) , and no family members were given a diagnosis of an m. ulcerans lesion > 23 months apart in a cohort spanning 18 years and nearly 2,000 combined years of elapsed time since diagnosis . this finding suggests that family members have been exposed to a source in the family s environment that persists only for a short period . second , with an incubation period for m. ulcerans disease estimated to be a median of 4.5 months ( 12 ) , the observation that the median time between diagnoses in family clusters was <3 months suggest that infections were not being transmitted between family members . further evidence against human - to - human transmission is apparent from whole - genome snp analysis , which showed that pairs of isolates from 2 ( 2a/2b and 1a/1b ) of 3 family clusters were not genetically identical . these findings support previous suggestions that m. ulcerans is unlikely to be transmitted from person to person ( 13 ) . unknown is the type of short - term exposure that leads to the close temporal relation of family clustered infections . opossums have been proposed as a source , either through contamination of the environment by infected feces or by an intermediate vector , such as mosquitoes , which transfer the infection from infected opossums to humans by a bite ( 7 ) . infected opossum(s ) in the family environment might cause cases of human infection , then subsequently die of the disease ( 14 ) , removing the source of infection . alternatively , transmission could be related to a short - term change in the environment involving soil or foliage as a result of such events as home construction and renovation , or planting and removing trees or grasses ( 13 ) . mosquitoes in the area might be transiently infected / contaminated with m. ulcerans and infect humans through bites during this time ( 15 ) . in summary , the incidence rate of lesions in another family member ( 5.69/1,000 person - years ) was higher than reported incidence rates during 20052009 in the general population of the bellarine peninsula ( 0.854.04 cases / year/1,000 population ) ( 7 ) . this finding suggests that genetic susceptibility or , more likely , localized exposure risk increases the likelihood of infection . the incidence of m. ulcerans disease family clusters in an observational cohort in southeastern australia was higher than in the general population of the disease - endemic area . however , when clusters occur , they are closely temporally related , which suggests a short - term risk for exposure and infection . epidemiologic and genetic evidence suggests human - to - human transmission is not the source of infection .
we conducted epidemiologic and genetic analyses of family clusters of mycobacterium ulcerans ( buruli ulcer ) disease in southeastern australia . 
 we found that the incidence of m. ulcerans disease in family members was increased . 
 however , the risk for exposure appeared short - term and not related to human - human transmission .