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).

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).

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.

DNA was extracted from pools of <15 individual mosquitoes (occasional pools had <23 mosquitoes) of the same sex and species by using the FastDNA Kit (revision no. 6540-999-1D04) and the FastPrep Instrument (Qbiogene Inc., Irvine, CA, USA) according to the manufacturer’s instructions. We adapted fluorescence-based real-time PCR technology to screen mosquitoes for 3 M. ulcerans–specific DNA sequences as described (23). Briefly, oligonucleotide primers and TaqMan MGB probes (Applied Biosystems, Foster City, CA, USA) labeled with fluorescent dyes 6FAM or VIC were designed that targeted 3 independent high-copy number repetitive sequences (IS2404 and IS2606 [24] and the ketoreductase B domain [KR] from pMUM001 [6]). The copy number of these targets per bacterial cell in the published sequenced of M. ulcerans, to which the outbreak strain is phylogenetically closely related, is 213 for IS2404, 91 for IS2606, and 30 for KR (8). Assays were conducted with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Each pool was first tested for IS2404 with an internal positive control to test for PCR inhibition and separate negative and positive controls. Samples were considered positive for a given target when they had a result above a previously determined critical threshold (23). Pools that were positive for IS2404 were then screened in duplicate with confirmatory assays to detect IS2606 and KR. For pools with sufficiently high signal strength, amplification and sequencing of variable number tandem repeat (VNTR) locus 9 were conducted by using a nested PCR. The first round PCR used 2 primers, MUVNTR9NF (5′-ACTGCCCAGACATGGCGA-3′) and MUVNTR9NR (5′-ACGCGAGGTGGAACAAAGC-3′), designed to flank the published VNTR locus 9 primer. First-round PCR products were used as template for a second-round PCR performed as described by Ablordey et al. (25). PCR products of the expected size were sequenced by using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. Precipitated reaction products were tested in a 3730S Genetic Analyzer (Applied Biosystems) (23). The maximum likelihood estimate (MLE) per 1,000 mosquitoes tested (bias corrected MLE) was calculated by using software recommended for this purpose by the Centers for Disease Control and Prevention (Atlanta, GA, USA) (26).

The climate in Point Lonsdale is temperate with a mean daily maximum temperature of 12.8°C in July (winter) and 22.4°C in January (summer). Average annual rainfall is 660 mm and is spread throughout the year (e.g., average 41.3 mm in January and 59.1 mm in July) (27). Most of the town is low-lying and close to sea level, and there are several natural and human-made swamps and water features in the vicinity (Figure 3). Natural vegetation includes dense clumps of coastal tea trees (Leptospermum laevigatum).

Point Lonsdale shares a beach with Queenscliff (Figure 3), a neighboring town 4 km to the east. Point Lonsdale and Queenscliff (postcode 3225) were included in the 2001 Australian Bureau of Statistics Census and had a resident population of 3,851 at that time, but there are also large numbers of visitors to this scenic area during summer holiday periods (e.g., 54,000 people visited the Queenscliff Visitor Information Centre in 2005; pers. comm.).

M. ulcerans infection was not found in the area before 2002. From January 2002 through April 2007, BU developed in 79 persons (48 residents and 31 visitors). Initially, all patients were local residents, but in 2004 the outbreak increased in intensity and began to include visitors as well as residents (Figure 4). All case-patients who could be accurately located either lived in or visited Point Lonsdale and the western edge of Queenscliff, and none were linked solely to the main township of Queenscliff.

Most case-patients were adults and many were elderly (Figure 5), although 14 of the 79 were children <18 years of age. Among visitors, there was a bimodal age distribution, with relatively low numbers of adults 20–50 years of age. An estimate of the age-specific attack rate for residents of Point Lonsdale/Queenscliff was obtained with reference to the 2001 Australian census. Because census data were not available for the 2 towns separately, the calculation assumes that the age distribution of Point Lonsdale and Queenscliff is similar. A similar analysis for visitors was not performed because appropriate denominators could not be determined. The risk appeared to increase strongly with age and was ≈7× higher for those >55 years of age than in those <55 years of age (p<0.001) (Figure 6).

The incubation period for residents and for most visitors could not be assessed because exposure to the BU-endemic area occurred repeatedly over a prolonged period. However, in 2006, one patient reported a 1-week contact history with Point Lonsdale (P.D.R. Johnson, unpub. data). Her case was diagnosed and reported 7 months after this exposure.

A total of 23,692 mosquitoes were captured in Point Lonsdale during a 25-month period; 96% were Aedes camptorhynchus (Thomson). Twelve other species comprised the remaining 4% (Table 1). Of 11,504 mosquitoes tested, 48 pools were positive for IS2404; of these, 13 pools were also positive for KR and IS2606. Forty-one of 48 pools were female Ae. camptorhynchus, 4 were positive pools of Coquillettidia linealis (Skuse), and 1 each were Anopheles annulipes (Walker s.l.), Culex australicus (Dobrotworsky and Drummond), and Ae. notoscriptus (Skuse). For 2 positive pools with particularly high M. ulcerans DNA concentrations, VNTR locus 9 was amplified and the sequence was identical to that of the local outbreak strain cultured from case-patients (23).

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).

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).

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).