Surveillance duplex ultrasound prompted interventions after carotid endarterectomy.

OBJECTIVE: Current societal guidelines recommend duplex ultrasound (DUS) surveillance beyond 30 days after carotid endarterectomy (CEA) for patients with risk factors for restenosis or who underwent primary closure. However, the appropriate duration of this surveillance has not yet been identified, and the rate at which DUS surveillance prompts intervention is unknown. Multiple calls for decreasing health care spending that does not provide value, including unnecessary testing, have been made. The purpose of this study was to examine the rate of intervention prompted by surveillance DUS on the ipsilateral or contralateral carotid artery after CEA and determine the value of continued surveillance by determining the rate of DUS-prompted intervention. METHODS: A single-center, retrospective chart review of all patients older than 18 years who had undergone CEA from August 2009 to July 2022 was performed. Patients with at least one postoperative duplex in our Intersocietal Accreditation Council-accredited ultrasound lab were included. Exclusion criteria were patients with incomplete medical charts or patients who underwent a concomitant procedure. The primary end point was return to the operating room for subsequent intervention based on abnormal surveillance DUS findings. Secondary end points were the number of postoperative surveillance duplexes, duration of surveillance, and incidence of perioperative stroke. The study participant data were queried for patients who had a diagnosis of stroke that occurred following their procedure. RESULTS: A total 767 patients, accounting for 771 procedures, were included in this study, which resulted in 2145 ultrasound scans. A total of 40 (5.2%) patients required 44 subsequent interventions that were prompted by DUS surveillance scans. The average number of ultrasound scans per patient was 2.8 (range: 0-14), and the average duration of surveillance was 26.4 months (range: 0-155 months). Of the 767 patients, 669 (87.2%) had a unilateral CEA. A total of 62 of 767 (8.1%) patients had planned endarterectomies on the contralateral side based on initial imaging, not prompted by interval DUS surveillance scans. Of 767 patients, 28 (3.7%) patients who underwent CEA had a subsequent procedure for progression of contralateral disease, which was prompted by duplex surveillance scans. The average duration between index CEA and intervention on contralateral carotid was 29.57 months (range: 3-81 months). A total of 11 patients, accounting for 12 procedures, underwent a subsequent procedure for restenosis of their ipsilateral carotid, prompted by duplex surveillance scans. The average duration between index CEA and reintervention on the ipsilateral carotid was 17.9 months (range: 4-70 months). Three of 767 (0.4%) patients in total were identified as having a perioperative stroke. CONCLUSIONS: The overall rate of ipsilateral reintervention after CEA is low. A small percentage of patients will progress their contralateral disease, ultimately requiring surgical intervention. These data suggest that regular duplex surveillance after CEA is warranted for patients with at least moderate contralateral disease; however, the yield is low for ipsilateral restenosis after 36 months based on this single institution study. Further study is needed to better delineate which patients need follow-up to decrease unnecessary testing while still targeting patients most at risk of restenosis or contralateral progression of disease.

Popliteal pseudoaneurysm in a young patient with multiple hereditary exostosis.

Multiple hereditary exostosis is an osteogenic disorder that causes outgrowths of cartilaginous bone tumors that are associated with adjacent neurovascular compressive injuries. We present the case of an adolescent male with multiple hereditary exostosis complicated by popliteal pseudoaneurysm formation who underwent excision of the osteochondroma and vein patch angioplasty repair of the artery. We highlight the rare association between this genetic disease and subsequent vascular complications and review the available literature of arterial complications of this disease.

Operative Case Volume for Vascular Integrated Residents and Fellows Following the Elective Surgery Shutdown due to COVID-19.

In response to the COVID-19 pandemic, nonemergent surgery was postponed in efforts to limit disease spread. To determine whether these changes affected vascular integrated resident (VR) and fellow (VF) operative volume, Accreditation Council for Graduate Medical Education (ACGME) case log data was reviewed. Case volume and standard deviation for each major category was for graduates of 2020 and 2021 were compared to the year prior to the pandemic, 2019. There were only 3 significant changes when comparing 2020/2021 to the prepandemic baseline of 2019, with increase in abdominal obstructive cases for VRs (8.1 in 2021 vs 5.9 in 2019, P = .021), an increase in upper extremity cases for VFs (18.9 in 2021 from 15.8 in 2019, P = .029), and a decrease in venous cases for VFs (39.6 in 2021 from 48.4 in 2019, P = .011). Postponing nonemergent surgery did not translate to significant changes in operative cases for graduating VRs and VFs.

Postamputation diagnosis of squamous cell carcinoma in a patient with lymphedema.

Chronic lymphedema is fraught with morbidity, including tissue loss. We present the case of a woman with long-standing lymphedema suffering from nonhealing ulcerations despite multiple interventions, who underwent below-knee amputation. Surgical pathology yielded a diagnosis of invasive squamous cell carcinoma. We highlight the uncommon association between lymphedema and squamous cell carcinoma, and the importance of routine pathological testing with lower extremity amputations.

Validating the Clinical Value of Temporal Artery Biopsy.

BACKGROUND: Giant cell arteritis (GCA) is a potentially devastating disease that may require treatment with high-dose steroids. Traditionally, diagnosis requires patients to meet at least 3 of 5 clinical criteria, one of which is a positive temporal artery biopsy (TAB). Vascular surgeons are often asked to perform TAB though it is not necessarily required for diagnosis or management. This study aimed to determine if TAB results altered management of patients with a concern for GCA by changing steroid use postoperatively in our health care system. METHODS: A retrospective review at a single-center tertiary care hospital was performed between 2007 and 2018. The inclusion criteria were patients greater than 18 years old with complete steroid treatment records who underwent a temporal artery biopsy due to concern for GCA. Steroid use and duration of treatment both pre- and post-operative were collected and analyzed. RESULTS: Eighty-three of 117 cases reviewed met inclusion criteria. Ninety-one percent (76) of patients had a negative biopsy. Twenty-nine percent (23) of negative biopsies met criteria for GCA prior to biopsy. Of those with a negative biopsy, steroids were continued in 68% (52) of patients after 30 days, 49% (37) after 90 days and 45% (34) after 180 days. Steroids were never started in 11% (6). One patient with a positive biopsy was discontinued on steroids due to intolerance. There was no statistically significant difference in duration of steroids between those with a positive and negative biopsy (average 610 and 787 days respectively; P = 0.682). Average follow up was 33 months. DISCUSSION: The duration of steroid use for patients with concern for GCA was not found to be altered by the performance of a TAB at our institution. Given the extremely low yield and absence of impact on steroid duration, TAB is not a useful diagnostic test at our institution. Similar reviews are recommended to determine the utility of TAB at other institutions that may differ in patient population or prescribing practices.

Temporizing stent graft for aortoenteric fistula with massive gastrointestinal hemorrhage.

OBJECTIVES: We present a case of a 59-year-old male with an actively bleeding aortoenteric fistula (AEF) that was temporized using an endovascular stent prior to staged open reconstruction. METHODS: Verbal informed consent was given by the patient's family for publication of this case report. The patient presented with pulseless electrical activity secondary to hemorrhagic shock due to a massive gastrointestinal bleed. His past surgical history included an aortobifemoral bypass (ABFB) that subsequently underwent extra-anatomic reconstruction with right axillofemoral artery bypass for right femoral infected pseudoaneurysm. Two months prior to presentation, he underwent a second revision with in-situ reconstruction for left limb graft infection. CTA now demonstrated actively bleeding AEF. He was emergently treated with endovascular stenting. Once stabilized, a two-stage revision with extra-anatomic bypass and aortic stump closure for management of his AEF was performed. RESULT: The patient was adequately stabilized using endovascular techniques followed by two-stage revision but unfortunately expired secondary to septic shock 20 days postoperatively. CONCLUSION: This case highlights the utility of endovascular stent graft to successfully obtain hemodynamic stability and optimization prior to open repair of AEFs.

Intraoperative blood pressure lability carries a higher risk of headache after carotid endarterectomy.

OBJECTIVE: Cerebral hyperperfusion syndrome (CHS) is a rare but potentially devastating complication after carotid endarterectomies (CEA). Its symptoms range from new-onset unilateral headache (HA) to intracranial hemorrhage (ICH). Risk factors for CHS in the literature to date have not yet yielded a consensus. This study examines intraoperative and postoperative blood pressure variations as potential risk factors for HA. METHODS: A single-center retrospective review at a tertiary care center from January 2010 to November 2019 was performed. Inclusion criteria were all patients undergoing CEA for symptomatic or asymptomatic carotid disease. Patients with incomplete charts were excluded. Primary endpoints were new-onset unilateral HA or postoperative ICH. Data on intraoperative and postoperative mean arterial pressure (MAP), systolic blood pressure (SBP), the mode of endarterectomy, shunt placement, and contralateral carotid status were collected. RESULTS: There were 735 patients who met the inclusion criteria: 430 patients underwent modified eversion CEA (59%) and 305 patients for patch angioplasty (42%). The incidence of HA was 19% (n = 142) in our total cohort. Of the 19% with HA, 1.5% (n = 11) demonstrated no relief with analgesics and strict blood pressure control; noncontrast head computed tomography scans were performed subsequently. One patient (0.1%) had an ipsilateral ICH. Univariate analysis demonstrated that greater intraoperative MAP peak had the highest risk for HA (odds ratio [OR], 1.014; 95% confidence interval [CI], 1.007-1.022; P = .0002), followed by intraoperative MAP variability (OR, 1.011; 95% CI,1.005-1.018; P ≤ .0008), and peak intraoperative SBP (OR, 1.01; 95% CI, 1.004-1.015; P = .0011). An unpaired Student t test identified change in intraoperative MAP (P < .005), change in the SBP (P < .005), and peak SBP (P < .001) were significantly associated with HA. Interestingly, there was no significant difference between postoperative MAP variability and HA (P = .1). The mode of endarterectomy showed no statistically significant difference in risk for developing HA (OR, 1.165; 95%; 95% CI, 0.801-1.694; P = .42). CONCLUSIONS: Greater intraoperative variability in blood pressures are significantly associated with a higher risk of HA. Adhering to stricter intraoperative blood pressure parameters and limiting blood pressure variability may be beneficial at decreasing the incidence of CHS and its complications.

Chromosome-level genome assembly reveals genomic architecture of northern range expansion in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae).

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bioinformatic tools for correction and further improvement. The new assemblies have dramatically improved contiguity and gaps compared to the originals: N50 values increased 26- to 36-fold, and the number of gaps were reduced by half. Ninety per cent of the content of the assemblies is now contained in 12 and 11 scaffolds for the female and male assemblies, respectively. Based on linkage mapping information, the 12 largest scaffolds in both assemblies represent all 11 autosomal chromosomes and the neo-X chromosome. These assemblies now have nearly chromosome-sized scaffolds and will be instrumental for studying genomic architecture, chromosome evolution, population genomics, functional genomics, and adaptation in this and other pest insects. We also identified regions in two chromosomes, including the ancestral-X portion of the neo-X chromosome, with elevated differentiation between northern and southern Canadian populations.

Traumatic thoracolumbar projectile with concomitant vertebral body and aortic injury.

Penetrating subdiaphragmatic aortic trauma is associated with high morbidity and mortality with studies having reported a 50%-70% associated mortality. We describe a case of a patient with a subdiaphragmatic aortic injury caused by a 7.4-cm common nail that traversed through his L1 vertebral body into the aorta. His aortic injury was managed jointly with vascular surgery and neurosurgery teams.

Poly-Specialty Application Practices of Medical Students Applying to Integrated Vascular Surgery Residency.

BACKGROUND: It is not uncommon for medical students seeking surgical residencies to apply to and rank two or more surgical specialties. Level of interest in a specialty is consistently cited as one of the most important factors for program directors when evaluating applicants for 0 + 5 integrated vascular surgery (IVS) programs. The purpose of this study was to examine trends in poly-specialty application submission to IVS and poly-specialty ranking of IVS to determine the percentage of applicants to IVS residencies with vascular surgery as their true preference. METHODS: Electronic Residency Application Service (ERAS) statistics for noninternational medical graduates from 2011 to 2017 were mined for trends in poly-specialty applications between IVS and other surgical disciplines. The poly-specialty application percentage, range, and standard deviation were determined. The National Resident Match Program (NRMP) results and data from 2011 to 2018 were also used to identify those U.S. seniors who ranked IVS programs as their preferred choice, defined as ranking vascular as the only choice or the first-choice specialty. This was compared with those who ranked a specialty other than vascular surgery first but had vascular surgery listed on their rank list. These data were also collected for applicants to orthopedic surgery, neurosurgery, otolaryngology, obstetrics and gynecology, integrated cardiothoracic surgery, and integrated plastic surgery. RESULTS: Between 2011 and 2017, applicants who submitted ERAS applications to IVS most often poly-specialty applied to IVS and general surgery (87%) followed by IVS and the following: preliminary surgery (71%), plastic surgery (22%), orthopedic surgery (19%), neurosurgery (17%), otolaryngology (16%), obstetrics and gynecology (12%), and urology (3%). The percentage of the applicant pool submitting rank lists with multiple specialties fell over the study period from 94% in 2011 to 67% in 2018. Between 2011 and 2018, an average of 14% of IVS applicants (n = 463), who submitted rank lists to the NRMP, ranked a specialty other than vascular as their true preference (range 7-23 SD 5). Only integrated cardiothoracic surgery had a higher percentage of applicants listing a different specialty as their true preference at 25% (range 18-36 SD 7). Nearly all (97-99%) applicants to orthopedic surgery, neurosurgery, otolaryngology, obstetrics and gynecology, and plastic surgery applied to that specialty as their true preference. CONCLUSIONS: IVS residency applicants were most likely to apply for poly-specialty via the ERAS to general surgery and IVS. Compared to the other surgical specialties, those who submitted rank lists to the NRMP listing integrated cardiothoracic and IVS had the highest likelihood of ranking another specialty higher. Care must be taken when evaluating applications to IVS residencies to determine the applicant's level of interest in vascular surgery as a career.

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death.

The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury.

Arterial protocol including prophylactic distal perfusion catheter decreases limb ischemia complications in patients undergoing extracorporeal membrane oxygenation.

OBJECTIVE: Venoarterial extracorporeal membrane oxygenation (ECMO) is a salvage therapy in patients with severe cardiopulmonary failure. Owing to the large size of the cannulas inserted via the femoral vessels (≤24-F) required for adequate oxygenation, this procedure could result in significant limb ischemic complications (10%-70%). This study evaluates the results of a distal limb perfusion arterial protocol designed to reduce associated complications. METHODS: We conducted a retrospective institutional review board-approved review of consecutive patients requiring ECMO via femoral cannulation (July 2010-January 2015). To prevent arterial ischemia, a distal perfusion catheter (DPC) was placed antegrade into the superficial femoral artery and connected to the ECMO circuit. Limb perfusion was monitored via near-infrared spectroscopy (NIRS) placed on both calves. Decannulation involved open repair, patch angioplasty, and femoral thrombectomy as needed. RESULTS: A total of 91 patients were placed on ECMO via femoral arterial cannula (16-F to 24-F) for a mean duration of 9 days (range, 1-40 days). A percutaneous DPC was inserted prophylactically at the time of cannulation in 55 of 91 patients, without subsequent ischemia. Of the remaining 36 patients without initial DPC placement, 12 (33% without DPC) developed ipsilateral limb ischemia related to arterial insufficiency, as detected by NIRS and clinical findings. In these patients, the placement of a DPC (n = 7) with or without a fasciotomy, or with a fasciotomy alone (n = 4), resulted in limb salvage; only one patient required subsequent amputation. After decannulation (n = 7), no patients had further evidence of limb ischemia. Risk factors for the development of limb ischemia identified by categorical analysis included lack of DPC at time of cannulation and ECMO cannula size of less than 20-Fr. There was a trend toward younger patient age. Overall ECMO survival rate was 42%, whereas survival in patients with limb ischemia was only 25%. CONCLUSIONS: Limb ischemia complications from ECMO may be decreased by prophylactic placement of an antegrade DPC. Without DPC, continuous monitoring using NIRS may identify limb ischemia, which can be treated subsequently with DPC and or fasciotomy.

Adaptive and neutral markers both show continent-wide population structure of mountain pine beetle (Dendroctonus ponderosae).

Assessments of population genetic structure and demographic history have traditionally been based on neutral markers while explicitly excluding adaptive markers. In this study, we compared the utility of putatively adaptive and neutral single-nucleotide polymorphisms (SNPs) for inferring mountain pine beetle population structure across its geographic range. Both adaptive and neutral SNPs, and their combination, allowed range-wide structure to be distinguished and delimited a population that has recently undergone range expansion across northern British Columbia and Alberta. Using an equal number of both adaptive and neutral SNPs revealed that adaptive SNPs resulted in a stronger correlation between sampled populations and inferred clustering. Our results suggest that adaptive SNPs should not be excluded prior to analysis from neutral SNPs as a combination of both marker sets resulted in better resolution of genetic differentiation between populations than either marker set alone. These results demonstrate the utility of adaptive loci for resolving population genetic structure in a nonmodel organism.

Taking the Lead: A Case Report of a Leiomyoma Causing Duodeno-Duodenal Intussusception and Review of Literature.

Background: Duodenal masses are rare entities and symptomatic presentation generally is due to abdominal pain or the presence of gastrointestinal bleeding. A number of published case reports in the literature have detailed various neoplasms that have caused an intussusception isolated to the duodenum. This is a particularly unusual phenomenon due to the location and fixation of this portion of the proximal small bowel to the retroperitoneum. We present here a case of duodeno-duodenal intussusception secondary to a leiomyoma. Case: A 65-year-old Caucasian male presented with intermittent bloody stools and syncope over a 9-month period secondary to a duodenal leiomyoma causing intussusception, which was treated through a pancreaticoduodenectomy. Conclusion: Intussusception of the duodenum is an uncommon entity and the diagnosis of a leiomyoma should be considered in the setting of a potential mass in the small intestine.

Neoadjuvant Chemotherapy and Appleby Procedure for Pancreatic Acinar Cell Carcinoma: A Case Report.

Background: Acinar cell carcinoma is a rare form of pancreatic cancer, accounting for 1-2% of all cases of exocrine pancreatic neoplasms in adults. Due to its rarity, no randomized controlled trials have been performed to determine the optimal treatment options. As such, high-quality case reports and case series are needed to help guide clinicians in the management of this deadly disease. Case Presentation: A 56-year-old Caucasian male presenting with abdominal pain and weight loss was diagnosed with stage III acinar cell carcinoma of the pancreatic body with celiac axis involvement. Although initially deemed unresectable, the patient responded favorably to nine cycles of 5-fluorouracil-based neoadjuvant chemotherapy. The tumor was successfully resected through distal pancreatectomy with en bloc splenectomy and en bloc celiac artery resection (Appleby procedure). Final pathology analysis showed negative resection margins and complete chemotherapeutic response within the pancreas, with residual tumor cells detected in only a single peripancreatic lymph node. Conclusion: 5-fluorouracil-based chemotherapy may be a promising option for the neoadjuvant treatment of locally unresectable acinar cell carcinoma. With sufficient expertise, negative surgical resection margins are possible even with vascular involvement. Due to the generally poor prognosis associated with acinar cell carcinoma, such aggressive treatment measures are warranted.

The first assessment of operative logs for traditional vascular fellowship track versus integrated vascular training programs.

OBJECTIVE: As vascular surgery training paradigms evolve, one measure of success is operative experience. This study assessed the initial operative experience of those graduating from new integrated programs (0+5) vs those from the traditional programs (5+2). METHODS: National operative case log data supplied by the Accreditation Council for Graduate Medical Education was compiled for vascular surgical residents graduating between 2010 and 2013. Mean case numbers for the 0+5 residents were compared with those for the 5+2 residents (experience from their general surgery residency plus vascular fellowship) for total vascular operations, open vascular operations, endovascular procedures, and total operative experience. RESULTS: The 5+2 trainees performed significantly more procedures than the 0+5 trainees (mean, 1605 vs 1015); however, they performed 12% less vascular procedures (mean, 758 vs 851). No significant differences in total number of open vascular operations (mean, 404 vs 411) or specific open operations for cerebral vascular disease, aneurysm, peripheral obstruction, and access were found. The increase in vascular procedures logged by 0+5 trainees was realized by a 24% increase in endovascular procedures, mainly involving diagnostic arteriography, caval filter placement, and balloon angioplasty. No significant differences were seen in endovascular aneurysm repair (mean, 63 vs 60) and stent placement (mean, 59 vs 60). CONCLUSIONS: This report summarizes the first data available for the 0+5 trainee operative experience. Compared with the traditional 5+2 trainees, the 0+5 trainees have (1) equivalent open vascular training and (2) overall superior endovascular training, although this was accounted via an increase in minor procedures. The overall operative experience remains greater for the 5+2 trainees secondary to 2 extra years of training. Further longitudinal studies will be needed to fully characterize the effect of the new 0+5 training paradigm.

The bacteriophage WORiC is the active phage element in wRi of Drosophila simulans and represents a conserved class of WO phages.

BACKGROUND: The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi) is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC. RESULTS: In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB. CONCLUSIONS: The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia.

Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations.

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility.