Comparable Clinical and Functional Outcomes Between Osteochondral Allograft Transplantation and Autologous Chondrocyte Implantation for Articular Cartilage Lesions in the Patellofemoral Joint at a Mean Follow-up of 5 Years.

PURPOSE: To assess clinical outcomes and return to sport (RTS) rates among patients that undergo osteochondral allograft (OCA) transplantation and autologous chondrocyte implantation (ACI) or matrix-induced autologous chondrocyte implantation (MACI), for patellofemoral articular cartilage defects. METHODS: A retrospective review of patients who underwent an OCA or ACI/MACI from 2010-2020 was conducted. Patient-reported outcomes (PROs) collected included: Visual Analog Scale for pain/satisfaction, Knee Injury and Osteoarthritis Outcome Score (KOOS), and RTS. The percentage of patients that met the Patient Acceptable Symptom State (PASS) for KOOS was recorded. Logistic regression was used to identify predictors of worse outcomes. RESULTS: A total of 95 patients were included (78% follow-up) with ACI or MACI performed in 55 cases (57.9%) and OCA in 40 (42.1%). A tibial tubercle osteotomy was the most common concomitant procedure for OCA (66%) and ACI/MACI (98%). Overall, KOOS pain was significantly poorer in OCA than ACI/MACI (74.7, 95% CI [68.1, 81.1] vs 83.6, 95% CI [81.3, 88.4], p= 0.012), while the remaining KOOS subscores were non-significantly different (all p>0.05). Overall, RTS rate was 54%, with no significant difference in return between OCA or ACI/MACI (52% vs 58%, p= 0.738). There were 26 (27%) reoperations and 5 (5%) graft failures in the entire group. Increasing age was associated with lower satisfaction in OCA and poorer outcomes in ACI/MACI, while larger lesion area was associated with lower satisfaction and poorer outcomes in ACI/MACI. CONCLUSION: Clinical and functional outcomes were similar in patients that underwent OCA or ACI/MACI for patellofemoral articular cartilage defects at a mean follow-up of 5 years. Patients who received OCA had a higher proportion of degenerative cartilage lesions and, among those with trochlear lesions, reported higher pain at final follow-up than their ACI/MACI counterparts. Overall, increasing age and a larger lesion size were associated with worse patient-reported outcomes.

Clinical and Functional Outcomes of Documented Knee Dislocation Versus Multiligamentous Knee Injury: A Comparison of KD3 Injuries at Mean 6.5 Years Follow-up.

BACKGROUND: Previous research has found that the incidence of neurovascular injury is greatest among multiligamentous knee injuries (MLKIs) with documented knee dislocation (KD). However, it is unknown whether there is a comparative difference in functional recovery based on evidence of a true dislocation. PURPOSE: To determine whether the knee dislocation-3 (KD3) injury pattern of MLKI with documented tibiofemoral dislocation represents a more severe injury than KD3 MLKI without documented dislocation, as manifested by poorer clinical outcomes at long-term follow-up. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A multicenter retrospective cohort study was performed of patients who underwent surgical treatment for KD3 MLKI between May 2012 and February 2021. Outcomes were assessed using the International Knee Documentation Committee (IKDC) score, Lysholm score, Tegner activity scale, and visual analog scale (VAS) for pain. Documented dislocation was defined as a radiographically confirmed tibiofemoral disarticulation, the equivalent radiology report from outside transfer, or emergency department documentation of a knee reduction maneuver. Subgroup analysis was performed comparing lateral (KD3-L) versus medial (KD3-M) injuries. Multivariable linear regression was conducted to determine whether documented dislocation was predictive of outcomes. RESULTS: A total of 42 patients (25 male, 17 female) were assessed at a mean 6.5-year follow-up (range, 2.1-10.7 years). Twenty patients (47.6%) were found to have a documented KD; they reported significantly lower IKDC (49.9 vs 63.0; P = .043), Lysholm (59.8 vs 74.5; P = .023), and Tegner activity level (2.9 vs 4.7; P = .027) scores than the patients without documented dislocation. VAS pain was not significantly different between groups (36.4 vs 33.5; P = .269). The incidence of neurovascular injury was greater among those with documented dislocation (45.0% vs 13.6%; P = .040). Subgroup analysis found that patients with KD3-L injuries experienced a greater deficit in Tegner activity level than patients with KD3-M injuries (Δ: -3.4 vs -1.2; P = .006) and had an increased incidence of neurovascular injury (41.7% vs 11.1%; P = .042). Documented dislocation status was predictive of poorer IKDC (ß = -2.15; P = .038) and Lysholm (ß = -2.85; P = .007) scores. CONCLUSION: Patients undergoing surgical management of KD3 injuries with true, documented KD had significantly worse clinical and functional outcomes than those with nondislocated joints at a mean 6.5-year follow-up. The current MLKI classification based solely on ligament involvement may be obscuring outcome research by not accounting for true dislocation.

Patients who undergo tibial tubercle anteromedialization with medial patellofemoral ligament reconstruction demonstrate similar rates of return to sport compared to isolated MPFL reconstruction.

PURPOSE: To investigate the rate of return to sports and sport psychological readiness between patients who underwent isolated MPFLR (iMPFLR) compared to a matched cohort of patients who underwent MPFLR with anteromedializing tibial tubercle osteotomy (MPFLR/TTO). METHODS: Patients who underwent primary MPFLR with or without TTO for recurrent patellar instability were retrospectively reviewed from 2012 to 2020 at a single institution. Preinjury sport and work information, Kujala, Tegner, Visual Analogue Score for pain, satisfaction and MPFL-Return to Sport after Injury (MPFL-RSI) score were collected. Two readers independently measured the tibial tuberosity-trochlear groove distance, Caton-Deschamps index and Dejour classification for trochlear dysplasia. Patients in iMPFLR and MPFLR/TTO groups were matched 1:1 on age, sex, body mass index and follow-up length. Multivariate regression analysis was performed to determine whether the MPFL-RSI was associated with a return to sport. RESULTS: This study included 74 patients at mean follow-up of 52.5 months (range: 24-117). These groups returned to sport at similar rates (iMPFLR: 67.6%, MPFLR/TTO: 73.0%, not significant [ns]), though iMPFLR patients returned more quickly (8.4 vs. 12.8 months, p = 0.019). Rates of return to preinjury sport level were also similar (45.9% vs. 40.5%, ns). Patients with Dejour B/C took more time to return to sport compared to patients with mild/no trochlear pathology (13.8 vs. 7.9 months, p = 0.003). Increasing MPFL-RSI score was significantly predictive of the overall return to sport (odds ratio [OR]: 1.08, 95% confidence interval [CI] [1.03, 1.13], p < 0.001) and return to preinjury level (OR: 1.07, 95% CI [1.04, 1.13], p < 0.001). Most patients in iMPFLR and MPFLR/TTO groups resumed work (95.7% vs. 88.5%, ns), though iMPFLR patients who returned to preinjury work levels did so more quickly (1.7 vs. 4.6 months, p = 0.005). CONCLUSION: Patients who underwent MPFLR with anteromedializing TTO demonstrated similar rates of return to sport and psychological readiness compared to an isolated MPFLR matched comparison group, though iMPFLRs returned more quickly. Patients with more severe trochlear pathology required more time to return to sports. LEVEL OF EVIDENCE: Level III.

Increased kinesiophobia leads to lower return to sport rate and clinical outcomes following osteochondral allograft transplantation of the knee.

PURPOSE: The purpose of this study is to describe the postoperative psychological state of patients following osteochondral allograft (OCA) transplantation in the knee and to determine whether patient-perceived kinesiophobia is associated with the rate of return to sport (RTS). METHODS: A retrospective review of the electronic medical record at a single institution was conducted for all patients that underwent OCA transplantation from January 2010 to 2020. Patient-reported outcomes including the visual analog scale (VAS), knee injury and osteoarthritis outcome score (KOOS) and the Tampa scale of kinesiophobia-11 (TSK-11) were collected. Patients were surveyed regarding their postoperative RTS status. RESULTS: A total of 38 patients (52.6% female) were included in our analysis. Overall, 24 patients (63.2%) returned to sport with 12 (50%) of these patients returning at a lower level of play. When comparing patients that return to sport to those that did not, patients that return had significantly superior KOOS pain (p = 0.019) and KOOS QOL (p = 0.011). Measures of kinesiophobia (TSK-11) were significantly higher among patients that did not return to sport (p = 0.014), while satisfaction (n.s.) and pain intensity (n.s.) were comparable between groups. Logistic regression models controlling for demographic factors, VAS pain scores and lesion size showed that for every one-point increase in TSK-11 kinesiophobia score, patients were 1.33 times more likely to return to sport at a lower level (p = 0.009). For every one-point increase in TSK-11 scores KOOS QOL decreased by 2.4 points (p < 0.001). CONCLUSION: Fear of reinjury decreases the likelihood that patients will return to their preoperative level of sport after OCA transplantation. Patients that do not return to sport report significantly greater fear of reinjury and inferior clinical outcomes, despite similar levels of satisfaction and pain compared to those that return. LEVEL OF EVIDENCE: Level III.

Patient-reported outcomes and return to pre-injury activities after surgical treatment of multi-ligamentous knee injuries in patients over 40-years-old: Average 5-years follow-up.

BACKGROUND: Multi-ligamentous knee injuries (MLKI) are potentially devastating injuries, though existing prognostic research among older patients who sustain MLKI is limited. The purpose was to investigate clinical outcomes and rates of return to pre-injury activities following surgical treatment of MLKI in patients at least 40 years old. METHODS: This study was a multi-center retrospective case series of patients who underwent surgical treatment for MLKI from 2013-2020 and were ≥ 40 years old at time of injury. Outcomes were assessed via e-mail and telephone using the International Knee Documentation Committee (IKDC) score, Lysholm score, Tegner activity scale, a satisfaction rating, and return to pre-injury sport and work surveys. Stepwise linear regression was used to assess the impact of preoperative characteristics on IKDC and Lysholm scores. RESULTS: Of 45 patients eligible for inclusion, 33 patients (mean age: 48.6 years [range: 40-72]) were assessed at a mean follow-up of 59.1 months (range 24-133). The cohort reported a mean IKDC of 63.4 ± 23.5, Lysholm of 72.6 ± 23.6, and Tegner of 3.8 ± 2.0. There was a 41.2% rate of return to sports, and 82.1% returned to work. Documented knee dislocation was predictive of poorer IKDC (ß:-20.05, p = 0.025) and Lysholm (ß:-19.99, p = 0.030). Patients aged > 50 were more satisfied compared to those 40-50 years old (96.2 ± 4.9 vs 75.6 ± 23.3, p = 0.012). CONCLUSIONS: Patients who sustained MLKI aged at least 40 at injury demonstrated fair clinical outcomes at a mean 5-year follow-up. Older patients who sustained MLKI reported a relatively high rate of return to work but were less likely to return to sports. LEVEL OF EVIDENCE: IV, Case series.

Posterior Tibial Slope in Patients Undergoing Bilateral Versus Unilateral ACL Reconstruction: MRI and Radiographic Analyses.

BACKGROUND: An increased posterior tibial slope (PTS) is a risk factor for primary anterior cruciate ligament (ACL) tears and graft failure, but the PTS has not been well-defined in those who have experienced bilateral ACL injuries. PURPOSE: The primary aim was to compare the PTS, as well as the rate of an elevated PTS (>12° on lateral radiography; >7° on magnetic resonance imaging [MRI]), between patients who have undergone bilateral ACL reconstruction (ACLR) versus unilateral ACLR. A secondary purpose was to examine whether these associations remained consistent on both plain radiography and MRI. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: We retrospectively identified patients who underwent primary ACLR at our institution from the years 2012 to 2020. Patients who underwent nonsimultaneous bilateral ACLR (n = 53) were matched to those who underwent unilateral ACLR (n = 53) by age, sex, and body mass index. Exclusion criteria were rotated lateral radiographs, MRI scans of inadequate quality, and concomitant ligament injuries or fractures. Those who had undergone unilateral ACLR with <5-year follow-up were further excluded. There were 3 blinded readers who measured the PTS on lateral radiographs, while the medial PTS (MPTS) and lateral PTS (LPTS) were measured on MRI scans. Bivariate regression was performed to determine the correlation between radiographic and MRI measurements. RESULTS: The PTS on radiography (11.26° vs 10.13°, respectively; P = .044) and the LPTS on MRI (7.32° vs 6.08°, respectively; P = .012) in the bilateral ACLR group were significantly greater than those in the unilateral ACLR group but not the MPTS on MRI (4.55° vs 4.17°, respectively; P = .590). The percentage of patients in the bilateral group with a radiographic PTS >12° was 41.0% compared with 13.2% in the unilateral group (P = .012). The bilateral group had a significantly higher rate of an LPTS >7° compared with the unilateral group (53.8% vs 32.1%, respectively; P = .016) but not for an MPTS >7° (P = .190). On MRI, the LPTS (6.90°± 2.73°) was significantly greater than the MPTS (4.41°± 2.92°) (P < .001). There was a weak correlation between MPTS and radiographic PTS measurements (R = 0.24; P = .021), but LPTS and radiographic PTS measurements were not significantly correlated (R = 0.03; P = .810). CONCLUSION: Patients who underwent bilateral ACLR had a significantly greater PTS on radiography and a significantly greater LPTS on MRI compared with those who underwent unilateral ACLR. The rate of a radiographic PTS >12° was 2.4 times greater among patients undergoing bilateral ACLR compared with those undergoing unilateral ACLR. PTS measurements on radiography demonstrated a weak to negligible correlation with PTS measurements on MRI, suggesting that future normative PTS values should be reported specific to the imaging modality.

Kinesiophobia and Pain Catastrophizing Leads to Decreased Return to Sport Following Autologous Chondrocyte Implantation but Does Not Affect Return to Work.

OBJECTIVE: To evaluate the effect of fear of reinjury on return to sport (RTS), return to work (RTW), and clinical outcomes following autologous chondrocyte implantation (ACI). DESIGN: A retrospective review of patients who underwent ACI with a minimum of 2 years in clinical follow-up was conducted. Patient-reported outcomes collected included the Visual Analog Scale (VAS) and Knee Injury and Osteoarthritis Outcome Score (KOOS). Kinesiophobia and pain catastrophizing was assessed using the Tampa Scale of Kinesiophobia-11 (TSK-11) and Pain Catastrophizing Scale (PCS), respectively. Patients were surveyed on their RTS and RTW status. RESULTS: Fifty-seven patients (50.9% female) were included in our analysis. Twenty-two (38.6%) patients did not RTS. Of the 35 patients (61.4%) who returned, nearly half (48.6%) returned at a lower level of play. TSK-11 (P = 0.003), PCS (P = 0.001), and VAS pain scores (P < 0.001) were significantly greater in patients that did not RTS than in those who returned. All KOOS subscores analyzed were significantly lower (P < 0.001) in patients that did not RTS than in those who returned at the same level or higher. Of the 44 (77.2%) patients previously employed, 97.7% returned to work. Increasing TSK-11 scores were associated with lower odds of returning to sport (P = 0.003). CONCLUSION: Fear of reinjury decreases the likelihood that patients will return to sport after ACI. Patients that do not return to sport report significantly greater levels of fear of reinjury and pain catastrophizing and lower clinical knee outcomes. Nearly all patients were able to return to work after surgery. LEVEL OF EVIDENCE: IV case series.

Return to Play After Knee Articular Cartilage Restoration: Surgical Options, Rehabilitation Protocols, and Performance Outcomes.

PURPOSE OF REVIEW: Numerous cartilage restoration techniques have proven to be effective in the treatment of articular cartilage defects. The ultimate goal of these procedures is to improve pain and function, thereby increasing the likelihood of a patient's return to physical activity. Postoperative rehabilitation is a key component for a successful and expedient return to activities. The purpose of this article is to review the current literature regarding common surgical options, rehabilitation protocols, and performance outcomes after operative treatment of articular cartilage defects. RECENT FINDINGS: Studies have demonstrated improved short- to long-term outcomes in a majority of techniques. However, the clinical benefits of microfracture are short-lived, which has led to the use of alternative procedures. Rehabilitation protocols are not standardized, but emphasis has been placed on bracing, weightbearing, early continuous passive range of motion, and strengthening to improve function. There is growing evidence to suggest that accelerated rehabilitation after matrix-induced autologous chondrocyte implantation may result in superior outcomes compared to delayed rehabilitation. Overall, most techniques result in satisfactory rates of return to play, though existing comparative studies typically include patients with heterogeneous pathology, complicating effective synthesis of outcomes data. In appropriately selected patients, cartilage restoration procedures after articular cartilage injury result in favorable patient-reported clinical outcomes and high rates of return to play. While studies emphasize the critical role that rehabilitation plays with respect to outcomes after surgery, there are substantial inconsistencies in protocols across techniques.

Angiotensin Receptor Blockers and Angiotensin-Converting Enzyme Inhibitors Have No Significant Relationship With Postoperative Arthrofibrosis After Shoulder Arthroscopy.

Purpose: To determine the effect of perioperative angiotensin II receptor blocker (ARB) or angiotensin-converting enzyme inhibitors (ACEi) on postoperative arthrofibrosis, as defined by requiring manipulation under anesthesia (MUA) or new diagnosis of adhesive capsulitis (AC) following arthroscopic shoulder procedures. Methods: Patients were retrospectively identified using Current Procedural Terminology surgical billing codes to identify patients who underwent any shoulder arthroscopic procedure at a single urban academic institution from 2012 to 2020 with a minimum 2-year follow-up. Patients were excluded if <30 years old at time of surgery, as these patients rarely use ARB and ACEi medications, or if they had pre-existing AC. Demographics, active medication prescriptions at the time of surgery, and medical comorbidities were recorded. Multivariable logistic regression was performed to determine the effect of ARB/ACEi on subsequent MUA or AC by 90 days, 1 year, and 2 years. Results: In total, 5,559 patients were included in the final analysis. A majority of the cohort (53.4%) underwent arthroscopic surgery between the ages of 50 to 69 years. Most patients were male (61.8%) and without obesity (67.3%). In total, 18.9% and 15.0% were taking an ARB or ACEi medication perioperatively, respectively. Within 2 years' postoperatively, 51 patients (0.9%) underwent subsequent ipsilateral MUA, and 174 patients (3.1%) developed AC. Patients taking ARBs had a 17.5% rate of postoperative arthrofibrosis within 2-years compared with 19.1% in those not on ARBs, although this difference was not significant (P = .58). Likewise, no significant difference was found between those taking ACEi versus not (15.0% vs 15.0%, P = .99). Individual generic ARB/ACEi subgroup analysis did not demonstrate any significant associations with rate of postoperative arthrofibrosis (P > .05). Conclusions: ARBs or ACEi did not significantly affect the rate of postoperative arthrofibrosis following shoulder arthroscopy, however female sex, diabetes mellitus, and Black/African American race were associated with an increased rate of necessitating MUA or developing AC within 2 years postoperatively. Level of Evidence: Level III, retrospective cohort study.

Patients who undergo bilateral medial patellofemoral ligament reconstruction return to sport at a similar rate as those that undergo unilateral reconstruction.

PURPOSE: Lateral patellar instability is a debilitating condition not only to athletes, but also to a wide range of highly active individuals. Many of these patients experience symptoms bilaterally, though it is unknown how these patients fair with return to sports following a second medial patellofemoral ligament reconstruction (MPFLR). The purpose of this study is to evaluate the rate of return to sport following bilateral MPFLR compared to a unilateral comparison group. METHODS: Patients who underwent primary MPFLR with minimum 2-year follow-up were identified from 2014 to 2020 at an academic center. Those who underwent primary MPFLR of bilateral knees were identified. Pre-injury sport participation and Tegner score, Kujala score, Visual Analog Score (VAS) for pain, satisfaction, and MPFL-Return to Sport after Injury (MPFL-RSI) scale were collected. Bilateral and unilateral MPFLRs were matched in a 1:2 ratio based on age, sex, body mass index, and concomitant tibial tubercle osteotomy (TTO). A sub-analysis was performed regarding concomitant TTO. RESULTS: The final cohort consisted of 63 patients, including 21 patients who underwent bilateral MPFLR, matched to 42 unilateral patients at mean follow-up of 47 ± 27 months. Patients who underwent bilateral MPFLR returned to sport at a rate of 62% at a mean of 6.0 ± 2.3 months, compared to a unilateral rate of 72% at 8.1 ± 4.2 months (n.s.). The rate of return to pre-injury level was 43% among bilateral patients and 38% in the unilateral cohort. There were no significant differences in VAS pain, Kujala, current Tegner, satisfaction, and MPFL-RSI scores between cohorts. Approximately half of those (47%) who failed to return to sport cited psychological factors and had significantly lower MPFL-RSI scores (36.6 vs 74.2, p = 0.001). CONCLUSION: Patients who underwent bilateral MPFLR returned to sports at a similar rate and level compared to a unilateral comparison group. MPFL-RSI was found to be significantly associated with return to sport. LEVEL OF EVIDENCE: III.

Posterior tibial tubercle measured by the sagittal TT-TG distance correlates with increased risk for patellofemoral chondral lesions.

PURPOSE: To evaluate the variation in tibial tubercle sagittal alignment in patients with and without patellofemoral (PF) cartilage wear. METHODS: This was a single-centre, retrospective review of patients that underwent a cartilage restoration procedure for isolated PF cartilage wear from 2014 to 2020. Patients were matched in a 1:2 ratio for age, sex and BMI to partial meniscectomy patients as controls. The sagittal TT-TG (sTT-TG) distance was measured on preoperative axial T2 magnetic resonance imaging (MRI) and was defined as the distance between a point at the nadir of the trochlear cartilage and the most anterior point of the tibial tubercle. RESULTS: One hundred and forty patients (47 cartilage restoration, 94 meniscectomy) were included. Mean age, BMI, and height for the total cohort were 34.01 ± 8.7, 26.6 ± 6.4, and 173.0 ± 17.7 respectively, with 78 males (55%) and 63 females (45%). There were no significant differences between groups for age, BMI or sex (n.s). The cartilage restoration group (- 2.5 mm ± 5.9) was found to have a significantly more posterior (negative) sTT-TG compared to the meniscectomy group (1.72 mm ± 6.7) (p < 0.001). Interrater reliability was excellent (ICC = 0.931, p < 0.001). Patients with less than - 3.4 mm sTT-TG were 2.74 times more likely to have a cartilage restoration procedure compared to those with greater than - 3.4 mm (OR 2.7, 95% CI 1.3-5.85). Patients with < - 10 mm posterior translation were 13.7× (CI 1.6-111.1) more likely to have a cartilage restoration procedure. CONCLUSION: Patients that underwent isolated cartilage restoration procedures had a significantly more posterior tibial tubercle than partial meniscectomy controls based on the sagittal TT-TG. The more posterior the tubercle, the more likely the patient had a cartilage restoration procedure. Surgeons should consider the sTT-TG measurement in patients presenting with anterior knee pain, particularly patellofemoral lesions. LEVEL OF EVIDENCE: III.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila.

BACKGROUND: Little is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells. RESULTS: The objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses. CONCLUSIONS: We demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.