Measuring intraoperative anesthetic parameters during hepatectomy with inferior vena cava clamping.

PURPOSE: Uncontrollable bleeding remained problematic in anatomical hepatectomy exposing hepatic veins. Based on the inferior vena cava (IVC) anatomy, we attempted to analyze the hemodynamic and surgical effects of the combined IVC-partial clamp (PC) accompanied with the Trendelenburg position (TP). METHODS: We prospectively assessed 26 consecutive patients who underwent anatomical hepatectomies exposing HV trunks between 2020 and 2023. Patients were divided into three groups: use of IVC-PC (group 1), no use of IVC-PC (group 2), and use of IVC-PC accompanied with TP (group 3). In 10 of 26 patients (38%), hepatic venous pressure was examined using transhepatic catheter insertion. RESULTS: IVC-PC was performed in 15 patients (58%). Operating time and procedures did not significantly differ between groups. A direct hemostatic effect on hepatic veins was evaluated in 60% and 70% of patients in groups 1 and 3, respectively. Group 1 showed significantly more unstable vital status and vasopressor use (p < 0.01). Blood or fluid transfusion and urinary output were similar between groups. Group 2 had a significantly lower baseline central venous pressure (CVP), while group 3 showed a significant increase in CVP in TP. CVP under IVC-PC seemed lower than under TP; however, not significantly. Hepatic venous pressure did not significantly differ between groups. Systolic arterial blood pressure significantly decreased via IVC-PC in group 1 and to a similar extent in group 3. Heart rate significantly increased during IVC-PC (p < 0.05). CONCLUSION: IVC-PC combined with the TP may be an alternative procedure to control intrahepatic venous bleeding during anatomical hepatectomy exposing hepatic venous trunks.

Single-Stage Surgical Treatment of Acute Type A Aortic Dissection and Blunt Abdominal Trauma: A Case Report.

A 53-year-old man suddenly developed chest and back pain while driving, resulting in an accident. Computed tomography revealed acute type A aortic dissection with malperfusion of the left lower extremity, retroperitoneal extravasation, hematoma in the anterior mediastinum, and ascites in the rectovesical pouch. Exploratory laparotomy before aortic repair revealed intestinal perforation and retroperitoneal bleeding, which were repaired, and an ascending aortic replacement was performed. Visceral trauma with active bleeding should be treated with priority, even if the need for systemic heparinization accompanies acute type A aortic dissection during surgery for aortic dissection.

Biological functions and structural biology of Plasmodium falciparum autophagy-related proteins: The under-explored options for novel antimalarial drug design.

Malaria remains a threat to global public health and the available antimalarial drugs are undermined by side effects and parasite resistance, suggesting an emphasis on new potential targets. Among the novel targets, Plasmodium falciparum autophagy-related proteins (PfAtg) remain a priority. In this paper, we reviewed the existing knowledge on the functions and structural biology of PfAtg including the compounds with inhibitory activity toward P. falciparum Atg8-Atg3 protein-protein interaction (PfAtg8-PfAtg3 PPI). A total of five PfAtg (PfAtg5, PfAtg8, PfAtg12, PfAtg18, and Rab7) were observed to have autophagic and/or non-autophagic roles. Available data showed that PfAtg8 has conserved hydrophobic pockets, which allows it to interact with PfAtg3 to form PfAtg8-PfAtg3 PPI. Additionally, 2-bromo-N-(4-pyridin-2-yl-1,3-thiazol-2-yl) benzamide was identified as the most powerful inhibitor of PfAtg8-PfAtg3 PPI. Due to the dearth of knowledge in this field, we hope that the article would open an avenue to further research on the remaining PfAtg as possible drug candidates.

Successful Right Hepatectomy for Recurrent Liver Tumor Originating from an Inferior Vena Cava Leiomyosarcoma: A Follow-Up Case Report.

BACKGROUND Leiomyosarcoma (LMS) of the inferior vena cava (IVC) is a rare malignancy, and complete resection may provide better patient survival. Hepatectomy for intrahepatic tumor recurrence has not been previously reported. CASE REPORT A 58-year-old woman underwent resection of an IVC-LMS with en bloc nephrectomy, adrenalectomy, and retroperitoneal soft tissue resection without IVC reconstruction 3 years 8 months ago. Twenty-nine months after the primary operation, a solitary intrahepatic liver tumor was found adjacent to the right and middle hepatic veins during imaging follow-up. The patient was diagnosed with LMS recurrence. As her liver functional parameters permitted major hepatectomy, right hepatectomy combined with resection of the vena cava wall leaving a tumor-free margin and securing the confluence of the middle hepatic vein were successfully accomplished via an anterior approach, without adverse events. Intrahepatic metastasis of LMS invading the vena cava wall has been diagnosed historically. Her postoperative course was uneventful, and at 1-year follow-up after the second surgery, she was observed to have no tumor relapse without any adjuvant treatment. Previous reports have shown that IVC-LMS is often observed, and operative risk or prognosis is based on the extension of the LMS toward the hepatic veins or cardiac atrium. CONCLUSIONS Radical hepatectomy for recurrent IVC-LMS has not been previously reported, and our case experience revealed that a challenging surgical intervention resulting in complete tumor removal can provide good survival outcomes.

Knowledge survey regarding blast wound education of student doctors at a local academic medical university in Japan.

Objectives: To improve knowledge about blast injury for medical student doctors or surgeons. In the modern uncertain era, education and training programs for blast injuries for medical student doctors or surgeons are recently necessary worldwide. Material and Methods: To understand primary corresponding ability to treat blast injuries, leading to improvement of the trauma education curriculum, a retrospective study by a knowledge survey was performed between 2018 and 2019. The subject had the title of Student Doctor (SD) at university. Results: The answers of 183 participants who answered the interview questionnaire with 16 questions were summarized. Although most SDs received basic lectures for trauma medicine and majority of SDs knew about mass casualty incidents and primary treatment, the existence of knowledge on soft targets is limited. One-fourth of the SDs knew the characteristics of blast wounds. Most SDs understood priority triage for a conscious person with massive bleeding from a limb with hemostasis to save lives. The 17% selected cardiopulmonary resuscitation first and 72% of SDs could explain hemorrhagic shock; however, only four could explain adequate hemostatic procedures. Most had no interest regarding necessity of their knowledge in the field of serious blast trauma wounds. Conclusion: Experience in trauma surgery training from stages in SDs and authorized education are important for raising students' knowledge of unexpected serious blast incidents.

A case of diaphragmatic hemangioma completely resected by partial diaphragmatic resection.

Diaphragmatic hemangiomas are rare tumors and the preferred resection range in surgical procedures is considered on a case-by-case basis. We report a case of diaphragmatic hemangioma that was completely resected by partial diaphragmatic resection. An 81-year-old man was referred for the examination of right diaphragmatic mass. Computed tomography revealed two contrast-enhanced nodules (diameter: 17 and 10 mm, respectively) on the right diaphragm. The nodules were completely resected by partial resection of the diaphragm via video-assisted thoracic surgery using an ultrasonic coagulation and incision device. Resection was performed leaving part of the muscular layer of the diaphragm. Histopathology confirmed the nodule to be hemangioma originating from the diaphragm and no hemangiomatous lesions were noted in the normal connective tissue in the resected stump. Partial diaphragmatic resection is a less invasive treatment method and may be a useful surgical procedure for diaphragmatic hemangioma.

Successful Surgical Repair of Type A Acute Aortic Dissection in a Patient with Vascular Ehlers-Danlos Syndrome.

Surgery for vascular complications of a patient with vascular Ehlers-Danlos syndrome (vEDS) is challenging due to the fragility of the associated tissues. In this study, we present a type A acute aortic dissection case in a patient with vEDS successfully treated via total arch replacement. A 42-year-old woman was transferred to our hospital 10 days after the onset of symptoms and underwent emergency surgery. Intraoperative findings revealed severe inflammatory changes without tissue fragility that is distinctive of vEDS. The postoperative course was uneventful except for left recurrent laryngeal nerve palsy, and 24 months after the operation, the patient has remained free from any arterial event.

Motor Progression and Nigrostriatal Neurodegeneration in Parkinson Disease.

OBJECTIVE: The motor severity in Parkinson disease (PD) is believed to parallel dopaminergic terminal degeneration in the striatum, although the terminal was reported to be virtually absent by 4 years postdiagnosis. Meanwhile, neuromelanin-laden dopamine neuron loss in the substantia nigra (SN) elucidated a variability at early stages and gradual loss with less variability 10 years postdiagnosis. Here, we aimed to clarify the correlation between motor impairments and striatal dopaminergic terminal degeneration and nigral neuromelanin-laden dopamine neuron loss at early to advanced stages of PD. METHODS: Ninety-three PD patients were divided into early and advanced subgroups based on motor symptom duration and whether motor fluctuation was present. Striatal dopaminergic terminal degeneration was evaluated using a presynaptic dopamine transporter tracer, 123 I-ioflupane single photon emission computed tomography (SPECT). Nigral neuromelanin-laden dopamine neuron density was assessed by neuromelanin-sensitive magnetic resonance imaging (NM-MRI). RESULTS: In patients with early stage PD (motor symptoms for ≤8 or 10 years), motor dysfunction during the drug-off state was paralleled by a decline in 123 I-ioflupane uptake in the striatum despite the absence of a correlation with reductions in NM-MRI signals in SN. Meanwhile, in patients with advanced stage PD (motor symptoms for >8 or 10 years and with fluctuation), the degree of motor deficits during the drug-off state was not correlated with 123 I-ioflupane uptake in the striatum, despite its significant negative correlation with NM-MRI signals in SN. INTERPRETATION: We propose striatal dopaminergic terminal loss measured using 123 I-ioflupane SPECT and nigral dopamine neuron loss assessed with NM-MRI as early stage and advanced stage motor impairment biomarkers, respectively. ANN NEUROL 2022;92:110-121.

Fragment-Based Drug Discovery for Trypanosoma brucei Glycosylphosphatidylinositol-Specific Phospholipase C through Biochemical and WaterLOGSY-NMR Methods.

Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) of Trypanosoma brucei, the causative protozoan parasite of African trypanosomiasis, is a membrane-bound enzyme essential for antigenic variation, because it catalyses the release of the membrane-bound form of variable surface glycoproteins. Here, we performed a fragment-based drug discovery of TbGPI-PLC inhibitors using a combination of enzymatic inhibition assay and water ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiment. The TbGPI-PLC was cloned and overexpressed using an Escherichia coli expression system followed by purification using three-phase partitioning and gel filtration. Subsequently, the inhibitory activity of 873 fragment compounds against the recombinant TbGPI-PLC led to the identification of 66 primary hits. These primary hits were subjected to the WaterLOGSY NMR experiment where 10 fragment hits were confirmed to directly bind to the TbGPI-PLC. These included benzothiazole, chlorobenzene, imidazole, indole, pyrazol and quinolinone derivatives. Molecular docking simulation indicated that six of them share a common binding site, which corresponds to the catalytic pocket. The present study identified chemically diverse fragment hits that could directly bind and inhibit the TbGPI-PLC activity, which constructed a framework for fragment optimization or linking towards the design of novel drugs for African trypanosomiasis.

[An adult case of neurocutaneous melanosis with acute exacerbation after a long asymptomatic period following excision of a melanocytic nevus].

Neurocutaneous melanosis is caused by postzygotic NRAS mutations in neural crest cells, resulting in large or multiple nevi in the skin and proliferation of leptomeningeal melanocytes in the central nervous system. The onset of neurological symptoms is usually before the age of 2 years, but it can also occur in adults. A 35-year-old male had been asymptomatic for a long time after excision of a large congenital melanocytic nevus, but he developed headache, disturbance of consciousness, and seizure. Methotrexate was ineffective, cerebral pressure was decreased by spinal drainage, and steroid pulse therapy was temporarily effective. Seizures and disturbance of consciousness worsened and the patient died on the 92nd day. Cerebrospinal fluid human melanin black-45 immunostaining and serum 5-S-cysteinyldopa (5-S-CD) were useful in diagnosing melanocytic proliferation, and serum 5-S-CD may be useful in predicting prognosis.

Current status of trauma surgery at a Japanese prefectural academic institute: improved organization in a regional prefecture.

PURPOSES: Balancing scheduled surgery and trauma surgery is difficult with a limited number of surgeons. To address the issues and systematize education, we analyzed the current situation and the effectiveness of having a trauma team in the ER of a regional hospital. METHODS: This retrospective study analyzed the demographics, traumatic variables, procedures, postoperative morbidities, and outcomes of 110 patients who underwent trauma surgery between 2012 and 2019. The trauma team was established in 2016 and our university hospital Emergency Room (ER) opened in 2012. RESULTS: Blunt trauma accounted for 82% of the trauma injuries and 39% of trauma victims were transported from local centers to our institute. The most frequently injured organs were in the digestive tract and about half of the interventions were for hemostatic surgery alone. Concomitant treatments for multiple organ injuries were performed in 31% of the patients. The rates of postoperative severe complications (over Clavien-Dindo IIIb) and mortality were 10% and 13%, respectively. Fourteen (12.7%) of 24 patients who underwent damage-control surgery died, with multiple organ injury being the predominant cause of death. CONCLUSION: Systematic education or training of medical students and general surgeons, as well as the co-operation of the team at the regional academic institute, are necessary to overcome the limited human resources and save trauma patients.

Influence of preoperative right ventricular function on left ventricular remodeling and survival after subvalvular repair for functional mitral regurgitation.

OBJECTIVES: The objective of this study was to analyze our surgical experiences with mitral valve plasty (MVP) combined with subvalvular procedures (SVPs) for functional mitral regurgitation (FMR) and to determine which preoperative factors affected clinical outcomes. METHODS: This study retrospectively analyzed 33 patients who underwent MVP combined with SVPs for FMR with a left ventricular ejection fraction lower than 40% and advanced remodeled left ventricles. The mean follow-up period was 49 ± 33 months. RESULTS: The preoperative mean right ventricular fractional area change (RVFAC) used to quantify right ventricular (RV) systolic function was 26 ± 11%. Sixteen patients (48%) had an RVFAC < 26%. One patient died during hospital stay, and nine more patients died of cardiac causes during follow-up. The 3- and 5-year rates of freedom from cardiac-related mortality were 78% and 68%, respectively. RVFAC was the significant predictor of cardiac-related mortality in a univariate analysis (risk ratio [RR] = 0.92, 95% confidence interval [CI] 0.85-0.99, p = 0.03) and demonstrated a non-significant tendency to predict cardiac-related mortality in the Cox multivariate analysis (RR = 0.94, 95% CI 0.86-1.003, p = 0.08). Continued reverse left ventricular remodeling was associated with an RVFAC ≥ 26%. At 3 years, there was also a significant difference in survival rates of cardiac-related mortality between patients with an RVFAC ≥ 26% and < 26% (94% vs. 61%; p = 0.03). CONCLUSIONS: Preoperative RV function affected left ventricular remodeling and cardiac-related mortality after MV surgery. MVP combined with SVPs for FMR provided promising results for patients without severe RV dysfunction.

Clinical Outcomes of a Customized Mitral Valve Plasty for Functional Mitral Regurgitation with a Low Ejection Fraction and Implications for Preoperative Right Ventricular Function.

PURPOSE: To evaluate clinical outcomes of customized mitral valve plasty (MVP) for the treatment of functional mitral regurgitation (FMR) with a low ejection fraction (EF) and to determine which preoperative factors affected the clinical outcome. METHODS AND RESULTS: MVP was adjusted according to the degree of left ventricle (LV) remodeling. We performed mitral annuloplasty (MAP) alone in 14 patients and added subvalvular procedures (SVPs) in 22 patients at a high risk of recurrent MR. During follow-up, reverse LV remodeling was obtained and the 3-year and 5-year non-recurrence rates of MR grade ≥2 were 94% and 89%, respectively. Two patients died during their hospital stay, and four more patients died of cardiac causes during follow-up. The 3-year and 5-year rates of freedom from cardiac-related mortality were 86% and 81%, respectively; no significant difference was observed between the two treatment groups. Right ventricular fractional area change (RVFAC) was a significant predictor of cardiac mortality. Patients with an RVFAC of <26% had significantly poorer cardiac-related mortality (71% at 3 years) than those with an RVFAC of ≥26% (95% at 3 years). CONCLUSION: Customized MVP provided durable mitral competence and reverse LV remodeling. Preoperative RV function was associated with cardiac-related mortality.

Successful treatment of left ventricular patch infection by latissimus dorsi muscle flap covering without patch removal.

Removal of the infected prosthesis is considered an essential procedure in the treatment of prosthetic graft infection following cardiovascular surgery. Here, we present a case of left ventricular patch infection following repair of left ventricular rupture that was successfully treated by coverage with a latissimus dorsi muscle flap without patch removal. A 61-year-old man underwent double-patch repair for left ventricular-free wall rupture following posterior myocardial infarction. He underwent drainage and omental transposition with re-sternotomy for postoperative mediastinitis by Candida albicans, followed by pericardial fenestration via left thoracotomy for infectious pericarditis; however, left ventricular patch infection was detected. Considering the high invasiveness of a reoperation for patch removal, we preserved and covered the patch using a left pedicled latissimus dorsi muscle flap via left thoracotomy. The postoperative course was uneventful, and the patient was asymptomatic with no signs of recurrence at 30 months.

[A Case of Positional Vertebral Artery Occlusion due to Physiological Cervical Extension with Occipital Condylar Spur].

Positional vertebral artery occlusion(PVAO)is a mechanical occlusion of the extracranial vertebral artery(VA)due to physiological movement of the head and neck. However, only a few cases of mechanical VA compression due to routine flexion-extension of the neck have been reported. We present a unique case of PVAO due to neck extension with an occipital condylar spur. A 78-year-old man was admitted to our hospital for sudden onset of right hemiparesis and dysarthria. Magnetic resonance imaging(MRI)revealed bilateral occipital and cerebellar infarctions and vessel occlusion extending from the VA to the basilar artery. Mechanic thrombectomy resulted in partial recanalization. Computed tomography angiography(CTA)performed the next day showed spontaneously recanalized left VA with some wall irregularity. CTA in the neck-extended position revealed a severely compressed left VA in its V3 segment, which was attributed to the left occipital condylar spur with degenerative changes of the condyle-C1 facet. Cervical MRI also showed a pseudotumor from the lower clivus to the odontoid process that indicated mechanical stress on the occipitocervical ligaments. An occiput to C2 fusion was performed to stabilize and avoid dynamic vascular compression. Postoperative CTA revealed no evidence of restricted flow with flexion or extension movements of the neck. It should be noted that physiological head and neck movements accompanied by condylar degenerative changes could be a cause of vertebrobasilar insufficiency.

Congenital Abdominal Aortic Aneurysm in a Four Year Old Girl.

INTRODUCTION: Abdominal aortic aneurysm (AAA) in neonates, infants, and children is uncommon, usually occurring as a result of infections, connective tissue disorders, vasculitis, or iatrogenic trauma. A case of idiopathic congenital AAA, an extremely rare disease of unknown origin, is described. REPORT: In March 2018, a 40 mm hypoechoic mass adjacent to the left kidney was detected incidentally by abdominal ultrasound for investigation of hypercalciuria in a four year old girl. Contrast enhanced computed tomography (CT) revealed an infrarenal fusiform AAA measuring 39 mm in maximum diameter, a 15 mm left renal artery aneurysm, a 14 mm right hypogastric artery aneurysm, and a 12 mm left hypogastric artery aneurysm. Cerebral magnetic resonance imaging revealed multiple intracranial aneurysms between 8 and 15 mm in diameter. Considering the size of the AAA and risk of rupture, surgical repair was planned. In May 2018, the congenital AAA was successfully repaired with a 10 mm Dacron aorto-aortic tube graft. Increases in the size of the left renal artery aneurysm and a left middle meningeal artery aneurysm were detected 12 and 14 months post-operatively, respectively. Coil embolisations were performed. An intracranial dural arteriovenous fistula (AVF) was discovered incidentally by cerebral angiography for treatment of the left middle meningeal artery aneurysm. Transarterial embolisation for AVF was also performed. At the 21 month post-operative follow up, the patient is doing well, and the untreated aneurysms have not grown. CONCLUSION: Long term outcomes after surgical repair for congenital AAA are unclear. Moreover, growth of residual aneurysms was detected post-operatively, so follow up with frequent multimodality imaging for multiple aneurysms is necessary.

[Successful Total Resection with Preceding Arterial Coil Embolization of Intradural Extramedullary Tumor at Craniovertebral Junction Encasing Dominant-side Vertebral Artery].

OBJECTIVE: The surgical resection of craniovertebral junction(CVJ)meningioma is challenging because of the neighboring brainstem, lower cranial nerves, and vertebral artery(VA). Moreover, encasement of the VA by the tumor can raise the risk of complications and require cautious manipulation during surgery. CASE: A 46-year-old woman presented with a one-year history of neck pain. She had temporal hemiplegia and numbness on her left side. Magnetic resonance imaging(MRI)showed a CVJ meningioma pushing the brainstem from the right vertebral side and encasing the right VA. Digital subtraction angiography(DSA)showed two feeding arteries arising from the right VA and a sunburst sign. The right VA was the dominant side but did not have the right posterior inferior cerebellar artery(PICA). The anterior spinal artery(ASA)was dominant in the left VA. We performed a balloon test occlusion(BTO)for 20 min and it did not cause any complications;therefore, we occluded the VA using endovascular coils. After 4 days, we removed the meningioma in the prone position, using a far-lateral approach and C1-laminectomy. The laterally located meningioma pushed the brainstem. After detaching the tumor from the dura, we cut the encased VA and the tumor was resected safely(Simpson grade II). Postoperatively, she developed temporal thermal hypoalgesia on the left side of her body. Magnetic resonance imaging showed a microinfarction in the medulla. CONCLUSION: If the VA test occlusion provides a clear result, pre-operative endovascular sacrifice of the VA encased by CVJ meningioma is a feasible treatment strategy.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Extended sandwich technique via the right atrial approach for post-infarction posterior ventricular septal rupture.

An 84-year-old man who underwent percutaneous coronary intervention for acute inferior myocardial infarction due to occlusion of the mid portion of the right coronary artery was transferred to our hospital because of post-infarction posterior ventricular septal rupture. We performed the extended sandwich technique via the right atrial approach as well as tricuspid and mitral valve replacement and permanent pacemaker implantation. Mild residual shunt was detected post-operatively, but the patient's condition was controlled well with diuretics.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.