Outcome of Total Surgical Debridement of Covid Associated Rhino-Orbito-Cerebral Mucormycosis Based on a New Surgical Staging System: A Cohort Study.

To propose a surgical staging system with management protocol for post-covid Rhino-orbito-cerebral mucormycosis (ROCM) with central skull base osteomyelitis. A prospective cohort study of a total of 193 post-covid ROCM patients was conducted between May 2021 and January 2022 at a tertiary care centre. Patients were assessed radiologically and staged from I to V. Follow up period was 16 months and the surgical outcome in terms of recurrent disease was assessed. A total of 193 patients (129 primary and 64 revision) were studied. Maxilla was found to be the epicenter of anterior disease (69.3%) and pterygoid wedge was noted to be the epicenter of posterior disease (85.6%). More than 65% of our patients, at the time of presentation, presented with involvement of the central skull base. Intracranial disease was noted in 13.9% of patients and the mortality rate was 6.2%. This staging system provides a systematic step-by-step protocol for the management of ROCM, with emphasis on meticulous disease clearance at the central skull base.

Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients.

Introduction: Comorbidities and immunosuppressive therapies are associated with reduced immune responses to primary COVID-19 mRNA vaccination in kidney transplant recipients (KTRs). In healthy individuals, prior SARS-COV-2 infection is associated with increased vaccine responses, a phenotype called hybrid immunity. In this study, we explored the potential influence of immune suppression on hybrid immunity in KTRs. Methods: Eighty-two KTRs, including 59 SARS-CoV-2-naïve (naïve KTRs [N-KTRs]) and 23 SARS-CoV-2-experienced (experienced KTRs [E-KTRs]) patients, were prospectively studied and compared to 106 healthy controls (HCs), including 40 SARS-CoV-2-naïve (N-HCs) and 66 SARS-CoV-2-experienced (E-HCs) subjects. Polyfunctional antibody and T cell responses were measured following 2 doses of BNT162b2 mRNA vaccine. Associations between vaccine responses and clinical characteristics were studied by univariate and multivariate analyses. Results: In naïve KTRs, vaccine responses were markedly lower than in HCs and were correlated with older age, more recent transplantation, kidney retransplantation after graft failure, arterial hypertension, and treatment with mycophenolate mofetil (MMF). In contrast, vaccine responses of E-KTRs were similar to those of HCs and were associated with time between transplantation and vaccination, but not with the other risk factors associated with low vaccine responses in naïve KTRs. Conclusion: In conclusion, hybrid immunity overcomes immune suppression and provides potent humoral and cellular immunity to SARS-CoV-2 in KTRs.

Mucosal antibody response and SARS-CoV-2 shedding in patients with COVID-19 related olfactory dysfunction.

Olfactory dysfunction (OD) was one of the most common symptom of infection with the Wuhan strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and could persist for several months after symptom onset. The pathogenesis of prolonged OD remains poorly understood but probably involves sustained viral replication associated with limited mucosal immune response to the virus. This prospective study was conducted to investigate the potential relationship between nasal SARS-CoV-2 viral load and antibody levels in patients with loss of smell. One hundred and five patients were recruited 2 weeks after presenting with confirmed coronavirus disease 2019 associated OD. Based on the identification sniffing test performed at enrollment, 52 patients were still anosmic or hyposmic and 53 were normosmic. SARS-CoV-2 was detectable in nasal wash of about 50% of anosmic and normosmic patients. Higher viral load was detected in anosmic patients with lower levels of SARS-CoV-2 specific nasal immunoglobulins (Ig) IgG and IgA. This association was not observed in normosmic patients. No relationship between nasal viral load and antibodies to endemic coronaviruses was observed. SARS-CoV-2 replication in the nasal cavity may be promoted by defective mucosal antibody responses in patients with OD. Boosting mucosal immunity may limit nasal SARS-CoV-2 replication and thereby help in the control of persistent OD.

Younger Children Develop Higher Effector Antibody Responses to SARS-CoV-2 Infection.

Background: The basis of the less severe clinical presentation of coronavirus disease 2019 (COVID-19) in children as compared with adults remains incompletely understood. Studies have suggested that a more potent boosting of immunity to endemic common cold coronaviruses (HCoVs) may protect children. Methods: To test this hypothesis, we conducted a detailed analysis of antibodies induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children aged 2 months to 14 years. Results: Younger children had higher titers of antibodies to SARS-CoV-2 receptor binding domain (RBD), S1 but not S2 domain, and total spike (S) protein, higher avidity RBD immunoglobulin G, and higher titers of neutralizing and complement-activating antibodies as compared with older children. In contrast, older children had higher titers of antibodies to HCoVs, which correlated with antibodies to the SARS-CoV-2 S2 domain but not with neutralizing or complement-activating antibodies. Conclusions: These results reveal a unique capacity of young children to develop effector antibody responses to SARS-CoV-2 infection independently of their immunity to HCoVs.

Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation.

Antibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSAs) are associated with a higher risk of AMR, not all patients with DSAs develop rejection, suggesting that the characteristics of alloantibodies determining their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we apply systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcγR-binding properties. Levels of afucosylated anti-A2 antibodies are elevated in seropositive patients, especially those with AMR, suggesting potential cytotoxicity via FcγRIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 drives potentiated binding to, slower dissociation from, and enhanced signaling through FcγRIII, a receptor widely expressed on innate effector cells, and greater cytotoxicity against HLA-A2+ cells mediated by natural killer (NK) cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and contribute to pathogenesis.

HIV-Associated Alterations of the Biophysical Features of Maternal Antibodies Correlate With Their Reduced Transfer Across the Placenta.

BACKGROUND: Human immunodeficiency virus (HIV) infection during pregnancy is associated with reduced transplacental transfer of maternal antibodies and increased risk of severe infections in children who are exposed and uninfected with HIV. The basis of this reduced transfer of maternal immunity has not yet been defined but could involve modifications in the biophysical features of antibodies. The objective of this study was to assess the impact of maternal HIV infection on the biophysical features of serum IgG and transplacental antibody transfer. METHODS: Maternal serum IgG subclass levels, Fc glycosylation, Fc receptor (FcR) binding, and transplacental transfer of pathogen-specific maternal IgG were measured in pregnant women with HIV (WWH) and pregnant women testing negative for HIV (WNH) in Cape Town, South Africa. RESULTS: Maternal antibody profiles were strikingly different between pregnant WWH and WNH. Antibody binding to FcγR2a and FcγR2b, IgG1 and IgG3 antibodies, and agalactosylated antibodies were all elevated in WWH, whereas digalactosylated and sialylated antibodies were reduced compared to pregnant WNH. Antibody features that were elevated in WWH were also correlated with reduced transplacental transfer of vaccine antigen-specific antibodies. CONCLUSIONS: HIV infection is associated with marked alterations of biophysical features of maternal IgG and reduced placental transfer, potentially impairing antimicrobial immunity.

A Feasible, Low-Cost, Capsicum and Tomato Model for Endoscopic Sinus and Skull Base Surgery Training.

To describe and standardize an easily available, viable, low-cost capsicum and Tomato model for endoscopic sinus and Skull Base surgery training. Rhinology fellows performed the following stimulated endoscopic sinus exercises using the capsicum and tomato models at our centre using a Karl Storz angled Endoscope and Medtronic Debrider with various angled blades. Each student dissected 10 specimens before training these procedures on human patients, and the benefit of the capsicum and tomato models training was evaluated. 10 rhinology fellows of comparable academic level participated in the training. All participants agreed that the capsicum and tomato model dissections improved their skills in using powered instruments and endoscopic instruments, 90% agreed that the dissections improved their hand-eye coordination, precision, the manoeuvrability of angled blades and confidence with respect to their further training in human patients. A novel technique of low-cost model using Capsicum and Tomato has been used for training fellows in endoscopic sinus and Skull Base surgery. However, no standardization of this training has been performed to ensure that it is a valuable tool for learning and skill-building. The standardized method described in this study increased the skills and confidence of the fellows before beginning their training on human patients. Moreover, our results demonstrate the feasibility of the model, considering its cost-effectiveness and easy availability. A novel technique of low-cost simulation exercises using capsicum and tomato have been described. Future studies with this model should be conducted to assess whether the resulting increase in skills prevents and reduces medical errors, increases patient safety, reduces training costs, and improves the quality of otolaryngological care.

Robust innate responses to SARS-CoV-2 in children resolve faster than in adults without compromising adaptive immunity.

SARS-CoV-2 infection in children is less severe than it is in adults. We perform a longitudinal analysis of the early innate responses in children and adults with mild infection within household clusters. Children display fewer symptoms than adults do, despite similar initial viral load, and mount a robust anti-viral immune signature typical of the SARS-CoV-2 infection and characterized by early interferon gene responses; increases in cytokines, such as CXCL10 and GM-CSF; and changes in blood cell numbers. When compared with adults, the antiviral response resolves faster (within a week of symptoms), monocytes and dendritic cells are more transiently activated, and genes associated with B cell activation appear earlier in children. Nonetheless, these differences do not have major effects on the quality of SARS-CoV-2-specific antibody responses. Our findings reveal that better early control of inflammation as observed in children may be key for rapidly controlling infection and limiting the disease course.

Predictive factors of smell recovery in a clinical series of 288 coronavirus disease 2019 patients with olfactory dysfunction.

BACKGROUND AND PURPOSE: The aim was to evaluate potential predictive factors of smell recovery in a clinical series of 288 patients presenting olfactory dysfunction (OD) related to coronavirus disease 2019 (COVID-19). Potential correlations were sought between epidemiological, clinical and immunological characteristics of patients and the persistence of OD at 60 days. METHODS: COVID-19 positive patients presenting OD were prospectively recruited from three European hospitals. Baseline clinical and olfactory evaluations were performed within the first 2 weeks after OD onset and repeated at 30 and 60 days. In a subgroup of patients, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies were measured in serum, saliva and nasal secretions at 60 days. RESULTS: A total of 288 COVID-19 patients with OD were included in the study. Two weeks after the onset of the loss of smell, 52.4% of patients had OD on psychophysical tests, including 113 cases (39.2%) of anosmia and 38 cases (13.2%) of hyposmia. At 60-day follow-up, 25.4% of the patients presented persistent OD. There was no significant correlation between sex, age, viral load on nasopharyngeal swab or COVID-19 severity and poor olfactory outcome. In a subgroup of 63 patients, it was demonstrated that patients with poor olfactory outcomes at 60 days had lower levels of salivary and nasal immunoglobulin G (IgG) and IgG1, but similar levels of antibodies in the serum. CONCLUSIONS: No clinical markers predicted the evolution of OD at 60 days. Patients with poor olfactory outcome at 60 days had lower saliva and nasal antibodies, suggesting a role for local immune responses in the persistence of COVID-19 related OD.

Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.

A Hybrid Approach Towards Successful Resection of a Huge Carotid Body Paraganglioma Using Coil Embolization and Traditional Surgical Techniques.

Carotid Body Paraganglioma (CBPGL), is a type of neuroendocrine tumor that should be managed promptly due to their malignant potential and locally aggressive nature making resection at a later stage difficult. The objective of this case report is to explore the benefit of coil embolization and describe the surgical techniques employed in successful resection of a huge CBPGL.

PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.

HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation, an enhanced effect at low concentration and an inhibitory effect only at higher concentrations, unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C.

Comprehensive preoperative staging system for endoscopic single and multicorridor approaches to juvenile nasal angiofibromas.

BACKGROUND: Juvenile nasal angiofibromas (JNA) is a benign lesion with high vascularity and propensity of bone erosion leading to skull base invasion and intracranial extension. It is known to involve multiple compartments, which are often surgically difficult to access. With evolution in surgical expertise and technical innovations, endoscopic and endoscopic-assisted management has become the preferred choice of surgical management. Over the last four decades, various staging systems have been proposed, which are largely based on the extent of nasal angiofibroma. However, no clear guidelines exist for the stage-appropriate surgical management. In this study, we aim to formulate a novel staging system based on the analysis of high quality preoperative imaging and propose detailed surgical guidelines related to disease stages as observed in 242 primary cases of JNA. METHODS: A retrospective analysis of the case records of 242 primary JNA cases was performed at our center. Patients were staged according to various existing staging systems as well as our own new staging system, and outcome variables were compared with respect to intraoperative blood loss, multiple staged operations, and tumor recurrences. Operative records were studied and precise endoscopic surgical guidelines were formulated for each stage. RESULTS: Comparing the intraoperative blood loss seen in stages of various classifications, it was found that intraoperative blood loss correlated best and statistically significantly with stages in the newly proposed Janakiram staging system when compared to the existing staging systems. Staged operations were performed in a total of 7/242 patients, and there was a significant association between the requirement of a staged operation and tumor extent (Fischer's exact test, P < 0.001). Tumor recurrence was seen in 22 cases and the pterygoid wedge was found to be the most frequent site of recurrence initially. As the extent of resection improved with better surgical technique over time, recurrences were only found in superior orbital fissure, around the internal carotid artery, and in the middle cranial fossa. CONCLUSION: This new Janakiram staging system is based on preoperative imaging data from one of the largest JNA case series reported thus far. Respective guidelines reliably stratify patients into treatment groups with definite surgical approaches and predicts outcome. Improved surgical approaches in the modern endoscopic era have redefined JNA management with improved outcome. This study shows the importance of precise presurgical imaging and the choice of the most suitable surgical approach in reducing morbidity and mortality in JNA surgery.

Trigeminocardiac reflex during endoscopic juvenile nasopharyngeal angiofibroma surgery: an appraisal.

Juvenile nasopharyngeal angiofibroma is a locally aggressive benign tumour which has propensity to erode the skull base. The tumour spreads along the pathways of least resistance and is in close proximity to the extracranial part of trigeminal nerve. Advancements in expanded approaches for endoscopic excision of tumours in infratemporal fossa and pterygopalatine fossa increase the vulnerability for the trigeminocardiac reflex. The manipulation of nerve and its branches during tumour dissection can lead to sensory stimulation and thus inciting the reflex. The aim of our study is to report the occurrence of trigeminocardiac reflex in endoscopic excision of juvenile nasopharyngeal angiofibroma. To describe the occurence of trigeminocardiac reflex during endoscopic endonasal excision of juvenile nasopharyngeal angiofibroma. We studied the occurrence of TCR in 15 patients (out of 242 primary cases and 52 revision cases) operated for endoscopic endonasal excision of JNA. The drop in mean arterial blood pressure and heart rate were observed and measured. To the best of our knowledge of English literature, this is the first case series reporting TCR as complication in endoscopic excision of JNA. occurence of this reflex has been mentioned in various occular, maxillofacial surgeries but its occurence during endoscopic excision of JNA has never been reported before. Manifestation of trigeminocardiac reflex during surgery can alter the course of the surgery and is a potential threat to life. It is essential for the anesthetist and surgeons to be familiar with the presentations, preventive measures and management protocols.

Imaging in Juvenile Nasopharyngeal Angiofibroma: Clinical Significance of Ramharan and Chopstick Sign.

Imaging plays an important role in the diagnosis, staging and prognosis of JNA. Certain radiological changes as seen on CECT were observed to be consistent in our case series. This study analysed preoperative and postoperative CECT of large series of JNA patients to evaluate the sites and pattern of spread of tumor. We evaluated the clinical significance of pterygoid wedge in preoperative and postoperative imaging and thus elucidating two new radiological signs. Retrospective analysis of the pre operative and post operative imaging data of 242 patients with JNA. The findings in the scan were clinically correlated with the endoscopic intraoperative findings. Preoperative evaluation of the pterygoid wedge revealed widening on the involved side in 99.1% of our cases which is 1.8 times greater compared to the uninvolved side. The possibility of residual/recurrent tumor was found to be significantly higher in those where the pterygoid wedge was not removed by drilling (p < 0.001) Drilling of the pterygoid wedge intra operatively, reduced the rate of residual/recurrence from 31.9 to 3.07%. Widening of the pterygoid wedge seen in the preoperative CECT, referred as RAM HARAN sign occurs in JNA. It has a significant diagnostic value as a radiological sign in JNA. Drilling of the pterygoid wedge intraoperatively reduces the rate of recurrence of JNA. Appearance of the two pterygoid plates on postoperative CECT, as two parallel lines, referred as Chopstick sign, has a remarkable prognostic value as a radiological sign in JNA.

The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa.

BACKGROUND: HIV-1 subtype C demonstrates several biological properties distinct from other viral subtypes. One such variation is the duplication of PTAP motif in p6 Gag. PTAP motif is a key player in viral budding. Here, we studied the prevalence of PTAP motif duplication in subtype C viral strains in a longitudinal study. METHODS: In a prospective follow-up study, 65 HIV-1 seropositive drug-naive subjects were monitored in two different clinical cohorts of India for 2 years with repeated sampling at 6-month intervals. The viral RNA was extracted from plasma, the gag segment was amplified and sequenced. From a subset of viral isolates the sequences of pol, env and LTR were sequenced. Using HIV-1 gag amino acid sequences available from public databases and additional sequences derived from the Indian and South-African cohorts, we examined the nature of PTAP motif duplication in subtype C. RESULTS: In 16% (8 of 50) of the primary viral strains of India, we identified a sequence duplication of the PTAP motif in Gag p6. The length of the sequence duplication varied from 6 to 14 amino acids in the viral isolates but remained fixed within a subject over a period of 24-36 month follow-up. In the duplicated motif, the core PTAP motif was invariable, but the flanking residues were highly variable. In an acute phase clinical cohort of South Africa, in a subset of 75 subjects, we found the presence of the PTAP duplication at a frequency of 29.3%. An analysis of the gag sequences from the extant databases showed that unlike other subtypes of HIV-1, subtype C has a natural propensity to generate the PTAP motif duplication at a significantly higher frequency and of greater length. Additionally, the global prevalence of PTAP duplication in subtype C appears to be increasing progressively over the past 30 years. CONCLUSION: We showed that in subtype C, the duplication of the PTAP motif in p6 Gag involves sequence stretches of greater length, and at a much higher frequency as compared to other HIV-1 subtypes. Given that subtype C naturally lacks the Alix binding motif, the acquisition of an additional PTAP motif may confer replication advantage on this HIV-1 subtype. Further investigation is warranted to examine the significance of PTAP motif duplication on the replicative fitness of HIV-1.

A Drastic Aftermath of Embolisation in Juvenile Nasopharyngeal Angiofibroma.

The role of preoperative embolization in alleviating intra operative haemorrhage in small to medium sized JNA is dubious. We report an unusual case of JNA who developed cerebral edema, hemiplegia and aphasia following glue embolisation and underwent frontotemporal craniectomy. This drastic aftermath of embolisation challenges the safety of preoperative embolisation in such lesions.

Endoscopic Excision of Non-embolized Juvenile Nasopharyngeal Angiofibroma: Our Technique.

To approach Juvenile nasopharyngeal angiofibroma extending to the sphenoid sinus, pterygoid wedge and minimal involvement of the pterygopalatine fossa (Radkowski Stage 2 A) with an endoscopic technique without embolization with no recurrence and minimal morbidity and mortality. This is a retrospective, descriptive study based on the medical records of 15 patients with histologically confirmed JNA who underwent endoscopic binostril four handed endoscopic excision in our centre without embolisation between 2010 and 2015. All 15 patients were young males with a mean age of 14.13 years who underwent endoscopic excision of JNA without embolisation. Nasal obstruction (100 %) and epistaxis (100 %) were the most common symptoms. Average surgery length was 1 h 41 min. Mean blood loss was 67.2 ml and none of patients required blood transfusion. All patients had crusting and septal defect postoperatively, only 3 (20 %) had synechiae. Mean hospitalization time was 3.66 days. 2 (13.33 %) of our patients had a residual tumor and one (6.66 %) had a relapse in pterygoid wedge. There were no cases of death or significant morbidity. The follow up period was 1 year. Endoscopic endo nasal bi nostril four handed technique can achieve complete resection without embolization in case of small to medium sized JNA s in the hands of an experienced surgeon with minimal blood loss, low rates of recurrence and morbidity.

Multiport Combined Endoscopic Approach to Nonembolized Juvenile Nasopharyngeal Angiofibroma with Parapharyngeal Extension: An Emerging Concept.

Background. Surgical approaches to the parapharyngeal space (PPS) are challenging by virtue of deep location and neurovascular content. Juvenile Nasopharyngeal Angiofibroma (JNA) is a formidable hypervascular tumor that involves multiple compartments with increase in size. In tumors with extension to parapharyngeal space, the endonasal approach was observed to be inadequate. Combined Endoscopic Endonasal Approaches and Endoscopic Transoral Surgery (EEA-ETOS) approach has provided a customized alternative of multicorridor approach to access JNA for its safe and efficient resection. Methods. The study demonstrates a case series of patients of JNA with prestyloid parapharyngeal space extension operated by endoscopic endonasal and endoscopic transoral approach for tumor excision. Results. The multiport EEA-ETOS approach was used to provide wide exposure to access JNA in parapharyngeal space. No major complications were observed. No conversion to external approach was required. Postoperative morbidity was low and postoperative scans showed no residual tumor. A one-year follow-up was maintained and there was no evidence of disease recurrence. Conclusion. Although preliminary, our experience demonstrates safety and efficacy of multiport approach in providing access to multiple compartments, facilitating total excision of JNA in selected cases.

The Evolving Profile of the Signature Amino Acid Residues in HIV-1 Subtype C Tat.

Using several HIV-1 tat exon 1 amino acid sequences available from public databases and additional sequences derived from a southern Indian clinical cohort, we compared the profile of the signature amino acid residues (SAR) between two different time periods, 1986-2004 and 2005-2014. The analysis identified eight positions as signature residues in subtype C Tat and demonstrated a changing pattern at four of these positions between the two periods. At three locations (histidine 29, serine 57, and proline 60), there appears to be a nonuniform negative selection against the SAR. The negative selection appears to be severe, especially against histidine 29 (p < .0001) and moderate against proline 60 (p < .0001). The negative selection against serine 57 is statistically insignificant and appears to have begun recently. At position 63, the frequency of signature residue glutamic acid increased over the past decade, although the difference was not significant. Importantly, at the three locations where the negative selection is in progress, the substitute amino acids are the generic residues present in most of the other HIV-1 subtypes. Our data demonstrate that viral evolution can subject specific amino acid residues to subtle and progressive selection pressures without affecting the prevalence of other amino acid residues.