Noninvasive Evaluation of Breast Tumor Response to Combined Ultrasound-Stimulated Microbubbles and Hyperthermia Therapy Using Quantitative Ultrasound-Based Texture Analysis Method.

OBJECTIVES: Quantitative ultrasound (QUS) is a noninvasive imaging technique that can be used for assessing response to anticancer treatment. In the present study, tumor cell death response to the ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) treatment was monitored in vivo using QUS. METHODS: Human breast cancer cell lines (MDA-MB-231) were grown in mice and were treated with HT (10, 30, 50, and 60 minutes) alone, or in combination with USMB. Treatment effects were examined using QUS with a center frequency of 25 MHz (bandwidth range: 16 to 32 MHz). Backscattered radiofrequency (RF) data were acquired from tumors subjected to treatment. Ultrasound parameters such as average acoustic concentration (AAC) and average scatterer diameter (ASD), were estimated 24 hours prior and posttreatment. Additionally, texture features: contrast (CON), correlation (COR), energy (ENE), and homogeneity (HOM) were extracted from QUS parametric maps. All estimated parameters were compared with histopathological findings. RESULTS: The findings of our study demonstrated a significant increase in QUS parameters in both treatment conditions: HT alone (starting from 30 minutes of heat exposure) and combined treatment of HT plus USMB finally reaching a maximum at 50 minutes of heat exposure. Increase in AAC for 50 minutes HT alone and USMB +50 minutes was found to be 5.19 ± 0.417% and 5.91 ± 1.11%, respectively, compared to the control group with AAC value of 1.00 ± 0.44%. Furthermore, between the treatment groups, ΔASD-ENE values for USMB +30 minutes HT significantly reduced, depicting 0.00062 ± 0.00096% compared to 30 minutes HT only group, showing 0.0058 ± 0.0013%. Further, results obtained from the histological analysis indicated greater cell death and reduced nucleus size in both HT alone and HT combined with USMB. CONCLUSION: The texture-based QUS parameters indicated a correlation with microstructural changes obtained from histological data. This work demonstrated the use of QUS to detect HT treatment effects in breast cancer tumors in vivo.

Dental caries, tobacco usage and associated risk factor of dental caries in patients visiting a government hospital in Western, Nepal.

BACKGROUND: This study was conducted to assess the prevalence of dental caries, tobacco usage, and associated risk factors for dental caries in patients who visited a government hospital in Western, Nepal. METHODS: This analytical cross-sectional study was conducted from January to April 2022. Patients above 18 years visiting the dental OPD of a government hospital, and who had provided informed consent were enrolled in the study using a convenience sampling technique. As the study also involved an illiterate population, in that case, informed consent was obtained from their respective legal guardian as well. A pretested standardized, close-ended questionnaire was administered by researchers to gather information regarding the associated risk factors and oral hygiene practices. Clinical examination was done for dental caries according to the criteria by the World Health Organization (WHO) using the "DMFT" index (WHO modification 1987). Bivariate and multivariable logistic regression was done and the odds ratio and p-value was calculated. For all tests, statistical significance was set at p < 0.05. RESULTS: A total of 219 participants completed the study with a mean age of 31.73 ± 12.46. The prevalence of dental caries and tobacco was found to be 80.36% and 5.02% respectively. Participants without health insurance had 2.35 times higher odds of dental caries (95% CI: 1.03-5.36). Not rinsing the mouth after eating sweets was associated with 3.07 times higher odds of dental caries (95% CI: 1.31-7.18). Those who hadn't visited a dentist in the past 12 months had lower odds (0.42; 95% CI: 0.18-0.94). Eating fresh fruit daily showed statistically higher odds (2.70; 95% CI: 1.04-6.99) of dental caries. Non-tobacco users had higher odds (14.19; 2.55-78.99) of dental caries. CONCLUSION: Dental caries is highly prevalent, while tobacco usage is relatively low. Factors associated with dental caries included lack of health insurance coverage, consumption of fruits once daily, recent dental visits within the past year, not rinsing the mouth with water after consuming sweets, and non-tobacco users.

Ultrasound-stimulated microbubbles enhancement of fractionated radiation for tumor treatment.

BACKGROUND: Radiation therapy (XRT) causes numerous biological changes in tumor microenvironment. Radiation vascular response, due to endothelial disruption, can influence treatment outcomes in a dose-dependent manner. Ultrasound-stimulated microbubbles (USMB) have also been demonstrated to create a vascular response in the tumor microenvironment and enhance tumor response when used in combination with XRT. Single doses of 8-10 Gy are known to induce activation of acid sphingomyelinase (ASMase)-induced ceramide production, causing vascular damage. Destruction of vasculature results in endothelial apoptosis followed by tumor cell death. The effect of tumor response is known to be synergistic by 10-fold higher cell kill observed when USMB is combined with radiation. METHODS: In this study, we used an USMB approach in combination with conventional low dose fractionated radiation to enhance endothelial cell responses to XRT in human PC3 prostate cancer xenograft model. Mice were divided into untreated, USMB therapy, fractionated XRT, and combined USMB therapy followed by XRT (USMB + XRT) groups. USMB therapy was delivered twice per week in the USMB-alone and combined USMB + XRT treatment groups over four weeks. Radiation treatments were delivered in fractions of 2 Gy/day (total 40 Gy in 20 fractions, BED10 = 48 Gy) in the XRT-alone and combined USMB + XRT groups. The treatment outcome was evaluated using histopathology, power Doppler, and immunohistochemistry assays. RESULTS: Tumor growth assessment showed that sizes of tumors increased in the control and the single treatment groups over a treatment period of four weeks, but significantly decreased with the combined treatments of USMB + XRT. Immunohistochemical analysis indicated a statistically significant vascular disruption in mice that received treatment involving a full 4-week schedule of combined (USMB + XRT) treatments. A statistically significant increase in vascular disruption was demonstrated through CD68 and trichrome fibrosis staining. Changes in local perfusion assessed using high-frequency power Doppler imaging demonstrated attenuated blood flow in the combined group. DISCUSSION AND CONCLUSIONS: This work demonstrates the efficacy of using USMB as a radiation sensitizer in a mouse model of human PC3 tumor xenograft. This radiation treatment enhancement modality has the advantage of targeting tumor vasculature with ultrasound stimulation that can be implemented prior to radiation treatment.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

Progress Toward Measles and Rubella Elimination - India, 2005-2021.

In 2019, India, along with other countries in the World Health Organization (WHO) South-East Asia Region,* adopted the goal of measles and rubella elimination by 2023,† a revision of the previous goal of measles elimination and control of rubella and congenital rubella syndrome (CRS) by 2020§ (1-3). During 2017-2021, India adopted a national strategic plan for measles and rubella elimination (4), introduced rubella-containing vaccine (RCV) into the routine immunization program, launched a nationwide measles-rubella supplementary immunization activity (SIA) catch-up campaign, transitioned from outbreak-based surveillance to case-based acute fever and rash surveillance, and more than doubled the number of laboratories in the measles-rubella network, from 13 to 27. Strategies included 1) achieving and maintaining high population immunity with at least 95% vaccination coverage by providing 2 doses of measles- and rubella-containing vaccines; 2) ensuring a sensitive and timely case-based measles, rubella and CRS surveillance system; 3) maintaining an accredited measles and rubella laboratory network; 4) ensuring adequate outbreak preparedness and rapid response to measles and rubella outbreaks; and 5) strengthening support and linkages to achieve these strategies, including planning and progress monitoring, advocacy, social mobilization and communication, identification and utilization of synergistic linkages of integrated program efforts, research, and development. This report describes India's progress toward the elimination of measles and rubella during 2005-2021, with a focus on the years 2017-2021.¶ During 2005-2021, coverage with the first dose of a measles-containing vaccine (MCV) administered through routine immunization increased 31%, from 68% to 89%. During 2011-2021, coverage with a second MCV dose (MCV2) increased by 204%, from 27% to 82%. During 2017-2021, coverage with a first dose of RCV (RCV1) increased almost 14-fold, from 6% to 89%. More than 324 million children received a measles- and rubella-containing vaccine (MRCV) during measles-rubella SIAs completed in 34 (94%) of 36 states and union territories (states) during 2017-2019. During 2017-2021, annual measles incidence decreased 62%, from 10.4 to 4.0 cases per 1 million population, and rubella incidence decreased 48%, from 2.3 to 1.2 cases per 1 million population. India has made substantial progress toward measles and rubella elimination; however, urgent and intensified efforts are required to achieve measles and rubella elimination by 2023.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety.

Background & objectives: Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS-CoV-2 used in these programmes. Methods: Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin-Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription-polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control. Results: SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per µl. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively. Interpretation & conclusions: Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories.

Effect of Ultrasound-Stimulated Microbubbles and Hyperthermia on Tumor Vasculature of Breast Cancer Xenograft.

OBJECTIVE: The objective of the present study was to investigate the treatment effects of ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) on breast tumor vasculature. METHODS: Tumor-bearing mice with breast cancer xenografts (MDA-MB-231), were exposed to different treatment conditions consisting of control (no treatment), USMB alone, HT alone, USMB with HT exposures of 10 and 50 minutes. Quantitative 3D Doppler ultrasound and photoacoustic imaging were used to detect tumor blood flow and oxygen saturation, respectively. In addition, histopathological analysis including TUNEL staining for cell death, and CD31 staining for the vessel count, was performed to complement the results of power Doppler and photoacoustic imaging. RESULTS: Results demonstrated a decrease in tumor blood flow as well as oxygenation level following 50 minutes HT treatment either alone or combined with USMB. In contrast, 10 minutes HT alone or combined with USMB had minimal effects on blood flow and tumor oxygenation level. Treatment with HT for 50 minutes caused drops in tumor oxygenation, which were not evident with USMB treatment alone. Additionally, results revealed an increase in cell death after 10 minutes HT with or without USMB and a decrease in vessel count compared to control. Unlike previous studies which demonstrated synergistic treatment effects combining USMB with other modalities such as radiation or chemotherapy, USMB and HT effects were not synergistic in the present study. CONCLUSION: The results here demonstrated HT and USMB both alone or together resulted in a significant reduction in tumor blood flow, tumor oxygenation, and vessel count with observed increases in cell death response.

Involvement of Ceramide Signalling in Radiation-Induced Tumour Vascular Effects and Vascular-Targeted Therapy.

Sphingolipids are well-recognized critical components in several biological processes. Ceramides constitute a class of sphingolipid metabolites that are involved in important signal transduction pathways that play key roles in determining the fate of cells to survive or die. Ceramide accumulated in cells causes apoptosis; however, ceramide metabolized to sphingosine promotes cell survival and angiogenesis. Studies suggest that vascular-targeted therapies increase endothelial cell ceramide resulting in apoptosis that leads to tumour cure. Specifically, ultrasound-stimulated microbubbles (USMB) used as vascular disrupting agents can perturb endothelial cells, eliciting acid sphingomyelinase (ASMase) activation accompanied by ceramide release. This phenomenon results in endothelial cell death and vascular collapse and is synergistic with other antitumour treatments such as radiation. In contrast, blocking the generation of ceramide using multiple approaches, including the conversion of ceramide to sphingosine-1-phosphate (S1P), abrogates this process. The ceramide-based cell survival "rheostat" between these opposing signalling metabolites is essential in the mechanotransductive vascular targeting following USMB treatment. In this review, we aim to summarize the past and latest findings on ceramide-based vascular-targeted strategies, including novel mechanotransductive methodologies.

Application of Ultrasound Combined with Microbubbles for Cancer Therapy.

At present, cancer is one of the leading causes of death worldwide. Treatment failure remains one of the prime hurdles in cancer treatment due to the metastatic nature of cancer. Techniques have been developed to hinder the growth of tumours or at least to stop the metastasis process. In recent years, ultrasound therapy combined with microbubbles has gained immense success in cancer treatment. Ultrasound-stimulated microbubbles (USMB) combined with other cancer treatments including radiation therapy, chemotherapy or immunotherapy has demonstrated potential improved outcomes in various in vitro and in vivo studies. Studies have shown that low dose radiation administered with USMB can have similar effects as high dose radiation therapy. In addition, the use of USMB in conjunction with radiotherapy or chemotherapy can minimize the toxicity of high dose radiation or chemotherapeutic drugs, respectively. In this review, we discuss the biophysical properties of USMB treatment and its applicability in cancer therapy. In particular, we highlight important preclinical and early clinical findings that demonstrate the antitumour effect combining USMB and other cancer treatment modalities (radiotherapy and chemotherapy). Our review mainly focuses on the tumour vascular effects mediated by USMB and these cancer therapies. We also discuss several current limitations, in addition to ongoing and future efforts for applying USMB in cancer treatment.

In vivo assessment of prostate cancer response using quantitative ultrasound characterization of ultrasonic scattering properties.

BACKGROUND: The study here investigated quantitative ultrasound (QUS) parameters to assess tumour response to ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) treatment in vivo. Mice bearing prostate cancer xenografts were exposed to various treatment conditions including 1% (v/v) Definity microbubbles stimulated at ultrasound pressures 246 kPa and 570 kPa and HT duration of 0, 10, 40, and 50 min. Ultrasound radiofrequency (RF) data were collected using an ultrasound transducer with a central frequency of 25 MHz. QUS parameters based on form factor models were used as potential biomarkers of cell death in prostate cancer xenografts. RESULTS: The average acoustic concentration (AAC) parameter from spherical gaussian and the fluid-filled spherical models were the most efficient imaging biomarker of cell death. Statistical significant increases of AAC were found in the combined treatment groups: 246 kPa + 40 min, 246 kPa + 50 min, and 570 kPa + 50 min, in comparison with control tumours (0 kPa + 0 min). Changes in AAC correlates strongly (r2 = 0.62) with cell death fraction quantified from the histopathological analysis. CONCLUSION: Scattering property estimates from spherical gaussian and fluid-filled spherical models are useful imaging biomarkers for assessing tumour response to treatment. Our observation of changes in AAC from high ultrasound frequencies was consistent with previous findings where parameters related to the backscatter intensity (AAC) increased with cell death.

SARS-CoV-2 detection in sewage samples: Standardization of method & preliminary observations.

Background & objectives: Since its first recognition in Wuhan, China, in December 2019, the SARS-CoV-2 has spread rapidly across the world. Though SARS-CoV-2 spreads mainly via the droplets of respiratory secretions, it was also detected in stool samples of patients, indicating active infection of the gastrointestinal tract. Presence of SARS-CoV-2 RNA in sewage samples was reported in February 2020, raising the possibility of using environmental water surveillance to monitor SARS-CoV-2 activity in infected areas. The aim of this study was to standardize the methodology for detection of SARS-CoV-2 from sewage and explore the feasibility of establishing supplementary surveillance for COVID-19. Methods: Sewage specimens were collected from six sites in Mumbai, India, using the grab sample method and processed using polyethylene glycol (PEG)-dextran phase separation method for virus concentration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the presence of SARS-CoV-2 RNA. Results: A total of 20 sewage samples collected from six different wards in Mumbai city, before the spread of SARS-CoV-2 infections and during May 11-22, 2020, were processed using the phase separation method. The WHO two-phase PEG-dextran method was modified during standardization. SARS-CoV-2 was found to concentrate in the middle phase only. All samples collected before March 16, 2020 were SARS-CoV-2 negative. Viral RNA was detected in sewage samples collected during the ongoing COVID-19 pandemic in all the six wards. Interpretation & conclusions: PEG-dextran phase separation method was effectively used to concentrate SARS-CoV-2 from domestic waste waters to detection levels. It would be feasible to initiate sewage surveillance for SARS-CoV-2 to generate data about the viral transmission in various epidemiologic settings.

SLE presenting as DAH and relapsing as refractory retinitis.

Dear Editor, A 17-year-old boy, diagnosed with Systemic Lupus Erythematosus (SLE), presented to ophthalmology department with gradual painless diminution of vision in both eyes (right more than left). He had already received 6 pulses of cyclophosphamide and steroids at monthly intervals one year back for diffuse alveolar hemorrhage (DAH) and was on maintenance oral 40 mg prednisolone and 3 grams mycophenolate mofetil (MMF). There was no history of oliguria, skin rash, joint pain, oral ulcers, photosensitivity or any neurological deficit in this presentation. There was no proteinuria, hematuria or worsening of renal function.

Diagnostic Assay Development for Poliovirus Eradication.

With poliovirus eradication nearing, few pockets of active wild poliovirus (WPV) transmission remain in the world. Intratypic differentiation (ITD) plays a crucial part in laboratory surveillance as the molecular detection method that can identify and distinguish wild and vaccine-like polioviruses isolated from acute flaccid paralysis cases or environmental sources. The need to detect new variants of WPV serotype 1 (WPV1) and the containment of all serotype 2 polioviruses (PV2) in 2015 required changes to the previous version of the method. The ITD version 5.0 is a set of six real-time reverse transcription-PCR (rRT-PCR) assays that serve as accurate diagnostic tools to easily detect and differentiate PV serotypes and genotypes. We describe the creation and properties of quantitation standards, including 16 control RNA transcripts and nine plaque-isolated viruses. All ITD rRT-PCR assays were validated using these standards, and the limits of detection were determined for each assay. We designed and pilot tested two new assays targeting recently circulating WPV1 genotypes and all PV2 viruses. The WPV1 assay had 99.1% specificity and 100% sensitivity, and the PV2 assay had 97.7% specificity and 92% sensitivity. Before proceeding to the next step in the global poliovirus eradication program, we needed to gain a better understanding of the performance of the ITD 5.0 suite of molecular assays and their limits of detection and specificities. The findings and conclusions in this evaluation serve as building blocks for future development work.

Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr) in human poliovirus receptor gene.

BACKGROUND & OBJECTIVES: It is important to understand the role of cell surface receptors in susceptibility to infectious diseases. CD155 a member of the immunoglobulin super family, serves as the poliovirus receptor (PVR). Heterozygous (Ala67Thr) polymorphism in CD155 has been suggested as a risk factor for paralytic outcome of poliovirus infection. The present study pertains to the development of a screening test to detect the single nucleotide (SNP) polymorphism in the CD155 gene. METHODS: New primers were designed for PCR, sequencing and SNP analysis of Exon2 of CD155 gene. DNAs extracted from either whole blood (n=75) or cells from oral cavity (n=75) were used for standardization and validation of the SNP assay. DNA sequencing was used as the gold standard method. RESULTS: A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150) of DNA samples tested by both SNP detection assay and sequencing. INTERPRETATION & CONCLUSIONS: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

Genetic diversity of enterovirus A71, India.

We have identified circulation of 3 genogroups of enterovirus (EV) A71 in India. A new genogroup (proposed designation G) was discovered during this study. We isolated genogroups D and G in wide geographic areas but detected subgenogroup C1 only in 1 focus in western India. A systematic nationwide search for EV-A71 is warranted.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

BACKGROUND & OBJECTIVES: The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. METHODS: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. RESULTS: No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. INTERPRETATION & CONCLUSIONS: rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

Hypoxic Ischemic Encephalopathy with Cervical Spinal Cord Injury: A Diagnostic Dilemma.

Perinatal spinal cord injury is a relatively uncommon, but a frequently misdiagnosed disorder. Improvements in obstetric care have certainly led to a decrease in the incidence of birth related spinal cord trauma but unfortunately the incidence of hypoxic-ischemic encephalopathy is still very high. The exact incidence of spinal cord trauma is difficult to determine because the spinal cord is not routinely examined in far and few neonatal autopsies done in India. Here, authors present a neonate who received treatment for birth asphyxia and then had extubation failure which made the clock tick towards cervical cord injury. This baby had a hemorrhagic contusion of cervical spinal cord.