In-vitro antiviral activity of Carica papaya formulations against dengue virus type 2 and chikungunya viruses.

Dengue and chikungunya are diseases of global health significance and currently, no antivirals are available to treat these arboviral diseases. Carica papaya leaves extract is traditionally used to treat thrombocytopenia in patients infected with the dengue virus. The current study was undertaken to study the antiviral activity of commercially available Carica papaya leaves extract (CPLE) based products and CPLE prepared in four formulations against dengue virus type 2 (DENV-2) and chikungunya virus (CHIKV). Maximum nontoxic concentrations of the commercially available CPLE based products and CPLE based formulations (Carica papaya leaves in powder form, Carica papaya leaves in lyophilized form, Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves) were used for screening the antiviral activity. The antiviral activity against DENV-2 and CHIKV were assessed post infection using focus forming unit assay. Effective formulations were tested under different conditions i.e. pretreatment, cotreatment and posttreatment. The virus output after treatment was assessed by real-time RT-PCR, immunofluorescence assay and focus forming unit assay. The results revealed Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves formulations showed significant inhibition in case of DENV while papaya leaves in powder form showed significant reduction in case of CHIKV. This study demonstrates the antiviral activity of CPLE formulations against DENV-2 and CHIKV infection in in-vitro system and needs further validation in in-vivo models.

Serotype and genotype diversity of dengue viruses circulating in India: a multi-centre retrospective study involving the Virus Research Diagnostic Laboratory Network in 2018.

OBJECTIVES: A retrospective study was undertaken to investigate the circulating dengue virus (DENV) serotypes and genotypes in India in 2018. METHODS: In total, 4963 samples referred to virus research diagnostic laboratories (n=21), the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) and ICMR-NIV field units (n=2) for diagnosis of dengue in 2018 were tested using a real-time reverse transcription polymerase chain reaction assay for the presence of DENV serotypes. Representative samples were sequenced for the envelope (E) gene. RESULTS: Regional diversity was observed with regard to the dominant circulating serotypes. DENV-2 was found to be the most common serotype in many states. Thrombocytopenia, petechiae and malaise were associated with DENV-2 infection. Phylogenetic analyses of DENV E gene sequences revealed the circulation of genotypes I and V of DENV-1, two lineages of DENV-2 genotype IV, DENV-3 genotype III and DENV-4 genotype I. CONCLUSIONS: This study found regional differences in the prevalence of circulating DENV serotypes in India, and provides baseline data for continuous molecular surveillance. Molecular surveillance may have implications for predicting large-scale outbreaks of dengue if regional shifts in the predominantly circulating serotypes and genotypes are detected during the early phase of the dengue season.

Use of whole blood over plasma enhances the detection of dengue virus RNA: possible utility in dengue vaccine trials.

In the present study, the utility of viral RNA isolated from whole blood over plasma for detection of dengue virus (DENV) was investigated in 80 samples referred for serotyping by DENV serotype-specific one-step real-time RT-PCR. DENV RNA was detected in 71.25% of the whole blood samples compared to 46.25% in the corresponding plasma samples. In secondary infections, DENV RNA was detected in 83.3% of whole blood samples, while it was detected in 40.5% of plasma samples (P = 0.0001). Non-structural protein 1 (NS1) antigen was detected in only 54.8% of the secondary infections. The detection rate of DENV RNA in whole blood is higher than in plasma. We suggest that one-step real-time RT-PCR using RNA from whole blood combined with an NS1 ELISA should be the choice for dengue diagnosis in dengue vaccine trials.

Clinical evaluation of an in-house-developed real-time RT-PCR assay for serotyping of dengue virus.

In the present study, an in-house-developed real-time RT-PCR (rRT-PCR) for serotyping of dengue virus (DENV) was evaluated for its performance, using 612 clinical samples. Compared to the composite reference standard, the in-house-developed rRT-PCR had an overall sensitivity of 97.5% and a specificity of 100%. The assay had a sensitivity of 100%, 95.6%. 96.9% and 100% for detecting DENV-1, DENV-2, DENV-3 and DENV-4, respectively. We recommend periodic evaluation of real-time RT-PCR assays for detecting DENV serotypes with a large number of samples and the use of at least two assays that target different regions of DENV genomes.

Spatio-temporal distribution analysis of circulating genotypes of dengue virus type 1 in western and southern states of India by a one-step real-time RT-PCR assay.

Dengue virus type 1 (DENV-1) Asian and American/African (AM/AF) genotypes were reported to be co-circulating in southern and western states of India based on envelope (E) gene sequencing of few representative samples. The objective of the present study was to develop a one-step real-time RT-PCR to discriminate between Asian and AM/AF genotypes of DENV-1 and investigate the spatio-temporal distribution of the DENV-1 genotypes in southern and western states of India. A one-step real-time RT-PCR to discriminate the Asian and AM/AF genotypes of DENV-1 was developed and validated using 40 samples (17 Asian and 23 AM/AF), for which the envelope (E) gene sequence data was available. DENV-2, DENV-3 and DENV-4 isolates, one each and DENV negative samples (n = 17) were also tested by the assay. Additional 296 samples positive for DENV-1 from selected Southern and Western states of India were genotyped using the real-time RT-PCR assay. Among the samples used for validation, the genotyping results were concordant with sequencing results for 39 samples. In the one discordant sample which was positive for AM/AF by sequencing, the genotyping assay tested positive for both Asian and AM/AF genotype. DENV-2, DENV-3 and DENV-4 isolates were not reactive in the assay. None of the DENV negative samples were positive (sensitivity 100% and specificity 98.2%). A total of 336 samples (40 samples with sequence data and 296 samples without sequence data) were used for spatio-temporal distribution analysis. The results revealed that the Asian genotype was the predominant genotype in Tamil Nadu and Kerala, the southern states. The AM/AF genotype was the predominant genotype in Maharashtra, a western state of India. In Nashik district of Maharashtra, Asian genotype was observed in 32.6% of DENV-1 samples during 2017 while the same decreased to 7.3% during 2018. In Pune district, Asian genotype was observed in 40.0% of DENV-1 samples during 2018 only. To conclude, a one step real-time RT-PCR has been developed for discriminating Asian and AM/AF genotypes of DENV-1. This assay can act as a complement to sequencing but not a substitute and can be utilized in resource limited settings for molecular surveillance of DENV-1. DENV-1 Asian genotype was the dominant genotype in South India while, AM/AF genotype was dominant in Western India.

Emergence of dengue virus type 1 and type 3 as dominant serotypes during 2017 in Pune and Nashik regions of Maharashtra, Western India.

India witnessed dengue outbreaks during 2017 in different parts with more than 180000 cases. There is no data on the serotypes/genotypes of dengue virus (DENV) associated with the 2017 outbreak season. The present study investigated DENV circulating in Pune and Nashik regions of Maharashtra, Western India at molecular level. IgM negative samples that were collected before 6th post onset days of illness were tested for DENV RNA and serotyped by real time RT-PCR based methods. Representative samples of each serotype were processed for virus isolation and envelope (E) gene sequencing. Among the 472 samples tested for DENV serotypes from Nashik, DENV-1 was observed in 36.2%, DENV-2 in 12.9%, DENV-3 in 35.4%, DENV-4 in 8.0%, and multiple serotypes in 7.4% of the samples respectively. In Pune region, among the 109 samples tested for DENV serotypes, DENV-1 was observed in 27.5%, DENV-2 in 11.0%, DENV-3 in 52.3%, DENV-4 in 4.6%, and multiple serotypes in 4.6% of the samples respectively. Comparison of serotype distribution from 2009 to 2017 from the Pune region revealed the emergence of DENV-3 as the dominant serotype followed by DENV-1 in 2017. In the Nashik region, both DENV-1 and DENV-3 were predominant in 2017. Phylogenetic analyses revealed co-circulation of American African (AM/AF) and Asian genotypes of DENV-1. DENV-1 Asian genotype was detected for the first time in the region. No genotype changes were observed for DENV-2 (cosmopolitan genotype), DENV-3 (genotype III) and DENV-4 (genotype I). For DENV-3, a unique amino acid substitution (I380T) was observed in the domain III of E protein of 2017 isolates and was not observed in earlier DENV-3 genotype III isolates. To conclude, the results suggest the emergence of DENV-1 with circulation of both Asian and AM/AF genotypes and DENV-3 with unique amino acid substitutions in Pune and Nashik regions. The study underscores the need for continuous molecular monitoring at a large scale to detect the changes in DENV serotypes/genotypes that might have implications for earlier prediction of dengue outbreaks and designing dengue vaccines and predicting its efficacy.

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method.

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein-Jenson (L-J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L-J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L-J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Mammographic image quality in relation to positioning of the breast: A multicentre international evaluation of the assessment systems currently used, to provide an evidence base for establishing a standardised method of assessment.

INTRODUCTION: Optimum mammography positioning technique is necessary to maximise cancer detection. Current criteria for mammography appraisal lack reliability and validity with a need to develop a more objective system. We aimed to establish current international practice in assessing image quality (IQ), of screening mammograms then develop and validate a reproducible assessment tool. METHODS: A questionnaire sent to centres in countries undertaking population screening identified practice, participants for an expert panel (EP) of radiologists/radiographers and a testing panel (TP) of radiographers. The EP developed category criteria and descriptors using a modified Delphi process to agree definitions. The EP scored 12 screening mammograms to test agreement then a main set of 178 cases. Weighted scores were derived for each descriptor enabling calculation of numerical parameters for each new category. The TP then scored the main set. Statistical analysis included ANOVA, t-tests and Kendall's coefficient. RESULTS: 11 centres in 8 countries responded forming an EP of 7 members and TP of 44 members. The EP showed moderate agreement when the scoring the mini test set W = 0.50 p < 0.001 and the main set W = 0.55 p < 0.001, 'posterior nipple line' being the most difficult descriptor. The weighted total scores differentiated the 4 new categories Perfect, Good, Adequate and Inadequate (p < 0.001). CONCLUSION: We have developed an assessment tool by Delphi consensus and weighted consensus criteria. We have successfully tabulated a range of numerical scores for each new category providing the first validated and reproducible mammography IQ scoring system.

Diagnostic potential of monoclonal antibodies against the capsid protein of chikungunya virus for detection of recent infection.

Chikungunya fever is self-limiting. However, neurological and hemorrhagic complications have been seen in recent outbreaks. The clinical manifestations of this disease are similar to those of dengue virus infection, indicating the need for differential diagnosis in areas such as India, which are endemic for both viruses. The aim of the present study was to develop monoclonal antibodies (MAbs) against Chikungunya virus (CHIKV) and assess their use in MAb-based IgM capture ELISA (MAC ELISA). The ELISA detects CHIKV-specific IgM antibodies, a marker of recent infection, in a patient's serum. One IgG1 and two IgM isotype hybrids were obtained. All of the subclones derived from the IgG1 hybrid recognized the C protein of CHIKV. The anti-C MAb ClVE4/D9 was the most promising as a detector antibody in MAC ELISA (C-MAb ELISA) yielding higher positive-to-negative (P/N) ratios. When compared with the CHIKV MAC ELISA kit developed by the National Institute of Virology (NIV), Pune (NIV MAC ELISA), the sensitivity of the test was 87.01 % with 100 % specificity. The positive and negative predictive values (PPV and NPV) were 100 % and 94.47 %, respectively. In precision testing, standard deviation (SD) and coefficient of variation (% CV) values of the C-MAb ELISA were within acceptable limits. The C-MAb ELISA detected anti-CHIKV IgM in serum of patients up to five months after the onset of infection, indicating that anti-C MAbs have strong potential for use in MAC ELISA to detect recent CHIKV infection.

Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

The trajectory of hope: pathways to find meaning and reconstructing the self after a spinal cord injury.

STUDY DESIGN: This is a qualitative study. OBJECTIVES: To evaluate and track the importance and the continuum of hope, and its trajectory, from the point of view of the individual with a spinal cord injury (SCI) and a rehabilitation psychologist. SETTING: This study was conducted in the Indian Spinal Injuries Centre, New Delhi, India, and in patients' homes in the National Capital Region, India. METHODS: Twenty individuals with an SCI were interviewed for the study at intervals of 2 weeks, 6 months, 1 year and 2 years since the time of the injury. Semi-structured interviews were conducted, in which the following theoretical research questions were investigated: 'What is the meaning, relevance and significance of hope in the individual's life following an SCI? Does the meaning and subject of hope change at different points in time?' RESULTS: Three distinctive themes markedly emerged in the trajectory of hope: (1) Hope for a complete recovery; (2) hope for self-reliance despite the injury; and (3) hope for an optimum quality of life. The make-up of each theme, its significance and contribution to recovery and/or rehabilitation, while tracking the influence of time since injury, family and friends, as well as other agencies and pathways, are discussed. CONCLUSION: After sustaining a life-altering injury, hope becomes the force that spurs individuals. Psychologists and rehabilitation counselors need to focus on instilling realistic hope, goal setting, sustaining motivation, enabling adaptive appraisals and problem-solving. Further recommendations include developing and testing interventions against the context of the continuum of hope.

The diagnostic value of white cell count, C-reactive protein and bilirubin in acute appendicitis and its complications.

INTRODUCTION: Inflammatory markers such as white cell count (WCC) and C-reactive protein (CRP) and, more recently, bilirubin have been used as adjuncts in the diagnosis of appendicitis. The aim of this study was to determine the diagnostic accuracy of the above markers in acute and perforated appendicitis as well as their value in excluding the condition. METHODS: A retrospective analysis of 1,169 appendicectomies was performed. Patients were grouped according to histological examination of appendicectomy specimens (normal appendix = NA, acute appendicitis = AA, perforated appendicitis = PA) and preoperative laboratory test results were correlated. Receiver operating characteristic (ROC) curve area analysis (area under the curve [AUC]) was performed to examine diagnostic accuracy. RESULTS: ROC analysis of all laboratory variables showed that no independent variable was diagnostic for AA. Good diagnostic accuracy was seen for AA when all variables were combined (WCC/CRP/bilirubin combined AUC: 0.8173). In PA, the median CRP level was significantly higher than that of AA (158mg/l vs 30mg, p<0.0001). CRP also showed the highest sensitivity (100%) and negative predictive value (100%) for PA. CRP had the highest diagnostic accuracy in PA (AUC: 0.9322) and this was increased when it was combined with WCC (AUC: 0.9388). Bilirubin added no diagnostic value in PA. Normal levels of WCC, CRP and bilirubin could not rule out appendicitis. CONCLUSIONS: CRP provides the highest diagnostic accuracy for PA. Bilirubin did not provide any discriminatory value for AA and its complications. Normal inflammatory markers cannot exclude appendicitis, which remains a clinical diagnosis.

Gene silencing of A-kinase anchor protein 4 inhibits cervical cancer growth in vitro and in vivo.

Earlier, we reported an association of A-kinase anchor protein 4 (AKAP4) expression in cervical cancer patient specimens, indicating its implications as an immunotherapeutic target. In this study, we investigated the possible role of AKAP4 in cervical carcinogenesis. AKAP4 messenger RNA and protein expression was assessed in four cervical cancer cell line models, C-33A, CaSki, HeLa and SiHa. Gene silencing approach was employed to investigate the potential role of AKAP4 in cellular growth, proliferation, colony-forming ability, migration and invasion in aggressive squamous cell carcinoma cells (SiHa). Further, the effect of downregulation of AKAP4 on tumor growth was examined in the cervical cancer xenograft model in nude mice. Our data clearly indicated that AKAP4 was expressed in all cervical cancer cells at the gene and protein level. We also observed distinct cytoplasmic and surface localization by indirect immunofluorescence and flow cytometry, respectively. Ablation of AKAP4 protein caused significant inhibition in cellular proliferation, colony-forming ability, migration and invasion ability of SiHa cells. Further, gene silencing of AKAP4 also resulted in reduced tumor growth in nude mice in vivo. Collectively, AKAP4 surface localization and its significant association with malignant properties of cervical cancer cells imply its clinical utility as an immunotherapeutic target.

Usefulness of multiplex PCR in the diagnosis of genital tuberculosis in females with infertility.

The purpose of this investigation was to evaluate the usefulness of multiplex polymerase chain reaction (m-PCR) in detecting uterine tuberculosis in women with infertility. In a prospective study, endometrial curetting from 620 females with infertility were investigated using laparoscopy, hysteroscopy, histopathology, smear microscopy, mycobacterial culture in BACTEC MGIT™ 960, and in-house m-PCR. The mean age of the women was 29.75 ± 4.66 years. The majority (596) sought medical attention for infertility; of them, 455 (76.34 %) presented with primary and 141 (23.65 %) with secondary infertility. A total of 158 (25.48 %) women were diagnosed as having uterine tuberculosis by at least one of the diagnostic methods. Among them, laparoscopy was positive in 46 (29.11 %), hysteroscopy in 77 (48.73 %), histopathology in only 8 (5.06 %), smear for acid fast bacilli in 4 (2.53 %), and liquid culture in 24 (15.18 %) patients. The in-house m-PCR was positive in 135 (85.44 %) women. Of these, 129 (95.55 %) samples were positive for Mycobacterium tuberculosis, while 6 (4.44 %) were positive for non-tuberculous mycobacterial DNA. Of the 129 M. tuberculosis PCR-positive women, 112 received anti-tubercular treatment and 23 of these conceived and fell pregnant after the completion of treatment. For the diagnosis of uterine tuberculosis, m-PCR was found to be the most efficient diagnostic tool compared to the other methods.

A randomised study evaluating the use of pyridoxine to avoid capecitabine dose modifications.

BACKGROUND: Pyridoxine is frequently used to treat capecitabine-induced hand-foot syndrome (HFS), although the evidence of benefit is lacking. We performed a randomised placebo-controlled trial to determine whether pyridoxine could avoid the need for capecitabine dose modifications and improve outcomes. METHODS: A total of 106 patients planned for palliative single-agent capecitabine (53 in each arm, 65%/35% colorectal/breast cancer) were randomised to receive either concomitant pyridoxine (50 mg po) or matching placebo three times daily. RESULTS: Compared with placebo, pyridoxine use was associated with an increased rate of avoiding capecitabine dose modifications (37% vs 23%, relative risk 0.59, 95% CI 0.29, 1.20, P=0.15) and fewer grade 3/4 HFS-related adverse events (9% vs 17%, odds ratio 0.51, 95% CI 0.15-1.6, P=0.26). Use of pyridoxine did not improve response rate or progression-free survival. CONCLUSION: Pyridoxine may reduce the need for capecitabine dose modifications and the incidence of severe HFS, but does not impact on antitumour effect.

Survival of hepatitis A and E viruses in soil samples.

Survival of hepatitis A virus (HAV) and hepatitis E virus (HEV) in soil samples spiked with respective viruses was analysed using real-time PCR. Virus-spiked soil samples were incubated at environmental temperature (ET) and 37°C and processed weekly. Both HAV and HEV were less stable at fluctuating ET than at 37°C. Of the 403 soil samples collected in the vicinity of Mutha river, India, 19.1% and 4.9% were found to be contaminated with HAV and HEV, respectively.

Ultrasound elastography as an adjuvant to conventional ultrasound in the preoperative assessment of axillary lymph nodes in suspected breast cancer: a pilot study.

AIMS: To compare the performance of ultrasound elastography with conventional ultrasound in the assessment of axillary lymph nodes in suspected breast cancer and whether ultrasound elastography as an adjunct to conventional ultrasound can increase the sensitivity of conventional ultrasound used alone. MATERIALS AND METHODS: Fifty symptomatic women with a sonographic suspicion for breast cancer underwent ultrasound elastography of the ipsilateral axilla concurrent with conventional ultrasound being performed as part of triple assessment. Elastograms were visually scored, strain measurements calculated and node area and perimeter measurements taken. Theoretical biopsy cut points were selected. The sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) were calculated and receiver operating characteristic (ROC) analysis was performed and compared for elastograms and conventional ultrasound images with surgical histology as the reference standard. RESULTS: The mean age of the women was 57 years. Twenty-nine out of 50 of the nodes were histologically negative on surgical histology and 21 were positive. The sensitivity, specificity, PPV, and NPV for conventional ultrasound were 76, 78, 70, and 81%, respectively; 90, 86, 83, and 93%, respectively, for visual ultrasound elastography; and for strain scoring, 100, 48, 58 and 100%, respectively. There was no significant difference between any of the node measurements CONCLUSIONS: Initial experience with ultrasound elastography of axillary lymph nodes, showed that it is more sensitive than conventional ultrasound in detecting abnormal nodes in the axilla in cases of suspected breast cancer. The specificity remained acceptable and ultrasound elastography used as an adjunct to conventional ultrasound has the potential to improve the performance of conventional ultrasound alone.