Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

BACKGROUND: The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. METHODS AND RESULTS: The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. CONCLUSIONS: It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

Evaluation of a flow cytometric test for G6PD-deficient erythrocytes.

OBJECTIVE: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked recessive disorder, is the commonest erythrocytic enzymopathy worldwide. Reliable diagnosis and severity prediction in G6PD-deficient/heterozygous females remain challenging. A recently developed flow cytometric test for G6PD deficiency has shown promise in precisely identifying deficient females. This paper presents our experiences with this test in a subtropical setting and presents a modification in flow cytometric data acquisition strategy. METHODS: The methaemoglobin reduction + ferryl Hb generation-based flow cytometric G6PD test was compared with the screening methaemoglobin reduction test (MRT) and confirmatory G6PD enzyme activity assay (EAA) in 20 G6PD-deficient males, 22 G6PD-heterozygous/deficient females and 20 controls. Stained cells were also assessed for bright/dim G6PD activity under a fluorescent microscope. RESULTS: Flow cytometry separated and quantified %bright cells in heterozygous/deficient females, objectively classifying them into 6 normal (>85% bright cells), 14 intermediate (10-85%) and two G6PD-deficient (<10% bright cells). Concordance with MRT was 89% (55/62 cases) and with EAA was 77% (48/62 cases). Fluorometrically predicted violet laser excitation (405-nm) with signal acquisition in the 425-475 nm region was a technical advancement noted for the first time in this paper. CONCLUSION: Flow cytometry/fluorescence microscopy represent technically straightforward methods for the detection and quantification of G6PD-deficient erythrocytes. Based on our results, we recommend their application as a first-line investigation to screen females who are prescribed an oxidant drug like primaquine or dapsone.

A flow cytometric cell-cycle assay using methyl green.

Methyl green (MG), a conventional, low-cost histological stain, was used to design a flow cytometric cell-cycle/DNA-ploidy assay. On fluorometry, MG absorbed maximally at 633-nm, showed negligible fluorescence in free-state, but emitted brightly when bound to DNA. Optimal dye and cell concentrations for staining and effects of time and photobleaching on stained cells were determined for a lyse-permeabilize-stain protocol. Linearity of DNA-binding, coefficients-of-variation of G0/G1-peaks and minimal carryover were confirmed. Assay results correlated highly with a propidium iodide-based kit in 29 acute lymphoblastic leukemia specimens. The MG-based DNA-ploidy assay represented an accurate and inexpensive alternative to conventional PI-based assays.

blaOXA-48 carrying clonal colistin resistant-carbapenem resistant Klebsiella pneumoniae in neonate intensive care unit, India.

Bacteria resistant to colistin, a last resort antibiotic reflect the pre-antibiotic era. In this study, colistin resistance carbapenem-resistant K. pneumoniae (COLR- CRKP) strains from neonate's intensive care unit were evaluated. Molecular analysis showed that all the four colistin resistant K. pneumoniae isolates were clonally related with strong biofilm formation ability and harbored blaSHV-34 and blaOXA-48 genes. Our result suggested the need of proper surveillance and adequate infection control to limiting the spread of these organisms.

Detection of AmpC ß-lactamase and adherence factors in uropathogenic Escherichia coli isolated from aged patients.

Escherichia coli mediated urinary tract infection has been reported to be most prevalent among patients of different class, gender and ages. Currently, multidrug resistant E. coli harboring several virulence factors are most perilous threats for patients especially for elders. The aim of this study was to determine the antibiotic resistance pattern, co-resistance and phenotypic virulence factors present in uropathogenic E. coli isolated from aged patients. Thirty-nine E. coli isolates were collected during May-June 2014 from patients between 50 to 80 years of age. Experiments have been carried out to determine the antibiotic resistance, co-resistances and phenotypic adherent factors present in each isolate. Clonal relatedness was also determined in the AmpC positive uropathogenic E. coli (UPEC). 97.43% isolates were found to be multidrug resistant and 41.02% of them were AmpC producer. AmpC producer group showed higher multiple antibiotic resistance index than AmpC non-producer (p value < 0.01) group. Interestingly, adherence factor Type 1 fimbriae were found among 84.61% of total isolates which were more prevalent in elderly female patients than males. Biofilm production studies revealed that 84.61% of total isolates are more common in elderly males. This study adds value for the proper empiric selection of antibiotic therapy as well as calls for continuous monitoring of the incidence of drug resistance virulent uropathogenic E. coli mediated urinary tract infection in elderly patients.

Listeria innocua infection in an old case of total knee replacement - an unusual case report.

Prosthetic implant-associated arthritis due to Listeria is mostly reported for Listeria monocytogenes. Here, we describe a patient who underwent total knee replacement 12 years ago and presented with pain, tenderness, redness and local rise in temperature in the right knee. Purulent fluid was aspirated. Upon microbiological analysis, culture yielded Listeria innocua. L. innocua is rare. Listeria is not reported as a contaminant and routine cultures may be negative. Because of the long interval between surgery and the onset of symptoms, clinical suspicion, radiological investigations and analysing multiple samples are of immense help.

Effect of Tele-Guided Imagery on Stress among Caregivers of Chronic Neurological Patients: A Randomized Controlled Trial.

Background and Objectives Chronic neurological conditions impose a burden on caregivers as they are a major contributor for disability-adjusted life years of patients. Stress is the common psychological concern of caregivers. This randomized controlled study examined the effect of tele-guided imagery on stress among caregivers of these neurological patients. Methods Thirty caregivers were selected for participation in study as per the inclusion criteria and were randomly assigned into experimental and control groups. Initial caregivers' mental health screening was performed with the mini-mental state examination tool. For outcome measure, perceived stress scale (PSS) score tool was utilized and both pre- and postintervention scores were noted. Effect of tele-guided intervention was compared with the control group. Results Significant difference was noted in the PSS scores among the caregivers receiving tele-guided imagery when compared with controlled group caregivers. Conclusion Tele-guided imagery is an effective tool for stress management among caregivers. Also, it improves the individual's mental health aspect.

Mucormycosis and aspergillosis: The deadly duo in COVID-19-a case report.

COVID-19 disease has been associated with fungal infections such as aspergillosis and mucormycosis, especially in diabetic patients who have suffered from a moderately severe form of COVID-19 infection and are treated with steroids. Though there are multiple case reports describing co-infection with mucormycosis during the second wave of the COVID outbreak, the report of a dual fungal infection along with superadded bacterial infection is rare. Here we report a case where the same patient had a fungal storm with aspergillosis and mucormycosis and superadded Klebsiella. She was treated aggressively with antifungal agents, antibiotics, surgical debridement, and other supportive care. She improved and was discharged from the hospital after a long stay. She is being followed up regularly in the outpatient department and doing well.

Coronavirus Disease 2019-Associated Mucormycosis Diagnosed by Fine-Needle Aspiration Cytology.

The temporal association of mucormycosis with coronavirus disease 2019 (COVID-19) has been termed COVID-19-associated mucormycosis (CAM). Because of its poor prognosis, early diagnosis and treatment are crucial. Although tissue samples for culture and histological evaluation are the cornerstones of diagnosis, the role of fine-needle aspiration cytology (FNAC) and cytologic findings are also significant. Here, we report a case of mucormycosis in a COVID-19-positive 34-year-old male diagnosed by FNAC and confirmed by fungal culture. To our knowledge, this is possibly the first and only case report of CAM diagnosed by FNAC.

PHi-RACE: PGIMER in-house rapid & cost effective classifier for the detection of BCR-ABL1-like acute lymphoblastic leukaemia in Indian patients.

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes.

Characterization of Immunophenotypic Aberrancies with Respect to Common Fusion Transcripts in B-Cell Precursor Acute Lymphoblastic Leukemia: A Report of 986 Indian Patients

Objective: Based on the immunophenotype, acute lymphoblastic leukemia (ALL) can be categorized into B-cell or T-cell lineages. B-cell precursor ALL (BCP-ALL) cases show various genetic/molecular abnormalities, and varying frequencies of chimeric fusion transcripts in BCP-ALL cases are reported from different parts of the world. We studied the immunophenotypic aberrancy profiles of a large number of BCP-ALL cases with respect to various common chimeric fusion transcripts. Materials and Methods: Flow cytometric immunophenotyping and multiplex reverse-transcription polymerase chain reaction assays were performed for 986 BCP-ALL cases. Results: Among 986 BCP-ALL cases, the incidence of various fusion transcripts was 38.36% in adult cases and 20.68% in pediatric cases. Adult BCP-ALL patients with t(9;22)(BCR-ABL1) fusion transcripts and expression of aberrant myeloid markers were significantly older at presentation (p=0.0218) with male preponderance (p=0.0246) compared to those without aberrant myeloid expression. In pediatric patients with the t(12;21)(ETV6-RUNX1) chimeric fusion transcript, aberrant expression of CD13 was observed in 39.13%, CD33 in 36.95%, and CD117 in 8.69% of patients, respectively. Pediatric BCP-ALL patients with the ETV6-RUNX1 fusion transcript and expression of aberrant myeloid markers were not significantly different compared to those without with respect to demographic and clinical/hematological characteristics (p=0.5955). Aberrant myeloid markers were rarely or never expressed in pediatric and adult BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts. Conclusion: Aberrant myeloid markers were frequently expressed among BCP-ALL patients with the t(9;22)(BCR-ABL1) and t(12;21) (ETV6-RUNX1) fusion transcripts. However, BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts rarely or never expressed aberrant myeloid markers. Aberrant myeloid CD markers can be used in predicting chimeric fusion transcripts at baseline so as to plan appropriate tyrosine kinase inhibitor therapy in cases of BCP-ALL with specific chimeric fusion transcripts. This study has delineated the relationship of chimeric fusion transcripts with the aberrant expression of myeloid markers in a large cohort of BCP-ALL cases.

Hepatitis E Genotype 1 Outbreak in Jharkhand, India: A Descriptive Analysis.

INTRODUCTION: A waterborne outbreak of hepatitis E virus occurred in the Jamshedpur city of Jharkhand from March 2018 to October 2018. The aim of the present study is to study the hepatitis E virus outbreak clinically, serologically and etiologically. METHODS: Five hundred and eighty-four clinically and biochemically documented cases were screened for the hepatotropic viral markers (HepatitisA, B, C, and E) by the ELISA. Demographic data such as gender, age, clinical diagnosis, location, outcome and time of admission were extracted from the online hospital management system. Water samples from the affected area were tested for HEV RNA detection. Genotyping of HEV virus was carried out by sequencing and phylogenetic analysis. RESULTS: Hepatitis E genotype 1 was confirmed as the major etiological agent in this outbreak due to the faecal contamination of drinking water supply while establishing illegal water connections. Mixed infection of HEV-HAV (5.31%) or HEV-HBV (0.91%) was also detected in the present series of acute viral hepatitis. CONCLUSION: The study highlights the importance of screening for both enterically transmitted hepatotropic viral markers as well as the parenterally transmitted hepatotropic viral markers during the outbreaks of acute viral hepatitis.

Results of Serosurveillance and Forecasting the Third Wave of COVID-19 in an Industrial District in India.

Prevalence of immunoglobulin G (IgG) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in the industrial district of East Singhbhum (Jharkhand, India) from July, August, November, and December 2020 and January 2021 after the first wave and in July 2021 after the second wave of coronavirus disease 2021 (COVID-19) infections may be utilized to find the possibility of a third wave of COVID-19 infections. Based on the trend of the loss of protective IgG antibodies after the first wave and the seropositivity of 75% in the district in July 2021, simple forecasting and proportional estimates of the seropositivity in the next eight months and the estimated maximum number of the cases was done. We also considered the seropositivity without vaccination in July 2021 (63%). Additionally, the trend of the weekly RT-PCR and rapid antigen testing for SARS-CoV-2 may also preemptively predict an imminent wave. Based on the East Singhbhum population and the vaccination coverage with at least one dose till July 2021 (Covishield or Covaxin), it is estimated that a 4-5% monthly vaccination coverage rate of new individuals will not allow the seropositivity to fall below 50% and hold at bay a major wave. Vaccination coverage of 3% or less would allow a continuous drop in acquired immunity in the district and can potentially cause a rise in cases, making the community susceptible to a future surge of infections. A 3-5% vaccination rate of new individuals is unlikely to see a drop in the community seropositivity below 50% and the number of new cases of COVID-19 infections going above 478 to 712 per month at least till March 2022. The assumptions are based on presuming that there will be no new mutant of SARS-CoV-2 that escapes the immunity provided by previous infection or vaccination over the next eight months. However, currently, there is no evidence to speculate on any new variant of concern causing a major wave globally. The B.1.617.2 (delta) variant was first identified in October 2020 and there was a lag of six months to the second surge of COVID-19 infections in East Singhbhum, primarily caused by this variant. Additionally, 3% and above, with a rising weekly trend of reverse transcription-polymerase chain reaction (RT-PCR) positivity for SARS-CoV-2 can provide at least four to eight weeks advance warning before the peak of the wave if an imminent future wave is impending.

A Case of Bilateral Central Retinal Artery Occlusion in a Post-COVID Rhino-Orbital-Cerebral Mucormycosis Patient.

Central retinal artery occlusion (CRAO) is a multifactorial disease, where inflammation and hypercoagulability are the major risk factors. It is a rare occurrence in this geographical area in patients diagnosed with sinus mucormycosis infection, which has emerged as one of the most fulminant, opportunistic secondary infection during post-COVID era. We report a case of a bilateral CRAO, in a 66-year-old, post-COVID, diabetic patient. A complete eye examination followed by radiological imaging of brain, orbit, and paranasal sinuses were done. Multidisciplinary approach was contemplated to reach a diagnosis of bilateral rhino-orbital-cerebral mucormycosis (ROCM). Intravenous liposomal amphotericin-B injection was started as a part of systemic management and an aggressive sinus debridement of both sides with amphotericin-B wash was also done. Despite an early diagnosis and intervention, the patient succumbed to her illness. All post-COVID patients presenting with the complaints of blurring of vision should be meticulously examined for the presence of any retinal abnormality in both the eyes as this may be a manifestation of an underlying secondary fungal infection. Early diagnosis of ROCM and management will help in reducing complications.

A Prospective Study on Rapidly Declining SARS-CoV-2 IgG Antibodies Within One to Three Months of Testing IgG Positive: Can It Lead to Potential Reinfections?

Background COVID-19 immunoglobulin G (IgG) antibodies have been considered to provide protective immunity and its immunoassays have been widely used for serosurveillance. In our serosurveillance on an industrial workforce of randomly selected 3296 subjects, COVID-19 IgG antibody positivity was reported in 7.37% (243) subjects. However, when 30 days later, eight of the 243 COVID-19 IgG antibody-positive individuals complained of symptoms suggestive of COVID-19 infection and were confirmed as COVID-19 infection by reverse transcription-polymerase chain reaction (RT-PCR), their COVID-19 IgG antibodies were retested. Seven of the eight previously IgG positive individuals had lost their protective antibodies. Methods Subsequently, a prospective clinical trial was planned by repeating the test for IgG antibodies on the remaining earlier positive 235 individuals at 45-65 days after their initial test. Only 201 of the 235 individuals consented and participated in the non-randomized single-arm observational trial. Results Only 28.36% (57/201) retained their IgG antibodies and 70.15% (141/201) had lost their IgG antibodies. Three cases reported equivocal results on retesting. Conclusions Our findings show that the protective COVID-19 IgG antibodies rapidly decline over one to three months. Further studies are needed with a quantitative assay over a period with neutralizing antibodies to establish if its decay can potentially lead to reinfections. Rapidly decaying protective IgG antibodies would impact herd immunity and vaccine durability. It is critical for the potential vaccines to generate both protective T- and B-cell immune responses in a sustained manner.

Bacteriological profile and antimicrobial resistance patterns of burn wound infections in a tertiary care hospital.

BACKGROUND: The bacterial infections that prevail in the burnt patients continue to be a critical complication in the burnt patients and vary with time and place. Identification of bacterial pathogens with information of their antimicrobial susceptibility of burn wounds can help clinicians to select appropriate medication procedure as in providing them with suitable antibiotic for empirical treatment. METHODS: Retrospective study of thirty-one months (Jan 2015 to July 2017) was designed to evaluate bacteria involved in burnt wound infection and its antimicrobial susceptibilities in a Burn Intensive Care Unit of Eastern India. Pus samples were cultured on cysteine Lactose electrolyte deficient agar (Hi-Media, India). Positive bacteria cultures were identified and tested for antimicrobial susceptibility using VITEK®2 (bioMerieux, Durham, NC, USA) and interpreted according to Clinical Laboratory Standards Institute guidelines. RESULTS: Two hundred and seventy-two wound swabs from burnt patients were received, out of which 62.8% (n = 185) were revealed as positive for the presence of bacteria. Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and E. coli were discovered to be the most common organisms in patients. Isolated bacteria were least resistant to TIGECYCLINE and COLISTIN. CONCLUSION: Data regarding the incidence of pathogens and their resistance patterns would benefit the clinicians to prescribe appropriate antibiotics, articulating policies for empirical antimicrobial therapy to control the different types of infections.

Penicillium on the Iris! An Unusual Presentation and Cause of Postoperative Anterior Uveitis.

Intraocular fungal infections may not present solely as postoperative anterior uveitis or as a focal anterior segment lesion. The present study describes a 50-year-old woman with well-controlled diabetes who presented with postoperative anterior uveitis three months after uncomplicated cataract surgery. A fuzzy lesion was observed on her iris. The patient underwent an anterior chamber wash and removal of the lesion, followed by intracameral treatment with voriconazole. Culture of the lesion showed that it was a species of Penicillium. The patient has remained stable after treatment. Three aspects of this case were unusual: a fungal lesion of unusual etiology and location, inflammation restricted to the anterior segment despite a fungal background, and the excellent response to treatment with a very favorable outcome.

Comparison of two commercial ELISA kits for detection of rubella specific IgM in suspected congenital rubella syndrome cases and rubella IgG antibodies in a serosurvey of pregnant women.

Enzyme linked immunosorbent assay (ELISA) for antibody identification, is important for laboratory confirmation of rubella infection in different settings. The Enzygnost rubella ELISA, widely used in the World Health Organization (WHO) Global Measles and Rubella Laboratory Network, is expensive and often unavailable. Qualitative and quantitative performance of the Euroimmun ELISA was compared with the Enzygnost ELISA, for detection of rubella specific IgM, using 283 sera collected from suspected congenital rubella syndrome (CRS) patients and IgG antibodies using 435 sera from a serosurvey among pregnant women. Good qualitative agreement was observed for detection of both rubella specific IgM (94.7% agreement and κ of 0.86) and IgG (96.3% agreement and κ of 0.84). Bland-Altman analysis for IgG yielded a mean difference of 0.781 IU/ml with 97.1% values within ±2 SD of the mean difference. Our study findings suggest that Euroimmun ELISA may be considered for detection of rubella specific IgM in suspected CRS cases and rubella specific IgG in surveillance studies.

Cryptococcus laurentii fungemia in a low birth weight preterm neonate: India.

In the last few years, Cryptococcus laurentii infections have increased, particularly in immunocompromised patients. Here we report the first case of C. laurentii fungemia in low birth weight preterm infant from India. A preterm neonate was admitted for feeding intolerance and respiratory distress. Blood culture ascertained the causative organism as C. laurentii. The isolated strain was found susceptible to amphotericin B and the patient recovered following the administration of amphotericin B (5mg/kg body weight) for 8days.

Sero-prevalence of rubella among pregnant women in India, 2017.

BACKGROUND: We conducted a sero-survey among pregnant women attending antenatal clinics of six hospitals which also function as sentinel sites for CRS surveillance, to estimate the prevalence of IgG antibodies against rubella. METHODS: We systematically sampled 1800 pregnant women attending antenatal clinics and tested their sera for IgG antibodies against rubella. We classified sera as seropositive (titre ≥10 IU/ml), sero-negative (titre <8 IU/ml) or indeterminate (titre 8-9.9 IU/ml) per manufacturer's instructions. In a sub-sample, we estimated the titers of IgG antibodies against rubella. IgG titer of ≥10 IU/mL was considered protective. RESULTS: Of 1800 sera tested, 1502 (83.4%) were seropositive and 24 (1.3%) were indeterminate and 274 (15.2%) were sero-negative. Rubella sero-positivity did not differ by age group, educational status or place of residence. Three hundred and eighty three (87.8%) of the 436 sera had IgG concentrations ≥10 IU/mL. CONCLUSION: The results of the serosurvey indicate high levels of rubella sero-positivity in pregnant women. High sero-prevalence in the absence of routine childhood immunization indicates continued transmission of rubella virus in cities where sentinel sites are located.