A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting.

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000-25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Wernicke encephalopathy after Roux-en-Y gastric bypass in a young patient.

We report a case of Wernicke encephalopathy (WE) in a woman in her 20s who had Roux-en-Y gastric bypass surgery for severe obesity, which resulted in a severe depletion of the patient's thiamine reserve and development of WE syndrome, we also emphasise the importance of prompt diagnosis of this serious complication in addition to the importance of adequate therapy.

Post-operative Day Zero Versus Day One Follow-Up for Uncomplicated Cataract Surgery.

Purpose To compare the postoperative outcomes and management of uncomplicated cataract surgery seen on postoperative day 0 (POD0) versus postoperative day one (POD1).  Methods A retrospective cohort study of patients who followed up within 0-14 days of their uncomplicated surgery (current procedural terminology code 66984) from December 2018 to March 2020. Those who had perioperative complications, those who had combined glaucoma filtering surgery as well as other minimally invasive glaucoma surgery (MIGS) procedures, and those who did not complete their first two follow-up visits within 14 days of their surgery were excluded. Visual acuity (VA), intraocular pressure (IOP), post-operative interventions, and complications of the first and second postoperative visits were collected. Results Of the 665 participants studied, the mean (standard deviation) age was 68 (11) years old and 60% were female (n=304) with a mean (SD) pre-op logarithm of the minimum angle of resolution (logMAR) VA of 0.715 (0.625). About one-third (32%) of patients were seen on POD0. Compared to POD1, a higher percent of patients with glaucoma were seen POD0 (23% vs 14%; p = 0.008). The mean VA on POD0 was 0.840 (0.653), which was significantly worse than the mean VA of 0.539 (0.599) on POD1 (p<0.0001). There was no significant difference in VA by the second post-op visit. IOP did not significantly differ between POD0 and POD1 groups at the first post-operative visit. The most common changes in the post-operative drop regimen were related to IOP and inflammation control. The rate of interventions did not significantly differ between groups (p>0.1). Patients who received intervention on POD0 were not seen significantly sooner at the next follow-up visit compared to those seen on POD0 without undergoing an intervention. The incidence of an IOP spike greater than 30mmHg on POD0 or POD1 was not significantly different between patients with and without underlying glaucoma (overall p = 0.2020; with glaucoma p= 0.1238; without glaucoma p=0.999). Those with a history of glaucoma were not more likely to receive intervention to lower IOP on POD0 versus those seen on POD1 (p = 0.999).  Conclusion It can be difficult to evaluate patients the day after their uncomplicated cataract surgery, and it is difficult to predict which patients may have post-operative complications. Our study shows no significant changes in management for patients seen on POD0 compared to POD1. Surgeons can expect significantly better visual acuity on POD1, but otherwise, post-operative outcomes were similar between patients seen on POD0 and those seen on POD1. Surgeons may offer the option of a POD0 visit for patients who underwent uncomplicated cataract surgery.

How many oral antidiabetic drugs before insulin?

Worsening of glycemic control in type 2 Diabetes mellitus occur on account of declining beta cell function. This calls for up titration of the chosen drug, addition of another agent with complementary action and eventually insulin usually after 2 or three OADs. Introduction of insulin has many issues which include parenteral route of administration, cost and enhancement of hypoglycemic tendency. We propose the addition of another OAD in lieu of insulin in whom glycemic control can be achieved equally well without insulin.

Pattern of variation in the mono- and dinucleotide repeat microsatellites associated with lynch syndrome in an Indian population.

BACKGROUND: Microsatellite instability (MSI) is associated with Lynch syndrome and hence is a surrogate marker for defective mismatch repair genes. The aim of this study was to investigate the degree of instability associated with each of the 5 microsatellite (MS) loci recommended by the National Cancer Institute (NCI), USA within an Indian population of mixed ethnicity and suffering from Lynch syndrome. METHODS: DNA from clinical samples originating from the study population (n = 130) were subjected to automated fragment analysis for all 5 MS loci, and data generated were analyzed to determine the frequency of variation of each of the MS in the resource population. RESULTS: Out of 130, 116 samples responded to polymerase chain reaction (PCR) for all 5 MS loci. 21 (16.15%) were MSI-high (MSI-H) while 27 (20.76%) and 68 (52.30%) were MSI-low (MSI-L) and microsatellite stable (MSS), respectively. D5S346 exhibited the highest instability (27 out of a total of 82 cases of instability recorded for all 5 MS in all 116 patients tested) followed by D2S123 (23/82), BAT26 (14/82), BAT25 (11/82), and D17S250 (7/82). CONCLUSION: MS D17S250 and BAT25 of the 5 MS panel recommended by the NCI are not informative enough and hence should be avoided for diagnosing Lynch syndrome in the Indian population.

Rare e14a3 (b3a3) BCR-ABL fusion in chronic myeloid leukemia in India: the threats and challenges in monitoring minimal residual disease (MRD).

OBJECTIVE: The primary objective of this work was to confirm the occurrence of rare BCR ABL fusion variant involving the a3 region of the ABL gene in a patient positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions and the challenges and threats that it poses in a routine laboratory setting which use commercial kits for monitoring the minimal residual disease. METHODS: A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. PCR amplicon generated from in the process was sequenced and analyzed. RESULTS: The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus of affected cells and presented a '1O1G2F' signal pattern. RT-PCR with probes from commercial kit designed to detect common breakpoints within the M- and m-BCR regions involving e13a2, e14a2 and e1a2 fusion variants respectively failed to generate any signal. Further investigation revealed presence of the rare e14a3 (b3a3) fusion. DISCUSSION: This is the first report of rare e14a3 fusion in the BCR ABL gene in a CML patient from India. The observation indicates the need for interrogating rare BCR ABL fusions when common breakpoint cluster regions are absent such that minimal residual disease (MRD), critical for disease monitoring, can be performed and false positive remission cases can be avoided. It also emphasizes the utility and significance of cytogenetics and FISH techniques in primary diagnosis of CML and use of RT-PCR based assays only for generating secondary information within special reference to MRD. CONCLUSION: The rare e14a3 (b3a3) fusion of the BCR ABL gene is present in Indian population as demonstrated from this first report and clinical laboratories using commercial kit that do not cover such rare fusions are likely to generate false result thereby declaring complete molecular remission in CML patients under therapy while conducting MRD assay using RT-PCR technology.

Distinct geographical clustering of HIV-1 and a signature amino acid at position 41 of the p24 unveiled by gag variability in India.

A portion of the gag gene cDNA for p24 protein from 30 Indian HIV-1 proviral DNA was amplified by PCR and sequenced. Phylogenetic analysis with reference samples of A1, A2, B, C, D, F1, F2, G, H, J, K, N and O subtypes revealed that 29 test samples aligned with subtype C reference strain while 1 matched with HIV-1 subtype A. Multiple alignment of predicted amino acid sequence of the Indian test samples and reference C subtype of HIV-1 samples from other countries indicated a molecular signature by way of rigid conservation of the amino acid 'S' at position 41 of the gag p24 protein in all Indian HIV-1 samples analyzed in this study as opposed to 'T' in the same position in C subtype sequences from other parts of the world. A phylogenetic analysis and visualization of the resulting tree in radial position showed distinct clubbing of all Indian C subtypes and formation of a cluster when compared to C subtype sequences from other countries with a single Chinese sample as an exception which was found in the Indian cluster. The use of a portion of p24 gene sequence as tool for subtyping as well as phylogenetic grouping with special reference to its geographical location is discussed.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.