Assessing and Minimizing the Effect of Malaria on SARS-CoV-2 Serodiagnostics

Malaria may affect the reliability of SARS-CoV-2 seroassay performance and limit understanding of SARS-CoV-2 epidemiology in malaria-endemic regions. We present our experience conducting SARS-CoV-2 serosurveillance in seasonal malaria-affected communities in Mali and discuss relevant literature regarding the effect of malaria on the performance of SARS-CoV-2 serodiagnostics, including approaches to minimize the effect of malaria-associated assay interference.

Optimization of an in silico protocol to characterize membrane PAINS

Membrane Pan-Assay INterference compoundS (PAINS) are a class of molecules that interact non-specifically with lipid bilayers and alter their physicochemical properties. An early identification of these compounds avoids chasing false leads and the needless waste of time and resources in drug discovery campaigns. In this work, we optimized an in silico protocol based on umbrella sampling (US)/MD simulations to discriminate between compounds with different membrane PAINS behavior. We showed that the method is quite sensitive to membrane thickness fluctuations, which was mitigated by changing the US-reference position to the P-atoms of the closest interacting monolayer. The computational efficiency was improved further by decreasing the number of umbrellas and adjusting their strength and position in our US scheme. The ISDM-calculated membrane permeability coefficients confirmed that resveratrol and curcumin have distinct membrane PAINS characteristics and indicate a misclassification of nothofagin in a previous work. Overall, we have presented here a promising in silico protocol which can be adopted as a future reference method to identify membrane PAINS.

The use of HRM shifts in qPCR to investigate a much neglected aspect of interference by intracellular nanoparticles

Genetic molecular studies used to understand potential risks of engineered nanomaterials (ENMs) are incomplete. Intracellular residual ENMs present in biological samples may cause assay interference. This report applies the high-resolution melt (HRM) feature of RT-qPCR to detect shifts caused by the presence of gold nanoparticles (AuNPs). A universal RNA standard (untreated control) sample was spiked with known amounts of AuNPs and reverse transcribed, where 10 reference genes were amplified. The amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves were analysed and detected interference caused by AuNPs, which differed according to the amount of AuNP present (i.e. semi-quantitative). Whether or not the assay interference was specific to the reverse transcription or the PCR amplification step was tested. The study was extended to a target gene-of-interest (GOI), Caspase 7. Also, the effect on in vitro cellular samples was assessed (for reference genes and Caspase 7). This method can screen for the presence of AuNPs in RNA samples, which were isolated from biological material in contact with the nanomaterials, i.e., during exposure and risk assessment studies. This is an important quality control procedure to be implemented when quantifying the expression of a GOI from samples that have been in contact with various ENMs. It is recommended to further examine 18S, PPIA and TBP since these were the most reliable for detecting shifts in the difference curves, irrespective of the source of the RNA, or, the point at which the different AuNPs interacted with the assay.

Optimization of an ammonia assay based on transmembrane pH-gradient polymersomes

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30–800 μM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

A Case of macro-TSH masquerading as subclinical hypothyroidism

A 47-year-old man was commenced on levothyroxine following a diagnosis of subclinical hypothyroidism with nonspecific symptoms. Despite increasing doses of levothyroxine, his thyroid-stimulating hormone (TSH) remained elevated and he was referred for further assessment as he was unable to tolerate further titration. On assessment, his thyroid function demonstrated an elevated TSH and elevated free-T4. The initial impression was of iatrogenic thyrotoxicosis, with possible underlying thyroid hormone resistance, TSHoma or assay interference. After discontinuation of levothyroxine, free-T4 normalised but TSH remained elevated. There was a normal response to thyrotropin-releasing hormone (TRH) testing. T3 suppression testing demonstrated free-T4 reduction but persistently high TSH. THRβ sequencing was normal. TSH measurement by alternative assays revealed discrepant results. Gel filtration chromatography revealed the presence of high-molecular weight TSH variant alongside normal TSH. Macro-TSH is a rare phenomenon with spuriously elevated TSH and which may mimic subclinical hypothyroidism. Recognition of macro-TSH avoids misdiagnosis and prevents inappropriate treatment.

Optimization of an Ammonia Assay based on Transmembrane pH-gradient Polymersomes

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using four-parameter logistic regression, the assay had a large quantification range (30–800 µM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

Discordant Thyroid Function Tests in Patients With HIV: A Case Report and Discussion of Diagnostic Approach

Background: TFT patterns correlate directly with clinical symptoms in most patients with thyroid disease. However, some patients have TFTs which are at odds to clinical findings, posing a diagnostic challenge. Such cases warrant careful evaluation with a structured, rational approach to avoid misdiagnosis and mistreatment. Clinical Case: A 48-year-old female liver transplant recipient with HIV presented with an asymptomatic multinodular goiter, low TSH and low/normal fT4. Her medications were nadolol, Bactrim, ursodiol, azithromycin, tacrolimus, efavirenz, emtricitabine, tenofovir and zolpidem. Thyroid US showed 3 dominant nodules with benign pathology on FNA. Uptake scan showed diffuse increased uptake consistent with Grave’s disease. She tried MMI for one year, then opted for RAI ablation and was started on LT4 with titration of dose. fT4 remained low/normal regardless of receiving LT4, MMI or no therapy over the following eight years. TSH was appropriately suppressed from 1.42 to 0.160 (0.45 - 4.500 mIU/L) when LT4 weekly dose was increased from 175 mcg to 350 mcg. On lower dose LT4 (150 mcg to 250 mcg weekly) her fT4 stayed low, 0.50 - 0.74 (0.82 - 1.777mg/dL) with normal TSH of 0.73 - 1.42. She remained clinically euthyroid throughout. Serial yearly thyroid US showed no significant change. Pituitary hormonal work-up (IGF-1, FSH, LH, ACTH, estradiol and cortisol) was appropriate for age. fT4 by direct dialysis assay was low with normal TSH. tT4, T3 uptake, rT3, TBG and TPO Ab were normal. HAMA antibodies were negative. LT4 was stopped and she remains clinically euthyroid with low fT4 0.56 and normal TSH 1.73. Conclusion: The pattern of low fT4 with normal TSH is uncommon. Potential causes that were systematically evaluated include non-thyroidal illness, central hypothyroidism and assay interference. Normal pituitary function and appropriate TSH suppression while on LT4 ruled out central hypothyroidism. Her overall profile suggested fT4 assay interference. Commonly known interference factors were ruled out; biotin, HAMA Ab, pregnancy, renal failure, TBG excess, familial dysalbuminemic hyperthyroxiemia and trans-thyretin-associated hyperthyroxidemia. Direct dialysis is the gold standard fT4 assay but does not detect agents displacing T4 from TBG. It is less commonly recognized that low fT4 with normal TSH has been described amongst clinically euthyroid persons with HIV, at a prevalence of 1.3%–6.8%. Low fT4 is associated with didanosine, stavudine, and ritonavir, but not her anti-virals. The significance of low fT4 in patients with HIV is unclear, because they do not have a higher rate of hypothyroid symptoms. In the absence of clinical hypothyroidism, such as in this patient, LT4 treatment is not recommended. This case uniquely spans eight years and therefore offers valuable insight into the appropriate work-up, management and long-term natural history of low fT4 in persons with HIV.