Native and Methylated Cyclodextrins with Positive and Negative Solubility Coefficients in Water Studied by SAXS and SANS

While the solubility of native α-, β-, γ-cyclodextrins (CDs) in water rises with temperature, the opposite is true for their methylated derivatives (mCDs; per-dimethylated β-CD and per-trimethylated γ-CD). The mCDs are well-soluble in cold water and crystallize upon heating, which we associate with the hydrophobic effect. To study the hydrophobic effect and hydration of CDs and mCDs dissolved in water (D2O), we performed small-angle X-ray and neutron scattering (SAXS and SANS) measurements. The experimental scattering curves were put on absolute scale and compared to scattering curves calculated from crystal structures using the cube method. The results of the comparison indicate that (i) in solution, CDs and mCDs are in monomeric form, (ii) van der Waals and solute excluded volumes can be related by introducing a shell of a thickness that correlates with the solute’s structure and solute−water interactions, and (iii) the SAXS curves calculated under the assumption of a uniform distribution of electron density in the solute molecules agree with experimental ones for CDs, but not for mCDs. The temperature and concentration dependence of SAXS curves is significant for mCDs and weak for CDs and is discussed in terms of solute−solute interactions. Specifically, these interactions become more attractive in solutions of mCDs with increasing temperature, concentration, or both, in accord with mCDs’ negative temperature coefficient of solubility in water

Distribution of the quantitative PCR results for monkeypox virus (blue) and orthopoxvirus (red) for the digital PCR (squares) and PCR/NAT (dots) methods.

Seven participants reported results in copies/mL. The colored lines indicate the mean values of the quantitative results for MPXV (blue) and OPXV (red) respectively, and the blue area indicates the 95% confidence interval of the mean value for the quantitative MPXV PCR results. A confidence interval for OPXV was not feasible.</p

Fig 2 -

Analysis of Ct/Cq values for (A) monkeypox virus PCR results and (B) for orthopoxvirus PCR results for different test systems. The grey boxes display all results for the respective sample, and the distributions of specific manufacturer-based collectives are illustrated as smaller, colored box plots in overlay with the total results. For all boxes, the whiskers stretch from the 1st quartile—1.5*(interquartile range) to the 3rd quartile + 1.5*(interquartile range).</p

Qualitative PCR results for the four samples of monkeypox EQA survey for A) MPXV DNA and B) OPXV DNA.

Qualitative PCR results for the four samples of monkeypox EQA survey for A) MPXV DNA and B) OPXV DNA.</p

Tables with MPXV qualitative results submitted by EQA participants.

Test kits that showed less than 5 results were aggregated into "other” (NS1 Table. The correct result for the corresponding samples is highlighted in light grey. The quota is defined as percentage of correct results in relation to all results for both the individual test kit (horizontally) and over all test kits for the corresponding sample (sum). (XLSX)</p

Aggregated information provided by the participating laboratories on their methods and target genes used for all analysis combined (qualitative and quantitative for MPXV and OPXV).

Aggregated information provided by the participating laboratories on their methods and target genes used for all analysis combined (qualitative and quantitative for MPXV and OPXV).</p

Sample properties.

BackgroundIn May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests.MethodsWe analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level.Results141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each.ConclusionThe EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.</div

Results of the Passing Bablok fit for the MPXV Ct/Cq results.

Results of the Passing Bablok fit for the MPXV Ct/Cq results.</p

Tables with OPXV qualitative results submitted by EQA participants.

Samples 418001, 418002 and 418004 are positive for MPXV DNA. Test kits that showed less than 5 results were aggregated into "other (NS1 Table. The correct result for the corresponding samples is highlighted in light grey. The quota is defined as percentage of correct results in relation to all results for both the individual test kit (horizontally) and over all test kits for the corresponding sample (sum). (XLSX)</p

Fig 1 -

Distribution of the qualitative PCR results for the four samples of the monkeypox EQA survey for A) monkeypox virus (MPXV) and B) orthopoxvirus (OPXV). Numbers in the columns represent the actual number of results for the corresponding category.</p