Accurate automated quantitative imaging of tortoise erythrocytes using the NIS image analysis system

The standard method for assessing blood cell characteristics using an ocular micrometer is time-consuming and limited. We used the Nikon NIS Elements imaging software and May-Grünwald-Giemsa staining to determine whether automated image analysis is suitable for rapid and accurate quantitative morphometry of erythrocytes. Blood was collected during four seasons from 126 geometric tortoises and the blood smears were evaluated for cell (C) and nuclear (N) characteristics of the erythrocytes. We measured area, length (L), width (W), perimeter, elongation and pixelation intensity, and calculated L/W and N/C areas. Erythrocyte size differed among cohorts; females, the larger sex, had smaller erythrocytes than either males or juveniles. Males had more elongated erythrocytes than females and erythrocytes of adults were more elongated than those of juveniles. Erythrocyte size and shape influence the efficiency of gas exchange owing to surface area to volume ratios, which are greater for small, elongated cells than for large, round cells. The high N/C ratio and low pixelation intensities of males and juveniles indicate that they may have had more immature erythrocytes in their circulation than females. The use of pixelation intensity to indicate the presence of immature erythrocytes was validated by seasonal differences that corresponded to the biology of the tortoises. Pixelation intensity was lowest in winter. We found that automated image analysis is a rapid and reliable method for determining cell size and shape, and it offers the potential for distinguishing among developmental stages that differ in staining intensity. The method should be useful for rapid health assessments, particularly of threatened species, and for comparative studies among different vertebrates.Web of Scienc

Diagnostic accuracy of a three-gene Mycobacterium tuberculosis host response cartridge using fingerstick blood for childhood tuberculosis: a multicentre prospective study in low-income and middle-income countries

BACKGROUND: Childhood tuberculosis remains a major cause of morbidity and mortality in part due to missed diagnosis. Diagnostic methods with enhanced sensitivity using easy-to-obtain specimens are needed. We aimed to assess the diagnostic accuracy of the Cepheid Mycobacterium tuberculosis Host Response prototype cartridge (MTB-HR), a candidate test measuring a three-gene transcriptomic signature from fingerstick blood, in children with presumptive tuberculosis disease. METHODS: RaPaed-TB was a prospective diagnostic accuracy study conducted at four sites in African countries (Malawi, Mozambique, South Africa, and Tanzania) and one site in India. Children younger than 15 years with presumptive pulmonary or extrapulmonary tuberculosis were enrolled between Jan 21, 2019, and June 30, 2021. MTB-HR was performed at baseline and at 1 month in all children and was repeated at 3 months and 6 months in children on tuberculosis treatment. Accuracy was compared with tuberculosis status based on standardised microbiological, radiological, and clinical data. FINDINGS: 5313 potentially eligible children were screened, of whom 975 were eligible. 784 children had MTB-HR test results, of whom 639 had a diagnostic classification and were included in the analysis. MTB-HR differentiated children with culture-confirmed tuberculosis from those with unlikely tuberculosis with a sensitivity of 59·8% (95% CI 50·8–68·4). Using any microbiological confirmation (culture, Xpert MTB/RIF Ultra, or both), sensitivity was 41·6% (34·7–48·7), and using a composite clinical reference standard, sensitivity was 29·6% (25·4–34·2). Specificity for all three reference standards was 90·3% (95% CI 85·5–94·0). Performance was similar in different age groups and by malnutrition status. Among children living with HIV, accuracy against the strict reference standard tended to be lower (sensitivity 50·0%, 15·7–84·3) compared with those without HIV (61·0%, 51·6–69·9), although the difference did not reach statistical significance. Combining baseline MTB-HR result with one Ultra result identified 71·2% of children with microbiologically confirmed tuberculosis. INTERPRETATION: MTB-HR showed promising diagnostic accuracy for culture-confirmed tuberculosis in this large, geographically diverse, paediatric cohort and hard-to-diagnose subgroups. FUNDING: European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung; German Center for Infection Research (DZIF)