Blood Stains

We did a prospective diagnostic accuracy study for cryptosporidiosis in children presenting to health-care facilities with diarrhoea or dysentery, in Jimma Medical Centre, Jimma, and Serbo Health Centre, Serbo, in southwest Ethiopia. Jimma Medical Centre is a tertiary referral hospital located in an urban area; Serbo Health Centre covers a rural area around the smaller town of Serbo, approximately 16 km from Jimma.
Study nurses screened children younger than 5 years of age at the paediatric outpatient departments and all inpatient wards at both centres for eligibility. Children were consecutively enrolled from 0800 h to 1800 h every day at Jimma Medical Centre and from 0800 h to 1700 h in Serbo Health Centre. Children were eligible if they had diarrhoea (three or more loose stools within the previous 24 h) or dysentery (at least one loose stool with stains of blood within the previous 24 h), regardless of whether these were the primary complaints leading them to seek health care. The exclusion criterion was inpatient admission for longer than 24 h before enrolment in the study. Written informed consent was obtained from the children's caregivers and we followed STARD guidelines.⁸
The embedded case-control study included children with diarrhoea from the 15 districts in and around Jimma and the eight districts surrounding Serbo. Community controls without diarrhoea in the previous 48 h were recruited by weekly frequency matching by geographical district of the household, age group, and enrolment week.²³ (link)
Jimma University Institutional Review Board (reference RPGC/610/2016), the Ethiopian National Research Ethics Review Committee (reference JU JURPGD/839/2017), and the Regional Committee for Medical and Health Research Ethics of Western Norway (reference 2016/1096) approved the study.

For initial development of the paper-based newborn SCD screening test, blood samples were collected from SCD (HbSS) patients at the Texas Children’s Hematology Center and from healthy volunteers (HbAA) into Vacutainer vials (K₂EDTA, BD Diagnostics, USA) using standard venipuncture technique. Samples were stored at 4 °C until use. Artificially reconstituted samples with a range of HbS levels were created by mixing ABO/Rh-matched, equal-hematocrit HbAA and HbSS blood samples at various ratios.
For test validation in Cabinda, blood samples were collected from newborns by heel-stick onto blood collection cards (Whatman 903 Protein Saver Card, GE Healthcare, USA) and into capillary blood collection tubes (Microvette, K₂EDTA, Sarstedt AG & Co, Germany). Eluted dried blood spot samples were tested with isoelectric focusing (IEF) following existing standard operating procedures7 (link). Liquid blood samples were refrigerated and used to perform the paper-based test within 7 days of collection. For all patients, the paper-based test was completed before IEF analysis. Local health workers interpreted the results of the paper-based test visually, using reference images of HbS-positive and HbS-negative bloodstains.

Blood samples from wild birds, 30 males and 50 females of Scopoli’s Shearwater (Calonectris diomedea), 20 males and 25 females of European Storm Petrel (Hydrobates pelagicus melitensis), 3 males and 3 females of Yelkouan Shearwater (Puffinus yelkouan), previously sexed by PCR with standard primers (CHD2550 and CHD2718) have been employed to test and validate the method. Further C. diomedea samples were employed (1200 of blood and 130 of feathers), among which males and females resulted nearly equally represented.
Samples were collected during the breeding seasons. Freshly sampled blood (one single drop of 1–3 μL or, alternatively, a few mm² of blood-spotted filter paper) was added to 50 μL of lysis solution (200 mM KOH, 20 mM Na2EDTA, 0.25% Triton X-100); in fresh blood, complete lysis occurs within a few seconds, and it results in sudden increase of the solution viscosity, due to free DNA. However, further incubation at ambient temperature for 1–2 min after lysis improves DNA and protein denaturation and solubilization (some blood proteins, such as proteases, are known to inhibit DNA amplification), as well as the dissociation of DNA/proteins complexes.
Dry blood stains absorbed onto filter paper (air-dried and stored up to several months at room temperature) were incubated in lysis buffer at 70–80 °C for 10 min or until the blood appeared dissolved (alternatively, incubation can be carried out overnight at room temperature); feathers (one per sample) require harsher conditions and were incubated at 98 °C for 5 min or, alternatively, at 80 °C for 15 min, followed by 1 min at 98 °C.
After a brief, thorough mixing, six volumes of a BTB neutralization solution (50 mM Tris-HCl, 0.0025% bromothymol blue) were added and the samples were briefly mixed. This makes the use of micropipettes not strictly necessary, and both solutions can be measured as drops (six drops of neutralization solution per drop of lysis solution). The pH indicator BTB was previously tested for its compatibility with amplification reactions and was included to check samples for proper pH values, compatible with downstream applications. In fact, the dye turns from yellow to blue/green around pH values of 7.6 (Figure 1A), the correct pH is easily monitored by the color of the solution (Figure 1B) and the resulting buffer system ensures suitable pH values even if slightly different volumes are added.