Detection of HIV-1 Infection in Dried Blood Spots from a 12-Year-Old ABO Bedside Test Card

Background and Objectives: We tested dried blood from an ABO bedside test card which had been stored at room temperature for 12 years, to prove that a patient with HIV-1 infection had been infected by blood transfusion. Materials and Methods: Immunoblots for HIV-1 antibodies and threefold PCRs with halfnested primers for the HIV-1 integrase gene were done with eluates from the dried blood spots. Results: HIV-1 antibodies and HIV-1 DNA could be detected in the sample from one unit of blood, but not from the two other units or from the recipient before transfusion. Conclusion: Further studies should be done on the validity of stored dried blood as an alternative to the storage of frozen donor serum for several years for ‘look-back’ studies.     

Validity of Estimation of Haemoglobin Content in Dried Blood Spot Samples

The ability to use indirect cyanmethemoglobin method using 20 µl of dried blood spots (DBSs) on filter paper for the analysis of haemoglobin (Hb) levels could be an important diagnostic tool for areas that have limited access to laboratory facilities. We assessed the validity of a method for Hb estimation in which a single drop of whole blood was directly taken on the filter paper. We collected 124 DBSs containing 20 µl of blood (filter paper A) and single drop of whole blood (filter paper B) from subjects living in Nainital, Uttrakhand. Estimation of Hb was done by indirect cyanmethemoglobin method in both the filter papers. A correction factor for predicting value of Hb from DBSs of single drop of whole blood was established. The Bland–Altman plot suggested that the difference in Hb values obtained by the single drop of blood and 20 µl of DBSs was within the 1.5 SD limits, suggesting high validity of the correction factor. The estimation of Hb using single drop of whole blood on filter paper after applying the correction factor provides results similar to indirect cyanmethemoglobin method using 20 µl of blood. Hence, single drop of whole blood on filter paper can be used as an alternate method for estimation of Hb in large scale community surveys.     

Stability and Feasibility of Dried Blood Spots for Hepatitis E Virus Serology in a Rural Setting

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In many low-income countries it causes large outbreaks and disproportionally affects pregnant women and their offspring. Surveillance studies to find effective preventive interventions are needed but are hampered by the lack of funding and infrastructure. Dried blood spots (DBS) offer an easier and more robust way to collect, transport, and store blood samples compared to plasma/serum samples, and could ease some of the barriers for such studies. In this study we optimize an HEV IgG ELISA for DBS samples and validate it on 300 paired DBS and plasma samples collected in rural areas of Bangladesh from participants in a HEV vaccine study. We demonstrate that HEV IgG in blood stored as DBS is stable for two months at up to 40 °C, and for five freeze-thaw cycles. The specificity was 97% and the overall sensitivity of the DBS assay was 81%. The sensitivity was higher in samples from vaccinated participants (100%) compared to previously infected participants (59%), reflecting a positive correlation between IgG titer and sensitivity. We found a strong correlation between DBS and plasma samples with an r2 of 0.90, but with a higher degree of difference between individual paired samples. Our study shows that DBS offers a stable alternative to plasma/serum for HEV IgG measurements and can facilitate serological studies, particularly in resource limited areas.     

Direct Detection of Oxy Asymmetrical Hemoglobin Hybrids in Blood Samples

Hb Chicago is a newly discovered hemoglobin variant which was present in a Black newborn baby and her father. The leucine residue at α136, which normally participates in the contact with the heme group, is replaced by a methionine residue. The two heterozygotes were clinically well with normal hematological data. Isolation of the α^x and α^A chains by reverse phase high performance liquid chromatography and hydrolysis of these chains with dilute formic acid at 110°C for 24 hours, followed by separation of the resulting peptides by reverse phase high performance liquid chromatography, greatly facilitated the final identification of the abnormality. The baby and both parents had a-G_γ-G_γ-globin gene arrangement on one chromosome (normal:-G_γ-A_γ which explains the high a G^γ values in the Hb F of these three persons.     

Impact of Mass Azithromycin Distribution on Malaria Parasitemia during the Low-Transmission Season in Niger: A Cluster-Randomized Trial

We assessed the effect of mass azithromycin treatment on malaria parasitemia in a trachoma trial in Niger. Twenty-four study communities received treatment during the wet, high-transmission season. Twelve of the 24 communities were randomized to receive an additional treatment during the dry, low-transmission season. Outcome measurements were conducted at the community-level in children < 1–72 months of age in May–June 2011. Parasitemia was higher in the 12 once-treated communities (29.8%, 95% confidence interval [CI] = 21.5–40.0%) than in the 12 twice-treated communities (19.5%, 95% CI = 13.0–26.5%, P = 0.03). Parasite density was higher in once-treated communities (354 parasites/μL, 95% CI = 117–528 parasites/μL) than in twice-treated communities (74 parasites/μL, 95% CI = 41–202 parasites/μL, P = 0.03). Mass distribution of azithromycin reduced malaria parasitemia 4–5 months after the intervention. The results suggest that drugs with antimalaria activity can have long-lasting impacts on malaria during periods of low transmission.     

Cost Savings with Rapid Diagnostic Tests for Malaria in Low-Transmission Areas: Evidence from Dar es Salaam, Tanzania

Rapid diagnostic tests (RDTs) for malaria may help rationalize antimalarial drug use. However, the economic effects of these tests may vary. Data on costs were collected from 259 patients in 6 health facilities by using exit and in-charge interviews and record reviews during a trial of RDT rollout in Dar es Salaam, Tanzania. The RDTs decreased patient expenditure on drugs (savings = U.S. $0.36; P = 0.002) and provider drug costs (savings = U.S. $0.43; P = 0.034) compared with control facilities. However, RDT introduction did not significantly reduce patients'' overall expenditures (U.S. $1.02, 95% confidence interval [CI] = $0.76–$1.36 versus U.S. $1.33 95% CI = $0.99–$1.77) and may increase total provider costs (U.S. $3.63, 95% CI = $3.40–$3.89 versus U.S. $2.32, 95% CI = $1.99–$2.69) compared with control facilities. Clinician''s compliance with test results was higher with RDTs than with routine microscopy (95% versus 82%; P = 0.002). The RDTs reduced drug costs in this setting but did not offset the cost of the tests, although they also resulted in non-monetary benefits, including improved management of patients and increased compliance with test results.     

Proficiency Testing of Human Leukocyte Antigen-DR and Human Leukocyte Antigen-DQ Genetic Risk Assessment for Type 1 Diabetes Using Dried Blood Spots

Background

The plurality of genetic risk for developing type 1 diabetes mellitus (T1DM) lies within the genes that code for the human leukocyte antigens (HLAs). Many T1DM studies use HLA genetic risk assessment to identify higher risk individuals, and they often conduct these tests on dried blood spots (DBSs) like those used for newborn bloodspot screening. One such study is The Environmental Determinants of Diabetes in the Young (TEDDY), a long-term prospective study of environmental risk factors. To provide quality assurance for T1DM studies that employ HLA genetic risk assessment, the Centers for Disease Control and Prevention (CDC) conducts both a voluntary quarterly proficiency testing (VQPT) program available to any laboratory and a mandatory annual proficiency testing (PT) challenge for TEDDY laboratories.

Methods

Whole blood and DBS samples with a wide range of validated HLA-DR and HLA-DQ genotypes were sent to the participating laboratories. Results were evaluated on the basis of both the reported haplotypes and the HLA genetic risk assessment.

Results

Of the reported results from 24 panels sent out over six years in the VQPT, 94.7% (857/905) were correctly identified with respect to the relevant HLA-DR or HLA-DQ alleles, and 96.4% (241/250) were correctly categorized for risk assessment. Significant improvement was seen over the duration of this program, usually reaching 100% correct categorization during the last three years. Of 1154 reported results in four TEDDY PT challenges, 1153 (99.9%) were correctly identified for TEDDY eligibility.

Conclusions

The different analytical methods used by T1DM research centers all provided accurate (>99%) results for genetic risk assessment. The two CDC PT programs documented the validity of the various approaches to screening and contributed to overall quality assurance.