Evaluation of dried blood spots collected on filter papers from three manufacturers stored at ambient temperature for application in HIV-1 drug resistance monitoring.

As more HIV-infected people gain access to antiretroviral therapy (ART), monitoring HIV drug resistance (HIVDR) becomes essential to combat both acquired and transmitted HIVDR. Studies have demonstrated dried blood spots (DBS) are a suitable alternative in HIVDR monitoring using DBS collected on Whatman 903 (W-903). In this study, we sought to evaluate two other commercially available filter papers, Ahlstrom 226 (A-226) and Munktell TFN (M-TFN), for HIVDR genotyping following ambient temperature storage. DBS were prepared from remnant blood specimens collected from 334 ART patients and stored at ambient temperature for a median time of 30 days. HIV-1 viral load was determined using NucliSENS EasyQ® HIV-1 v2.0 RUO test kits prior to genotyping of the protease and reverse transcriptase regions of the HIV-1 pol gene using an in-house assay. Among the DBS tested, 26 specimens had a viral load ≥ 1000 copies/mL in all three types of filter paper and were included in the genotyping analysis. Genotyping efficiencies were similar between DBS collected on W-903 (92.3%), A-226 (88.5%), and M-TFN (92.3%) filter papers (P = 1.00). We identified 50 DR-associated mutations in DBS collected on W-903, 33 in DBS collected on A-226, and 48 in DBS collected on M-TFN, resulting in mutation detection sensitivities of 66.0% for A-226 and 88.0% for M-TFN when compared to W-903. Our data indicate that differences among filter papers may exist at this storage condition and warrant further studies evaluating filter paper type for HIVDR monitoring.

Follow-up of infants diagnosed with HIV - Early Infant Diagnosis Program, Francistown, Botswana, 2005-2012.

The 2011 prevalence of human immunodeficiency virus (HIV) among pregnant women in Botswana was 30.4%. High coverage rates of HIV testing and antiretroviral prophylaxis have reduced the rate of mother-to-child transmission of HIV in Botswana from as high as 40% with no prophylaxis to <4% in 2011. In June 2005, the national Early Infant Diagnosis (EID) Program began testing HIV-exposed infants (i.e., those born to HIV-infected mothers) for HIV using polymerase chain reaction (PCR) at 6 weeks postpartum. During 2005-2012, follow-up of all HIV-infected infants diagnosed in all 13 postnatal care facilities in Francistown, Botswana, was conducted to ascertain patient outcomes. A total of 202 infants were diagnosed with HIV. As of September 2013, 82 (41%) children were alive and on antiretroviral therapy (ART), 79 (39%) had died, and 41 (20%) were either lost to follow-up, had transferred, or their mothers declined ART. Despite success in preventing mother-to-child transmission in Botswana, results of the EID program highlight the need for early diagnosis of HIV-infected infants, prompt initiation of ART, and retention in care.

Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

Hospitalization and mortality among primarily nonbreastfed children during a large outbreak of diarrhea and malnutrition in Botswana, 2006.

BACKGROUND: In 2006, a pediatric diarrhea outbreak occurred in Botswana, coinciding with heavy rains. Surveillance recorded a 3 times increase in cases and a 25 fold increase in deaths between January and March. Botswana has high HIV prevalence among pregnant women (33.4% in 2005), and an estimated 35% of all infants under the age of 6 months are not breastfed. METHODS: We followed all children <5 years old with diarrhea in the country's second largest referral hospital at the peak of the outbreak by chart review, interviewed mothers, and conducted laboratory testing for HIV and enteric pathogens. RESULTS: Of 153 hospitalized children with diarrhea, 97% were <2 years old; 88% of these were not breastfeeding. HIV was diagnosed in 18% of children and 64% of mothers. Cryptosporidium and enteropathogenic Escherichia coli were common; many children had multiple pathogens. Severe acute malnutrition (kwashiorkor or marasmus) developed in 38 (25%) patients, and 33 (22%) died. Kwashiorkor increased risk for death (relative risk 2.0; P = 0.05); only one breastfeeding child died. Many children who died had been undersupplied with formula. CONCLUSIONS: Most of the severe morbidity and mortality in this outbreak occurred in children who were HIV negative and not breastfed. Feeding and nutritional factors were the most important determinants of severe illness and death. Breastfeeding is critical to infant survival in the developing world, and support for breastfeeding among HIV-negative women, and HIV-positive women who cannot formula feed safely, may prevent further high-mortality outbreaks.

Early diagnosis of human immunodeficiency virus in infants using polymerase chain reaction on dried blood spots in Botswana's national program for prevention of mother-to-child transmission.

BACKGROUND: Botswana has high antenatal human immunodeficiency virus (HIV) prevalence (33.4%). The public health system provides free services for prevention of mother to child transmission of HIV (PMTCT) and antiretroviral therapy, which can reduce vertical HIV transmission from 35% to <5%. Infant HIV diagnosis is challenging in resource-limited settings, and HIV prevalence among HIV-exposed infants in Botswana is unknown. Dried blood spot (DBS) polymerase chain reaction (PCR) provides a feasible method to assess PMTCT programs and identify HIV-infected children. METHODS: We trained staff in 15 clinics and a hospital to obtain DBS on HIV-exposed infants age 6 weeks to 17 months receiving routine care. Samples were sent to the national HIV reference laboratory. Roche Amplicor 1.5 DNA PCR testing was performed. RESULTS: Between June-December 2005, 1931 HIV-exposed infants age 6 weeks to 17 months were tested for HIV, of whom 136 (7.0%) were HIV infected. Among infants