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1.  Laboratory Challenges Conducting International Clinical Research in Resource-Limited Settings 
There are many challenges to performing clinical research in resource-limited settings (RLS). Here we discuss several of the most common laboratory issues that must be addressed. These include issues relating to organization and personnel, laboratory facilities and equipment, standard operating procedures, external quality assurance, shipping, laboratory capacity and data management. While much progress has been made, innovative ways of addressing some of these issues are still very much needed.
PMCID: PMC3893068  PMID: 24321984
good laboratory practice; quality management; resource limited settings; hiv infection; tuberculosis
2.  Workshop Summary: Novel Biomarkers for HIV Incidence Assay Development 
Reliable methods for measuring human immunodeficiency virus (HIV) incidence are a high priority for HIV prevention. They are particularly important to assess the population-level effectiveness of new prevention strategies, to evaluate the community-wide impact of ongoing prevention programs, and to assess whether a proposed prevention trial can be performed in a timely and cost-efficient manner in a particular population and setting. New incidence assays and algorithms that are accurate, rapid, cost-efficient, and can be performed on easily-obtained specimens are urgently needed. On May 4, 2011, the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), sponsored a 1-day workshop to examine strategies for developing new assays to distinguish recent from chronic HIV infections. Participants included leading investigators, clinicians, public health experts, industry, regulatory specialists, and other stakeholders. Immune-based parameters, markers of viral sequence diversity, and other biomarkers such as telomere length were evaluated. Emerging nanotechnology and chip-based diagnostics, including algorithms for performing diverse assays on a single platform, were also reviewed. This report summarizes the presentations, panel discussions, and the consensus reached for pursuing the development of a new generation of HIV incidence assays.
PMCID: PMC3358102  PMID: 22206265
3.  Diminished Human Immunodeficiency Virus Type 1 DNA Yield from Dried Blood Spots after Storage in a Humid Incubator at 37°C Compared to −20°C▿  
Journal of Clinical Microbiology  2008;46(9):2945-2949.
Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ∼85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.
PMCID: PMC2546762  PMID: 18614660
4.  Evaluation of Dried Blood Spots for Human Immunodeficiency Virus Type 1 Drug Resistance Testing▿  
Journal of Clinical Microbiology  2006;45(2):517-521.
Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at −20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at −20°C or at −70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that −20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.
PMCID: PMC1829056  PMID: 17166967
5.  Topoisomerase Sequences of Coagulase-Negative Staphylococcal Isolates Resistant to Ciprofloxacin or Trovafloxacin 
Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of ≥4 μg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, ≥4 μg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to ≤2 μg of trovafloxacin per ml and 32% were susceptible to ≤1 μg of trovafloxacin per ml. Sixteen isolates, including twelve that expressed fluoroquinolone resistance, were chosen for detailed analysis. Identification of species by rRNA sequencing revealed a preponderance of Staphylococcus haemolyticus and S. hominis among fluoroquinolone-resistant strains. Segments of genes (gyrA and grlA) encoding DNA gyrase and DNA topoisomerase IV were sequenced. Considerable interspecies variation was noted, mainly involving noncoding nucleotide changes. Intraspecies variation consisted of coding changes associated with fluoroquinolone resistance. As for S. aureus, ciprofloxacin resistance (MIC, ≥8 μg/ml) and increased trovafloxacin MICs (0.25 to 2 μg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of ≥8 μg/ml also occurred, but these required an additional mutation in grlA.
PMCID: PMC89335  PMID: 10390214
6.  Topoisomerase Mutations in Trovafloxacin-Resistant Staphylococcus aureus 
A total of 201 Staphylococcus aureus isolates were surveyed for susceptibility to ciprofloxacin and trovafloxacin. Of 66 methicillin-resistant isolates, 89% were ciprofloxacin resistant and 6% were also trovafloxacin resistant. Trovafloxacin-resistant strains had unusual patterns of quinoline resistance mutations in DNA topoisomerase genes, including two mutations in the A subunit (encoded by grlA) of topoisomerase IV.
PMCID: PMC105881  PMID: 9687420

Results 1-6 (6)