In-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple PCR primer approach.

Co-infections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of co-infections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.

Microbiological quality assessment of natural therapeutic herbal drug products prepared locally in Dar es Salaam, Tanzania.

One hundred and eighty one liquid and 183 powder preparations were purchased from different herbalists practising in open air markets in Hala District Dar es Salaam Region and analysed for total viable aerobic plate counts. Isolated microorganisms were identified by conventional microbiological and biochemical methods. The study revealed that the level of bacterial contamination in both liquid and powder drug samples was significantly higher (P <0.01) compared to the acceptable levels of 105/cfc/g. For fungi, the level of contamination in powders was highly significant (P <0.02) while in liquids the levels were not significant (P <0.2) when compared to the acceptable levels of 104/cfc/g. In both preparations, two potentially pathogenic organisms, Klebsiella spp. and Pseudomonas spp., were isolated. The level of contamination with these bacteria was significantly high (P <0.01) in both preparations. Other pathogenic micro notorganisms recovered were Candida spp. and Escherichia coll The level of contamination with these agents was significantly high (P < 0.01) in the powders. These results indicate that liquid and powder herbal preparations commonly sold by traditional herbalists in open air markets in Dar es Salaam are frequently contaminated by bacteria and fungi. Some of these microbial contaminants are potential pathogens of man, and may thus predispose the patients taking these preparations to the risk of acquiring infections. These contaminated medicinal preparations may also facilitate transfer of strains of microorganisms which harbour antibiotic resistance genes. In view of these findings, it is recommended to conduct further studies to find out how these herbalists collect, prepare, store and dispense the drugs. Attempts should also be made to train traditional herbalists the fundamental aspects of Good Manufacturing Practices, with a view to improving their skills in proper handling of drugs in order to minimise chances of contamination.