Summaryobjective: Recently developed molecular gametocyte detection techniques have shown that submicroscopic Plasmodium falciparum gametocytes are common in symptomatic patients and can infect mosquitoes. The relevance for the infectious reservoir of malaria in the general population remains unknown. In this study, we investigated submicroscopic asexual parasitaemia and gametocytaemia in inhabitants of an area of hypoendemic and seasonal malaria in Tanzania.methods: Two cross-sectional malariometric surveys were conducted in the dry and wet seasons of 2005 in villages in lower Moshi, Tanzania. Finger prick blood samples were taken to determine the prevalence of P. falciparum parasites by microscopy, rapid diagnostic test and real-time nucleic acid sequence-based amplification (QT-NASBA).results: 2752 individuals participated in the surveys, of whom 1.9% (51/2721) had microscopically confirmed asexual parasites and 0.4% (10/2721) had gametocytes. In contrast, QT-NASBA revealed that 32.5% (147/453) of the individuals harboured asexual parasites and 15.0% (68/453) had gametocytes. No age dependency or seasonality was observed in submicroscopic parasite carriage.discussion: Molecular detection techniques reveal that carriage of submicroscopic asexual parasite and gametocyte densities is relatively common in this low transmission area. Submicroscopic gametocytaemia is likely to be responsible for maintaining malarial transmission in the study area.

Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 l of venous blood to achieve this sensitivity, compared to 50 l of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.Routine clinical diagnosis of malaria is usually based on microscopic detection of Plasmodium parasites in blood smears. However, this technique is relatively laborious when large numbers of samples need to be quantified simultaneously. Furthermore, the detection limit of microscopy, 1 to 20 parasites per l of blood, may not always be sufficiently sensitive. Parasite densities below the detection level of microscopy may play an important role in Plasmodium population dynamics and the epidemiology of the disease; therefore, the availability and use of more-sensitive detection techniques is a prerequisite for many research projects. With the rapid developments in the field of molecular biology, several nucleic acid-based amplification methods, including PCR, reverse transcriptase PCR, and nucleic acid sequence-based amplification (NASBA) (2, 3, 7-9, 11), are now available for detection of Plasmodium parasites. Because many studies require accurate and sensitive quantification of parasites, most of these techniques have been adapted for quantitative analysis (1,3,6,7,11). In preparation for vaccine trials and epidemiological studies directed at evaluation of interventions, the present study was designed to evaluate the newly developed real-time quantitative NASBA (QT-NASBA) and to compare the assay to another quantitative nucleic acidba...

Introduction Routinely monitoring the HIV viral load ( VL ) of people living with HIV ( PLHIV ) on anti‐retroviral therapy ( ART ) facilitates intensive adherence counselling and faster ART regimen switch when treatment failure is indicated. Yet standard VL ‐testing in centralized laboratories can be time‐intensive and logistically difficult in low‐resource settings. This paper evaluates the outcomes of the first four years of routine VL ‐monitoring using Point‐of‐Care technology, implemented by Médecins Sans Frontières ( MSF ) in rural clinics in Malawi. Methods We conducted a retrospective cohort analysis of patients eligible for routine VL ‐ testing between 2013 and 2017 in four decentralized ART ‐clinics and the district hospital in Chiradzulu, Malawi. We assessed VL ‐testing coverage and the treatment failure cascade (from suspected failure (first VL >1000 copies/ mL ) to VL suppression post regimen switch). We used descriptive statistics and multivariate logistic regression to assess factors associated with suspected failure. Results and Discussion Among 21,400 eligible patients, VL ‐testing coverage was 85% and VL suppression was found in 89% of those tested. In the decentralized clinics, 88% of test results were reviewed on the same day as blood collection, whereas in the district hospital the median turnaround‐time for results was 85 days. Among first‐line ART patients with suspected failure (N = 1544), 30% suppressed ( VL <1000 copies/ mL ), 35% were treatment failures (confirmed by subsequent VL ‐testing) and 35% had incomplete VL follow‐up. Among treatment failures, 80% (N = 540) were switched to a second‐line regimen, with a higher switching rate in the decentralized clinics than in the district hospital (86% vs. 67%, p < 0.01) and a shorter median time‐to‐switch (6.8 months vs. 9.7 months, p < 0.01). Similarly, the post‐switch VL ‐testing rate was markedly higher in the decentralized clinics (61% vs. 26%, p < 0.01). Overall, 79% of patients with a post‐switch VL ‐test were suppressed. Conclusions Viral load testing at the point‐of‐care in Chiradzulu, Malawi achieved high coverage and good drug regimen switch rates among those identified as treatment failures. In...

The TLC assay was found to be sensitive, specific, and robust in the detection of subtherapeutic nevirapine concentrations in saliva specimens obtained from African HIV-infected adults. It is an attractive alternative to HPLC for therapeutic drug monitoring of nevirapine in resource-limited settings.

No abstract