Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Background

The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT).

Methodology and Results

Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%.

Conclusions/Significance

The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.     

采矿业对赞比亚的社会经济及环境的影响

采矿业是赞比亚一类非常重要的工业 ,大量的开采和选矿作业给周边地区带来了严重的环境问题。导致了该区域内地表水、空气、土壤的污染 ,并对当地人民的身体健康带来威胁。本文主要分析铜矿开采业对赞比亚的社会经济及环境的影响 ,进一步探讨可以保持当地采矿业的可持续发展的有效措施。     

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

An on-line adaptive glucose feeding system incorporating patterns recognition for glucose concentration control in glutamate fermentations

In glutamate fermentation, intermittent feeding is the most widely used glucose feed strategy. This feeding strategy causes severe fluctuations of glucose concentration and osmotic pressure in fermentation broth, which deteriorates the viability of the cell and reduces glutamate production in turn. In order to maintain glucose concentration at stable and constant levels, an on-line prediction and feedback control system based an empiric mass balance model was developed. However, the control system did not work properly and sometimes glucose concentration could even decline to 0 level (glucose exhaustion), as the model parameter varies in different runs. As a result, a novel model-based adaptive feedback control system incoporating with an artificial neural network (ANN) based pattern reconition unit for on-line diagnosizing the fault of glucose exhaustion was proposed and applied for glutamate fermentation. This adaptive control system could accurately detect glucose exhaustion when it occurs, and then immediately updates the control parameter based on some pre-defined rule. With the proposed control system, glucose was automatically fed, and its concentration could be maintained at desired levels constantly. As a result, glutamate concentration was 17 ~ 30% higher than that of the traditional fermentations using the intermittent glucose feed strategy.     

Density‐dependent reproduction and pollen limitation in an invasive milkweed,Calotropis procera (Ait.) R. Br. (Apocynaceae)

Calotropis procera (Ait.) R.Br. (Apocynaceae), an invasive woody milkweed, has expanded its range in northern Australia affecting rangeland and pastoral productivity. While self‐compatibility should enhance the species range expansion, spread of C. procera is limited by the availability of larger wasp and bee species that are able to vector its solid pollinia. Pollination efficiency is thus likely dependent on both pollinator abundance and plant density. Calotropis procera flowers year round in Australia but fruiting is limited to the warm months of the year when pollinators are most abundant, indicating that seasonal regulation of reproduction may be due to pollinator limitation. We examine the propositions that C. procera reproduction is regulated by the interaction between plant population density and pollinator pressure and that low pollinator pressure causes low per capita plant fecundity. All pollinators belonged to Order Hymenoptera and pollinator composition was similar at six of the seven sites. Fruit production per plant (fecundity) was lower above and below intermediate densities (350–550 plants ha^?1) of flowering plants with evidence of a weak Allee effect at lower plant density. Pollinator visitation rates per plant were low at high and low plant densities, and greatest at intermediate densities, while pollen supplementation experiments showed that C. procera is pollen limited (Pollen Limitation Index_fruit = 0.9) even at intermediate densities. Pollen limitation caused by low pollinator pressure at low plant densities and pollinator satiation at high plant densities may account for these fruit production trends. Management should be conducted in the colder months when pollinator pressure is low and plants are not reproducing. In addition, where stand eradication cannot be achieved in one attempt, management should reduce flowering plants to below intermediate densities where the fecundity per plant is low.     

Plant genomics in Africa: present and prospects

Plants are the world’s most consumed goods. They are of high economic value and bring many health benefits. In most countries in Africa, the supply and quality of food will rise to meet the growing population’s increasing demand. Genomics and other biotechnology tools offer the opportunity to improve subsistence crops and medicinal herbs in the continent. Significant advances have been made in plant genomics, which have enhanced our knowledge of the molecular processes underlying both plant quality and yield. The sequencing of complex genomes of African plant species, facilitated by the continuously evolving next-generation sequencing technologies and advanced bioinformatics approaches, has provided new opportunities for crop improvement. This review summarizes the achievements of genome sequencing projects of endemic African plants in the last two decades. We also present perspectives and challenges for future plant genomic studies that will accelerate important plant breeding programs for African communities. These challenges include a lack of basic facilities, a lack of sequencing and bioinformatics facilities, and a lack of skills to design genomics studies. However, it is imperative to state that African countries have become key players in the plant genome revolution and genome derived-biotechnology. Therefore, African governments should invest in public plant genomics research and applications, establish bioinformatics platforms and training programs, and stimulate university and industry partnerships to fully deploy plant genomics, particularly in the fields of agriculture and medicine.     

Cellular phone‐based photoplethysmographic imaging

We present study results on visible light reflection photoplethysmographic (PPG) imaging with a mobile cellular phone operated in video imaging mode. PPG signal components around 0.1 Hz attributed to the sympathetic component of the heart rate, 1 Hz as the heart rate and 2 Hz as heart rate high order harmonic were quantified on the index finger of a healthy volunteer. The green channel reported PPG signals throughout the sampled area. The blue and red channel returned plethysmographic information, but the signal strength was highly position specific. Our results obtained with a cellular phone as the data acquisition device are encouraging, especially in the broad context of personal or home‐based care and the role of cellular phone technology in medical imaging. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A Low-Cost Ultrasound Program Leads to Increased Antenatal Clinic Visits and Attended Deliveries at a Health Care Clinic in Rural Uganda

Background

In June of 2010, an antenatal ultrasound program to perform basic screening for high-risk pregnancies was introduced at a community health care center in rural Uganda. Whether the addition of ultrasound scanning to antenatal visits at the health center would encourage or discourage potential patients was unknown. Our study sought to evaluate trends in the numbers of antenatal visits and deliveries at the clinic, pre- and post-introduction of antenatal ultrasound to determine what effect the presence of ultrasound at the clinic had on these metrics.

Methods and Findings

Records at Nawanyago clinic were reviewed to obtain the number of antenatal visits and deliveries for the 42 months preceding the introduction of ultrasound and the 23 months following. The monthly mean deliveries and antenatal visits by category (first visit through fourth return visit) were compared pre- and post- ultrasound using a Kruskal-Wallis one-way ANOVA. Following the introduction of ultrasound, significant increases were seen in the number of mean monthly deliveries and antenatal visits. The mean number of monthly deliveries at the clinic increased by 17.0 (13.3–20.6, 95% CI) from a pre-ultrasound average of 28.4 to a post-ultrasound monthly average of 45.4. The number of deliveries at a comparison clinic remained flat over this same time period. The monthly mean number of antenatal visits increased by 97.4 (83.3–111.5, 95% CI) from a baseline monthly average of 133.5 to a post-ultrasound monthly mean of 231.0, with increases seen in all categories of antenatal visits.

Conclusions

The availability of a low-cost antenatal ultrasound program may assist progress towards Millennium Development Goal 5 by encouraging women in a rural environment to come to a health care facility for skilled antenatal care and delivery assistance instead of utilizing more traditional methods.     

Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus

Cellulase production was investigated in a culture of a strain of Acremonium cellulolyticus. The medium components were optimized for the improvement of cellulase production. The maximum production of cellulolytic enzymes was obtained in a medium containing (grams per liter) 50 Solka Floc, 5 (NH4)2SO4, 24 KH2PO4, 4.7 potassium tartrate hemihydrate, 1.2 MgSO4.7H2O, 1 Tween 80, 4 urea, 0.01 ZnSO4.7H2O, 0.01 MnSO4.6H2O, and 0.01 CuSO4.7H2O, with a pH of 4.0. In the flask culture, 15.5 filter paper units (FPU)/mL of maximum cellulase activity was obtained, 17.42 FPU/mL in a 7-L bioreactor, and 13.08 FPU/mL in a 50-L scale bioreactor for 4-8 d at 30 degrees C. Average production rates were 1.94 FPU/mL.d in flasks, 2.86 FPU/mL.d in the 7-L bioreactor, and 2.56 FPU/mL.d in the 50-L bioreactor. Cellulase production on a small scale was successfully reproduced in the 50-L pilot scale bioreactor. Saccharification activity from A. cellulolyticus was compared with cellulolytic enzymes produced by other strains. The A. cellulolyticus culture broth had a comparable saccharification yield in comparison with those of other Trichoderma enzymes (GC220 or Cellulosin T2) under the same total cellulase activity. Its saccharification yield (percent of released reducing sugar to used dried substrate) was 60%, and its glucose content was 83%.     

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.