LAMP for human African trypanosomiasis: a comparative study of detection formats

Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl₂ before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl₂, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl₂ adversely affects reaction sensitivity and was unpopular with several observers.     

Proteomic fingerprinting for the diagnosis of human African trypanosomiasis

Papadopoulos et al. recently reported the discovery of a diagnostic serum proteomic signature for human African trypanosomiasis (HAT), using a combination of surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry and data-mining algorithms. This novel approach, coupled with biochemical characterization of the proteins that contribute to the signature, provides powerful new tools for the development of improved diagnostic tests, disease staging and identification of potential novel drug targets in HAT.     

WSSV和IHHNV二重实时荧光PCR检测方法的建立

根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。     

PathogenMip assay: a multiplex pathogen detection assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

Therapy of human African trypanosomiasis: current situation

This paper is a review of the current situation of the treatment of human African trypanosomiasis. The existing approved drugs are old, toxic and/or expensive. Therapeutic failures are common. Several factors may contribute to the problems of chemotherapy, including differences in the epidemiology of the disease, difficulties in the diagnosis and staging of the infection, availability, distribution and pharmacologic properties of drugs, standardization of treatment regimens, response to therapy, follow-up period, and relapses and clinical trials. The new therapeutic approaches include the development and approval of new drugs, the use of new therapeutic regimens, the study of drug combinations, and the development of new formulations.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

Contributions and limits of the diagnosis of human African trypanosomiasis

Human African trypanosomiasis, or sleeping sickness, is still a worrying problem in Africa. Sleeping sickness is a disease for which a systematic monitoring is necessary, particularly for the trypanosomiasis caused by Trypanosoma brucei gambiense, which is characterized by a long asymptomatic stage. In the absence of specific clinical signs, mass screening of populations remains the only way to control the disease and to avoid its spreading. The lack of sensitivity and specificity of the diagnosis tests classically used led to the development of molecular tools. PCR amplification of parasite specific sequences has considerably improved the diagnostic of the parasitic infection, the stage diagnosis as well as the post-therapeutic follow-up. But there are limits with a use in routine and research is still necessary to make PCR a real tool for control of sleeping sickness.     

Diagnostic and neuropathogenesis issues in human African trypanosomiasis

Human African trypanosomiasis, also known as sleeping sickness, is caused by protozoan parasites of the genus Trypanosoma, and is a major cause of human mortality and morbidity. The East African and West African variants, caused by Trypanosma brucei rhodesiense and Trypanosoma brucei gambiense, respectively, differ in their presentation but the disease is fatal if untreated. Accurate staging of the disease into the early haemolymphatic stage and the late encephalitic stage is critical as the treatment for the two stages is different. The only effective drug for late stage disease, melarsoprol, which crosses the blood-brain barrier, is followed by a severe post-treatment reactive encephalopathy in 10% of cases of which half die. There is no current consensus on the diagnostic criteria for CNS involvement and the specific indications for melarsoprol therapy also differ. There is a pressing need for a quick, simple, cheap and reliable diagnostic test to diagnose Human African trypanosomiasis in the field and also to determine CNS invasion. Cerebrospinal fluid and plasma analyses in patients with Human African trypanosomiasis have indicated a role for both pro-inflammatory and counter-inflammatory cytokines in determining the severity of the meningoencephalitis of late stage disease, and, at least in T. b. rhodesiense infection, the balance of these opposing cytokines may be critical. Rodent models of Human African trypanosomiasis have proved very useful in modelling the post-treatment reactive encephalopathy of humans and have demonstrated the central role of astrocyte activation and cytokine balances in determining CNS disease. Such animal models have also allowed a greater understanding of the more direct mechanisms of trypanosome infection on CNS function including the disruption of circadian rhythms, as well as the immunological determinants of passage of trypanosomes across the blood-brain barrier.     

Molecular-beacon multiplex real-time PCR assay for detection of Vibrio cholerae

A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.     

New drugs for the treatment of human African trypanosomiasis: research and development

Chemotherapy of human African trypanosomiasis is problematic because of the high frequency of severe adverse events, the long duration and high cost of treatment, and an increasing number of treatment-refractory cases. New cost-efficient, easy-to-use drugs are urgently needed. Whereas basic research on potential drug targets is anchored in academia, the complex, highly regulated and very expensive process of preclinical and clinical drug development is almost exclusively in the hands of pharmaceutical companies. Jennifer Keiser, August Stich and Christian Burri here review, from the angle of industrial drug research and development, the past ten years of research activities at different stages of the development of trypanocidal drugs, and assess future prospects. The absence of compounds in clinical development Phases I-III indicates no new drugs will become available in the next few years.     

Prevalence of human African trypanosomiasis in the Democratic Republic of the Congo

Human African Trypanosomiasis (HAT) is a major public health problem in the Democratic Republic of the Congo (DRC). Active and passive surveillance for HAT is conducted but may underestimate the true prevalence of the disease. We used ELISA to screen 7,769 leftover dried blood spots from a nationally representative population-based survey, the 2007 Demographic and Health Survey. 26 samples were positive by ELISA. Three of these were also positive by trypanolysis and/or PCR. From these data, we estimate that there were 18,592 people with HAT (95% confidence interval, 4,883-32,302) in the DRC in 2007, slightly more than twice as many as were reported.     

Update on field use of the available drugs for the chemotherapy of human African trypanosomiasis

Despite the fact that eflornithine was considered as the safer drug to treat human African trypanosomiasis (HAT) and has been freely available since 2001, the difficulties in logistics and cost burden associated with this drug meant that the toxic melarsoprol remained the drug of choice. The World Health Organization responded to the situation by designing a medical kit containing all the materials needed to use eflornithine, and by implementing a training and drugs distribution programme which has allowed a transition to this much safer treatment. The introduction of the combination of nifurtimox and eflornithine (NECT) has accelerated the shift from melarsoprol to the best treatment available, due to reduced dosage and treatment time for eflornithine that has significantly lessened the cost and improved the burden of logistics encountered during treatment and distribution. The decrease in the use of more dangerous but cheaper melarsoprol has meant a rise in the per patient cost of treating HAT. Although NECT is cheaper than eflornithine monotherapy, an unexpected consequence has been a continuing rise in the per patient cost of treating HAT. The ethical decision of shifting to the best available treatment imposes a financial burden on HAT control programmes that might render long-term application unsustainable. These factors call for continuing research to provide new safer and more effective drugs that are simple to administer and cheaper when compared to current drugs.     

Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.     

Accuracy of five algorithms to diagnose gambiense human African trypanosomiasis

Background

Algorithms to diagnose gambiense human African trypanosomiasis (HAT, sleeping sickness) are often complex due to the unsatisfactory sensitivity and/or specificity of available tests, and typically include a screening (serological), confirmation (parasitological) and staging component. There is insufficient evidence on the relative accuracy of these algorithms. This paper presents estimates of the accuracy of five algorithms used by past Médecins Sans Frontières programmes in the Republic of Congo, Southern Sudan and Uganda.

Methodology and Principal Findings

The sequence of tests in each algorithm was programmed into a probabilistic model, informed by distributions of the sensitivity, specificity and staging accuracy of each test, constructed based on a literature review. The accuracy of algorithms was estimated in a baseline scenario and in a worst-case scenario introducing various near worst-case assumptions. In the baseline scenario, sensitivity was estimated as 85–90% in all but one algorithm, with specificity above 99.9% except for the Republic of Congo, where CATT serology was used as independent confirmation test: here, positive predictive value (PPV) was estimated at <50% in realistic active screening prevalence scenarios. Furthermore, most algorithms misclassified about one third of true stage 1 cases as stage 2, and about 10% of true stage 2 cases as stage 1. In the worst-case scenario, sensitivity was 75–90% and PPV no more than 75% at 1% prevalence, with about half of stage 1 cases misclassified as stage 2.

Conclusions

Published evidence on the accuracy of widely used tests is scanty. Algorithms should carefully weigh the use of serology alone for confirmation, and could enhance sensitivity through serological suspect follow-up and repeat parasitology. Better evidence on the frequency of low-parasitaemia infections is needed. Simulation studies should guide the tailoring of algorithms to specific scenarios of HAT prevalence and availability of control tools.     

Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.     

CCL18 in a multiplex urine-based assay for the detection of bladder cancer

The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak?) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p?=?0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to 0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.