Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are tsetse fly-transmitted protozoan parasites and human pathogens that cause human African trypanosomiasis (HAT; sleeping sickness). Causing substantial morbidity throughout sub-Saharan Africa, death by HAT is likely if a patient is untreated.1–3 The trypanosomes replicate at the tsetse fly bite site before disseminating from the skin through the hemolymphatic system to the spleen, liver, lymph nodes, skin, eyes, endocrine system, and the heart (stage 1).
The development of clinical neurological signs and symptoms that characterize stage 2 HAT can take weeks to months with T. b. rhodesiense (east African) and months to years with T. b. gambiense (west African) infections. Once central nervous system disease is established, the parasites are shielded from most trypanocidal drugs, the majority of which do not penetrate the blood–brain barrier (BBB). For treatment of stage 2 neurologic HAT, toxic BBB-permeable trypanocidal drugs are used.1–3 Melarsoprol, effective for stage 2 T. b. gambiense and T. b. rhodesiense HAT, can cause a lethal severe posttreatment reactive encephalopathy brain inflammation. Less toxic eflornithine is active only against gambiense HAT and resistance to both drugs is increasing.
The early identification and treatment of the largely asymptomatic human reservoir is absolutely critical if any program to control gambiense HAT is to be successful. Although parasite detection in the blood by wet blood film examination is frequently successful in T. b. rhodesiense infections because of the high levels of parasitemia, this method is insensitive in T. b. gambiense infections that constitute 90–95% of all HAT cases.1–3 Because of the often-silent neurological involvement, stage determination still relies on lumbar puncture to examine cerebrospinal fluid (CSF) for trypanosomes and/or changes suggestive of chronic meningoencephalitis. Single or double centrifugation of CSF is required to concentrate parasites for microscopic detection. In the absence of visible parasites, the conventional criterion for stage 2 classification requires CSF lymphocyte cell counts that are scored according to arbitrary cutoffs ranging from > 5, > 10, or even 20 cells/μL.4,5
Loop-mediated isothermal amplification (LAMP) is a proven cost-effective, simple, and rapid DNA amplification method that uses four or six primers for the detection of DNA with high sensitivity and specificity.6,7 The basic four LAMP primers consist of the outer forward (F3), outer backward (B3), forward inner (FIP), and backward inner (BIP) primers, and when present, the loop forward (LF) and loop backward (LB) primers. The major advantages of LAMP include 1) the reaction proceeds under isothermal conditions and detection can be conducted within 60 minutes, 2) it requires simple heating devices such as a water bath, laboratory heat block, or portable tube-LAMP scanners8, and 3) target amplification is indicated by i) turbidimetric measurements,9 or ii) by the addition of fluorescent dyes,10–12 or iii) after addition of hydroxynaphthol blue to yield a reaction product color change from purple (no DNA) to blue (DNA positive).12
Based on protocols for polymerase chain reaction (PCR) diagnosis of HAT, and recognizing the potential of the use for the diagnosis of HAT and other infectious diseases, we described LAMP for the detection of African trypanosomes.13 Highly sensitive, T. brucei subspecies LAMP reactions that recognize Trypanosoma brucei brucei, T. b. rhodesiense, T. b. gambiense, and Trypanosoma evansi,13,14 or are specific for T. b. rhodesiense,15 have been developed. Although specific for T. b. gambiense, the detection limit of LAMP targeting the T. b. gambiense-specific glycoprotein (TgsGP) is ∼100 fg (equivalent to one parasite).16 Trypanosoma brucei gambiense LAMP (TBG1) targeting the high copy number T. b. gambiense 5.8S ribosomal RNA internal transcribed spacer 2 (5.8S-ITS2) gene (GenBank accession no. AF306777) has high analytical sensitivity to as little as 1 fg DNA (equivalent to 0.01 parasite)17; however, none of the TBG1 primer binding sites span the CCCA (C3A) (557–560 bps) insertion site described by Agbo and others.18 Sequence analysis revealed two regions in the 5.8S-ITS2 gene containing C3A sequences (Figure 1A). The first C3A fragment (461–464 bp) on the 5.8S-ITS2 gene is conserved among T. brucei species infective to animals and humans. However, the second C3A fragment (557–560) is conserved in T. b. gambiense.18 Using PrimerExplorer software version 4 software (http://primerexplorer.jp/e/), we identified a 5.8S-ITS2-targeted LAMP primer set that targets the parasite-specific C3A tetranucleotide at the 5′ end of primer F3 in TBG4 (Figure 1B). Comparisons between the TBG4 LAMP primers to the published TBG1 LAMP sequences that target the same 5.8S-ITS2 gene show that only TBG4 targeted the specific second T. b. gambiense C3A fragment (Figure 1C).
LAMP reactions using a commercially available kit (Eiken Chemical Co., Tochigi, Japan) were optimized for reagent concentration, reaction time, and temperature in real time in a Loopamp real-time turbidimeter LA320C (Teramecs, Tokyo, Japan) as previously described.19 Briefly, the reaction contained 12.5 μL of 2 × LAMP reaction buffer (40 mM Tris-HCl [pH 8.8], 20 mM KCl, 16 mM MgSO4, 20 mM [NH4]2SO4, 0.2% Tween 20, 1.6 M Betaine, 2.8 mM of each deoxyribonucleotide triphosphates [dNTPs]), 1.0 μL TBG1,17 or TBG4 (Figure 1) primer mix (5 pmol each of F3 and B3, 40 pmol each of FIP and BIP, and for TBG4 20 pmol each of LF and LB), 1 μL (8 units) Bst DNA polymerase (New England Biolabs, Tokyo, Japan), and template DNA. Final volumes were adjusted to 25 μL with distilled water and the reaction temperature was kept at 63°C, optimal for TBG1 LAMP and TBG4 LAMP (Figure 2). We considered precipitation occurring after a reaction time of 60 minutes to be nonspecific artifacts. It is important to note that TBG4 LAMP specificity appears to diminish with assay temperatures ≥65°C monitored in real time by turbidity and after addition of hydroxynaphthol blue (not shown). Furthermore, the reactions were assayed in four blocks with each block containing eight samples with two no template controls for every six samples assayed. A positive response from any no template control negated the entire 32-reaction run and the samples were reassayed.
Our initial analytical specificity studies showed that TBG4 LAMP recognized genomic DNA from T. b. gambiense (DAL 069, DAL 972, IL 3258, 348BT) but not genomic DNA from T. b. rhodesiense (Type 1 T. b. rhodesiense strains LouTat 1A, GYBO, IL1501) or T. b. brucei (427, 927). TBG4 LAMP also did not recognize DNA from the eukaryotic protozoan parasites (Babesia microti, Plasmodium falciparum, and Toxoplasma gondii) and prokaryotic bacteria (Borrelia burgdorferi, Borrelia crocidurae, Ehrlichia chaffeensis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus), DNA from normal human blood and blood spiked with DNA of pathogens whose clinical presentation might mimic HAT (E. coli) or DNA from clinical blood samples of bacteremic patients from The Johns Hopkins Hospital Microbiology laboratory (with approval of the Johns Hopkins Medicine Institutional Review Board) (Figure 2B). TBG1 LAMP has high analytical sensitivity and is capable of detecting 1 fg DNA (equivalent to 0.01 parasite).17 We tested TBG1 and TBG4 LAMP using the same assay conditions. We found the detection limit of TBG1 LAMP for T. b. gambiense genomic DNA was ≥1 fg (not shown). TBG4 LAMP was 10-fold more sensitive than TBG1 LAMP; that is, the detection limit for T. b. gambiense genomic DNA was at least 0.1 fg of T. b. gambiense DNA, equivalent to 0.001 parasite (Figure 2C and D).
Early and accurate HAT diagnosis presents the highest likelihood that effective anti-trypanosome treatment can be administered before the onset of neurological signs. While based on TBG1 LAMP, TBG4 LAMP was designed to target the T. b. gambiense-specific C3A tetranucleotide sequence present in the 5.8S-ITS2 gene. With sequence information for the 5.8S-ITS2 gene region in T. b. rhodesiense being unavailable, it is remotely possible that some rhodesiense isolates may contain the T. b. gambiense-specific C3A tetranucleotide. Just how well TBG4 LAMP compares to other LAMP17,20 or PCR-based assays under actual clinical and field situations with regard to sensitivity and specific identification of T. b. gambiense remains to be determined. Based on previous work,19 we predict that the detection of intact free-swimming T. b. gambiense in blood or CSF by TBG4 LAMP will be dramatically enhanced after detergent lysis to facilitate release of their DNA prior to assay. This is especially important when sample size is limited. Circulating cell-free DNA (cfDNA) released into the bloodstream as a result of cell death, necrosis, or by release by viable cells has been found in many conditions including a variety of inflammatory and autoimmune diseases, cancer,20 and during trypanosome infections.16 The detection of low levels of T. b. gambiense-derived cfDNA by TBG4 released from damaged and dying African trypanosomes into the circulation may help provide for very early HAT detection.
In conclusion, further development and controlled laboratory and field validation of LAMP-based tests will facilitate HAT diagnosis and monitoring during the early stages of disease prior to serological reactivity in tests such as card agglutination test for trypanosomiasis, as well as in the early detection of neurological HAT. We believe the studies outlined here define the next steps in achieving prompt, accurate diagnosis of HAT. We hope that improved diagnostic testing could ultimately inform appropriate treatment of HAT and thereby reduce mortality from both disease and treatment.
We thank John Mansfield (University of Wisconsin at Madison, WI) for the gift of Trypanosoma brucei rhodesiense LouTat 1A.
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