Semi-quantitative measurement of asymptomatic L. infantum infection and symptomatic visceral leishmaniasis in dogs using Dual-Path Platform® CVL.

Infection with Leishmania causes diseases with variable presentation. The most severe form is visceral leishmaniasis (VL), caused by either L. donovani or L. infantum. Despite efforts to eliminate VL, to date, molecular detection in resource-poor settings have lacked the accuracy and rapidity that would enable widespread field use and the need for accurate, sensitive assays to detect asymptomatic Leishmania infection has become apparent. The domestic dog serves as the primary reservoir host of L. infantum. Study of this reservoir population provides an opportunity to evaluate the sensitivity and specificity of diagnostics for well-defined, symptomatic, canine visceral leishmaniasis (CVL) and asymptomatic L. infantum infection. Blood samples from an L. infantum-endemic population of US hunting dogs were evaluated with Dual-Path Platform (DPP®) CVL compared to those obtained via direct detection methods (culture- and Leishmania-specific quantitative polymerase chain reaction, qPCR) and immunofluorescence anti-Leishmania antibody test (IFAT). Statistically significant correlations were found between DPP® CVL development time and clinical status, culture status, circulating DNA levels, and IFAT titer. DPP® CVL results correlated with both clinical severity of disease and serological evidence of asymptomatic L. infantum infection. By precisely documenting the minimum time required for the development of a clear positive result in DPP® CVL, this test could be used in a rapid, semi-quantitative manner for the evaluation of asymptomatic and symptomatic CVL. Our results also indicate that a similar test could be used to improve our understanding of human VL.

Accurate Serodetection of Asymptomatic Leishmania donovani Infection by Use of Defined Antigens.

Infection with Leishmania donovaniis typically asymptomatic, but a significant number of individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200 million people in areas where it is endemic. While diagnosis of acute VL has been simplified by the use of cost-effective confirmatory serological tests, similar standardized tools are not widely available for detecting asymptomatic infection, which can be 4 to 20 times more prevalent than active disease. A simple and accurate serological test that is capable of detecting asymptomaticL. donovaniinfection will be useful for surveillance programs targeting VL control and elimination. To address this unmet need, we evaluated recombinant antigens for their ability to detect serum antibodies in 104 asymptomaticL. donovani-infected individuals (qualified as positive forL. donovani-specific antibodies by direct agglutination test [DAT]) from the Mymensingh district of Bangladesh where VL is hyperendemic. The novel proteins rKR95 and rTR18 possessed the greatest potential and detected 69% of DAT-positive individuals, with rKR95 being more robust in reactivity. Agreement in results for individuals with high DAT responses, who are more likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard antigen that is used to confirm clinical diagnosis of VL but that is now becoming widely used for surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests amenable to rapid or high-throughput screening may enable simple and accurate detection of asymptomatic infection. Such tests will be important tools to measureL. donovaniinfection rates, a primary goal in surveillance and a critical measurement with which to assess elimination programs.

G quadruplexes are genomewide targets of transcriptional helicases XPB and XPD.

G4 motifs are greatly enriched near promoters, suggesting that quadruplex structures may be targets of transcriptional regulation. Here we show, by ChIP-Seq analysis of human cells, that 40% of the binding sites of the transcription-associated helicases, XPB and XPD, overlap with G4 motifs. The highly significant overlap of XPB and XPD binding sites with G4 motifs cannot be explained by GC richness or parameters of the genomewide analysis, but instead suggests that these proteins are recruited to quadruplex structures that form in genomic DNA (G4 DNA). Biochemical analysis demonstrates that XPD is a robust G4 DNA helicase and that XPB binds G4 DNA. XPB and XPD are enriched near the transcription start site at 20% of genes, especially highly transcribed genes. XPB and XPD enrichment at G4 motifs characterizes specific signaling pathways and regulatory pathways associated with specific cancers. These results identify new candidate pathways for therapies targeted to quadruplexes.

Development and comparative evaluation of two antigen detection tests for Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management. METHODS: In this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One-Way ANOVA was used to assess the discrimination capacity of the tests and Cohen's kappa was used to assess their correlation. RESULTS: The Leishmania Antigen Detect ELISA demonstrated >90% sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88% on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post-treatment. For the Leishmania Antigen Detect ELISA, positivity was high at day 0 at 95%, falling to 21% at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91% at Day 0 and more patients, remaining positive at Days 30 and 180. DISCUSSION: The Leishmania Antigen Detect and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options. CONCLUSION: The ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.

G4 motifs correlate with promoter-proximal transcriptional pausing in human genes.

The RNA Pol II transcription complex pauses just downstream of the promoter in a significant fraction of human genes. The local features of genomic structure that contribute to pausing have not been defined. Here, we show that genes that pause are more G-rich within the region flanking the transcription start site (TSS) than RefSeq genes or non-paused genes. We show that enrichment of binding motifs for common transcription factors, such as SP1, may account for G-richness upstream but not downstream of the TSS. We further show that pausing correlates with the presence of a GrIn1 element, an element bearing one or more G4 motifs at the 5'-end of the first intron, on the non-template DNA strand. These results suggest potential roles for dynamic G4 DNA and G4 RNA structures in cis-regulation of pausing, and thus genome-wide regulation of gene expression, in human cells.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Complementary roles for exonuclease 1 and Flap endonuclease 1 in maintenance of triplet repeats.

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.

Activities of human exonuclease 1 that promote cleavage of transcribed immunoglobulin switch regions.

Eukaryotic exonuclease 1 functions in replication, recombination, mismatch repair, telomere maintenance, immunoglobulin (Ig) gene class switch recombination, and somatic hypermutation. The enzyme has 5'-3' exonuclease, flap endonuclease, and weak RNaseH activity in vitro, but it has been difficult to reconcile these activities with its diverse biological functions. We report robust cleavage by human exonuclease 1 of transcribed G-rich DNA sequences with potential to form G loops and G4 DNA. Predicted Ig switch recombination intermediates are substrates for both exonucleolytic and 5' flap endonucleolytic cleavage. Excision is nick-dependent and structure-dependent. These results lead to a model for exonuclease 1 function in class switch recombination in which cleavage at activation-induced deaminase (AID)-initiated nicks produces gaps that become substrates for further attack by AID and subsequent repair.

Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision.

The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1.

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment.

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19-100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16-94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55-89.16%) for VL samples, 100% (95%CI, 69.15-100%) for RVL samples, and 52.5% (95% CI, 36.13-68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98-100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

The efficiency of hypoxanthine excision by alkyladenine DNA glycosylase is altered by changes in nearest neighbor bases.

Alkyladenine DNA glycosylase (AAG) excises a structurally diverse group of damaged purines including hypoxanthine, 1,N(6)-ethenoadenine, 3-methyladenine, and 7-methylguanine from DNA to initiate base excision repair at these sites. Excision occurs in an enzyme.DNA complex in which the damaged base is flipped out of the DNA helix into the enzyme active site. To determine whether local DNA sequence could affect the overall efficiency of excision of hypoxanthine from DNA, single-turnover kinetics of excision, AAG.DNA binding, and melting temperatures were measured for DNA substrates that differed in the base pairs immediately 5' and 3' to hypoxanthine. When Hx was flanked by a 5'G and a 3'C, the efficiency of excision was reduced dramatically in comparison to a duplex containing a 5'T and 3'A. The reduction in excision efficiency was largely due to a decrease in binding affinity of AAG for DNA. The overall effect of GC versus TA nearest neighbors was to magnify the difference in the efficiencies of excision of Hx from pairs with thymine and difluorotoluene from a factor of 5 to a factor of about 100. In general, DNA substrates that were more stable as indicated by higher melting temperatures gave reduced efficiencies of excision of Hx. These results are discussed in terms of a model in which the relative stabilities of base-flipped versus unflipped complexes contribute the overall efficiency of excision and substrate specificity of AAG.

Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease.

We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could serve as targets of the antibody response and be useful as diagnostic indicators. To optimize reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient's sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as well as supporting a diagnosis of Chagas disease.

Evaluation of diagnostic performance of rK28 ELISA using urine for diagnosis of visceral leishmaniasis.

BACKGROUND: Recombinant fusion proteins are now commonly used to detect circulating antibodies for the serodiagnosis of visceral leishmaniasis (VL) in Asia, Africa and the Americas. Although simple, these tests still require blood collection and their use in remote settings can be limited due to the need of collection devices, serum fractionation instrument and generation of biohazardous waste. The development of an accurate and non-invasive diagnostic algorithm for VL, such as could be achieved with urine, is desirable. METHODS: We enrolled 87 VL patients and 81 non-VL individuals, including 33 healthy endemic controls, 16 healthy non-endemic controls, 16 disease controls and 16 tuberculosis (TB) patients. We compared the efficacy of recombinant antigens rK28, rK39 and rKRP42 for the diagnosis of VL when either serum or urine were used to develop antibody-detection ELISA. RESULTS: As expected, each of the antigens readily detected antibodies in the serum of VL patients. rK28 ELISA showed the highest sensitivity (98.9 %), followed by rK39 and rKRP42 ELISA (97.7 and 94.4 %, respectively); overall specificity was > 96 %. When urine was used as the test analyte, only a marginal drop in sensitivity was observed, with rK28 ELISA again demonstrating the greatest sensitivity (95.4 %), followed by rK39 and rKRP42 ELISA, respectively. Again, the overall specificity was > 96 %. CONCLUSIONS: Our data indicate the potential for using urine in the diagnosis of VL. Detection of antibodies against rK28 demonstrated the greatest sensitivity. Together, our results indicate that rK28-based antibody detection tests using urine could provide a completely non-invasive tool amenable for diagnosis of VL in remote locations.

Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs.

Distinct activities of exonuclease 1 and flap endonuclease 1 at telomeric g4 DNA.

BACKGROUND: Exonuclease 1 (EXO1) and Flap endonuclease 1 (FEN1) are members of the RAD2 family of structure-specific nucleases. Genetic analysis has identified roles for EXO1 and FEN1 in replication, recombination, DNA repair and maintenance of telomeres. Telomeres are composed of G-rich repeats that readily form G4 DNA. We recently showed that human EXO1 and FEN1 exhibit distinct activities on G4 DNA substrates representative of intermediates in immunoglobulin class switch recombination. METHODOLOGY/PRINCIPAL FINDINGS: We have now compared activities of these enzymes on telomeric substrates bearing G4 DNA, identifying non-overlapping functions that provide mechanistic insight into the distinct telomeric phenotypes caused by their deficiencies. We show that hFEN1 but not hEXO1 cleaves substrates bearing telomeric G4 DNA 5'-flaps, consistent with the requirement for FEN1 in telomeric lagging strand replication. Both hEXO1 and hFEN1 are active on substrates bearing telomeric G4 DNA tails, resembling uncapped telomeres. Notably, hEXO1 but not hFEN1 is active on transcribed telomeric G-loops. CONCLUSION/SIGNIFICANCE: Our results suggest that EXO1 may act at transcription-induced telomeric structures to promote telomere recombination while FEN1 has a dominant role in lagging strand replication at telomeres. Both enzymes can create ssDNA at uncapped telomere ends thereby contributing to recombination.

Effects of hydrogen bonding within a damaged base pair on the activity of wild type and DNA-intercalating mutants of human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N(6)-ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.