Evaluation of point-of-care tests for cutaneous leishmaniasis diagnosis in Kabul, Afghanistan.

BACKGROUND: Kabul (Afghanistan) is a major focus of cutaneous leishmaniasis (CL) caused by Leishmania tropica. Microscopy remains the reference test for diagnosis despite its low performance. We evaluated whether Loopamp™ Leishmania Detection Kit (Loopamp) and CL Detect™ Rapid Test (CL Detect), detecting Leishmania DNA and antigen, respectively could improve CL diagnosis. METHODS: A diagnostic accuracy study with prospective inclusion was conducted in a leishmaniasis reference clinic in Kabul. Slit skin samples from CL suspects were analysed by microscopy. Samples taken with a dental broach were tested with CL Detect, Loopamp, and PCR. All samples were transferred to the Academic Medical Center (AMC, the Netherlands) for PCR and Loopamp analyses. The diagnostic performance of the tests was evaluated against a reference combining microscopy and PCR. FINDINGS: 274 CL suspects were included in the study. In Kabul, CL Detect had a 65·4% sensitivity [95% Confidence Interval (CI): 59.2-71.2%] and a 100% specificity [95% CI: 80.5-100%], while these were 87.6% [95%CI: 82.9-91.3%] and 70.6% [95% CI: 44.0-89.7%] for Loopamp. At AMC the Loopamp's sensitivity (92.2% [95% CI: 88.2-95.2%]) and specificity (94.1% [95% CI: 71.3-99.8%]) were higher. An algorithm where CL Detect negative suspects would be tested by Loopamp yielded a 93.4% sensitivity [95% CI: 89.6-96.1%] and a 94.1% specificity [95% CI: 71.3-99.8%] when Loopamp's performance at AMC was used. INTERPRETATION: The high specificity of CL Detect and the performance of Loopamp allow their use in a diagnostic algorithm that would minimize the number of CL patients referred for confirmation. FUND: Federal Ministry of Education and Research, Germany.

Use of malaria rapid diagnostic tests by community health workers in Afghanistan: cluster randomised trial.

BACKGROUND: The World Health Organisation (WHO) recommends parasitological diagnosis of malaria before treatment, but use of malaria rapid diagnostic tests (mRDTs) by community health workers (CHWs) has not been fully tested within health services in south and central Asia. mRDTs could allow CHWs to diagnose malaria accurately, improving treatment of febrile illness. METHODS: A cluster randomised trial in community health services was undertaken in Afghanistan. The primary outcome was the proportion of suspected malaria cases correctly treated for polymerase chain reaction (PCR)-confirmed malaria and PCR negative cases receiving no antimalarial drugs measured at the level of the patient. CHWs from 22 clusters (clinics) received standard training on clinical diagnosis and treatment of malaria; 11 clusters randomised to the intervention arm received additional training and were provided with mRDTs. CHWs enrolled cases of suspected malaria, and the mRDT results and treatments were compared to blind-read PCR diagnosis. RESULTS: In total, 256 CHWs enrolled 2400 patients with 2154 (89.8%) evaluated. In the intervention arm, 75.3% (828/1099) were treated appropriately vs. 17.5% (185/1055) in the control arm (cluster adjusted risk ratio: 3.72, 95% confidence interval 2.40-5.77; p < 0.001). In the control arm, 85.9% (164/191) with confirmed Plasmodium vivax received chloroquine compared to 45.1% (70/155) in the intervention arm (p < 0.001). Overuse of chloroquine in the control arm resulted in 87.6% (813/928) of those with no malaria (PCR negative) being treated vs. 10.0% (95/947) in the intervention arm, p < 0.001. In the intervention arm, 71.4% (30/42) of patients with P. falciparum did not receive artemisinin-based combination therapy, partly because operational sensitivity of the RDTs was low (53.2%, 38.1-67.9). There was high concordance between recorded RDT result and CHW prescription decisions: 826/950 (87.0%) with a negative test were not prescribed an antimalarial. Co-trimoxazole was prescribed to 62.7% of malaria negative patients in the intervention arm and 15.0% in the control arm. CONCLUSIONS: While introducing mRDT reduced overuse of antimalarials, this action came with risks that need to be considered before use at scale: an appreciable proportion of malaria cases will be missed by those using current mRDTs. Higher sensitivity tests could be used to detect all cases. Overtreatment with antimalarial drugs in the control arm was replaced with increased antibiotic prescription in the intervention arm, resulting in a probable overuse of antibiotics. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01403350 . Prospectively registered.

Prevalence of Zoonotic and Vector-Borne Infections Among Afghan National Army Recruits in Afghanistan.

OBJECTIVE: To measure prevalence of prior/current Plasmodium vivax and Plasmodium falciparum (PV and PF), Brucella spp. (BR), dengue virus (DENV), Leishmania donovani (visceral leishmaniasis; VL), and Crimean-Congo hemorrhagic fever (CCHF) virus exposure among Afghan National Army (ANA) recruits. METHODS: Randomly chosen, nationally representative serum samples from consenting men aged 18-40 years and who were screened between February 2010 and January 2011 were tested, with ∼25 samples/province. Samples were screened for PV and PF antigens and VL antibody with rapid diagnostic tests. Reactive malaria screening results were confirmed with polymerase chain reaction assay. Enzyme-linked immunosorbent assays were used to screen for CCHF and DENV antibodies; reactive DENV samples were confirmed with the plaque-reduction neutralization test. BR screening and confirmatory testing was performed with slide and tube agglutination, respectively. Correlates of BR titres >1:80 were analyzed using logistic regression. RESULTS: Of 809 participants contributing specimens, 62% had previously lived outside Afghanistan, predominantly in Pakistan and Iran. CCHF (4.1%, n = 33), DENV (2.1%, n = 17), and VL (1.0%, n = 8) antibody prevalence was low. For PV and PF, only 7 out of 56 reactive samples had detectable nucleic acid. For BR, 8.0% (n = 65) of samples had screening titers >1:40, of which 83.1% had confirmatory titers >1:80. Participants from Kabul and surrounding provinces had lower odds (OR = 0.19, 95% CI: 0.04-1.00) of BR antibody compared with other regions. CONCLUSIONS: BR exposure was relatively common with a nearly national distribution, whereas geographic distribution for other pathogens aligned roughly with the expected vector distribution. Public health protection measures should include vector control, food safety, and enhanced diagnostics for acute febrile illness.

Status of insecticide resistance in high-risk malaria provinces in Afghanistan.

BACKGROUND: Insecticide resistance seriously threatens the efficacy of vector control interventions in malaria endemic countries. In Afghanistan, the status of insecticide resistance is largely unknown while distribution of long-lasting insecticidal nets has intensified in recent years. The main objective of this study was thus to measure the level of resistance to four classes of insecticides in provinces with medium to high risk of malaria transmission. METHODS: Adult female mosquitoes were reared from larvae successively collected in the provinces of Nangarhar, Kunar, Badakhshan, Ghazni and Laghman from August to October 2014. WHO insecticide susceptibility tests were performed with DDT (4 %), malathion (5 %), bendiocarb (0.1 %), permethrin (0.75 %) and deltamethrin (0.05 %). In addition, the presence of kdr mutations was investigated in deltamethrin resistant and susceptible Anopheles stephensi mosquitoes collected in the eastern provinces of Nangarhar and Kunar. RESULTS: Analyses of mortality rates revealed emerging resistance against all four classes of insecticides in the provinces located east and south of the Hindu Kush mountain range. Resistance is observed in both An. stephensi and Anopheles culicifacies, the two dominant malaria vectors in these provinces. Anopheles superpictus in the northern province of Badakhshan shows a different pattern of susceptibility with suspected resistance observed only for deltamethrin and bendiocarb. Genotype analysis of knock down resistance (kdr) mutations at the voltage-gated channel gene from An. stephensi mosquitoes shows the presence of the known resistant alleles L1014S and L1014F. However, a significant fraction of deltamethrin-resistant mosquitoes were homozygous for the 1014L wild type allele indicating that other mechanisms must be considered to account for the observed pyrethroid resistance. CONCLUSIONS: This study confirms the importance of monitoring insecticide resistance for the development of an integrated vector management in Afghanistan. The validation of the kdr genotyping PCR assay applied to An. stephensi collected in Afghanistan paves the way for further studies into the mechanisms of insecticide resistance of malaria vectors in this region.

Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

Pharmacokinetics and Toxicity in Rats and Monkeys of coDbait: A Therapeutic Double-stranded DNA Oligonucleotide Conjugated to Cholesterol.

Increased DNA repair activity in cancer cells is one of their primary mechanisms of resistance to current radio- and chemotherapies. The molecule coDbait is the first candidate in a new class of drugs that target the double-strand DNA break repair pathways with the aim of overcoming these resistances. coDbait is a 32-base pair (bp) double-stranded DNA molecule with a cholesterol moiety covalently attached to its 5'-end to facilitate its cellular uptake. We report here the preclinical pharmacokinetic and toxicology studies of subcutaneous coDbait administration in rodents and monkeys. Maximum plasma concentration occurred between 2 to 4 hours in rats and at 4 hours in monkeys. Increase in mean AUC0-24h was linear with dose reaching 0.5 mg·h/ml for the highest dose injected (32 mg) for both rats and monkeys. No sex-related differences in maximum concentration (Cmax) nor AUC0-24h were observed. We extrapolated these pharmacokinetic results to humans as the subcutaneous route has been selected for evaluation in clinical trials. Tri-weekly administration of coDbait (from 8 to 32 mg per dose) for 4 weeks was overall well tolerated in rats and monkeys as no morbidity/mortality nor changes in clinical chemistry and histopathology parameters considered to be adverse effects have been observed.

Genome-wide mapping of meiotic DNA double-strand breaks in Saccharomyces cerevisiae.

DNA double-strand breaks (DSBs) initiate meiotic recombination in eukaryotes. We describe two strategies that use microarrays to determine the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) approach that targets the Spo11 protein, which remains covalently attached to DSB ends in certain mutant backgrounds. The second approach involves BND cellulose enrichment of the single-strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11 removal.

Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae.

DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Delta mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (> or = 50 kb) "DSB-hot" regions that are separated by "DSB-cold" domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Delta mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)-associated DSBs that accumulate in processing-capable, repair-defective dmc1Delta and dmc1Delta rad51Delta mutants. DSBs were observed at known hot spots, but also in most previously identified "DSB-cold" regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Delta shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Delta, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.

Phylogenomics of type II DNA topoisomerases.

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.

DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus.

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.