Evaluation of Less Invasive Sampling Tools for the Diagnosis of Cutaneous Leishmaniasis.

Background: Diagnosis of cutaneous leishmaniasis (CL) usually relies on invasive samples, but it is unclear whether more patient-friendly tools are good alternatives for diverse lesions when used with polymerase chain reaction (PCR). Methods: Patients with suspected CL were enrolled consecutively in a prospective diagnostic accuracy study. We compared dental broach, tape disc, and microbiopsy samples with PCR as index tests, using PCR with skin slit samples as reference test. Subsequently, we constructed a composite reference test including microscopy, the 3 index tests and skin slit PCR, and we compared these same tests with the composite reference test. We assessed diagnostic accuracy parameters with 95% confidence intervals for all comparisons. Results: Among 344 included patients, 282 (82.0%) had CL diagnosed, and 62 (18.0%) CL absence, by skin slit PCR. The sensitivity and specificity by PCR were 89.0% (95% confidence interval, 84.8%-92.1%) and 58.1% (45.7%-69.5%), respectively, for dental broach, 96.1% (93.2%-97.8%) and 27.4% (17.9%-39.6%) for tape disc, and 74.8% (66.3%-81.7%) and 72.7% (51.8%-86.8%) for microbiopsy. Several reference test-negative patients were consistently positive with the index tests. Using the composite reference test, dental broach, and skin slit had similar diagnostic performance. Discussion: Dental broach seems a less invasive but similarly accurate alternative to skin slit for diagnosing CL when using PCR. Tape discs lack specificity and seem unsuitable for CL diagnosis without cutoff. Reference tests for CL are problematic, since using a single reference test is likely to miss true cases, while composite reference tests are often biased and impractical as they require multiple tests.

Aetiologies of acute undifferentiated febrile illness at the emergency ward of the University of Gondar Hospital, Ethiopia.

OBJECTIVE: Causes of acute febrile illness (AFI) often remain undetermined in developing countries, due to overlap of symptoms and limited available diagnostics. We aimed to assess the aetiology of AFI in adults in a referral hospital in northwest Ethiopia. METHODS: While all participants were tested for malaria by rapid diagnostic test (RDT), microscopy was only done on physician's request. Dengue virus (DENV) infections were detected using an RDT and ELISAs and dengue, yellow fever and chikungunya cases were identified by PCR. Bacterial aetiologies were investigated using blood culture and PCR. RESULTS: The aetiology of acute infection was identified for 20.5% of 200 patients enrolled. Eleven percent tested positive for Plasmodium, while microscopy was only requested for half of the identified malaria cases. For 4.0% of the Plasmodium-infected patients, an acute or past DENV (co-)infection was detected. We found 7.5% acute and 13.0% past DENV - all serotype 3 - infections. Bacterial infections were observed in 4.5% of the patients. CONCLUSION: Malaria is still a considerable aetiology of AFI and dengue is underrecognised. There are areas where both diseases occur concomitantly, and the DENV-3 serotype presumably spreads from Sudan to northern Ethiopia. As only 20.5% of the aetiologies were identified, a broader testing platform is required.

Evaluation of the CL Detect Rapid Test in Ethiopian patients suspected for Cutaneous Leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is common in Ethiopia, mainly affecting impoverished populations in rural areas with poor access to health care. CL is routinely diagnosed using skin slit smear microscopy, which requires skilled staff and appropriately equipped laboratories. We evaluated the CL Detect Rapid Test (InBios, Washington, USA), which is supplied with a dental broach sampling device, as a diagnostic alternative which could be used in field settings. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the diagnostic accuracy of the CL Detect Rapid Test on skin slit and dental broach samples from suspected CL patients at the Leishmaniasis Research and Treatment Center in Gondar, Ethiopia. A combined reference test of microscopy and PCR on the skin slit sample was used, which was considered positive if one of the two tests was positive. We recruited 165 patients consecutively, of which 128 (77.6%) were confirmed as CL. All microscopy-positive results (n = 71) were also PCR-positive, and 57 patients were only positive for PCR. Sensitivity of the CL Detect Rapid Test on the skin slit was 31.3% (95% confidence interval (CI) 23.9-39.7), which was significantly higher (p = 0.010) than for the dental broach (22.7%, 95% CI 16.3-30.6). Sensitivity for both methods was significantly lower than for the routinely used microscopy, which had a sensitivity of 55.5% (IQR 46.8-63.8) compared to PCR as a reference. CONCLUSIONS/SIGNIFICANCE: The diagnostic accuracy of the CL Detect Rapid Test was low for skin slit and dental broach samples. Therefore, we do not recommend its use neither in hospital nor field settings. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov as NCT03837431.

Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum.

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Miltefosine for the treatment of cutaneous leishmaniasis-A pilot study from Ethiopia.

BACKGROUND: Cutaneous leishmaniasis (CL) in Ethiopia, caused by Leishmania aethiopica, is often severe and hard to treat compared to CL caused by other species elsewhere. Miltefosine is the only oral anti-leishmanial drug, with a favorable side-effect profile compared to routinely available sodium stibogluconate (SSG), but evidence about its use for L. aethiopica is lacking. METHODOLOGY AND PRINCIPAL FINDINGS: In an observational cohort study, treatment outcomes, safety and adherence among CL patients who required systemic treatment and received miltefosine for 28 days in Boru Meda Hospital and University of Gondar Hospital were studied. Patient cure was defined as 100% flattening for non-ulcerated lesions and 100% flattening and 100% re-epithelization for ulcerated lesions. Outcomes were documented for day 28, 90 and 180, both per site, and pooled, adjusting for site as a fixed effect with effect coding. Among 94 included patients (32 in Gondar, 62 in Boru Meda), median lesion duration was 12 months, median size six cm, and mucosal involvement (46.8%) and diffuse (30.9%) lesions were common. Adherence to miltefosine was good, and side-effects were tolerable. Initial outcomes at day 28 were promising, with 68.8% and 94.0% of patients having good improvement or cure in Gondar and Boru Meda respectively. In Boru Meda, outcomes were good with 72.7% and 72.9% cure at day 90 and day 180 respectively. In Gondar, results were less promising, with only 12.5% and 26.7% cure at day 90 and day 180, although confidence intervals were wide. In pooled estimates, 48.7% of patients reached cure at day 180, and 32.3% relapsed. Outcomes were better in Boru Meda Hospital, for smaller lesions and for mucosal lesions. CONCLUSIONS/SIGNIFICANCE: Based on miltefosine's good initial response, tolerable side-effects, tablet-form, we propose to include miltefosine for future clinical trials using extended treatment schedules, combination therapy, or targeting specific subgroups. TRIAL REGISTRATION: ClinicalTrials.gov NCT04004754.

Early diagnosis and follow-up of acute schistosomiasis in a cluster of infected Belgian travellers by detection of antibodies and circulating anodic antigen (CAA): A diagnostic evaluation study.

BACKGROUND: In order to evaluate the diagnostic value of schistosome circulating anodic antigen (CAA) detection, serum and urine CAA-levels were determined in a single cluster of 34 Belgian tourists at three timepoints within a period of 14 weeks following proven Schistosoma exposure in South Africa and compared with two in-house antibody assays. METHODS: Samples were collected 4-5 and 7-8 weeks post-exposure and subsequently 5-6 weeks following praziquantel treatment. Schistosoma antibodies were detected by an adult worm antigen-immunofluorescence assay (AWA-IFA) and a soluble egg antigen-enzyme-linked immunosorbent assay (SEA-ELISA), while CAA concentrations were determined by the Up-Converting reporter Particle labelled Lateral Flow (UCP-LF) test. RESULTS: Antibodies were detected in 25/34 (73%) travellers pre-treatment and in 27/34 (79%) post-treatment, with the AWA-IFA showing better performance than the SEA-ELISA. Pre-treatment, CAA was detected in 13/34 (38%) and 33/34 (97%) of the travellers in urine and serum, respectively. Post-treatment, all except one traveller became serum CAA negative. This in contrast to the detected antibodies, as well as the previously reported diagnostic results of this cluster. CONCLUSIONS: The UCP-LF CAA serum assay has been demonstrated as the most sensitive method for the diagnosis of early Schistosoma infections and post-treatment monitoring in travellers.

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia.

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.

Acute Schistosomiasis With a Schistosoma mattheei × Schistosoma haematobium Hybrid Species in a Cluster of 34 Travelers Infected in South Africa.

BACKGROUND: Diagnosis of schistosomiasis remains elusive soon after infection. We evaluated several diagnostic methods in a cluster of travelers with simultaneous freshwater exposure in South Africa. METHODS: Eosinophil count, schistosome antibody tests, stool and urine microscopy, and serum Dra1 PCR assays were performed at weeks 4-5 (early symptomatic phase), 7-8 (praziquantel treatment), and 13-14 (after treatment). Sequencing was done on serum samples from 3 patients to identify the species. RESULTS: Of the 34 travelers (16 adults and 18 children), 32 developed symptoms 2-6 weeks after exposure. A raised eosinophil count (>750/µL) was seen in 12 of 33 at weeks 4-5, and in 22 of 34 at weeks 7-8. Schistosoma antibodies were detected in 3 of 33 at weeks 4-5 and in 12 of 34 at weeks 7-8 and weeks 13-14. The Dra1 PCR result was positive in 24 of 33 travelers at weeks 4-5, in 31 of 34 at weeks 7-8, in 25 of 34 at weeks 13-14, and at least once in all. Ova were absent in all urine and stool samples obtained. Sequencing identified Schistosoma mattheei nuclear and Schistosoma haematobium mitochondrial DNA, indicative of a hybrid species. CONCLUSIONS: The Dra1 PCR confirmed the diagnosis in all exposed travelers at a much earlier stage than conventional tests. The causative species is probably an S. mattheei × S. haematobium hybrid.

Diagnostic accuracy of direct agglutination test, rK39 ELISA and six rapid diagnostic tests among visceral leishmaniasis patients with and without HIV coinfection in Ethiopia.

Diagnosis of a first-time visceral leishmaniasis (VL) infection in Ethiopia is established by use of a rapid diagnostic test (RDT) detecting antibodies against rK39, direct agglutination test (DAT) and microscopy according to the national algorithm. The performance of individual tests and algorithm is variable and depends on several factors, one being HIV status. Limited data are available on the performance of tests in VL-HIV coinfected patients. Assessment of the performance of DAT (ITM-A), rK39 ELISA (Serion) and six RDT (Onsite Leishmania Ab CTK, Antigen ICT Xinjier, IT Leish Biorad, Kalazar Detect Inbios, rK39 IgG1 Coris, rk28 IgG1 Coris) for the diagnosis of VL was done on a panel of 91 stored serum and plasma samples of 'first-episode' suspected VL patients, with HIV coinfection (n = 51) and without (n = 40). A combined reference standard was used: either positive microscopy on tissue aspirates, or in case of negative microscopy, positive PCR results on the aspirate slide. Additionally, endemic healthy controls (n = 20), non-endemic controls (n = 10) and patients with confirmed malaria infection (n = 10) were tested for specificity evaluation. Sensitivities ranged from 69.2% for DAT (applied cut-off ≥ 1/3200) to 92.2% for the Onsite RDT, whereas specificities ranged from 20.0% for Kalazar Antigen ICT to 100% for IT Leish and rK39 IgG1. Sensitivities from all assays decreased upon stratification according to HIV status but was only significantly different for rK39 Serion ELISA (p-value 0.0084) and the Onsite RDT (p-value 0.0159). In conclusion, performance of commercially available assays for VL on samples from Northern-Ethiopian patients varied widely with a substantial decrease in sensitivity in the VL-HIV coinfected group. Clear guidelines on minimal performance criteria of individual tests and algorithms are needed, as well as which reference standard should be used to determine the performance.

Evaluation of C-reactive protein and myxovirus resistance protein A to guide the rational use of antibiotics among acute febrile adult patients in Northwest Ethiopia.

OBJECTIVES: In low-resource settings, treatment is often given empirically without knowledge of the aetiology due to a lack of diagnostics. In the search for reliable rapid tests to guide treatment work-up, this study was performed to determine whether two biomarkers could differentiate bacterial from non-bacterial infections in acute febrile patients. METHODS: Adults with acute fever were recruited at a referral hospital in Ethiopia. The QuikRead Go test was used to quantify C-reactive protein (qCRP) and the FebriDx test was used for combined qualitative detection of the bacterial CRP marker with myxovirus resistance protein A (MxA), a viral biomarker. RESULTS: Of the 200 patients included in this study, most presented with 2-3 days of fever, headache, and joint pain. Antibiotics were prescribed for 83.5% and antimalarials for 36.5%, while a bacterial infection was only confirmed in 5% and malaria in 11%. The median qCRP level for confirmed bacterial infections was 128 mg/l. The FebriDx and QuikRead Go test had an overall agreement of 72.0%. CONCLUSIONS: An over-prescription of antibiotics for febrile patients was observed, even for those with low CRP levels and without a confirmed bacterial infection. The added value of the FebriDx was limited, while the combined use of rapid tests for qCRP and malaria should be considered for the management of acute febrile illness and antibiotic stewardship.

Epidemiology, clinical pattern and impact of species-specific molecular diagnosis on management of leishmaniasis in Belgium, 2010-2018: A retrospective study.

BACKGROUND: Species-directed therapy of leishmaniasis has been recommended for travelers since 2014, but little is known about species distribution and treatment practices in non-endemic countries. We aimed to describe leishmaniasis cases in Belgium since species typing became available and evaluate its impact on patient management. METHOD: Retrospective analysis of all patients diagnosed by PCR at our national reference laboratory from 2010 to 2018. Species were typed by Hsp-70 sequencing. RESULTS: We identified 18 visceral leishmaniasis (VL) and 147 (muco)cutaneous leishmaniasis ((M)CL) cases. VL was exclusively due to L. infantum and consistently treated with liposomal amphotericin B, with four observed failures. (M)CL was caused by ten different species. Of 62 cases diagnosed and species typed after 2014 with timing information, 28 (45.2%) were treated before the species result was available. Therapy was not species-directed in 10/32(28.1%) of those treated after species identification. Patients treated according to the guidelines tended to have a favorable outcome more often than those who were not (36/44, 81.8% versus 8/19, 57.9%; p = 0.045). CONCLUSIONS: In contrast to VL, various species caused (M)CL in our setting and species result was often not considered for treatment. Outcome tended to be better however when therapy was species-directed.

Sexual Transmission of Visceral Leishmaniasis: A Neglected Story.

For visceral leishmaniasis (VL), a major vector-borne parasitic disease, an alternative sexual transmission route is well documented in dogs but evidence is lacking in humans. Here, we discuss the current knowledge and key questions to be answered as it may be an additional obstacle in ongoing VL elimination programs.

Minimally Invasive Microbiopsies as an Improved Sampling Method for the Diagnosis of Cutaneous Leishmaniasis.

Current sampling methods to diagnose cutaneous leishmaniasis are invasive and painful. An alternative and minimally invasive microbiopsy device was evaluated in a diverse range of cutaneous leishmaniasis lesions in Ethiopia. Using polymerase chain reaction-based diagnosis, the microbiopsy outperformed the routine skin slit sample by detecting more patients while pain scores were significantly lower.

Point-of-Care Biomarkers to Guide Antibiotic Prescription for Acute Febrile Illness in Sub-Saharan Africa: Promises and Caveats.

Empiric malaria treatment in Sub-Saharan Africa has significantly decreased with the scaling-up of malaria rapid diagnostic tests; this coincided with a pronounced increase in empiric antibiotic prescriptions. In high-income countries, guidance for antibiotic prescriptions using biomarkers such as C-reactive protein (CRP) and procalcitonin (PCT) has reduced antibiotic use while safe-guarding patient safety. Importantly, several low-cost point-of-care CRP/PCT tests are currently available. However, only a few studies on the role of CRP/PCT in differentiating bacterial vs viral infections in acute febrile illness have been conducted in Sub-Saharan Africa. Studies from Central and West Africa (most of which is malaria-endemic) are particularly scarce, and only 1 has included adults. The evidence base for point-of-care use of CRP/PCT biomarkers in acute fever in Sub-Saharan Africa should be urgently built. Before engaging in clinical trials to assess clinical impact, pilot studies should be conducted to address key knowledge gaps including recommended CRP/PCT cutoff values and the effect of malaria coinfection.

Promising application of monoclonal antibody against chikungunya virus E1-antigen across genotypes in immunochromatographic rapid diagnostic tests.

BACKGROUND: Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. METHODS: Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. RESULTS: The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0-95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. CONCLUSIONS: Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).

Host transcriptomic signature as alternative test-of-cure in visceral leishmaniasis patients co-infected with HIV.

BACKGROUND: Visceral leishmaniasis (VL) treatment in HIV patients very often fails and is followed by high relapse and case-fatality rates. Hence, treatment efficacy assessment is imperative but based on invasive organ aspiration for parasite detection. In the search of a less-invasive alternative and because the host immune response is pivotal for treatment outcome in immunocompromised VL patients, we studied changes in the whole blood transcriptional profile of VL-HIV patients during treatment. METHODS: Embedded in a clinical trial in Northwest Ethiopia, RNA-Seq was performed on whole blood samples of 28 VL-HIV patients before and after completion of a 29-day treatment regimen of AmBisome or AmBisome/miltefosine. Pathway analyses were combined with a machine learning approach to establish a clinically-useful 4-gene set. FINDINGS: Distinct signatures of differentially expressed genes between D0 and D29 were identified for patients who failed treatment and were successfully treated. Pathway analyses in the latter highlighted a downregulation of genes associated with host cellular activity and immunity, and upregulation of antimicrobial peptide activity in phagolysosomes. No signs of disease remission nor pathway enrichment were observed in treatment failure patients. Next, we identified a 4-gene pre-post signature (PRSS33, IL10, SLFN14, HRH4) that could accurately discriminate treatment outcome at end of treatment (D29), displaying an average area-under-the-ROC-curve of 0.95 (CI: 0.75-1.00). INTERPRETATION: A simple blood-based signature thus holds significant promise to facilitate treatment efficacy monitoring and provide an alternative test-of-cure to guide patient management in VL-HIV patients. FUNDING: Project funding was provided by the AfricoLeish project, supported by the European Union Seventh Framework Programme (EU FP7).

Cutaneous Leishmaniasis in Syrian Refugee Children: a Case Series.

Cutaneous leishmaniasis is the most common presentation of infection by protozoan parasites of the genus Leishmania. Patients with cutaneous leishmaniasis may have one or several disfiguring skin lesions that resemble other dermatologic diseases. Old World cutaneous leishmaniasis is a major public health problem in the World Health Organization Eastern Mediterranean Region. Conflict and ensuing collapse of health systems leads to migration of leishmaniasis patients from countries like Syria. Pediatricians in nonendemic countries should be aware of this disease entity. We identify knowledge gaps and summarize treatment options for cutaneous leishmaniasis.

Detection of Zika Virus Replication in Human Semen by Reverse-Transcription Polymerase Chain Reaction Targeting of Antisense Ribonucleic Acid.

BACKGROUND: Persistence of Zika virus (ZIKV) ribonucleic acid (RNA) in semen is common after infection. METHODS: We designed a reverse-transcription polymerase chain reaction assay that targets antisense ZIKV RNA (asRNA) to assess ZIKV replication competence in ZIKV RNA-positive semen samples. RESULTS: We detected ZIKV asRNA in semen of 9 of 19 men (47.4%) diagnosed with ZIKV infection. All asRNA-positive samples had high ZIKV loads (cycle threshold values <26) and were obtained within 21 days of symptom onset. CONCLUSIONS: The sensitivity of the asRNA assay for detection of ZIKV replication was higher than that of conventional virus isolation methods (47.4% vs 21.1%, P = .032).