Time of incubation of agar-plate culture for the diagnosis of Strongyloides stercoralis infection.

PURPOSE: Agar-plate culture (APC) remains the most sensitive parasitological technique for S. stercoralis diagnosis. Although it was first described three decades ago, the time of incubation of the plates is neither a commonly described feature nor usually standardized. The aim of the study was to analyze the required time to detect S. stercoralis larvae in APC. METHODS: A prospective laboratory-based study including all patients with at least one positive APC was performed. The plates were incubated at room temperature for 7 days. Clinical, analytical and parasitological features including results of the direct visualization of the stool (DV) after formalin-ether concentration and time-to-detection (TTD) of the larvae in APC were recorded. RESULTS: A total of 141 samples from 75 patients had a positive APC. In 49 of them (65.3%) three or more stool samples were processed for direct visualization (DV) and APC. Of these 49 patients, 8 (16.3%) were also diagnosed with DV and 41 (83.7%) were diagnosed only with APC. In 38 samples from 23 (30.7%) patients, the TTD was below 2 days, while in 27 samples from 13 (17.3%) patients, the larvae were detected on the 6th and 7th day. CONCLUSION: Direct visualization failed to detect S. stercoralis in most of the patients that were diagnosed with APC. Incubation periods below 2 and 5 days would miss an important percentage of infections. At least 7 days of incubation of the APC are required to detect presumably low-burden chronic infections in non-endemic countries.

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum.

BACKGROUND: The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Primer caso de rinosinusitis fúngica no invasiva por Aspergillus melleus / First case of non-invasive fungal rhinosinusitis by Aspergillus melleus

No disponible

Prevalence of submicroscopic malaria infection in immigrants living in Spain.

BACKGROUND: The importance of submicroscopic malaria infections in high-transmission areas could contribute to maintain the parasite cycle. Regarding non-endemic areas, its importance remains barely understood because parasitaemia in these afebrile patients is usually below the detection limits for microscopy, hence molecular techniques are often needed for its diagnosis. In addition to this, the lack of standardized protocols for the screening of submicroscopic malaria in immigrants from endemic areas may underestimate the infection with Plasmodium spp. The aim of this study was to assess the prevalence of submicroscopic malaria in afebrile immigrants living in a non-endemic area. METHODS: A prospective, observational, multicentre study was conducted. Afebrile immigrants were included, microscopic observation of Giemsa-stained thin and thick blood smears, and two different molecular techniques detecting Plasmodium spp. were performed. Patients with submicroscopic malaria were defined as patients with negative blood smears and detection of DNA of Plasmodium spp. with one or both molecular techniques. Demographic, clinical, analytical and microbiological features were recorded and univariate analysis by subgroups was carried out with STATA v15. RESULTS: A total of 244 afebrile immigrants were included in the study. Of them, 14 had a submicroscopic malaria infection, yielding a prevalence of 5.7% (95% confidence interval 3.45-9.40). In 71.4% of the positive PCR/negative microscopy cases, Plasmodium falciparum alone was the main detected species (10 out of the 14 patients) and in 4 cases (28.6%) Plasmodium vivax or Plasmodium ovale were detected. One patient had a mixed infection including three different species. CONCLUSIONS: The prevalence of submicroscopic malaria in afebrile immigrants was similar to that previously described in Spain. Plasmodium vivax and P. ovale were detected in almost a third of the submicroscopic infections. Screening protocols for afebrile immigrants with molecular techniques could be useful for a proper management of these patients.

Comparative study of two commercial tests for Strongyloides stercoralis serologic diagnosis.

Background: Serological diagnosis of Strongyloides stercoralis is often limited by its low specificity due to cross-reactivity with other parasitic nematodes. Novel serological tests assumed to be more specific have been recently developed. The aim of our study was to compare two commercial tests based on different antigens for S. stercoralis diagnosis in humans from a non-endemic area. Methods: A retrospective laboratory-based study was conducted in the Hospital Universitario 12 de Octubre, Madrid, Spain. Samples from patients with a requested S. stercoralis serology from January 2013 to October 2016 were tested with two commercial enzyme-linked immunosorbent assay (ELISA) tests (crude larval suspension ELISA [CrAg-ELISA] and recombinant antigen ELISA [NIE-ELISA]). Sensitivity, specificity and predictive values were calculated using primary and composite gold standards. The κ index was calculated. Results: A total of 249 samples from 233 patients were tested (κ=0.735). The CrAg-ELISA yielded sensitivities from 89.2% (95% confidence interval [CI] 80.7 to 94.2) to 94.7% (95% CI 75.4 to 99.0) and the NIE-ELISA from 72.3% (95% CI 58.2 to 83.1) to 78.9% (95% CI 56.7 to 91.5). Specificity ranged from 72.3% (95% CI 58.2 to 83.1) to 89.3% (95% CI 83.1 to 93.4) for the CrAg-ELISA and from 85.1% (95% CI 72.3 to 92.6) to 93.6% (95% CI 88.2 to 96.6) for the NIE-ELISA. Conclusions: The NIE-ELISA is more specific than the CrAg-ELISA, but its low sensitivity limits its use in S. stercoralis screening. New diagnostic tests are needed for the diagnosis of S. stercoralis.

Usefulness of the ARCHITECT Chagas® assay as a single test for the diagnosis of chronic Chagas disease.

OBJECTIVES: Imported Chagas disease (CD) is an emerging health problem in Europe due to immigration from endemic countries. Although WHO currently recommends two different serological methods to establish diagnosis, new tools like the ARCHITECT Chagas assay have potential for use as a single diagnostic test. Our objective was to determine an optimal signal-to-cut-off (S/CO) value for the ARCHITECT Chagas assay to diagnose CD with a single test. METHODS: A retrospective study conducted at the 12 de Octubre University Hospital (Madrid, Spain). All patients with requests for Chagas screening between January 2014 and August 2017 were consecutively included. All samples were routinely tested with the ARCHITECT assay. Negative samples (S/CO < 0.8) required no further testing. Immunochromatographic testing (ICT) and/or indirect immunofluorescence (IFI) was used to confirm samples with S/CO ≥ 0.8. Receiver operator characteristic (ROC) curve analysis determined the ARCHITECT S/CO value that yielded 100% specificity and positive predictive value. SPSS software, version 22.0 was used for data analysis. RESULTS: A total of 4153 samples were analysed; 361 (8.69%) gave a reactive ARCHITECT Chagas result. 261/361 (72.3%) were women; median age was 38 years old (2-79). 92.8% were Bolivian. A total of 307 (85.0%) were confirmed as cases of Chagas; 52 (14.4%) were not infected; two (0.6%) were not evaluable. Seroprevalence was 7.39%. An S/CO ≥ 3.80 yielded 100% specificity (95% confidence interval [CI], 0.93-1.00) and 100% positive predictive value (95% CI, 0.99-1.00). CONCLUSIONS: Using S/CO ≥ 3.80, the ARCHITECT Chagas could be used as a single test for diagnosis of chronic CD in Bolivian immigrants. Patients with S/CO between 0.80 and 3.80 would require additional testing.

The value of xpert MTB/RIF-generated CT values for predicting the smear status of patients with pulmonary tuberculosis.

BACKGROUND: Smear microscopy is used to assess the patient's infectiousness at the time of initial diagnosis of pulmonary tuberculosis. However, its limited sensitivity and specificity highlights the need for new diagnostic strategies. The aim of our study was to assess the diagnostic accuracy of GX Ct value as a predictor of smear status and its usefulness to quantify mycobacterial load. METHODS: All GX-positive sputum samples during a seven-year period were included in the study. Correlations among Ct values, smear status and TTD on liquid culture were calculated. An optimal Ct value for ruling in infectious patients was established. Clinical and radiological variables were also analyzed. RESULTS: Sixty-eight samples from 65 patients were included. Ct value and TTD yielded a positive correlation (ρ = 0.714; p < 0.05), while Ct and smear grade yielded an inverse correlation (r = -0.71). An optimal Ct value for ruling in smear positive patients was established at 21.1 cycles (90.5% sensitivity, 61% specificity, 81% PPV and 78% NPV). CONCLUSIONS: Our study confirms the value of GX Ct levels for quantifying mycobacterial load and demonstrates the added value of Ct as a predictor of positive smear status, especially at Ct values below 21.