Bayesian latent class estimation of sensitivity and specificity parameters of the PMS-PCR test for the diagnosis of cattle sub-clinically infected with Mycobacterium avium subsp. paratuberculosis.

The objective of this study was to estimate the performance of the peptide magnetic separation PCR test (PMS-PCR) for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) in sub-clinically infected dairy cattle. Twenty-one herds were randomly selected from a source population of 131 commercial dairy herds with a known history of MAP infection, located in the De Los Rios and De Los Lagos regions, in southern Chile. In the selected herds, all milking cows with ≥2 parities and without any clinical signs were sampled, collecting feces and blood-serum samples. The PMS-PCR test was used to analyze the fecal samples, while serum samples were analyzed using a commercial ELISA kit. A Bayesian latent class model was used to estimate the sensitivity (Se) and specificity (Sp) of the diagnostic tests. A total of 1381 animals were sampled in the 21 selected dairy herds, with an average sample size of 65 animals per herd (range 10-721). The PMS-PCR test had a greater Se than the ELISA test, with a median of 85.5 % (posterior probability interval (PPI) 95 %: 79.3-91.0%), while the ELISA test presented a median of 21.7 % (95 % PPI: 18.3-25.4%). On the other hand, the ELISA test had a better Sp than the PMS-PCR test, with a median of 97.7 % (95 % PPI: 96.6-98.5%), whereas PMS-PCR presented a median of 90.8 % (95 % PPI: 88.3-93.9%). Model results showed that PMS-PCR has a better Se than all available tests for MAP diagnosis in subclinical animals. However, this test should be used with care in herds with high infection rates, where a high MAP environmental load is expected, potentially increasing the frequency of false positive cases due to the pass-through phenomenon.

Is the Occurrence of Ankyloglossia in Newborns Associated with Breastfeeding Difficulties?

Background: Ankyloglossia is characterized by abnormal tongue movements that can possibly interfere with breastfeeding due to incorrect latching, pain, nipple fissure, and ineffective suction. Objective: To determine the prevalence of ankyloglossia in newborns and its association with exclusive breastfeeding and early breastfeeding difficulties. Materials and Methods: This is an analytical cross-sectional study conducted in seven public maternity hospitals in the city of Recife, PE, Brazil. The study sample consisted of 822 mothers/newborns of both genders. The diagnosis of ankyloglossia was confirmed by comparing two previously standardized and validated lingual frenulum assessment tools. Information on the mother's socioeconomic profile and breastfeeding difficulties were also collected. The data were analyzed using bivariate and multivariate logistic regression models. Results: The prevalence of ankyloglossia was 2.6% when using the Bristol Tool and 11.7% with the Assessment Tool for Lingual Frenulum Function (Neonatal Tongue Screening Test-NTST). The agreement between the two assessment tools was 2.2%, with a significant difference between them (p < 0.001). There was an association between the occurrence of ankyloglossia and breastfeeding difficulties (odds ratio = 1.99), but no association with exclusive breastfeeding practice was found. Conclusions: The diagnostic tools used herein revealed different prevalence rates of ankyloglossia in newborns. This condition was associated with breastfeeding difficulties, and the NTST was more effective in determining such an association.

Multiplex malaria antigen detection by bead-based assay and molecular confirmation by PCR shows no evidence of Pfhrp2 and Pfhrp3 deletion in Haiti.

BACKGROUND: The Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens. METHODS: From September 2012 through February 2014, dried blood spots (DBS) were collected in Haiti from 9317 febrile patients presenting to 17 health facilities in 5 departments throughout the country as part of a bed net intervention study. All DBS from RDT positive persons and a random sampling of DBS from RDT negative persons were assayed for P. falciparum DNA by nested and PET-PCR (n = 2695 total). All PCR positive samples (n = 331) and a subset of PCR negative samples (n = 95) were assayed for three malaria antigens by a multiplex bead assay: pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and HRP2/3. Any samples positive for P. falciparum DNA, but negative for HRP2/3 antigens were tested by nested PCR for Pfhrp2 and Pfhrp3 gene deletions. RESULTS: Of 2695 DBS tested for Plasmodium DNA, 345 (12.8%) were originally found to be positive for P. falciparum DNA; 331 of these had DBS available for antigen detection. Of these, 266 (80.4%) were positive for pAldo, 221 (66.8%) positive for pLDH, and 324 (97.9%) were positive for HRP2/3 antigens. Seven samples (2.1%) positive for P. falciparum DNA were not positive for any of the three antigens by the bead assay, and were investigated for potential Pfhrp2/3 gene deletion by PCR. These samples either successfully amplified Pfhrp2/3 genes or were at an estimated parasite density too low for sufficient DNA to perform successful genotyping. CONCLUSIONS: Malaria positive samples in multiple Haitian sites were found to contain the HRP2/3 antigens, and no evidence was found of Pfhrp2/3 deletions. Malaria RDTs based on the detection of the HRP2/3 antigens remain a reliable P. falciparum diagnostic tool as Haiti works towards malaria elimination.

Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

BACKGROUND: Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs). METHODOLOGY: We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs. PRINCIPAL FINDINGS: All diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Proof of Concept for a Portable Platform for Molecular Diagnosis of Tropical Diseases: On-Chip Ready-to-Use Real-Time Quantitative PCR for Detection of Trypanosoma cruzi or Plasmodium spp.

Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecular-based tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device and a silicon chip, named Q3-Plus. In addition, a ready-to-use reaction format, where all qPCR reagents are stored on plate or on chip, was compared with the traditional freezer-stored format. No significant differences were observed in detecting T. cruzi or Plasmodium spp. DNA between thermocyclers, as well as between reagents' formats, for storage periods of up to 28 days (at 2°C to 8°C or 21°C to 23°C, respectively). When challenged with patients' samples, the Q3-Plus system performed as efficiently as the standard equipment for Plasmodium spp. DNA detection, showing it to be a valuable solution to malaria point-of-care diagnostics. Detection of T. cruzi DNA in chronic patients' samples using the Q3-Plus system yielded approximately 50% efficiency relative to the ABI7500. These results are essential to support future endeavors to bring molecular diagnostics to the point of care, where most needed.

Determination of baseline values for routine ophthalmic tests in bearded capuchin (Sapajus libidinosus).

BACKGROUND: Establish baseline values for ophthalmic diagnostic tests in Sapajus libidinosus. METHODS: Ophthalmic diagnostic tests, namely Schirmer tear test 1 (STT-1), intraocular pressure (IOP), B-mode ultrasound, culture of the bacterial conjunctival microbiota, and conjunctival exfoliative cytology, were performed in 15 S. libidinosus. RESULTS: Mean values found were as follows: 2.50 ± 2.94 mm/min for the STT-1; 13.3 ± 3.32 mm Hg for the IOP; 2.47 ± 0.41 mm for the depth of the anterior chamber; 2.86 ± 0.96 mm for the axial length of the lens; 10.97 ± 0.48 mm for the depth of the vitreous chamber; and 16.32 ± 1.24 mm for the axial length of the eyeball. The bacterial genus most frequently found was Staphylococcus spp. Conjunctival cytology showed intermediate epithelial, squamous superficial epithelial, and keratinized cells. CONCLUSIONS: Determination of baseline values for eye measurements and ophthalmic tests will assist in the diagnosis of eye diseases in S. libidinosus monkeys.

Automated microscopy for routine malaria diagnosis: a field comparison on Giemsa-stained blood films in Peru.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains a major form of diagnosis in malaria case management, and is a reference standard for research. However, as with other visualization-based diagnoses, accuracy depends on individual technician performance, making standardization difficult and reliability poor. Automated image recognition based on machine-learning, utilizing convolutional neural networks, offers potential to overcome these drawbacks. A prototype digital microscope device employing an algorithm based on machine-learning, the Autoscope, was assessed for its potential in malaria microscopy. Autoscope was tested in the Iquitos region of Peru in 2016 at two peripheral health facilities, with routine microscopy and PCR as reference standards. The main outcome measures include sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. METHODS: A cross-sectional, observational trial was conducted at two peripheral primary health facilities in Peru. 700 participants were enrolled with the criteria: (1) age between 5 and 75 years, (2) history of fever in the last 3 days or elevated temperature on admission, (3) informed consent. The main outcome measures included sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. RESULTS: At the San Juan clinic, sensitivity of Autoscope for diagnosing malaria was 72% (95% CI 64-80%), and specificity was 85% (95% CI 79-90%). Microscopy performance was similar to Autoscope, with sensitivity 68% (95% CI 59-76%) and specificity 100% (95% CI 98-100%). At San Juan, 85% of prepared slides had a minimum of 600 WBCs imaged, thus meeting Autoscope's design assumptions. At the second clinic, Santa Clara, the sensitivity of Autoscope was 52% (95% CI 44-60%) and specificity was 70% (95% CI 64-76%). Microscopy performance at Santa Clara was 42% (95% CI 34-51) and specificity was 97% (95% CI 94-99). Only 39% of slides from Santa Clara met Autoscope's design assumptions regarding WBCs imaged. CONCLUSIONS: Autoscope's diagnostic performance was on par with routine microscopy when slides had adequate blood volume to meet its design assumptions, as represented by results from the San Juan clinic. Autoscope's diagnostic performance was poorer than routine microscopy on slides from the Santa Clara clinic, which generated slides with lower blood volumes. Results of the study reflect both the potential for artificial intelligence to perform tasks currently conducted by highly-trained experts, and the challenges of replicating the adaptiveness of human thought processes.

Comparison of the Purelyse® - IS6110 nested PCR with the Xpert® MTB/RIF test in clinical samples with suspected tuberculosis.

Tuberculosis (TB) has a high incidence, prevalence and mortality in the world. Due to its high level of transmission and long-term pharmacological treatment, it is important to have sensitive and specific diagnostic tests. Recently, the PureLyse® system, which is a novel DNA extraction method, was proposed to be an important tool for molecular diagnosis of TB. Here, we compare the PureLyse® system followed by an IS6110 nested PCR (PureLyse® - IS6110 nested PCR) with the Xpert® MTB/RIF test for Mycobacterium tuberculosis complex (MTBC) identification in 40 clinical samples. Among the 40 samples, 26 samples were positive and 14 negative for the Xpert® MTB/RIF test as well as for the PureLyse® - IS6110 nested PCR. According to the Xpert® MTB/RIF test, positive samples presented different bacillary concentrations from "High" to "Very low" and rifampin resistance was observed in 5 samples. The concordance of both molecular methods makes the PureLyse® - IS6110 nested PCR suitable for MTBC detection in patients for low-income resources.

Malakit: an innovative pilot project to self-diagnose and self-treat malaria among illegal gold miners in the Guiana Shield.

BACKGROUND: Illegal gold miners in French Guiana, a French overseas territory ('département') located in Amazonia, often carry malaria parasites (up to 46.8%). While the Guiana Shield Region aims at malaria elimination, the high prevalence of Plasmodium in this hard-to-reach population in conjunction with frequent incorrect use of artemisinin-based anti-malarials could favour the emergence of resistant parasites. Due to geographical and regulatory issues in French Guiana, usual malaria control strategies cannot be implemented in this particular context. Therefore, new strategies targeting this specific population in the forest are required. METHODS: Numerous discussions among health institutions and scientific partners from French Guiana, Brazil and Suriname have led to an innovative project based on the distribution of kits for self-diagnosis and self-treatment of Plasmodium infections. The kit-distribution will be implemented at "resting sites", which are areas across the border of French Guiana regularly frequented by gold miners. The main objective is to increase the appropriate use and complete malaria treatment after a positive malaria diagnosis with a rapid test, which will be evaluated with before-and-after cross-sectional studies. Monitoring indicators will be collected from health mediators at the time of kit distribution and during subsequent visits, and from illegal gold miners themselves, through a smartphone application. The project funding is multisource, including Ministries of Health of the three countries, WHO/PAHO, and the European Union. RESULTS: This project will start in April 2018 as a 18 month pilot study led by the Clinical Investigation Centre of Cayenne. Results should be available at the end of 2019. DISCUSSION: This innovative approach may have several limitations which should be taken into account, as potential side effects, kit misuse or resale, declarative main criteria, or no Plasmodium vivax curative treatment. Close monitoring is thus needed. CONCLUSIONS: This project may be the best available solution to a specific and important public health challenge in the Guiana Shield. If the use of self-diagnosis and self-treatment approach is effective, this strategy could be sustained by health institutions in the region.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin.

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients.

BACKGROUND: Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. CONCLUSION: Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.

Normas técnicas del sub-programa de ultrasonografía precoz nivel 1, y nivel 2 del período 22 a 26 semanas, para unidades de ultrasonografía comunal: actualización 2005 / Technical standars of early ultrasonography sub-program level 1, and level 2 of period 20 to 26 weeks, for communal ultrasonography units: update 2005

Como una forma de promover el desarrollo de unidades de ultrasonografía comunal en el país, hemos decidido publicar nuestras actuales Normas Técnicas, fruto de la experiencia acumulada y publicada por más de 20 años.

Diagnostic tests in rheumatology

The diagnosis of the rheumatic disorders may be easy or may be complicated; some clinical syndromes overlap considerably, some laboratory tests may be equivocal. However, a judicious mix of clinical features and laboratory as well as radiological data will in most cases lead to a satisfactory working diagnosis (AU)