Agglutination Tests

E. coli strain PCN033 was isolated from the brain of a diseased pig from Hunan Province, China [10 (link)]. E. coli strain PCN061 was isolated from the lung of a diseased pig from Hunan Province, China. These two strains were routinely cultured in Luria–Bertani (LB) medium at 37 °C. According to Johnson et al. [12 (link)] and Ding et al. [11 (link)], ExPECs were defined as E. coli isolates containing two or more virulence markers: papA/papC, sfa/foc, afa/dra, kpsMTII and iutA. PCN033 and PCN061 were examined in PCR for the presence of the above virulence markers. Serotyping, phylogenetic grouping and virulence analysis in mice model of these two strains were performed in previous study [9 (link)]. The serotypes of these strains were identified by serum agglutination assay using specific O-antigen antiserum in the China Institute of Veterinary Drugs Control, Beijing, China. The phylogenetic groups of PCN033 and PCN061 strains were determined based on PCR detection of the chuA and yjaA genes and DNA fragment TSPE4.C2 [83 (link)].

Sera were processed from blood samples collected from subjects during house visits. The microscopic agglutination test (MAT) was performed to evaluate for serologic evidence of a prior Leptospira infection [34] . A panel of five reference strains (WHO Collaborative Laboratory for Leptospirosis, Royal Tropical Institute, Holland) and two clinical isolates [6] (link) were used which included L. interrogans serovars Autumnalis, Canicola and Copenhageni, L. borgspetersenii serovar Ballum, and L. kirschneri serovar Grippotyphosa. The use of this panel had the same performance in identifying MAT-confirmed cases of leptospirosis during surveillance in Salvador [6] (link),[16] (link) as did the WHO recommended battery of 19 reference serovars [34] . Screening was performed with serum dilutions of 1∶25, 1∶50 and 1∶100. When agglutination was observed at a dilution of 1∶100, the sample was titrated to determine the highest titer.

The surveillance methods used in our population-based study have been described previously.¹⁰ (link) In brief, nurses assessed all individuals who presented as an outpatient or who were admitted to one of the six health facilities in the study area (Basse, Gambissara, Demba Kunda, Fatoto, Garawol, and Koina). Enrolment involved standardised screening of patients for referral to a clinician in Basse. Clinicians used standardised criteria to identify patients with suspected pneumonia, sepsis, or meningitis, and requested blood culture, lumbar puncture, or chest radiography according to protocol (appendix pp 7–9, 17). Aspiration of pleural fluid of lung aspiration was performed for patients with a pleural effusion or dense peripheral consolidation radiologically. We defined invasive pneumococcal disease as suspected pneumonia, sepsis, or meningitis with isolation. Vaccine failure was defined as invasive pneumococcal disease following two or more doses of PCV covering the homologous serotype, given more than 14 days before the event.11 (link), 12 (link) Weight was recorded on a digital scale (TANITA, Arlington Heights, IL, USA) and height with a ShorrBoard (Weigh and Measure, Olney, MD, USA). Rapid malaria tests (ICT Diagnostics, Cape Town, South Africa) were done on all patients with suspected pneumonia, sepsis, or meningitis from August to December (the malaria transmission season) each year and in a 10% random sample from January to July each year. This random sample was chosen by random selection of the final digit of the patients' surveillance identity number (0–9) and during the dry season any patient whose identity number ended in zero had a malaria test. Samples were not collected between Oct 5 and Nov 3, 2010, when the field station flooded.
Blood, lung aspirate, cerebrospinal fluid, pleural fluid, and other microbiological samples were processed in Basse using conventional microbiological culture and identification techniques.¹³ (link)
S pneumoniae was identified by morphology and optochin sensitivity. All pneumococcal isolates were confirmed at the WHO Regional Reference Laboratory (MRC Fajara, The Gambia), and serotyped with a latex agglutination assay using factor and group-specific antisera (Statens Serum Institut, Copenhagen, Denmark). Serotypes 6A and 6B were differentiated from 6C by PCR.¹⁴ (link) Serotyping of 10% of isolates was repeated at the National Institute for Communicable Diseases in South Africa (Johannesburg, South Africa). The laboratories in Basse and Fajara submitted to external quality assurance throughout the study (UK National External Quality Assessment Service [Sheffield, UK], the WHO Reference Laboratory in Denmark, and the Royal Australasian College of Pathologists [Sydney, Australia]).

RDTs were selected to include locally commercially available tests approved by the Colombian regulatory authority (Instituto Nacional de Vigilancia de Medicamentos y Alimentos [INVIMA]): SD Bioline Syphilis 3.0 (Standard Diagnostics Inc, Kyonggi-do, Korea) and Alere Determine Syphilis TP (Abbott Diagnostics Medical Co, Ltd). Both tests use an immunochromatographic platform with a strip showing a colored test line if treponemal antibodies are detected in the specimen and a colored control line if the test is working properly. RDTs were run according to manufacturer’s instructions, read after 20 minutes and were considered positive if both the test and control lines were colored, even if the test line was faint. They were considered negatives if only the control line was colored and invalid when only the test or neither of the lines were colored.
RDTs were performed at the point-of-care using capillary blood (CB) from a finger prick and at a reference laboratory using serum. A volume of 20μL of CB or 10μL of serum with its corresponding buffer for Bioline and 50μL of CB with its corresponding buffer or serum without buffer for Determine were used. RDTs on CB were read by one of 5 operators at the point-of-care and, on sera at the reference laboratory by three evaluators, two microbiologists and one physician, RDTs on sera were considered positive or negative when at least two evaluators agreed on the assessment of the test and were considered invalid when at least one evaluator considered the test invalid. Invalid tests were repeated once.
Diagnosis of syphilis is challenging due to the lack of a reliable reference standard; however, serology tests remains as the most widely implemented tests for syphilis [21 (link), 22 (link)]. Considering that the index tests under study detect treponemal antibodies, we decided to use two TT as reference standard: Treponema pallidum haemagglutination test (TPHA) and enzyme linked immunoassay (ELISA) for syphilis. TPHA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) and T. pallidum ELISA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) were performed using 10 μL of serum for each test. As with index tests, selection of the reference standard kits was based on availability and approval by INVIMA. We decided to use composite reference standard due to the limitations of each individual test such as higher false positive rate of immunoassays and lower sensitivity of agglutination assays during latent syphilis [23 (link)]. The reference standard was considered positive when both tests were positive, negative when both tests were negative and undetermined when the two results were discordant or one of the tests was invalid.

Bacterial isolates were obtained from each skin lesion by applying a “Film Stamp” for 10 s to the affected dorsal skin (8 cm² area [=2 cm × 4 cm]) (9 (link)). After an overnight culture on tryptic soy agar plates at 37°C, the colonies grown were characterized by color and diameter, and colony numbers were expressed as colony-forming units (CFUs) per 8 cm² skin area. Microscopic examination of Gram-stained colonies and the PS Latex (Eiken Chemical, Tokyo, Japan) slide agglutination test were also carried out to identify these organisms. S. aureus was finally identified from the reaction profile based on the 20 biochemical tests included in the API STPH system (Biomérieux, Marcy-I’Etoile, France).

Direct Agglutination Test (DAT) based on L. donovani promastigotes was produced by KIT Biomedical Research—Amsterdam, The Netherlands. IT-Leish based on rK39 antigen was produced by DiaMed AG—Cressier-sur-Morat, Switzerland, and Bio-Rad Laboratories—Marnes-la-Coquette, France. Kalazar-Detect based on rK39 antigen was produced by InBios International—Seattle, WA, USA. All tests were performed at Instituto de Medicina Tropical, Faculdade de Medicina, USP, by the time the samples were collected, according to the manufacturer’s recommendations.
For DAT, samples were two-fold diluted from 1:100 (final 1:200) through 1:102,400 (final 1:204,800), and a cut-off of 1:3,200 was adopted [18 (link), 23 (link), 26 (link), 27 (link)]. All healthy endemic control samples were DAT negative (titer < 1:3,200). However, VL patients with negative DAT were included in the study as long as they were positive in the parasitological test.