Immunochromatography

The Infectious Diseases and Beliaghata General Hospital (ID&BGH), in Kolkata, a 770 bedded hospital, provides treatment for about 20,000 to 25,000 hospitalized patients with acute diarrhoea annually. In the present systematic active surveillance, every fifth patient with diarrhoea or dysentery without other associated illness on two randomly selected days of the week was enrolled as study subjects from cases admitted at the ID&BGH. This study was conducted between November 2007 and October 2009. The dehydration status of each diarrhoea case was classified as no, some or severe dehydration according to WHO guidelines. The clinical, demographic and laboratory data was checked manually and entered into pre-designed data entry proforma developed in visual basic with inbuilt entry validation checking facilitated programme in structure query language (SQL) server by dual entry method by trained data entry professionals. Data was randomly checked and matched to derive consistency and validity for analysis. The edited data was exported and a final analysis was performed using the SPSS.17.0 software (SPSS Inc., Chicago, IL, USA).
This study was approved by the duly constituted Institutional Ethics Committee (IEC). As per the recommendation of IEC, individual informed consent was obtained from each patient enrolled in this study and confidentiality was maintained. Faecal specimens were collected in McCartney bottles using sterile catheters or as rectal swabs in Cary Blair medium and were examined within 2 hrs for 24 enteric pathogens comprising bacterial, viral and parasitic pathogens using a combination of conventional, immunological and molecular methods (Fig. 6). PCR targeting ompW and toxR were performed for the species confirmation of V. cholerae and V. fluvialis, respectively [31 (link),32 (link)]. Confirmed strains of V. parahaemolyticus, Shigella spp and Salmonella spp were serotyped using commercially available antisera (Denka Seiken, Tokyo, Japan, BioRad, Marnes-la-Coquette, France). V. cholerae strains were serotyped using antisera prepared in NICED. Representative strains of V. cholerae O1 were examined by MAMA-PCR to determine the type of cholera toxin B subunit gene (ctxB) [33 (link)]. Three different lactose-fermenting colonies isolated from each sample were picked from MacConkey agar plate and included in the multiplex PCR assay for the detection of different DEC that include enterotoxigenic E. coli (ETEC, inclusive of both heat-labile and heat-stable enterotoxin producers), enteropathogenic E. coli (typical and atypical EPEC) and enteroaggregative E. coli (EAEC) [34 (link)]. Simplex PCR was also performed for the detection of enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) [35 (link),36 (link)].
Antimicrobial susceptibility testing was performed by disk diffusion (Kirby- Bauer method) using commercially available disks (Becton Dickinson Co., Sparks, MD, USA) with interpretation stipulated by the Clinical and Laboratory Standard Institute [37 ]. Two hundred and thirty representative (one third from the total number of strains) V. cholerae O1 strains covering all the months and all the Shigella strains were included in the testing. Rotavirus was detected by polyacrylamide gel electrophoresis and silver staining [38 (link)]. Norovirus [Group I and II (NVGI and NVGII)], Sapovirus and Astrovirus were detected by RT-PCR using random primers for reverse transcription and specific primers for polymerase chain reaction [24 (link),39 (link)]. Different viruses were detected according to the appropriate amplicon sizes observed in agarose gels stained with ethidium bromide. Adenovirus was detected by the commercially available RotaAdeno VIKIA kit (biomereux, France), which is a qualitative test-based on immunochromatography in lateral flow format [40 (link)]. For detection of enteric parasites, faecal samples were processed separately for microscopic and molecular analysis. For microscopic analysis, the samples were first concentrated using formalin ethyl acetate concentration method [41 ] and an aliquot of each sample was preserved in 10% formalin and stored at 4°C for subsequent use. Aliquots of fresh stool specimens were also preserved at -80°C for ELISA and PCR assays. All the faecal samples were screened using a highly sensitive antigen capture ELISA (Tech Lab, Blacksburg, USA) and PCR for the detection of Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Faecal samples were processed by microscopy using iodine wet mount staining and trichome staining procedure for Blastocystis hominis [42 ].
Using the surveillance data, an estimate of the total number of cases specific for each pathogen in two consecutive years was extrapolated. From the monthly enrolled cases, the isolation rate of different pathogens was calculated for that particular month. An estimate of total number of cases with particular pathogen for a particular month was then extrapolated by multiplying the total admitted cases with particular isolation rate of the pathogenic with an assumption that similar isolation rate would be among non-enrolled cases. In this way, pathogen-specific total number of yearly estimated cases was calculated.
The risk age group was also explored for predominant enteric pathogens such as V. cholerae O1, Rotavirus, shigellae and G. lamblia by Multinomial Logistic Regression (MLR) analysis [43 (link),44 (link)]. This analysis helps to determine the likelihood age of the patient associated with any enteric pathogen. The age groups were classified into 8 categories viz. <1 year, 1-2 years, >2-5 years, >5-14 years, >14-30 years, >30-45 years, >45-60 years and >60 years and were coded from 1 to 8, respectively. Infection caused by an enteric pathogen was coded as '1' for the pathogen present and '2' for its absence. The extreme values of the classified age group was fixed as a reference category.

This prospective observational study was conducted between December 16, 2019 and March 25, 2020. The original plan was to collect samples until May 31, 2022 but the study was suspended early on March 25, 2020 when the spread of coronavirus disease 2019 (COVID-19) began in Japan [6 (link)–8 (link)]. Due to the continued COVID-19 pandemic, the study was not resumed, and the analysis was conducted on the samples collected by March 25, 2020. We selected seven internal medicine clinics, pediatrics, and otorhinolaryngology clinics in Nagasaki Prefecture that could participate, the Urabe Otorhinolaryngology Clinic, Iida Naika Syounika Clinic, Ohisama Pediatric Clinic, Nishida Gastrointestinal Intermedicine Clinic, Onitsuka Internal Medicine Clinic, Hirose Clinic, and Tomonaga Medical Clinic. We also included a hospital that participated in the previous study, the Japanese Red Cross Nagasaki Genbaku Hospital. In eight medical facilities, we included patients who visited or were hospitalized with influenza-like illness (ILI), as defined by the World Health Organization’s case definition [9 ]. Patients were excluded if they were administered anti-influenza agents within one month before sampling. After obtaining informed consent, nasopharyngeal swabs and gargle samples were collected. Two nasopharyngeal swabs (1PY1502P; Japan Cotton Swab Industry, Limited, Tokyo, Japan) were collected from all the patients by a healthcare provider. Gargle samples were collected from patients whom the physician judged to be able to perform gargling. In gargle samples, the patients gargled for 5 s with 20 mL of water (water for injection; Hikari Pharmaceutical CO., LTD. Tokyo, Japan), which was collected. Gargle samples were stored at −20 °C in a container (Multi-purpose container, 70 mL; Sarstedt, K.K., Tokyo, Japan) until further analysis. One of the swabs was used in each medical facility for detecting influenza by DIAs using silver amplification immunochromatography (FUJI DRI-CHEM IMMUNO AG Cartridge FluAB; Fujifilm, Kanagawa, Japan) [10 (link)], according to manufacturer’s instruction. Another nasopharyngeal swab and gargle samples were stored at −20 °C in a sealable tube (PP screw cap test tube; Maruemu Corporation, Osaka, Japan) without media until further analysis. The physicians determined the clinical diagnosis based on medical history, physical findings, and DIAs results, from which they produced a clinical report for each patient. Since TRCsatFLU was not approved in Japan when this study was conducted, and it was necessary to prevent the use of TRCsatFLU results for the diagnosis of influenza at medical facilities, nasopharyngeal swabs and gargle samples were transferred to Tosoh Corporation for performing TRCsatFLU and RT-PCR. All information, such as clinical report forms and TRCsatFLU and RT-PCR results, was summarized and analyzed at Nagasaki University Hospital. If the results of TRCsatFLU were different from those of RT-PCR, the samples were analyzed by sequencing at Tosoh Corporation.

The GBD estimates the incidence of infectious meningitis for each country (specific objective a). Meningitis was defined as a “disease caused by inflammation of the meninges, the protective membrane surrounding the brain and spinal cord, and that is typically caused by an infection in the cerebrospinal fluid (CSF). Symptoms include headache, fever, stiff neck, and sometimes seizures” (13 (link)). Infectious meningitis is then classified into four groups: meningococcal, H. influenzae type B, pneumococcal, and others.
A systematic review of surveillance systems reports, scientific literature claims data-inpatient visits, and inpatient hospital data, published up to the end of 2013, was done. Cases were recorded with ICD-9 and ICD-10 codes: N. meningitidis (36-36.9 and A39-A39.9), H. influenzae (320 and G00.0), and S. pneumoniae (320.1 and G00.1). General incidence, and per infectious agent, were generated by Bayesian meta-regressions based on 1,348 non-fatal outcomes sources. To differentiate incident from prevalent cases, the lethality, rate of long-term complications, and sequelae fraction (epilepsy, vision impairment, hearing loss, motor and cognitive impairment, intellectual disability, and behavioral problems) were also computed (13 (link)).
The MenAfriNet Consortium and the WHO record suspected meningitis cases per epidemiological week (for the analysis of epidemic curves by subregion and when assessing the yearly trends of BM incidence in relationship with climate variables (specific objective b), this is the level of certainty). Suspected cases are defined as: “any person with sudden onset of fever (>38.5°C rectal or 38°C axillary) and one of the following signs: neck stiffness, altered consciousness or other meningeal signs” and “any toddler with sudden onset of fever (>38.5°C rectal or 38°C axillary) and one of the following signs: neck stiffness, flaccid neck, bulging fontanel, seizure or other meningeal signs” (43 ).
Some of the cases underwent a lumbar puncture for confirmation in CSF. Basic cytochemical and microbiological analysis contribute to a probable level of certainty: “any suspected case with a macroscopic aspect of its CSF turbid, lousy or purulent; or with a microscopic test showing Gram-negative diplococcus, Gram-negative bacillus, Gram-positive diplococcus; or with leukocytes count more than 10 cells/mm³” (12 , 43 ) and “any infant with CSF leukocyte count >100/mm³ or 10–100 cells/mm³ and either and elevated protein (100 mg/dL) or decreased glucose (< 40 mg/dL) level” (12 , 43 ).
Finally, a smaller proportion reached the confirmed definition: “isolation or identification, in CSF or blood, of the causal pathogen (N. meningitidis, H. influenzae type B, S. pneumoniae, etc.) from the CSF of a suspected/probable case by culture, polymerase chain reaction, immunochromatographic dipstick or latex agglutination test” (12 , 43 ).
Suspected cases are reported by providers at a health facility and informed to the district surveillance officer each week, who then compile and notify the data to the provincial and national instances. Notification must be done even in absence of cases and throughout the year. This information is merged by the national instance of each country and then sent to the WHO, and their partners, on a weekly or monthly basis (if no epidemic is registered). Laboratory tests' results must be also included (12 , 43 ).