Cryptosporidiosis

To illustrate the analytical process we used data collected from a birth cohort of children observed over three years in a semi-urban community in south India. The aim of the study was to examine the change in serum IgG levels measured by ELISA units to the immunodominant gp15 antigen as a consequence of the first episode of symptomatic cryptosporidial infection [1 (link)]. A total of 452 children were recruited over an 18-month period starting in March 2002; 373 children completed the 3-year follow-up. Field-workers visited each child twice-a-week to record any morbidity. Surveillance stool samples were collected every two weeks and diarrheal stool samples were collected with each episode of diarrhea [2 (link)]. The diarrheal stool samples were examined for the presence of Cryptosporidium spp. by microscopy and the positive samples were subjected to PCR-RFLP for genetic characterization [3 (link)]. Fifty-three children in this cohort experienced a total of 58 episodes of confirmed cryptosporidial diarrhea, out of which 47 episodes were due to C. hominis (see details elsewhere [3 (link)]). For illustrative purposes, we used data from 40 children whose first episode of cryptosporidial diarrhea was due to C. hominis infection. For these 40 children we utlilized the results of ELISA testing in two surveillance stool samples collected before and after the child's first episode of cryptosporidial diarrhea. The original data are provided in supplemental material, which include information on IR values, sampling date and child's age (see Additional file 1). The details on measuring serum IgG levels to the gp15 antigen and normalization of ELISA units can be found elsewhere [1 (link),4 (link)]. For the purpose of this study, serum IgG levels are used as a measure of immune response.
The main objective for the performed statistical analysis is to derive inferences from a change in the immune responses measured in ELISA units at those two time points. In statistical terms we aim to detect the difference in the markers of immune responses in a study with a pre-post design delivering two repeated measurements for each subject.
In this tutorial we use the following notations: Y_i - values for immune responses for i- child; each Y_i consists of two values: Y_t1 - first measurement and Y_t2 - second measurement, where t₁ - time of first measurement; t₂ - time of second measurement. A degree of change on an individual level is defined in three ways: as an absolute difference, ΔY_i = Y_t2 - Y_t1, an absolute difference of log-transformed values, ΔY_i = lnY_t2 - lnY_t1 and log-fold change, ΔY_i = ln(Y_t2 /Y_t1). We also specify t_E as the time of the event of interest. Additional relevant information to the presented illustration includes age at measurement and date of measurements. Sections below demonstrate the importance of this information in better understanding the variability in immune responses.

Prospective longitudinal birth cohorts (“Cryptosporidiosis and Enteropathogens in Bangladesh”; ClinicalTrials.gov identifier NCT02764918) were established at 2 sites in Bangladesh. Mirpur, Dhaka is a relatively poor, urban neighborhood [12 (link), 13 (link)], and Mirzapur is a rural subdistrict located 60 km northwest of Dhaka, as previously described [14 (link)]. Further site descriptions are provided in Table 1 and the Supplementary Methods.
Pregnant women in their second trimester were identified. In Mirpur, field research assistants performed a census. In Mirzapur, study participants were identified using a demographic surveillance system previously established by the Bangladeshi government [14 (link)]. Interested women meeting study criteria and providing informed consent underwent clinical examination, urinalysis, and gestational age confirmation by ultrasound. Exclusion criteria were age <18 years, gestational age ≥7 months, hypertension, edema, proteinuria, or intent to migrate from the study area (Figure 1). After delivery, infants assessed by the study medical officer (SMO) within the first 7 days of life were eligible for enrollment. For logistical reasons at Mirpur, the monthly maximum enrollment was 27.
The SMO collected demographic and socioeconomic data using a structured questionnaire at enrollment. Thereafter, field research assistants performed twice-weekly, in-home visits to interview caregivers and collect information regarding child morbidity and diarrhea. The SMO assessed all children monthly. Caregivers were encouraged to bring the child to the study clinic whenever the child developed symptoms of any illness, not only diarrhea. If acutely ill during an in-home visit, the child was referred to the study clinic. If the SMO determined that further treatment was needed, care was provided free of charge at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) Mirpur Treatment Centre, the icddr,b Dhaka Hospital in Mirpur, or the Kumudini Hospital in Mirzapur.
Height and weight were measured for mothers at the infant enrollment visit. Infant length (to nearest 0.1 cm using length board and plastic tape) and weight (kilograms, measured with electronic scale; TANITA, HD-314) were obtained every 3 months. Weight-for-age z score (WAZ) and length-for-age z score (LAZ) were determined using World Health Organization Anthro software (version 3.2.2). The change in LAZ (Δ-LAZ) was calculated by subtracting the enrollment LAZ from that at 24 months.

The aim was to describe and analyse trends in the Cryptosporidium species and gp60 genotypes identified in human outbreaks of cryptosporidiosis in England and Wales from January 2009 to December 2017. Definitions used to define an outbreak were: an incident in which two or more people experienced a similar illness and linked in time or place, or a greater than expected rate of Cryptosporidium reports compared with the usual background rate for a place and time. Cryptosporidium outbreaks were extracted from the eFOSS database and from records for those that also came to the attention of the national CRU during outbreak investigations. The proportions of outbreak routes of transmission were compared between the two databases by uncorrected Chi square and a P-value of 0.05 was regarded as significant. The databases were reconciled by PHE centre, setting/place name, postcode, dates of first and last known cases, and populated with Cryptosporidium species and gp60 subtypes identified in the stools of cases and any additional samples tested. The outbreaks were analysed for trends in vehicles and settings, season, and associated Cryptosporidium species and gp60 subtypes. The CRU archive and the NCBI nucleotide DB and PubMed were searched for previous reports of subtypes found.
To identify species, Cryptosporidium positive stools were sent by primary diagnostic laboratories to the national CRU, generally within 5 days of collection [13 (link)]. Oocysts were separated from faecal material by salt flotation, disrupted by boiling, and DNA extracted using proteinase K digestion and a spin column kit (QIAamp DNA mini kit, Qiagen, Hilden, Germany) as described previously [13 (link)]. Samples were screened for C. parvum and C. hominis using a duplex real-time PCR assay [46 (link)] and other species were sought using a nested PCR targeting the SSU rDNA gene [47 (link)]. A nested PCR targeting the gp60 gene was used to subtype C. parvum and C. hominis samples known or suspected to be part of outbreaks as described previously [48 (link)]; to simplify workflow a cocktail of single round PCR primers was developed and used from 2015, as described previously [49 (link)]. PCR amplicons were subjected to bidirectional sequencing (Applied Biosystems 3500XL) and sequence similarities searched for in the NCBI Blastn website tools. Gp60 subtypes were confirmed by manual identification of trinucleotide repeats and other repeat sequences (Fig. 1). The findings were contextualised at the time to inform outbreak investigations and updated for this article.
In animal contact outbreaks, animals were sampled by a Veterinary Investigation Officer if requested by the outbreak control team and tested using immunofluorescence microscopy (Crypto-cel, Cellabs) at the Animal and Plant Health Agency’s central laboratory, Weybridge. Cryptosporidium-positive samples were sent to the CRU for genotyping as described above. In recreational and drinking water outbreaks, sampling and testing was undertaken as described in [7 ] if requested by the outbreak control team. Cryptosporidium positive microscope slides sent to the CRU for genotyping were processed as described previously [37 (link)] until 2015. After 2015 DNA extraction from slides was done using a chelex-based method as described previously [50 ].

In this layer, a lightweight protocol must be deployed, because the proposed IoT-based irrigation system is made up of constrained devices in terms of memory and energy. Characteristics of lightweight cryptography were highlighted in ISO/IEC 29192 and ISO/IEC JTC 1/SC 27. Lightweight properties are evaluated based on chip size and energy consumption, and small code and/or RAM size in case of software implementation [21 (link)]. So, the Expeditious Cipher (X-cipher) was selected [31 ]. The X-cipher was proposed in 2011 and is considered a lightweight high-throughput encryption protocol. The cipher sub-keys are generated using the well-studied SHA-512 and Whirlpool 512 hash functions. The two hash functions are cascaded in a pseudo-random manner that depends on the user key to enhance the cipher security. The cipher utilizes a variable-size user key. The encryption rate can attain 512 bits per cycle in the case of hardware implementation with parallelization encryption paths. Eight rounds are recommended for the proper operation of the Expeditious Cipher (X-cipher). The circuitry of the encryptor and decryptor are identical, as such, a high code density and small implementation area are achieved, as required by lightweight cryptography. An additional important feature of the X-cipher is the separation of key scheduling process from the encryption process, which allows the change of cipher design, key, and block size by simply changing the hash function. The Expeditious Cipher (X-cipher) complete encryption algorithm is presented in [31 ]. NodeMCU has a cryptography module called crypto that contains various functions for working with cryptographic algorithms. The AES 128-bit is supported in ECB and CBC modes, in addition to several hash functions, namely, MD5, SHA-1, SHA-256, SHA-384, and SHA-512. The MQTT module implemented in C language is available on the NodeMCU ESP8266 board to run the MQTT client to publish the temperature and humidity readings to the MQTT broker running on Raspberry Pi. So, because the proposed Expeditious Cipher (X-cipher) depends on the SHA-512 hash function that is already implemented in the crypto module and the modulo 2 addition between the hashed key and the plain text, the selected lightweight algorithm can be compiled easily on the NodeMCU board. Table 2 compares the operational features of AES (traditional encryption protocol), PRESENT (lightweight standard protocol), X-cipher (selected lightweight protocol), and the lightweight encryption protocol proposed recently in the literature.

The site is located within a lowland composed by eroded outcrops of ancient igneous deposits, with the presence of river terraces and well oxidised sandy deposits underlain by sedimentary formations of laterite. Within the region, various formations of complex metamorphic outcrops (crypto-crystalline silicates and siliceous stone formations) occur. Furthermore, Shaw and Daniels¹ (pg. 190, Figure 3) show the presence of quartzite veins which appear approximately 5-10 km West of Iho Eleru. Furthermore, various deposits of considerably eroded quartzite and vein quartz cobbles are present within local seasonal river valleys.
Iho Eleru is a rock shelter found toward the base of an igneous inselberg, found on the western margin of the Ikere Batholith. Topographically, the main human activity area would have been behind the drip line area of the shelter, within the main upper leveled platform area (trenches D & F, XVII-XXVII). On its southern and eastern edges, steep inclined taluses slope downwards. On its western edge, a very steep incline slopes upwards toward the upper levels of the inselberg, where other sheltered areas are found. On the northern edge of the platform area, the rock shelter closes following the two overhanging igneous outcrops, forming a small tunnel (Central Tunnel) with an SW-NE orientation. The stratigraphy at Iho Eleru shows very irregular igneous beds upon which the archaeological sediments sit. The depths of the stratigraphy vary from 0 cm (subsurface outcrop) to over 2m in depth (Trench S). The sediments have post-erosional origin, where materials from the upper levels of the inselberg fall during particularly wet periods, depositing within the platform area and surrounding slopes. In turn, similar activities cause an erosion of the platform area sediments, causing the sloped areas to have inverted stratigraphies. For this reason, during our re-analysis of the chronometric dates of the site, only charcoal samples that were retrieved in the upper level area were selected. Nevertheless, the resulting ¹⁴C dates still show discrepancies and inverted dates. This has been interpreted as being caused by heavy erosional processes, mostly during wet season periods, which cause Iho Eleru rock shelter to become a main channelling area for water moving downstream of the upper levels of the inselberg.
Shaw and Daniels state that the stratigraphical excavation was conducted with an initial layer (spit 1) that was excavated with a total depth of 25 cm, and subsequent spits of 15 cm. However, later descriptions of the excavation show that each and every spit per square was considered to be 15 cm in depth. This contradiction might be caused by a use of a 25 cm deep spit only in specific areas of the rock shelter, however such information could not be found by the authors of this paper. For these reasons, a consistent depth of 15 cm per spit was considered and applied for the upper plateau area which was evaluated in this study, and which is consistent with the original descriptions, interpretations and excavation diagrams by Shaw and Daniels (1984).
In the platform area of the rock shelter, Shaw and Daniels describe 4 main sediment types:
These units appear to be very distinct in terms of their sedimentary properties and likely reflect changes in the local environment surrounding the rock shelter. We propose some thoughts regarding possible site formation factors operating at Iho Eleru and describe how these may relate to changes observed in our paleoclimatic modeling.