Molecular Detection of Pathogens “Causing Most of Infants’

Severe and Fatal Diarrhea” in Infant Drinking Water and their

Relationship with Water Quality Determinants in Eastern
Ethiopia: Loop-Mediated Isothermal Amplification (LAMP)
based Study
Samuel Mebrahtom1*, Alemayehu Worku2, Daniel J.Gage3, Heven Seme4, Adugna Abera4

1- Ethiopian Institute of Water Resources, Addis Ababa University, Addis Ababa, Ethiopia
2- School of Public Health, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
3- Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, United States of America
4- Bacterial, Parasitic and Zonotic Diseases Research Directorate, Ethiopian Public Health Institute, Addis Ababa,
Ethiopia.

*samrontom@gmail.com

Abstract
Background
Waterborne pathogens such as Rotavirus, Cryptosporidium, Shigella and Toxin-producing
strain of E. coli were reported to be the most common causes that responsible for most severe
and fatal diarrhea among the world’s infants. As per epidemiological studies, a number of
children died from an illness caused by these pathogenic agents that thrive in contaminated
drinking water. This study aimed to detect the existence of these pathogens in infant’s drinking
water samples using LAMP technique and analyzing their relationship with water quality
determinants in Eastern Ethiopia.

Methods
This study employed molecular testing-based cross-sectional study design in Eastern Ethiopia.
A total of 410 water samples were collected from infant point-of-use at household level and 37
samples from the corresponding water sources from June, 2020 to May, 2021. A 1000ml liter of
water samples were collected in a sterile bottle with exclusives of any chemical additives. The
loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of the
targeted pathogens. Primers were designed for target DNA/RNA of each target organism and
optimization done. Data were collected from the households mothers/care takers of infants
using pre-coded and pre-tested structured questionnaires and water sample were taken from
infant point-of consumption and corresponding water sources. These data were entered using
CSpro version 5.1 and transform to SPSS version 23 to establish frequency distribution and
analyze the relationship among variables by applying phi coefficient correlation and binary
logistic regression.

Findings
Out of the 410 water samples tested from infant point-of-use, the prevalence of
Cryptosporidium Oocysts, Shigella species, toxin-produce strain of E-coli and Rotavirus found
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to be 117(28.5%), 123(30.0%), 108(26.3%) and 132(32.2%), respectively. The simultaneous
isolates of overall pathogens were found in 13.2% water samples. Among the 37 water sources,
10 (27.0%) positive for Cryptosporidium Oocyst, 12(32.4%) for Shigella species, 11(29.7%) for
Toxin produce stain of E.coli and 14 (37.8%) for Rotavirus. Positive significant correlation was
observed between infant point-of-consumption and water sources towards the presences of
each targeted pathogens. Households using unimproved water source showed strong
significant association to the presence of cryptosporidium Oocyst (p=0.000, AOR: 4.02, 95% CI:
2.29, 7.04), Shigella species (p=0.000, AOR: 4.21, 95% CI: 2.43, 7.29) and Toxin produce strain
of E.coli (p=0.004, AOR: 2.14, 95% CI: 1.27, 3.59). However, no statistical significant association
was observed in unimproved water source with the occurrence of rotavirus. The presence of
overall (four) pathogens shows significant association with households sourced water from
unimproved supplies (p=0.001, AOR: 3.96, 95% CI: 1.73, 9.07). Fetched drinking water other
than freshly (on the day of our visit) collected water showed less likely significant relationship
with the presence of cryptosporidium, toxin produce strain of E.coli and rotavirus.

Conclusion

Infants demonstrated high levels of exposure to consume contaminated drinking water by

those recognized pathogens that causes the most severe and fatal diarrhea. Unimproved water
source remained the only statistical significant in relation to the simultaneous presence of
these pathogens. Significant positive correlation observed between point-of-consumption and
water sources. Therefore, effort should be made on new development and rehabilitation of
improved water sources with its overall source protection safety and health education to the
mothers/caretakers are essential.

Key words: Cryptosporidium, Shigella, Toxin-producing strain of E. coli, Rotavirus, Infant,

Drinking water at point-of-use, Water Source, LAMP

1. Background

Waterborne diseases are caused by extensive range of pathogenic microbes which include
viruses, bacteria and parasites, responsible for high morbidity and mortality of infants and
children(1). The etiologic agents causing diarrhea are typically transmitted by the fecal-oral
route from an infected person to the mouth of another person upon ingestion of contaminated
water, food, objects, or hands(2). Infant’s exposures to etiologic agents and develop diseases or
even death is much more severe than the general population(3). Some of these agents cause
immediate effect, while others may not be noticed for many years.

According to the large new studies, there are pathogenic microbes responsible for causing the
most severe and fatal diarrhea among the Worlds infants (<1 year of age). These studies found
that the most common causes were a virus referred as rotavirus; a protozoan called
Cryptosporidium; and two bacteria, Shigella and a toxin-producing strain of E. coli(4–6). These
four etiologic agents - together accounted for more than 60% of diarrheal deaths in children <5
Page 2 of 33
years worldwide as well as in the case of Ethiopia (7). Along with a wide range of water access
problems faces nations, poor drinking water quality also continue to pose a major threat to
human health(8), and remains one of the most significant challenges. In addition, the role of
drinking water as a carrier of disease causing microbes is a major public health concern in
many developing worlds. Evidence suggested that a number of children died from an illness
caused by these pathogenic agents that thrive in contaminated drinking water(9,10).
Reportedly, unsafe water is continuing to be responsible for 72.1% of diarrhea deaths in
children younger than 5 years old (11). The global initiative to reduce the diarrhea death shall
be focus on the most common aetiology that dominantly causes the death among infants and
children.

Microbial drinking-water quality testing on the most common etiologic agents plays an
essential role in taking appropriate public health measures. For this case, the molecular-based
techniques that involve direct DNA or RNA detection have been essential for the correct
identification of species-specific of pathogenic agents in drinking water samples. Several
molecular methods such as PCR, Real Time PCR or multiplex PCR have been developed which
could able to detect the pathogens accurately, however, these methods have some drawback
like low detection limit(12). In recent years, new advanced molecular techniques have been
developed that guided the use of methods that amplify the nucleic acid material at isothermal
conditions. Among these techniques, Loop-mediated isothermal amplification (LAMP) is a
powerful and novel nucleic acid amplification method, which is simple, highly sensitive and
specific, less time-consuming than PCR-based methods, and less prone to inhibition from DNA
preparations(13,14). It is ranked as the most frequently used method(15), and also an
important constituent for the efficient screening and testing of drinking water samples in
resource limited settings(16).

To date, there are no studies in Ethiopia as well as other sub-Saharan countries in particular
that examined molecular detected pathogens from infants used drinking water along with the
corresponding water sources as well as their relationship with water quality determinants. And
as a result, there is lacking of information on this regards. The occurrence of pathogenic
microbes in infant drinking water is affected by combination of a wide range natural and
human influences on water quality(17), many of which are inadequately understood.
Therefore, the present study aimed to detect the existence of Rotavirus, Cryptosporidium
Oocyst, Shigella species, and Toxin produce E. coli in infant’s drinking water samples at point-of
consumption and the corresponding water sources using LAMP technique and analyzing their
association with the quality of drinking water determinants in Eastern Ethiopia. This study will
contribute to fill aforementioned gaps and functioning as a platform for future research. This
paper, thus, helps policy makers to design the right intervention to improve the quality of
infant’s uses drinking water at household level as well as water sources so as to achieving
reduction of illness and death related to water-borne pathogens that are causing the most
severe and fatal diarrhea in infants.

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2. Materials and Methods
2.1 Statistical Methodology
2.1.1 Study area

This study was conducted in four districts that include Chiro, Mieso, Amibara and Awash
fentale, which are located in Eastern part of Ethiopia. Chiro and Mieso districts are resided in
the West Hararghe administrative zone of Oromia while Amibara and Awash Fentale districts
are enrolled in zone 3 administration of Afar region. As of 2016, the projected population of
each districts estimated to be Chiro (223,167), Mieso (176,200), Amibara (80,531) and Awash
Fentale (37,892). As per the nation context, about 3.4% of the total populations are under the
age of one year. This study covered with an overall area of 5,186.14 sq. km. The study area has
two main climate seasons: a dry season and a rainy season. The dry season occurs from October
to February whereas the rainy seasons have two periods – the period from June to September
is the main rainy season and some rainy period usually from March to May. Acute respiratory
infection, Malaria, and Diarrheal diseases are the most common reportable disease in the areas.

2.1.2 Study Design

This study employed molecular testing-based cross-sectional study design which applied LAMP
technique for the simultaneous detection of four microbial pathogens (Cryptosporidium,
Shigella, Toxin produce strain of E. coli and Rotavirus) from infant’s drinking-water sample at
point-of-use in the households and the corresponding water sources conducted during the
period from June, 2020 to May, 2021 in the four districts in Eastern Ethiopia.

2.1.3 Study Population

The study population was comprised of infants (<1 year of age). The sampling unit was
households with infants, and the basic sampling population units (i.e. elements from which
required information ascertained) were their mothers/primary caregivers.

2.1.4 Sample Size Determination

The sample size (i.e. the number of households to be included in the study to represent the
population of interest) was calculated using Openepi version 3.03a with the single population
proportion. The sample size was calculated considering an assumption that the estimated
prevalence of feacally contaminated drinking water sample of 80% households with children in
the overall study group at the baseline conducted in one of the districts in Eastern Ethiopia(18),
an error risk parameter of 1.96 (for an error risk of 5% i.e. 95% confidence limits), a desired
precision of 5% and design effect of 1.5, which resulted the sample size of 369 households with
infants, and 15% of non-response rate is added, and thus, 424 drinking-water samples were
taken from infant’s drinking water at point-of-use in the households.

Page 4 of 33
2.1.5 Sampling Technique

A two-stage cluster sampling along with probability proportional to the population size
sampling technique was employed for this study. In the first sampling stage, thirty “kebeles”
(the small administrative unit) or clusters were allocated to each targeted districts on the basis
of proportional allocation to the available “kebele” size, followed by selection of eligible
“kebeles” using lottery method. The second sampling stage comprised of households with the
infant within the selected “kebele” using simple random sampling.

2.1.6 Data Collection Instrument and Methods

Data and water sample were collected by trained enumerator using pre-coded structured
questionnaires and water sample leveling format.

2.1.7 Study Variables

2.1.7.1 Independent (Predictors) variables of the study: Primary source of drinking water
(improved or unimproved), drinking water storage hygienic status, practices of water
treatment at point-of-use, water fetched time, water point location and presence of residual
chlorine.

2.1.7.2 Dependent (Outcome) Variables of the study: Presence of microbial pathogens

(Cryptosporidium Oocyst, Shigella species, Toxin produce strain of E.coli and Rotavirus)

2.1.8 Data Quality Control

Data quality was kept by regulating both random and systemic error. Data quality was
maintained through properly designed data collection tool. The data collectors as well as
supervisors recruited with the relevant educational background and language proficiency.
Training was held to the data and water sample collection procedures, and pre-test carried out
in a community with similar characteristics. The data collection procedures developed and the
collected data were reviewed by principal investigator. Any identified errors were discussed
and immediate measure has been taken.

2.1.9 Data Management and Analysis:

The raw data were entered into CSPro version 6.1 then transformed to SPSS version 23 for
analysis. Descriptive statistics such as frequency distribution and cross-tabulation were made
to summarize the study variables. Phi coefficient correlation was done to analyze the
association between point-of consumption and water sources towards the presence of
pathogens. Binary logistic regression was applied to establish crude and adjusted odds ratios
with 95% confidence intervals for the association between water quality determinants and
each targeted pathogens. In bivariate conditional logistic regression, each variable that showed
p-value <0.25 were considered as eligible for multivariable logistic regression. Variables that
Page 5 of 33
demonstrated p-value <0.05 in the multivariable logistic regression were declared as
significantly associated with the outcome variable (presence of targeted pathogens).

2.2 Molecular – LAMP Laboratory Methodology

2.2.1 Selection of Target genes

For the detection of each pathogenic microbe, a specific gene target region was selected. The
selection was based on either gene most commonly used for identification of the target genes
that expressed proteins unique to the organism of choice that helped to differentiate the
presence of each pathogenic microbe. The specific targeted genes encoding the 18S rRNA
presented in Cryptosporidium spp., ipaH gene in Shigella, stx2A and stx2B gene in Toxin
produce strain of E.coli, and the VP7 glycoprotein gene for Rotavirus were chosen. These genes
served as sites for specific primer design that were used in LAMP assays.

2.2.2 Primer Design

LAMP primers were designed based on the targeted genes specific to four targeted waterborne
pathogens. The primer sequences for LAMP amplification were designed based on the
Cryptosporidium 18S rRNA gene Accession number GenBank: L16996.1, Shigella ipaH gene
accession number GenBank: M76443.1, Toxin produce strain of E.coli stx2A and stx2B gene
accession number GenBank: FN252458.1, and Rotavirus VP7 glycoprotein gene accession
number GenBank: AB018697.1 from NCBI. The primers were designed by means of the Primer
Explorer Version 5 software, available on https://primerexplorer.jp/e/. The software was used
to design one set of primers (five to six primers per target organism) (see Table 1).

Page 6 of 33
Table 1: Primers used for LAMP assays for Cryptosporidium, Shigella, Toxin produce strain of
E.coli, and Rotavirus
Primer Name Primer Sequence Target Gene
Cryptosporidium 18S rRNA
FIP CCTCGTTCAAGATCAATAATTGCAA-ATGGGTAATCTTTTGAATATGCA
BIP TCCTAGTAAGCGCAAGTCATCAG-ATTCAATCGGTAGGAGCG
F3 GTATATATTCCTGTTTCGAAGGA
B3 TCCGAATAATTCACCGGATC
LB GCTGATTACGTCCCTGCCCTTTG
Corresponding nucleotide position of Cryptosporidium parvum 18s rRNA gene, 1746bp (Accession No. 16996.1)

Shigella ipaH
FIP AAGCTCCGCAGAGGCACTGA-CACGCAATACCTCCGGATTC
BIP AGCAGTCTTTCGCTGTTGCTGC-CCGGAGATTGTTCCATGTGA
F3 GCCTTTCCGATACCGTCTCT
B3 TGATGGACCAGGAGGGTT
LF TGCAGCGACCTGTTCACG
LB CACTGAGAGCTGTGAGGACCG
Corresponding nucleotide position of Shigella flexneri ipaH gene, 1312bp (Accession No. M76443.1)

Toxin produce Stx2A and

strain of E.coli stx2B
FIP AGACGAAGATGGTCAAAACGC-GCAGTTATTTTGCTGTGGA
BIP CGGGTTCGTTAATACGGCAA-CGGGCACTGATATATGTGT
F3 TCGGTGTCTGTTATTAACCA
B3 TGGAAACCGTTGTCACAC
LF TGATAGACATCAAGCCCTCGTA
Corresponding nucleotide position of Escherichia coli stx2A gene for Shiga toxin 2 A-subunit and stx2B gene for Shiga toxin
2 B-subunit, strain CB10686, 1450bp (Accession No. FN252458.1)

Rotavirus VP7
FIP TTGGTTGCTAGCTTCAATTGGATAA-CGCATATGCTAACTCTACTCAA glycoprotein
BIP TGGTGAATGGAAAGATACATTGTCA-TGTACTCTTTAAAGTAGACCGAT
F3 CCAATAACAGGATCAATGGATAC
B3 GGATCGACAGAAAATTCAACAA
LB TGTTTCTTACAAAAGGCTGGCCAAC
Corresponding nucleotide position of Human rotavirus A gene for VP7, 981bp (Accession No. AB018697.1)

2.2.3 Oligonucleotide primers Synthesis

A synthetic laboratory made DNA or RNA strand or oligonucleotide primers for each targeted
genes were prepared based on the designed primers sequence. All oligonucleotides primers
were synthesized by Africa's Genomics Company - Inqaba Biotec East Africa Ltd. The synthesis
report for all oligonucleotides is provided in Annex 1.

Page 7 of 33
2.2.4 Drinking Water Sample Collection
The drinking water samples were collected from two spots; one from the main drinking vessels
of infant at point-of consumption in the household and another from the corresponding water
source. One liter of water samples were taken in a volume of 1000ml sterile bottle with the
exclusive of any chemical additives. As per a recommendation of WHO, the collected water
samples were stored in a dark cool box at 4ºC ±1ºC and transported within sex hours from time
of collection before the next step (filtration) processed.

2.2.5 Filtration

Once the water sample (one liter) is received, filtration was carried out for each sample using
Zeta Plus 1MDS electropositive microfilter media disc with 47-mm sized, which capable of
simultaneously capturing and recovering of multiple microbes from water sample(19). The
1MDS 47-mm disc filter were placed in clean and sterile bronze filtration support disc housing
and fitted with the filter funnel for each filtration experiment. The water sample was then
poured into the filter funnel and pumped until all (one liter) samples become clear. Afterward,
the filtration unit was opened and the 1MDS filters disc removed using sterilized forceps and
placed in a sterilized petri dish, and then stored at 4°C in the dark for not more than 12 days
before undergoing elution process.

2.2.6 Elution
The elution of adsorbed pathogenic microbes from the filter disc surface was performed by
using eluting solution. For a single 1MDS disc filter to be eluted, 200ml eluting/backflush
solution were prepared by mixing (50ml 1.5% beef extract with 0.05M glycerin - autoclaved at
1210c for 15min + 50ml 0.01% Tween 80 + 50ml 0.1% Sodium polyphosphate (NaPP) + 50ml
0.001% Antifoam agent to reduce foaming effect). The pH of eluting solution was adjusted to 8
using accumet pH benchtop meter (Thermo Fischer Scientific, Canada), which could be reduce
potential bactericidal effects(19). The adsorbed filter disc were placed in clean and sterile
bronze filtration support disc housing and fitted with the filter funnel. Before the elution
pumping process begun, the eluent solution was allowed to contact the filter disc for 10
minutes. Afterward, 200ml eluent solution was poured into the filter funnel and pumped into
the sterilized filter housing in the reverse direction of sample flow until the eluent solution was
become clear. At last, the 200ml eluent was poured into four sterile 50ml falcon tube to be
centrifuged for having concentration of targeted pathogens from filtered eluate.

2.2.7 Concentration of Targeted Pathogens from the Filter eluate (Centrifuge)

The eluted effluent underwent a secondary concentration stage in a multiple centrifugation

steps before nucleic acid extraction. The subdivided eluate into four sterile 50ml falcon tubes
centrifuged at 4,100×g for 30 min. Thereafter, the tubes were removed from the centrifuge
carefully without shaking and the supernatant had been discarded ~25ml from each centrifuge
falcon tubes using a serological pipette. By pipetting up/down each tube, the aliquots of the re-
Page 8 of 33
suspended materials were transferred into two 50ml tube and a further centrifugation under
the same conditions followed. The supernatant was then carefully aspirated to 35ml and the
remaining was gently shacked and transferred into two sterile 15-ml tubes and centrifuged at
4,100×g for 30 min. Again, the supernatant was aspirated to ~12 ml from each of the 15ml
tubes to waste without disturbing the pellets. The remaining supernatant (3ml) was
transferred into one of a single 15ml tube and centrifuged at 4,100×g for 30 min. At an end, the
supernatant was carefully aspirated until approximately 300μl of the supernatant remained in
each of the 15ml tubes and stored at –20°C until DNA/RNA extraction.

2.2.8 DNA/RNA Extraction

Genomic DNA for Cryptosporidium, Shigella and Toxin produce strain of E. coli and RNA for
Rotavirus was extracted from 300μl eluate (concentrated water samples) using DaANGene
RNA/DNA Purification Kit (MDSS GmbH, Germany), according to the manufacturer’s
instructions. The RNA genome for Rotavirus was converted to cDNA using First-Strand cDNA
Synthesis protocols-E6300(20). The extracted DNA/RNA as well as the converted cDNA
samples was then stored at –20°C until amplification is done by LAMP.

2.2.9 LAMP Reactions

2.2.9.1 Reaction mixture for LAMP (Operated on ice)

The reaction mixture comprised of all the composition necessary to make new strands of
DNA/cDNA in the LAMP process. The reaction mixture composed of the following reagents; 1)
Master Mix - containing 1x Thermopol reaction buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM
Tris-HCl, 2.0 mM MgSO4, 0.1% Triton X-100; New England Biolab), 6mM MgSO4 (100mM;New
England Biolabs Inc.), 0.8M betaine (Glentham Life Sciences Ltd, Corsham, UK), 1.4mM dNTP’s
(10mM; New England Biolabs Inc.), Bst 2.0 DNA polymerase large fragment (8U; New England
Biolabs Inc), 50x LAMP fluorescent dye (New England Biolabs Inc.), Primer mixture [1.6μM FIP,
1.6μM BIP, 0.4μM LPF, 0.4μM LFB, 0.2μM F3 and 0.2μM B3; Africa's Genomics Company, Inqaba
Biotec East Africa Ltd], Nuclease free H2O (Fisher BioReagents) and 2) Template genomic
DNA/cDNA. The reaction mixture for each targeted pathogens was prepared on ice containing
box to a final volume of 25μl (see Table 2).

2.2.9.2 Optimization of LAMP assay for each pathogen

To establish best amplification reaction conditions, the concentrations, temperature and time
of reaction mixture were optimized for the LAMP assay. The LAMP assay for each targeted
pathogenic microbe was tested under different conditions of concentration, amplification
temperature and time, that is, 60 to 65°C for 1h followed by heat inactivation step at 80°C for 5
to 10min. The genomic DNAs/RNA-cDNA extracted from the positive control of each targeted
pathogens and DNA/RNA extracted from nuclease free water as a negative control were used
for the optimization of LAMP. After several optimization reactions, the LAMP reaction mixture
conditions for each targeted pathogens were found to be suitable (see Table 2). The optimum
temperature and time for best amplification result of Shigellla species, Cryptosporidium Oocyst,
Page 9 of 33
and Rotavirus were seen at 60°C for 1 hour followed by heat inactivation of 80°C for 5min
while Toxin produce strain of E.coli showed at 64°C for 1 hour and inactivated at 80°C for 5min
and no amplification result was observed in negative control.

Table 2: Reaction mixture of LAMP for each targeted pathogen

1. Prepare primer Mix Quantity (μl)
Primer Mixture Ingredient Cryptosporidium Shigella Toxin produce Rotavirus
(Per reaction) strain of E.Coli
1.6μM FIP 0.4μL 0.8μL 1.0μL 1.0μL
1.6μM BIP 0.4μL 0.8μL 1.0μL 1.0μL
0.4μM LPF - 0.4μL 1.0μL 1.0μL
0.4μM LFB 0.2μL 0.4μL - -
0.2μM F3 0.05μL 0.1μL 1.0μL 1.0μL
0.2μM B3 0.05μL 0.1μL 1.0μL 1.0μL
Total 2.5μL 2.6μL 5.0μL 5.0μL
2. Create the Reaction Mix
Reaction Mix Ingredients
(Per reaction)
1X Isothermal Buffer 2.5μL 2.5μL 2.5μL 2.5μL
6mM MgSO4 1.5μL 1.5μL 1.5μL 1.5μL
1.4mM DNTPs 3.5μL 3.5μL 3.5μL 3.5μL
0.8M Betaine 4.0μL 4.0μL 4.0μL 4.0μL
Bst 2.0 DNA polymerase 1.0μL 1.0μL 1.0μL 1.0μL
Nuclease free H2O 5.0μL 6.4μL 2.0μL 2.0μL
Total 20 μL 18.9 μL 14.5 μL 14.5μL
3. Template DNA 3μL 3μL 5.0μL -
4. Template cDNA - - - 5.0 μL
5. LAMP fluorescent dye 0.5 μL 0.5 μL 0.5 μL 0.5 μL
Overall reaction mixture 25μL 25μL 25μL 25μL

2.2.9.3 LAMP Operational procedure and reaction

The final concentration of the LAMP reaction mixture of 25μl was dispensed into each labeled
Loopamp Reaction Tube (EIKEN Chemical Co.,Ltd. Tochigi, Japan). The reaction was carried out
by inserting the reaction tubes in a Bio-RAD iCycler Thermal Cycler (Bio-Rad Laboratories, Inc.
USA). Based on optimization result of Shigella, Cryptosporidium and Rotavirus, the reaction
was run by heating at 95°C for 3 minutes and subsequently incubation at 60°C for 60 minutes
followed by inactivated at 80°C for 5min and lastly cooled at 4°C for 5 minutes to terminate the
reaction. The same procedure was undertaken for the detection of toxin produce strain of E.coli
by activating at 64°C for 60 minutes and inactivated at 80ºC for 5min.

Page 10 of 33
2.2.9.4 Detection Using Electrophoresis of the LAMP products
After completion of LAMP, amplified DNA/cDNA were detected by visualized under U.V. Trans-
illuminator (SYNGENE, Synoptics Ltd, UK), and photographed and the numbers were noted
down for all positive samples.

2.2.10 Positive and Negative Control

To determine the positive control, the live pathogens strain, which was obtained from
Ethiopian Institute of Public Health (EPHI) and Ethiopian Bio-diversity Institute (EBI) bio-
banking repositories laboratory, was diluted in 1000ml distilled water and filtration, elution,
centrifuging and DNA/RNA extraction under the same sample processing conditions followed.
A negative control was done using nuclease free water instead of the DNA template. The
positive control and a negative control were included in every LAMP reaction to ensure that
false-negative and false-positive results were eliminated.

2.2.11 Sensitivity and Specificity of LAMP

To support the analytical sensitivity of the LAMP assay, it was indicated in the previous studies
that exploring the high sensitivity of LAMP than that of PCR, signified the detection limit of
LAMP assay by tested with serially diluted DNA samples of Cryptosporidium Oocyst(21),
Shigella species(22), Toxin produce strain of E.coli(22–24) and RNA-cDNA of Rotavirus(25).

The specificity of the LAMP assays for Shigella was examined with three closely related
bacterial species: Salmonella species, Vibrio Cholera and Shiga Toxin produce E.coli. The result
demonstrated a positive for only Shigella species and negative for aforementioned bacterial
species. The specificity of Toxin produce strain of E.coli specific LAMP was performed by
testing it with three other bacterial species: Salmonella species, Vibrio Cholera and Shigella
species, which solely resulted positive for Toxin produce strain of E.coli and negative for all the
above-mentioned bacterial pathogens. The LAMP assay for Cryptosporidium Oocyst and
Rotavirus was examined with Giardia cysts and Adenovirus, respectively, which demonstrated
only positive for Cryptosporidium Oocyst and Rotavirus. Therefore, the assay established in
this study was found to detect only sequence of the targeted pathogens and no cross-reaction
with other pathogenic microbes, representing its high specificity.

2.2.12 Quality control for LAMP

For the laboratory quality assurance of lamp assay, the ICR microbial laboratory manual was
followed (26). The operating procedures were kept exactly as stated in the protocol. All LAMP
laboratory equipment was maintained in safe and working condition. Each DNA/RNA
extraction and LAMP testing performed in a secured separate room set-up to avoid
contamination and applied by knowledgable and skilled personnel’s. The sample positive result
was confirmed by comparing it with the reference of the positive control. All tests performed
was photographed and recorded directly into electronic registry format.

Page 11 of 33
2.3 Operational Definitions

Oligonucleotide: The term “oligonucleotide” or “oligo” usually refers to a synthetic laboratory-

made DNA or RNA strand(27).

Molecular methods: can be defined as those methods that target macromolecules containing
information about the identity of the microorganisms that produce them(28).

Loop-mediated isothermal amplification (LAMP): is a rapid molecular method in which

reaction performed in a single-tube under isothermal condition for the amplification of nucleic
acid (DNA or RNA) by using Bst DNA polymerase with strand displacement for the detection of
certain pathogens(29)

Optimization: means the identification of optimum concentrations, temperature and time at

which the LAMP functions to its best capacity both in terms of yield and specificity.

Primer: is a short single-stranded DNA fragment used in molecular laboratory techniques in

which pair of primers hybridizes with the sample DNA and defines the region that will be
amplified, resulting in millions and millions of copies in a very short timeframe(30).

Drinking Water Treatment: Boiling, Add bleach/chlorine, Water filter (ceramic, sand,
composite) and Solar disinfection(31)

Safe Water Storage: Water stored in plastic, clay or metal pot narrow mouth (usually diameter
of 3cm or less), have a lid or secured cover and a tap (spigot), cleaned and kept cover(32),
while its vice versa, could be taken as unsafe water storage.

Improved water source: Piped water into dwelling/yard/plot, Public tap/Standpipe,

Tubewell/borehole, Protected dug well, Protected spring, Rainwater and Bottled water;
whereas unimproved water source includes Unprotected dug well, Unprotected spring,
Surface water (river, dam, lake, pond, stream, canal, irrigation channels), Cart with small
tank/drum, Tanker-truck(31).

2.4 Ethical Consideration

Ethical approval for the study was provided by the Addis Ababa University-Ethiopian Institute
of Water Resources Ethics Review board (EIWR No. 134/08/16). All interviewed
mothers/caregivers of infants were informed about the objectives of the study and verbal
consent was obtained before data and water sample collection. Personal data such as name and
contact information about the respondent and water sample, is kept and be encrypted to
protect privacy and confidentiality. The water samples were tested and analyzed anonymously
and original paper records were stored in a locked file cabinet. The personal identifiers are
removed from study documents, and also computer-based files were stored in a password to
protect the participants’ confidentiality.

Page 12 of 33
3. Results
3.1 General Characteristics of Study Population and Households

A total of 410 households with infants were included in the study, yielded a response rate of
97.0%. The mean age (+ SD) of the study subject was 8.35 (± 2.59) months. Majority of the
study population was fall within the age group of 6-12months at 73.4%. The sex composition
comprised of 217 (52.9%) males and 193 (47.1%) females with an overall sex ratio of 1.1:1.

Among the 410 water samples collected, the highest proportion of households primarily
sourcing drinking water for their infant were from piped water public tap/standpipe (37.3%),
followed by unprotected spring (28.8%), protected spring (12.0%) and piped water in their
own yard or premises (10.2%). About 32.7% of the households collected water from
unimproved water sources. Nearly all (99.8%) of the household reportedly that they did not
practiced any form of water treatment before their infant to drink. Majority of the households
(94.9%) observed to be stored their water in unsafe manner. About 71.7% of the collected
water samples were taken from the households that were fetched at the present day prior to
the survey. Most of the households (83.7%) fetched their water from the water point found at
public space. Almost all (99.8%) of the water sample were found with residual chlorine of
0.1mg/l (see Table 3).

Page 13 of 33
Table 3: Frequency distribution of study population and households characteristics
Frequency Percentage
Characteristics (n) (%)
Age of Infant
<6months 109 26.6
6-12 months old 301 73.4
Sex of Infant
Male 217 52.9
Female 193 47.1
Primary water Source type
Piped water into dwelling 4 1.0
Piped water into yard or plot 42 10.2
Piped water, public tap/standpipe 153 37.3
Piped water kiosk or retailer 7 1.7
Protected dug well 8 2.0
Protected spring 49 12.0
Bottled water 13 3.2
Unprotected dug well 1 0.2
Unprotected spring 118 28.8
River water 2 0.5
Irrigation canal 13 3.2
Water Source Improvement
status
Improved water source 276 67.3
Unimproved water source 134 32.7
Household Point-of-use water
treatment Practices
Do not treat 409 99.8
Treat Water 1 0.2
Drinking Water Storage
Unsafe 389 94.9
Safe 21 5.1
When water is fetched?
Today 294 71.7
Yesterday 87 21.2
Days before yesterday 29 7.1
Where the water point is found?
In the dwelling 17 4.1
Private yard/plot 37 9.0
Neighbor’s yard/shared compound 13 3.2
Public Space 343 83.7
Residual chlorine
0.1mg/l 409 99.8%
0.3mg/l 1 0.2%

Page 14 of 33
3.2 LAMP Result of Targeted Pathogens in Infants Point-of-use Water Sample

Of the total 410 water samples tested using LAMP technique, 28.5% [95% CI, 24.2- 32.9] were
found positive for Cryptosporidium Oocysts, about 30.0% [95% CI, 25.6- 34.4] were positive for
Shigella species, 26.3% [95% CI, 22.1- 30.6] were positive for toxin-produce strain of E-coli and
32.2% [95% CI, 27.7- 36.7] were positive for Rotavirus. Totally, 13.2% [95% CI, 9.9- 16.4]
water samples were positive for simultaneously detected of all the above mention pathogens in
the same water sample (see figure 1).

Figure 1: Prevalence of waterborne pathogens from infant drinking water samples

Prevalence of Waterborne pathogens from Water Sample

Type of Microbial pathogens

Rotavirus
32.2%

Shigella species
30.0%

Cryptosporadium
28.5%

Toxin Produce Strain of E.Coli

26.3%

Overall pathogens existed

13.2%

0 10 20 30 40
Percentage

Figure 2. LAMP detection of Shigella species in water sample with fluorescent dye added seen with
ultraviolet light; NC: negative control; PC: positive control; sample number 9 is positive

Page 15 of 33
3.3 LAMP Result of Targeted Pathogens in infants used water sources

A total of 37 water samples taken from various types of water sources that used by the
household for their children consumption in different geographic areas has been included in
the study. These include public tap/standpipe designated from the borehole 16(43.2%),
unprotected spring 11(29.7%), protected spring 5(13.5%), reservoir designated from borehole
2(5.4%) and 1(2.7%) for each protected dug well, irrigation canal and river. About 35.1% of the
water samples were observed to be drawn from unimproved water sources. Most of the water
sources (83.8%) reported to be not regularly cleaned. The majority (97.3%) of the water
source did not treat in a regular basis and about 73.0% did not have catchment protection or
fence.

Out of the 37 water source sample tested by LAMP, 10 (27.0%) positive for Cryptosporidium
Oocyst, 12(32.4%) for Shigella species, 11(29.7%) for toxin produce stain of E.coli and 14
(37.8%) for Rotavirus. Among the tested water sources, 6 of 16 (37.5%) public tap/standpipe
designated from the borehole found to be positive for the cryptosporidium, toxin produce
strain of Ecoli and rotavirus, whereas, 7 of 16 (43.8%) were positive for shigella species. Of the
11 water samples taken from unprotected spring, 2(16.7%) were positive for cryptosporidium
and toxin produce strain of E.coli, while 2(18.2%) and 5(41.7%) positive for shigella and
rotavirus, respectively. Only one from five protected spring were positive for Cryptosporidium
and Shigella species, while two samples were positive for Toxin produce E.coli. Among the
water sample taken from the two reservoirs designated from the borehole, one sample were
positive for the cryptosporidium, shigella species and toxin produce strain of E.coli, while the
two samples were observed to be positive for rotavirus. Sample taken from protected dug well
was only positive for rotavirus (see table 4).

Table 4: Water sources samples tested by LAMP

Total Positive Result of Microbial Pathogens

water Cryptosporidium Shigella Toxin Rotavirus
Type of Water Sources sample Oocysts species produce
taken E.Coli
Piped water, public tap/standpipe 16 6 7 6 6
Reservoir designated from borehole 2 1 1 1 2
Protected dug well 1 0 0 0 1
Protected spring 5 1 1 2 0
Unprotected spring 11 2 2 2 5
River water 1 0 1 0 0
Irrigation canal 1 0 0 0 0
Overall water sources 37 10 12 11 14

Page 16 of 33
3.4 Correlation of targeted pathogens presence between Water source and point-of-
consumption

The phi coefficient correlation showed a positive significant relationship between the primary
water source and infant point-of-consumption at household’s level towards the presences of
targeted pathogens. Accordingly, the occurrence of cryptosporidium in water source sample was
statistically significant with moderate positively correlated to the presence of cryptosporidium
from drinking water at point-of-use (Phi= 0.527; p=0.000). Similarly, there was significant positive
correlation on the occurrence of shigella species between the water source and at point-of-use
(Phi= 0.524; p=0.000). The existence of toxin produce strain of E.coli in water source sample
resulted significantly correlated with water sources (Phi= 0.424; p=0.000). Rotavirus shows
significant with negligible positive association between the water source and point of use at
household level (Phi= 0.113; p=0.023).

3.5 Multivariable’s Conditional Logistic Regression Analysis of Targeted Pathogens

with selected explanatory water quality determinants

In binary logistic regression, bivariate analysis indicated that unimproved water source and
retention time of water before use were significantly associated with the occurrence of
cryptosporidium, shigella and Toxin produce strain of E.coli. While, unsafe water storage,
retention time of water before use and fetching water at public space shows significant
association with the presence of rotavirus. The existence of the overall pathogens significantly
associated with unimproved water source and retention time of water before an infant to drink.

After adjustment, in the multivariable conditional logistic regression, households with infants
who used water from unimproved sources were significantly more likely to increase odds of
the presence of cryptosporidium Oocyst (p=0.000, AOR: 4.02, 95% CI: 2.29, 7.04), Shigella
species (p=0.000, AOR: 4.21, 95% CI: 2.43, 7.29) and Toxin produce strain of E.coli (p=0.004,
AOR: 2.14, 95% CI: 1.27, 3.59). However, no statistical significant association was observed in
households using unimproved water source with the occurrence of rotavirus. The household’s
water sample tested positive for the cryptosporidium was significantly less likely among those
who fetch water days before yesterday (p=0.001, AOR: 0.03, 95% CI: 0.00, 0.26) and yesterday
(p=0.010, AOR: 0.51, 95% CI: 0.30, 0.85) with respect to those who fetch water at the present
day prior to the survey. Similarly, the presence of toxin produce strain of E.coli shows less
likely relationship with fetched water on the days before yesterday (p=0.008, AOR: 0.18, 95%
CI: 0.05, 0.64) than the present day, while rotavirus showed on those households fetched water
yesterday (p=0.002, AOR: 0.46, 95% CI: 0.28, 0.76). There is no any significant relationship
with drinking water storage status and the place where the households fetching water to the
presence of each targeted microbial pathogens in drinking water samples (see Table 5).

In the final model, the simultaneous existence of the overall (four) pathogens among water
samples shows significant association with households sourced water from unimproved which
Page 17 of 33
were approximately four times (p=0.001, AOR: 3.96, 95% CI: 1.73, 9.07) higher than those
households that used from improved water sources. The water sample taken from yesterday
fetched water of the households were less likely to present all targeted pathogens than
households fetched the present day prior to survey (p=0.009, AOR: 0.44, 95% CI: 0.23, 0.81)
(see Table 5).

Table 5: Multivariable logistic regression analysis for the presence of targeted pathogens in
water sample and determinates of water quality in Eastern Ethiopia
Adjusted odds Ratio (95% CI)
Factors Model 1 Model 2 Model 3 Model 4 Final Model
Water Source
Improvement
status
Unimproved 4.02 (2.29, 7.04)* 4.21 (2.43, 7.29)* 2.14 (1.27, 3.59)* 3.96 (1.73, 9.07)*
Improved 1 1 1 1
Drinking
Water
Storage
Unsafe 1.78 (0.68,4.68)
Safe 1
When water
is fetched
Days before 0.03 (0.00,0.26)* 0.18 (0.05,0.64)* 1.08 (0.46,2.53)
yesterday - -
Yesterday 0.51 (0.30,0.85)* 0.66 (0.39,1.11) 0.65 (0.39,1.09) 0.46 (0.28,0.76)* 0.44 (0.23,0.81)*
Today 1 1 1 1 1

Where the
water point is
found?
Public Space 1.07 (0.59,1.97)
In the dwelling/
yard 1

Factors that hadn’t p-value of <0.2 from bivariate analysis & not eligible in multivariable
Model 1: Association of factors with presence of Cryptosporidium oocest in the water
sample
Model 2: Association of factors with presence of Shigella species in water sample
Model 3: Association of factors with presence of Toxin Produce strain of E.coli in water
sample
Model 4: Association of factors with presence of Rotavirus in water sample
Final Model: Association of factors with presence of all targeted pathogens
(Cryptosporidium, Shigella, Toxin produce strain of E.coli and Rotavirus) simultaneously in
the water sample
*statistically significant at p<0.05

Page 18 of 33
Discussion

The present study drawn to detect four targeted water-borne microbial pathogens from infant
use drinking water and explores their association with the determinants of water quality.
These pathogens were a protozoan named as Cryptosporidium Oocyst; the two bacterial
etiologic agents known as Shigella species and toxin-producing strain of E.coli as well as a virus
called as Rotavirus. These pathogens are identified and reported as extremely responsible for
casing the most severe and fatal diarrhea among the infants, which contribute the high burden
of infant illness and death(6).

Due to the severity and fatality of the pathogens, it would be significant to test drinking water
by detecting these pathogens simultaneously from the infant drinking water sample. This work
is utmost important for the reason that drinking water is the basis for the reservoir and source
of transmission of etiologic agents that causing diseases. The conventional methods in testing
water quality are used in the isolation of indicator organisms such as total coliform,
thermotolerant coliform or E.coli, which inward unable to detecting specific pathogenic species
in drinking water(33,34). The existences of these microbial indicators are not a confirmation of
pathogen presence(35), meanwhile, its absence is not a guarantee of the absence of pathogens
in drinking water(28). As studies suggested, the absence of indicator organisms in water
quality analysis could not be implying the water is necessarily safe, particularly from protozoa
and viruses(36).

Molecular analytical techniques are useful tools for evaluating the microbial quality of water
(37). Among a wide range of molecular techniques, nucleic acid based amplifications methods
comprise of PCR have been developed to detect microbial pathogenic species directly in
drinking water samples(38). This method consists of concentration of the organism of interest
from the water sample, extraction of the nucleic acid from the target organism, amplification of
the genomic segments chosen and quantification of the amplified genomic segments(39).
Lately, a novel technology termed loop-mediated isothermal amplification (LAMP) has been
developed which has a high sensitivity and accuracy in replicating high amount of DNA in
isothermal conditions(29). Therefore, this study was proposed to detect presence of the
targeted pathogens in infant drinking water using loop-based isothermal amplification
technique and analyzed its association with drinking water quality determinants.

As the result indicated, almost one-third of the households providing water for the
consumption of their infants at point-of-use were positive for rotaviruses. Regardless of the
specific study subject, this finding was higher than the studies reported previously in
elsewhere(40–44). On the other hand, three-eighth of rotavirus presence in drinking water
sources found in this study was much higher than previously reported results(45–54), while
lower than the study in Ghana(55), where 48.1% of water samples tested by multiplex RT-PCR
were positive for rotaviruses. This disparity of the prevalence status might be explained by the
use of different technique for rotavirus detection having dissimilar sensitivity and specificity.

Page 19 of 33
Remarkably, our result appeared to be relatively the highest prevalent than the others detected
microbial pathogens both in water samples that was tested from children used drinking vessels
at households level and those of the corresponding water sources. This might be attributed to
the highest spreading phenomena of rotaviruses in the environment of the study area.
Rotavirus are excreted in very large quantities in the feces of infected subjects, at a rate of up to
1011 virus particles per gram(41), and it is very resistant in the environment and
physicochemical treatment process that able to stability of the virus and present in large
amount in environmental water(40). Even studies indicated as rotavirus survive well enough in
conventionally chlorine based treated drinking water to make it a possible vehicle for their
transmission(56). In addition, rotavirus has high infectivity with increased risk of transmission
in comparison with protozoa and bacteria(57).

Furthermore, our study revealed no significant association between households using drinking
water from unimproved water source and the presence of rotavirus in water sample as does
observed in other detected protozoa (cryptosporidium) and bacteria (Shigella and toxin
produce strain of E.coli) pathogens. This makes reasonably to assume that household’s access
to improved water source is not a guarantee to be always safe(58). The only significant
relationship was observed in retention time, implying household’s fetched water yesterday
prior to the survey has protective effect for the existence of rotavirus in water sample. This
might be due to the influence of temperature and water movement such as large, slow-moving
or stagnant sources on rotavirus survival and transmission(59).

Our study also detected Cryptosporidium Oocyst in almost two in every seven household’s
drinking water supplied to the infants. This may have implications for poor drinking water
quality status in the study area as WHO categorizes cryptosporidium as a reference pathogen
for the assessment of drinking water quality(51). Regardless of the population group that
consumed the water, the result of our study is much higher than previously findings of the
studies conducted in somewhere else(61,62). In the corresponding water sources, three-
eleventh of the sample were found positive for cryptosporidium Oocyst, which is higher than
the prevalence study reported in Tigray at 5%(63), and slightly higher than a 2008 study in
Addis Ababa at 21%(64), and less than the study in Dire-Dawa(65) and the 2012 study in Addis
Ababa found as all(100%) water samples were positive(66). Our result appeared to be lower
than the study conducted elsewhere, such as in Egypt(67), Shanghai(61), and Switzerland(68).
On the contrary, higher than the study conducted in four countries of Southeast Asia(69). The
authors that reviewed varies studies showed the prevalence of cryptosporidium species in
drinking water ranged from 1.4%-100%(70). Numerous reasons can cause the observed
differences. One of the reasons for this rate discrepancy might be attributed to the practices of
animal grazing that have been made in the area, and the Oocysts excretion of infected hosts in
the environment can have a chance to making the causal contamination of water sources.
Another reason can be that the detection technique might also influence the magnitude of the
result.

Page 20 of 33
Cryptosporidium enlighten as sophisticated protozoan parasite that exhibits various
characteristics which support its extended survival in environment(71). It can be transmitted
through water in the Oocyst form, and more resistant to environmental conditions and
disinfection(72,73). The infective dose of cryptosporidium is extremely low(74). Evidences
suggested as Cryptosporidium requires as few as 10–30 Oocysts per 100 liters of water for the
possible existence of an outbreak(75). In our study, it can be assumed as detected at least one
Oocyst in one liter of infant’s drinking water, which alarmingly putting infants at serious health
risk in Eastern Ethiopia. It is evident that the households whose water was drawn from
unimproved water source for their infant use had significant relationship with the presence of
Cryptosporidium Oocysts by four-fold than those households sourcing water from improved
for their infant consumption. This is obliquely in line with a study suggested as household’s use
of an improved water source has protective effect to the presence of cryptosporidium in
drinking water(62). Studies suggested that Cryptosporidium Oocystes were found more in
surface water than the other water sources(76), and can survive for months(60), indicating
chance for several moments to ingest. This certainly attributed to the fact that unimproved
water source is more exposed to contamination by human and animals wastes. Our study also
further investigated that those households fetched drinking water other than the day of our
survey had less likely significant relationship with the presence of cryptosporidium Oocyst in
drinking water. The possible reason for this observable finding might be the contribution of the
poor quality of water sources in the study area.

In terms of Shigella species detection, our results showed that over two in every seven (30%) of
the households water samples tested for Shigella species were positive. Apart from the specific
study subject in the households, this result is higher than the previous inland studies
investigation(77,78)and somewhere else (79–82), while slightly lower than a study in
Egypt(83). This indicates that the drinking water in the study area has high chance of
contamination due to poor water quality. On the other hand, Shigella species were detected
nearly one-third of the water sample tested from the corresponding water sources. This finding
is higher than the study conducted in Jijiga at 8% (78), 4.8% in ziway(84) town and 6.5% in
rural districts of Shashemene(77) in Ethiopia. Several studies in elsewhere(85–89) have also
shown lower contamination prevalence of Shigella species in drinking water sources than the
present study, while other study not recorded in the sample at all(86). The highest prevalence
either at point-of-consumption or water sources in our study might be due to using of
unimproved water source, lack of water source protection and unhygienic practices in the
study area. In addition, the testing methods differences among the studies might be able to
influence the prevalence of pathogens detected. Most of the earlier studies detected this
pathogen using the biochemical and other tests, which differ greatly with molecular technique
in sensitivity and specificity.

Furthermore, households that had collected water from unimproved water sources were
strongly associated with the presence of Shigella species by four times than those collected
from improved sources. This finding is supported by the view with other study that
Page 21 of 33
unimproved water sources are more likely exposed to fecal contamination than improved
water sources(90).

The other detected bacteria type, known as toxin produce strain of E.coli, was found positive in
over one-fourth of the drinking water sample from infant point-of-consumption in the
households. This finding appeared to be slightly higher than inland study(91), and much higher
prevalent than the studies in elsewhere(92–94), and much lower than the result from other
study in Bangladesh(95). With regard to water sources sample, one-third were contaminated
with toxin produce strain of E.coli. This finding is higher than the studies in Ziway(84) and
Modjo(96) town of Ethiopia, Uganda(85), South Africa(97), and Brazil(98–100), and nearly
similar with the study conducted in India(101), while much lower than studies in northern
Ghana(102) and southeast of the island of Puerto Rico(103). Similar reasons that provided for
other pathogens could explain for the prevalence variance among studies. Markedly, our study
used LAMP to detect pathogens has high sensitive and specific than other methods(21–24).

Our study also confirmed that households getting water from unimproved sources for infant
consumption was more likely to be presented toxin produce strain of E.coli by two fold than
improved sources. This finding consistent to the other way round with the result reported in
the previous studies, which suggested that improved water sources significantly reduce the
detection of E.coli virulence genes in stored drinking water(104). This is because getting water
from improved sources associated could be reduced environmental contamination.

A positive significant correlation was observed between water samples tested from infant
point-of-use at household level and those of the corresponding water sources towards the
presence of Cryptosporidium, Shigella and Toxin produce strain of E.coli. This could be
explained by the fact that the water sources might be subjected to feacal contamination, which
responsible for the pathogens to be existed in household’s drinking water. However, rotavirus
shows negligible positive correlation. This circumstance might be explained by the fact that this
pathogen holds additional favorable condition to be existed in drinking water at the household
level through home-based contamination.

More generally, our results showed one-eighth of the household’s infant used drinking water
were positive for the combinations of four pathogens that simultaneously isolated from same
water samples. The simultaneous presence of these pathogens in drinking water closely linked
with the status of the water source. This is implying that households collecting water from
unimproved sources for infant consumption were nearly four-times more likely to have the
existence of these pathogens as compared to those sourcing water from improved supplies.
Unimproved water sources exposed to fecal as well as other matters contamination and
consecutively could allow different pathogens to be existed in drinking water. Studies indicated
that the decline in water quality between the source and point-of-use is proportionally greater
where source water is largely uncontaminated, which are often “improved” water
sources(105). Nearly similar result with our study setting, about 35.1% of Ethiopian

Page 22 of 33
community still uses drinking water from unimproved sources(106), which should be given
due attention to intervene improved water source development and rehabilitation. In addition,
the uncommon use of any form of water treatment at household level in the study area may
also contribute the existence of these pathogens in drinking water.

Strength and limitation:

One of the strengths of this study is that we used LAMP techniques, which is the most powerful
and novel nucleic acid amplification method that could be detecting pathogenic agents from
drinking water with high sensitivity and specificity. Outstandingly, this is the first study to
simultaneously detect combinations of four waterborne pathogens from infants used drinking
water sample. Despite this, out study could provide new useful information for building
evidence based health policy and strategies, better outlook in planning for the best solutions,
and generate ideas for further research.

This study has certain limitations that include the study design that we used, which
demonstrated a snapshot of water sample test result. This showed the contamination of infants
used drinking water by the targeted pathogenic agents at one-time event occurrence in the
study area. Although one-time sampling information is very useful, it does not allow us to
capture the burden of their presence in drinking water during the infantile period. In addition,
since no studies are observed on molecular detection of pathogenic agents from infant’s
drinking water samples, comparisons between studies were difficult.

Conclusion

The detection of pathogenic microbe’s genomes from drinking water sample in our study has
significant role towards infant health. Infants demonstrated high levels of exposure to consume
contaminated drinking water by those recognized pathogens that causing the most severe and
fatal diarrhea. The high level of prevalence in the present study seems to be robustly influenced
by the technique used to detect the pathogens. With the exception of rotavirus, unimproved
water sources remained the only strong significant determinants for contamination of drinking
water by these pathogens at the point of consumption. The presence of pathogens in infants
used drinking water at household level is positively correlated with the water sources from
which it is drawn, implies that the existence of pathogens at point-of-use mainly depends on its
source of the water. Therefore, effort should be made on new development and rehabilitation
of improved water sources. Water safety protection and other sanitary measures should be
implemented to mitigate contamination from human and animal wastes. Persistence health
education must be undertaken to increase awareness of mothers or caretakers of infants on the
prevention of waterborne pathogens in drinking water aimed at keeping the water to be safe
for infants.

Page 23 of 33
Declarations

Abbreviations
AOR Adjusted Odds Ratio
cDNA Complement Deoxyribonucleic acid
CI Confidence Interval
COR Crude Odds Ratio
DNA Deoxyribonucleic acid
E.coli Escherichia coli
EIWR Ethiopian Institute of Water Resources
LAMP Loop-mediated Isothermal Amplification
NCBI National Center for Biotechnology Information
PCR Polymerase Chain Reaction
RNA Ribonucleic acid
SD Standard Deviation
SPSS Statistical package for Social Sciences

Acknowledgments

The authors would like to thank Addis Ababa University, EIWR, Ethiopian public health
association (EPHI), Ethiopian Bio-diversity Institution (EBI) for their technical and in-kind
resources assistant. Our deepest acknowledge to the overall districts health offices (Chiro,
Mieso, Amibara and Awash Fentale) for their dedication and commitment in providing
information and facilitate the survey. We also extend our sincere thank to the study
participants such as data collectors, supervisors and the respondents.

Author Contributions

S.M, A.W, D.G involved in conceptualization, data curation, format analysis, investigation,
methodology, supervision, validation, visualization, writing-review and editing. SM contributed
in writing-original draft. S.M, HS, AA contributed in project administration, resource, molecular
laboratory (LAMP) technical methodology, software and writing-review and editing.

Funding Statement
We have no received specific funding for this particular work

Availability of data and materials

We confirm that all the relevant data are fully available. The dataset are accessible by
contacting the corresponding author and provided upon a reasonable request.

Consent for publication

Not applicable

Page 24 of 33
Competing interests
We have no competing interests exist

Authors reporting experiments on human data

We confirm that all methods were carried out in accordance with relevant guidelines and
regulations along with the approval.

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