Studies on waterborne parasites from natural environment and domestic water sources in Uganda are very scarce and unpublished. Water dwelling free-living amoebae (FLA) of the genus
Free-living amoeba (FLA) of the genera
Studies worldwide have documented the common
The FLA of the genera
The risks associated with FLA infection in Uganda are not known, the reason why this study is being carried out. The associations are expected to be high because significant portions of these communities are using natural environmental water and domestic tap water sources without any definite measures for protecting water systems in QEPA and KLA from these organisms [
The study was conducted in a rural area, Queen Elizabeth Protected Area (QEPA) in western Uganda, and an urban area, Kampala (KLA) City, Uganda. The QEPA is located at 00 12S, 30 00E (latitude: 0.2000; longitude: 30.0000) and is 1,978 sq. km in size. The protected area is known for its wildlife, including Cape buffaloes (
Kampala is the capital city of Uganda lying at latitude 0°18′58′′N, longitude 32°34′55′′E, with 72.97 mi2 (189 km2) and divided into five boroughs each consisting of a concentration of slums with a rapidly growing population. Currently estimated at 1,659,600 [
This study does not require an ethical statement.
The study involved one year of field work and one year of confirmatory laboratory phase. Laboratory phase involved analysing water samples for FLA. The water sources considered were those from natural environmental and domestic tap water sources. The sampling sites were purposively selected based on their benefit, convenience, and importance to public health.
In QEPA, selection of the study area was based on certain landmarks that included the following: (1) along the Kyambura River, (2) Kazinga Channel banks, (3) Kazinga Midchannel, (4) fish landing sites (FLS), and (5) community piped tap water. In Kampala, the location of sampling sites was based on previous waterborne disease outbreak occurrences. We selected areas in central Kampala (Banda, Kisenyi, Katanga, Kasubi, Kazo, Bwaise, Lubigi, and Makerere) that were reported to have undergone disease outbreak in the last 5 years and collected samples from piped tap water and natural environmental surface water (swamp and stream).
We used nonnutritive medium (Page Amoeba Saline solution of 2.5 mM NaCl, 1 mM KH2PO4, 0.5 mM Na2HPO4, 40 mM CaCl2, and 20 mM MgSO4) seeded with 0.1 mL of a heat inactivated 48-hour culture of
All DNA was extracted from culture positive plates by chemical lysis and purification [
Amplification of 18S ribosomal DNA (18S rDNA) from
The reactions were carried out with a DreamTaq PCR kit (Thermo Scientific DreamTaq, USA). A 25
Positive gel samples were extracted and the DNA purified with QIAquick Gel Extraction Kit (Qiagen Inc., Netherlands). Partial 18S rDNA segment of the amoeba isolates was exposed to cycle sequencing with JDP1/JDP2 and CRN5/1137 as sequencing primers [
Data was entered into Excel, from which it was extracted and analysed using SPSS (IBM, USA). Variables were summarised by the use of mean and standard error of the mean (SEM). Application of univariate analysis to compare prevalence across sampling sites was executed using cross-tabulation with a
The water samples were collected during cold rainy (November, March, and July) and cool dry (January, May, and September) seasons. Overall, FLA parasite prevalence in both study sites was higher during the rainy season except for
Overall seasonal prevalence of FLA.
The prevalence and mean (SEM) of the parasites from different sources are shown in Table
The overall prevalence of the FLA.
Parasite | QEPA | KLA | ||||
---|---|---|---|---|---|---|
Freq ( | Prev (%) | Mean ± SE | Freq ( | Prev (%) | Mean ± SE | |
| 143 | 35 | 1.9 ± 0.2 | 82 | 28.3 | 2.8 ± 0.5 |
| 55 | 13.5 | 0.4 ± 0.1 | 48 | 16.6 | 1.4 ± 0.3 |
| 77 | 18.9 | 1.2 ± 0.1 | 67 | 23.1 | 1.4 ± 0.3 |
Freq: frequency. Prev: prevalence.
Prevalence of parasites across water source (natural and tap water) considered is presented in Table
Prevalence and mean across water sources.
Parasite | QEPA | KLA | ||
---|---|---|---|---|
Tap water | Environmental water | Tap water | Environmental water | |
| ||||
(+) (%) | 36 (43) | 107 (33) | 48 (28.2) | 15 (23.1) |
Mean ± SEM | 2.26 ± 0.4 | 8.92 ± 1.6 | 5.2 ± 1.2 | 2.4 ± 1.3 |
| ||||
(+) (%) | 19 (22.6) | 58 (17.9) | 38 (22.4) | 10 (15.4) |
Mean ± SEM | 1.20 ± 0.15 | 4.93 ± 0.92 | 4.4 ± 1.0 | 0.9 ± 0.16 |
| ||||
(+) (%) | 12 (14.3) | 43 (13.3) | 25 (14.7) | 9 (13.8) |
Mean ± SEM | 0.5 ± 0.15 | 1.58 ± 0.6 | 3.5 ± 1.1 | 0.5 ± 0.3 |
Following FLA sequencing, the products were blasted and compared with the GenBank results from NCBI (Figure
Isolated free-living amoeba, accession numbers, water source, and associated diseases in humans.
Accession | Source | FLA | Diseases | Characteristics |
---|---|---|---|---|
KU884884 | Tap KLA Bwaise | | Encephalitis [ | Mental status changes, hemiparesis, meningismus, and ataxia [ |
KU894799 | Tap QEPA Katunguru | | Keratitis [ | Blinding infection of the cornea [ |
KU894800 | Tap QEPA Katunguru | | Keratitis [ | Blinding infection of the cornea [ |
KU894801 | Tap QEPA Albertine | | Keratitis [ | Blinding infection of the cornea [ |
KU894802 | | | Keratitis [ | Blinding infection of the cornea [ |
KU894803 | | | Keratitis [ | Blinding infection of the cornea [ |
KU894804 | | | Keratitis and encephalitis [ | Disseminated disease, blindness, and CNS function impairment [ |
KU894805 | | | Keratitis [ | Blinding infection of the cornea [ |
KU894806 | | | Unknown | — |
KU894807 | | | Unknown | — |
KU894808 | | | Unknown | — |
KU894809 | | | Unknown | — |
KU894810 | | | Unknown | — |
KU894811 | | | Unknown | — |
KU894812 | | | Unknown | — |
KU894813 | | | Unknown | — |
KU894814 | | | Keratitis [ | Blinding infection of the cornea [ |
FLS: fish landing sites; KCB: Kazinga Channel banks; KLA: Kampala; QEPA: Queen Elizabeth Protected Area. R. Kyambura: the Kyambura River.
Estimation of evolutionary divergence between sequences.
Percent identity | |||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| | | | | | | | | | | | | | | | | | | | | | | | | | | | ||||
| |||||||||||||||||||||||||||||||
Divergence | | 53.2 | 53.6 | 53.4 | 52.8 | 53.8 | 53.8 | 51.4 | 52.1 | 56.5 | 34.5 | 36.0 | 31.5 | 52.2 | 25.6 | 53.3 | 25.4 | 53.7 | 25.2 | 25.1 | 24.8 | 24.3 | 25.0 | 25.2 | 30.5 | 29.9 | 30.5 | 30.9 | | KU884884 | |
| 239.1 | 97.3 | 95.6 | 96.0 | 94.4 | 94.4 | 53.3 | 55.4 | 54.9 | 32.9 | 34.6 | 30.7 | 55.4 | 26.4 | 52.5 | 23.9 | 91.5 | 23.8 | 23.5 | 25.6 | 24.2 | 24.6 | 24.6 | 29.3 | 28.9 | 29.9 | 31.0 | | KU894799 | ||
| 220.3 | 1.9 | 95.7 | 95.0 | 94.0 | 94.5 | 52.3 | 54.4 | 55.0 | 32.8 | 34.8 | 30.6 | 54.5 | 26.6 | 52.5 | 23.8 | 91.7 | 23.6 | 23.4 | 25.5 | 24.0 | 24.3 | 24.7 | 29.1 | 28.9 | 29.8 | 31.1 | | KU894800 | ||
| 205.1 | 3.0 | 3.7 | 94.1 | 93.4 | 95.6 | 52.8 | 55.2 | 55.0 | 32.3 | 34.6 | 30.6 | 55.0 | 26.4 | 52.5 | 24.2 | 89.8 | 23.8 | 23.5 | 25.5 | 24.0 | 24.8 | 25.2 | 29.4 | 29.0 | 30.1 | 31.1 | | KU894801 | ||
| 218.7 | 4.0 | 4.2 | 3.3 | 94.0 | 94.4 | 52.9 | 55.0 | 54.4 | 33.3 | 34.2 | 30.3 | 55.0 | 26.6 | 52.6 | 23.6 | 89.7 | 23.6 | 23.4 | 25.6 | 23.9 | 24.4 | 24.8 | 29.1 | 28.9 | 29.9 | 31.1 | | KU894802 | ||
| 212.0 | 4.8 | 6.0 | 5.3 | 4.0 | 94.0 | 52.1 | 54.0 | 55.8 | 34.2 | 35.6 | 31.8 | 54.0 | 27.8 | 53.8 | 24.7 | 88.7 | 24.3 | 24.0 | 26.4 | 25.0 | 25.0 | 25.5 | 29.8 | 29.4 | 30.5 | 31.7 | | KU894803 | ||
| 226.2 | 3.3 | 4.5 | 1.7 | 4.3 | 5.7 | 50.9 | 53.0 | 55.4 | 33.2 | 35.3 | 31.5 | 53.0 | 27.5 | 53.6 | 25.1 | 89.0 | 24.6 | 24.3 | 26.3 | 24.8 | 25.4 | 25.8 | 29.9 | 29.8 | 30.7 | 32.6 | | KU894804 | ||
| 131.0 | 145.9 | 140.9 | 142.4 | 144.7 | 144.1 | 138.8 | 79.5 | 63.2 | 35.7 | 37.0 | 32.9 | 80.7 | 27.7 | 54.6 | 28.3 | 56.2 | 27.7 | 27.7 | 25.6 | 25.6 | 25.6 | 26.4 | 30.2 | 29.9 | 30.5 | 31.0 | | KU894805 | ||
| 127.1 | 104.3 | 103.0 | 101.4 | 103.2 | 104.8 | 100.6 | 48.4 | 65.9 | 45.5 | 42.0 | 36.2 | 96.0 | 43.0 | 66.6 | 32.2 | 58.3 | 32.3 | 32.2 | 41.6 | 38.1 | 31.5 | 32.2 | 35.6 | 35.3 | 36.4 | 35.2 | | KU894806 | ||
| 133.5 | 181.9 | 177.7 | 163.7 | 179.3 | 167.1 | 158.4 | 50.0 | 38.9 | 47.4 | 53.7 | 49.1 | 67.0 | 41.5 | 66.6 | 38.9 | 58.1 | 38.1 | 38.3 | 39.5 | 39.5 | 47.9 | 48.6 | 47.1 | 47.7 | 48.2 | 48.5 | | KU894807 | ||
| 141.0 | 165.3 | 163.6 | 165.3 | 149.0 | 147.2 | 156.4 | 64.0 | 26.4 | 48.3 | 65.2 | 59.6 | 47.8 | 67.7 | 65.1 | 55.3 | 31.3 | 54.5 | 54.5 | 65.4 | 58.5 | 54.9 | 55.6 | 58.8 | 59.1 | 59.2 | 58.9 | | KU894808 | ||
| 136.8 | 169.1 | 170.9 | 163.3 | 167.7 | 156.2 | 153.4 | 53.1 | 34.2 | 31.6 | 42.4 | 83.6 | 43.2 | 69.5 | 58.3 | 68.5 | 33.3 | 68.9 | 68.9 | 68.9 | 67.5 | 73.3 | 74.2 | 92.8 | 89.8 | 86.3 | 84.6 | | KU894809 | ||
| 121.6 | 139.1 | 140.4 | 139.2 | 138.4 | 133.2 | 131.4 | 47.2 | 33.8 | 30.4 | 42.2 | 7.9 | 37.4 | 70.1 | 53.4 | 69.7 | 29.1 | 68.3 | 68.3 | 68.1 | 66.4 | 74.4 | 75.2 | 84.7 | 86.0 | 86.4 | 95.8 | | KU894810 | ||
| 127.9 | 102.9 | 102.6 | 101.4 | 101.7 | 103.4 | 98.7 | 45.6 | 6.9 | 32.9 | 21.1 | 29.4 | 29.6 | 45.2 | 68.7 | 32.8 | 58.4 | 32.5 | 32.3 | 43.6 | 36.8 | 32.3 | 33.2 | 36.5 | 36.2 | 36.9 | 36.0 | | KU894811 | ||
| 133.0 | 123.8 | 125.7 | 124.7 | 119.2 | 115.6 | 118.0 | 46.3 | 6.6 | 36.2 | 24.8 | 24.1 | 27.2 | 0.0 | 64.8 | 74.2 | 24.7 | 72.8 | 72.8 | 96.4 | 82.7 | 73.0 | 73.2 | 68.6 | 68.6 | 69.4 | 70.5 | | KU894812 | ||
| 135.7 | 150.7 | 152.3 | 147.4 | 142.3 | 135.0 | 139.5 | 56.3 | 13.2 | 39.9 | 18.8 | 30.8 | 30.4 | 7.6 | 4.6 | 49.3 | 50.6 | 47.2 | 47.2 | 61.7 | 54.2 | 48.6 | 49.4 | 51.5 | 51.7 | 52.9 | 52.9 | | KU894813 | ||
| 121.1 | 135.9 | 139.0 | 136.2 | 136.6 | 130.2 | 127.5 | 36.3 | 31.1 | 39.0 | 43.2 | 24.7 | 26.3 | 29.0 | 29.2 | 31.9 | 22.0 | 95.2 | 95.2 | 71.5 | 70.1 | 69.9 | 70.3 | 68.2 | 68.1 | 69.1 | 71.1 | | KU894814 | ||
| 206.5 | 4.5 | 4.7 | 4.6 | 6.8 | 6.8 | 5.1 | 141.5 | 103.9 | 156.8 | 149.9 | 145.3 | 122.7 | 103.1 | 114.0 | 135.8 | 124.7 | 23.2 | 23.0 | 25.0 | 23.5 | 24.2 | 24.6 | 29.0 | 28.7 | 29.7 | 29.4 | | KM189419.1_Acanthamoeba_sp. | ||
| 120.8 | 135.2 | 140.0 | 134.8 | 134.5 | 131.5 | 127.9 | 35.2 | 30.0 | 38.6 | 42.8 | 23.5 | 26.4 | 27.8 | 28.4 | 31.8 | 1.0 | 126.3 | 99.5 | 75.6 | 74.0 | 74.0 | 74.1 | 72.2 | 71.9 | 72.8 | 69.0 | | JQ271689.1_Hartmannella_vermiformis | ||
| 122.2 | 139.9 | 144.9 | 139.4 | 139.0 | 135.6 | 131.8 | 35.2 | 30.5 | 38.1 | 42.8 | 23.5 | 26.4 | 28.3 | 28.4 | 31.8 | 1.0 | 130.3 | 0.6 | 75.6 | 74.0 | 74.1 | 74.2 | 72.2 | 72.1 | 72.9 | 69.0 | | KT185625.1_Vermamoeba_vermiformis | ||
| 132.9 | 126.7 | 131.1 | 127.2 | 123.2 | 119.7 | 121.1 | 47.0 | 7.8 | 36.7 | 25.3 | 23.4 | 27.5 | 0.7 | 0.4 | 5.3 | 29.5 | 119.3 | 27.8 | 27.8 | 85.5 | 76.1 | 76.0 | 71.8 | 71.8 | 72.3 | 68.5 | | AF293895.1_Echinamoeba_exundans | ||
| 131.8 | 130.7 | 135.2 | 131.1 | 130.7 | 123.1 | 124.2 | 46.2 | 14.5 | 36.4 | 35.8 | 25.2 | 29.0 | 16.1 | 14.9 | 17.1 | 31.5 | 123.0 | 29.9 | 29.9 | 14.9 | 75.4 | 75.0 | 69.7 | 69.9 | 70.1 | 67.4 | | AJ489261.1_Echinamoeba_thermarum | ||
| 129.9 | 144.7 | 151.7 | 140.1 | 143.8 | 139.9 | 135.9 | 45.8 | 28.7 | 18.9 | 38.9 | 16.4 | 18.9 | 22.9 | 24.9 | 24.2 | 29.5 | 131.1 | 27.8 | 27.6 | 24.1 | 25.0 | 97.0 | 76.4 | 78.0 | 77.3 | 75.0 | | AY364635.1_Nuclearia_pattersoni | ||
| 132.3 | 146.0 | 153.2 | 141.3 | 145.1 | 138.8 | 137.0 | 45.0 | 27.8 | 18.9 | 38.4 | 15.6 | 18.3 | 22.4 | 24.6 | 23.9 | 28.8 | 132.1 | 27.1 | 26.9 | 23.8 | 25.2 | 2.5 | 76.9 | 78.5 | 78.0 | 75.8 | | AF484687.1_Nuclearia_simplex | ||
| 136.8 | 164.1 | 170.6 | 160.8 | 162.5 | 153.9 | 154.5 | 53.6 | 34.9 | 31.9 | 42.7 | 0.0 | 10.3 | 29.3 | 28.4 | 31.4 | 28.4 | 145.2 | 26.6 | 26.6 | 27.1 | 29.8 | 20.5 | 20.0 | 96.1 | 91.7 | 85.5 | | AF411276.1_Bodomorpha_minima | ||
| 139.2 | 176.8 | 187.5 | 172.4 | 170.2 | 163.2 | 162.6 | 52.7 | 34.8 | 31.5 | 42.6 | 1.6 | 9.9 | 29.1 | 28.3 | 30.8 | 28.1 | 155.6 | 26.6 | 26.3 | 27.0 | 29.3 | 19.1 | 18.6 | 2.3 | 93.6 | 86.7 | | EU647174.1|_Cercomonadida | ||
| 134.2 | 156.9 | 162.9 | 153.9 | 152.2 | 147.9 | 145.5 | 50.6 | 31.1 | 32.6 | 42.3 | 6.0 | 10.3 | 27.1 | 26.8 | 28.8 | 26.1 | 139.3 | 25.2 | 25.0 | 26.0 | 29.0 | 20.3 | 19.6 | 7.2 | 5.8 | 87.2 | | EU709140.1_Cercozoa_sp. | ||
| 124.5 | 135.8 | 136.0 | 134.8 | 136.4 | 131.2 | 128.2 | 50.7 | 34.8 | 32.0 | 44.7 | 8.0 | 0.2 | 31.4 | 27.9 | 32.2 | 26.8 | 119.7 | 26.7 | 26.7 | 27.9 | 28.8 | 19.2 | 18.6 | 10.5 | 10.0 | 10.5 | | AY748806.1_Cercomonas_agilis | ||
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| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Phylogenetic tree based on neighbor-joining, showing the divergence of FLA. Comparison with closely related species from the GenBank database sequences with their accession numbers (GenBank NCBI).
The confirmed
All the parasites identified in this study were matched with the reported diseases they cause in humans (Table
We investigated the presence of FLA in natural and domestic (tap) water in QEPA and KLA. The prevalence and mean (±SEM) of
The primer pair JDP1/JDP2 that was used is more specific in the amplification of
Infective trophozoites of FLA in the environment are maintained and spread by water during rainy seasons when there is a run-off of water containing human and animal faecal matter from land into the water bodies, which eventually end up at the points where communities fetch and utilise the water. Most FLA prevalence and mean intensities are higher in the rainy season than dry season [
The findings from the present study indicate that there is reasonably high contamination of both natural and domestic water systems with
Celsius
Amoebic keratitis
Central nervous system
Environment
Free-living amoeba
Fish landing site
Grams
Granulomatous amoebic encephalitis
Human immunodeficiency virus/acquired immune deficiency syndrome
Kazinga Channel bank
Kazinga Channel middle
Kampala
Liter
Milligrams
Milliliters
Millimolar
National Center for Biotechnology Information
Number
Protected area
Primary amoebic meningoencephalitis
Picogram
Picomoles
Prevalence
Queen Elizabeth Protected Area
Revolutions per minute
Specific gravity
United Nations Educational, Scientific and Cultural Organization
Unidentified amoeba
Volts
Microseconds.
There are no competing interests.