Methanol Extract of Three Medicinal Plants from Samburu in Northern Kenya Show Significant Antimycobacterial, Antibacterial and Antifungal Properties
We determined the antimycobacterial, antibacterial, and antifungal potential of medicinal plants used by the Samburu Community of Northern Kenya, following an ethnobotanical survey. Using BACTEC MGIT 960 system, we assessed plant extract effects on four mycobacterial strains, i.e., Mycobacterium tuberculosis, M. Kansasii, M. fortuitum, and M. smegmatis. For Candida albicans, Salmonella typhi, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia, we determined zones of inhibition, Minimum Inhibitory Concentrations (MICs) and minimum bactericidal/fungicidal concentrations (MBCs/MFCs) using standard procedures. Preliminary phytochemistry on the extracts was also carried out using standard procedures. The extracts from Scadoxus multiflorus and Acacia nilotica gave strong antimycobacterial activity (zero GUs) against slow growing mycobacteria strains in all the concentrations tested. Scadoxus multiflorus was also active against M. tuberculosis. Boscia angustifolia was active against M. tuberculosis (183 GUs). Acacia nilotica showed strong antimicrobial activity against E. coli (with of MIC 4.69 mg mL-1 and MBC of 18.75 mg mL-1), P. aeruginosa (with both MIC and MBC of 18.75 mg mL-1), K. pneumoniae, and C. albicans (with MIC of 9.38 mg mL-1 and MBC of 18.75 mg mL-1). Thylachium africanum showed good antimicrobial activity against S. aureus (with MIC of 18.75 mg mL-1 and MBC of 37.5 mg mL-1) and P. aeruginosa (with both MIC and MBC of 4.69 mg mL-1). Preliminary phytochemistry identified six phytochemicals to which tannins was common to all plant extracts. The data suggests that methanolic extracts of at least three plant species could be a rich source of antimicrobial agents. These results provide an indication of merit in their ethnomedicine use by the local communities.
to cite this article:
R.M. Mariita, C.K.P.O. Ogol, N.O. Oguge and P.O. Okemo, 2011. Methanol Extract of Three Medicinal Plants from Samburu in Northern Kenya Show Significant Antimycobacterial, Antibacterial and Antifungal Properties. Research Journal of Medicinal Plants, 5: 54-64.
Plants form an integral part of life in many indigenous African communities
as a readily and cheaply available alternative to allopathic medicines (Wagate
et al., 2010). They have been used since prehistoric times to alleviate
and treat diseases (Potterat and Hamburger, 2008). In
Africa, traditional medicine is of great value and more than 70% of the people
refer to traditional healers concerning health issues (Tijjani
et al., 2009). Traditionally, the Samburu community of Northern Kenya
utilizes plants for both food and therapeutic purposes. It is estimated that
about 85% of the Samburu people use medicinal plants for primary medicare (Omwenga
et al., 2009).
With the emergence of new diseases and drug resistance to infections such as
HIV/AIDS, malaria, tuberculosis, diarrheal diseases and skin problems; traditional
medicine should be given more attention in modern research and development (Asres
et al., 2001; Jeruto et al., 2008;
Ani et al., 2009). Multiple drug resistance in
human pathogenic microorganisms has developed due to indiscriminate use of commercial
antimicrobial drugs commonly used in the treatment of infections (Aliero
et al., 2008). In addition to this problem antibiotics are sometimes
associated with adverse effects on host including hypersensitivity, immune suppression
and allergic reactions (Nebedum et al., 2009).
Among such infections is TB which is a deadly infectious disease that annually
kills about 3 million people worldwide (Camacho-Corona et
al., 2008). TB is now highly associated infection of persons suffering
from Human Immunodeficiency Virus (HIV). There is also a major therapeutic problem
due to the worldwide emergence of Escherichia coli, Klebsiella pneumoniae,
Haemophilus and many other β-lactamase producers (Khan
et al., 2009).
A number of studies have been conducted in different countries in the last
few years to prove that plant has antimicrobial activity (Khanna
and Kannabiran, 2008). Because of unmatched availability of chemical diversity,
natural products, either as pure compounds or as standardized plant extracts,
provide unlimited opportunities for new drug leads (Parekh
and Chanda, 2007). Now with 78% of the new chemical entities being natural
or natural product-derived molecules, there has been a promising alternative
treatment of infectious diseases using medicinal plants (Lokhande
et al., 2007).
Traditionally, herbalists use plant extracts to treat ailments but with no
knowledge of scientific base of their activities (Andy et
al., 2008). The Samburu pastoralists are among the few communities in
Kenya that have retained immense knowledge on ethnobotany. This knowledge is
however dwindling rapidly due to changes towards a less traditional lifestyle,
overgrazing and overexploitation of plant resources. In an earlier survey (Omwenga
et al., 2009), a number of plants were identified as important to
the Samburu community for medicinal purposes. Present study thus aimed at assessing
the effect of plants extracts from selected candidate species on common
pathogens. Specifically, we investigated antimycobacterial (antituberculosis),
antibacterial and antifungal activities of methanol crude extracts from eight
plant species. We further undertook a preliminary phytochemical assessment to
provide clues of active secondary compounds in the plants.
MATERIALS AND METHODS
Study site: The ethnobotanical survey was undertaken among five community
groups inhabiting eastern Samburu District in northern Kenya between June 2008
and July 2009 (Fig. 1). Samples were collected between Ewaso
Nyiro River and the Mathews Range from five communities namely Lodungokwe, Namunyak,
Ngilai West, Ngutuk Ongiron, and Nkaroni in July 2009 with the help of the volunteers
from different countries.
Plant material: An ethnobotanical survey was carried out between 2008
and 2009 through the use of questionnaires, in-depth interviews and market visits
(Omwenga et al., 2009). The herbalists were identified
with the help of the local administration. Information gathered from the survey
included plant vernacular names, the parts used and the diseases treated. The
plants were identified and taxonomically grouped at the Department of Pharmacy
and Complimentary Alternative Medicine, Kenyatta University, Nairobi, Kenya;
where voucher specimens were also deposited.
|| Map of the study area showing Samburu community groups studied
From this initial study, we selected eight species (Appendix 1) for extraction
Preparation of plant extracts: The plant samples collected were chopped
into small pieces, shade dried and grounded using hammer type milling machine
(Meecan, CM/L-1364548, India) at the Department of Pharmacy/CAM, Kenyatta University,
Nairobi, Kenya. The powdered material was transferred into and extracted in
the soxhlet extractor using methanol for 72 h (Aiyelaagbe
and Osamudiamen, 2009). The extracts were filtered through a Whatmann filter
paper No. 42 (125 mm) and concentrated using a rotary evaporator (Laborota 4000,
SN 090816862, Germany) with the water bath set at 40°C (Edeoga et al., 2005), then dried in a dessicator over anhydrous CuSO4. The powdered
residue were transferred into vials and stored at 4°C in airtight vials
Test microorganisms: The four species of mycobacteria used for the assays were obtained from the Center for Respiratory Diseases Research (CRDR), Kenya medical Research Institute (Kemri), Nairobi, Kenya. These included Mycobacterium tuberculosis, M. kansasii, M. smegmatis and M. fortuitum. Salmonella typhi (clinical isolate), Klebsiella pneumoniae (clinical isolate), Pseudomonas aeruginosa (ATCC 25852), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 20591) and Candida albicans (ATCC EK138), a yeast like fungi, were obtained from Kenyatta National Hospital in Nairobi, Kenya and used in the antibacterial and antifungal activity tests, respectively.
Growth media: Mycobacteria strains were inoculated in parallel solid medium (Lowenstein Jensen) and the liquid (mycobacterial growth indicator tube: MGIT 960) media. Bacteria were grown in Mueller Hinton agar (Oxoid) and C. albicans in Potato Dextrose Agar (PDA).
Antimycobacterial susceptibility test using BACTEC MGIT
960 system: This was used in the antimycobacterial activity assessment of
the plant extracts. The extracts were dissolved in 0.01% DMSO to final concentrations
of 0.5, 1.0 and 2.0 mg mL-1. A stock solution of 2.0 mg mL-1
of isoniazid was used as the positive and 0.01% DMSO as the negative control,
respectively. Into the 7 mL BBL MGIT tubes,
0.8 mL of the mixture of growth OADC (containing Oleic acid, Bovine albumen,
Dextrose and Catalase) supplement (added to provide essential substances for
rapid growth of mycobacteria) and BBL MGIT
PANTA (a mixture of antimicrobial agents) were added. Then 1 mL of the extract
was added into the BBL MGIT tubes containing
the supplement to attain appropriate concentrations of 0.5, 1.0 and 2 mg mL-1.
Mycobacterium suspension (adjusted to 0.5 McFarland standard) was introduced
into the BBL MGIT tubes. The strains included
M. tuberculosis (Mtb), M. kansasii (Mk), M. fortuitum (Mf)
and M. smegmatis (Ms). The BACTEC MGIT 960 system was
loaded using manufactures instructions and incubated at 37°C. Culture
vials which remained negative for a minimum of 42 days (maximum 56 days) were
removed and recorded as negative, while growth units (GUs) for the positive
ones were recorded appropriately (Becton and Company, 2007).
The same was done for the controls. Results were provided as positive/negative
and numerical Growth Units (GUs) using a non-radiometric evaluation technique
(Becton and Company, 2007).
Evaluation of antibacterial and antifungal activity: The antibacterial
and antifungal activities of extracts from the 8 plant species were assayed
in vitro by agar Disc Diffusion (DD) method (Parekh
and Chanda, 2007). Filter paper discs (6 mm) were impregnated with the plant
extracts. Mueller Hinton agar and Potato Dextrose Agar (PDA) were prepared using
manufactures instructions for purposes of culturing the bacteria and fungi
respectively. Normal saline solution was used to dilute a 24 h culture of the
bacterial type culture or clinical isolate to attain a 0.5 McFarland standard.
Spread plate method was used to culture 100 μL of the microbial suspension
that was introduced into the Petri dishes (Meite et al.,
2009). Eighteen dry sterile discs (6 mm diameter) were soaked in the plant
extract (made by dissolving 300 mg of the extracts in 1000 μL of methanol)
air dried and placed on the spread plates at reasonable distances. Discs impregnated
with methanol and airs dried were used as negative controls and various standard
conventional antibiotics (Amoxicillin (Hangzhou Ruijian Chemical Co., Ltd.,
batch 490805241); Ciprofloxacin (Chengdu Ware Yuanheng Pharmaceutical Co., Ltd.,
batch 20070907); Fluconazole (Pfizer Ltd., UK batch 30) as positive controls.
The plates were then incubated at 35°C for 24 h. This was replicated three
times for each pathogen.
Candida albicans was cultured by taking 100 μL from the broth and spreading on PDA. The culture was incubated at 25°C for 72 h. The cork boarer was used to pick a section of the young mycelium which was placed at the centre of the PDA plate and the dry discs which were impregnated with 100 μL of the plant extracts placed at a distance around the inoculum mycelium. The inoculum was incubated at 25°C for 72 h. Fluconazole and dry discs treated with methanol were also used as positive and negative controls, respectively. All tests were performed in triplicate. Microbial growth inhibition was determined by measuring the zones of inhibition using a transparent ruler.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal
Concentration (MBC/MFC): The Minimum Inhibitory Concentration (MIC) which
is the least amount of antimicrobial agent that will inhibit visible growth
of an organism after an overnight incubation was determined using the microtitre
dilution broth method in 96-well micro plates. This was done only where the
plant extract showed strong antibacterial activity by the disk diffusion method
(≥9-15 mm) (Rani and Khullar, 2004). The wells were
filled with 50 μL of the Nutrient broth for bacterial strains and Potato
dextrose broth for C. albicans. The extract was then prepared by taking
300 mg of the plant extract and mixing it with 1000 μL of DMF (0.01% Dimethyl
formanide) for complete dissolution of the extract. Then 50 μL of the plant
extract was dispensed into the first well before serial dilutions were done
by transferring 50 μL of nutrient or potato dextrose broth containing the
extract from the first well to the second well, and from the second well to
the third well through the fourth well. Fifty microlitres of the test isolate
was then dispensed into each well. One well (without extract or drug) was used
as negative control of the growth of the microorganisms in the medium whereas
another well with 50 μL of the antibiotic (Amoxicillin/Ciprofloxacin/fluconazole)
was used as positive control. Incubation was done at 37°C for 24 h. The
MIC values were determined as the lowest concentrations of the extract capable
of inhibiting microbial growth.
For the determination of MBC/MFC, wells where there was no growth were subcultured on nutrient agar and PDA. The lowest concentration of the plant extracts that did not yield any colony on the solid medium (Nutrient or PDA agar) after sub culturing and incubating for 24 h for bacterial strains and 72 h for C. albicans was taken as the MFC/MBC. All tests were performed in triplicates.
Test for alkaloids (Wagners method): Alkaloids presence was determined
by dissolving and filtering 200 mg plant extract in 10 mL methanol followed
by filtration using Whatmann filter paper No. 42 (125 mm) filters. One thousand
microlitres (1 mL) of the filtrate was then mixed with 6 drops of Wagners
reagent (Obadoni and Ochuko, 2001). Creamish, brownish-red
or orange precipitate indicated the presence of alkaloids. A low (+) reaction
was recorded if the addition of the reagent produced a faint turbidity; a moderate
(++) reaction was recorded if alight opalescence precipitate was observed and
a high (+++) reaction was recorded if a heavy yellowish-white precipitate was
Test for cardiac glycosides (Keller-Killani test): Five milliliter of
each extracts were treated with 2 mL of glacial acetic acid containing one drop
of ferric chloride solution. This was underlayed with 1 mL of concentrated sulphuric
acid. A brown ring at the interface indicated a deoxysugar characteristic of
cardenolides. A (+) reaction was recorded when a faint green-blue color was
observed (indicating low concentrations of detectable cardiac glycosides); a
(++) reaction was recorded when a medium green-blue colour was observed (indicating
moderate concentrations of detectable cardiac glycosides) and a (+++) reaction
was recorded when a deep green-blue colour was observed (indicating high concentrations
of detectable cardiac glycosides) (Aiyelaagbe and Osamudiamen, 2009).
Test for flavonoids: Five milliliters of dilute ammonia solution were added to a portion of the aqueous filtrate of each plant extract followed by addition of concentrated H2SO4. A yellow coloration observed in each extract indicated the presence of flavonoids. The yellow colouration disappeared on standing. A (+) reaction was reported in pale yellow colour; (++) in moderate yellow and (+++) in strong yellow coloration, indicating low, moderate or high concentration of flavonoids respectively in the plant extract (Edeoga et al., 2005).
Test for saponins: To 0.5 mg of extract was added 5 mL of distilled water in a test tube. The solution was shaken vigourously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigourously, after which it was observed for the formation of an emulsion (Aiyelaagbe and Osamudiamen, 2009). A (+) sign was recorded when the froth reached a height of 50 mm; a (++) sign with the height of 0.6-1 cm and a (+++) sign with a height of more than 100 mm to indicate low, moderate or high concentration of saponins, respectively in the plant extract.
Test for tannins: About 0.5 mg of the extract was boiled in 10 mL of water in a test tube and then filtered. A few drops of 0.1% ferric chloride were added and observed for brownish green or a blue-black colouration (Edeoga et al., 2005). A (+) reaction was recorded when a slight precipitate was observed; a (++) reaction was recorded when a medium precipitate was observed and a (+++) reaction was recorded when a heavy precipitate was observed. The reactions were used to indicate the presence of different concentrations of detectable tannins, with (+) representing low, (++), moderate and (+++) high levels of tannins.
Test for terpenoids (Salkowski test): To 0.5 mg each of the extract was added 2 mL of chloroform. Concentrated H2SO4 (3 mL) was carefully added to form a layer. A reddish brown colouration at the interface indicated the presence of terpenoids (Aiyelaagbe and Osamudiamen, 2009; Edeoga et al., 2005). A (+) reaction was recorded when a faint reddish brown coloration was observed; a (++) reaction was recorded when a medium reddish brown coloration was observed and a (+++) reaction was recorded when a deep reddish brown coloration was observed.
At the highest concentration of 2.0 mg mL-1, all plant extracts showed high activity (with Zero GUs) against mycobacteria strains tested (Table 1), except for M. smegmatis which had 17 GUs against C. africana. Acacia nilotica and S. multiflorus extracts were effective against fast growing mycobacteria at all concentrations. The S. multiflorus extract was also active against M. tuberculosis at 1.0 mg mL-1 concentrations. At 0.5 mg mL-1, B. angustifolia gave appreciable inhibition against M. kansasii (501 GUs) and M. tuberculosis (183 GUs) compared to the negative control (10597 and 18683 GUs, respectively). Grewia simi and C. africana showed measurable effects at 1.0 or 0.5 mg mL-1.
||Antimycobacterial activity (GUs) of eight plant species identified
to have medicinal properties by Samburu herbalists in Northern Kenya. The
test used was BACTEC MGIT 960 system
|Gus: Numerical growth units, Mk: Mycobacteria kansasii,
Mtb: M. tuberculosis, Mf: M. fortuitum, Ms: M. smegmatis,0:
indicates complete inhibition, ND: Not done, Positive control: Isoniazid,
Negative control: Dimethyl sulphoxide. *Note: The higher the growth index,
the less inhibitory the extract is to mycobacteria (compared to negative
|| Antibacterial activity of five plant species identified to
have medicinal properties by Samburu herbalists in northern Kenya
|1: S. typhi, 2: S. aureus, 3: E. coli, 4:
P. aeruginosa, 5: K. pneumoniae, 6: C. albicans. Positive
controls: Fluconazole for C. albicans, Zeftazidime for S. typhi,
Ciprofloxacin for K. pneumoniae and Amoxicillin for S. aureus,
E. coli and P. aeruginosa. Values are means of triplicates
All the extracts showed varying degrees of antibacterial and antifungal activity
against the test organisms (Table 2) with some plant extracts
showing strong antimicrobial activity with zones of inhibition of between 9.00
and 12.00 mm. Acacia nilotica extract showed strong antimicrobial activity
against E. coli, P. aeruginosa, K. pneumoniae and C. albicans
with inhibition zones of between 9.00 and 12.0 mm. Thylachium africanum
showed strong antibacterial activity against S. aureus and P. aeruginosa
(zones of inhibition of 10.66 and 10.00 mm, respectively). The extracts of B.
angustifolia and S. multiflorus gave strong antibacterial activity
only against S. aureus (zone of inhibition of 9.00 mm) and S. typhi
(zone of inhibition of 9.00 mm), respectively, but G. simi gave weak
antimicrobial activity (zones of inhibition of between 6.00-8.00 mm) against
all the test microorganisms.
||Minimum inhibitory concentrations and minimum bactericidal/fungicidal
concentrations (mg mL-1) produced by the medicinal plants against
various bacterial test cultures
|ND: Not done
||Preliminary phytochemical screening of eight plant species
identified to have medicinal properties by Samburu herbalists in Northern
|+++: Present in high concentration, ++: Moderately present,
+: Trace, -: Absent. Four medicinal plant species (C. quadrangularis,
A. nilotica, A. etbaica and S. multiflorus) exhibited
presence of all the six phytochemicals with moderate to high concentrations
being recorded in A. nilotica and S. multiflorus
The A. nilotica extract showed strong MICs (4.69 mg mL-1)
and MBC of 18. mg mL-1 for E. coli, MIC and MBC of 18.75 mg
mL-1 for P. aeruginosa and K. pneumoniae respectively
(Table 3). Thylachium africanum was most active against
P. aeruginosa with an MIC of 4.69 mg mL-1 and an MBC of similar
concentration. Boscia angustifolia and S. multiflorus were the
least active extracts against S. aureus and S. typhi with MICs
of 37.5 mg mL-1 and MBCs of 75 mg mL-1, respectively.
Preliminary phytochemistry indicated that the extracted samples showed presence of most six phytochemicals tested for (Table 4), including alkaloids, cardiac glycosides, flavonoids, saponins, tannins and terpenoids. Tannins were present in extracts of all the eight while terpenoids in only four plant species.
The activity of S. multiflorus and A. nilotica extracts against mycobacterial strains showed that the plants contain pharmacologically active substances. The results were comparable to those of the standard drug (Isoniazid) in BACTEC MGIT 960 system. The two extracts appeared particularly active against M. smegmatis and M. fortuitum where they were potent at 0.5 mg mL-1. Scadoxus multiflorus was also active against M. tuberculosis at 0.5 mg mL-1. Other plant extracts gave varying results, with B. angustifolia giving moderate activity.
General antibacterial and antifungal results were also notable (Table 2), with A. nilotica extracts showing strong antimicrobial activity against E. coli (inhibition zone of 12.00 mm), P. aeruginosa (inhibition zone of 11.66 mm), K. pneumoniae (inhibition zone of 9.00 mm) and C. albicans (inhibition zone of 10.33 mm). Minimum inhibitory concentrations and minimum bactericidal/fungicidal concentrations (mg mL-1) produced by the medicinal plants against various bacterial test cultures showed strong antimicrobial activity (Table 3), with A. nilotica extracts showing strong antimicrobial activity against E. coli (MIC of 4.69 mg mL-1 and MBC of 18.75 mg mL-1), P. aeruginosa (both MIC and MBC of 18.75 mg mL-1), K. pneumoniae (both MIC and MBC of 18.75 mg mL-1) and C. albicans (MIC of 9.38 mg mL-1 and MBC of 18.75 mg mL-1).
A zone of inhibition ≥ 9-15 mm is an indication of strong antimicrobial
activity (Rani and Khullar, 2004). These findings are
in agreement with the findings of Tijjani et al.
(2009), whereas they contradict those they reported as being hardly susceptible
to plant extracts whose doses were as low as 200 mg mL-1. The activity
of A. nilotica is in agreement with the findings of Khan
et al. (2009) where it was found to give the most potent antimicrobial
extract against K. pneumoniae, C. albicans, P. aeruginosa and E.
coli, but contrasts with their findings that it is active against S.
aureus and S. typhimurium. From a study carried out by Haj
Ali and Yagoub (2007), the inhibitory effects of the A. nilotica
fruit extracts on Staphylococcus aureus, Escherichia coli, Proteus
vulgaris, Klebsiella pneumoniae and Pseudomonas aeruginosa
were compared with those of selected antibiotics. The ethanol extract of A.
nilotica fruit was either equally or more effective than the test antibiotics,
which contrasts our current study.
The eight species used as medicinal plants by Samburu communities showed presence
of four to six phytochemicals. Present study is in concurrence with others (Aliero
et al., 2008) that have shown S. multiflorus bulbs to contain
alkaloids, flavonoids, tannins, saponin and cardiac glycosides. All the six
phytochemicals identified are known to show medicinal activity as well as exhibiting
physiological effects (Edeoga et al., 2005). For
instance, flavonoids have antidiarrhoeal effects (Meite
et al., 2009), while plants rich in saponins have immune boosting
and anti-inflammatory properties (Aliero et al.,
2008; Meite et al., 2009). Tannins have antibacterial
potential due to their ability to react with proteins to form stable water soluble
compounds that kill bacteria by directly damaging their cell membranes (Aliero
et al., 2008). The MspA in M. fortuitum and M. smegmatis
which is absent in slow-growers could be the reason for low inhibitory activity
of the A. nilotica and S. multiflorus extract against the fast growers
(Sharbati-Tehrani et al., 2005).
The high microbial effects exhibited by S. multiflorus and A. nilotica may thus be attributed to the high concentration of phytochemicals detected in their extracts, particularly alkaloids and flavonoids. The presence of these phytochemical components in the two plant species is an indication that they may have medicinal properties as used by the Samburu herbalists.
The results of the investigation revealed that most of the medicinal plants contain pharmacologically active substances with antimycobacterial, antibacterial and antifungal properties. It also points out that there is a possibility of getting effective compounds from natural sources, which can be of value in the fight against tuberculosis and other infectious diseases. The study also provides support for the use of these plants in the management of infectious diseases in the Samburu community and elsewhere.
The authors acknowledge the funding of the project by Earthwatch Institute and the volunteers who participated in the ethnobotanical survey, collection and processing of samples and preliminary analysis. Many thanks to the Catholic Hospital Wamba, Samburu, from whose Lab. preliminary analysis of the samples was done, and the Kenya Medical Research Institutes (Kemri) Centre for Respiratory Diseases Research (CRDR) for allowing Mariita Richard to work from their Level III TB lab. Authors also thank the plant taxonomist, Mr. Karimi Lucas of Department of Pharmacy, Kenyatta University, Kenya, for identifying the plant materials.
||Selected Medicinal plants used by the Samburu Community of
Kenya to treat mycobacterial, selected bacterial and fungal diseases
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