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e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 15, Issue 5 Ver. IV (May. 2016), PP 73-77
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Efficacy of Some Anti-Malaria Drugs and Antibiotics on
Plasmodium and Salmonella Species Isolated From Patients
Attending A University Health Center in Nigeria
Nwosu, A.O.,*1, Oraekeyi, N.P., 1 Abdulrazaq, M.1, Damshit, J.D.,
1
Barki, J.L.., 1 Ogbonna, C.I.C., 2 And Mawak, J.D.2
1
Health services unit University of Jos, Plateau State Nigeria
Department of Plant Science and Technology, Faculty of Natural Sciences Unijos Plateau state Nigeria.
3
Department of Microbiology, Faculty of Natural Sciences, University of Jos, Nigeria
2
Abstract
Background: Malaria is the highest killer of people in Africa, and Nigeria in particular. It kills more people
than the deadly human immunodeficiency virus, (HIV). In fever investigation, two important diagnoses to be
ruled out are typhoid and malaria. An association between malaria and typhoid fever was established and
named “typho-malaria fever”.
Method: Blood and stool specimens were collected from a total of 200 male and female patients aged 5-60
years attending University of Jos Health centre. Apparently healthy individuals (controls) and patients with
symptoms of fever were examined for malaria parasitaemia and salmonella species by microscopy and
stool/blood culture.
P-alaxin, Artemether/Lumefantrine, Artesunate/Amodiaquine, and ciprofloxacin,
Cefuroxime &Azithromycin were the anti-malaria and anti-biotic drugs used respectively. The microscopic film
for positive malaria parasite cases and the sensitivity of the Salmonella isolates were recorded.
Results: A total of 90 out of 200 (45.0%) specimen were positive for the 1st malaria test (pre-administered
results) and 39 (19.5%) were positive for 2nd malaria test (post-administered results). While 12 out of 200
(6.0%) subjects‟ yielded Salmonella species. The data obtained which were subjected to statistical analysis
using ANOVA at (p<0.05) and chi square test.
Conclusion: Densely populated malaria parasite mean showed anti-malaria drug resistance in vivo, while
cultural isolated Salmonella organism showed precise drug sensitivity invitro. In other words, heavy & multiple
re-infection of malaria parasite exhibited some degree of resistance to the anti-malarial drugs while salmonella
in vitro culture results represented sensitivity to the anti-typhoid drugs used.
Keywords: Malaria, Typhoid fever, anti-malaria, antibiotics, efficacy.
I.
Introduction
Malaria and typhoid fever are among the most endemic diseases in Africa. In the investigation and
diagnosis of fever in Nigeria, two important diagnoses to be ruled out are malaria and typhoid. Both diseases
have been associated with poverty and under development with significant death rates [1]. An association
between malaria and typhoid fever was described in the medical literature in the United States Army [2].
Malaria is one of the most serious health problems facing the world today. The World Health
Organization estimates that over 300 million new cases and approximately 2-3 million deaths of malaria occur
each year. Malaria is endemic in tropical Africa, with an estimated 90% of the total malaria incidence and
deaths occurring in the continent, particularly among pregnant women and children.
Malaria is a mosquito –borne infectious disease caused by a eukaryotic protist of the genus
plasmodium. There are currently six species of genus Plasmodium known to infect humans; Plasmodium
falciparum, P.vivax, P. ovale, P. curtisi, P. ovale walker, P. malariae and P. knowlesi ([3][4][5]). Plasmodium
falciparum is the species responsible for most of the mortality and morbidity associated with the disease.
Malaria and typhoid fever often present with mimicking symptoms especially in their early stages. These
include; fever, headache, vomiting, stomach-ache and body pain. The situation often presents a diagnostic
confusion [6]. It is sometimes difficult to differentiate clinically, the presentation of typhoid fever from that of
malaria without laboratory support and diagnosis. However, differentiation can be achieved using cultural
laboratory diagnostic tests [7]. According to [8] in a study publication on epidemiological studies of malaria and
typhoid infections in Abeokuta, Ogun State Nigeria, the prevalence of typhoid infection was 17.4% but did not
differ between sexes although significantly higher among older children (P=0.0001). Their observation from the
longitudinal study carried out between October 2000 and September 2001 in Abeokuta, Ogun State Nigeria
showed that out of the 3997 feverish cases recorded, 82.4%and 32.8% were respectively due to malaria and
typhoid.
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Efficacy of Some Anti-Malaria Drugs and Antibiotics on Plasmodium and Salmonella Species..
Worldwide drug resistance constitutes an impediment to the control of malaria [9]. This has led to the
need for constant surveillance and monitoring for changes in the sensitivity of malaria parasites to different
malaria drugs [10]. The effect of anti-malaria drug is generally characterized by the inhibition of parasite growth
and consequently the parasite is multiplied. As a result of widespread resistance, malaria therapy now consist
combination of two or more drugs that attack different biochemical processes in plasmodium [11]. Recently, the
federal ministry of Health Nigeria has implemented the new treatment policy on malaria which includes the use
of Artemisinin-based combination therapy (ACT) as the new first line drug for the treatment of uncomplicated
malaria [12]. New discovery could help turn antibiotic into anti malaria drugs. This will go a long way in
alleviating the problems faced with drug resistance which is in line with Melbourne researchers making progress
towards new anti-malaria drugs. This was after revealing how an antibiotic called emetine blocks the molecular
machinery that produces the protein required for malaria parasite survival [13]. This study was carried out to
determine the efficacy of some common malaria drug and antibiotics on patients in terms of drug sensitivity.
More studies have proved that high prevalence of malaria is an established fact. Furthermore, within the last
decade an unusually high number of illnesses have been diagnosed as malaria coexisting with typhoid fever
[4].This established fact of co-existence of malaria and typhoid has led to this present study in search of one
drug that will eliminate, if not eradicate these co-existing organism.
Typhoid and paratyphoid fever are caused by Salmonella species which are water borne disease that
causes infection of the intestinal tract and blood streams, [14].
An estimated 17 million cases of typhoid were reported worldwide each year, resulting in 0.6 million deaths
[15]. The main route of transmission is by person-to-person spread, either in hospitals or in community. The
incubation period of enteric fever bacilli is usually 10 – 20 days, with diarrhea, starting 3 – 4 days after onset of
fever and lasting an average of 6 days. ([16],[17]). Asymptomatic persons may also harbor Salmonella
unknowingly. Both remains potentially infectious for weeks or months some become infectious and some
becomes life-long excreters. Although typhoid and malaria are caused by two very different organisms: – gram
negative bacilli (Salmonellae) and a protozoan (Plasmodium) .They are transmitted via different mechanisms.
This two important diseases share rather similar symptomatology. Malaria and typhoid fever often present with
mimicking symptoms especially in the early stages of typhoid fever. The similar symptoms include; fever,
headache, vomiting, stomach ache and body pain. The situation often presents a diagnostic confusion. [4].
However, it is sometimes very difficult to differentiate clinically, the presentation of typhoid fever from that of
malaria without laboratory support and diagnosis.
II.
Materials And Method
Study Area/Location
The study was conducted in the University of Jos Clinic (UHS) located in Jos North Local Government
Area of Plateau State, Nigeria. The Clinic serves the general medical needs of the entire University Staff,
Students, and Staff dependants, patients living in and around the University environment.
Study population:
A Total of 200 patients (male & female) comprising of in- patients presenting at the clinic with signs
and symptoms suggestive of typhoid fever or malaria symptoms and 50 apparently healthy individuals visiting
the Clinic for other health/ related purposes were recruited for the study. They were aged 5-60 years.
Ethical consideration:
The protocol for the study was reviewed and approved by Ethical Review Committee of Jos University
Teaching Hospital (JUTH) Jos Nigeria. In addition, informed consent was sought and obtained from the Patients
or Parents of children. A structured questionnaire was used to obtain the biodata of the participants.
Collection of specimens:
Samples used in this study were blood and stool samples. Five milliliter (5ml) of blood samples were
aseptically collected by vein- puncture. Stool specimens were collected into clean wide mouth glass bottles.
Processing of specimens; Preparation of blood films:
A drop measuring six microlitre (6µl) of blood was placed on a clean grease free glass slide, to make a
thick blood film by spreading it onto the glass slide. This was allowed to air dry and stained using fields stain A
& B.
The thin blood film was made by placing a drop measuring 2µl drop of blood onto a grease free glass
slide and a blood spreader placed to touch the blood at an angle of 450C. With a quick and fast movement, the
blood was spread onto the slide to have a head, body and tail and allowed to air dry and stained using Leishman
stain [18].
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Efficacy of Some Anti-Malaria Drugs and Antibiotics on Plasmodium and Salmonella Species..
Stool:
Pea size of the stool sample (About 1gm) was inoculated into 10mls of Selenite F (liquid media) and
incubated at 370C for 24hrs. Stool samples were cultured following standard procedures for Salmonella species‟
isolation as stated in [19].This was further sub cultured onto Deoxycholate citrate agar (DCA) and Salmonella
Shegella (SS) agar. They were also incubated for 24hours at370C. The standard method of inoculating plate and
the pour plate method of setting up sensitivity as stated in the Medical Laboratory Manual For Tropical
Countries were used. Salmonella species were identified on the basis of cultural, microscopic and biochemical
characterization ([19][20]).
Separation of serum:
The two milliliter (2mls) of the blood specimens was allowed to clot and spun for 5 minutes at 2500
revolution per minute (rpm). After retracting, the Serum was separated and stored in the fridge until required.
III.
Laboratory Analysis
Parasitological examination of slides for Malaria parasite:
The thick and thin films were examined under the X 100 (oil emersion) power objective for the
presence of any peripheral parasitaemia. Microscopically confirmed Malaria parasite subjects (pre-drug test)
were administered anti-malaria drugs. Patients administered antimalarial drugs (Artemether/Lumefantrine;
Artesunate Amodiaquine; P-Alaxin) were rescreened for malaria parasitaemia after four days to re-determine the
efficacy of the drugs.
Serological Test for Typhoid fever:
The sera were screened for antibodies to Salmonella using the commercial antigen suspension reagents
(Biosystem, febrile Serodiagnostics). A drop of the serum was placed on a tile mixed with a drop of the antigen,
rocked in a mixer for 3-4 minutes and observed for agglutination. Significant widal tests were recorded when
the somatic (0) and flagella (H) gave agglutination reaction tiles of
.
Bacteriological Test for Typhoid:
Stool Culture:
The stool sample inoculated into 10mls of Selenite f and incubated aerobically at 370C for 24hrs was
plated out onto these solid media. This was aseptically sub cultured into Deoxycholate citrate agar (DCA) and
Salmonella –Shigella agar (SSA) using a sterile wire loop, a loop full was picked, sub cultured onto the media
and incubated aerobically at 370C for 24hrs after which they were observed for growth. Suspected Salmonella
colonies were identified as Salmonella enteric serovar typhi using standard microbiological techniques [21].
Blood Culture:
Two milliliter of the blood samples were inoculated into 18mls of prepared brain heart infusion broth
and mixed by rotating in between the palms for 1 minute. These were then incubated aerobically at 37 0c for
24hrs. This was later sub-cultured onto Salmonella Shigella agar using a sterile wire loop and incubated
aerobically at 370c for 24hrs. The brain heart infusion broth media were further incubated for another eight days
after which any slow growing organism will be isolated by further sub-culturing as above. The identified
Salmonella bacilli were set up for drug sensitivity testing (Ciprofloxacin; Cefuroxime and Azithromycin drugs
tested in the sensitivity single disc prepared).
Sensitivity Testing:
Pour plate method for sensitivity testing: The isolated salmonella Spp were inoculated into 10mls of
peptone water and incubated at 370c for 2hrs before pouring onto the dry Mueller Hinton sensitivity agar media
plate. Then the excess was drained off from the top of the agar media plate. The already prepared antibiotic
single disc were placed onto the plate and incubated aerobically at 37 0c for 24hrs. The zone of clearance were
measured and recorded in millimeters. The three single disc used were; Ciprofloxacin, Azithromycine and
Cefuroxime.
IV.
Results
Table 1 represents the first microscopic laboratory examination of malaria parasites and widal
screening test for Febrile Patients and control subjects. Ninety (45.0%) of the febrile and fifteen (30.0%) of the
control subjects out of the two hundred study subject and fifty control subjects respectively were positive for
malaria parasites and widal for typhoid. Those subjects with significant titer of ≥ ¹/80 and positive for malaria
otherwise called typhomalaria fever cases were 110 (55.0%) and 20 (40.0%) of the study control subjects out of
200 and 50 cases respectively.
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Efficacy of Some Anti-Malaria Drugs and Antibiotics on Plasmodium and Salmonella Species..
The “real gold” standard test of cultural isolated salmonella organism and microscopic malaria parasite positive
cases were 102 (51.0%) and 23(46.0%) for the control subjects
Table 1: First Microscopic Examination of Malaria Parasite (MP), Widal Screening, Culture Isolated
Salmonella species and Control Subjects.
Infection
Positive for malaria parasite (MP)
Positive for widal + MP
Positive for MP + Isolated Salmonella Spp.
Negative for MP &Cultures for Salmonella Spp.
Total
Subjects (%)
Febrile Patient N= 200
90(45.0)
110 (55.0)
102 (51.0)
90 (45.0)
200(100)
Control Subjects N= 50
15(30.0)
20(40.0)
23(46.0)
27(54.0)
50(100)
Table 2 shows the results of second microscopic examination of malaria parasites and sensitivity
pattern of isolated salmonella species. Four days after the subjects have taken antimalarial Drugs, their blood
samples were retested (post drug test) for MP, microscopically confirmed Positive cases were 39 out of 90
positive cases of pre drug test. Eight out of the fifteen (pre drug test) subjects of the control were also positive
for post drug mp test. Twelve Salmonella isolated subjects showed sensitivity pattern by inhibition clearance
zones of 2-4 milliliter for ciprofloxacin being the most sensitive drug, followed by 10 subjects for ceftrioxone
and 8 for Azithromycin, while 7 subjects showed very minimal sensitivity or resistance to the drugs used in this
assay.
Table 2: Second microscopic examination results for Malaria parasite and sensitivity tests for isolated
Salmonella spp.
Infection
Positive for malaria parasite only
Salmonella Isolate sensitive to *Cip. (2 – 4mm)
Salmonella Isolate sensitive to *Cef. (2 – 3mm)
Salmonella Isolate sensitive to *Azith. (2mm)
Salmonella Isolate of sensitivity less than 2mm
Negative for MP
Total
Subjects (%) Typhomalarial fever
Febrile
Control
39(38.2)
8(34.78)
12(11.76)
4(17.39)
10(9.8)
1(4.34)
8(7.84)
1(4.34)
9(8.82)
2(8.69)
26(25.49)
7(30.43)
102(100)
23(100)
* – Ciprofloxacin=cip, Cefuroxime=cef, Azithromycine-Azith.
V.
Discussion
Epidemiological observation suggests that strategies that control Malaria also decrease the rates of
bacterial infections & multiplications [22].Although malaria and typhoid are said to be endemic in Nigeria, this
study shows that Malaria is far more likely to cause fever than typhoid fever. [23] observed the malaria high
prevalence and reliability of cultural isolation of salmonella especially in invitro drug sensitivity. A reliable
malaria parasite result can be established from a confirmed microscopic positive test .This is in agreement that
microscopy is the” gold standard” for malaria parasite diagnosis.([24][25]).This has helped to rule out the
confusion faced by clinicians in handling Typhomalarial cases. Cultural diagnosis and isolation of typhoid fever
shows the unreliable nature of widal test, which is basically used in the diagnosis of typhoid fever in the Tropics
and Nigeria in particular. A reliable diagnosis of typhoid fever is based on cultural isolation of Salmonella
organism ([26],[20]). This study is in line with the publications of Pearson et al., [27] and Prasanna [25] and
2000 confirming Blood culture as the gold standard for typhoid diagnosis. Also in line with that of Prasanna
[25] that provided information on malaria and typhoid co-infection otherwise named „typhomalarial fever‟
being a major public health problem. Patients presenting with fever clinically suspected to be either malaria or
typhoid should be subjected to laboratory diagnosis for confirmatory tests. Early results confirming malaria
positive and salmonella Sensitivity to antibiotics should be administered with the sensitive drugs, in order to
prevent drug resistance and other side effects, faced by self medication that can lead to many deaths / casualties.
In conclusion, the result in this study indicates that Artemisinin drugs are the best for the treatment of
uncomplicated malaria infection, especially in co- infections with malaria. This will further led to the
eradication of the bacteria from the body. All these are in agreement with the World Health Organization
(WHO) [24], recommending that ACT is the first line treatment of malaria infection. Today, WHO recommends
Artemisinin –based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by P.
falciparum. ACTs has been integral to the remarkable recent success in global malaria control, and there is
blood consensus that protecting the efficacy of these ACT medicine combinations is an urgent priority [24]. The
most effective drug established from this study being Artemether/Lumefantrine, this concurring with the
publications of Donelly et al., [5]. Furthermore, the prompt treatment of typhoid disease with antibiotics
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Efficacy of Some Anti-Malaria Drugs and Antibiotics on Plasmodium and Salmonella Species..
especially with ciprofloxacin reduces the case fatality [28], especially when the right sensitive drug is
administered timely.
Finally, chemotherapeutic and diagnostic approach, utilizing the microscopically confirmed Malaria
parasites positive tests, and culturally isolated salmonella drug sensitivity tests, with regards to their
chemotherapeutic drug application and efficacy, will influence strongly the institution of „Evidence based
policy‟ in the management of drug resistant malaria and typhoid fever. In the University of Jos clinic as a whole
and Africa at large, Together we can prevent wrong diagnosis, by helping to save more lives from early death.
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