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ADVERSE PREGNANCY OUTCOMES ASSOCIATED WITH MALARIA AND TYPHOID CO-INFECTION IN PREGNANT WOMEN ATTENDING ANTENATAL CLINICS IN ANAMBRA STATE, SOUTH- EASTERN NIGERIA. *Ukibe S.N1, Ikeako L.C2, Mbanugo J.I3, Obi-Okaro A.C4, Eneanya C.I3, Ukibe N.R5 and Abor N.A5 1.

Dept of Prosthesis and Orthopaedic Technology, School of Health Technology, Federal University of Technology, Owerri, Nigeria

2.

Dept of Obstetrics and Gynaecology, Faculty of Medicine, Anambra State University Teaching Hospital, Awka, Nigeria. 3.

Dept of Parasitology and Entomology, Faculty of Bioscience, Nnamdi Azikiwe University, Awka campus, Nigeria. 4.

Dept of Community Medicine, Nnamdi Azikiwe University Teaching Hospital, Nnewi campus, Nigeria

5.

Dept of Human Biochemistry, Faculty of Health Science, Nnamdi Azikiwe University, Nnewi campus. 1.

Corresponding author Tel: +2348035000346 Email: [email protected]

Abstract Background: Malaria in pregnancy has been associated with adverse pregnancy outcomes. Its co-infection with typhoid fever is indeed a double tragedy. Objective: The present study was designed to evaluate the pregnancy outcomes in malaria and or malariatyphoid co-infected pregnant women attending antenatal clinics in Anambra State, South Eastern Nigeria. Methodology: This was a cross sectional study involving 700 pregnant women recruited during routine antenatal visits by voluntary participation between January 2012 and March 2013. Blood samples were collected under aseptic conditions. Malaria infection was determined by immunodiagnostic methods and confirmed by Giemsa staining of thick and thin smears. Typhoid infection was determined by stool culture for Salmonella Typhi.. Pregnancy outcomes were obtained from hospital records in the various hospitals used for the study. Rsults: The result showed that the preterm delivery rate (PTD) was 6.3%, while 6 (0.9%) of the women had spontaneous abortions. Four of the women (0.57%) had intra-uterine deaths (IUDs) while 46 (6.6%) had low birth weight babies (LBW) indicating LBW rate of 6.6%. All the women with these complications were either malaria infected or malaria-typhoid co-infec ted. Conclusion and Recommendation: Malaria and its co- infection with typhoid are responsible for most of the adverse pregnancy outcomes in the study area. Use of insecticide treated bed nets (ITNs), environmental sanitation and provision of basic amenities including portable water will go a long way to reverse this trend. Key Words: Malaria, typhoid, co-infection, pregnancy outcome, Southeast, Nigeria. 1|A M E R I C A N I J Volume 2 2015 Issue 2 FEB-MAR AIJCSR

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Introduction

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January 2012 to March 2013 from five major private and public health institutions in the state namely General

Malaria is an enemy of pregnant women with reported prevalence rates in Nigeria ranging from 4.8-80% [1, 2, 3, 4, 5, 6]. In our recent study [7], we reported a prevalence rate of 73.1% in Anambra State, Nigeria. The physiological changes associated with pregnancy state are marked with lowered immunity to infections including malaria and typhoid fever [8, 9]. It is pertinent to note that both malaria and typhoid fever are tropical diseases and are endemic in Sub-Saharan Africa. The high endemicity of malaria, coupled with that of typhoid fever has been associated with high incidence of co- morbidity states. In our recent study [10], we reported a malaria-typhoid co-infection rate of 20% in pregnant women.

hospital/Anambra State University Teaching Hospital (ASUTH) Awka, Divine hospital and maternity Awka, Christ the King Specialist hospital Awka, Life specialist hospital Nnewi and General hospital, Onitsha respectively. To arrive at the sample size, the annual 3% growth rate for the female population in Nigeria was determined as at 2004. The population of women of reproductive age which is 49% of all female population was also determined. Pregnant women constitute 5% of women of reproductive age and this was also determined. The figures obtained were substituted in the formula for calculation of sample size. Informed consent was obtained from the participants after due permission had been sought for and obtained from the hospital authorities. The faculty board of Ethical

The concurrent infection of the pregnant woman by

Committee approved the study.

malaria and typhoid fever is indeed a double tragedy since both diseases have been associated with adverse pregnancy outcomes which include maternal anaemia [11, 12, 13], low

Sample Collection:

birth weight babies [14, 15], and spontaneous abortions [16,

3ml of whole blood was collected through venepuncture

17]. The present study was therefore designed to evaluate

from each participant under sterile condition and 1ml placed

the pregnancy outcomes in malaria infected or malaria-

into EDTA bottle for malaria parasite examination. 2ml was

typhoid co-infected pregnant women attending antenatal

placed into a sterile plain bottle to retract and then

clinics in Anambra State, South-Eastern Nigeria.

centrifuged to produce serum for Widal test examination.

Materials and Methods

METHODS

Study area: The study was conducted in hospitals chosen from three different Local government areas in Anambra

Determination of Malaria infection

state, Nigeria, namely Awka South, Onitsha North and Nnewi North. Anambra state is located in the South Eastern zone of Nigeria with an estimated population of 4.9million people according to 2006 census [18]. The state is bounded by Delta state to the West, Enugu state to the East, Kogi state to the North and Imo state to the South [19]. The study locations: Awka, Onitsha and Nnewi are the three major

P. falciparum antigen Rapid Test Device: Principle- The principle of P. falciparum antigen detection is based on a rapid chromatographic immunoassay for the qualitative detection of circulating P. falciparum antigen in the whole blood. This method utilizes Gold conjugate to selectively detect Plasmodium antigen.

cities of the state. Procedure-The

procedure

was as described

by the

manufacturer. (Paracheck, Orchid Biomedical System, Vena Study population- A total of 700 pregnant women aged

Goa, India). Briefly, 10µl of the whole blood specimen of

between 17 and 46 years, who came for routine antenatal

the participant was transferred into appropriately labeled

services were recruited by voluntary participation between

specimen cassettes containing sample well. Subsequently, 3 2|A M E R I C A N I J

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drops of buffer supplied by the manufacturer was added into

tubes were incubated in a 56 C water bath for 18 hrs before

the sample wells. After 15mins the results were read.

reading. A titre of >1/80 to either antigen in a single serum specimen was taken to be indicative of typhoid fever . The

Interpretation- The test device has inherent quality control

service of a specialist Laboratory Scientist was employed.

that validates the result. The presence of two pink lines at the region of the control and test sample signifies presence

Stool Culture for Salmonella

of P.falciparum malaria infection while the presence of only 1pink line in the control region signifies absence of P.

Materials needed:

falciparum malaria infection.

Giemsa Stained thick and thin blood smear for microscopic Detection of P.falciparum parasites [20]. -Placing a small drop of blood in the centre of each slide made thick films,

1.

Selenite- F Broth

2.

Salmonella Shigellae Agar (SSA)

3.

Tripple Sugar Iron Agar (TSIA)

4.

Urea Agar Base

The media were prepared according to the manufacturer’s instruction.

- Using another slide, the blood was spread out to cover an

Step 1: Sample Collection:

area four times of its original area to get a satisfactory thin film.

The stool samples were collected in sterile universal

- The films were allowed to dry thoroughly for 30 mins at room temperature and then stained with Giemsa stain freshly diluted with buffered water of PH 6.8 (1:10 Dilution)

containers. About 2-3 gm of the stool was used each time. Step 2: 10ml of Selenite- F broth was added into 3gm of the stool sample in a sterile container and mixed vigorously for

- The films were allowed to stain for 45 minutes and then

hard stool but gently for soft or watery stool. The mixture

washed with clean distilled or buffered water and allowed to

was incubated at 37OC for 24hrs.

air dry in a draining trough

Step3: Using a sterile wire loop, a loopful of the sample was

- The blood films were then examined microscopically using

inoculated onto SSA and streaked to get discreet colonies. It

the x10 oil immersion objective.

was then incubated at 37OC for 24hrs.

Interpretation: Malaria parasites, pigments and species were

Step 4: The plates were read. If lactose fermenters (LF) (ie

identified as ring forms using standard charts and reported

pink colours) were seen on the SSA, no further test was

as +, ++, +++.

done, but if non- lactose fermenters (creamy or colourless colonies) were seen, they were subcultured onto a fresh SSA

Widal Test Examination

medium to obtain a pure growth for biochemical test.

Widal agglutination test was performed on all malaria

Step5: From the pure growth, Gram Staining was done. A

positive

antigen

single colony was collected using a sterile wire loop and

suspension (Plasmatec Laboratory Products Ltd, Antec

sub-cultured onto TSIA Slant in a test tube, and also stabed

Diagnostics Ltd, U.K) containing O and H antigens of S.

into the medium. The isolate was sub-cultured onto Urea

Typhi and Paratyphi respectively. A serial dilution of 1 in 50

Agar Slant in a Bijou bottle. They were incubated for 24hrs

to 1 in 400 of serum in 0.85% saline was made, and 0.3ml of

at 37OC. The results were read and interpreted with the help

each dilution was added to 0.3ml of antigen suspension.

of a standared chart. The culture was done at the

blood

samples

using

commercial

Positive and negative serum controls were included. The 3|A M E R I C A N I J Volume 2 2015 Issue 2 FEB-MAR AIJCSR

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microbiology department of Nnamdi Azikiwe University

Table 2: Birth Weight of Babies delivered by pregnant

Teaching Hospital Nnewi.

women in Anambra State,South-Eastern Nigeria.

Determination of Pregnancy Outcome Frequency

Percentage

0-2.4

46

6.6%

2.5-3

270

38.6%

3.1-3.5

190

27.1%

3.6-4

173

24.7%

4.1-4.5

20

2.9%

of variance (ANOVA) were used to assess significant

>4.5

1

0.1%

differences.. The level of significance was considered at P≤

Total

700

100%

Pregnant women were followed up till the time of deliveries. Relevant pregnancy outcomes were extracted from hospital

Birth (Kg)

weight

records including documented birth weight of babies.

Statistical Analysis: The version 16 of SPSS package was used for statistical analysis. The independent variables obtained in this study were expressed as mean (±SD). Chi square (x2) and analysis

0.05. Range=0.4-4.6Kg

Results Table1: Pregnancy Outcome in Malaria infected, Malaria-typhoid co-infected, and Malaria negative/typhoid negative (Control) women in Anambra State, Nigeria.

Birth (Kg)

Outcome

Groups (%)

N =700

Malariainfected (%)

MalariaTyphoid (%)

Control (%)

Total (%)

340 (48.6%)

190 (27.1%)

116 (16.6%)

646 (92.6%)

24 (3.4%)

20 (2.9%)

0 (0)

44 (6.3%)

4 (0.6%)

2 (0.29%)

0 (0)

6 (0.9%)

Normal delivery (ND) Preterm delivery (PTD) Spont. Abortion (SPA) Intrauterine death (IUD)

1 (0.1%)

Table 3: Comparism of birth weights (bwt) of babies delivered by Malaria infected, Malaria-typhoid coinfected and Control women in Anambra state, SouthEastern Nigeria.

3 (0.43%)

0 (0)

4 (0.57)

Weights

Groups and frequency

Malaria-infected n=356 (A) (%)

Malaria-Typhoid n=156 (B) (%)

MP/Typhoid negative (Control )n=188 (C) (%)

0-2.4

30 (4.29)

16 (2.29)

0 (0)

2.5-3

160 (22.86)

74 (10.57)

12 (1.71)

3.1-3.5

145 (20.71)

56 (8)

20 (2.86)

3.6-4

20 (2.86)

10 (1.42)

134 (19.14)

4.1-4.5

1(0.14)

0 (0)

21 (3)

>4.5

0 (0)

0 (0)

1 (0.14)

N=700, Range=0.4-4.6 Mean Bwt = 2.98±0.57

2.95±0.51

F- Value=

41.25,

3.58±0.39

A vs B. P > 0.05 (Not significantl) 4|A M E R I C A N I J Volume 2 2015 Issue 2 FEB-MAR AIJCSR

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involve prematurity resulting from preterm deliveries as was evident in this study. Preterm delivery rates have varied Table 1 shows that 646(92.6%) of the pregnant women delivered normally (operative delivery inclusive). The preterm delivery (PTD) rate was 6.3% while 6 (0.9%) of the women had spontaneous abortions. Four of the women had intra uterine deaths (IUDs). All the women with these adverse complications of pregnancy (Preterm deliveries, Spontaneous abortions, or intra-uterine foetal deaths) were either malaria infected or malaria- typhoid co- infected. None of the women in the control group had these complications.

from one country to another. Whereas the World Health Organization [28] estimates that 15 million babies are born preterm annually, Nigeria is ranked 3rd behind India and China as countries with the highest prevalence rates of preterm births.[28]. Previous studies in Nigeria [29, 30] have reported preterm delivery rates ranging from 6-12% which is comparable to the rate observed in the present study. A recent study in Uganda [31] associated malaria with intra-uterine growth retardation which eventually resulted to low birth weight babies. Such babies were

Table 2 shows that 6.6% of the women had babies with

reported to have fewer chances of survival hence the high

birth weights below 2.5Kg suggesting a LBW Prevalence

perinatal mortality rates reported in these areas. The

rate of 6.6%. More than 38% of the women had babies with

complication of malaria with typhoid fever renders a deadly

birth weights within the range of 2.5-3Kg while only a few

blow to the pregnant woman with a worsening of the above

of them (2.9%) had babies with birth weights above 4kg.

scenario.

Table 3 shows that there was a significant difference

High maternal anaemia rates which have been reported

between the birth weights of babies born by Malaria infected

in several studies conducted in Nigeria [32, 33, 34, 35, 36,

women (A) and the Control(C) (P

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