Al-Kufa University Journal for Biology / VOL.8/ NO.

2/ Year: 2016
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Bacteriological study of puerperal sepsis in Al-Najaf city

Dr.kareem Thamir Muhammad Abbas Dr. Adel Ibadi
College of medicine MS.c Student College of science
Kufa university Kufa university

Abstract:.
In this study ,which is a case control study ,the aim was to study the puerperal sepsis in al-
Najaf city and to find out the different factors that is related to the occurrence of this problem .
A total of 100 post partum women suffering from puerperal sepsis who were attending the
outpatient clinics of AL –Hakeem and AL-Zahraa maternity and child Hospital ,and another 100
post partum women who were free from any signs or symptoms of puerperal sepsis ,but they
were attending different outpatient clinics in AL-Hakeem ,AL-Zahra'a and many other health
service centers ,for the sake of their babies vaccination or for any other cause ,were chosen as a
control group .It was concluded from the study that the age of most incidence of puerperal sepsis
was the (20-24) years ; when the parity was taken in consideration it was found that the
primiparous were the mostly affected in contrast to the control group where the multiparas were
the mostly predominant .
Most of the patient group were delivered in a general hospital while most of control group had
been delivered in homes.
It was found that E.coli was the mostly predominant microorganism isolated from the patients
group 23% by streptococcus agalactiae and proteus vulgaris 5% for each.

1-Introduction:.
Puerperal sepsis is an infection of the genital tract at the time interval between rupture of
amniotic membrane and the 42nd day following delivery or abortion. Two or more of the
following manifestations must be present to define the condition. The manifestations are: pelvic
pain, fever of (38-.45oC) or more, abnormal vaginal discharge with foul odor and delayed
reduction of uterine size (Dolea& Stein,2003) .
According to World Health Organization (WHO) puerperal sepsis is defined as an infection of
the genital tract occurring at any time between the rupture of membranes or labor and the 42nd
day post partum in which 2 or more of the following are present: pelvic pains, fever (that is oral
temperature 38,5°C or higher on any occasion, abnormal vaginal damage (example presence of
pus), abnormal smell or foul odor of discharge, delay in the rate of reduction of the size of the
uterus (less than 2 cm per day during the first 8 days). (World Health Organization 2009)
One of the predisposing conditions usually leading to the Puerperal sepsis is the home delivery
in unhygienic conditions and should be included at the top of the list. Other factors include low
socioeconomic condition, anemia, parity, prolonged period of delivery after rupture of the
amniotic membrane, frequent per vaginal examinations and prolonged labor (Chisembele
,2004).
Many causative organisms of Puerperal sepsis like Streptococcus pyogenes and other Beta-
haemolytic Streptococci, Enterococcus faecalis, Anaerobic cocci, , Proteus species, Escherichia
coli with other Coliforms(Cheesbrough,2000).
In 1879, Louis Pasteur identified the Streptococcus as the causative organism for Puerperal
sepsis.
Since the early 1930s, when Rebecca Lancefield reported her grouping system for hemolytic
Streptococci, Group A Streptococcus was widely acknowledged as the major pathogen

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associated with Puerperal sepsis. Group B Streptococcus (GBS) was initially thought to be a
commensal until 1937, Shet et al in India reported 7 cases of GBS associated Puerperal sepsis
with 3 deaths (Shet& Ferrieri ,2004) .
Aims of the study :.

1-Isolation and identification of the causative microorganisms of puerperal sepsis.

2-Identification of the effect of the age, parity and place of delivery on the
occurrence of puerperal sepsis in both the patient and the control groups with comparison.

2-Material&Method :.
2-1 Patients and methods
Three hospitals in Al- Najaf (Al-Hakeem , Al-Sadur and Al-Zahra'a ) were included in this
study. At the period between November 2011 and October 2012 ,One hundred women suffering
from puerperal sepsis were included in the present study after being diagnosed by a specialist
gynecologist .Also the same number of newly delivered women who were free of puerperal
sepsis were taken as a control group . Two vaginal swabs were taken from each patient ( by the
gynecologist or an expert nurse), one sample was directly rubbed on the surfaces of blood agar
,maCconky agar , chocolate agar,Colmbia agar,Hicrom agar and manitol salt agar then incubated
aerobically (for blood , macconkey and manitol salt agar plates only) and anaerobically(only the
chocolate agar plates and also with Co2) at 37C for 24 hours. The other swab was used for direct
smear to search for any Trichomonas vaginalis and or Candida albicans.
Agreement in written was taken from every patient and control subject as an important
requirement to perform any further steps in the study.
2-2 .Identification of Microorganisms:.
2.2.1. Detection of Trichomonas vaginalis.
In wet preparation showing of a typical jerky movement ,trophozoites these indicated present
of T .vaginalis.

2.2..2. Detection of Candida albicans

Three method were used for isolate and identification of this microorganisms as follows:
2-2-2-1. Microscopical Examination
Wet preparation and Gram stain were used for detection of Candida spp.. Candida cells were
seen as rounded to avoid shape ,some time typical budding and Gram positive cells (Betty et al.,
2002).
2-2-2-2. Culture Method
vaginal swab was cultured on hicrom candida agar plates (selective media for the Candida
spp.) and incubated at 37˚C for 24-48h. to isolate a pure colony, for germ tube test.
2-2-2-3 Germ Tube Test
A- Avery small inoculums of Candida spp. were suspended ,with 0.5ml of rabbit plasma.
B-Incubated at 37˚C for no longer than 3hours.
C- A drop of suspension was placed on microscopical slide, examined under high power (X400)
for detection of Candida albicans (Betty et al., 2002).

2-3. Identification of Streptcoccus spp.

Vaginal swab was cultured on to blood agar plate and incubated for 24-48h. at 37˚C in candle
Jar (5%CO2), the culture was examined for study colony morphology and type of heamolysis
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caused by various streptococcus spp. these are classified into their species by using classical
biochemical tests as shown in figure (3-1) the sub culturing on brain heart infusion agar for
antibiotic test ( Levinson and Jawetz ,2002).

2-4. Identification of Staplylococcus spp.

Vaginal swab was cultured onto blood agar plates at 37˚C for 24h. under aerobic condition
the culture was examined for study colony morphology and type of heamolysis.The various
staphylococcus spp. are classified into their species by using classical biochemical test as shown
in figure (3-1).
A subculture on brain heart infusion agar for antibiotic test ( Collee et al., 1996).

2-5.Identification of Neisseria gonorrhoeae

2-5-1. Microscopical Examination
Smear was made from cervical swabs, stained by Gram stain .Presence of Gram negative
diplococci extra or inter a polymorphanuclear,indicate the presence of N.gonorhoea.

2-5-2. Culture Method

Vaginal swab was cultured on barin infusion heated blood agar plate, and incubated at 37˚C
for 24-48h. in candal jar (5% CO2), N.gonorrhoeae isolates were identified by study of colonial
morphology ,Gram stain ,and using classical biochemical tests as shown in Figure (3-1). A
subculture in the same media for isolation pure colony for antibiotic test ( WHO,1999).
2-6. Identification of Gardnerella vaginalis
Three method were used for isolation and identification of this microorganisms.
2-6- 1. Swift test
The test was carried out by adding a drop of 10% KOH on fresh cervical secretion, amino
odor indicated as positive result of Granderella Vaginalis (Patrick et al., 1999).
2-6-2. Detection of Clue Cells
A gram stain was used to detect the presence of clue cells under oil emersion lens. The
epithelial cells were covered by coccobacilli bacteria, (Patrick et al., 1999).
2-6-3. Culture Method
Cervical swab was cultured on to Columbia agar, and incubated at 37˚C for 24-48h. in a
candle Jar(5%CO2).
This culture was examined for study of morphology colonial and beta-hemolysis the
identification colonies were using classical biochemical test .

2-7. Identification of Enteric bacteria

Vaginal swab was cultured on MacCankey’s agar the differential ability of this media is
based on lactose fermentation, the identification colonies were using classical biochemical
test,sub culture on E.M.B for isolation of typical E.coli colonies that produce metallic sheen
(MacFaddin, 2000;Levinson and Jawetz, 2002).

2-8. Identification of type Bacteria

API-20E test strip (from bioMerieux, Inc.) is used to identify the enteric gram negative rods
and gram positive Bacteria (although API makes a variety of other test strips for yeast, Staph,
anaerobes, etc.) 20 separate test compartments are on the strip, all dehydrated. A bacterial
suspension is used to rehydrate each of the wells. Some of the wells will have color changes due
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to pH differences: others produce end products that have to be identified with reagents. A profile
number is determined from the sequence of + and - test results, then looked up in a codebook
having a correlation between numbers and bacterial species.

PROCEDURE:
Prepare a suspension of the bacteria in the saline tube
1. Inoculate a large colony (2-3mm diameter)of the bacterium (pure culture) into the
0.85% NaCl solution, making sure that the suspension is homogenous and without clumps of
floating bacteria.

2. Use a McFarland barium sulfate standard #3 to quantities the suspension and to produce a
standard inoculums size.
Inoculate the API strip
1. Holding the strip at a slight angle up from the table top, you will now inoculate the
bacterial suspension into each well with the sterile pipette.

2. Touch the end of the pipette to the side of the cupule, allowing capillary action to draw the
fluid into the well as you slowly squeeze the bulb. This should eliminate any bubbles forming in
the wells. Each well should be filled bubbles forming in the wells. Each well should be filled up
to the neck (see diagram).
3. CIT, VP, and GEL have boxes around their names. These test wells will be filled all the way
up to the top of the well.
4. LDC, ODC, ADH, H2S, and URE are filled as described in step B, but they will then be filled
up to the top with sterile mineral oil.

Incubate the strip in its chamber

1. The bottom of the incubation chamber has small indented wells in the bottom: fill it with
water just enough to fill these indentations.
2. Place the strip into this bottom. There should not be so much water that it slops onto the API
strip.
3. Place the top of the incubation chamber over the bottom, and label it.
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4. Place the strip at 37o C for 18-24 hours.

INTERPRETATION:
1. Add the proper reagents to the compartments:
o 1 drop of Kovac's to the IND (read within a couple of minutes)
o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes)
o 1 drop of FeCl3 to TDA
2. Read all other tests as described (chart below) without reagents using the table below..
3. Record results on the diagram handed out to you in lab. The oxidase test reaction should be
negative, and is added as the last test result.
4. Take your result sheet to the computer to INPUT YOUR REACTIONS: The API 20E website
database will provide you with potential organisms.

Statistical Analysis :.
Statistical analysis was done by using SPSS(statistical package for social sciences) version
20.In which we use Chi square (X2) test for categorical data .We set Pvalue <0.05 as significant
(Charles et al.,1987).
Results&Discusions:.
There were one hundred patients with puerperal sepsis included in the present study with
different ages and another 100 newly delivered ( within 42 days post partum ) without puerperal
sepsis who were chosen as a control group. They were divided into different groups according to
their ages as it will be shown in table-1.We noticed from the table that most of those who had
puerperal sepsis were present in the age groups (20-24) and (25-29) years significantly more
than those in the healthy control group p<0.005.
Table (1) Patients with puerperal sepsis divided into different age groups with 5- years
interval.
Age in years Puerperal percentage Control percentage
sepsis
<20 7 7% 12 12%
20-24 47 47% 35 35%
25-29 20 20% 17 17%
30-34 15 15% 28 28%
35-39 7 7% 8 8%
40-45 4 4% 0 0%

X2=11.312 P=0.045

When the parity was taken as a comparative variable between those with puerperal sepsis and
the other without it was found that those who were primiparas had been more affected by
puerperal sepsis more than others. when this was compared with the control group it was shown
that, though all the control subjects were free from puerperal sepsis, yet, most of them fall in the
multi and grand-multiparas group and the difference was significant p=0.001. as it is shown in
table (2).

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Table (2) The frequency of occurrence of puerperal sepsis according to the state of parity
of the patient.
parity Puerperal percentage Control Percentage%
sepsis

primiparas 50 50% 27 27%

multiparas 25 25% 45 45%

grandmultiparous 25 25% 28 28%

X2=12.754 P=0.0001

One of the predisposing factor usually leading to Puerperal sepsis is hospital delivery. Table 3
determines that those who underwent hospital delivery were more commonly affected
(50%)with puerperal sepsis than those who had been delivered in home ( 23% )while those
delivered at a private hospitals had( 27%),when the control group was taken in consideration it
was shown that most of them 56% were delivered at home by a mid wife and 14% at a private
hospital while 30% were delivered at a general hospital. The factor of place of delivery was of
great influence on the occurrence of puerperal sepsis as we could noticed that most of those with
puerperal sepsis had been delivered in a public hospital significantly more than in case of the
control group, P was less than 0.001.

Table( 3): The frequency of occurrence of puerperal sepsis according to the place of
delivery.

Place of delivery Puerpera Percentage% control Percentage%

l sepsis

Home delivery 23 23% 56 56%

General Hospital 50 50% 30 30%

delivery

Private hospital 27 27% 14 14%

delivery

X2=22.907 P= < 0.001

The vaginal swab culture and sensitivity showed that the comments microorganism isolated were
E.coli and staph. aureus 23%and14% respectively. Strep. spp and Proteus spp were found in 5%
of cases for each, and klebsiella spp.in 4%of the cases while Candida albicans was found in 10%
of the cases.Thirty nine percent of the cases of puerperal sepsis showed mixed type of growth.
As showen in table No 4.

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Table (4): the frequency of isolation of different bacterial species from cases of puerperal
sepsis.

Place of delivery Puerperal Percentage% control Percentage%

sepsis

Home delivery 23 23% 56 56%

General Hospital 50 50% 30 30%

delivery

Private hospital 27 27% 14 14%

delivery

Puerperal sepsis is an important cause of hospitalization due to its clinical morbidity. Usually the
infection is of polymicrobial origin with a mixture of both Aerobic and Anaerobic
organisms(Stevens,2006). a large number of patients particularly from lower socio-economic
states seek admission for proper management from the sequeleae of puerperal sepsis .

When the age of the patients with puerperal sepsis was taken in to consideration table(1) It was
shown from the present study that the age mostly affected by puerperal sepsis was that 20-24
years .

When is normally the fertile age in our society ,this result is in agreement with what was
registered by (Shamshad et al. ,2010) who found that the mothers who developed puerperal
sepsis were mostly 66.3% and he attributed the causes to that the young inexperienced mothers
land up in the hands of traditional birth attendants and mostly deliver outside health facility due
to lack of education regarding health care, antenatal visits and delivery in well equipped well
staffed medical facility . of course this explanation is fit for the community of the researcher
,while in our case ,regarding the place of delivery and its impact on the occurrence of puerperal
sepsis the condition is differ ,as we found most of the cases with puerperal sepsis . occurred
among those who delivered in the public hospitals more than in those who delivered outside the
hospital ((domiciliary)) , and we attributed the causes to the lack of infection control policy in
our hospitals and to the dirty labor theaters that are present even in the most famous public
hospitals.

Another study also support the age and parity tend to be younger ,70% less than 30 years age
and 77% having parity of 2 or less ( deGroot & Makapoo,1998; Vallely et al .,2005).

The place of delivery had its' impact on the occurrence of puerperal sepsis as it was shown in
table (3) which determined that those who were delivered at general hospital ranked the highest
percent of puerperal sepsis in compares to other places(50%) while those who were delivered at
private hospitals and at home had 27% and 23 % respectively .

These result are in agreement with what was registered by Simoes.,et al 2005 who found that
puerperal sepsis complication rate was significantly higher in case of surgical delivery (caesarian

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section ) than in those delivered at home 20.21 versus 11.79 % respectively and the differences
he found was highly significant p o.ooo1.

When the type of commonest bacteria isolation in puerperal sepsis is taken in consideration table
(4)

it was found that the commonest bacterial isolation is E.coli which represented 23% of the
isolates followed by staph. aureus 14% then proteus valgaris ,strepto coccus aglactiae 5% for
both and lastly klebsiella pnemoniae 4%.

These result ,little bit less than what was registered by (Ahmed et al. ,2008 )who found that
staph .aureus isolates was 61.9% and E.coli was 14.28% while streptococcus spp was 11.90 %.

These little differences could be attributed to the sample size differences between the two
researches (Ahmed et al. ,2008) applied only 50 cases while the present study depended 100
cases.

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