Ali Hattem Bayati¹, Yasmeen J. Al-Bayaa², Sevan Najem Alwan³ and Ibrahim Isam Al-Karkhi⁴

¹Department, Technical College of Health, Sulaimani Polytechnic University, Sulaimani city, Kurdistan Region/Iraq.

^2,3Department of Microbiology , Baghdad Medical College, Baghdad, Iraq.

⁴Memorial University of Newfoundland, NL, Canada.

Corresponding  Author E-mail: dr.alkarkhi@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1101

Abstract

Among many viral causes of miscarriage, maternal infections caused by Cytomegalovirus and Epstein-Barr virus infections are important causes. This study aimed to detect the possible occurrence of Cytomegalovirus and Epstein-Barr virus infections in placental tissues from patients with spontaneous abortion using immunohistochemistry and in situ hybridization techniques. Immuno-histochemistry technique and chromogenic in situ hybridization assay was used to detect placental infection with Cytomegalovirus and Epstein-Barr virus in 40 women with spontaneous miscarriage and in 40 healthy delivery n Baghdad/Iraq. Equal detection rates of Epstein-Barr virus in placental tissues by either CISH or IHC were (22.5%), yet the validity results of Epstein-Barr virus - VCA by IHC as compared to Epstein-Barr virus - EBER by CISH have displayed a sensitivity of 44.4% and 83.9%, respectively. The detection rates of Cytomegalovirus -DNA by CISH and Cytomegalovirus -protein by IHC were (30%), (37.5 %), respectively. The results of Cytomegalovirus -DNA -ISH as compared to this Cytomegalovirus - IHC-protein had revealed a sensitivity and specificity of 41.7% and 64.3%, respectively. Cytomegalovirus and Epstein-Barr virus are important causes of placental infections among miscarriage females in Baghdad, and Cytomegalovirus might be detected in placenta of normal delivery. Although CISH technique considered as the gold standard method for detecting of latent Epstein-Barr virus and /or Cytomegalovirus infection were IHC has showed a compatibility to that technique and might reach rates of  high sensitivity and specificity similar to it.

Keywords

Cytomegalovirus; Epstein-Barr virus; immunohistochemistry; chromogenic in situ hybridization; miscarriage; pregnancy; placenta

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Copy the following to cite this article: Bayati A. H, Al-Bayaa Y. S, Alwan S. N, Al-Karkhi I. I. Detection of Cytomegalovirus and Epstein Barr Virus in Placental Tissues of Aborted Women. Biomed Pharmacol J 2017;10(1)

Copy the following to cite this URL: Bayati A. H, Al-Bayaa Y. S, Alwan S. N, Al-Karkhi I. I. Detection of Cytomegalovirus and Epstein Barr Virus in Placental Tissues of Aborted Women. Biomed Pharmacol J 2017;10(1) Available from: http://biomedpharmajournal.org/?p=13545

Introduction

The abortions reasons in several circumstances yet are mysterious, nevertheless the bacterial toxicities signify a main reason in abortion, where germs seems to be the utmost elaborate pathogens (Khameneh et.al., 2014) and (Oliver and Overton ,2014).

Between numerous germs, Humanoid Herpes germ toxicities of placenta which could be hurtful in pregnant foremost to complaints in the growth of fatality, early delivery, failure, or main irregularities, besides it could yield to a prolonged or frequent parental infection. In specific, CMV through pregnancy could spread the placenta   by viremia, subsequent to main and recurring toxicity, or by rising way as of the cervix, regularly subsequent recurrence. The germ with a smallest mid herpes viruses, Epstein-Barr virus which usually related with irregular abortions (Avgil and Ornoy, 2006) and (Di Stefano et.al., 2008).

The goal of the current research is to investigate the differences in the presence of two herpetic virus-related toxicities inside placental soft tissue as of caseswhose spontaneously abortion through determination of infections with CMV, and EBV involving the placenta as a possible causes for subsequent abortion by using the following techniques: Immunohistochemistry and In situ hybridization; as well as comparing the two techniques for detection of CMV, and EBV.

Method

This retrospective study made the use of paraffin contain placental soft tissue that composed from histo-pathological annals of Training Labs form the hospital of AL-Yarmouk in Baghdad / Iraq and belongs to forty (40) feminine cases with miscarriages as patient’s collection; the range of the ages were (19-43 years), and 40 placental soft tissue of normal delivery as a control group. Depiction Rabbit and Mouse with certain HRP \DAB inspection IHC Kit ab80436 (2013) Abcam was used for inspection of CMV –protein and Epstein Barr – viral capsid antigen (EBV –VCA) specific  primary antibodies . CISH Implementation AP-NBT kit for detection of EBV (EBER – RNA) and CMV (DNA) for chromogenic in situ hybridization (CISH) using biotin – labeled Zyto Fast CISH probe was purchased from ZytoFast/Germany Cat. Numbers (T-1070-40 – 2011). Numerical investigation: Investigation of statistics was achieved by means of the obtainable numerical set of (SPSS-22).

Results

For IHC technique, the expression of EBV –VCA IHC signs were identified as a brown color staining localized at nuclear (Figure1, A). The placental soft tissue tasters of aborted women presented 22.5% (9 cases from 40), whereas not any of well control placental soft tissue displayed EBV – VCA antigen expression. The maximum ratio of EBV-IHC shows a modest sign 66. 7 % (6 cases from 9)  as depicted in Table 1.

Figure 1: Microphotograph staining of trophoblastic placental tissues from miscarriage patients (red arrow): A- EBV IHC staining in cell nucleus intensity. B- IHC staining for CMV   at cell nucleus. C- Negative IHC staining for EBV. D- EBV-ERER  CISH positive signals in the cell nucleus of  trophoblastic placental  tissues  showed score 2 and moderate  intensity. E-CMV -DNA staining CISH of trophoblastic placental tissues in the cell nucleus score 1 and weak intensity. F- Negative CISH staining for EBV. Click here to View figure

Table 1: Immuno-histochemical scoring & signal intensity data of EBV –VCA

Groups		Miscarriage Set		Control Set		P value
		Number	%	Number	%
EBV IHC Positively Score	Negatively	31	77.50	40.00	100.00
	Positively	9	22.50
	(Score I)	7	77.80
	(Score II)	2	22.20
	(Score III)
EBV IHC Positively  Intensity	(Weakly / I)	3	33.30
	(Moderately / II)	6	66.70
	(Strongly / III)

The general appearance of CMV protein at nuclear localization (Figure1, B) was distinguished in 37.5% of the placental soft tissue of failure group and in 5% of healthy placental soft tissue. A great percent (around 60%) among placental soft tissue in the failure group had feeble score (score I). The uppermost positive  CMV-IHC reactions in those with miscarriage group were showed strong  signal intensity 66.7%  .Statistically, noteworthy changes (p <0.05) was distinguished associating with the infection degree  among miscarriage and control group (Table 2).

Table 2: CMV-protein of IHC in placental soft tissue

Groups		Miscarriage Set		Control Group		P value
		No.	%	Number	%
CMV IHC Positively Score	Negatively	25.00	62.50	38.00	95.00	1x 10-4*
	Positively	15.00	37.50	2.00	5.00
	(Score I)	9.00	60.00	2.00	100
	(Score II)	1.00	6.70
	(Score III)	5.00	33.30
CMVIHC Positively Intensity	(Weakly / I)	1.00	6.70	2.00	100.00
	(Moderately / II)	4.00	26.70
	(Strongly / III)	10.00	66.70

*Very clear differences among the ratios by (Pearson Chi-square test at 0.05 level)

The chromo-genic are in situ hybridization (CISH) system, the whole percent of positive CISH of EBERS between placental soft tissue in the miscarriage group was 22.5%, while none of the 40 control placental tissues have revealed positive CISH- signals for this EBV marker (figure 1, D). The scoring of EBER RNA- CISH reactions  was in its highest percentage  66.7%  of all positive cases  in the low score (score I), while the their signal  intensity of the  color development where the percentage of  EBER- RNA –CISH reactions that showed weak, moderate, and strong  intensities were 4(44.5%), 3(33.3%), and 2(22.2%) respectively (Table 3).

Table 3: Distribution of scoring and intensity of CISH actions for EBER RNA- in placental tissues of miscarriage and control group.

Groups		Miscarriage  Group			Control Group		P value
		No		%	No	%
EBV CISH Positively Score	Negatively	31.00		77.50	40.0	100.00
	Positively	9.00		22.50
	(Score I)	6		66.70
	(Score II)	3		33.30
	(Score III)
EBV CISH Positively Intensity	(Weakly / I)		4	44.4
	(Moderately / II)		3	33.3
	(Strong / III)		2	22.2

CMV DNA was noticed in (30%) from the studied placental soft tissue in the miscarriage set, whereas placental tissues of healthy delivered women have revealed in (15%). Regarding the signal scoring, the most frequently detected CMV – positive  placental tissue in the  miscarriage group were observed to have the lower score (score I), (50%), 58.3 % revealed  weak  sign strength ,numerically, no difference observed    (p >0.05) as depicted in (Table 4).

Table 4: Distribution of HCMV DNA CISH result of the examined placental tissues of miscarriage and their counterpart control tissues

CMV CISH signal Score & Signal intensity		Miscarriage Group		Control Group		P value
		Number	%	Number	%
CMV CISH Positive Score	Negative	28.00	70.00	34.00	85.00	0.108
	Positive	12.00	30.00	6.00	15.00
CMV CISH Positive Intensity	Score  I	6.00	50.00	5.00	83.30	0.308
	Score  II	3.00	25.00	1.00	16.70
	Score  III	3.00	25.00
	weak         / I	7.00	58.30	6.00	100.00
	Moderate /II	5.00	41.70
	strong      / III

The Validity of CISH and IHC techniques for diagnosing placental tissues of miscarriage patient infected with EBV showed that of the total 40 placental tissues in the miscarriage group, nine have been identified to express CISH reaction for EBERs and IHC reaction VCA. No numerically important differences between these results of the two detection methods (P<0.005). The statistical observed sensitivity and specificity were  44.4% and 83.9%, for CISH and IHC for detection of EBV in placental tissues of miscarriage patients respectively (Table 5), while the validity of CISH and IHC techniques  in  diagnosis CMV placental infections showed that the percentage of positive –reaction results of  CMV –DNA -CISH in placental tissues of miscarriage patients was 30%  while  CMV-IHC  protein expression  in placental tissues of miscarriage  group was 37.5%  .  The number of cells in serial sections that showed positive IHC was slightly greater than those have showed CISH positive reaction. Labeling results of both in situ hybridization and immunohistochemistry were showed in five cases. Statistically the observed sensitivity and specificity were 41.7% and 64.3%, for CISH and IHC for detection of CMV in placental tissues of miscarriage patients, respectively (Table 6).

Table 5:  Validity of EBV- CISH and EBV- IHC

EBV IHC Score	Miscarriage Group
	EBV CISH
	Negative (n=31)		Positive (n=9)
	No	%	No	%
Negative	26.00	83.90	5.00	55.60
Positive	5.00	16.10	4.00	44.40
P value		0.073

Table 6: Validation of CMV for DNA CISH and for CMV protein IHC among the placental miscarriage tissues.

CMV IHC Score	Miscarriage Group
	CMV CISH Score
	Negative (n=28)		Positive (n=12)
	No	%	No	%
Negative	18	64.30	7.00	58.30
Positive	10	35.70	5.00	41.70
P value		0.722

Discussion

EBV could be the reason of infection to placenta in pregnancy, with consequent complications fetus. Primary and secondary EBV infections may both occur during pregnancy. However, main infections with EBV seems trans placental diffusion were occasional, whereas secondary maternal EBV infections weren’t rare (Avgil and Ornog  2006).

Most of laboratory tests that were performed to diagnose EBV infections (including infections during pregnancy) use molecular techniques (like PCR) and serological tests for EBV antigens or antibodies specific to EBV by ELISA, immunofluorescent technique, or rapid tests (Christian et al., 2012).  In this study, 9 out of 40 (22.5%) of the examined placental tissues were positive for EBV (EBER) by CISH   technique. Also the appearances of EBV-VCA were distinguished at nuclear localization in 22.50% (9 out of 40). In the current study which is  presented here,  a partial measurable CISH method was used for  scoring of bleach growth,  according to the number of placental cells infected with EBV and the results showed that most of infections have  of score I, and this  might represents the comparative mild to reasonable EBV infections of placental tissue through pregnancy as well as these findings were  supported by measurement of color intensity of positive cases were most of the examined  slides were having mild signal  intensity.

In the present study, the positive placental tissues of miscarriage group may indicate recently infected, or lytically reactivated EBV infection, however, the presence of EBER as detected by CISH, does not always correspond with the presence of VCA by IHC.

Immunohistochemistry was applied for detection VCA of EBV in placental tissue samples and it showed compatibility of those obtained by ISH and this might indicate a high compassion and specially the IHC-VCA   test, since the EBER in situ hybridization for detecting of latent EBV infection is considered the gold standard method and as supported by the finding of Truong et. al., 2009. IHC has also been used where  in this regard ,  Xiao and his worker, 2014 have  showed  also  strong agreement for both techniques which  were used  for detection EBV in patients with lupus nephritis.

The present result are supported by Xuan-Hong (2011) who  suggested  that the  possibility of the EBV virus to  transmission  from the  uterus to the fetus , which were  resulting in stillbirth , abortion , or congenital defects.

The current study provide a novel information about the histological localization of EBV nucleic acid in placental tissues using CISH, were  the overall frequency of EBV infection is 22.5 % of the examined placentae, the present result  is higher than those reported by other abroad studies such as that  done by Gervasi and his colleagues (Gervasi et al.,2012 ) who found  viral genomic  sequences of EBV (0.1%) of their examined cases of  the amniotic fluid ( in the mid trimester of pregnancy)  by using quantitative real-time PCR for the presence of EBV-genome sequences. Recent studies done by Song and his Colleagues in 2015, used ISH for detection EBV in lympho-proliferative disorders, nasopharyngeal carcinoma and related malignancies with final color or fluorescence detection however, there is no reported data on detection of EBV by ISH during pregnancy and this signify a novelty for this study(Song, 2015 ).

Human cytomegalovirus is an significant etiological agent of intrauterine infection, which may lead to some thoughtful consequences in pregnant women such as miscarriage, stillbirth, cerebellar malformation and fetus development retardation, as depicted in Staar et., al 2012.

The results of this study was in line with a serological study done in Iraq to discover the association of TORCH infections  in women with spontaneous abortions , CMV Ab prevalence  of  positive  cytomegalovirus  CMV(38.50%) in 2009 and ,  the lowest percentage were observed  only  (29.10%) of cases  in 2010. This could suggest that the exposure to CMV infection was declined over this period in Iraq.   (Majeed, 2011)

The study that was achieved in Baghdad by (Maysara et., al 2012)  to evaluate the prevalence of seropositivity of specific IgM antibody for CMV by ELISA, CMV specific IgM antibody was detected in 15.7% of the 108 women with history of abortion.

In Australia, (Jenna et., al 2015) conclude to  the result that the total CMV DNA was distinguished in 5.0 % of  placenta soft tissue of miscarriage women along with the  infections were confirmed by using the  immune-histochemical assay and viral  proteins.

Additional seroepidemiological research was made in Havana which was made by (Aimée et., al 2015) at maternity hospital by means of marketable ELISA kits has shown that the prevalence of active cytomegalovirus infection was detected in 16.70 %,.

Our results disagree using a  previous outcomes  done by  Sharief,( 2005) , who showed an absolute positive  results of aborted women with a  primary abortion i.e  reveled  highest percentage (100% )  which was  considered  to carry a  high titer of IgG. In developed countries such as  United States it was  stated that HCMV infection at rate of  80% was observed  at age 35-40 years old women who  have anti- HCMV IgG  in comparison to  50% -80% HCMV infection in the  younger age  women  (Gold, and Nankervis, 2007).  The hematogenous way of CMV diffusion in the placenta can clarify the principal infection to this microbe in the variable villi.

The current study revealed that sensitivity of IHC is slightly higher than that of ISH for histological detection of CMV, this might be due to CMV protein over expression (which was studie here) can be detected by IHC but not by ISH, similar result were also obtained by LU DY and his colleagues (2009) found similar findings that the result of IHC is higher than those of ISH; in our opinion the CISH is more difficult to done and this might slightly affect the sensitivity of the procedure.

Yiska et., al (2011) did an Immunohistochemical analysis to infected units to  parental decidua exposed the appearance of CMV immediate-early and pp65 early-late virus-related genetic factor and gB as well which stated late later infection. The control tissues were found negative by immunohistochemical staining. These findings indicate that HCMV, in the infected placental tissues undergoes a full replication cycle.

While this research dominant proportion of control group (6 from 40 using CISH) and (2 from 40 using IHC), this reflect to CMV positivity in these placental soft tissues in controller groups may reflect the need for following the studies values of the infections of such pregnancies both on toddlers and mothers subsequently later the deliveries cause the abortion consequences of placental CMV infection thru pregnancy and postnatal consequence should be well thought-out as well.

The present research follow  the inclusion patients with abortions, and omitted the other results of  infected pregnancy like fetal inherited anomalies and illnesses, it represent a restriction to this study which was established by recording as well as the difficult understanding of CMV infection in the control placental tissue  group.

In study in Iraq , Alwan and  Sera  (2011) have  indicated  that CMV infection rate was (46.6%),These results were  higher than our results ,  this different in the  CMV prevalence rate  may be due to different in the sample  size included in the study group   as well as may be due  to substantial diffusion of infection in local population , perhaps    that CMV  is endemic in Iraq during that period ,  or may be CMV infections  are un common in a particular region  .

The current results obtained from  both techniques   for EBV have revealed that among   Forty  placental tissues  from  miscarriage patients ,   EBER- CISH and VCA-IHC have been found in 22.5% . While  the  detection rates of EBV-CISH and EBV-IHC in placental tissue sections were equivalent  yet the validity results  EBER by ISH where compared to  VCA by IHC  had displayed a sensitivity and specificity of 44.40 % and 83.90 %, respectively,

Also in this study the evaluation of the efficacy of these two procedures but for detection the presence of CMV infection in placental tissues from miscarriage patients. Both CISH and IHC for diagnosing  nuclear inclusions  of  this virus in placental tissues have showed that , CMV-DNA- CISH and CMV-protein -IHC were found in 30% , 37.5 %  respectivly. The comparism  of  the results of  both technique  revealed  a sensitivity and specificity of 41.7% and 64.3%, respectively. The numbers of infected cells scored were slightly higher with IHC than with in CISH technique. These results show that the use of both in CISH and IHC for EBERs and the EBV- VCA as well as CMV –DNA and CMV proteins are both specific, as well as sensitive methods.

The current result of this study are matching with Niedobitek et al.,(1988) result . They used ISH, IHC, and morphological analysis of tissue from patients with AIDS who have widespread CMV infections.  They showed that the evaluation of the results in ISH and IHC were significantly extra sensitive than the morphological analysis.  It was further shown that IHC has shows a higher CMV infection in cells than in CISH. They concluded that IHC as technique appeared to be more suitable to be used.

Several authors have denoted that ISH was considered as a method of optimal to detect EBERs –EBV presence inside tissues section and this is because of  the huge data of duplicates of EBERs present in infected cell where  these works have  showed Positive staining in the nuclei of the EBV-infected cells (Weiss and Chen , 2013;  Hänel et.al., 2001; Margaret  and Weihua , 2008 ).

In addition our data were match with the fact that ISH was considered as the gold standard for precisely identifing the sites of expression of EBV as it and was highly sensitive and specific; however, some authors denoted that ISH is expensive (Lerner et al. ,1981). The results of the current study showed that the two detection techniques had strong agreement, which in turn indicated that the results of both were reliable.

In conclusion CMV and EBV are important causes of placental infections among miscarriage females in Baghdad, and CMV might be detected in placenta of normal delivery. Although  CISH technique considered as the gold standard method for detecting of  latent EBV and /or CMV infection were IHC has showed a  compatibility  to  that technique  and might reach rates of  high sensitivity and specificity similar to it.

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