Modes of Perinatal Infection, Selected Agents and Fetal/Placental Pathology

	Bacteria	Viruses	Other	Placenta	Fetal
Hematogenous	Listeria, syphilis, E. coli	CMV, HSV, VZV, HIV, parvovirus B19, rubella, enterovirus, dengue, HHV-8, West Nile	Toxoplasma Chagas	acute or chronic villitis, chronic vasculitis, necrotizing funisitis	sepsis, viremia, disseminated infection, tissue necrosis (liver, lung)
Ascending	Group B Strep., E. coli, Listeria	CMV, HSV, HIV	Mycoplasma, Ureaplasma, Candida	acute chorio-amnionitisacute funisitis, vasculitis, necrotizing funisitis	cutaneous, pneumonia, gastroenteritis, sepsis, meningitis
Intrapartum	Group B Strep., Listeria, E. coli	CMV, HSV, HIV, HBV, HPV	Candida	chorio-amnionitis	cutaneous, eye, rarely systemic, meningitis
Postnatal	Group B Strep.E. coli	CMV, HSV, HIV, HHV-8enterovirus, dengue		normal	misc., agent specific

Ascending Infection
Most cases of ascending infection are bacterial in origin. Organisms colonizing the cervix, vagina or perineum may infect the maternal endometrium or fetal membranes at the cervical os. While it is true that ruptured membranes carry an increased risk for infection of the amniotic cavity, most cases of premature rupture of membranes are secondary to the membranes being already weakened by infection. Bacteria can cross intact membranes [4]. The structure of the fetal membranes is complex. Amnion and chorion completely surround the gestational sac. The chorion of the free membranes is in direct contact with the maternal decidualized endometrium and the attached chorion is exposed to the maternal blood flow within the subchorionic intervillous space. The chorion has 3 layers and is thicker than the amnion which has 5 thinner layers but greater tensile strength [5].

Most infants born with a clinical or histologic diagnosis of chorioamnionitis are not septic, but may have localized infection in the lungs or within the gastrointestinal tract from aspirated or swallowed contaminated amniotic fluid. The more severe the inflammation within the membranes, the more likely the infant will be septic. Even more predictive for fetal sepsis is the presence of a fetal inflammatory reaction within the umbilical cord (funisitis) or chorionic plate vessels (vasculitis) [2, 6] . Necrotizing chorioamnionitis and three-vessel inflammation of the umbilical cord are associated with an increased incidence of preterm birth and perinatal death [6, 7] . Only 20-33% of women with histologically important inflammation of the membranes are symptomatic [6, 7] . While many infants are not infected they are affected by the fetal inflammatory response syndrome, which may be independently responsible for morbidity and/or mortality [8]

Acute Chorioamnionitis
Benirschke & Kaufmann feel that chorioamnionitis (which they use as synonymous with acute chorioamnionitis) is always (their bold) caused by infection [3]. Other forms of injury to the placenta, such as infarction will also illicit an acute inflammatory reaction, but will be localized to the area of injury. Chorioamnionitis is much more common in preterm placentas; and is responsible for at least 50% of all preterm deliveries [9, 10] . At term, acute chorioamnionitis is only present in 4-10% of placentas [11, 12] .

The gross appearance of the membranes is a very poor judge of inflammation. The membranes are usually macroscopically normal. Infection may result in slight clouding (loss of translucency) of the membranes, granularity or a dull appearance which may be obscured by formalin fixation. Only severe inflammation will result in grossly, thickened cloudy membranes that may be yellow or green due to the pyocyanin within the white blood cells, obscuring the fetal vessels on the chorionic plate. Infection is frequently associated with membrane edema.

The intensity or grade of inflammation used to define chorioamnionitis differs from publication to publication [13, 14] . It has been described very generally as dense, or as specifically as one focus of at least five neutrophils or ten or greater neutrophils in ten nonadjacent fields at 400x magnification within free or attached membranes [13, 14] Defined criteria for examination of membranes in premature preterm rupture of membranes have been reported [15]. A recent publication from the Perinatal Section of the Society for Pediatric Pathology suggests a definition for both grade and stage of acute inflammation [16].

Stage or progression of inflammation is also inconsistent. Naeye uses three histologic stages of chorioamnionitis (I, II, III) to indicate subchorionic inflammation, chorionic inflammation and full thickness inflammation of both chorion and amnion, respectively [17]. Redline also uses 3 stages, but they are slightly different [16]. Early infection may be confined to the maternal decidua near the cervical os, exemplified by a diffuse sprinkling of maternal neutrophils that is often associated with some decidual necrosis. Membrane rupture occurs overlying the cervical os, probably due to unequal stretch or unequal wall tension or possibly due to a loss of decidual blood supply in that area [18]. Some acute decidual inflammation is seen in 85% of term deliveries, especially near the zone of membrane rupture [2]. Inflammation marginated at the junction of the cellular and fibroblastic chorion indicates migration towards the amniotic cavity and is a feature of intra amniotic fluid infection. Maternal neutrophils marginate within the subchorionic fibrinoid, prior to migrating in the chorion and amnion on their way towards the amniotic fluid cavity. Chellam suggests that this is equivalent to margination of inflammatory cells within a vascular space and therefore may represent the earliest maternal response to infection within the amniotic cavity [19]. The inflammatory infiltrate progresses through the chorion (chorionitis) and amnion (chorioamnionitis), and eventually into the amniotic fluid. Since inflammation is chemotactically attracted to stimulus within the amniotic fluid, thickening of the membranes either by squamous metaplasia or placement of the chorionic plate vessels, will focally interrupt this process.

Romero et al, have proposed a four stage, clinical progression of ascending infection [20]. Stage I is an overgrowth of organisms in the vagina and/or cervix. Stage II is localized inflammation of the intrauterine cavity localized to the decidua. Stage III is intra amniotic infection. Stage IV is infection of the fetus, through breathing or swallowing the contaminated amniotic fluid, or through cutaneous infection, including conjunctiva. It would be appropriate to add a stage V to this to scheme to indicate systemic fetal infection (sepsis or meningitis).

Location and appearance of the inflammation has lead to assumptions about duration of infection. In general the inflammatory response is neutrophilic, regardless of the duration of infection; hampering our ability to determine duration of infection. Intact neutrophils have led some to estimate infection of <24 hours [21], as the average life span of neutrophils outside the vascular space is 24 hours [22]. The problem with this correlation is that the neutrophils that migrate into the avascular tissues of the amnion may not disintegrate as quickly as in ischemia tissues which have been studies in acute myocardial infarction. Naeye reports that maternal inflammation is subchorial intervillositis in the first 48 hours of an infection and that only in the next few subsequent days do the neutrophils migrate into the chorionic plate [17]. The maternal inflammatory cells continue their progress towards the amniotic fluid cavity. The time necessary for complete progress from the maternal space to the intra amniotic fluid space has been estimated at 5-7 days, but may occur more quickly with highly virulent organisms [17]. Fox says that it would be imprudent to try to date a chorioamnionitis with any degree of precision [1].

Cultures of the placenta are of limited value, in part due to antibiotic use during labor and especially due to contamination through a vaginal delivery. There is no correlation between organisms isolated from the cervical canal or from the placenta and fetal infection [23]. Microorganisms were not isolated significantly more often from placentas in cases with chorioamnionitis than those without [24]. There was a correlation between increased severity of membrane inflammation and fetal vascular inflammation with increasing incidence of positive cultures [24]. In preterm labor, bacteria can be isolated from amniotic fluid of intact membranes or from placental cultures with variable success [25]. Approximately 70% of placental cultures are positive when there is histologic chorioamnionitis [25]. Any cultures should be sent directly from labor delivery. There is a decreased recovery of organisms with refrigeration and prolonged interval between delivery and cultures. It has been proposed that cultures are most reliable when obtained from the space between the amnion and chorion, thus eliminating surface contamination. Cultures for aerobes, anaerobes, Mycoplasma and Ureaplasma will increase the yield.

Subacute Chorioamnionitis
Recently Ohyama et al, described subacute chorioamnionitis as a specific entity that is associated with prematurity, development of chronic lung disease [26]. In his study 54% of placentas with chorioamnionitis had this form of inflammation, while in my experience it is present in <20%. Subacute chorioamnionitis was defined as a mixed degenerative neutrophil and mononuclear cell infiltrate. Amnion necrosis was present in 40%. Subacute chorioamnionitis was felt to be an indicator of persistent intrauterine inflammation, possibly caused by organisms with low virulence. Rarely an antibiotic treated acute chorioamnionitis may have mixed acute and chronic inflammation.

Chronic Chorioamnionitis
Chronic chorioamnionitis is often associated with chronic inflammatory lesions elsewhere within the placental or decidual tissues, but can occur as an isolated phenomena. It has rarely been described in conjunction with viral infections; HSV [27] , rubella [28], and toxoplasmosis [29]. Chronic chorioamnionitis tends to be most often associated with villitis of undetermined etiology [30]. It is usually focal and rarely involves the amnion connective tissue and does not result in necrosis of the amnion epithelium [31].

Fetal Vasculitis and Funisitis
Fetal inflammatory response of the umbilical cord and chorionic plate vessels occurs after the maternal response and usually suggests a more well established infection. It may be attenuated of absent in midgestation, although I have seen a significant funisitis as early as 18 weeks gestation and rare neutrophils even earlier.

Funisitis can rarely be grossly identified. The umbilical cord may be quite edematous. Within the extra fluid within the cord substance, large accumulations of neutrophils may be visible as white rings incompletely around the fetal vessels. The fetal response to infection within the amniotic fluid is towards the amnion surface (amniotropic), similar to an Ochterlony-type reaction. Concentric inflammation may be the result of cord injury. Funisitis may be segmental due to positioning of the cord within the uterus [21], thus the recommendation for examination of two sections of umbilical cord from different areas.

An acute fetal inflammatory response is most often a response to ascending bacterial infection. Chronic vasculitis or funisitis is less common, but may occur in cases of hematogenous acquired viral infection [32]. In severely macerated fetuses, caution should be used in diagnosing funisitis, as degeneration of the vascular smooth muscle may falsely give the impression of inflammation. Acute inflammation of the chorionic plate vessels, may precede inflammation of the umbilical vessels. Gross examination rarely demonstrates an amniotropic intravascular density. Inflammation of the chorionic plate vessels is usually obscured by the associated chorioamnionitis. Inflammation within the amniotic fluid is mixed maternal and fetal. Most of the fetal cells come from the chorionic plate vessels [33]. Chronic fetal vasculitis is most often a nonspecific response, associated with villitis of undetermined etiology. Inflammation of the chorionic plate, may therefore be a combination of maternal and fetal inflammation. The fetal inflammatory response frequently includes eosinophils as an acute reaction, not only in the preterm [19], but in the term baby as well. This may be due to the small pool of neutrophils and the presence of large amounts of eosinophilic extramedullary hematopoiesis within the liver. The most severe consequence of inflammation of the chorionic plate vessels is thrombosis.

As with chorioamnionitis, no uniform definition has been accepted but van Hoeven defined funisitis are presence of neutrophils within the vessel wall, with or without extension into the substance of Wharton's jelly, simple margination of neutrophils was excluded from their definition [6]. Redline has also proposed 3 stages and 2 grades, but combines the chorionic plate and umbilical vessels. The umbilical vein becomes inflamed first, then the arteries [34]. Inflammation begins as margination of neutrophils at the endothelium with progressive movement through the muscle wall into the cord substance.

Necrotizing Funisitis
Necrotizing or sclerosing funisitis is evidence of a prolonged fetal inflammatory response. Fetal neutrophils that have migrated out of the umbilical vessels towards to amnion surface undergo degeneration, necrosis, and finally calcification. Lack of lymphatic drainage of the umbilical cord results in accumulation of debris. The etiology has been attributed to a number of organisms, which have in common, the ability to result in prolonged infection without spontaneous uterine contractions. In many cases no infectious cause is identified. The most common etiology is syphilis [35], but it has also been reported in HSV [36], and cultures have been positive with common organisms such as group B Streptococcus and Gardnerella, many with a history of prolonged rupture of membranes [37]. There was no statistically significant correlation between the degree of necrotizing funisitis and fetal outcome; however poorer fetal outcome is suggested with severe necrotizing funisitis, including a high rate of intrauterine growth restriction, stillbirth and necrotizing enterocolitis and chronic lung disease [26, 38] .

Other Causes of Inflammation of the Umbilical Cord
While acute chorioamnionitis is thought to be exclusively due to infection, funisitis may be due to other things. In the case of cord compression, inflammation may be secondary to tissue injury. Theoretically, the inflammation cells should be evenly distributed around the injured vessel, not in the usual amniotropic pattern. Meconium laden macrophages have been identified within Wharton's jelly and are associated with smooth muscle injury and inflammation of the umbilical cord vessels (39). Meconium associated inflammation is usually more severe in the cord than the membranes. This may be due to direct injury of the cord vessels by meconium. It has been postulated that the muscle injury is secondary injury due to vasoconstriction. The presence of meconium within the fetal lung may also set up an inflammatory response which is then manifest within the cord vessels (40).

Intrauterine Acquired Maternal Hematogenous Infection
Infectious agents within the maternal blood may infect the placental villi from the intervillous space. In order for this to happen, there must be maternal bacteremia, viremia or parasitemia. Rarely villitis may be seen as a late complication associated with chorioamnionitis as the result of fetal sepsis and reinfection of the placental villi. Most cases of villitis are idiopathic, referred to as villitis of undetermined etiology (VUE) [41]. Infectious etiologies are most often viral. Infection may stay confined to the placental tissues are enter the fetal blood stream through the villous capillaries. There are several proposed mechanisms for how the infectious agents gain entrance to the fetal system. The two most commonly accepted theories include either the direct transport of infected maternal cells or free pathogen into the fetal circulation, or contiguous infection of a placental cell with subsequent infection of fetal cell. Placental factors that control this include physical barriers (villus trophoblast epithelium), immune mechanisms (probably related to both maternal and fetal immune competence) especially the phagocytic and killing capabilities of the Hofbauer cell.

The gross appearance of a placenta with villitis is usually normal. There may rarely be a granular, hydropic or pale appearance of the parenchyma. Increased thickness of the basal plate has been noted [42], and is most commonly seen in basal villitis. In cases of abscess formation, white nodules may be visible on the basal plate or cut surface. Pallor or friability of the villi may be due to widespread necrosis, atrophy and fibrin deposition, but is nonspecific and may be due to prolonged retention after fetal demise, or immaturity of the placenta [41, 43] . Villitis is usually considered a microscopic diagnosis. Examination of four blocks of placenta is sufficient for detection of the majority of cases of villitis [44].

Various classifications of villitis have been proposed. Altshuler has described villitis based on tissue response as proliferative, necrotizing, reparative and stromal fibrosis. It was proposed that these may be progressive stages of villitis with necrosis as the initial acute stage and fibrosis the end point of the various forms [45]. Villitis can be described by the type of inflammation; acute (neutrophilic), chronic (lymphocytic, histiocytic, lymphohistiocytic, plasmacytic, mixed), or granulomatous (including Langhans type giant cells). Categorizing the villitis on the type of inflammation is more useful in determining the etiology. The villous inflammation is a combination of maternal and fetal inflammatory cells [46]. The number of inflammatory cells within the villus necessary to diagnose villitis has not been specified.

Most cases of villitis are thought to be an immune reaction against fetal/placental antigens and are referred to as nonspecific villitis or VUE. The lymphocytes within the stroma are predominantly helper/inducer CD3+ or CD4+, T-cells, with few suppressor, CD8+ T-cells, CD45/LCA positive and CD68 macrophages [47, 48, 49, 50] and no B-cells or plasma cells. The placenta has a limited ability to respond specifically to various antigenic stimuli. The histology of villitis is sufficiently varied to suggest that the entity designated as VUE may well be a response to more than one antigen/organism. Anchoring villi at the placental base are intimately associated with maternal tissues and do not have a protective syncytiotrophoblast layer. In this way the anchoring villi may be more susceptible to maternal immune stimulus and do contain more inflammatory cells compared to non-basal villi in normal placentas [48]. Involvement of the stem villi occurs in about 10% of cases. This is usually associated with a higher grade of villous inflammation. Intercellular adhesion molecule 1 (ICAM-1) is not normally present on villous trophoblast, but becomes so in the inflamed villi. ICAM is involved in lymphocyte-endothelial interactions [50].

VUE has been reported in 5%-18% [3, 51] of placentas. The lower incidence is from random placentas and the higher incidence is from placentas that were selected for examination, based on abnormal maternal or fetal features. The only consistent finding in infants is IUGR [3]. There is some correlation between severity of villitis and severity of IUGR [52]. Fetal/neonatal IgM has not been found to be elevated, as you would expect for an in utero exposure [3].

Villitis attributable to specific infectious agents are the exception, probably accounting for only 5% of all villitides [2]. Acute villitis is seen in maternal sepsis with organisms such as E. coli, group B Streptococcus and Listeria monocytogenes. The inflammatory infiltrates in VUE and known infectious etiologies are similar [49] and has provided "circumstantial evidence that VUE is the result of chronic infection" [52]. Most investigators are less convinced of an underlying pathogen in most cases of VUE. The most significant difference in VUE and the specific villitides appears to be the presence of significant numbers of plasma cells which should be a warning to rule out infectious agents, particularly CMV, syphilis EBV and HSV. Infectious etiologies of villitis are often associated with a significant increase in the number of nucleated red blood cells within the villous vessels. Granulomatous villitis, may be seen in infection with organisms that cause granulomatous inflammation elsewhere; as in mycobacterium, toxoplasmosis, HSV and varicella.

Special Stains and Molecular Techniques in the Diagnosis of Infectious Agents.
There have been considerable advances in the use of molecular techniques to rapid detection of pathogens, especially those that are fastidious or non-culturable. Some of the rewards of diagnostic molecular techniques include rapid turnaround time, increased specificity, enhanced sensitivity, ability to identify esoteric microorganisms, quantitation, genotyping, and monitoring of drug resistance. Qualitative assays determine presence or absence of a nucleic acid consistent with the presence of an infectious agent. Polymerase chain reaction (PCR) replaces the conventional process of biologic amplification (growth in culture) with enzymatic amplification, allowing identification of as few as 100 copies of a particular DNA sequence [53]. Reverse transcription PCR may be useful in detection of RNA virus infections including hepatitis C virus, HIV and enteroviruses [54, 55] . Because of the sensitivity, contamination is a constant threat and specimen integrity is very important. These techniques are labor intensive and expensive. Most of the newer techniques allow for extraction of DNA from fixed tissues, it is still preferable in some cases to have fresh or frozen samples. It must be remembered that a positive PCR does not mean the same thing as a positive culture (viability), or a rise in antibody titer (response to recent infection). Quantitative assays are useful for correlating the number of nuclei acid molecules with clinical disease and monitoring responses to therapy. Sequence analysis can be used to subtype or detect drug resistance [53]. More readily accessible to most laboratories is immunohistochemistry (IHC) to identify specific antigens (CMV, parvovirus, toxoplasmosis, syphilis, GBS) within formalin-fixed, paraffin-embedded tissue samples [56, 57, 58, 59, 60] . I have found CMV and parvovirus IHC to be the most useful, especially identifying positive cells in autolyzed tissues.

Less sophisticated methods may also be useful in identifying infectious agents. Many are readily identifiable on routine hematoxylin and eosin (H&E) stain. Tissue gram stains such as Brown and Bren (B&B) are useful for Gram-positive organisms but Brown & Hopps (B&H) or the Gram-Twort method is preferable for Gram-negative organisms. Silver impregnation methods (Steiner & Steiner, Warthin-Starry) will stain virtually all bacteria and most fungi; and are therefore not specific but sensitive methods for identifying small numbers of bacteria. The advantage of silver is that very thin organisms are coated with the metal, making them visible with light microscopy, making is particularly applicable to Fusobacterium and syphilis. Treponema spirochetes are identified within the necrotic debris of the necrotizing funisitis of the umbilical cord, but organisms have also been identified within cord which is free of inflammation [58]. If the patient has been treated within a few hours of delivery, no organisms will be seen. Histologic examination alone may be as specific and nearly as sensitive as the PCR performed on formalin-fixed, paraffin-embedded tissues. Steiner & Steiner stain and Warthin-Starry stains are difficult to perform and yield fewer positive results compared to PCR (61).

Specific Perinatal Viral Infections

Cytomegalovirus (CMV)
CMV is the most common viral infection acquired in utero. CMV may also be acquired during the birth or with injection of breast milk. The placenta is grossly unremarkable or may be hydropic and pale. Meconium staining of the fetal membranes is frequently seen [62, 63] . The villi may have appropriate maturation or reflect immaturity. The villitis is usually distinctively lymphoplasmacytic but may also be absent or granulomatous [62, 63] . CMV has an affinity for endothelium that will result in vascular injury and hemosiderin deposition within the villous stromal or Hofbauer cells. Syncytiotrophoblast necrosis may result in increased perivillous fibrin deposition [63]. Calcification of sclerotic blood vessels, stroma or decidua may be seen [64]. The severity of the fetal infection is usually reflected in the severity of the placental changes [63, 65] .

Characteristic viral inclusions, are present in 43-75% of the cases [63, 64, 65] , and are identifiable within Hofbauer cells or villous endothelium and rarely syncytiotrophoblast or cytotrophoblast on routine H&E stain. Virus may be detected in cells without characteristic Cowdry inclusions by immunohistochemistry [65, 66] in situ hybridization [67], polymerase chain reaction [68] or immunofluorescence [62]. In situ hybridization is less specific as it detects unencapsulated, intranuclear viral DNA where as immunohistochemistry detects DNA contained within complete CMV nucleocapsid of replicating virus. Nuclear and cytoplasmic staining is seen [65, 69] . CMV probably accounts for only 1-6% of all villitides [68]. Endometrial and cervical cells are frequently culture positive in seropositive women. Infection of the placenta may occur directly from the infected endometrium.

Seropositivity varies from 59-80% and reactivation occurs in 1-15%. Maternal risk for infection during gestation is approximately 2%. Risk for transmission to the fetus is greatest with primary infection [35%], equal during all trimesters, worse with earlier infection. Fetal infection during reactivation is rare (1-3%), there is also consideration for reinfection with a different strain. 0.5-3% of newborns are infected (many postnatally through breast milk) with CMV; 93% are asymptomatic. Those asymptomatic during the neonatal period have 10-15% incidence of long term sequelae, including neurosensory deafness (progressive, uni or bilateral), chorioretinitis, neurologic abnormalities, and learning disabilities. Symptomatic infants may be stillborn, neonatal death, premature, low birth weight, have thrombocytopenia, purpura, jaundice, hematomegaly, anemia and intracranial calcifications (ependyma is especially vulnerable). Outcome for symptomatic infants is poor with 60% hearing loss, 45% mental retardation, 35% cerebral palsy and 15% chorioretinitis. Neuroimaging may show multifocal lesions in the deep parietal white matter, with or without cysts, ventricular dilatation and gyral abnormalities.

Co-Infection of CMV and HIV
There has not be an increase in CMV infection during pregnancy in our cohort of HIV positive women and this is supported by others [70, 71] . Caselli, et al reported an infant co-infected with CMV and HIV who was asymptomatic at birth [72]. We have had two cases of congenital CMV associated with intrauterine fetal demise in HIV infected women with advanced disease. Both had serologic confirmation of prior CMV infection and thus are recurrent cases with severe consequences to the fetus. HIV status of the fetuses was not determined.

Human Immunodeficiency virus (HIV)
There are no specific features of the placenta in HIV infected women [73, 74] . There is a higher rate of acute chorioamnionitis in HIV infected women, particularly those who have AIDS-defining characteristics [75]. Increased transmission of HIV to the infant has also been associated with histologic chorioamnionitis [75, 76] .

The chorioamnionitis and funisitis appears to be secondary to usual ascending bacterial infection. Rarely acute or chronic villitis has been reported [74]. Viral load within the placenta is very low and routine IHC techniques for demonstration of p24 antigen and gp41 antigen consistently are negative. In situ hybridization and in situ PCR have been more successful in demonstrating virus within placental tissues [77]. The placenta acts as a good barrier to infection and demonstration of viral antigens in placental tissues does not predict fetal infection [78] cells found to contain HIV include syncytiotrophoblast, cytotrophoblast, Hofbauer cells and endothelial cells [78, 79] .

Herpes Simplex Virus (HSV)
Perinatal infection may occur with either HSV-1 or HSV-2. Herpes is the only virus that has been well documented to cause in utero and ascending types of infection. Most infants are infected during primary genital infection, via an ascending route or at the time of delivery; infection rate is 50-75%. The risk for ascending infection is increased with prolonged rupture of membranes. In utero infection occurs rarely during the viremic phase of a primary HSV-2 infection [80]. Recurrent genital infection carries a very low risk for fetal infection (5%) [81]. The gross placenta is usually normal, or in the case of primary in utero infection, may be hydropic.

Necrotizing funisitis containing neutrophils and nuclear debris admixed with eosinophils, lymphocytes and plasma cells has been reported [82]. When two rings were present the inner contained neutrophils and eosinophils and the outer ring nuclear debris, lymphocytes and plasma cells [82]. Acute necrotizing chorioamnionitis, with necrosis of the amniotic epithelium may also be seen [82]. Focal lymphoplasmacytic infiltrates in the decidua basalis with multiple foci of bland decidual necrosis both basalis and capsularis. Multifocal coagulative necrosis of villi with minimal or absent inflammation is reported [83].

I have seen one placenta and fetus with confirmed transplacental infection. Necrotizing endovasculitis with rare cells with characteristic intranuclear inclusions were seen in the umbilical cord or large stem vessels. Large numbers of "smudged"were noted in the Wharton's jelly, just beneath the amnion epithelium. Both of these cell types were positive by IHC. Such transplacental infection may result in diffuse skin lesions and severe hepatic necrosis. In the case of ascending infection, a few multinucleated giant cells were found within the connective tissue of the membranes. Similar findings have been reported by others [84, 85] . Acute or plasmacytic chorioamnionitis has been also reported [2].

HSV is easily cultured and grows very quickly in many different cells lines. Direct immunofluorescence performed on scrapings from lesions is sensitive and specific. Immunohistochemistry is widely available and useful [86]. In situ hybridization is sensitive for cells in tissue sections and lesion scrapings, but not widely available. PCR is highly sensitive and specific, especially for detection of viral DNA within small brain biopsies or CSF. None of these techniques has been applied to the placenta in large numbers [86, 87, 88] .

Babies with congenital HSV infection are usually asymptomatic at delivery. Onset of fever and seizures occurs within the first few days of life. Persistent fever in the presence of antibiotics should suggest HSV. Skin, eye or mucosal lesions may be present, and are most commonly seen in the area of the presenting part (scalp), often mistaken as lesion from fetal scalp electrode. Disseminated disease affects the fetal liver primarily, but necrotic lesions will also be present in lungs and adrenal glands. Seizures are usually secondary to hepatic encephalopathy. Pregnancy is infrequently associated with an increased risk for disseminated disease and maternal death due to fulminant hepatic failure.

Human Herpes Virus 8 (HHV-8)
Selected populations with a high prevalence of HHV-8 have been studied to determine whether vertical transmission occurs. 15% of seropositive women had HHV-8 DNA detectable within their peripheral blood mononuclear cells (PBMC). 0-16% of baby samples drawn at birth showed HHV-8 DNA within PBMC, suggesting infrequent perinatal transmission [89]. Acquisition of antibody to HHV-8 occurs in older children, implying a horizonal route as the more common form of transmission [90, 91, 92] . A high incidence of co-infection of HHV-8 and HIV suggests sexual transmission.

Epstein-Barr Virus (EBV)
EBV infection is relatively common among women of child-bearing age. Congenital EBV has been described resulting from maternal infection in the first trimester and less commonly in the third trimester [93]. The placenta from first trimester infection, has been reported to show slight to moderate chronic chorionitis, lymphohistiocytic and plasmacytic with large atypical vacuolated cells probably Hofbauer cells [94]. Inflammation of the fetal vessels has been described as endovasculitis, perivasculitis with occasional vascular obliteration [94]. We have had one case of IUGR with PCR positive amniotic fluid for EBV. The placenta showed chronic intervillositis with a rare intervillous macrophage positive with in situ hybridization for EBER.

Hepatitis B virus (HBV)
Hepatitis B virus usually infects infants perinatally either through transfusion during labor or as a result of exposure to vaginal secretions during delivery [95]. Rare cases of intrauterine transmission can occur [96]. Lucifora et al showed strong reactivity of the Hofbauer cells and villous endothelium, with IHC but no pathologic changes were noted [97]. A jaundiced patient with HBV showed a deep yellow-green staining of the villous Hofbauer cells, trophoblast and membranes [3].

Hepatitis C virus (HCV)
The prevalence of HCV in the general population is 0.2%, but is 1.5% in HIV positive individuals [98]. Vertical transmission of HCV occurs only in the presence of positive HCV-RNA and is higher in HIV-positive mothers than HIV-negative mothers. There is also a higher rate of HIV transmission in the presence of HCV infection. Rarely infants will clear viremia, while most will remain viremic. Liver dysfunction has been noted as early a 1 year of age. Breast milk is not known as a vehicle for HCV transmission.

Varicella- Zoster Virus (VZV)
Placentas from three women who had onset of symptoms of varicella from 27 weeks before delivery to one day postpartum, were examined by Qureshi and Jacques. One placenta showed extensive basal chronic villitis with a lymphohistiocytic infiltrate and occasional multinucleated giant cells [99]. Garcia examined three macerated stillborns that showed tiny, yellow, scattered lesions on the placental surface that were large areas of granulomatous villitis. Homogeneous intranuclear inclusions were found in the decidual cells [100]. First trimester varicella infection with subsequent fetal demise demonstrates, acute, chronic or granulomatous villitis with eosinophilic "glassy" appearing nuclei, confirmed by IHC [101, 102] . PCR has been utilized for prenatal diagnosis using chorionic villus sampling [103].

In utero infection is associated with skin scarring, disseminated foci of necrosis, hypoplasia or flexion contractures of limbs and fetal demise [104]. Mothers who develop a varicella rash 5 days before to 2 days after delivery and this carries a great risk to the neonate. Up to 20% of these infants will develop neonatal varicella which carries a 20-30% mortality rate. This is associated with stillbirth and preterm delivery, hemorrhagic pneumonia, and neonatal mortality but no structural defects. Higher rate of maternal pneumonia and mortality are also noted during pregnancy. VZIG can prevent up to 50% of infections and reduces severity in remainder.

Rubella
Most of the placental findings of congenital rubella are those associated with early spontaneous or elective terminations. Focal damage of the syncytiotrophoblast, cytotrophoblast and endothelium with eosinophilic inclusions in trophoblast, decidua and Hofbauer cells have been reported [105]. Acute and chronic villitis may be present [106, 107, 108] placental lesions do not correlate completely with fetal infection [109]. Identification of rubella viral RNA is sensitive in early pregnancy, but may disappear from the placenta in a term pregnancy, infected earlier [110].

Garcia et al, described sparse focal areas of hyalinization and lymphocytic infiltrate in umbilical vessels and stem vessels [105]. Rubella viral intracytoplasmic or intranuclear eosinophilic round inclusions were seen in cytotrophoblast and decidual cells. Focal, basal, necrotizing villitis, was seen. Indirect immunofluorescence done on formalin-fixed, paraffin-embedded tissues and was positive in most cases within the amnion epithelium, chorionic vessels, and subchorionic fibrin [105]. A review of all cases submitted with a diagnosis of congenital rubella were examined at Texas Children's Hospital and the AFIP and no viral inclusions were identified within placental tissues on routine H&E stains.

There are an estimated 100,000 cases of congenital rubella worldwide, primarily in developing countries with low immunization rates. Congenital rubella syndrome is characteristically IUGR, failure to thrive, mental retardation, cardiac defects (PDA, ASD, VSD), hepatosplenomegaly, thrombocytopenia, microcephaly, encephalitis, mental retardation and cataracts. The most significant sequelae are blindness and deafness. Risk for fetal infection is greatest during the first month of gestation (50%) and essentially 0% after 20 weeks gestation. Pathology is that of widespread chronic inflammation and scarring, with vasculitis and calcifications noted only in the brain.

Measles
Chandwani, et al. showed two placenta with focal chronic villitis and intervillositis. Multinucleated giant cells containing intranuclear inclusions were confirmed as paramyxovirus on electron microscopy [111].

Parvovirus B19
Parvovirus B19 is a known cause of fetal anemia, nonimmune hydrops fetalis and intrauterine fetal demise. It has been predicted that up to 20-30% of hydrops may be due to parvovirus [112] The virus binds to an antigen of the P-system blood group known as P antigen or globoside, which is present on erythrocytes, erythroblasts, megakaryocytes, endothelial cells, placental trophoblasts and fetal liver and heart cells [112]

The placenta grossly is enlarged, thickened and pale, relatively nonspecific features of hydrops [113]. The severity of the anemia in fatal cases, is so severe as to render the placenta nearly colorless.

Microscopically the villi are massively edematous. The capillaries are usually packed with nucleated cells, often so immature as to not have hemoglobinized cytoplasm and are nearly indistinguishable from myeloblasts. The most characteristic cell is a nucleated red blood cell with well hemoglobinized cytoplasm. The retained nucleus contains a characteristic eosinophilic inclusion with a band of dense chromatic at the periphery. Early nuclear inclusions are basophilic. While theoretically possible for infection of multiple cell type, the erythroblast is the only cell microscopically infected [113, 114, 115] . Giant pronormoblasts may be an example of the toxic effects of the virus in a cell that is not fully permissive for viral replication [116].

Immunohistochemistry for parvovirus is very useful in suspicious cases, as all phases of nuclear inclusions are positive and degenerative changes can be ruled out [117, 118] . Most sensitive methods such as PCR, rarely identifies a case not already characterized by the intranuclear inclusions [115, 118, 119] .

Congenital parvovirus was once thought to be an all or none phenomena; but in utero infection can occur without adverse consequences. The classic presentation is hydrops fetalis with marked fetal anemia. The bone marrow is hypocellular and there may be increased extramedullary hematopoiesis. Intranuclear inclusions are seen in immature red blood cells throughout all organs. Ascitic fluid may be bile stained. Rare infants who are symptomatic at birth have shown a neonatal hepatitis-like picture. Infected, asymptomatic infants do not show any long term sequelae.

Enterovirus
Enteroviruses including coxsackie virus, echovirus, enterovirus, and poliovirus may cause intrauterine infection which may result in abortion, neonatal illness or possibly congenital anomalies, but most babies are asymptomatic [120, 121] . The possibility of long term neurologic sequelae has yet to be confirmed [54, 122] . Diagnosis relies on suspicion and a positive cell culture, however molecular techniques are becoming more available, such as RT-PCR, and RT in-situ PCR, for detection of viral RNA.

Garcia et al, have review the only large series of placentas from enteroviral infections [123, 124] Microscopically there was mild acute and chronic chorionitis. Fetal vasculitis was present in all cases, sometimes associated with myocytolysis, edema, perivascular infiltrate and neutrophils marginated within the vessel wall. Villitis involving single or multiple villi could be acute or chronic and have massive areas of necrosis associate with intervillositis. IHC detected positivity in the cytoplasm of the cytotrophoblast, but by electron microscopy virus was found in the syncytiotrophoblast [123].

Human papilloma virus
HPV has been identified in syncytiotrophoblast of first trimester spontaneous abortions, but not in third trimester placental tissue, by PCR [125]. Perinatal infection occurs during the delivery process with the upper respiratory tract most severely affected. Recurrent laryngeal papillomas necessitating multiple resections and rarely transformation to squamous cell carcinoma has occurred.

West Nile Virus (WNV)
WNV is a single-stranded RNA flavivirus and spread by the mosquito. Infection during pregnancy is associated rarely with spontaneous abortion or neonatal illness. Chorioretinitis and cystic lesions of the brain have been reported [126].

Dengue Infection
Dengue is a also a flavivirus and is spread by a mosquito vector. Vertical transmission of dengue has been documented with primary and secondary infection resulting in neonatal fever, thrombocytopenia and elevated liver enzymes [127, 128] . All cases have been associated with maternal febrile illness at the time of delivery.

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