The use of direct immunofluorescence and nested polymerase chain reaction in diagnosing perinatal infections of Chlamydia trachomatis

Results. The presence of C. trachomatis, irrespective of the origin of the swabs (ocular or from the throat), was confirmed in 62 newborns, amounting to 57.6% of the tested population. The occurrence of C. trachomatis in ocular swabs was confirmed in 35 children (32.7%). In the material taken from the throat, there were 48 newborns considered chlamydia-positive (44.9%). In the specimens taken from both the ocular and pharyngeal locations, there was a higher proportion of positive results while using the nested-PCR method in comparison to the DIF test. The specificity of the DIF method with reference to the nested-PCR was 67.9% for ocular swabs. In the material taken from the throat, the sensitivity of the DIF method with reference to the nested-PCR was 75.0% and the specificity was 62.1%.


Introduction
Infections of Chlamydia trachomatis (C.trachomatis), caused by oculogenital serotypes D-K, are among the most common sexually transmitted diseases (STDs).It is estimated that there are approx.100 million new cases of C. trachomatis infection every year worldwide. 13][4] Moreover, it is suggested that pregnant women in this age group undergo testing for C. trachomatis during their 3 rd trimester of pregnancy. 4he oculogenital serovars (D-K) of C. trachomatis in newborn infants may be responsible for developing conjunctivitis and interstitial pneumonia, with conjunctivitis occurring more frequently.These include perinatal infections that take place during the passage of the newborn infant through the infected mother's cervix. 5,6However, 1 case of C. trachomatis conjunctivitis was reported in a newborn infant delivered by cesarean section, which argues in favor of intrauterine infection by the continuity of tissues. 6The risk of C. trachomatis perinatal infection in newborn infants is estimated at approx.30%. 5 chlamydial etiology should be considered if the mother was infected in the past.Neonatal conjunctivitis occurs in 18-50% of children of infected mothers, with frequent occurrence in preterm infants, who are at risk, since chlamydia infections may cause premature labor. 7The infection develops up to 3 weeks after birth and may become chronic.Its characteristic symptoms include mucopurulent discharge from the conjunctival sac with accompanying swelling and redness, but the infection can also be asymptomatic.Conjunctivitis, if left untreated, may lead to blindness. 8hlamydia trachomatis conjunctivitis and pneumonia coexist in up to 1/2 of ill newborn infants.Interstitial pneumonia develops between the 3 rd and the 12 th week of age, and can vary in intensity.In most cases, the disease is mild, but breathing disorders may at times require oxygen therapy.High levels of eosinophils can be reported in the blood.A large number of B lymphocytes and plasma cells in the blood, which is characteristic for C. trachomatis pneumonia, results in high levels of immunoglobulin M (IgM) antibodies in the blood.The changes can be seen in spirometry and in radiological examination.The symptoms include a dry cough, a low-grade fever and rapid breathing. 6,7,9It is suspected that C. trachomatis infection may be related to sudden infant death syndrome (SIDS). 7,10nfrequently, C. trachomatis in newborn infants can cause infections of the vagina, rectum, nasopharynx, or middle ear.

Aim of the study
The aim of the study was to estimate the frequency of infection caused by C. trachomatis in newborn infants.

Material and methods
The experimental material consisted of swabs collected from newborn infants at the 1 st Department and Clinic of Gynecology and Obstetrics (Wroclaw Medical University, Poland).A total of 109 children were tested.Four swabs were collected from every newborn infant: 2 from the eye and 2 from the throat.We used a direct immunofluorescence test (DIF) -Chlamydia Pathfinder (Bio-Rad Laboratories, Marnes-la-Coquette, France) and the nested polymerase chain reaction technique (nested-PCR) -PCR-Chlamydia trachomatis test (DNA Gdańsk, Poland) in order to detect chlamydia infection.Two hundred and eighteen tests were conducted by DIF (109 eye swabs and 109 throat swabs), and the same number of tests was done by nested-PCR.All tests were performed in the Chlamydia Research Laboratory, Department of Basic Sciences, Wroclaw Medical University, Poland.
The quality of the tested material is of great diagnostic importance in the case of C. trachomatis.Due to the fact that chlamydiae are obligate intracellular parasites, the obtained material must contain epithelial cells.Detecting bacteria in the absence of epithelial cells provides unreliable results.Neither eye fluid nor saliva constitute suitable test material.
Since epithelium was not detected in the swabs taken from 2 patients, we did not take into account the results from these samples.Altogether, we analyzed 428 results.
The swabs were collected by authorized personnel in accordance with the appropriate procedures.
Comparing the results of the eye swabs to those of the throat swabs, the measure of compliance was applied.Comparing the results of the DIF method in reference to the gold standard -the PCR method -the results of compliance, sensitivity, specificity, and positive and negative predictive value were specified, and 95% confidence intervals (CI) were estimated for these results.Compliance measurements were also analyzed with McNemar's test.
A probability of p < 0.05 was determined to be significant and a level of p < 0.01 was determined to be highly significant.

Results
We found chlamydia in both the eye and throat in 19.6% of all newborn infants (21 patients).Chlamydia was not detected at all in 42.1% of newborn infants (45 patients).
In the material taken from both the eye and throat, there was a significantly higher percentage of positive results obtained by nested-PCR (34/107 for the eye, 45/107 for the throat) than by DIF (1/107 for the eye; 12/107 for the throat) (Table 1).
The presence of C. trachomatis in eye swabs was reported in 35 newborns, i.e., in 32.7% of patients.There was only 1 positive result in DIF.In this sample, the nested-PCR results were negative, which is equivalent to the lack of consistently positive results in eye swabs.We reported 72 consistently negative results (68.2%) and 35 inconsistently negative results (32.7%) (Table 2).
In the medical interview, the respondents reported the occurrence of additional factors that could increase the likelihood of neonatal chlamydia.Newborn infants without such additional factors accounted for 57.0% of all patients (61 patients).We isolated several groups on the basis of a history of diabetes in the mother or the occurrence of specific symptoms accompanying chlamydia infection in the child (or even previously confirmed C. trachomatis infection).Among participants with no family history, chlamydia was reported in 36 patients (33.6%).
In the medical interview, 17 mothers of newborn infants (15.9%) who were tested during pregnancy or before pregnancy reported previous C. trachomatis infection.Chlamydia was detected in newborns in 23.5% of cases, whereas in 3 out of 4 cases, positive results were obtained from both the eye and throat, but only by nested-PCR (Table 3).
We reported symptoms of respiratory tract infections in 7 newborn infants, including 42.9% (3 newborns) with positive nested-PCR results taken from the throat, andin 1 case -from the eye.In 3 newborns, we did not report C. trachomatis in either the eye or throat.
In 6 newborn infants, we reported symptoms related to eye infection, whereas in 3 newborns (50.0%), we reported positive results from the eye, and in 1 case from the throat alone.In 2 patients, we found negative results despite the occurrence of symptoms.
In patients with eye or respiratory symptoms, we obtained positive results mainly by nested-PCR.In most of these cases, in both the throat and eye, DIF testing gave negative results (in all 6 newborns with ocular symptoms and in 6 out of 7 newborns with respiratory symptoms).
We carried out a follow-up examination in 16 newborn infants (15.0%).Chlamydia trachomatis was confirmed in 9 cases (56.3%) by nested-PCR, while there were no positive DIF results.
The compatibility (accuracy) of diagnosis for both methods equaled 87.85% (95% CI = 80.12-93.37%),which refers only to the "negative" results.However, in any case, the "positive" results were not consistent in both measurements.This is mainly due to the fact that the DIF method for eye swabs showed only 1 positive result.Among the results obtained by the DIF method for the throat, 12 positive results were reported.The results of McNemar's compliance test indicated a significantly high level of inconsistency of the results for both measurements (χ 2 = 7.69; degree of freedom (df) = 1; p = 0.0055) (Table 4).
The compatibility (accuracy) of diagnosis for both methods equaled 67.29% (95% CI = 57.54-76.05%),which refers only to the "negative" results.However, in any case, the "positive" results were not consistent in both measurements.This is mainly due to the fact that the DIF method for the eye swabs showed only 1 positive result.Among the results obtained by the PCR method for the eye, there were 34 positive results.Accordingly, the sensitivity of the DIF method for the eye compared to the PCR method for the eye equaled 0%; the specificity equaled 98.63%  (95% CI = 92.60-99.96%).The positive predictive value was 0% and the negative predictive value was 67.92% (95% CI = 58.16-76.66%).The results of McNemar's compliance test indicated a significantly high lack of consistency of results for both measurements (χ 2 = 29.26;df = 1; p < 0.0001).

Discussion
Researchers rarely raise the subject of the occurrence of C. trachomatis in newborn infants, but studies carried out on newborn infants born to healthy mothers are even less frequent.This may result from the fact that conjunctivitis, the most common form of chlamydia in newborn infants, usually presents mild symptoms and can resolve spontaneously.Another reason may be associated with the reluctance of some parents to take material from their children if there is no such need.Swabs, washes or aspirates from the nasopharynx and eyes are commonly used in diagnosing C. trachomatis infections in children.Competent eye or throat swabbing, even if it causes the child's discomfort, is necessary to achieve research reliability.
In 2012 and 2013, in order to diagnose perinatal infections, Frej-Mądrzak et al. examined the material collected from 55 children. 11The authors chose DIF as the research technique.The material included 33 eye swabs, 19 throat swabs and 11 urethra swabs.The authors reported 1 positive result in the throat swab (1.8%).

Table 1 .
Test results for the presence of Chlamydia trachomatis by the method used and the source location of the sample

Table 2 .
Consistency of results obtained by nested-PCR and DIF DIF -direct immunofluorescence test; PCR -polymerase chain reaction.

Table 3 .
The presence of the risk factors of Chlamydia trachomatis infection in the tested material and the incidence of positive results

Table 6 .
Summary of the results of DIF -eye and PCR -eye

Table 4 .
Summary of the results of DIF -eye and DIF -throat

Table 5 .
Summary of the results of PCR -eye and PCR -throat

Table 7 .
Summary of the results of DIF -throat and PCR -throat DIF -direct immunofluorescence test; PCR -polymerase chain reaction.