Abstract
The aim of this study was to assess the diagnostic value of IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time PCR on Guthrie cards to retrospectively establish the diagnosis of congenital toxoplasmosis (CT). To this purpose, Guthrie cards were collected from 18 infants born to mothers with primary Toxoplasma gondii infection during pregnancy. Moreover, the analytical sensitivity of T. gondii PCR was assessed by testing mock dried blood specimens set up with several known DNA dilutions. IgM WB was demonstrated to be the most sensitive method. When the results of T. gondii DNA detection and specific IgM recovery were combined, retrospective CT diagnosis by using Guthrie cards was established in 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or serologic results were found in the group of 12 uninfected infants, demonstrating the excellent specificity of the three methods (95% confidence interval, 78.1% to 99.5%). The findings of the present study suggest that, in cases of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after the perinatal period, in particular the combination of recovery of specific IgM antibodies and T. gondii DNA amplification, could be helpful. Nevertheless, since suboptimal conditions of storage of dried blood specimens can seriously affect sensitivity, negative results cannot rule out CT diagnosis. In contrast, because of the excellent specificity shown by IgM serologic testing and T. gondii DNA amplification on Guthrie cards, positive results obtained by either of the two methods should be considered diagnostic.
INTRODUCTION
Toxoplasma gondii primary infection during pregnancy can result in abortion, stillbirth, perinatal death, or congenital infection. Congenital toxoplasmosis (CT) is mostly asymptomatic at birth, but infected infants are at high risk of ocular and neurological sequelae during childhood or early adulthood (1, 2). Serology has a pivotal role in CT diagnosis: recovery of IgM and/or IgA in a newborn's serum or a different pattern of IgG reactivity between a mother and her infant at birth are considered evidence of congenital infection, as is the lack of IgG antibody titer decrease within the first year of life (3, 4). Western blotting (WB) has provided significant advances in the early diagnosis of CT (3, 5–7), but its use for infant follow-up is limited to the first 3 months of life.
Therefore, when visual or neurological abnormalities occur after the first year of life, the retrospective diagnosis of CT is challenging, mostly when the serological status of the mother during pregnancy is unknown, as is the case when prenatal screening is not performed (8).
Dried-blood-spot (DBS) sampling is a form of biosampling where blood samples are blotted and dried on filter paper. In particular, preprinted collection cards known as Guthrie cards have been designed for mass screening for inborn errors of metabolism (9). In the last 2 decades, many studies focused their attention on the usefulness of DBS card analysis as an alternative method to diagnose and monitor several congenital infectious diseases (10) or as a viable option for surveillance in settings where the collection of serum samples remains challenging (11, 12), since it allows the evaluation of specific antibody levels (13–15) as well as the presence of nucleic acids (16–18).
DBS analysis has been mainly proposed as a method to estimate prevalence of CT at birth (1, 19, 20) or as an alternative method for neonatal screening for CT. Recently, the Danish national neonatal CT screening program based on DBS analysis came to its end in 2007, as it was found not to be cost-effective (21).
At present, only a few studies have evaluated the usefulness of DBS testing for retrospective CT diagnosis and investigated the recovery of specific IgM to distinguish between congenital and acquired toxoplasmosis in children presenting with symptoms later in childhood (15) or the clinical usefulness of the measurement of T. gondii IgG avidity on DBS specimens in the postnatal diagnosis of congenital toxoplasmosis (22).
In any case, it is noteworthy that only conventional automated assays have been used for CT diagnosis with DBS specimens (10, 15, 19–21), while, curiously, no study has been designed to evaluate WB performance with eluted DBS samples. Moreover, detection of T. gondii DNA has never been attempted on DBS samples, even as a control for other congenital infectious diseases, such as those caused by cytomegalovirus or rubella. Finally, very few data on the usefulness of IgA testing on eluted Guthrie cards are available (23), despite the wide use of IgA enzyme immunoassays (EIAs) for laboratory CT diagnosis (3, 5).
The aim of the present study was to assess the diagnostic value of the following three methods to retrospectively establish the diagnosis of CT using DBS specimens: IgM WB, DNA amplification by real-time PCR, and IgA EIA. Their performances were evaluated to find out which technique was worth using and whether it was possible to increase the sensitivity by combining the results obtained by different methods.
To this purpose, Guthrie cards from a group of infants born to mothers with primary T. gondii infection during pregnancy were collected. Results obtained on eluates from DBS specimens were retrospectively compared to the results obtained by testing infants' sera at birth or during their follow-up. Moreover, analytical sensitivity of T. gondii PCR was assessed by testing mock DBS specimens set up with several known DNA dilutions.
(This work was presented in part at the 23rd European Congress of Clinical Microbiology and Infectious Diseases. April 27 to 30, 2013, Berlin, Germany.)
MATERIALS AND METHODS
Study group.
Eighteen infants born to mothers who acquired toxoplasmosis during pregnancy were entered in the study. Gestational ages at maternal seroconversion ranged between 10 and 38 weeks (mean, 20.6 ± 8.1 weeks). Maternal infection during pregnancy was routinely treated with spiramycin until delivery. In the case of a fetal infection documented by ultrasonographic findings consistent with toxoplasmosis and/or a positive amniotic fluid PCR (performed between 18 and 23 weeks of gestation), spiramycin was replaced by a combination of sulfadiazine, pyrimethamine, and folinic acid until 38 weeks of gestation.
All the enrolled infants were born between January 2010 and June 2012, at St. Orsola-Malpighi Hospital, Bologna, Italy. The enrolled infants underwent follow-up for at least 12 months to determine whether maternal infection had been vertically transmitted. Written informed consent was obtained from both parents to collect and test stored the neonatal DBS card for every child and to collect demographic, clinical, and microbiological data for infected and uninfected infants. The study protocol was reviewed by the institutional ethics committee at our center.
Guthrie card storage conditions.
In the Centralized Laboratory of St. Orsola-Malpighi Hospital, Guthrie cards are routinely stored at 8 to 15°C for 1 year and then at 4°C for another 4 years. For this study, three blood spots were used for each child: specifically, one was used for T. gondii DNA detection, another one for IgM recovery, and the remaining one for IgA recovery.
Serologic analysis.
All the sera from infants born to mothers who acquired T. gondii infection during pregnancy were tested by enzyme immunoassays (EIAs) and enzyme linked fluorescent assays (ELFAs). In particular, Enzygnost Toxoplasmosis IgG, Enzygnost Toxoplasmosis IgM (Siemens Healthcare Diagnostics, Marburg, Germany), and Vidas Toxo IgM (bioMérieux, Marcy l'Etoile, France) were used.
Moreover, for IgA detection, infants' sera were tested with reagents and instruments from Siemens Healthcare Diagnostics (Marburg, Germany), as previously reported (24). Serum samples and Enzygnost Toxoplasmosis IgM-negative and -positive controls were first prediluted 1:21 in Enzygnost sample buffer POD, then 1:2 diluted with Enzygnost RF absorbent reagent, and incubated at room temperature for 15 min. Diluted sera and controls were transferred into Enzygnost Toxoplasmosis IgG plates (100 μl/well, 1:42 final dilution). The plates were placed in a BEP III analyzer, and all the subsequent processing steps were performed fully automatically by the instrument, using anti-human IgA/POD conjugate (anti-EBV/IgM II) as the secondary antibody. Samples with an optical density (OD) of >0.250 were considered positive, whereas sera with OD values below 0.180 were considered negative, and sera with values ranging between 0.180 and 0.250 were considered borderline.
Finally, all mother-child serum pairs were tested by WB at 7 to 10 days after birth (Toxoplasma IgG/IgM WB; LDBio Diagnostics, Lyon, France). WB allows a comparison between IgG and IgM immunological profiles of the newborn and those of its mother (4, 6, 7). When required, WB was also performed on infants' sera drawn during the first trimester of follow-up, by comparing infants' profiles at later time points to their own previous profiles.
Management of infants born to mothers with primary infection during pregnancy.
Infants born to mothers with T. gondii infection during pregnancy underwent clinical examination, T. gondii serologic testing (at 1, 3, 6, 9 and 12 months of age), laboratory tests, cerebral ultrasonography, and fundoscopy in the first year of age.
Diagnosis of CT was established in infants with positive specific anti-T. gondii IgM and/or IgA in the first 3 months of life and/or different IgG profiles compared to those of their mothers at 7 to 10 days after birth (6, 25).
All the infants diagnosed with CT were treated for 12 months with pyrimethamine and sulfadiazine and showed persistent positive anti-T. gondii IgG at 6 months after the end of therapy. Infected infants were enrolled in a long-term follow-up to detect visual or neurodevelopmental sequelae. Uninfected infants underwent serological follow-up until 1 year of age; they showed decreasing levels of specific IgG antibodies and were seronegative at 12 months of age.
IgM recovery from Guthrie cards.
For IgM recovery from DBS samples, a previously described protocol (26) was adapted to LDBio Toxoplasma IgG/IgM WB testing, as follows. First, considering that a 6.35-mm (1/4-in.) filter paper disc contains about 6 μl of dried serum (27), it follows that 12 μl of eluted serum could be obtained from each single spot. Each spot was cut into 4 pieces and put in a microcentrifuge tube with 250 μl of elution buffer (PBS–0.1% Tween). Tubes were incubated overnight at 4°C in agitation on an automatic shaker and then centrifuged for 10 min at 2,200 × g. IgM WB analysis was performed by diluting the eluate with LDBio Toxoplasma WB sample buffer to a final volume of 588 μl, in order to maintain a 1:49 dilution, as suggested by the manufacturer for IgM testing.
Analytical assessment of LDBio Toxoplasma IgM WB on DBS samples.
A panel of simulated DBS samples was prepared and consisted of one IgM-positive and one IgM-negative DBS specimen. The positive samples were prepared by using 12 μl of a Virotrol ToRCH-M sample (Bio-Rad Laboratories, Hercules, CA, USA) and washed red blood cells, as described elsewhere (28). Negative controls consisted of red blood cells and 12 μl of the Enzygnost Toxoplasmosis IgM negative control. The mock specimens were stored at 4°C for up to 2 weeks before use, and then elution was performed as described above. At the end of elution, an IgM WB assay was performed. Results of positive and negative DBS controls were compared to those obtained by analyzing a fresh Virotrol ToRCH-M sample or an Enzygnost Toxoplasmosis IgM negative control by IgM WB assay. Both the mock specimens and fresh controls were run in duplicate.
IgA recovery from Guthrie cards.
For IgA recovery from DBS samples, each blood spot was cut into 4 pieces and put in a microcentrifuge tube with 252 μl of Enzygnost sample buffer POD, in order to maintain a 1:21 dilution. Tubes were incubated overnight at 4°C in agitation on an automatic shaker and then centrifuged for 10 min at 2,200 × g. The eluates were diluted 1:2 with Enzygnost RF absorbent reagent and incubated at room temperature for 15 min. Diluted specimens were then processed by the Enzygnost system for IgA, as described above.
Analytical assessment of the IgA Enzygnost system with DBS samples.
A panel of simulated DBS samples was prepared and consisted of three specimens: one IgA positive, one IgA negative, and one blank, for evaluating the background due to red cells. The mock specimens were run in quintuplicate. The positive samples were prepared by using 12 μl of the Enzygnost Toxoplasmosis IgM positive control and washed red blood cells, whereas negative samples consisted of red blood cells and 12 μl of the Enzygnost Toxoplasmosis IgM negative control, and the blank controls consisted exclusively of red cells. The mock specimens were stored at 4°C for up to 2 weeks before use, and then elution was performed as described above. At the end of the elution, IgA testing was performed. After the results of blank samples were subtracted, positive and negative DBS controls were compared to fresh Enzygnost Toxoplasmosis IgM positive and negative controls run in the same IgA assay.
T. gondii DNA amplification from Guthrie cards.
Nucleic acids were extracted from DBS specimens by using the Versant kPCR sample preparation system (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). In detail, the following procedure was used: 1/2 in. (about 1.2 cm) of each dried blood spot sample was inserted into a tube containing 800 μl of nuclease-free water, vortexed for 5 s to thoroughly mix the sample, and incubated at 80°C for 30 min. At the end of the incubation, the diluted blood samples were centrifuged in microtubes at 2,500 × g for 1 min, and 300 μl of supernatant was transferred into a properly labeled 5-ml tube. Finally, the tubes were processed on a Versant kPCR sample preparation system by using Versant sample preparation 1.0 reagents, following the manufacturer's instructions. An input volume of 100 μl was eluted in a 100-μl final volume. After nucleic acid extraction, the Toxoplasma Q-PCR Alert kit (ELITech Group, Puteaux, France) was used for the amplification of a specific highly repeated target region of the T. gondii genome (AF 146527). A region of the β-globin gene was used as a target for the internal control.
Analytical sensitivity of the Toxoplasma Q-PCR Alert kit on DBS samples.
The limit of detection (LOD) for DBS samples was determined by analyzing in quintuple replicates three different dilutions of TG12-07, a sample for quantitated quality control for molecular diagnostics (QCMD) belonging to the panel of the QCMD 2012 Toxoplasma gondii DNA EQA Programme (QCMD; www.QCMD.org). This specimen consisted of a cultured Toxoplasma gondii type II strain at a concentration of 80 ± 30 organisms/ml. The LOD was defined as the lowest concentration to give a reproducible positive result. Since the target region AF 146527 is repeated about 300-fold in each single microorganism, it could be estimated that TG12-07 contained 24,000 ± 9,000 copies/ml (24 ± 9 copies/μl). Three different quantities (20, 10, and 5 μl) of sample TG12-07 (containing 480 ± 180, 240 ± 90, and 120 ± 45 copies, respectively) were diluted with 10 μl of a T. gondii-negative fresh whole-blood sample, spotted on filter paper, and stored at 4°C for up to 2 weeks. The mock DBS samples were then processed for DNA extraction and amplification as described above. Considering that after initial water dilution only 1/8 of the volume was used for DNA extraction, it follows that the contents of the three different mock eluates were 60 ± 22, 30 ± 11, and 15 ± 6 copies, respectively. In order to prepare mock specimens of fresh blood, three different quantities (20, 10, and 5 μl) of TG12-07 were diluted with 10 μl of a T. gondii-negative fresh whole-blood sample. The final volume was adjusted to 800 μl with Versant sample preparation 1.2 blood pretreatment buffer, and the tubes were immediately processed on the Versant kPCR sample preparation system by setting an input volume of 100 μl and a final elution volume of 100 μl.
Statistical analysis.
Statistical analyses were performed using Stata/SE version 12.1 (StataCorp LP, College Station, TX, USA). Sensitivity and specificity with 95% confidence intervals were calculated. Continuous variables were expressed as means ± standard deviations (SD). Data were compared using Student's t test; a P value of <0.05 was considered significant.
RESULTS
Analytical assessment of LDBio Toxoplasma IgM WB on DBS samples.
Loss of WB sensitivity and/or a decrease of specificity on DBS samples due to the elution procedure was excluded. Results for positive and negative DBS controls showed profiles identical to those obtained by analyzing a Virotrol ToRCH-M sample or an Enzygnost Toxoplasmosis IgM negative control by IgM WB assay. In Fig. 1, the results for one panel tested are reported.
FIG 1.
Analytical assessment of LDBio Toxoplasma IgM WB on DBS samples. Results of negative and positive DBS controls were compared to those of the Enzygnost Toxoplasmosis IgM negative control and a Virotrol ToRCH-M sample, respectively. Lane 1, negative DBS control. Lane 2, Enzygnost Toxoplasmosis IgM negative control. Lane 3, positive DBS control. Lane 4, Virotrol ToRCH-M sample.
Analytical assessment of IgA Enzygnost system on DBS samples.
Problems due to the elution procedure were excluded. Indeed, after OD results of mock blank samples (mean ± SD, 0.035 ± 0.010) were subtracted, positive and negative DBS controls showed values similar to those obtained by analyzing fresh Enzygnost Toxoplasmosis IgM positive and negative controls.
In particular, mock negative specimens showed OD values ranging between 0.009 and 0.028 (mean ± SD, 0.016 ± 0.008), whereas the fresh Enzygnost Toxoplasmosis IgM negative control yielded OD values ranging between 0.012 and 0.027 (mean ± SD, 0.018 ± 0.008) (P = 0.584). Mock-positive specimens showed OD values ranging between 1.380 and 1.620 (mean ± SD, 1.508 ± 0.104), whereas the fresh Enzygnost Toxoplasmosis IgM positive control yielded OD values ranging between 1.410 and 1.630 (mean ± SD, 1.532 ± 0.083) (P = 0.696).
Analytical sensitivity of Toxoplasma Q-PCR Alert kit with DBS samples.
Mock DBS samples prepared with 20 μl and 10 μl, respectively, of TG12-07 gave positive results when analyzed with the Toxoplasma Q-PCR Alert kit. In particular, considering the lower dilution of TG12-07, the target region was detected with cycle threshold values ranging between 37.42 and 38.14 (mean ± SD, 37.82 ± 0.38), whereas the internal control was amplified with cycle threshold values ranging from 29.40 to 31.55 (mean ± SD, 30.32 ± 0.90).
The LOD for DBS specimens was extremely low and consistent with the sensitivity declared by the manufacturer (about 10 copies/reaction). Considering that in 100 μl of the lower dilution of TG12-07 there were 30 ± 11 T. gondii DNA copies, it follows that in the 20-μl PCR mix there were 6.0 ± 2.2 copies/reaction.
When mock fresh blood samples were tested, the results were comparable to those obtained with mock DBS specimens, with the LOD again being 6.0 ± 2.2 copies/reaction. In particular, when the three replicates prepared with 10 μl of TG12-07 were tested, the target region was detected with cycle threshold values ranging between 37.96 and 38.74 (mean ± SD, 38.03 ± 0.52), whereas the internal control was amplified with cycle threshold values ranging from 28.71 to 32.00 (mean ± SD, 30.06 ± 1.24). There were no significant differences in T. gondii or β-globin cycle threshold values between mock dried specimens and fresh samples (P = 0.507 and P = 0.714, respectively).
Perinatal findings.
CT diagnosis was excluded in 12 out of 18 children (Table 1, cases 7 to 18): they had IgM/IgA-negative results at birth and during follow-up, and their specific IgG titers progressively decreased, so that all 12 children were seronegative at 12 months of age.
TABLE 1.
Gestational ages of seroconversion and treatment for women enrolled and corresponding infants' serologic results at birth
| Infant group | Case | Gestational age of maternal seroconversion (wks) | Maternal treatment | Clinical signs or symptoms at birth | Result of IgM WB analysis | Comparison of IgG WB between mother and childa | Result of IgA EIA |
|---|---|---|---|---|---|---|---|
| CT cases | 1b | 15 | Sulfadiazine, pyrimethamine and folinic acid | + | + | Different (110, 95, 85, 76, 27, 25 kDa) | + |
| 2 | 16 | Spiramycin | + | + | Different (110, 30, 27 kDa) | − | |
| 3c | 38 | Spiramycin | − | − | Same | − | |
| 4 | 25 | Spiramycin | + | + | Different (110, 95, 25 kDa) | + | |
| 5 | 25 | Spiramycin | − | + | Same | − | |
| 6 | 32 | Spiramycin | − | + | Different (30, 27, 25 kDa) | − | |
| Uninfected infants | 7 | 10 | Spiramycin | − | − | Same | − |
| 8 | 13 | Spiramycin | − | − | Same | − | |
| 9 | 16 | Spiramycin | − | − | Same | − | |
| 10 | 11 | Spiramycin | − | − | Same | − | |
| 11 | 11 | Spiramycin | − | − | Same | − | |
| 12 | 26 | Spiramycin | − | − | Same | − | |
| 13 | 24 | Spiramycin | − | − | Same | − | |
| 14 | 27 | Spiramycin | − | − | Same | − | |
| 15 | 25 | Spiramycin | − | − | Same | − | |
| 16 | 21 | Spiramycin | − | − | Same | − | |
| 17 | 11 | Spiramycin | − | − | Same | − | |
| 18 | 25 | Spiramycin | − | − | Same | − |
Molecular masses of additional bands detected in neonatal serum are given in parentheses.
Fetal infection was documented during gestation by ultrasonographic findings and a positive amniotic fluid PCR performed at 21 weeks of gestational age.
Diagnosed 1 month after birth because of IgM WB assay positivity.
In contrast, CT was diagnosed in the remaining 6 infants. In particular, 5 newborns (Table 1, cases 1, 2, and 4 to 6) were IgM positive by WB at 7 to 10 days after birth. The remaining infant (Table 1, case 3) was infected very late in utero; his mother seroconverted at 38 weeks. This infant was diagnosed only at his first follow-up visit, because of the finding of IgM positivity by WB. At birth, this infant was negative for both IgM and IgA and showed an immunological profile identical to that of his mother.
Three out of 6 (50.0%) congenitally infected infants were symptomatic at birth (cerebral calcifications in one case and moderate ventriculomegaly associated with cerebral calcifications in the other 2 cases). The remaining 3 (50.0%) infants showed the subclinical form of CT, without clinical signs or symptoms at birth or during the follow-up period. All 6 CT infants were born to mothers who had been treated during pregnancy and underwent 1-year anti-protozoan therapy. No cases of abnormal psychomotor development or visual impairment were recorded in the symptomatic or asymptomatic group of CT-infected infants during the follow-up period.
Retrospective diagnosis on Guthrie cards.
Results of IgM WB and PCR testing on Guthrie cards in relation to storage time are shown in Table 2. In particular, the cards' storage time ranged from 6 to 29 months (mean ± SD, 16.2 months ± 8.4 months) for the 6 infected infants (Table 2). Guthrie cards obtained from uninfected infants (Table 2) had been stored for at least 1 year (mean ± SD, 20.8 ± 7.0 months; range, 12 to 33 months), which is not statistically different from the storage time for cards from the group of CT cases (P = 0.239).
TABLE 2.
T. gondii DNA amplification and IgM recovery on Guthrie cards of infants born to mothers with T. gondii infection during pregnancy in relation to card storage time
| Infant group | Case | Time of DBS storage (mos) | Result for Guthrie card |
|
|---|---|---|---|---|
| Anti-T. gondii IgM WB | T. gondii PCR | |||
| CT cases | 1 | 8 | + | − |
| 2 | 15 | + | − | |
| 3 | 6 | − | − | |
| 4 | 29 | − | + | |
| 5 | 19 | − | − | |
| 6 | 20 | − | − | |
| Uninfected infants | 7 | 12 | − | − |
| 8 | 14 | − | − | |
| 9 | 22 | − | − | |
| 10 | 20 | − | − | |
| 11 | 13 | − | − | |
| 12 | 13 | − | − | |
| 13 | 24 | − | − | |
| 14 | 27 | − | − | |
| 15 | 25 | − | − | |
| 16 | 13 | − | − | |
| 17 | 13 | − | − | |
| 18 | 13 | − | − | |
Of the five children classified with CT at birth because of IgM positivity, two had positive serologic IgM results on Guthrie cards (cases 1 and 2) (Table 2). It is noteworthy that the IgM reactivity profile was consistent with the one detected at birth and that these cards were stored for 8 and 15 months, respectively. When used on DBS specimens, IgM WB performed with 33.3% sensitivity (95% confidence interval, 14.4% to 58.8%). Moreover, T. gondii DNA amplification was positive in case 4 (Table 2) (T. gondii and internal control cycle thresholds were 35.44 and 30.08, respectively). It is also noteworthy that the internal β-globin control was amplified from each DBS specimen, with cycle threshold values ranging from 29.80 to 32.05 (mean ± SD, 30.32 ± 1.37), similar to those observed with mock dried specimens.
Combining results of T. gondii DNA detection and specific IgM recovery, retrospective CT diagnosis on Guthrie cards was established for 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or IgM WB results were found in the controls, indicating excellent specificity (95% confidence interval, 78.1% to 99.5%) of both methods on dried blood specimens. Regarding the PCR method on Guthrie cards from uninfected infants, the internal control was amplified from each DBS specimen, with cycle threshold values ranging from 28.50 to 33.05 (mean ± SD, 30.21 ± 1.94), similar to those observed when mock specimens or specimens from infected infants were tested.
Finally, IgA results of tests on Guthrie cards of the 6 CT cases are shown in Table 3. Since IgA testing was carried out several months later than other tests, the cards' storage time ranged between 22 and 45 months (mean ± SD, 32.2 ± 8.4). Of the two cases with positive IgA at birth, only one (case 1) showed a reactive IgA result on the corresponding DBS, although it was much weaker than the OD value obtained at birth (an OD of 0.280 for the dried spot versus an OD of 0.630 for the fresh sample). Since no positive results were found in the group of uninfected infants (95% confidence interval, 78.1% to 99.5% specificity), IgA testing also demonstrated excellent specificity.
TABLE 3.
IgA recovery on DBS specimens in relation to Guthrie card storage time
| Infant group | Case | Time of DBS storage (mos) | Result of anti-T. gondii IgA EIA on Guthrie card |
|---|---|---|---|
| CT cases | 1 | 24 | + |
| 2 | 31 | − | |
| 3 | 22 | − | |
| 4 | 45 | − | |
| 5 | 35 | − | |
| 6 | 36 | − | |
| Uninfected infants | 7 | 28 | − |
| 8 | 30 | − | |
| 9 | 38 | − | |
| 10 | 36 | − | |
| 11 | 29 | − | |
| 12 | 29 | − | |
| 13 | 40 | − | |
| 14 | 43 | − | |
| 15 | 41 | − | |
| 16 | 29 | − | |
| 17 | 29 | − | |
| 18 | 29 | − |
DISCUSSION
CT diagnosis in children presenting evocative symptoms after the perinatal period can be difficult in the absence of information on their mothers' antenatal serostatus (5). This situation is mainly observed in countries where maternal screening is not performed (29). Thus, the possibility of testing stored Guthrie cards could be helpful in distinguishing acquired from congenital toxoplasmosis (1, 15, 22).
DBS testing has been proposed as an alternative method of microbiologic analysis for different infections, since it presents many advantages, such as minimal invasiveness of sampling, relative ease of collection, the possibility of storage for long periods, and a good agreement with serum or plasma testing (12–14, 30). Many studies have evaluated the diagnostic potential of DBS cards testing for retrospective diagnosis of congenital infections during childhood, in particular those due to cytomegalovirus (31, 32). In this regard, it has been demonstrated that DNA recovery is more frequent in symptomatic cases, probably because the viral load is higher in the presence of clinical evidence (33, 34).
Nevertheless, it should be emphasized that the retrospective use of DBS specimens for diagnosis of different congenital infections has raised questions regarding the stability of nucleic acids and antibodies on filter paper (15, 31), as well as the sensitivity of DNA recovery methods and amplification techniques (32, 34–36).
The importance of having powerful extraction methods and sensitive PCR assays is particularly relevant for the diagnosis of T. gondii infection, which is characterized by a very low and transient parasitemia (5). To our knowledge, no data about the recovery of T. gondii DNA from DBS specimens are available. In the present study, the protocol of DNA extraction by the Versant kPCR sample preparation system, combined with amplification by the Toxoplasma Q-PCR Alert kit, was characterized by a very low experimental LOD, comparable to that observed for fresh blood specimens.
Nevertheless, during the retrospective study of Guthrie cards obtained from CT-infected children, only one DBS sample was positive by real-time PCR. It can be speculated that in this particular case the presence of symptoms, possibly correlated to a higher parasitemia, contributed to the positive result. In any case, the other two DBS specimens from symptomatic patients were negative for T. gondii DNA amplification. It is not surprising that only one of the three symptomatic cases was positive by PCR: relatively low sensitivity values are reported in the literature for postnatal CT diagnosis by PCR analysis on placenta, cord blood, or newborns' peripheral-blood specimens (37). Recently, Olariu et al. (38) demonstrated that even when maternal treatment has not been administered, PCR sensitivity in newborns' whole-blood specimens is less than 30%. Regarding maternal treatment, it is useful to remember that the rationale for the use of spiramycin is to reduce the parasite burden in the mother, since this molecule hardly crosses the placental barrier, whereas pyrimethamine-sulfonamide does so efficiently (3), and that apart from decreasing vertical transmission, the associated goal of prenatal treatment is to reduce fetal damage or sequelae in newborns, provided that transmission has occurred.
In any case, for infants born to T. gondii-infected mothers, regardless of their treatment status, whole-blood PCR at birth or during the follow-up period is not currently recommended, because of its lack of sensitivity (3, 37).
Taking into account all these limitations, it is our opinion that negative T. gondii DNA amplification results on Guthrie cards cannot retrospectively rule out CT diagnosis; however, a positive result should direct pediatricians to the correct management of infected children. Since CT symptoms can be confounding with other congenital infections, carrying out molecular diagnosis on Guthrie cards could be helpful in differential diagnosis. DNA extracted from DBS specimens could indeed be used not only for T. gondii detection but also for the detection of a broad spectrum of vertically transmitted pathogens. In particular, with the Versant kPCR sample preparation system, the extraction protocol requires DNA elution in a volume sufficient for several amplification procedures.
When the results of our serologic assays on Guthrie cards are evaluated, different questions arise. In particular, it is important to emphasize that many factors contributed to the final results, not only the analytical performances of the laboratory methods or the elution protocols used (11), but the conditions of storage of Guthrie cards may have seriously affected them, since the stability of antibodies is strongly influenced by temperature and humidity (15, 39).
This has particular importance when a retrospective study is conducted on DBS samples stored for different periods of time. Policies and practices regarding the storage and use of residual newborn screening specimens vary considerably not only among different countries but also among single regions or within federal states. In 2010 in Italy, a document by the National Bioethics Committee and National Committee for Biosecurity, Biotechnologies, and Life Sciences presented recommendations for the storage of Guthrie cards, suggesting that in addition to a mandatory 2 years of storage, longer-term storage is possible (40). Moreover, although in the same document the importance of low-temperature storage is highlighted, there is not any requirement regarding storage temperature.
It has been demonstrated that the ideal conditions for storage of Guthrie cards would be at −20°C in plastic bags with silica desiccant (39). Tan et al. reported that the detection of IgM anti-T. gondii was significantly reduced in Guthrie cards stored for more than 300 days at room temperature compared with those stored at 4°C or below (15).
In our setting, Guthrie cards are stored for the first year at 8 to 15°C and then at 4°C for another 4 years.
When comparing results obtained by IgM WB testing on DBS specimens to those obtained by WB testing at birth, we were able to confirm the same pattern of reactivity in 2/5 cases. In both cases, the intensity of the bands was weaker than the intensity observed at birth. Also, IgA testing was less sensitive on Guthrie cards than on fresh samples at birth. Since the experimental mock DBS specimens examined by both IgM and IgA assays showed reactivities comparable to those of the fresh samples, it follows that at least one critical factor in the progressive antibody deterioration was the condition and time of storage. In particular, the three DBS specimens showing discrepant IgM results compared to perinatal findings were stored for 29, 19, and 20 months, whereas the discrepant IgA result was observed on a Guthrie card stored for 45 months.
Further clinical studies would be advisable to evaluate the clinical and cost-effectiveness of cold storage for the retrospective serologic testing of Guthrie cards.
Finally, it is important to emphasize that even in the ideal scenario of testing only well-preserved Guthrie cards, retrospective DBS testing cannot detect all cases of CT infection. Such an analysis may not be useful for all infants that are IgM and IgA negative at birth, as has been observed for newborns of mothers reporting seroconversion during the very early or very late periods of gestation (41, 42) and/or at least 30% of infants born to women receiving treatment in utero, in particular when pyrimethamine and sulfonamide were used instead of spiramycin (3, 5, 43).
Actually, prenatal screening proved to be the most effective strategy to identify maternal infection cases, allowing prompt antenatal treatment and the enrollment of the corresponding infants in follow-up programs (44, 45). However, in settings of low T. gondii seroprevalence, universal prenatal screening is not recommended, and education is the most accepted cost-effective prevention method (46).
The findings of the present study suggest that, in the case of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after perinatal period could be helpful, in particular when the recovery of specific IgM antibodies and T. gondii DNA amplification are combined in order to improve the sensitivity. In our study, the addition of T. gondii DNA detection to IgM recovery allowed us to detect an infection that would have been missed if IgM WB had been the only method used on DBS specimens, giving the retrospective diagnosis on Guthrie cards a 50% sensitivity. Moreover, when the cost-benefit of T. gondii PCR on DBS specimens is considered, it should be emphasized that the extracted DNA can be used for several investigations, a very important parameter in differential diagnosis.
Regarding IgA testing on Guthrie cards, even if the immunoenzymatic assay proved to be very specific and quite easy to perform, it does not seem to add valuable information to IgM WB testing alone, as IgM WB testing is more sensitive. In addition, it should be considered that, after the routine screening for inborn errors of metabolism, there are often no more than a couple of dried spots left, so it is important to choose which of the three methods mentioned above should be performed. It follows that, since IgA assays are widely used for laboratory CT diagnosis (3, 5, 38, 47), IgA testing on DBS could be taken into account in settings where IgM WB testing is not available.
In conclusion, the protocol suggested above, which includes IgM WB testing and T. gondii DNA amplification on Guthrie cards, proved helpful for retrospective CT diagnosis, but it is important to consider that since suboptimal conditions of DBS specimen storage can affect sensitivity, negative results cannot rule out CT diagnosis. On the other hand, positive results obtained by either of the two methods should be considered diagnostic.
ACKNOWLEDGMENTS
We are grateful to Roberto Motta, Director of Centralized Laboratory of St. Orsola-Malpighi Hospital, for his helpful collaboration and to Alessandra Moroni of Microbiology Laboratory of St. Orsola-Malpighi Hospital for providing excellent support during this study.
Footnotes
Published ahead of print 4 June 2014
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