Employing digital PCR for enhanced detection of perinatal Toxoplasma gondii infection: A cross-sectional surveillance and maternal-infant outcomes study in El Salvador

Abstract

Toxoplasma gondii is a parasitic infection that can be transmitted in utero, resulting in fetal chorioretinitis and other long-term neurological outcomes. If diagnosed early, pregnancy-safe chemotherapeutics can prevent vertical transmission. Unfortunately, diagnosis of acute, primary infection among pregnant women remains neglected, particularly in low-and-middle-income countries. Clinically actionable diagnosis is complex due to the commonality of infection during childhood and early adulthood which spawn long-last antibody titers and historically unreliable direct molecular diagnostics. The current study employed a cross-sectional T. gondii perinatal surveillance study using digital PCR, a next generation molecular diagnostic platform, and a maternal-fetal outcomes survey to ascertain the risk of vertical toxoplasmosis transmission in the Western Region of El Salvador. Of 198 enrolled mothers at the time of childbirth, 6.6% had evidence of recent T. gondii infection—85% of these cases were identified using digital PCR. Neonates born to these acutely infected mothers were significantly more likely to meconium aspiration syndrome and mothers were more likely to experience labor and delivery complications. Multivariable logistic regression found higher maternal T. gondii infection odds were associated with the presence of pet cats, the definitive T. gondii host. In closing, this study provides evidence of maternal T. gondii infection, vertical transmission and deleterious fetal outcomes in a vulnerable population near the El Salvador-Guatemala border. Further, this is the first published study to show clinical utility potential of digital PCR for accurate diagnosis of congenital toxoplasmosis cases.

Citation:Lynn MK, Rodriguez Aquino MS, Cornejo Rivas PM, Miranda X, Torres-Romero DF, Cowan H, et al. (2024) Employing digital PCR for enhanced detection of perinatal Toxoplasma gondii infection: A cross-sectional surveillance and maternal-infant outcomes study in El Salvador. PLoS Negl Trop Dis 18(5): e0012153. https://doi.org/10.1371/journal.pntd.0012153

Editor:Gabriel Rinaldi, Aberystwyth University - Penglais Campus: Aberystwyth University, UNITED KINGDOM

Received:May 13, 2023; Accepted:April 16, 2024; Published: May 20, 2024

Copyright: © 2024 Lynn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability:The data presented in this study are available through the BioStudies database under accession number S-BSST1323.

Funding:This research received funding from the UofSC Maternal-Child Health Catalyst Graduate Scholars program sponsored by the Health Resources and Services Administration (T1CMC35361 to MKL) and the UofSC Office of the Vice President of Research’s SPARC grant program to MKL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Toxoplasma gondii is a highly diverse apicomplexan protozoan responsible for the zoonotic infection toxoplasmosis [1–3]. This infection is prevalent globally, and over one third of the world’s population is thought to be infected, with the majority of infections presenting asymptomatically [1,4–7]. Transmission occurs when humans ingest tissue cysts from undercooked meat or sporulated oocysts from the environment. Oocysts are spread through the environment via the feces of the definitive feline host species and may contaminate food or water [2]. When chronically infected, cysts remain within brain and muscle tissues for the lifetime of the host [3]. In low and middle income countries with consistently warm temperatures, the risk of toxoplasmosis infection may be higher as oocysts can persist for longer periods within the environment [2]. The infection circulates among a wide range of mammalian hosts and can also be transmitted vertically through the placenta [7].

Human congenital T. gondii transmission is a major global health issue, attributable to a worldwide burden of 1.2 million disability-adjusted life years (DALYS) [5]. The foremost concern for congenital disease is primary, acute infection during pregnancy—where the woman’s first lifetime infection occurs during her pregnancy [8]. Immunocompromised women may also transmit the parasite across the placenta with a reactivated infection [8]. Infection within earlier stages of pregnancy is associated with more severe symptoms and disease sequelae later in the child’s life [5,7,9]. Conversely, the risk of vertical transmission increases the further along in pregnancy in which T. gondii is primarily acquired, with the highest risk related to maternal seroconversion between 10 and 24 weeks of gestational age [9].

Though not well-defined, the mechanism of vertical transmission has been associated with specific fetal and maternal immune responses to the parasite and inflammatory response derived placental damage [1]. Clinical outcomes of congenital transmission can be severe, impacting the central nervous system, resulting in stillbirth, neonatal death, encephalitis, chorioretinitis, and intracranial calcifications [2,4,10]. Other commonly reported symptoms from clinical studies include microcephaly, seizures, hepatosplenomegaly, thrombocytopenia, rash, jaundice and anemia [4,5]. Classically indicative hallmarks of congenital toxoplasmosis include intracranial calcifications, chorioretinitis, and hydrocephalus. However in practice, this triad of symptoms only presents in a small number of cases, with hydrocephalus occurring in approximately 4% of vertically infected, symptomatic neonates [9]. The most common disease presentations are related to ocular infection and vision difficulties [9]. Most congenital infections will show no symptoms at the time of birth; however, 30% will nonetheless develop lasting symptoms of ocular disease by age 12 [9].

Global seroprevalence among pregnant women is approximately 33% and 1% for IgG and IgM, respectively, though significant variation exists by world region and national income [5,9,10]. Diagnostic challenges exist for T. gondii particularly surrounding temporality of infection [11,12]. T. gondii specific IgM may react to protozoal antigens in the absence of infection and may persist for months or years [12]. IgG avidity tests help to distinguish recent from past infections, as antigen-antibody binding strength increases over time after primary infection. Molecular methods have also been used to determine active infection to assist in clarifying acute infections, due to the complexity of assessing serologic test results [12].

A paucity of research exists regarding T. gondii maternal or congenital infection in Central America. Meta-analysis estimates suggest national IgG seroprevalence in El Salvador is nearly 52% and IgM seroprevalence is approximately 0.9%[10]. Prenatal screening guidelines from the El Salvador Ministry of Health recommend all women be tested for HIV and syphilis during prenatal care visits [13]: toxoplasmosis is only tested for in patients with consistent clinical symptoms. Therefore, few maternal toxoplasmosis epidemiology and transmission risk studies have been conducted in El Salvador, and the majority are limited to the gray literature. These studies demonstrate between 17–27% IgG seroprevalence and up to 15% IgM seroprevalence among women of childbearing age [14,15]. The incidence of neonatal infection is also not well described. However, fatal congenital toxoplasmosis has been recently reported in the eastern department of Usulután [16]. Studies of microcephalic infants and low birth-weight neonates presenting to a national hospital reported 7% and 1% seropositivity for T. gondii respectively [17,18]. This study aimed to assess the prevalence of active T. gondii infection and associated maternal and neonatal outcomes among women presenting for childbirth at the referent public hospital in Sonsonate, El Salvador.

Methods

Ethics statement

The use of banked sera samples for Toxoplasma gondii testing was approved by the National Ethics Committee of El Salvador (IRB N° 0005660). Adult participants provided written informed consent. For minors of age <18 years, both written informed participant assent and parental/guardian consent were obtained for participation.

Participant recruitment

From March to September 2022, 198 patients ≥15 years presenting for labor and delivery at Hospital Nacional General “Dr. Jorge Mazzini Villacorta” (Hospital Mazzini), the referent public hospital in Sonsonate, El Salvador, agreed to participate in the study. Following consent, participants donated blood samples for infectious disease testing, as well as a small urine sample to detect indicators of possible pre-eclampsia. Maternal sera for these 198 participants were available for T. gondii serology testing and their labor and delivery health records were available for clinical-epidemiologic analysis.

Serologic and Molecular Methods

Banked serum samples were screened for the presence of IgG and IgM antibodies against T. gondii by enzyme-linked immunosorbent assay (ELISA) (Calbiotech, El Cajon, CA, USA). IgG avidity testing was further performed to distinguish past versus recent infection using Avidity Toxoplasma gondii IgG ELISA kit (Abcam, Waltham, MA, USA). To confirm infection, digital polymerase chain reaction (dPCR) testing was performed following DNA extraction from whole blood samples mixed with DNA/RNA Shield preservative using QIAamp 96 Virus QIAcube HT Kit for DNA and RNA (Qiagen, Germantown, MD, USA). Presence of parasite DNA was determined using a next-generation digital polymerase chain reaction (dPCR) assay on the QIAcuity Four Digital PCR system (Qiagen, Germantown, MD) and Qiagen 24-well 26k Nanoplates (Qiagen, Germantown, MD). Previously validated primers and probes targeting REP529, the highly conserved and repetitive 529 bp fragment of the parasite genome were used [19–21]. Reaction mix was prepared using Qiagen QIAcuity Probe PCR kit (Qiagen, Germantown, MD), AluI cutter restriction enzyme (New England Biosystems, Ipswich, MA) and primers and FAM probe from Integrated DNA Technologies (IDT, Coralville, IA). Genomic DNA from Toxoplasma gondii strain RH (ATCC 50174D) was purchased from American Type Culture Collection (Manassas, VA USA) for use as a positive control. Digital PCR Partition classification was conducted via Umbrella in RStudio, R version 4.3.0 using a hard thresholding value of p0,A ≥ 0.95 and nuclease free water non-template controls [22]. The following reaction mixture prepared: 10 uL Qiagen master mix,4 uL 10 mM forward/reverse primers, 2 uL 10 mM FAM probe, 1 uL AlUI restriction enzyme, 33 uL nuclease free water, and 5 uL template DNA. Gradient PCR was conducted to determine the following optimal cycle conditions: 95.0° for 5 min then 50 cycles of 95.0° for 30 seconds, 56.0° for 1 min, and 72.0° for 30 seconds. A 1:10 serial dilution (1 – 1x10-6 μg/μL) of positive standards was used to determine the method detection limit for our system per the method described by Hunter et al [23]. The specific dPCR target sequences are listed in S1 Table. The following formula was then used to assess the limit of detection:

Results of these calculations are shown in Fig 1, finding a final limit of detection to be 0.011 copies per uL.

[Figure omitted. See PDF.]

Fig 1. Calculation results for the limit of detection.

The final limit of detection for the dPCR reaction was determined to be 0.011 copes per uL.

https://doi.org/10.1371/journal.pntd.0012153.g001

All samples underwent serology testing; however, not all samples underwent dPCR or avidity testing in cases of insufficient remaining biological samples. The number of banked samples that underwent each testing platform is described in Fig 2.

[Figure omitted. See PDF.]

Fig 2. Summary of testing algorithm and classification of recent T. gondii infection within study population.

Among Salvadoran perinatal women at the time of parturition, 17 individuals had evidence of recent T. gondii infection within the last 16 weeks. Due to differing quantities of banked sera and whole blood, some individuals’ samples did not receive dPCR testing and results relied on previously published clinical diagnostic algorithms of serology and avidity testing.

https://doi.org/10.1371/journal.pntd.0012153.g002

Interpretation of results

Participant samples were classified as having evidence of recent infection, defined as infection within the last 16 weeks, through assessing the combination of dPCR, IgG and IgM serology, and IgG avidity testing results as previously described [24–26]. Due to the complexity of elucidating infection timing in endemic settings, this study relied on the previously published infectious disease guidelines for interpretations of positive (+), negative (-) and equivocal (E) serology and avidity test results [24]. All banked whole blood samples with sufficient volume after DNA extraction underwent digital PCR (N = 190). Digital PCR was then used in combination with serologic results to determine evidence of recent infections. Specifically, recently infected mothers in this study were defined as meeting at least one of the following criteria:

* Sera sample determined IgG positive AND IgM result either positive or equivocal AND low avidity testing result.

* Sera sample determined IgM positive AND whole blood sample detected parasite DNA via dPCR

A simplified flow diagram of our testing and interpretation algorithm is presented in Fig 2 for clarity.

Statistical and spatial analysis

Fisher’s exact test was used to detect univariate associations between recent T. gondii infection, and 1) maternal and neonatal health outcomes abstracted from clinical charts, and 2) risk factors for infection obtained from the participant questionnaires. Multivariable logistic regression was used to determine statistically significant odds ratios to describe the relationship between recent toxoplasmosis infection and risk factors for infection. All statistical analyses were performed in RStudio version 4.1.1 (RStudio, PBC, Boston, MA).

Results

This perinatal cohort demonstrated high historical toxoplasmosis burdens, with an overall 51.5% IgG seroprevalence (N = 102), and 20.2% IgM seroprevalence (N = 40). Among perinatal samples, 10.1% (N = 20) were seropositive for both IgG and IgM. The distribution of IgG seropositive case counts may be found in S1 Fig. Among all 198 perinatal samples, we identified two participants that meet classic diagnostic criteria for a recent T. gondii infection. One participant was IgG(+) and IgM(+) with low avidity and one participant IgG(+) and IgM(E) with low avidity. We found 31.1% (N = 59/190) of perinatal samples tested via dPCR were positive for T. gondii DNA, with 5.8% IgM(+) (N = 11/190) shown in Fig 2. In total, 6.6% of women (n = 13) had evidence of recent T. gondii infection within the last 16 weeks prior to childbirth, Fig 3. The method detection limit for the measured parasite DNA concentration was determined to be 0.01 copies/μL, which corresponds to < 1 positive partition per dPCR plate. Measured parasite DNA ranged from 0.04 to 0.56 copies per microliter. The detection of parasite DNA within these samples coupled with serologic results provides evidence of potential early infection among perinatal women. The majority of participants with evidence of recent toxoplasmosis infection were distributed throughout Sonsonate department, particularly Nahuizalco (N = 3) and Izalco (N = 3), shown in Fig 3.

[Figure omitted. See PDF.]

Fig 3. Cases of Salvadoran perinatal women with evidence of recent toxoplasmosis were primarily from two underserved municipalities in Sonsonate (Izalco, Nahuizalco).

*ArcGIS map layers for El Salvador department and municipality boundaries are credited to Esri and Michael Bauer Research GmbH 2022, Dirección General de Estadística y Censos. https://www.esri.com/partners/michael-bauer-resear-a2T70000000TNZ3EAO.

https://doi.org/10.1371/journal.pntd.0012153.g003

Demographics and clinical history of the entire participant group can be found in Table 1. Among the entire cross-sectional perinatal group, median maternal age was 26 years (range: 15–45 years), and most resided in Sonsonate department. The majority of women reported overall good health status, though a small proportion had prior clinical history of miscarriage and pregnancy complications. Other relevant clinical histories present at the time of childbirth were gestational diabetes (N = 4), and history of seizure (N = 2). The majority of participants in this study lived in very low socioeconomic conditions with nearly 30% having significant substandard housing classified by barren earth floor, no electricity, or no potable water source in the home. Approximately 24% reported having pet cats and 15% of participants allowed animals to be inside the home, Table 1.

[Figure omitted. See PDF.]

Table 1. Demographics and clinical profiles of Salvadoran perinatal women and results of Fisher’s exact test.

https://doi.org/10.1371/journal.pntd.0012153.t001

Clinical outcomes during the current pregnancy and neonatal birth outcomes are described in Table 2. Approximately 17% had current pregnancies considered high-risk and most women delivered via Cesarean section (c-section). More than 22% of this perinatal group had at least one complication during pregnancy and 10% of neonates had at least one negative health outcome at the time of birth. Labor and delivery complications included maternal vaginal infection at the time of birth, premature rupture of the membranes (PROM), perinatal asphyxia, and neonatal injury. More than 20% of neonates were admitted to the neonatal intensive care unit (NICU) for varying reasons, including acute respiratory distress syndrome (ARDS), low APGAR scores, premature birth, and low birthweight. Among perinatal women with evidence of recent toxoplasmosis infection, two had complications during pregnancy including threat of miscarriage and placenta previa. Four women in this group had complications at parturition including breeched birth and PROM. Neonatal birth outcomes among those with evidence of a recent infection included MAS and ARDS, shown in Table 2. Four infants among those with evidence of recent infection were admitted to the NICU.

[Figure omitted. See PDF.]

Table 2. Current pregnancy and birth outcomes of Salvadoran perinatal women revealed significant associations between recent T. gondii infection and neonatal birth outcomes via results of Fisher’s exact test.

https://doi.org/10.1371/journal.pntd.0012153.t002

Fisher’s exact test revealed statistically significant univariate relationships between recent T. gondii infection and any labor and delivery complication (P = 0.053), shown in Table 2. Recent infection was also associated with neonatal MAS (P = 0.037) by Fisher’s exact test and multivariable logistic regression (OR:10.8, 95%CI:1.32, 80.0). Multivariable logistic regression revealed participants reporting pet cats had 4.94 times the probability of recent T. gondii infection (95%CI: 1.50, 17.14). Whereas, reporting increased wild animal species around the home was protective against recent infection (OR = 0.57, 95%CI: 0.31, 0.94).

Discussion

This study found 6.6% of perinatal women from a vulnerable region of western El Salvador had evidence of recent T. gondii infection, defined in this study as infection within 16 weeks of parturition [24]. These 13 women’s neonates were significantly more likely to experience MAS, highlighting the impact of this perinatal infection. Results of this manuscript present evidence for the need to implement education campaigns and further serial screening studies of toxoplasmosis in pregnancy and congenital infection in Central America. According to Salvadoran and neighboring countries clinical guidelines for perinatal infection testing, HIV and syphilis tests are conducted on every woman receiving perinatal care; however, toxoplasmosis is only tested when women present symptomatically at the time of their perinatal care visit. As more than 80% of maternal infection present subclinically or with non-specific symptomology, our results provide evidence that primary or reactivated maternal infections may be going undiagnosed in highly vulnerable populations [27].

None of the neonates in this study had hallmark signs of congenital T. gondii infection at birth (hydrocephaly, intracranial calcifications, retinochoroiditis [9,27]); however, most congenitally infected neonates are asymptomatic at birth [9]. Clinical disease develops in approximately a quarter of initially asymptomatically infected neonates within the first year of life [28]. In the current study, neonates were clinically managed according to local medical guidelines, which do not require eye or neurological exam. In contrast, infected children born with clinically apparent disease are at an increased risk for severe long-term sequelae [28]. Within our study population, significant relationships between recent T. gondii infection and negative birth outcomes were noted, specifically MAS and complications at the time of birth. MAS has previously been associated with maternal infection or inflammatory response in pregnancy [29]. These findings warrant prospective evaluation as early identification and chemotherapeutic treatment are associated with improved long-term outcomes [30,31].

Statistical analysis revealed those mothers reporting pet cat ownership had nearly 5 more likely to show evidence of recent T. gondii infection. Domestic cats serve as a definitive host and an important contributor to human transmission, along with consumption of game meat and contaminated drinking water [32–34]. Transmission foci have further been previously reported in under-resourced populations, typically associated with environmental contamination clusters [32,35]. The current study found recent maternal T. gondii infection was associated with reporting fewer wildlife species seen around the home. This is likely an indicator of rural living where greater wildlife diversity is found. Given the distribution of cases combined with high multi-dimensional poverty in these two departments (e.g. high rates of crowding, and low access to potable water and sanitation services [36]), further environmental studies should evaluate the potential sylvatic sources leading to maternal infection locally. Additionally, higher case counts were observed in Nahuizalco and Izalco departments. Future studies should focus in these areas to investigate pet ownership, hand and food washing behaviors, as well as meat consumption behaviors among residents to better clarify the epidemiologic picture of toxoplasmosis in this region.

This pilot study identified 51.5% IgG seroprevalence among perinatal women in this study, consistent with previously estimates of IgG seroprevalence among pregnant women across El Salvador [10] and Latin American countries [37–39]. In contrast, this study group demonstrated a 10.1% IgM seroprevalence, a significantly higher rate than national estimates of 0.9% IgM perinatal seroprevalence, although the grey literature (e.g. university student theses) suggest that IgM seroprevalence varies greatly across the country [10,14,15]. Serologic methods for determining infection timing are difficult to interpret, as IgM can persist for months to years following recent infection and may further react with T. gondii antigens in the absence of veritable infection [11,12,40]. For this reason, we utilized both IgG avidity testing and dPCR to better assess acute infection in our study population. Toxoplasmosis avidity tests are typically performed when IgM and IgG serologic results are positive after repeated serial collections during the pregnancy [12]. T. gondii IgG avidity tests assess the strength of antigen-antibody binding, which may help to narrow down the timing of infection [11]—IgG avidity increases with time after exposure and changes from low binding to high binding approximately 6 months after primary infection [11]. In contrast, dPCR is an emerging technology for direct quantification of parasitic infections, with increased sensitivity and specificity for detection of low level-parasitemia [41,42]. Though T. gondii dPCR studies are limited, results have shown a six-fold higher positivity detection rate of T. gondii among a group of known positive meat samples and a 14- to 160- fold higher detection rate among banked clinical samples [25,42]. Conventional PCR has also been previously used to investigate acute infection in blood donors and pregnant women [26,43]. For this investigation of dPCR clinical utility potential, we conservatively classified those dPCR (+) and IgM (+) to avoid the potential for identification of latent infection using this highly sensitive molecular technique. As serologic results can be difficult to interpret, our findings support the utility of dPCR molecular methods in combination with serologic testing for T. gondii screening during pregnancy [12].

This study had a few limitations worth noting. Given the complexity of T. gondii diagnosis in the context of vertical transmission, serial samples are traditionally recommended; however, this study’s design was cross-sectional. To combat this concern, we applied conservative diagnostic profile requirements to define a positive case: 1) dPCR (direct detection of parasitic DNA) plus a positive IgM serologic result or a 2) positive antibody result with low avidity, criteria consistent with standard clinical guidelines. Due to the heightened sensitivity of dPCR compared to traditional and qPCR methods, it is further possible that detected parasite DNA represents exposure to the T. gondii protozoan in the absence of veritable clinical infection. Further validation studies of dPCR are necessary to determine the utility of this next generation molecular method in the clinical setting. Additionally, as a next-generation molecular technique, the application of dPCR may be costly. Therefore, the potential employment of this technique may be limited outside of reference laboratories in low resource settings. This study was also limited by the inability to capture some important infection risk factors, as the original survey was designed for a maternal Chagas disease surveillance study and participants not prospectively contacted for additional T. gondii transmission risk factor assessment. Lastly, we were unable to follow neonates to determine congenital infection in this study; however, the results of this study have been presented and shared with the hospital director and the Ministry of Health for follow-up care.

Conclusion

Serology-based diagnosis of acute primary toxoplasmosis infection is complex, thus more effective and practical case diagnostics are critical in reducing the burden of congenital infections. This study provides supporting evidence that dPCR should be further explored for improved detection of acute T. gondii infection during pregnancy. Though cost may currently present limitations in lower resourced settings, future studies should be conducted to validate dPCR as a reliable infection testing method. The comparatively higher sensitivity of this method could aid in early detection of cases and reduce the need for multiple serologic tests in world regions that routinely screen for T. gondii throughout pregnancy. The current study identified high contemporary risk for undiagnosed congenital toxoplasmosis in the Western Region of El Salvador. The importance of this work highlights the need for education campaigns and enhanced future screening studies of pregnant women and their neonates in El Salvador and surrounding countries. Education and prenatal screening may present the opportunity for seronegative or immunocompromised pregnant women to avoid unnecessary exposure and for acutely infected women to obtain chemotherapeutics to prevent congenital transmission.

Supporting information

S1 Table. Targeted sequences used in digital PCR for molecular Toxoplasma gondii detection.

https://doi.org/10.1371/journal.pntd.0012153.s001

(DOCX)

S1 Fig. Case counts of toxoplasmosis IgG among perinatal women.

Positive cases were highest in Acajutla, Izalco, and San Fransico Menendez municipalities in two departments in El Salvador with high multidimensional poverty. *ArcGIS map layers for El Salvador department and municipality boundaries are credited to Esri and Michael Bauer Research GmbH 2022, Dirección General de Estadística y Censos. https://www.esri.com/partners/michael-bauer-resear-a2T70000000TNZ3EAO.

https://doi.org/10.1371/journal.pntd.0012153.s002

(TIF)

References

1. 1. Gómez-Chávez F, Cañedo-Solares I, Ortiz-Alegría LB, Flores-García Y, Figueroa-Damián R, Luna-Pastén H, et al. A proinflammatory immune response might determine Toxoplasma gondii vertical transmission and severity of clinical features in congenitally infected newborns. Frontiers in immunology. 2020;11:390. pmid:32231666

* View Article

* PubMed/NCBI

* Google Scholar

2. 2. El Bissati K, Levigne P, Lykins J, Adlaoui EB, Barkat A, Berraho A, et al. Global initiative for congenital toxoplasmosis: an observational and international comparative clinical analysis. Emerging microbes & infections. 2018;7(1):1–14.

* View Article

* Google Scholar

3. 3. Shwab EK, Zhu X-Q, Majumdar D, Pena HF, Gennari SM, Dubey JP, Su C. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology. 2014;141(4):453–61. pmid:24477076

* View Article

* PubMed/NCBI

* Google Scholar

4. 4. Barros M, Teixeira D, Vilanova M, Correia A, Teixeira N, Borges M. Vaccines in Congenital Toxoplasmosis: Advances and Perspectives. Frontiers in Immunology. 2021;11:621997.

* View Article

* Google Scholar

5. 5. Maldonado YA, Read JS, Byington CL, Barnett ED, Davies H, Edwards KM, et al. Diagnosis, treatment, and prevention of congenital toxoplasmosis in the United States. Pediatrics. 2017;139(2). pmid:28138010

* View Article

* PubMed/NCBI

* Google Scholar

6. 6. Rajapakse S, Weeratunga P, Rodrigo C, de Silva NL, Fernando SD. Prophylaxis of human toxoplasmosis: a systematic review. Pathogens and global health. 2017;111(7):333–42. pmid:28948861

* View Article

* PubMed/NCBI

* Google Scholar

7. 7. Kota AS, Shabbir N. Congenital Toxoplasmosis. 2019.

* View Article

* Google Scholar

8. 8. Rostami A, Riahi SM, Contopoulos-Ioannidis DG, Gamble HR, Fakhri Y, Shiadeh MN, et al. Acute Toxoplasma infection in pregnant women worldwide: A systematic review and meta-analysis. PLOS Neglected Tropical Diseases. 2019;13(10):e0007807.

* View Article

* Google Scholar

9. 9. Dubey J, Murata F, Cerqueira-Cézar C, Kwok O, Villena I. Congenital toxoplasmosis in humans: an update of worldwide rate of congenital infections. Parasitology. 2021;148(12):1406–16. pmid:34254575

* View Article

* PubMed/NCBI

* Google Scholar

10. 10. Bigna JJ, Tochie JN, Tounouga DN, Bekolo AO, Ymele NS, Youda EL, et al. Global, regional, and country seroprevalence of Toxoplasma gondii in pregnant women: a systematic review, modelling and meta-analysis. Scientific reports. 2020;10(1):1–10.

* View Article

* Google Scholar

11. 11. Teimouri A, Mohtasebi S, Kazemirad E, Keshavarz H. Role of Toxoplasma gondii IgG avidity testing in discriminating between acute and chronic toxoplasmosis in pregnancy. J Clin Microbiol. 2020;58(9):e00505–20. pmid:32321784

* View Article

* PubMed/NCBI

* Google Scholar

12. 12. Garnaud C, Fricker-Hidalgo H, Evengård B, Álvarez-Martinez MJ, Petersen E, Kortbeek LM, et al. Toxoplasma gondii-specific IgG avidity testing in pregnant women. Clinical Microbiology and Infection. 2020;26(9):1155–60. pmid:32334096

* View Article

* PubMed/NCBI

* Google Scholar

13. 13. MINSAL. Lineamientos técnicos para la atención de la mujer en el período preconcepcional, prenatal, parto, puerperio y al recién nacido. Servicios en SSR para atención de emergencias o desastres. In: Salud GyDe, editor. 2021.

* View Article

* Google Scholar

14. 14. PÉREZ MATA E, GONZÁLEZGONZÁLEZ V, ROMERO MEDRANO . DETERMINACIÓN DE LA PRESENCIA DE INMUNOGLOBULINAS IgG E IgM EN INFECCIÓN POR Toxoplasma gondii EN MUJERES DE 15 A 45 AÑOS QUE CONSULTAN LA UNIDAD DE SALUD DE CONCEPCIÓN BATRES DEPARTAMENTO DE USULUTÁN, PERIODO DE AGOSTO A SEPTIEMBRE DE 2012. Universidad de El Salvador Sitema Bibliotecario: Universidad de El Salvador 2012.

* View Article

* Google Scholar

15. 15. Guevara Diaz I, Navarrete Chavez M, Lazo Cruz J. DETECCIÓN DE ANTICUERPOS ANTI Toxoplasma gondii DE TIPO IgM, EN HEMOCOMPONENTES PROVENIENTES DE DONANTES MUJERES EN EL ÁREA DE BANCO DE SANGRE DEL HOSPITAL NACIONAL SAN JUAN DE DIOS DE SAN MIGUEL. PERIODO: AGOSTO Y SEPTIEMBRE DE 2013. Universidad de El Salvador Sistema Biblioteario Repositorio Universidad de El Salvador; 2013.

* View Article

* Google Scholar

16. 16. Rivas H. Reportan muerte de niño por toxoplasmosis. La Prensa. 7 May 2019.

* View Article

* Google Scholar

17. 17. Silva Garcia RC, Rosales W, Martinez Romero L. PERFIL CLINICO Y EPIDEMIOLÓGICO DE LACTANTES MENORES O IGUAL A 1 AÑO CON MICROCEFALIA QUE SON EVALUADOS EN LA CONSULTA EXTERNA DE NEUROLOGÍA DEL HOSPITAL NACIONAL DE NIÑOS BENJAMÍN BLOOM DE ENERO DE 2015 A DICIEMBRE DE 2016 [Posgrado de especialidades medicas]: Universidad de El Salvador; 2018.

* View Article

* Google Scholar

18. 18. Ramírez Peña S. PERFIL EPIDEMIOLÓGICO DEL RECIÉN NACIDO DE BAJO PESO AL NACER ENTRE 2,000 Y 2,499 g QUE INGRESA AL SERVICIO DE NEONATOS DEL HOSPITAL NACIONAL ESPECIALIZADO DE MATERNIDAD “DR. RAÚL ARGUELLO ESCOLÁN”. ENERO A JUNIO 2013. Universidad de El Salvador Sistema Bibliotecario Repositorio: Universidad de El Salvador; 2014.

* View Article

* Google Scholar

19. 19. Gutierrez-Loli R, Ferradas C, Diestra A, Traianou A, Bowman N, Bok J, et al. Development of a novel protocol based on blood clot to improve the sensitivity of qPCR detection of Toxoplasma gondii in peripheral blood specimens. The American journal of tropical medicine and hygiene. 2019;100(1):83. pmid:30457102

* View Article

* PubMed/NCBI

* Google Scholar

20. 20. Homan W, Vercammen M, De Braekeleer J, Verschueren H. Identification of a 200-to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. International journal for parasitology. 2000;30(1):69–75. pmid:10675747

* View Article

* PubMed/NCBI

* Google Scholar

21. 21. Marino AMF, Giunta RP, Salvaggio A, Castello A, Alfonzetti T, Barbagallo A, et al. Toxoplasma gondii in edible fishes captured in the Mediterranean basin. Zoonoses and public health. 2019;66(7):826–34. pmid:31278858

* View Article

* PubMed/NCBI

* Google Scholar

22. 22. Jacobs BK, Goetghebeur E, Vandesompele J, De Ganck A, Nijs N, Beckers A, et al. Model-based classification for digital PCR: your umbrella for rain. Analytical chemistry. 2017;89(8):4461–7. pmid:28350455

* View Article

* PubMed/NCBI

* Google Scholar

23. 23. Hunter ME, Dorazio RM, Butterfield JS, Meigs-Friend G, Nico LG, Ferrante JA. Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA. Molecular ecology resources. 2017;17(2):221–9. pmid:27768244

* View Article

* PubMed/NCBI

* Google Scholar

24. 24. Cherry J, Demmler-Harrison GJ, Kaplan SL, Steinbach WJ, Hotez PJ. Feigin and Cherry’s Textbook of Pediatric Infectious Diseases E-Book: 2-Volume Set: Elsevier Health Sciences; 2013.

* View Article

* Google Scholar

25. 25. Mancusi A, Giordano A, Bosco A, Girardi S, Proroga YT, Morena L, et al. Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples. Parasitology Research. 2022;121(5):1467–73. pmid:35230549

* View Article

* PubMed/NCBI

* Google Scholar

26. 26. Saki J, Foroutan M, Khodkar I, Khodadadi A, Nazari L. Seroprevalence and molecular detection of Toxoplasma gondii in healthy blood donors in southwest Iran. Transfusion and Apheresis Science. 2019;58(1):79–82. pmid:30579749

* View Article

* PubMed/NCBI

* Google Scholar

27. 27. Ahmed M, Sood A, Gupta J. Toxoplasmosis in pregnancy. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2020;255:44–50. pmid:33075679

* View Article

* PubMed/NCBI

* Google Scholar

28. 28. Auriti C, Bucci S, De Rose DU, Coltella L, Santisi A, Martini L, et al. Maternal&ndash;Fetal Infections (Cytomegalovirus, Toxoplasma, Syphilis): Short-Term and Long-Term Neurodevelopmental Outcomes in Children Infected and Uninfected at Birth. Pathogens. 2022;11(11):1278.

* View Article

* Google Scholar

29. 29. Monfredini C, Cavallin F, Villani PE, Paterlini G, Allais B, Trevisanuto D. Meconium aspiration syndrome: a narrative review. Children. 2021;8(3):230. pmid:33802887

* View Article

* PubMed/NCBI

* Google Scholar

30. 30. Wallon M, Kodjikian L, Binquet C, Garweg J, Fleury J, Quantin C, Peyron F. Long-term ocular prognosis in 327 children with congenital toxoplasmosis. Pediatrics. 2004;113(6):1567–72. pmid:15173475

* View Article

* PubMed/NCBI

* Google Scholar

31. 31. Garweg JG, Kieffer F, Mandelbrot L, Peyron F, Wallon M. Long-Term Outcomes in Children with Congenital Toxoplasmosis-A Systematic Review. Pathogens. 2022;11(10). pmid:36297244

* View Article

* PubMed/NCBI

* Google Scholar

32. 32. Ducrocq J, Ndao M, Yansouni CP, Proulx JF, Mondor M, Hamel D, et al. Epidemiology associated with the exposure to Toxoplasma gondii in Nunavik’s Inuit population using the 2017 Qanuilirpitaa cross-sectional health survey. Zoonoses Public Health. 2021;68(7):803–14. pmid:34254450

* View Article

* PubMed/NCBI

* Google Scholar

33. 33. Galal L, Stragier C, Boumédiène F, Hamidović A, Maugrion O, Dardé ML, Mercier A. Combining spatial analysis and host population genetics to gain insights into the mode of transmission of a pathogen: The example of Toxoplasma gondii in mice. Infect Genet Evol. 2020;78:104142. pmid:31841702

* View Article

* PubMed/NCBI

* Google Scholar

34. 34. Espindola BD, Fernandes FD, Roman IJ, Samoel GVA, Barcelos RAD, Döhler AR, et al. Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages. Rev Bras Parasitol Vet. 2022;31(3):e009322. pmid:36074435

* View Article

* PubMed/NCBI

* Google Scholar

35. 35. Antinarelli LMR, Silva MR, Guimarães R, Terror MS, Lima PE, Ishii JSC, et al. Rural residence remains a risk factor for Toxoplasma infection among pregnant women in a highly urbanized Brazilian area: a robust cross-sectional study. Trans R Soc Trop Med Hyg. 2021;115(8):896–903. pmid:33347595

* View Article

* PubMed/NCBI

* Google Scholar

36. 36. Salvador GdE. Medición Multidimensional de la Pobreza In: (SETEPLAN) STydPdlP, Ministerio de Economía atdlDGdEyCM-D, editors. Portal de Transparancia Insistuto de Acceso a la Información Pública; 2017. p. 45.

* View Article

* Google Scholar

37. 37. Hicks VJ, Sánchez C, López MR, Gottschlich A, Grajeda LM, Balish A, et al. Seroprevalence of high incidence congenital infections among pregnant women in Coatepeque, Guatemala and surrounding areas, 2017–2018. PLoS Negl Trop Dis. 2023;17(4):e0011248. pmid:37093863

* View Article

* PubMed/NCBI

* Google Scholar

38. 38. Medeiros JF, Rabelo ESAC, Rocha N, Georg AV, Melli P, Quintana SM, Duarte G. Seroprevalence of Toxoplasmosis in Puerperal Women Treated at a Tertiary Referral Hospital. Rev Bras Ginecol Obstet. 2023;45(2):59–64.

* View Article

* Google Scholar

39. 39. Flores C, Villalobos-Cerrud D, Borace J, Fábrega L, Norero X, Sáez-Llorens X, et al. Epidemiological Aspects of Maternal and Congenital Toxoplasmosis in Panama. Pathogens. 2021;10(6). pmid:34204401

* View Article

* PubMed/NCBI

* Google Scholar

40. 40. Ambou Frutos I, Lastra Pérez L, Vilches Lescaille D, Osorio Illas L, Ramos López M, Rodríguez Ahuar N. Manifestaciones clínicas asociadas al síndrome de TORCH. Revista Cubana de Oftalmología. 2018;31(1):132–44.

* View Article

* Google Scholar

41. 41. Pomari E, Piubelli C, Perandin F, Bisoffi Z. Digital PCR: A new technology for diagnosis of parasitic infections. Clinical Microbiology and Infection. 2019;25(12):1510–6. pmid:31226445

* View Article

* PubMed/NCBI

* Google Scholar

42. 42. Nabet C, Brossas JY, Poignon C, Bouzidi A, Paris L, Touafek F, et al. Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study. J Mol Diagn. 2023. pmid:37068735

* View Article

* PubMed/NCBI

* Google Scholar

43. 43. Berredjem H, Aouras H, Benlaifa M, Becheker I, Djebar MR. Contribution of IgG avidity and PCR for the early diagnosis of toxoplasmosis in pregnant women from the North-Eastern region of Algeria. African Health Sciences. 2017;17(3):647–56. pmid:29085392

* View Article

* PubMed/NCBI

* Google Scholar

Citation: Lynn MK, Rodriguez Aquino MS, Cornejo Rivas PM, Miranda X, Torres-Romero DF, Cowan H, et al. (2024) Employing digital PCR for enhanced detection of perinatal Toxoplasma gondii infection: A cross-sectional surveillance and maternal-infant outcomes study in El Salvador. PLoS Negl Trop Dis 18(5): e0012153. https://doi.org/10.1371/journal.pntd.0012153

References

1. Gómez-Chávez F, Cañedo-Solares I, Ortiz-Alegría LB, Flores-García Y, Figueroa-Damián R, Luna-Pastén H, et al. A proinflammatory immune response might determine Toxoplasma gondii vertical transmission and severity of clinical features in congenitally infected newborns. Frontiers in immunology. 2020;11:390. pmid:32231666

2. El Bissati K, Levigne P, Lykins J, Adlaoui EB, Barkat A, Berraho A, et al. Global initiative for congenital toxoplasmosis: an observational and international comparative clinical analysis. Emerging microbes & infections. 2018;7(1):1–14.

3. Shwab EK, Zhu X-Q, Majumdar D, Pena HF, Gennari SM, Dubey JP, Su C. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology. 2014;141(4):453–61. pmid:24477076

4. Barros M, Teixeira D, Vilanova M, Correia A, Teixeira N, Borges M. Vaccines in Congenital Toxoplasmosis: Advances and Perspectives. Frontiers in Immunology. 2021;11:621997.

5. Maldonado YA, Read JS, Byington CL, Barnett ED, Davies H, Edwards KM, et al. Diagnosis, treatment, and prevention of congenital toxoplasmosis in the United States. Pediatrics. 2017;139(2). pmid:28138010

6. Rajapakse S, Weeratunga P, Rodrigo C, de Silva NL, Fernando SD. Prophylaxis of human toxoplasmosis: a systematic review. Pathogens and global health. 2017;111(7):333–42. pmid:28948861

7. Kota AS, Shabbir N. Congenital Toxoplasmosis. 2019.

8. Rostami A, Riahi SM, Contopoulos-Ioannidis DG, Gamble HR, Fakhri Y, Shiadeh MN, et al. Acute Toxoplasma infection in pregnant women worldwide: A systematic review and meta-analysis. PLOS Neglected Tropical Diseases. 2019;13(10):e0007807.

9. Dubey J, Murata F, Cerqueira-Cézar C, Kwok O, Villena I. Congenital toxoplasmosis in humans: an update of worldwide rate of congenital infections. Parasitology. 2021;148(12):1406–16. pmid:34254575

10. Bigna JJ, Tochie JN, Tounouga DN, Bekolo AO, Ymele NS, Youda EL, et al. Global, regional, and country seroprevalence of Toxoplasma gondii in pregnant women: a systematic review, modelling and meta-analysis. Scientific reports. 2020;10(1):1–10.

11. Teimouri A, Mohtasebi S, Kazemirad E, Keshavarz H. Role of Toxoplasma gondii IgG avidity testing in discriminating between acute and chronic toxoplasmosis in pregnancy. J Clin Microbiol. 2020;58(9):e00505–20. pmid:32321784

12. Garnaud C, Fricker-Hidalgo H, Evengård B, Álvarez-Martinez MJ, Petersen E, Kortbeek LM, et al. Toxoplasma gondii-specific IgG avidity testing in pregnant women. Clinical Microbiology and Infection. 2020;26(9):1155–60. pmid:32334096

13. MINSAL. Lineamientos técnicos para la atención de la mujer en el período preconcepcional, prenatal, parto, puerperio y al recién nacido. Servicios en SSR para atención de emergencias o desastres. In: Salud GyDe, editor. 2021.

14. PÉREZ MATA E, GONZÁLEZGONZÁLEZ V, ROMERO MEDRANO . DETERMINACIÓN DE LA PRESENCIA DE INMUNOGLOBULINAS IgG E IgM EN INFECCIÓN POR Toxoplasma gondii EN MUJERES DE 15 A 45 AÑOS QUE CONSULTAN LA UNIDAD DE SALUD DE CONCEPCIÓN BATRES DEPARTAMENTO DE USULUTÁN, PERIODO DE AGOSTO A SEPTIEMBRE DE 2012. Universidad de El Salvador Sitema Bibliotecario: Universidad de El Salvador 2012.

15. Guevara Diaz I, Navarrete Chavez M, Lazo Cruz J. DETECCIÓN DE ANTICUERPOS ANTI Toxoplasma gondii DE TIPO IgM, EN HEMOCOMPONENTES PROVENIENTES DE DONANTES MUJERES EN EL ÁREA DE BANCO DE SANGRE DEL HOSPITAL NACIONAL SAN JUAN DE DIOS DE SAN MIGUEL. PERIODO: AGOSTO Y SEPTIEMBRE DE 2013. Universidad de El Salvador Sistema Biblioteario Repositorio Universidad de El Salvador; 2013.

16. Rivas H. Reportan muerte de niño por toxoplasmosis. La Prensa. 7 May 2019.

17. Silva Garcia RC, Rosales W, Martinez Romero L. PERFIL CLINICO Y EPIDEMIOLÓGICO DE LACTANTES MENORES O IGUAL A 1 AÑO CON MICROCEFALIA QUE SON EVALUADOS EN LA CONSULTA EXTERNA DE NEUROLOGÍA DEL HOSPITAL NACIONAL DE NIÑOS BENJAMÍN BLOOM DE ENERO DE 2015 A DICIEMBRE DE 2016 [Posgrado de especialidades medicas]: Universidad de El Salvador; 2018.

18. Ramírez Peña S. PERFIL EPIDEMIOLÓGICO DEL RECIÉN NACIDO DE BAJO PESO AL NACER ENTRE 2,000 Y 2,499 g QUE INGRESA AL SERVICIO DE NEONATOS DEL HOSPITAL NACIONAL ESPECIALIZADO DE MATERNIDAD “DR. RAÚL ARGUELLO ESCOLÁN”. ENERO A JUNIO 2013. Universidad de El Salvador Sistema Bibliotecario Repositorio: Universidad de El Salvador; 2014.

19. Gutierrez-Loli R, Ferradas C, Diestra A, Traianou A, Bowman N, Bok J, et al. Development of a novel protocol based on blood clot to improve the sensitivity of qPCR detection of Toxoplasma gondii in peripheral blood specimens. The American journal of tropical medicine and hygiene. 2019;100(1):83. pmid:30457102

20. Homan W, Vercammen M, De Braekeleer J, Verschueren H. Identification of a 200-to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. International journal for parasitology. 2000;30(1):69–75. pmid:10675747

21. Marino AMF, Giunta RP, Salvaggio A, Castello A, Alfonzetti T, Barbagallo A, et al. Toxoplasma gondii in edible fishes captured in the Mediterranean basin. Zoonoses and public health. 2019;66(7):826–34. pmid:31278858

22. Jacobs BK, Goetghebeur E, Vandesompele J, De Ganck A, Nijs N, Beckers A, et al. Model-based classification for digital PCR: your umbrella for rain. Analytical chemistry. 2017;89(8):4461–7. pmid:28350455

23. Hunter ME, Dorazio RM, Butterfield JS, Meigs-Friend G, Nico LG, Ferrante JA. Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA. Molecular ecology resources. 2017;17(2):221–9. pmid:27768244

24. Cherry J, Demmler-Harrison GJ, Kaplan SL, Steinbach WJ, Hotez PJ. Feigin and Cherry’s Textbook of Pediatric Infectious Diseases E-Book: 2-Volume Set: Elsevier Health Sciences; 2013.

25. Mancusi A, Giordano A, Bosco A, Girardi S, Proroga YT, Morena L, et al. Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples. Parasitology Research. 2022;121(5):1467–73. pmid:35230549

26. Saki J, Foroutan M, Khodkar I, Khodadadi A, Nazari L. Seroprevalence and molecular detection of Toxoplasma gondii in healthy blood donors in southwest Iran. Transfusion and Apheresis Science. 2019;58(1):79–82. pmid:30579749

27. Ahmed M, Sood A, Gupta J. Toxoplasmosis in pregnancy. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2020;255:44–50. pmid:33075679

28. Auriti C, Bucci S, De Rose DU, Coltella L, Santisi A, Martini L, et al. Maternal&ndash;Fetal Infections (Cytomegalovirus, Toxoplasma, Syphilis): Short-Term and Long-Term Neurodevelopmental Outcomes in Children Infected and Uninfected at Birth. Pathogens. 2022;11(11):1278.

29. Monfredini C, Cavallin F, Villani PE, Paterlini G, Allais B, Trevisanuto D. Meconium aspiration syndrome: a narrative review. Children. 2021;8(3):230. pmid:33802887

30. Wallon M, Kodjikian L, Binquet C, Garweg J, Fleury J, Quantin C, Peyron F. Long-term ocular prognosis in 327 children with congenital toxoplasmosis. Pediatrics. 2004;113(6):1567–72. pmid:15173475

31. Garweg JG, Kieffer F, Mandelbrot L, Peyron F, Wallon M. Long-Term Outcomes in Children with Congenital Toxoplasmosis-A Systematic Review. Pathogens. 2022;11(10). pmid:36297244

32. Ducrocq J, Ndao M, Yansouni CP, Proulx JF, Mondor M, Hamel D, et al. Epidemiology associated with the exposure to Toxoplasma gondii in Nunavik’s Inuit population using the 2017 Qanuilirpitaa cross-sectional health survey. Zoonoses Public Health. 2021;68(7):803–14. pmid:34254450

33. Galal L, Stragier C, Boumédiène F, Hamidović A, Maugrion O, Dardé ML, Mercier A. Combining spatial analysis and host population genetics to gain insights into the mode of transmission of a pathogen: The example of Toxoplasma gondii in mice. Infect Genet Evol. 2020;78:104142. pmid:31841702

34. Espindola BD, Fernandes FD, Roman IJ, Samoel GVA, Barcelos RAD, Döhler AR, et al. Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages. Rev Bras Parasitol Vet. 2022;31(3):e009322. pmid:36074435

35. Antinarelli LMR, Silva MR, Guimarães R, Terror MS, Lima PE, Ishii JSC, et al. Rural residence remains a risk factor for Toxoplasma infection among pregnant women in a highly urbanized Brazilian area: a robust cross-sectional study. Trans R Soc Trop Med Hyg. 2021;115(8):896–903. pmid:33347595

36. Salvador GdE. Medición Multidimensional de la Pobreza In: (SETEPLAN) STydPdlP, Ministerio de Economía atdlDGdEyCM-D, editors. Portal de Transparancia Insistuto de Acceso a la Información Pública; 2017. p. 45.

37. Hicks VJ, Sánchez C, López MR, Gottschlich A, Grajeda LM, Balish A, et al. Seroprevalence of high incidence congenital infections among pregnant women in Coatepeque, Guatemala and surrounding areas, 2017–2018. PLoS Negl Trop Dis. 2023;17(4):e0011248. pmid:37093863

38. Medeiros JF, Rabelo ESAC, Rocha N, Georg AV, Melli P, Quintana SM, Duarte G. Seroprevalence of Toxoplasmosis in Puerperal Women Treated at a Tertiary Referral Hospital. Rev Bras Ginecol Obstet. 2023;45(2):59–64.

39. Flores C, Villalobos-Cerrud D, Borace J, Fábrega L, Norero X, Sáez-Llorens X, et al. Epidemiological Aspects of Maternal and Congenital Toxoplasmosis in Panama. Pathogens. 2021;10(6). pmid:34204401

40. Ambou Frutos I, Lastra Pérez L, Vilches Lescaille D, Osorio Illas L, Ramos López M, Rodríguez Ahuar N. Manifestaciones clínicas asociadas al síndrome de TORCH. Revista Cubana de Oftalmología. 2018;31(1):132–44.

41. Pomari E, Piubelli C, Perandin F, Bisoffi Z. Digital PCR: A new technology for diagnosis of parasitic infections. Clinical Microbiology and Infection. 2019;25(12):1510–6. pmid:31226445

42. Nabet C, Brossas JY, Poignon C, Bouzidi A, Paris L, Touafek F, et al. Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study. J Mol Diagn. 2023. pmid:37068735

43. Berredjem H, Aouras H, Benlaifa M, Becheker I, Djebar MR. Contribution of IgG avidity and PCR for the early diagnosis of toxoplasmosis in pregnant women from the North-Eastern region of Algeria. African Health Sciences. 2017;17(3):647–56. pmid:29085392