Estudio comparativo de la detección de SARS CoV-2 por RT-PCR en muestras de hisopado nasofaringeo y saliva un estudio piloto en Bolivia

Objetivos: El muestreo de hisopado nasofaríngeo para la detección de SARS CoV-2 es un método estándar para el diagnóstico de COVID-19, pero su recolección generalmente ocasiona incomodidad en el paciente y expone a un mayor riesgo al personal de salud. La muestra de saliva parece ser una buena alternativa con respecto a las muestras de hisopado nasofaringeo, no es invasiva, reduce el riesgo de contaminación del personal sanitario y permite la auto recolección. Este estudio tiene por objetivo comparar la capacidad de detectar al SARS CoV-2 por RT-PCR en un mismo paciente, a partir de muestras de saliva y de hisopado nasofaríngeo para analizar la concordancia de los resultados obtenidos entre ambas muestras. Métodos: Treinta muestras de saliva y de HNF de pacientes con síntomas de COVID-19 que ingresaron al servicio de emergencia del Hospital Clínico Viedma fueron tomadas en paralelo. Ambas muestras fueron analizadas por RT-PCR para la detección de SARS CoV-2. La concordancia de resultados fue calculada por el coeficiente de kappa de Cohen. Resultados: Nuestros resultados muestran que existe una buena concordancia (Índice Kappa 0,730; IC del 95%: 0,486 - 0,974) entre los dos tipos de muestras analizadas. Conclusiones: La saliva parece ser una muestra fiable y efectiva para la detección del SARS CoV-2.

Objectives: Nasopharyngeal swab sampling for the detection of SARS-CoV-2 is a standard method for the diagnosis of COVID-19, but its collection usually causes discomfort in the patient and exposes healthcare workers to a higher risk. Saliva seems to be a good alternative to nasopharyngeal swabs, as it is non-invasive, reduces the risk of contamination of healthcare workers, and allows self-collection. This study aims to compare the ability to detect SARS-CoV-2 by RT-PCR in the same patient using saliva and nasopharyngeal swab samples to analyze the concordance of the results obtained between the two samples. Methods: Thirty saliva and nasopharyngeal swab samples from patients with COVID-19 symptoms who were admitted to the emergency department of the Viedma Clinical Hospital were taken in parallel. Both samples were analyzed by RT-PCR for the detection of SARS-CoV-2. The concordance of results was calculated using the Cohen's Kappa coefficient. Results: Our results show that there is good concordance (Kappa index 0.730; 95% CI: 0.486-0.974) between the two types of samples analyzed. Conclusions: Saliva seems to be a reliable and effective sample for the detection of SARS-CoV-2.

DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells.

AIM: Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2 O2 is instrumental for the sodium channel upregulation observed in H441 cells. METHODS: In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used. RESULTS: Cells forming epithelial domes induced by dexamethasone (0.1 µmol L-1 , 24 hours) and by 5-aza-2'-deoxytidine (1 µmol L-1 , 48 hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2 O2 . While single application of 0.2 mmol L-1 H2 O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1 mmol L-1 ) activated acutely amiloride-sensitive whole-cell currents from 3.91 ± 0.79 pA pF-1 to 4.76 ± 0.98 pA pF-1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2 O2 on ENaC gene expression. CONCLUSION: Our observations suggest that tonic production of H2 O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2 O2 -dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine ß-cells.

Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from ß-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat ß-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 µM, the Cl(-) current is outwardly rectifying, and at 2 µM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.

Analysis of aquaporin expression in liver with a focus on hepatocytes.

A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.

Immunocytochemistry of GLUT2, uptake of fluorescent desnitroso-streptozotocin analogs and phosphorylation of D-glucose in INS-1E cells.

The non-invasive imaging of GLUT2-expressing cells remains a challenge. As streptozotocin, and similarly alloxan, may be transported into cells by GLUT2, the major aim of the present study was to assess the possible use of fluorescent desnitroso-streptozotocin analogs for in vitro labeling of GLUT2-expressing cells. INS-1E cells, human embryonic kidney (HEK) cells, rat isolated pancreatic islets, rat hepatic cells, rat exocrine pancreatic cells and tumoral insulin-producing BRIN-BD11 cells were incubated in the presence of two distinct fluorescent desnitroso-streptozotocin analogs, probes A and B. The immunocytochemistry of GLUT2 in INS-1E cells and the phosphorylation of D-glucose by INS-1E cell homogenates were also examined. The uptake of probes A and B (12.0 µM) by INS-1E cells yielded apparent intracellular concentrations approximately one order of magnitude higher than the extracellular concentration. The two probes differed from one another by the absolute values for their respective uptake and time course, but not so by the pattern of their concentration dependency. Comparable results were recorded in HEK cells, rat isolated pancreatic islets and hepatocytes. Vastly different findings were recorded, however, in rat exocrine pancreatic cells, which do not express GLUT2. Moreover, an unusual concentration dependency for the uptake of each probe was observed in tumoral BRIN-BD11 cells. It is proposed that suitable fluorescent desnitroso-streptozotocin analogs may be used to label GLUT2-expressing cells.

A new role for aquaporin 7 in insulin secretion.

UNLABELLED: BACGROUNS/AIMS: Several insulinotropic agents were recently reported to cause ß-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7(+/+) or AQP7(-/-) mice. METHODS: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. RESULTS: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in ß-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7(+/+) or AQP7(-/-) mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7(-/-) mice. Yet, exposure of ß-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7(+/+) and AQP7(-/-) mice. Both the cell swelling and change in membrane potential were only impaired in AQP7(-/-) cells when exposed to 50 mM glycerol. CONCLUSION: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.

Pancreatic beta-cells: Role of glycerol and aquaglyceroporin 7.

Pancreatic ß-cells originate from gut endoderm during development. Pancreatic endocrine cells represent about 10% of the mature pancreatic cells, and ß-cells represent the majority of endocrine cells. ß-cells secrete insulin in response to elevation of nutrient concentrations. Insulin maintains glucose homeostasis by stimulating glucose uptake into muscle and adipose tissue. Aquaglyceroporin 7, permeable to water, glycerol and urea, is expressed in pancreatic ß-cells and was recently described as being involved in the control of insulin secretion.

Expression of the electrogenic Na+-HCO3--cotransporter NBCe1 in tumoral insulin-producing BRIN-BD11 cells.

BACKGROUND/AIMS: The expression of the electrogenic Na+-HCO3--cotransporter NBCe1 was recently documented in rat pancreatic islet B-cells, it being speculated that such a protein participates in the extrusion of bicarbonate generated by the oxidative catabolism of nutrients from insulin-producing cells. Considering the prevalence of a Crabtree effect in tumoral insulin-producing cells, the possible presence of NBCe1 was now investigated in BRIN-BD11 cells, an insulin-producing cell line established by electrofusion of normal pancreatic B-cells with immortalized RINm5F cells. METHODS: The possible presence of NBCe1 in BRIN-BD11 cells was investigated by RT-PCR, western blot analysis and immunocytochemistry. The release of insulin and net uptake of 22Na+ were also measured in the BRIN-BD11 cells. RESULTS: RT-PCR, western blot analysis and immunocytochemistry documented the presence of NBCe1 in BRIN-BD11 cells. A reported inhibitor of NBCe1, i.e. tenidap, (50-100 microM), inhibited basal and hypotonicity-induced insulin release from the BRIN-BD11 cells, whilst increasing the net uptake of 22Na+ by the same cells. The latter effect was, in relative terms, more pronounced in the presence than absence of ouabain. CONCLUSION: BRIN-BD11 cells, like normal pancreatic islet B-cells, express NBCe1, with predominance of the B variant of this electrogenic Na+-HCO3--cotransporter.

Contrasting effects of glycerol and urea transport on rat pancreatic beta-cell function.

BACKGROUND/AIMS: Pancreatic beta-cell function is influenced by changes in cell volume. Such volume changes depend on water permeability of the plasma membrane, conferred in part by aquaporins. Islet cells express aquaporin 7 (AQP7), which is permeable to urea and glycerol in addition to water. We therefore investigated the effects of glycerol and urea on rat pancreatic beta-cell function. METHODS: Electrical activity and whole-cell current were studied using the perforated patch technique. Cell volume was measured by video-imaging and insulin release by radioimmunoassay. Aquaporin 7 expression was studied by RT-PCR, Western blot and double fluorescent immunolabelling. RESULTS: The isosmotic addition of glycerol and urea resulted in depolarization of the plasma membrane and electrical activity, accompanied by beta-cell swelling, activation of the volume-regulated anion channel (VRAC) and insulin release. However, the effects of glycerol, in contrast to urea, persisted throughout exposure to the osmolyte. Glycerol also caused beta-cell activation when added hyperosmotically. A non-metabolizable glycerol analogue had comparable effects to urea on beta-cells. The expression of AQP7 was demonstrated in rat beta-cells. CONCLUSION: Glycerol and urea can activate beta-cells via their rapid uptake across the beta-cell plasma membrane, possibly via AQP7. This results in cell swelling, VRAC activation, electrical activity and insulin release. Glycerol appears to exert an additional effect, possibly related to its intracellular metabolism.

Expression of the electrogenic Na+-HCO3--cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells.

It was recently proposed that, in rat pancreatic islets, the production of bicarbonate accounts for the major fraction of the carbon dioxide generated by the oxidative catabolism of nutrient insulin secretagogues. In search of the mechanism(s) supporting the membrane transport of bicarbonate, the possible role of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells was investigated. Expression of NBCe1-A and NBCe1-B in rat pancreatic islet cells was documented by RT-PCR, western blotting, and immunocytochemistry. The latter procedure suggested a preferential localization of NBCe1-B in insulin-producing cells. Tenidap (3-100 microM), previously proposed as an inhibitor of NBCe1-A-mediated cotransport in proximal tubule kidney cells, caused a concentration-related inhibition of glucose-stimulated insulin secretion. It also inhibited 2-ketoisocaproate-induced insulin release and to a relatively lesser extent, the secretory response to L: -leucine. Tenidap (50-100 microM) also inhibited the metabolism of D: -glucose in isolated islets, increased (22)Na net uptake by dispersed islet cells, lowered intracellular pH and provoked hyperpolarization of plasma membrane in insulin-producing cells. This study thus reveals the expression of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells, and is consistent with the participation of such transporters in the process of nutrient-stimulated insulin secretion.

Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta, Argentina.

Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPD as belonging to T. cruzi IId. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II.

Comparison of Trypanosoma cruzi lineages and levels of parasitic DNA in infected mothers and their newborns.

To better understand the factors involved in maternal-fetal transmission of Trypanosoma cruzi, we compared DNA levels-obtained by use of quantitative real-time PCR and parasitic genotypes determined by PCR amplification followed by hybridization-in Bolivian mothers and their congenitally infected newborns. Mothers and their neonates displayed markedly different parasitic DNA levels, as most maternal estimated parasitemias (> 90%) were < 10 parasites/mL, whereas those of 76% of their newborns were > 1,000 parasites/mL. Comparison of T. cruzi TcII sublineages infecting mothers and newborns showed identity, without evidence of mixed infection in mothers or neonates. Analysis of minor variants of TcIId-genotyped parasites using sequence class probes hybridizing with hypervariable domains of kDNA minicircles showed discrepancies in half of mother/newborn pairs.

Trypanosoma cruzi: typing of genotype (sub)lineages in megacolon samples from bolivian patients.

Visceral dystrophy, a clinical complication of Chagas' disease, is more frequent in southern cone countries in South America, where Trypanosoma cruzi II (TcII) lineage predominates in human infection. As this major TcII lineage is not homogeneous population and its (sub)lineages are not geographically distributed evenly, therefore, we investigated the possible relationship between parasite (sub)lineages in megacolon patients. We typified the T. cruzi lineages and (sub)lineages in megacolon samples from 18 patients using kDNA probes specific of lineage TcI, TcIIb, TcIId and TcIIe. The majority of the samples (16/18) were (sub)lineage TcIId positive. However, two samples were positive for (sub)lineage TcIIb. Two synthetic probes discriminated variants of lineage TcIId. Proportion of TcIId variants encountered were 6/16, 6/16 and 4/16, similar to the distribution of Chagasic populations in Bolivia. Our data suggest that there is no preferential tropism of one particular lineage or variant of T. cruzi II in megacolon pathology.

Amniotic fluid is not useful for diagnosis of congenital Trypanosoma cruzi infection.

Although Trypanosoma cruzi can be transmitted transplacentally and induce congenital infection, no data are available about the presence of this parasite in human amniotic fluid. We examined 8, 19, and 4 amniotic fluid samples (collected at delivery or by aspiration of gastric content of neonates) from control uninfected mothers (M-B-), infected mothers delivering uninfected newborns (M+B-), and mothers of confirmed congenital cases (M+B+), respectively. Polymerase chain reaction (PCR), using nuclear and kinetoplastic DNA primers (Tcz1-Tcz2 and 121-122), were negative for all control M-B- samples, but positive for 5 of 19 M+B- and 2 of 4 M+B+ samples. To determine the number of parasites in the positive samples, real-time PCR using S35/S36 kinetoplastic DNA was performed. Only one M+B+ sample presented a high parasitic DNA amount, whereas the other six PCR-positive samples displayed traces of T. cruzi DNA. In conclusion, the release of parasites in amniotic fluid is probably a rare event that cannot be helpful for the routine diagnosis of congenital Chagas disease.

Congenital Chagas disease in Bolivia is not associated with DNA polymorphism of Trypanosoma cruzi.

This study aims to typify the Trypanosoma cruzi (sub)lineage(s) in umbilical cord blood of congenitally infected Bolivian newborns, using PCR amplifications of "Region Markers", mini-exon or kDNA fragments followed by hybridization or sequencing. New probes were also designed to distinguish three variants within the TcIId sublineage. The IIb, IId, or IIe T. cruzi sublineages, as well as different variants of the IId sublineage, were detected in infected neonates, whereas mixed infections were not found. The frequencies of the IId sublineage were similar in neonates (95.1%) and adults of the same area (94.1%). The IId-infected newborns displayed either asymptomatic, or severe and fatal clinical forms of congenital Chagas disease, as well as low or high parasitemia. Altogether these data show that T. cruzi DNA polymorphism, based on the presently available markers, is not associated with the occurrence of congenital infection or the development of severe clinical forms of congenital Chagas disease.

Glucose activation of the glucagon receptor gene: functional dissimilarity with several other glucose response elements.

The glucagon receptor (GLR) expression is positively regulated by glucose. This regulation is allowed by the presence, in the promotor of the rat GLR gene, of a sequence feature similar to the two E-boxes motifs constituting the carbohydrate response elements (ChoRE) described for several glycolytic and lipogenic enzyme genes. Using reporter gene assays, we demonstrated here that, despite structural homologies with these ChoREs, the GLR gene glucose response element presents various functional dissimilarities. Testing glucose analogs, we demonstrated that, as for other genes, the glucose must be first phosphorylated. However, at variance with others homologue genes, our data showed the implication of the nonoxidative branch of the pentose phosphate pathway in the transmission of the glucose signal and lack of inhibition by adenosine monophosphate (AMP)-kinase. Furthermore, the activity of our reporter gene was strongly stimulated by butyrate, propionate, and acetate. This observation contrasts with fatty-acid-induced inhibition of the glucose activation, observed for all other genes containing homolog ChoREs. We also showed that glucose and butyrate influence the reporter gene expression via different features.

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles.

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.

[Comparison of PCR methods for the diagnosis of congenital Trypanosoma cruzi infection]. / Comparación de métodos de PCR para el diagnóstico de la infección congenita por Trypanosoma cruzi.

PCR is a potentially interesting diagnostic tool to detect congenital T. cruzi infection. We have compared the sensitivity and capacity of a battery of T. cruzi PCR primers to detect the complete spectrum of known T. cruzi lineages, in order to improve and simplify the detection of infection in neonatal blood. We found that the primers Tcz1/Tcz2, targeting the 195 bp satellite repeat, detected all the parasitic lineages with the same sensitivity For all other tested primers (nDNA primers: BP1/BP2, 01/02, Pon1/ Pon2 and Tca1/Tca2; kDNA primers: S35VS36, 121/122), either, the intensity of amplicons varied according to T. cruzi lineages, or the assess were less sensitive. In order to better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested with parasitological methods. Reliability of our PCR test was demonstrated since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 out of 263 from babies, parasitologically negative, born from infected mothers) were negative. As our PCR method is simple, reliable, robust and cheap, it appears suitable for the detection of T. cruzi infection in neonatal blood.