Conceived and designed the experiments: AGS MB LO MS TD FG SSE CB AL JL. Performed the experiments: MB TD AMMJ CC FA VV YQ SD GH IZ RHL EV TT ZSL LG DN MMR JEL JDR PZ MF MIJ GC CB. Analyzed the data: AGS MB LO MS TD AMMJ CC YQ SD GH IZ RHL EV TT ZSL LG DN MMR JEL JDR MF MIJ GC NA AMDC CIG KAV PY FT CR PD OTC CA GR PB AA FG AD CB AL. Contributed reagents/materials/analysis tools: AGS GH RHL AA AD AL JL. Wrote the paper: AGS MB LO MS TD SD CA PB CB AL. Organized and coordinated the multicentric study: AGS.
The authors have declared that no competing interests exist.
A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of
An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing
This study represents a first crucial step towards international validation of PCR procedures for detection of
A century after its discovery, Chagas disease, caused by the parasite
A century after its discovery
Since 1990, a series of international initiatives based on vector control, systematic screening of blood donors in all endemic countries, and detection and treatment of congenital transmission have been launched for control and elimination of Chagas disease. These strategies have led to significant reduction in the number of infected people worldwide. According to information from 21 countries where the disease is endemic, the number of infected persons today is estimated to be 7,694,500, most of them at the chronic stage of disease
Traditional parasitological procedures, such as xenodiagnosis and haemoculture are laborious and time-consuming and show poor sensitivities in cases of low-level parasitaemias, limiting their usefulness in diagnosis and monitoring of drug efficacy
In this context, the assessment of the performances of currently available PCR tests for detection of
The participating laboratories were selected on the basis of their expertise in current processing of clinical samples for PCR detection of
Aiming to explore the highest extent of currently used PCR tests for detection of
Lb/Test | Extraction Method | Target | Primer Names | PCR | Master Mix | Cycles |
LbA | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 35 |
LbB | Solvent extraction | kDNA | S35 - S36 | Conventional | In-House | 30 |
LbC/1 | Solvent extraction | kDNA | S35 - S36 | Conventional | In-House | 32 |
LbC/2 | Solvent extraction | Sat-DNA | tcz1 - tcz2 | Conventional | In-House | 40 |
LbC/3 | Solvent extraction | 24s | D71-D71 | Conventional | In-House | 40 |
LbC/4 | Solvent extraction | CO II-DNA | Tcmit 31-40 | Conventional | In-House | 48 |
Lb/C5 | Solvent extraction | CO II-DNA | Nested Tcmit 10-21 | Conventional | In-House | 48 |
Lb/C6 | Solvent extraction | SL-DNA | Tcc- Tc1-Tc2 | Conventional | In-House | 30 |
Lb/D1 | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 36 |
Lb/D2 | Solvent extraction | Sat-DNA | TczF-TczR | Real Time | Qiuantitect (Kt) | 50 |
Lb/D3 | Solvent extraction | Sat-DNA | TczF-TczR | Conventional | In-House | 41 |
LbE | Chelex Resine | kDNA | 121-122 | Conventional | In-House | 35 |
LbF/1 | Roche Silica gel col (Kt) | Sat-DNA | cruzi1-2 | Real Time | Roche (Kt) | 45 |
LbF/2 | Roche Silica gel col (Kt) | kDNA | 32f-148r | Real Time | Roche (Kt) | 45 |
LbG/1 | Qiagen DNeasy Tissue kit (Kt) | kDNA FAM – IPC | 32f-148r | Real Time | Apllied Biosystem (Kt) | 55 |
LbG/2 | Qiagen DNeasy Tissue kit (Kt) | kDNA FAM | 32f-148r | Real Time | Apllied Biosystem (Kt) | 55 |
LbG/3 | Qiagen DNeasy Tissue kit (Kt) | kDNA VIC | 32f-148r | Real Time | Apllied Biosystem (Kt) | 55 |
LbG/4 | Qiagen DNeasy Tissue kit (Kt) | Sat-DNA | cruzi1-2 | Real Time | Apllied Biosystem (Kt) | 45 |
LbH/1 | Favorgen Glass fibers col (Kt) | kDNA | 121-122 | Conventional | GoTaq (Kt) | 33 |
LbH/2 | Favorgen Glass fibers col (Kt) | kDNA | 121-122 | Conventional | In-House | 33 |
LbI/1 | Favorgen Glass fibers col (Kt) | kDNA | 121-122 | Conventional | In-House | 40 |
LbI/2 | Favorgen Glass fibers col (Kt) | kDNA | S35 - S36 | Conventional | In-House | 40 |
LbJ | Solvent extraction | Sat-DNA | Tcz1-Tcz2 | Conventional | In-House | 40 |
LbK/1 | Silica gel col (Kt) | Sat-DNA | cruzi1-2 | Real Time | In-House | 40 |
LbK/2 | Silica gel col (Kt) | kDNA | 121-122 | Conventional | In-House | 40 |
LbL/1 | Blood mini Kit (Kt) | Sat-DNA | cruzi1-2 | Conventional | In-House | 40 |
LbL/2 | Blood mini Kit (Kt) | Sat-DNA | Satellite DNA based kit | Conventional | OligoC-T Coris (Kt) | 40 |
LbM | Silica gel col (Kt) | kDNA | TC1-TC2 | Conventional | In-House | 40 |
LbN/1 | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 40 |
LbN/2 | Solvent extraction | Sat-DNA | Tcz1-Tcz2 | Conventional | In-House | 35 |
LbO | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 40 |
LbP/1 | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 35 |
LbP/2 | CTAB (IH) | kDNA | 121-122 | Conventional | In-House | 35 |
LbQ | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 37 |
LbR | Roche Silica gel col (Kt) | kDNA | 121-122 | Conventional | In-House | 40 |
LbS/1 | Qiagen Silica gel col (Kt) | 18s | Tc18s F3-R4 | Conventional | AmpliTaq Gold (Kt) | 40 |
LbS/2 | Qiagen Silica gel col (Kt) | Sat-DNA | cruzi1-2 | Real Time | Platinum qPCR (Kt) | 40 |
LbS/3 | Qiagen Silica gel col (Kt) | 18s | Tc18s F1042- R1144 | Real Time | Platinum qPCR (Kt) | 40 |
LbS/4 | Qiagen Silica gel col (Kt) | kDNA | 121-122 | Conventional | AmpliTaq Gold (Kt) | 40 |
LbT | ATGEN kit (Kt) | kDNA | 121-122 | Real Time | Invitrogen (Kt) | 40 |
LbU/1 | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 40 |
LbU/2 | Solvent extraction | 24s | D71-D72 | Conventional | In-House | 32 |
LbV/1 | Silica gel col (Kt) | kDNA | 121-122 | Conventional | In-House | 40 |
LbV/2 | Silica gel col (Kt) | Sat-DNA | Tcz1-Tcz2 | Conventional | In-House | 30 |
LbW | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 40 |
LbX | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 35 |
LbY | Solvent extraction | kDNA | 121-122 | Conventional | In-House | 35 |
LbZ | Silica gel col (Kt) | Sat-DNA | cruzi1-2 | Real Time | TaqMan Univ (Kt) | 45 |
LbX/1-6, Laboratory and test identification, kDNA, minicircle DNA; Sat-DNA, satellite DNA; 24s, 24sa rDNA; 18s, 18s rDNA; SL, Spliced Leader; kDNA FAM, kDNA TaqMan probe labeled with FAM; kDNA VIC, kDNA TaqMan probe labeled with VIC; IPC, TaqMan Exogenous Internal Positive Control (Applied Biosystems).
The organizing laboratory (LabMECh, INGEBI, Buenos Aires) was in charge of preparing characterised samples in three different sets (A, B and C), as described below.
Set A. This set consisted of ten-fold serial dilutions of
Set B. This set contained seronegative human blood samples treated with Guanidine Hidrochloride 6M-EDTA 0.2 M buffer, pH 8.00
Set C. This was a panel of 42 pre-characterized archived clinical blood samples stored in Guanidine Hidrochloride-EDTA buffer, including 10 from seronegative patients and 32 from seropositive patients from endemic regions of Argentina, Bolivia, Brazil and Paraguay. The seropositive panel was composed by patients at different phases of
Samples from patients were obtained with written informed consent and approval of the Ethics Committee of the Rivadavia Hospital, Government of Buenos Aires city, Argentina and the Serodiagnostic Laboratory for Chagas Disease, Federal University of Goias, Goiania, Brazil. Furthermore, all samples were tested by two PCR tests performed on duplicate at the organizing laboratory, namely a hot-start PCR targeting kDNA according to Burgos et al
Each sample from set A, B and C was aliquoted and distributed into 1 ml Screw Top bar-coded tubes (Matrix Trackmates, UNITEK, USA) to each package. The packages were sent refrigerated to the participating laboratories (World Courier, Arg). Each laboratory received 50 µls of Set A and 500 µls of samples belonging to sets B and C.
Methods LbD2, LbD3 and LbQ: Solvent DNA extraction was carried out from 100 µl of Guanidine Hidrochloride-EDTA blood aliquots. Briefly, 100 µl aliquots were taken and well mixed with 100 µ l of phenol-chloroform-isoamylic alcohol (25∶24∶1) (phenol Tris–EDTA pH 8, USB Corporation, USA). After centrifugation for 3 min at 13000 rpm 150 µl of distilled water were added. The solution was mixed and centrifuged for 3 min at 13000 rpm. The aqueous phase was transferred to a clean tube, and a final extraction with 200 µ l of chloroform was performed. After centrifugation for 3 min at 13000 rpm the aqueous phase was transferred to a clean tube and mixed with 40 µg of rabbit liver glycogen (Sigma, USA). The DNA was precipitated with 200 µl of isopropyl alcohol during 30 minutes at −20°C. Then the solution was centrifuged at 13000 rpm for 15 min. The pellet was washed with 500 µl of 70% ethanol and centrifuged again 15 min at 13000 rpm. After discard the ethanol the pellet was allowed to dry during 10 min at 37°C. Finally the pellet was suspended in 50 µl 10 mM Tris-HCl, pH 8.5. DNA solution was stored at −20°C. Method LbF1: DNA isolation used a commercial kit (High Pure PCR Template preparation kit, Roche Applied Science) according to the manufacturer's protocol. DNA solution was stored at −20°C.
Method LbD3 was carried out in a MJR PTC-100 thermocycler (MJ Research, Watertown, MA, USA). Master mix was composed by 1X Taq platinum amplification buffer, 250 µM deoxynucleotide triphosphate solution (dNTPs), 3 mM MgCl2 solution, 1,5 U Taq Platinum (Invitrogen, Brazil), 0.5 µM sat-DNA specific primers TCZ-F (
Method LbQ was carried out in a MJR PTC-100 thermocycler (MJ Research, Watertown, MA, USA). Master mix was composed by 1X Taq platinum amplification buffer, 200 µ M dNTPs, 3 mM MgCl2 solution, 1,5 U Taq Platinum (Invitrogen, Brazil), 10 µM kDNA specific primers 121 (
Method LbD2 was conducted using a Rotor Gene 3000 (Corbett Research, Sydney, Australia) Real Time thermocycler. Each PCR reaction contained 1X Qiagen QuantiTect Sybr-Green PCR Master Mix (Qiagen), 0.5 µM SatDNA specific primers TCZ-F (
Method LbF1 was conducted using a Rotor Gene 3000 (Corbett Research, Sydney, Australia) Real Time thermocycler. Each PCR reaction contained 1X PCR FastStart Universal Probe Master Master Mix (Roche), 0.75 µM SatDNA specific primers cruzi 1 (
The possibility of contamination of the PCR reagents and of the solutions used to prepare DNA was carefully examined through the use of appropriate controls. Also two dilutions from DNA purified from Cl- Brener strain were analyzed in each round as strong positive and detection limit control, respectively.
An access database form was distributed to the participants to standardize reporting of results. Those laboratories performing more than one PCR test per sample sent a separate report for each test. The results were analyzed by using SAS Software and Microsoft Excel. Due to the exploratory nature of the study, a descriptive analysis of results is provided.
For set A, the following parameters were evaluated: 1) specificity (Sp): the proportion of negative PCR results in the three negative samples, 2) coherence: (Co) the ability of reporting positive PCR findings in a consecutive way, from the highest to the lowest detected DNA concentration for each series of DNA dilutions of parasite stocks and 3) the detection limits (DL) for each stock. A test was defined as Good Performing Method (GPM) if it was 100% specific and coherent and capable of detecting 10 fg/ul or less DNA for all parasite DTU stocks.
For set B the same parameters were evaluated: Sp, Co and DL. A test was defined as GPM if it was 100% specific and coherent and capable of detecting 5 parasite equivalents/mL of Guanidine Hidrochloride-EDTA treated blood or less.
For each sample of set C, a consensus PCR result was obtained on the basis of the reports by GPM tests in sets A and B, as done in other PCR interlaboratory studies
The sensitivity, specificity, accuracy and kappa index of the different PCR tests were calculated by using 1) the above mentioned consensus PCR results and 2) the serological diagnosis as the reference methods.
Inter-observer kappa coefficients were calculated using GraphPad Software on-line statistical calculators (
Twenty six laboratories reported PCR results, using one to six different PCR tests (
A total of 48 PCR tests were reported for set A samples and 44 of them for sets B and C. Twenty eight tests targeted minicircle DNA, 24 of them amplified the 330 bp variable region and 4 amplified a 118 bp fragment from the constant region (Lb F2 and Lb G1 to G3,
Set A | Set B | GPM | |||||||||
Lb/Test | Sp | Co | DL | Co | DL | Co | DL | Sp | Co | DL par/ml | |
LbA | Y | Y | 0.1 | N | 0.01 | N | 0.01 | Y | N | 0.005 | N |
LbB | Y | N | 0.001 | N | 0.001 | Y | 0.1 | Y | N | 0.005 | N |
LbC/1 | N | N | 0.001 | N | 0.001 | Y | 0.1 | N | Y | ND | N |
LbC/2 | Y | Y | ND | N | 1 | Y | 10 | N | |||
LbC/3 | Y | Y | ND | Y | ND | Y | ND | NA | NA | NA | N |
LbC/4 | Y | Y | ND | Y | ND | Y | ND | NA | NA | NA | N |
Lb/C5 | N | Y | 1 | N | 0.1 | N | 0.001 | NA | NA | NA | N |
Lb/C6 | Y | Y | ND | Y | ND | Y | ND | NA | NA | NA | N |
Lb/D1 | N | N | 0.005 | N | |||||||
LbF/2 | Y | Y | 1 | N | 0.01 | Y | 1 | N | |||
LbG/1 | Y | Y | 0.1 | Y | 1 | Y | ND | N | |||
LbH/1 | Y | Y | 1 | Y | 10 | N | 0.001 | Y | N | 0.05 | N |
LbH/2 | Y | Y | 1 | N | 0.1 | Y | 10 | Y | N | 0.05 | N |
LbI/1 | Y | Y | 1 | N | 0.001 | Y | 10 | N | |||
LbJ | Y | Y | 0.01 | N | 0.001 | N | 0.001 | Y | N | 0.5 | N |
N | |||||||||||
LbL/1 | Y | Y | ND | Y | 10 | Y | 1 | N | |||
LbL/2 | Y | Y | ND | Y | ND | Y | 1 | N | |||
LbM | N | Y | 0.001 | Y | 0.001 | N | 0.001 | Y | Y | ND | N |
LbN/1 | Y | Y | 0.1 | Y | ND | N | 0.1 | Y | N | 0.005 | N |
LbN/2 | Y | Y | 1 | Y | ND | Y | 10 | N | |||
LbO | Y | Y | 10 | N | 1 | Y | ND | N | |||
N | |||||||||||
N | |||||||||||
N | |||||||||||
LbR | Y | N | 0.00005 | N | |||||||
LbS/1 | Y | Y | 1 | Y | ND | Y | ND | Y | Y | ND | N |
N | |||||||||||
LbS/3 | Y | Y | 10 | Y | 10 | Y | ND | Y | Y | ND | N |
LbS/4 | N | N | 0.00005 | N | |||||||
LbT | N | N | 0.001 | Y | 1 | N | 0.01 | N | N | 0.05 | N |
LbU/1 | N | Y | 0.001 | Y | 0.001 | Y | 0.001 | Y | N | 0.005 | N |
LbU/2 | N | Y | 0.001 | Y | 0.001 | Y | 0.001 | N | N | 0.005 | N |
N | |||||||||||
LbV/2 | Y | Y | 0.1 | Y | ND | Y | 10 | N | |||
N | |||||||||||
LbX | N | N | 0.01 | N | 0.001 | N | 0.1 | Y | N | 0.0005 | N |
LbY | N | Y | 1 | Y | 1 | Y | 0.1 | Y | Y | ND | N |
LbZ | N | N | 0.0005 | N |
LbX/1-6, Laboratory and test identification; Bold Type, Good Performing Methods in sets A or B; GPM, Good Performing Methods in sets A and B; Sp, 100% of specificity in all controls without
Out of the 41 tests based on kDNA (28 tests) or Sat-DNA sequences (13 tests), 25 (51.2%) provided specific and coherent results for all three parasite stocks (Sp = Y, Co = Y,
Distribution of detection limits (DL) of specific and coherent PCR tests targeted to Sat-DNA (A) and kDNA sequences (B) for detecting serial dilutions of purified DNA from 3 parasite stocks (Set A) representative of
Analysis of
Analysis of
Analysis of
Overall, the reported PCR tests were less sensitive for detecting DNA from the
Out of the 44 PCR tests reported for spiked Guanidine Hidrochloride-EDTA blood samples, the three tests targeting sequences other than Sat-DNA or kDNA were not further analyzed, because they failed to detect the most concentrated sample (S1 and S3 tests) or showed false positive findings in the non-spiked control (U2 test) (Set B,
Twenty five out of 41 PCR tests based on kDNA and Sat-DNA sequences showed specific, coherent results and detection limits of at least 5 par/ml (GPM, bold fonts,
Out of the 44 PCR tests performed on clinical samples of Set C, a 18s-rDNA PCR (S3) and a SL-DNA PCR (C6) tests did not detect any positive sample and the 24s α rDNA-PCR test (U2) had only 40% of specificity. Consequently, they were not included for subsequent analysis. The levels of agreement among the 41 remaining PCR tests on the reports for each clinical sample are presented in
CLINICAL CASES | kDNA PCR n = 28 | Sat-DNA PCR n = 13 | GPM n = 16 | |||||||||||
ID | G | Ag | Status | Region | EN | pos/tot | % | Cons | pos/tot | % | Cons | pos/tot | % | Cons |
1 | F | NA | cChHD-HTx | Arg- Uk |
Uk | 26 | 92,9 | 12 | 92,3 | 15 | 93,8 | |||
2 | M | NA | cChHD-HTx | Arg-Chaco |
Yes | 26 | 92,9 | 10 | 76,9 | 15 | 93,8 | |||
3 | F | 54 | Mega III cChH | Br- MG | Yes | 20 | 71,4 | 12 | 92,3 | 12 | 75,0 | |||
4 | F | 42 | Pregnant | Arg- Salta |
Yes | 18 | 64,3 | 12 | 92,3 | 14 | 87,5 | |||
5 | F | 25 | Pregnant | Bo-Uk | Yes | 18 | 64,3 | 11 | 84,6 | 12 | 75,0 | |||
6 | M | 20 | Blood donor | Br-BA | Yes | 19 | 67,9 | 10 | 76,9 | 10 | 62,5 | |||
7 | F | 41 | cChHD | Br-BA | Yes | 18 | 64,3 | 9 | 69,2 | 11 | 68,8 | |||
8 | F | 31 | Pregnant | Bo-Uk | Yes | 16 | 57,1 | 10 | 76,9 | 11 | 68,8 | |||
9 | F | NA | Pregnant | Par-Uk2 | Yes | 15 | 53,6 | 11 | 84,6 | 12 | 75,0 | |||
10 | F | 22 | Ex-pregnant | Br-BA | Yes | 17 | 60,7 | 10 | 76,9 | 12 | 75,0 | |||
11 | F | 41 | Chronic CD | Br-BA | Yes | 16 | 57,1 | 9 | 69,2 | 11 | 68,8 | |||
12 | F | 24 | Ex-pregnant | Br-Go | Yes | 15 | 53,6 | 9 | 69,2 | 10 | 62,5 | |||
13 | F | 32 | Pregnant | Arg-Co | Yes | 16 | 57,1 | 8 | 61,5 | 8 | 50,0 | |||
14 | F | 35 | Ex-pregnant | Br-Ceara | Yes | 17 | 60,7 | 9 | 69,2 | 12 | 75,0 | |||
15 | F | 47 | cChHD | Br-Go | Yes | 17 | 60,7 | 8 | 61,5 | 9 | 56,3 | |||
16 | F | NA | Pregnant | Par-Uk | Yes | 14 | 50,0 | 8 | 61,5 | 9 | 56,3 | |||
17 | M | 55 | CD | Br-MG | Yes | 15 | 53,6 | 7 | 53,8 | 10 | 62,5 | |||
18 | M | 33 | cChHD | Br-BA | Yes | 15 | 53,6 | 7 | 53,8 | 8 | 50,0 | |||
19 | F | 66 | Mega II + CBBB | Br-BA | Yes | 15 | 53,6 | 5 | 38,5 | N E G | 9 | 56,3 | ||
20 | F | 18 | Ex-pregnant | Br-Go | Yes | 16 | 57,1 | 4 | 30,8 | N E G | 8 | 50,0 | ||
21 | F | 18 | Pregnant | Arg-Sg | Yes | 15 | 53,6 | 6 | 46,2 | N E G | 8 | 50,0 | ||
22 | F | 43 | Indeterminate CD | Br-BA | Yes | 13 | 46,4 | N E G | 7 | 53,8 | 6 | 37,5 | N E G | |
23 | F | 57 | Blood donor | Br-Piaui | Yes | 11 | 39,3 | N E G | 8 | 61,5 | 9 | 56,3 | ||
24 | F | 46 | Blood donor | Br-BA | Yes | 7 | 25,0 | N E G | 6 | 46,2 | N E G | 6 | 37,5 | N E G |
25 | F | 25 | Pregnant | Par-Uk |
Yes | 10 | 35,7 | N E G | 5 | 38,5 | N E G | 5 | 31,3 | N E G |
26 | F | 32 | Pregnant | Par-Uk |
Yes | 11 | 39,3 | N E G | 5 | 38,5 | N E G | 6 | 37,5 | N E G |
27 | F | 36 | Pregnant | Arg-Chaco | Yes | 11 | 39,3 | N E G | 5 | 38,5 | N E G | 5 | 31,3 | N E G |
28 | F | 36 | Pregnant | Arg-Chaco | Yes | 9 | 32,1 | N E G | 4 | 30,8 | N E G | 2 | 12,5 | N E G |
29 | M | 59 | cChHD | Br-Piaui | Yes | 9 | 32,1 | N E G | 2 | 15,4 | N E G | 5 | 31,3 | N E G |
30 | F | 29 | Mega-II | Br-Go | Yes | 7 | 25,0 | N E G | 4 | 30,8 | N E G | 5 | 31,3 | N E G |
31 | F | NA | Pregnant | Par-Uk |
Yes | 9 | 32,1 | N E G | 1 | 7,7 | N E G | 1 | 6,3 | N E G |
32 | F | 28 | Pregnant | Arg-Sg | Yes | 2 | 7,1 | N E G | 3 | 23,1 | N E G | 1 | 6,3 | N E G |
33 | M | 38 | Routine | Br-Go | No | 4 | 14,3 | N E G | 1 | 7,7 | N E G | 1 | 6,3 | N E G |
34 | M | 51 | Routine | Br-Uk | Yes | 5 | 17,9 | N E G | 1 | 7,7 | N E G | 1 | 6,3 | N E G |
35 | M | NA | Blood donor | Arg-BAs | No | 6 | 21,4 | N E G | 1 | 7,7 | N E G | 1 | 6,3 | N E G |
36 | F | 39 | Routine | Br-Go | Yes | 8 | 28,6 | N E G | 1 | 7,7 | N E G | 1 | 6,3 | N E G |
37 | F | 37 | Routine | Br-Go | No | 6 | 21,4 | N E G | 1 | 7,7 | N E G | 2 | 12,5 | N E G |
38 | M | NA | Blood donor | Arg-BA | No | 7 | 25,0 | N E G | 2 | 15,4 | N E G | 3 | 18,8 | N E G |
39 | F | 36 | Routine | Br-Bh | Yes | 7 | 25,0 | N E G | 4 | 30,8 | N E G | 3 | 18,8 | N E G |
40 | F | 40 | Routine | Br-Go | Yes | 8 | 28,6 | N E G | 4 | 30,8 | N E G | 5 | 31,3 | N E G |
41 | F | 40 | Routine | Br-Go | Yes | 9 | 32,1 | N E G | 6 | 46,2 | N E G | 5 | 31,3 | N E G |
42 | F | 58 | Routine | Br-Go | Yes | 11 | 39,3 | N E G | 6 | 46,2 | N E G | 6 | 37,5 | N E G |
Patients 1 to 32 are seropositive and 33 to 42 seronegative. 28 kDNA tests and 13 Sat DNA tests were performed for each sample.
kDNA, minicircle DNA; Sat-DNA, satellite DNA; GPM Good performing Methods in panels A and B; ID, sample identification number; G, Gender; Ag, age in years; EN, Endemic precedence; %: Percentage of positive results; Cons, Consensus PCR result; F, female; M, male; NA, not available; 28 kDNA tests and 13 Sat DNA tests were performed for each sample.
The individual performance of the 41 PCR tests was evaluated in comparison with the consensus PCR results reached by the 16 GPM in sets A and B (18 PCR positive, 20 PCR negative samples) and in comparison with serologic diagnosis (10 seronegative, 32 seropositive samples) (
Test | PCR performance vs consensus GPM K+S | PCR performance versus Serology | |||||||||
Lb/Test | PCR | Target | Se | Sp | Acc | kappa | Se | Sp | Acc | kappa | BPM |
N = 18 | N = 20 | N = 38 | N = 38 | N = 32 | N = 10 | N = 42 | N = 38 | ||||
LbA | C | K | 33.3 | 60.0 | 47.4 | −0.1 | 31 | 70 | 40.5 | 0.0 | N |
LbB | C | K | 72.2 | 35.0 | 52.6 | 0.1 | 66 | 30 | 57.1 | 0.0 | N |
LbC/1 | C | K | 0.0 | 100.0 | 52.6 | 0.0 | 0 | 100 | 23.8 | 0.0 | N |
LbC/2 | C | S | 66.7 | 15.0 | 39.5 | −0.2 | 69 | 10 | 54.8 | −0.2 | N |
Lb/D1 | C | K | 94.4 | 45.0 | 68.4 | 0.4 | 81 | 40 | 71.4 | 0.2 | N |
Y | |||||||||||
Y | |||||||||||
N | |||||||||||
Y | |||||||||||
LbF/2 | RT | K | 72.2 | 90.0 | 81.6 | 0.6 | 53 | 90 | 61.9 | 0.3 | N |
LbG/1 | RT | K | 100.0 | 60.0 | 78.9 | 0.6 | 84 | 60 | 78.6 | 0.5 | N |
N | |||||||||||
N | |||||||||||
N | |||||||||||
LbH/1 | C | K | 27.8 | 80.0 | 55.3 | 0.1 | 22 | 80 | 35.7 | 0.0 | N |
LbH/2 | C | K | 22.2 | 80.0 | 52.6 | 0.0 | 16 | 80 | 31.0 | 0.0 | N |
LbI/1 | C | K | 83.3 | 40.0 | 60.5 | 0.2 | 78 | 50 | 71.4 | 0.3 | N |
N | |||||||||||
LbJ | C | S | 55.6 | 60.0 | 57.9 | 0.2 | 59 | 70 | 61.9 | 0.2 | N |
N | |||||||||||
N | |||||||||||
LbL/1 | C | S | 88.9 | 45.0 | 65.8 | 0.3 | 84 | 60 | 78.6 | 0.4 | N |
LbL/2 | C | S | 83.3 | 60.0 | 71.1 | 0.4 | 72 | 60 | 69.0 | 0.3 | N |
LbM | C | K | 66.7 | 50.0 | 57.9 | 0.2 | 59 | 50 | 57.1 | 0.1 | N |
LbN/1 | C | K | 66.7 | 80.0 | 73.7 | 0.5 | 47 | 60 | 50.0 | 0.0 | N |
LbN/2 | C | S | 72.2 | 80.0 | 76.3 | 0.5 | 47 | 70 | 52.4 | 0.1 | N |
LbO | C | K | 66.7 | 55.0 | 60.5 | 0.2 | 47 | 30 | 42.9 | −0.2 | N |
N | |||||||||||
N | |||||||||||
Y | |||||||||||
LbR | C | K | 88.9 | 55.0 | 71.1 | 0.4 | 81 | 70 | 78.6 | 0.5 | N |
N | |||||||||||
LbS/4 | C | K | 55.6 | 90.0 | 73.7 | 0.5 | 47 | 100 | 59.5 | 0.3 | N |
LbT | RT | K | 50.0 | 75.0 | 63.2 | 0.3 | 41 | 80 | 50.0 | 0.1 | N |
LbU/1 | C | K | 16.7 | 95.0 | 57.9 | 0.1 | 9 | 90 | 28.6 | 0.0 | N |
N | |||||||||||
LbV/2 | C | S | 44.4 | 100.0 | 73.7 | 0.5 | 28 | 100 | 45.2 | 0.2 | N |
N | |||||||||||
LbX | C | K | 100.0 | 50.0 | 73.7 | 0.5 | 88 | 60 | 81.0 | 0.5 | N |
LbY | C | K | 77.8 | 50.0 | 63.2 | 0.3 | 75 | 80 | 76.2 | 0.5 | N |
LbZ | RT | S | 50.0 | 90.0 | 71.1 | 0.4 | 38 | 100 | 52.4 | 0.2 | N |
LbX/1-6, Laboratory and test identification; BPM, Best Performing Methods; Consensus GPM K + S: consensus findings of GPM by kDNA and Satellite DNA PCRs; C, Conventional PCR, RT, Real Time PCR; K, kDNA; S, Satellite DNA; Se, sensitivity; Sp, specificity; Acc, accuracy; kappa, kappa index; N, negative; Y, affirmative; 25–75p, 25th-75th percentiles; Bold type, Good Performing Methods (GPM) in sets A and B.
Four GPM showed the best operational parameters in set C (
The performance of these four tests was further evaluated at the coordinating laboratory on a subset of samples from seropositive and seronegative patients, analysed in four independent experiments (
A. LbD2 ; B.LbD3, C. LbF1 and D. LbQ. The methods are described in
N° Positive PCR/N° tested samples | |||||||
ID | LbD2 | LbD3 | LbF1 | LbQ | % pos | Cons | |
2/4 | 3/4 | 2/4 | 2/4 | 75 | pos | ||
4/4 | 4/4 | 4/4 | 4/4 | 100 | pos | ||
4/4 | 3/4 | 4/4 | 3/4 | 90,6 | pos | ||
2/4 | 3/4 | 3/4 | 3/4 | 59,4 | pos | ||
0/4 | 0/4 | 0/4 | 0/4 | 0 | neg | ||
0/4 | 0/4 | 0/4 | 0/4 | 0 | neg | ||
0/4 | 0/4 | 0/4 | 0/4 | 0 | neg | ||
0/4 | 0/4 | 0/4 | 0/4 | 0 | neg | ||
28/32 | 29/32 | 29/32 | 28/32 |
ID, sample identification number; LbX/1-6, Laboratory and test identification; % pos, Percentage of Positivity; Cons, Consensus PCR Result; pos, positive; neg, negative.
PCR vs Consensus PCR of |
PCR vs Serology | |||||||
Lb/Test | Se | Sp | Acc | kappa | Se | Sp | Acc | kappa |
N = 20 | N = 12 | N = 32 | N = 32 | N = 20 | N = 12 | N = 32 | N = 32 | |
75 | 100 | 87,5 | 0.8 | 60 | 100 | 75 | 0.5 | |
81,25 | 100 | 90,6 | 0.8 | 65 | 100 | 78 | 0.6 | |
81,25 | 100 | 90,6 | 0.8 | 65 | 100 | 78 | 0.6 | |
75 | 100 | 87,5 | 0.8 | 60 | 100 | 75 | 0.5 |
LbX/1−6, Laboratory and test identification; Se, sensitivity; Sp, specificity; Acc, accuracy; kappa, kappa index.
PCR technology has been widely used for the diagnosis and monitoring of disease progression and therapy outcome in many infectious diseases
Out of the 48 PCR tests reported by 26 laboratories, those targeting ribosomal, miniexon or CO II subunit gene sequences were not sensitive enough when challenged against 10 fg/µl or less of purified DNA from the 3 tested parasite stocks, to merit further consideration. Thus, these methods appeared not suitable for sensitive molecular diagnosis of Chagas disease in clinical settings. However, these parasitic targets are been widely used for genotyping parasite discrete typing units
In set A, GPM included kDNA and sat-DNA PCR tests in similar proportions. However, Sat-DNA PCR tests were less sensitive than kDNA-PCR tests to detect
Regarding
Set B allowed evaluation of the influence of DNA extraction procedures in the PCR performance. A 72.2% of DNA extraction methods based on commercial kits led to GPM in set B, whereas 57.8% of phenol-chlorophorm extracted DNA led to GPM reports. These findings indicated that Guanidine Hidrochloride-EDTA blood was suitable for DNA extraction using kits based on lysis buffers containing Guanidine salts.
Out of the 25 GPM in set B, 14 had a sensitivity of 0.05 par/ml, which should be adequate for diagnosis of infection in chronic patients
Analysis of PCR performance in set C clinical samples showed that the four best performing tests presented strong concordance with respect to consensus PCR results obtained by the 16 tests defined as GPM in sets A plus B (kappa index between 0.7 and 0.8). Out of them, three tests targeted sat-DNA sequences and only one targeted kDNA. These data are in agreement with previous works showing that PCRs targeting Sat-DNA performed better than PCRs targeting kDNA sequences
Moreover, two of the sat-DNA best performing tests used Real Time PCR, one with a Sybr Green fluorescent dye (LbD2) and the other one with a TaqMan probe (LbF1). It must be pointed out that LbD2 and LbD3 tests were performed by the same laboratory. Out of the 16 GPM performed by 11 different laboratories, 3 laboratories performed two methods (LbD, LbK and LbP) and one lab developed 3 tests (LbG). These data point to laboratory dependence concerning PCR performance, which may be due to multiple factors including technical expertise, correct use of quality controls, instrumentation and reagents. For example, tests LbF1, LbS2 (GPM) and LbZ (not GPM) were all based on sat-DNA Real Time PCR using the same primer pair (cruzi 1 – cruzi 2), differing in the trade marks of the DNA extraction and Master Mix kits. Some tests shown as GPM in sets A+B had very low sensitivities in set C (LbK2, LbP2, LbV1,
A major drawback of most PCR tests is that they do not contain an internal amplification control (IAC). An IAC is a non target DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence
Some other tests shown as GPM in sets A+B had very low specificities (LbI2, LbW, LbG2, LbG3,
The four BPM methods were transferred to the Coordinating laboratory, where they were evaluated in a subset of clinical samples, each one tested in four independent assays, obtaining good concordance and confirming the performance reported by the participating laboratories in the previous international study (
We thank the collaboration of Sonia O. Lafon, linked to logistic work and delivery of sample panels to the participating laboratories.
This article is dedicated to the memory of Dr Mariano J. Levin, head of LabMECh, INGEBI-CONICET, died on February 28th, 2010, who dedicated his life to scientific research in Chagas disease and supported with great commitment and passion this collaborative work.