Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam

Figures & data

Table 1.  Description of four H5N1 HPAI-infected Vietnamese poultry flocks sampled in 2009.

		Total numbers of birds, in flock/sampled/infected/uninfected (parentheses indicate numbers of carcasses among sampled birds)
Location: abbreviated identifier; name (type of poultry holding, species)	Date sampled	Chickens	Pekin ducks	Muscovy ducks	VI-positive specimens (n=52), denoted by infected species, number of isolates and anatomical site^a	VI-positive isolates sequenced in H5 gene (n=29) to identify infecting clade with species and number of birds
A: Thai Nguyên; Duong Van Loc (backyard, mixed)	8 April 2009	80(10)/15 (0)/5/10	2/0/NA/NA	200(14)/10(2)/3(2)/7	Muscovy ducks 3F and chickens 5T, 4C, 5F	2.3.2 Muscovy ducks, n=3; 2.3.4 chickens, n=5
B: Lang Son Hoang; Van Thanh (backyard, mixed)	18 April 2009	120(15)/10(0)/8/2	80(4)/15(2)/0/15(2)	None	Chickens 3T, 1C, 7F	2.3.2 chickens, n=7
C: Thai Nguyên; Pham Thi Lan (industrial, chickens)	29 April 2009	>11,000(>3000)/21(5)/14^b(1)/7(4)	None	None	Chickens 2T, 3C, 7F	2.3.2 chicken, n=1; 2.3.4 chickens, n=6
D: Quang Ninh Nguyen; Van Hai (backyard, mainly ducks)	22 June 2009	50(0)/0(0)/NA/NA	1400(193)/23(5)/12(2)/11(3)	10(0)/0(0)/NA/NA	Pekin ducks 3T, 2C, 7F	2.3.2 Pekin ducks, n=7
Infected and uninfected status at bird level determined by AI RRT-PCRs as described in text and Table 2. NA, not applicable. ^aAnatomical site: T, trachea; C, cloaca; F, feathers. ^bThe 14 include one bird judged as positive on the basis on one indeterminate RRT-PCR result (tracheal swab) by the “bead” M-gene RRT-PCR.

Figure 1.  Distribution of Ct values for different types of clinical specimens from chickens tested by both M-gene RRT-PCRs and the H5 HA2 RRT-PCR. Specimens from all 46 chickens included (a) tracheal swabs, (b) cloacal swabs and (c) feathers. Ct values (vertical axis) for M gene ‘wet’ and ‘bead’ RRT PCR results are shown by diamond and square symbols respectively, H5 HA2 RRT PCR Ct values are shown by triangles. Filled symbols indicate chicken specimens that were tested by VI, where dot and grey fills indicate VI positive and VI negative results respectively. The total numbers of “No Ct” results for each RRT PCR are stated above the corresponding test, with parentheses indicating numbers of VI negative and positive results by ^a and ^b respectively. Numbers of indeterminate RRT PCR results (Ct 36.01–39.99) are also stated above each graph (broken horizontal line indicates Ct 36), while numbers of positive RRT PCR results are stated beneath each graph for the corresponding test. The range of Ct values are listed for the positive results for the corresponding RRT PCR.

Figure 2.  Distribution of Ct values for different types of clinical specimens from ducks tested by both M-gene RRT-PCRs and the H5 HA2 RRT-PCR. Specimens from all 48 ducks included (a) tracheal swabs, (b) cloacal swabs and (c) feathers. All were tested by the three RRT PCRs described in Fig 1, and details are as explained in the footnote to the preceding figure. Fig 2 includes VI results for 32 duck specimens.

Table 2.  Sensitivity of RRT-PCR testing of each of the three clinical specimens to identify an infected bird.

	“Wet” M-gene RRT-PCR			“Bead” M-gene RRT-PCR			H5 HA2 RRT-PCR
Poultry type	T	C	F	T	C	F	T	C	F
Chickens	10/26 (39%)	11/26 (42%)	23/26 (89%)	19/26 (73%)	18/26 (69%)	24/26 (92%)	21/26 (81%)	19/26 (73%)	25/26 (96%)
Ducks	7/15 (47%)	1/15 (7%)	14/15 (93%)	13/15 (87%)	7/15 (47%)	15/15 (100%)	14/15 (93%)	7/15 (47%)	15/15 (100%)
Sensitivity for each RRT-PCR determined by the number of positive results (Ct <36; Figures 1 and 2) on a given type of clinical pecimen, compared with the number of infected chickens (n=26) and ducks (n=15), as determined in Table S2 T, tracheal swab; C, cloacal swab; F, feathers.

Table 3.  Results from a Bayesian model to estimate the sensitivities of the three main AI RRT-PCRs.

		Sensitivity of RRT-PCR^b
Poultry group	Combination of specimens tested^a	“Wet” M gene	“Bead” M gene	H5 HA2
Chickens	T and C	0.54 (0.36, 0.7)	0.83 (0.67, 0.94)	0.86 (0.71, 0.96)
	T, C and F	0.90 (0.77, 0.98)	0.97 (0.88, 1.0)	0.97 (0.88,1.0)
	F only	0.90 (0.76, 0.97)	0.93 (0.82, 0.99)	0.97 (0.86,1.0)
Ducks	T and C	0.58 (0.36, 0.79)	0.91 (0.72, 0.99)	0.91 (0.73, 0.99)
	T, C and F	0.91 (0.74, 0.99)	0.96 (0.81, 1.0)	0.96 (0.81,1.0)
	F only	0.91 (0.74, 0.99)	0.96 (0.81,1.0)	0.96 (0.81,1.0)
^aT, tracheal swab; C, cloacal swab; F, feathers. ^bData presented as median (2.5, 97.5 percentiles).

Table 4.  VI sensitivity determined by the numbers of successful isolates relative to the numbers of bird-level AI RRT-PCR-diagnosed infected animals.

	VI sensitivity
Poultry group	Tracheal swab	Cloacal swab	Feathers
Chickens	10/15 (67%)	8/15 (53%)	19/20 (95%)
Ducks	3/8 (38%)	2/7 (29%)	10/10 (100%)

Figure 3.  Phylogenetic analysis of the HA gene of 125 H5N1 HPAI isolates.

Figure 4.  M-gene RRT-PCR primer and probe binding sequences.

Figure 5.  Primer and probe binding sequences for H5 HA2 and CS RRT-PCRs.

Table 5.  Comparison of the H5 CS and H5 HA2 RRT-PCRs among the H5N1 HPAI-infected chickens (n = 26) and ducks (n = 15).

	H5 HA2 RRT-PCR
H5 CS RRT-PCR	+	Ct 36.01 to 39.99^a	−
a) Infected chickens (n=78 clinical specimens):
+	42	0	0
Ct 36.01 to 39.99^a	4	0	0
−	20	1	11
b) Infected ducks (n=45 clinical specimens):
+	6	0	0
Ct 36.01 to 39.99^a	1	0	0
−	29	0	9
^aIndeterminate Ct range.

Table S1.  Accession numbers of sequenced genes and virus isolates.

Location (Table 1)	Species^a	Bird number (part of isolate name in Fig. 3)	Clinical specimen^b sampled for successful VI & sequencing	HA sequence accession number	Clade	NA sequence accession number	M gene sequence accession number	Residue 31 in M2 protein
A	ch	18129	F	JN055363	2.3.4	ND	ND
A	M dk	18151	F	JN055364	2.3.2	ND	JN055394	N
A	ch	18083	C	JN055365	2.3.4	ND	JN055395	S
A	ch	18105	T	JN055366	2.3.4	ND	ND
A	ch	18106	T	JN055367	2.3.4	ND	ND
A	M dk	18152	F	JN055368	2.3.2	ND	ND
A	M dk	18153	F	JN055369	2.3.2	ND	ND
A	ch	18082	C	JN055370	2.3.4	ND	ND
B	ch	20346	C	JN055371	2.3.2	ND	JN055396	N
B	ch	20396	T	JN055372	2.3.2	ND	ND
B	ch	20388	F	JN055373	2.3.2	ND	ND
B	ch	20390	F	JN055374	2.3.2	ND	ND
B	ch	20392	F	JN055375	2.3.2	ND	ND
B	ch	20395	F	JN055376	2.3.2	ND	ND
B	ch	20389	F	JN055377	2.3.2	ND	ND
C	ch	27262	C	JN055378	2.3.4	JN055392	JN055398	N
C	ch	27265	C	JN055379	2.3.2	JN055393	JN055397	S
C	ch	27270	C	JN055380	2.3.4	ND	ND
C	ch	27319	F	JN055381	2.3.4	ND	ND
C	ch	27306	F	JN055382	2.3.4	ND	ND
C	ch	27309	F	JN055383	2.3.4	ND	ND
C	ch	27310	F	JN055384	2.3.4	ND	ND
D	P dk	27329	C	JN055385	2.3.2	ND	ND
D	P dk	27354	T	JN055386	2.3.2	ND	JN055399	N
D	P dk	27339	C	JN055387	2.3.2	ND	ND
D	P dk	27373	F	JN055388	2.3.2	ND	ND
D	P dk	27372	F	JN055389	2.3.2	ND	ND
D	P dk	27374	F	JN055390	2.3.2	ND	ND
D	P dk	27386	F	JN055391	2.3.2	ND	ND
Genetic polymorphism in M2 protein indicating sensitivity (S31) or resistance (N31) to adamantane-derived antiviral drugs also indicated. ^aSpecies indicated by ch=chicken, M dk=Muscovy duck, P dk=Pekin duck. ^bClinical specimen denoted by T=tracheal swab, C=cloacal swab, F=feathers. ND=not done.

Table S2.  Bird-level diagnosis of AI infection by a positive result on a clinical specimen(s) by at least one of the two M-gene and the H5 HA2 RRT-PCRs.

	Numbers of infected birds identified by different permutations of positive clinical specimens by AI RRT-PCRs^a
	All three clinical specimens positive or two positive with one indeterminate	Two positive clinical specimens or one positive with one indeterminate		One positive clinical specimen or one indeterminate
Species	T, C, & F	T & F	C & F	T alone	C alone	F alone	Total number of infected plus (+) uninfected^e birds
Chickens	18(1^b)	4 (1^c)	1	1^d	1	2	26 + 19^e(and one indeterminate^d)
Pekin ducks	10(3^b)	1	0	0	0	1	12 + 26^e
Muscovy ducks	3	0	0	0	0	0	3 + 7^e
^a Testing by both M-gene RRT-PCRs plus H5 HA2 RRT-PCR. ^bNumber of birds where the cloacal swab gave an indeterminate result by one of the three AI RRT-PCRs. However, the tracheal swabs and feathers included positive AI RRT-PCR results. ^cOne tracheal swab from one chicken gave an indeterminate result by the M-gene “wet” RRT-PCRs. ^dThis tracheal swab gave a single indeterminate RRT-PCR result by “bead” M-gene RRT-PCR, all other AI RRT-PCR results on all three clinical specimens were negative. ^eTo classify a given bird as uninfected, all three clinical specimens must be “No Ct” by all three AI RRT-PCRs.

Table S3.  M-gene RRT-PCR Ct values for the chicken specimens that were tested by VI.

		Virus isolation
M-gene RRT-PCR		+	−
+	“Wet”	22.24 to 35.69 (n=30 [30]), 27.09 & 25.65	(35.56) (n=1 [1])
+	“Bead”	16.95 to 35.62 (n=37 [37]), 24.83, 23.21	29.75 to 35.58 (n=9 [9]), 32.54, 31.91
−	“Wet”	No Ct values (n=3 [3])	No Ct values (n=17 [6])
−	“Bead”	No Ct values (n=0 [0])	No Ct values (n=12 [1])
Ct 36.01 to 39.99^a	“Wet”	36.48 to 38.57 (n=4 [4]), 37.65 & 37.78	37.61 to 39.56 (n=4 [4]), 38.73 & 38.87
Ct 36.01 to 39.99^a	“Bead”	None	(37.97) (n=1 [1])
Results are divided into different categories for the two M-gene RRT-PCRs and VI. Each entry states Ct range, mean and median values, respectively (or single Ct values shown in parentheses for two instances). Results for the two M-gene RRT-PCR tests are for the 59 chicken samples tested by VI, in which 56 of the 59 samples gave an amplification by at least one of the two methods. ^aAdditional rows similarly indicate range, mean and median Ct values observed for the indeterminate M-gene RRT-PCR results, both “wet” and “bead”. “n” denotes number of samples within stated Ct range (Figure 1), and square brackets indicate the number of samples that were positive by H5 HA2 and N1 RRT-PCRs.

Table S4.  M-gene RRT-PCR Ct values for the duck specimens that were tested by VI.

		Virus isolation
M-gene RRT-PCR		+	−
+	“Wet”	20.39 to 35.43 (n=12 [12]), 26.22, 25.55	(35.33 & 35.95) (n=2 [2])
+	“Bead”	18.13 to 32.78 (n=15 [15]), 24.55, 23.21	31.97 to 34.83 (n=6 [5]), 33.93, 34.41
−	“Wet”	No Ct values (n=0 [0])	No Ct values (n=15 [2])
−	“Bead”	No Ct values (n=0 [0])	No Ct values (n=11 [0])
Ct 36.01 to 39.99^a	“Wet”	(36.32, 36.45 & 36.71) (n=3 [3])	(36.29 & 39.43) (n=2 [2])
Ct 36.01 to 39.99^a	“Bead”	NA	(36.69 & 38.67) (n=2 [2])
Results are divided into different categories for the two M-gene RRT-PCRs and VI. Each entry states Ct range, mean and median values, respectively (or individual Ct values, maximum of three shown in parentheses). Results for the two M-gene RRT-PCR tests are for the 34 chicken samples tested by VI, in which 32 of the 34 samples gave an amplification by at least one of the two methods. ^aAdditional rows similarly indicate range, mean and median Ct values observed for the indeterminate M-gene RRT-PCR results, both “wet” and “bead”. “n” denotes number of samples within stated Ct range (Figure 2), and square brackets indicate the number of samples that were positive by H5 HA2 and N1 RRT-PCRs.