Archives of Medical Research - (2015) -

1 53
2 54
3 ORIGINAL ARTICLE 55
4 56
5 Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies in Mexican 57
6 Health Care Workers Before and After Vaccination 58
7 59
8 Q8 Guadalupe Aguilar-Madrid,a Juan Arturo Castel
an-Vega,b Cuauht
emoc Arturo Ju
arez-P
erez,a 60
9 Rosa Mar
ıa Ribas-Aparicio,b Iris Estrada-Garc
ıa,b Laura Baltierra-Jasso,b Nict
e Cervantes-Serv
ın,c 61
10 62
Vanessa M
endez-Ortega,b Luis C. Haro-Garc
ıa,d Francisco Ra
ul S
anchez-Rom
an,e
11 f b 63
12 Vianney Ortiz-Navarrete, Luis H. Fabila-Castillo, Anastasia Maga~ na-Hern
andez,b 64
13 Adolfo Ch
avez-Negrete,g Fabio Abdel Salamanca-G
omez,h and Alicia Jim
enez-Albertob 65
14 a
Occupational Health Research Unit, Centro M
edico Nacional ‘‘Siglo XXI’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico 66
15 b
Departments of Microbiology and Immunology, Escuela Nacional de Ciencias Biol
ogicas, Instituto Polit
ecnico Nacional (IPN), Mexico City, Mexico 67
c
16 Immunology Research Unit, Hospital for Infectious Diseases, La Raza National Medical Center, Instituto Mexicano del Seguro Social, Mexico City, Mexico
d
68
Academia de Salud Comunitaria, Promoci
on a la Salud. Universidad Aut
onoma de la Ciudad de M
exico, Mexico City, Mexico
17 e 69
Department of Workplace Health, Disability Division, Centro M
edico Nacional ‘‘Siglo XXI’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico
18 f
Department of Molecular Biomedicine, Centro de Investigaci
on y Estudios Avanzados, Instituto Polit
ecnico Nacional, Mexico City, Mexico 70
19 g
Education and Research, Specialties Hospital, Centro M
edico Nacional ‘‘Siglo XXI’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico 71
20 h
Coordinaci

on de Investigaci
on en Salud, Centro M
edico Nacional ‘‘Siglo XXI’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico 72
21 Received for publication September 19, 2014; accepted March 9, 2015 (ARCMED-D-14-00527). 73
22 74
23 75
24 Background and Aims. In April 2009, a new strain of influenza A(H1N1) was identified 76
25 in Mexico and in the U.S. In June 2009, WHO declared this a pandemic. Health care 77
26 workers constituted a risk group for their close contact with infected individuals. The 78
27 aim was to estimate seropositivity for A(H1N1)pdm09 in health staff at the Instituto 79
28 Mexicano del Seguro Social. 80
29 Methods. A two-stage cross-sectional study, before and after vaccination in the same 81
30 workers, was performed on a random sample of health-care workers. A socio- 82
31 occupational questionnaire was applied and serum antibodies against influenza 83
32 A(H1N1)pdm09 were determined through neutralization of retroviral pseudotypes; two 84
33 logistic regression models for both were constructed. 85
34 Results. The average (median/mean) age of 1378 participants from 13 work centers was 86
35 41.7 years and 68.7% (947) were women. Seroprevalence for the first stage was 26.5% 87
36 (365) (7.4e43%) vs. 20.8% (11) in a control group from the blood bank; for the second 88
37 stage, the vaccinated group was 33% (215) (18.2e47%) and 27% (196) (11.6e50%) for 89
38 the unvaccinated group. In regression models, seropositivity was associated with occupa- 90
39 tional exposure to suspected influenza infected patients, being physicians, and being 91
vaccinated.
40 92
41 Conclusions. Seropositivity against pandemic virus is similar to what was reported, both 93
42 for vaccinated (2.8e40.9%) and unvaccinated (18.8e64.7%). Low seroprevalence in the 94
vaccinated group indicates that between 67% and 73% were susceptible to infection.
43 95
Given the relatively low vaccine-induced seropositivity, it is imperative to increase,
44 hygiene and safety for health staff and at-risk populations, and strengthen epidemio- 96
45 logical surveillance. Ó 2015 IMSS. Published by Elsevier Inc. 97
46 98
Key Words: Influenza A(H1N1)pdm09, Seroprevalence, Vaccination, Health care workers,
47 99
Retroviral pseudotypes.
48 100
49 101
50 102
Address reprint requests to: Guadalupe Aguilar-Madrid, Occupational Cuauht
emoc, CP 06720, M
exico, D.F., M
exico; Phone: (þ52) (55)
51 103
Health Research Unit, Centro M
edico Nacional Siglo XXI-IMSS, Av. 5761-0725; FAX: ---; E-mail: gpeaguilarm@gmail.com Q1
52 Cuauht
emoc #330. Edificio C, Primer piso, Col. Doctores. Delegaci
on 104

0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.arcmed.2015.03.001

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 2 Aguilar-Madrid et al./ Archives of Medical Research - (2015) -

105 Introduction Epidemiological Surveillance Department of the IMSS 160

106 between April 24 and August 20, 2009 from data reported 161
In April 2009, a new strain of influenza A(H1N1) was iden-
107 by IMSS Regional Offices. A random selection of 13 work 162
tified in Mexico, which spread worldwide. In June 2009, the
108 centers from the three levels of medical assistance was 163
WHO declared a pandemic. On July 11, 2009, a Phase 6
109 done, taking into account that in Mexico City in 2009 there 164
pandemic alert was emitted, the first in 40 years, which
110 were 129 IMSS work centers (38 third-level centers, 48 165
continued until August 2010 (1).
111 second-level centers and 43 first-level centers). In each 166
During the 2009 influenza pandemic, the need to iden-
112 work center we obtained a list of all HCW staff and a 167
tify the highest risk of seropositivity among health staff
113 random selection was made within work categories. The 168
became urgent (2). The Instituto Mexicano del Seguro
114 calculated sample size for a cross-sectional study in Mexico 169
Social (IMSS) provides medical care to O38 million regis-
115 City for each of the groups was as follows: a) medical 170
tered users throughout Mexico. Its role in the pandemic was
116 staff—the total population of in 2009 consisted of 17,679 171
crucial in determining how the pandemic developed.
117 physicians, 92 of whom were considered as suspicious of 172
A total of 316,067 health care workers (HCWs) were em-
118 having influenza. Thus, the first stage sample (prevaccina- 173
ployed by the IMSS (3). A high proportion of these workers
119 tion) included 498 workers and the second stage (postvac- 174
were at risk of contracting influenza; therefore, ascertaining
120 cination) 420; b) nursing staff—a total population of 175
A(H1N1)pdm09 virus seroprevalence became a priority as
121 27,189 nurses were reported, 39 were influenza cases. Thus, 176
the pandemic progressed (4). A(H1N1) pdm09 vaccine
122 the first stage sample (prevaccination) included 680 177
arrived in Mexico in two shipments: during November 2009
123 workers and the second stage (postvaccination) 624; c) 178
and January 2010. IMSS HCWs vaccination began on
124 other staff (laboratory, general services, food and cleaning 179
November 27, 2009 and ended on February 26, 2010. Safety
125 services)—a total population of 4341 workers were re- 180
and hygiene methods were the only way to prevent persons
126 ported, nine were influenza cases. Thus, the first stage sam- 181
from contracting the virus before the HCWs were vaccinated.
127 ple (prevaccination) included 320 workers and the second 182
The aim of this study was to determine seroprevalence of
128 stage (postvaccination) 288. The total sample size was 183
antibodies to pandemic influenza A(H1N1)pdm09 virus in
129 1498 workers in the first stage, taking into account a non- 184
Mexican health care workers before and after vaccination.
130 participation rate of 10%. For the second stage, the same 185
131 Materials and Methods workers who participated during the first stage were 186
132 included, independent of whether or not they had been 187
133 A cross-sectional study in a random representative sample vaccinated. A loss rate of 8% occurred in this study; there- 188
134 of IMSS HCWs in Mexico City was performed. All occu- fore, 1378 participants remained at the end of the study and 189
135 pational categories of the three levels of medical care pro- this was the total number analyzed. 190
136 vided by the IMSS were included from cases reported in The sample included 1,378 workers: 466 physicians 191
137 HCWs until late May 2009. On November 6, 2009 protocol and medical residents, 624 nurses, and 288 workers from 192
138 number 2009-785-099 was authorized and approved by the other areas (janitorial, maintenance, administrative staff 193
139 IMSS National Research and Ethics Commission. and dietitians). The participation rate during the first 194
140 The study consisted of two stages: the first before vacci- stage was 99% and for the second stage was 92%. The 195
141 nation against A(H1N1)pdm09 virus and the second stage loss rate was 8% from the first to the second stage due 196
142 after the vaccination campaign, which included vaccinated to different reasons such as change of work center, 197
143 and unvaccinated HCWs who had participated in the first disability, absenteeism, retirement, change of city, or 198
144 stage. During both stages, the same number of workers not localizing the participant for the second stage. Partic- 199
145 participated. Participants also signed an informed consent ipants worked at 13 centers distributed throughout the 200
146 letter and completed a questionnaire dealing with environ- three health care levels: High Specialty Medical Units 201
147 mental and occupational hazards. A venous blood sample of Siglo XXI National Medical Center: Specialty Hospital 202
148 was taken from each worker to determine A(H1N1) (SH-SXXI-NMC) and Pediatric (PH-SXXI-NMC); La 203
149 pdm09 virus seroconversion. Raza National Medical Center: the General Hospital 204
150 The first stage began on November 25, 2009 and ended (GH-NMC-R), Specialty Hospital (SH-NMC-R), Infec- 205
151 on January 15, 2010. The second stage took place from tious Diseases (ID-NMC-R), Gynecology Hospital-3 206
152 January 3 to April 15, 2010, 6 weeks after vaccination (GYH-3-NMC-R), and Outpatient Clinic (OC-NMC-R). 207
153 against A(H1N1)pdm09. Vaccine given to HCWs was a Also, Magdalena de las Salinas hospitals: Hospital of 208
154 monovalent vaccine containing a single antigen derived Obstetrics and Gynecology-3 (HOG3-MS), Trauma and 209
155 from the influenza strain A/California/7/2009 (H1N1) (5). Orthopedics Hospital (TaOH-MS), Hospital of Obstetrics 210
156 and Gynecology and General Practice Unit-13 (GYH/ 211
Sample Size
157 GPU-13), General Hospital and GPU-26 (DGH/GPU- 212
158 Sample calculation was based on estimated prevalence 26), the GPU-57 (DGH/GPU-57), and the Epidemio- 213
159 of influenza AH1N1 among HCWs reported to the logical Surveillance Department (ESD) (Table 2). 214

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 Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies 3

215 In addition, this study included a convenient sample 04/2009. All plasmids necessary for pseudotyped retrovirus 270
216 group for comparison of 53 blood bank donors with the production were kindly donated by Dr. Weiss (see Ac- 271
217 aim of estimating seroprevalence of A(H1N1)pdm09 in knowledgements). The 294T/17 cell line was cultured 272
218 workers who did not work in Health Services in order to in Dulbecco’s modified Eagle’s medium (DMEM)-high 273
219 compare it with the first stage health care workers, which glucose supplemented with L-glutamine, nonessential 274
220 were included with the following criteria: being Mexican amino acids (MEM), penicillin/streptomycin, and 10% fetal 275
221 workers, not working in Health Services, and being blood bovine serum. The retroviral pseudotypes were produced by 276
222 donors at the IMSS Blood Bank from November 15 to co-transfection of 293T/17 cells with plasmids and Fu- 277
223 November 26, 2009 (in Mexico the massive vaccination GENE 6 (Roche) as previously described (6). 278
224 against AH1N1 began November 27, 2009). 279
225 In order to establish baseline prevalence of antibodies Pseudotype neutralization assay (PPN). Serum samples in- 280
226 against pandemic virus in HCW, a group involving blood activated at 56 C for 30 min were prediluted (1:20) in com- 281
227 donors was taken as a control group; inclusion criteria plete DMEM and then mixed with an equal volume of HA 282
228 included being a worker not from the health system, signing pseudotypes, which had been previously diluted in com- 283
229 informed consent letter, and not having been vaccinated plete DMEM supplemented with 32 mg/mL of polybrene 284
230 against pandemic influenza. to obtain 1  104 to 105 RLU/mL (relative luminescence 285
231 units per milliliter), so the final dilution of the serum was 286
232 Q7 Measuring Instruments 1:40. After 1 h at 37 C, 100 mL of the pseudotype-serum 287
233 mixtures were transferred to a monolayer of 293T cells 288
234 Questionnaires. In both stages, questionnaires were 289
completed by the subjects themselves, which explored soci- grown for 18 h with an initial inoculum of 1  104 cells.
235 Luminescence reading was taken 48 h after infection. As 290
236 odemographic data, medical history, previous training in 291
infection control, respiratory signs and symptoms, previous in the microneutralization assay with active viruses (7), sera
237 that showed an RLU reduction of 50% or more compared to 292
238 antiviral treatment and prophylaxis, vaccination against 293
seasonal influenza and to A(H1N1)pdm09 virus, work pseudotype control were considered positive. Data are from
239 duplicate analysis of the serum samples and were reported 294
240 and environmental history: direct contact with patients, 295
coworker and/or family under pandemic influenza suspi- as either positive or negative.
241 296
242 cion, and community background; personal protective 297
equipment use and frequency of general infection control Data Analysis
243 298
244 and hygiene practice. Workers from the 13 hospitals who Questionnaire data and neutralizing antibody results against 299
245 agreed to participate signed an informed consent letter. influenza A(H1N1)pdm09 virus HA were entered twice in 300
246 Excel (Microsoft, Inc.) and statistical analysis was per- 301
247 Blood samples. A 5-mL venous blood sample was formed in Stata software v.11. Measures of central tendency 302
248 collected in tubes with no anticoagulant. Samples were and dispersion for continuous variables as frequencies and 303
249 transported to laboratories in medical coolers with anti- percentages for categorical variables were calculated. For 304
250 freeze during a 90-min period for processing. Serum was comparison in the first and second stages, between vacci- 305
251 obtained and stored in cryotubes at 70 C. Sera separating nated and unvaccinated, between the different categories, 306
252 process was performed at the Pediatric Medical Research and for seroprevalence, c2 statistic was calculated. For 307
253 Unit in the ‘‘Siglo XXI’’ IMSS National Medical Center mean differences, Student t test was used along with 308
254 Immunochemistry Laboratory and the Medical Research ANOVA. Two unconditional logistic regression models 309
255 in Immunology and Infectious Diseases Unit of the ‘‘La Ra- were constructed in order to ascertain odds ratios (OR) of 310
256 za’’ IMSS. the probability of obtaining a positive result for A(H1N1) 311
257 pdm09 antibodies. For both models, residual analyses and 312
258 Serum analysis. Sera were transferred in vials to the tests of goodness of fit were performed. 313
259 Biological Production and Control Laboratory of the 314
260 Escuela Nacional de Ciencias Biol
ogicas, Instituto Polit
ec- 315
Results
261 nico Nacional (IPN) to determine specific antibodies 316
262 against influenza A(H1N1)pdm09 virus hemagglutinin us- A total of 1 378 HCWs from all occupational categories 317
263 ing a neutralization assay with HA-pseudotyped retroviral participated in the two stages of the study. Of these, 318
264 vectors (6). 68.7% (947) were women and 31.3% (431) men. Average 319
265 age was 41.7  9 (19e71) years. In women it was 41  320
266 Plasmids and cell lines for pseudotype retrovirus produc- 10.4 years vs. 42  8.6 years in men ( p !0.05) (Table 1). 321
267 tion. Genes encoding the HA, the M2 matrix protein and Vaccine uptake against pandemic influenza A(H1N1) 322
268 the neuraminidase (NA) of the influenza A(H1N1)pdm09 pdm09 virus among the participants was 47.3% (652) 323
269 virus came from the influenza virus strain A/California/ (29.2e70%). Women were more likely to agree to 324

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 4 Aguilar-Madrid et al./ Archives of Medical Research - (2015) -

325 Table 1. Average age and occupational characteristics of health care workers (IMSS 2009e2010) 380
326 381
All participants from the two-stages Comparison group
327 382
328 Total Mean (SD) Median (minemax) Total Mean (SD) Median (minemax) 383
329 384
Age (years) 1,378 41.7 (9) 43 (19e71) 56 32.6 (10) 31 (18e61)
330 Men 431 41 (10.4) 41.5 (19e66) 41 34.3 (10) 33 (18e61) 385
331 Women 947 42 (8.6) 44 (19e71) 15 28 (7) 28 (20e38) 386
332 Seniority at IMSS (years) 1,336 15.5 (8.6) 18 (0.01e36) 387
333 Seniority in current category (years) 1,341 12.7 (9) 13 (0.01e36) 44 6 (8.5) 2 (1e40) 388
Seniority in service (years) 1,336 6.8 (7.6) 3.5 (0.01e36)
334 389
Seniority at work center (years) 1,336 9.8 (8.6) 8 (0.01e36) 44 6 (8.5) 2 (1e40)
335 Number of contacts with suspected 1,378 5 (10) 1 (0e71) 390
336 cases of A(H1N1)pdm09 in workplace 391
337 392
SD, standard deviation; minemax, minimum and maximum values.
338 393
339 394
340 vaccination 65.2% (425); nurses and consulting-room assis- first and second stages in vaccinated workers (Table 2, 395
341 tants was the category with highest vaccination uptake at Figure 2). 396
342 42% (276) followed by physicians at 38% (254) (Tables 2 In the first stage, antibody seroprevalence rates by sex 397
343 and 3). were not statistically different. However, in the second 398
344 In the first stage, the overall antibody seroprevalence stage, vaccinated men showed a greater seroprevalence rate 399
345 rate for A(H1N1)pdm09 was 26.5% (365) with a range of (37.4% [5]) than women (30.6% [130]). Similarly, physi- 400
346 7.4e43%, which was greater than the control group, i.e., cians showed the highest seroprevalence in both stages. In 401
347 20.8% (13) ( p !0.05). Lowest seroprevalence (7.4%) those vaccinated against seasonal influenza, differences 402
348 was found at the SH-SXXI-NMC; highest seroprevalence were statistically significant in the first stage. In those sub- 403
349 was found at the ESD and the DGH/GPU-57, with 43 and jects who had five or more contacts with patients suspected 404
350 Q2 38.5%, respectively (Table 2, Figure 2). of being infected with influenza A(H1N1)pdm09, preva- 405
351 In the second stage, seropositivity rate in vacci- lence in the first stage was 32.7% (103) vs. 24.6% (262) 406
352 nated HCWs against A(H1N1)pdm09 was 33% (215) for those with fewer than five contacts ( p !0.05). Results 407
353 (18.2e47%) and 27% (196) (11.6e50%) in the unvacci- were similar in the second stage where prevalence was 39% 408
354 nated ( p 5 0.015). Highest seroprevalence was found at (61) in vaccinated group with at least five contacts as 409
355 the PH-SXXI-NMC with 46% (33). Moreover, significant opposed to 31% (154) for those with fewer contacts 410
356 differences were present among the seroprevalence between (Table 3). 411
357 412
358 413
359 Table 2. Seroprevalence of influenza A(H1N1)pdm09 in vaccinated and unvaccinated health care workers (IMSS 2009e2010) Q6 414
360 415
First stage Second stage
361 416
362 Pre-vaccinated Vaccinated against A(H1n1)pdm09 Unvaccinated 417
363 418
Work center n** Positive n (%) Positive n (%) Total Positive n (%) Total
364 419
365 1. SH-SXXI-NMC 95 7 (7.4) 7 (18.4) 38 11 (19.3) 57 420
366 2. HP-SXXI-NMC 107 39 (36.5) 33 (46) 72 7 (20) 35 421
367 3. GH-La Raza-NMC 336 108 (32) 39 (27) 144 53 (27.6) 192 422
4. SH-NMC-La Raza 89 16 (18) 8 (23.5) 34 12 (21.8) 55
368 5. ID-NMC-La Raza 86 28 (32.6) 19 (44) 43 5 (11.6) 43
423
369 6. GYH-3-NMC-La Raza 131 28 (21.4) 12 (26) 46 23 (27) 85 424
370 7. GYH No.3-MS 100 31 (31) 23 (47) 49 18 (35.3) 51 425
371 8. TaOC-MS 88 19 (21.6) 10 (21.7) 46 7 (16.7) 42 426
372 9. GYH/UMF-13 93 19 (20.4) 21 (44.7) 47 23 (50) 46 427
10. DGH/GPU-26 74 6 (8) 15 (32.6) 46 9 (32) 28
373 11. DGH/GPU-57 65 25 (38.5) 20 (43.5) 46 8 (42) 19
428
374 12. ESD 65 28 (43) 4 (21) 19 7 (15.2) 46 429
375 13. CMN OC. La Raza 49 11 (22.4) 4 (18.2) 22 13 (48.2) 27 430
376 Overall seroprevalence in HCW 1 378 365 (26.5)* 215 (33)* 652 196 (27)* 726 431
377 Seroprevalence, worker blood-bank donors 53 11 (20.8)* 432
378 *p !0.05 using Student t test. 433
379 **The number of patients during both stages of the study was the same. 434

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 Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies 5

435 Table 3. Seroprevalence of A(H1N1)pdm09 based on job characteristics of vaccinated and unvaccinated health staff (IMSS 2009e2010) 490
436 491
First stage Second stage
437 492
438 Unvaccinated Vaccinated Unvaccinated 493
439 n** Positive n (%) Positive n (%) Total (%) Positive n (%) Total
494
440 495
441 Sex** 1 378 365 (26.5) 215 (33)* 652 196 (27) 726 496
442 Men 431 112 (26) 85 (37.4) 227 58 (28.4) 204 497
Women 947 253 (27) 130 (30.6) 425 138 (26.4) 522
443 498
Category*
444 Physicians 466 144 (31)* 89 (35)** 254 5 7 (27)** 212 499
445 Nursing and related staff 624 152 (24.4) 85 (30.8) 276 85 (24.4) 348 500
446 Other categories (janitorial staff, maintenance, 288 69 (24) 41 (33.6) 122 54 (32.5) 166 501
447 administrative staff) 502
!5 contacts in the workplace with patients 1,063 262 (24.6)* 154 (31)** 495 162 (28.5)** 568
448 503
probably infected with A(H1N1)pdm09*
449 O5 contacts in the workplace with probable 315 103 (32.7) 61 (39) 157 34 (21.5) 158 504
450 cases of A(H1N1)pdm09 infection 505
451 Services at lower risk of infection 503 119 (23.6)* 67 (30)* 222 71 (25.3)** 281 506
452 Services at greater risk of infection 870 244 (28) 147 (34.3) 429 124 (28.2) 441 507
Without prophylactic treatment against A(H1N1)pdm09* 1,254 323 (25.8)* 198 (33)* 596 187 (28.4)* 658
453 508
With prophylactic treatment against A(H1N1)pdm09 124 42 (34) 17 (30.4) 56 9 (13.2) 68
454 Previously vaccinated against seasonal influenza 509
455 Unvaccinated* 295 65 (22)* 31 (33)** 93 50 (25)* 202 510
456 Vaccinated 1,083 300 (27.7) 184 (33) 559 146 (27.8) 524 511
457 Did not use PPE during pandemic 61 19 (31)** 11 (33.3)** 33 6 (21.4)** 28 512
Did use PPE during pandemic 687 189 (27.5) 122 (37) 329 99 (27.6) 358
458 513
459 *p !0.05 using Student’s t test; **The number of patients on both stages of the study was the same. 514
460 515
461 516
Jobs were classified according to their risk of contact heads of departments, epidemiologists, physicians of pro-
462 517
level with patients suspected of being infected with motion and prevention services, medical residents, and un-
463 518
pandemic influenza. Low-risk category included general of- dergraduate interns. They were also classified on the basis
464 519
fice assistants (GOAs), graphic designer, administrative of the type of service provided at each workplace and the
465 520
personnel, and janitorial and maintenance staff, medical as- increased risk represented by contact with possible patients
466 521
sistants, physical therapists, laboratory technician, food infected by A(H1N1)pdm09 virus. A first group consisted
467 522
handlers, radiographers, and dietitians. The high-risk cate- of emergency service personnel, ER staff, admissions staff,
468 523
gory consisted of general practitioners and specialists, surgical staff, inhalotherapy staff, intensive care staff, and
469 524
470 525
471 526
472 527
473 528
474 529
475 530
476 531
477 532
478 533
479 534
480 535
481 536
482 537
483 538
484 539
485 540
486 Figure 1. Comparison of cumulative cases of suspected influenza in 541
Mexico City from weeks 1 to 15 (2008e2009, Mexico). Source: Bolet
ın
487 Epidemiol
ogico Semanal. SINAVE (National Epidemiological Surveil- Figure 2. Comparison of seroprevalence for A(H1N1)pmd09 virus in 542
488 lance System), DGE (General Directorate of Epidemiology), Department vaccinated and unvaccinated health care workers (IMSS 2009e2010). *p 543
489 of Health, Mexico 2008 and 2009. !0.05 (differences between first stage and second stage vaccinated). 544

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 6 Aguilar-Madrid et al./ Archives of Medical Research - (2015) -

545 outpatient clinic staff. A second group included those in all Table 4. Unconditional logistic regression models for seroprevalence 600
546 the other services throughout the care levels. of the novel pandemic influenza A(H1N1)pdm09 virus in health care 601
workers (2009e2010)
547 During the first stage, in medical care services with high 602
548 and low risk of pandemic virus infection, there was 28% OR 95% CI 603
549 (244) vs. 23.6% (119) seroprevalence rate, respectively. In 604
First stage
550 the second stage (after vaccination), these were 34.3% 605
O5 contacts in the workplace with suspected 1.47 1.11e1.94
551 (147) vs. 30% (67), respectively, in vaccinated group cases of pandemic A(H1N1)pdm09 virus 606
552 ( p !0.05). Prevalence in physicians was greater in both infection 607
553 stages compared to other categories: 31% (144) unvacci- Nurses, medical assistants 0.95 0.68e1.32 608
554 nated in the first stage and 35% (89) in the vaccinated group Physicians 1.31 0.93e1.84 609
Seniority at current work center (in years) 1.01 0.99e1.02
555 in the second stage (Table 3). 610
Second stage
556 In the second stage of the study, in both those vaccinated Vaccinated against the pandemic 1.30 1.03e1.64 611
557 and unvaccinated against seasonal influenza virus and in A(H1N1)pdm09 virus 612
558 those who did receive the influenza A(H1N1)pdm09 virus O5 contacts in the workplace with suspected 1.18 0.94e1.50 613
559 vaccine, we found a seroprevalence rate of 33%. However, cases of pandemic A(H1N1)pdm09 virus infection 614
Service at greater riska of contact with patients 1.14 0.90e1.46
560 in those who did not receive the pandemic vaccine, we 615
suspected of being infected with the
561 found a statistically significant difference between those A(H1N1)pdm9 virus 616
562 who were vaccinated against seasonal influenza and those 617
563 who were not (Table 3). On the other hand, the incidence OR, odds ratio; CI, confidence interval.
a
618
Emergency service personnel, ER staff, admissions staff, surgical staff, in-
564 density ratio (IDR) 5 1.26 [95% CI: 0.73e2.15] indicates halotherapy staff, intensive care staff, and outpatient clinic staff working at
619
565 a greater probability of being seropositive to pandemic vi- the primary care level. 620
566 rus among HCWs in the IMSS system and an attributable 621
567 risk (ARe) of seropositivity to the A(H1N1)pdm09 virus 622
568 of 22% due to occupational exposure during pandemic (14). than reported by La Torre in nurses and auxiliary workers 623
569 In the second stage, seropositivity to A(H1N1)pdm09 in Italy (15). Furthermore, rates are higher than those re- 624
570 virus in vaccinated vs. unvaccinated was 33% vs. 27%, ported in HCWs in UK, Germany, France, and Italy 625
571 respectively, i.e., just 6.5% more in the vaccinated group. (15e29%) and those reported by Amodio et al. in 2010 626
572 Moreover, the calculated IDR (1.215 [95% CI: 1.02e1.41]) (16) where 18% of HCWs were vaccinated. In 2010, the 627
573 shows that seropositivity probability is greater in vaccinated Centers for Disease Control and Prevention (CDC) (13) 628
574 persons. A 17.45% ARe of seropositivity also was found, reported that estimated vaccination coverage against 629
575 which was attributed to vaccination against pandemic virus. A(H1N1) 2009 in high-risk Caucasian adults was 53.7% 630
576 In the first stage of the two unconditional logistic regres- (13), which is higher than our study. Seropositivity to 631
577 sion models constructed, the odds ratios (OR) and 95% A(H1N1)pdm09 virus was 5.7% higher in HCWs than the 632
578 confidence intervals (CI) were 1.47 (95% CI: 1.11e1.94) control group. This is a major warning given that only 633
579 for workers with more than five contacts with patients sus- 47.3% of the HCWs were vaccinated. 634
580 pected of being infected with the pandemic virus; in the Comparing results for antibody seropositivity against 635
581 physicians’ category, we found OR 5 1.31 (95% CI: pandemic virus (26.5%) in IMSS HCWs, a range from 636
582 0.93e1.84), and for seniority at current work center (in 2.8e40.9% in unvaccinated individuals is found in reports 637
583 years), OR 5 1.01 (95% CI: 0.99e1.02) (Table 4). In the published by several international studies. Few of these 638
584 second-stage model, those vaccinated are more likely to HCWs studies carried out comparison of seropositivity 639
585 generate antibodies against pandemic A(H1N1)pdm09 vi- before and after vaccination against pandemic virus. If 640
586 rus, with an OR of 1.30 (1.03e1.64). Workers who had con- comparisons were carried out, seropositivity was in the 641
587 tact with more than five patients suspected of being infected range of 18.8e64.7% range (Table 5) (8,9,11,12,17e23). 642
588 with pandemic influenza had an OR of 1.18 (95% CI: As in this study, Xu et al. (11) estimated a similar sero- 643
589 0.94e1.50). Those who worked during the pandemic in positivity among HWCs (21.5%). Bandaranayake et al. 644
590 those services at greatest risk of contact had an OR of (2010) (18) also reported 29.9% (38) seroprevalence among 645
591 1.14 (95% CI: 0.90e1.46) (Table 4). In both models, as- physicians, 27.5% (55) in nurses and 25% (46) among other 646
592 sumptions were met in residual analysis and goodness-of- HCWs. Authors estimate that 29.5% of New Zealand 647
593 fit tests. HCWs have immunity against influenza A(H1N1)pdm09 648
594 virus (18). 649
595 Our results are within the seroprevalence rates range 650
Discussion
596 published worldwide and are similar to the national sero- 651
597 Our seroprevalence study is the only one carried out in prevalence rate reported by the Secretar
ıa de Salud (SSa) 652
598 Mexico that included IMSS HCWs during the 2009 influ- 2010 (3). However, other reports by the same SSa reported 653
599 enza pandemic. Vaccination prevalence rate was greater a 71% seroprevalence rate in the vaccinated population, 654

ARCMED1997_proof ■ 18-3-2015 20-1-57

 Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies 7

655 Table 5. Comparison of seroprevalence of antibodies against influenza A(H1N1)pdm09 worldwide and in Mexico. 2009e2011 710
656 711
Seroprevalence of antibodies
657 Author (reference 712
658 number), year Type of Vaccination Unvaccinated % 713
659 and country N population Technique rate Vaccinated (n) (CI) 714
660 Apisarnthanarak (17), 22 HCWs HI 32% (7) 715
661 2010, Thailand 716
662 Bandaranayake (18), 171 HCWs HI 29.3% (primary care) 717
663 2010, New Zealand 25.3% (secondary care) 718
Chan (19), 2010, Taiwan 295 HCWs controls HI 20.9%
664 719
244 2.9%
665 Vernon (20), 2010, 127 Military Personnel HI 11% (14) 720
666 Singapore and HCWs 721
667 Olalla Sierra (21), 263 HCWs (4 hospitals) HI 19.4% (1.5e51.8) 18.8e64.7% 25.1 (5.6e83) 722
668 2011, Spain 223 723
Smith (22), 2011, UK 493 HCWs Microneutralization 10.3% (n 5 51)
669 724
(CI: 7.7 to 13)
670 Tandale (9), 2012, India 495 (Aug) HCWs HI 2.8% (14) 725
671 524 (Oct) 4.8 (25) 726
672 386 (Nov) 12% (46) 727
673 Xu (11), 2011, China 2 632 HCWs physicians HI 23.1% (303) 728
and nurses (13.7e24.4)
674 729
Yang (23), 2011, China 116 HCWs HI 37% (43)
675 110 40.9% (45) 730
676 Bandaranayake (17), 521 General population HI 26.7% (23.4e29.9) 731
677 2010, New Zealand 732
678 Deng (8), 2010, China 710 General population HI 51.4% 13.8% 733
Xu (11), 2011, China 50 111 General population HI 16.6% (n 5 7799) 34.8% 21.5
679 734
MacVernon (12), 2010, 496 (AprileSept. 2009) Healthy adult blood HI 12%
680 Australia 779 (Oct.eNov. 2009) donors 22% 735
681 Mexican Department of General population HI 71% 30% (21e41%) 736
682 Health, INDRE, 737
683 June 28 (10), 2010 738
Aguilar Madrid, current 1,271 HCWs PPN 47.3% (29.2e70%) 33% (215) 27% (196)
684 739
study, M
exico (18.2%) (11.6e50%)
685 Aguilar Madrid, current 53 Healthy working PPN 20.8% (11) 740
686 study, M
exico adult blood donors 741
687 742
HCWs, health care workers; HI, hemagglutinin inhibition test; PPN, pseudotype neutralization assay.
688 743
689 744
690 which is higher than that observed in our study and beyond employees) (3,10). Because of these inconsistencies by 745
691 the global range (Table 6) (8,9,11,12,17e23). the SSa, we cannot assess the actual number of HCWs in 746
692 Based on data of Hsieh (24) and SSa reports (25), we Mexico who were vaccinated during the pandemic (26). 747
693 consider that failure to declare the alert earlier probably Similarly, the 71% seroprevalence in the vaccinated group 748
694 contributed to further spread of the virus among HCWs reported by the SSa is greater than that in our study 749
695 (Figure 1) due to the fact that 90% of the hospitals studied (47.3%) and those reported globally (1.5e65%) 750
696 failed to take safety and hygiene measures; therefore, (8,9,11,12,17e23). 751
697 HCWs were probably at an increased risk of infection dur- Regarding seroprevalence in those vaccinated against 752
698 ing that period. This may have had implications for anti- both seasonal and pandemic influenza, there was a 33% 753
699 body levels found in HCWs as the sampling was seropositivity rate vs. 27.8% for those unvaccinated during 754
700 conducted in the pandemic second wave (week 48 in the pandemic, but vaccinated against seasonal influenza. 755
701 2009), so the HCWs had a 37-week average exposure. We believe that there was no cross-reaction in those vacci- 756
702 In addition, the SSa reported that vaccination rate had nated against both influenza viruses. However, several 757
703 been very low, i.e., between 1.5 and 51.8% (26). In our studies have assessed vaccine efficacy against both seasonal 758
704 study, the uptake was within this range (47.3%). However, and A(H1N1)pdm09 influenza (27). 759
705 as we see in Table 6, there is a disagreement between the Logistic regression models in both stages showed that 760
706 SSa report of 99.8% (391,917) and the IMSS records contact with patients suspected of being infected with 761
707 because the number of HCWs considered by SSa is higher pandemic influenza increased the likelihood of seroposi- 762
708 than the number of workers in the IMSS records (316,067 tivity for antibodies against pandemic virus such as 763
709 764

ARCMED1997_proof ■ 18-3-2015 20-1-58

8 Aguilar-Madrid et al./ Archives of Medical Research - (2015) -

belonging to high-risk categories (physicians and auxiliary

87 13,081, 890 13, 071, 188 100

100
79
14
97
%
nurses, nurses, and nurse managers) and services with

Source: Health Sector. INDRE-DGE/DH. Seroprevalence of influenza H1N1 Survey. June 28, 2010. ‘‘Informaci
on para el levantamiento de la alerta epidemiol
ogica de influenza A(H1N1).’’ Special
12, 206 810

26, 932 184

greater direct patient contact (emergency services, intensive

1,582, 121

Source: Bolet
ın Epidemiol
ogico Semanal. SINAVE (National Epidemiological Surveillance System), DGE (General Directorate of Epidemiology), Department of Health, Mexico 2008 and 2009.
Dose

72, 065
care, etc.). This means that occupational exposure increases

Total
the likelihood of infection in HCWs.
Our results confirm the evidence that global epidemio-

632, 036 84 12,253, 879

92 20,416, 207 20,822, 333 102 1,630, 106 1,630, 106 85 27,811 319
logical surveillance of HCWs will make it possible to better

82 1,991 860
Target

501 690
define seasonal patterns in the spread of the influenza A
virus and to plan vaccination periods accordingly as well
as to stress the health and safety measures necessary for
%

HCWs and other high-risk groups (children and elderly per-

Mothers or guardians
of infants !6 months

659, 325

sons). We believe that both measures should be given the

97,825
Dose

same priority, especially given the low rates shown for

influenza vaccination in Mexico and worldwide.
Also, hemagglutinin inhibition (HI) and microneutrali-
755,989

99 754,568
91 119,549
Target

zation (MN) using live virus are validated tests for determi-
N/A

nation of influenza neutralizing antibodies (28). However,

HI lacks sensitivity and has to be standardized for each
106
%

new reagent batch (virus, antisera, erythrocytes, etc.). The

MN assay has the disadvantage of handling live virus,
9,964, 076

100 9,918, 392 9,864, 246

requiring a Biosafety Level 3 facility for highly pathogenic

43 1,088, 394 994, 011
Dose
2e64 years

or exotic strains (28). Thus, there are many studies that sup-
port the use of retroviral pseudotypes bearing hemag-
glutinin (HA) as a substitute for influenza viruses in
452, 132 66 1,572, 026 1,572, 026 100 9,409, 421
Target

neutralization assays whose application allows the work

N/A

in a Biosafety Level 2 environment. In such studies, it

C
ordova Montoya. June 28, 2010. CeNSI;

has been demonstrated that neutralization of retroviral

pseudotypes is as sensitive and specific as the HI and MN
%
Infants 6-23 months

using live virus, proving to be a valid alternative for the

2,693 676
951, 424
170, 226

detection and quantification of neutralizing antibodies

Dose
Table 6. Application of vaccine in at-risk persons (8) (DGE-INDRE, Mexico 2009e2010)

against different influenza virus subtypes (6,27,29,30). In

the same way, Temperton and colleagues demonstrated that
the results of assays using retroviral pseudotypes bearing
1,096, 890 95 1,193 203 858, 034 72 2,918 528
Target

367, 187 85 951,424

38, 715 51 395,078
N/A

HA for determining H5N1 neutralizing antibodies in sera

from humans that recovered from infection and sera from
animals experimentally immunized correlated with those
%

from HI and MN (29). In another study, neutralization of

Pregnant women

Commission for the Prevention y Control of Influenza. Jos
e Angel

retroviral pseudotypes showed significant correlation with

Dose

HI and MN when evaluating the immunogenicity of an

H5N1 vaccine (27) and due to its performance it is being

proposed for serodiagnosis, seroepidemiology and for the
Target

95 687,964

99 429,485

N/A
90 75,754

evaluation of vaccines (30).

In our study, the 50% cutoff point used to consider sero-
%

positivity was chosen according to Garc
ıa et al. (30) who

Health care workers

compared an optimized pseudotype-based neutralization

423, 629

391, 917
281, 344
Dose

test with the microneutralization test to detect H5 neutra-

lizing antibodies in well-characterized human sera. They
found that a neutralization cutoff value of 50% results in
1,154, 955
444, 890

396, 438
313, 627

the best diagnostic performance for their pseudotype-

Target

N/A

based assay.
During the A(H1N1)pdm09 pandemic influenza, labora-
tory diagnosis was a fundamental and decisive tool in
Of Health
Department
Institution

pandemic treatment, control, and surveillance (31). This

ISSSTE
Others

was apparently a significant failure in the way the epidemic

IMSS

Total

was dealt with as well as the fact that national laboratories

ARCMED1997_proof ■ 18-3-2015 20-1-58

 Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies
(Instituto de Diagn
ostico y Referencia Epidemiol
ogicos,
InDRE) were unprepared for the emergence of the new
influenza pandemic strains. Standardizing and improving
laboratory methods worldwide could benefit the work of
laboratories by connecting them as a unitary network
ensuring comparability of results (31).
In conclusion, antibody seropositivity rate against
A(H1N1)pdm09 virus in both stages was very low in the
vaccinated and unvaccinated IMSS HCWs of the Valley
of Mexico. This means that is essential for SSa authorities
to assess vaccination impact and health and safety measures
in at-risk populations and to establish a HCW surveillance
program in Mexico’s health care system.
The low antibody seroprevalence against influenza
A(H1N1)pdm09 virus even after HCWs vaccination sug-
gests that the vaccine was poorly immunogenic. It is likely
that general hygiene measures implemented during and af-
ter the epidemiological alert on April 2009 in Mexico may
have contributed to reduce the virus attack rate.
Acknowledgments
Our thanks to all health workers at the IMSS for their invaluable
participation; the medical and nursing staff of the IMSS Workers
Health Promotion and Prevention Programs; the directors of health
care units; the heads of IMSS Research Units at the CMNSXXI,
Pediatric Hospital and Centro M
edico Nacional La Raza Infec-
tious Diseases Hospital for granting use of their laboratories, to
the field staff and chemists, and the nurses that participated in
the project’s field work.
Physicians: Roc
ıo Zavala Y., Miguel Rechy L., Jos
e Uribe R.,
Jos
e L. Ju
arez M., Maricela Orozco G., Eva Ochoa S., Rosario
Figueroa O., Sandra Mel
endez B., Arturo Pliego H., Martha E.
Luengas E., V
ıctor Soriano, Leobardo Chiu M., Jorge I. Garc
ıa
F., Martha Callejas M., Benjam
ın Acosta C., V
ıctor H. Borja A.,
C
esar Bonilla, Francisco Javier Torres, and Francisco Blanco F.
We also thank Dr. Carol D. Weiss for plasmids and for help
with the Pseudotype Neutralization Assay (PPN).
This study received support from Consejo Nacional de Ciencia
y Tecnolog
ıa (CONACYT), PAHO-ME-LOA-0900017.001, Fon-
do Sectorial de Investigaci
on en Salud y Seguridad Social 2009-
Q3 02, No. 126774 (also part of CONACYT), CONACYT Institu-
tional Grant 16/2009 and from the Instituto Polit
ecnico Nacional
(IPN) through SIP grants 20130044, 20131349, and 20131346.
AJ-A, IEG and JAC-V are recipients of Comisi
on de Operaci
on
y Fomento de Actividades Acad
emicas (COFAA) and Programa
de Est
ımulos al Desempe~no de los Investigadores (EDI) fellow-
ships. RMR-A was a recipient of COFAA and Programa de
Est
ımulo al Desempe~no Docente (EDD) fellowships, all granted
by the IPN-Mexico. EA-F thanks CONACyT-Mexico and IPN
for graduate studies fellowships.
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