Journal of Infection (2008) 56, 191e196

www.elsevierhealth.com/journals/jinf

Haemophilus influenzae type-b and non-b-typeinvasive diseases in urban children (<5 years)of Bangladesh: Implications for therapy andvaccination

Mahbubur Rahman a,*, Shahadat Hossain a, Abdullah Hel Baqui a,Shereen Shoma a, Harunur Rashid a, Nazmun Nahar b,Mohammed Khalequ Zaman a, Farida Khatun c

a International Centre for Diarrhoeal Disease Research, Dhaka, Bangladeshb Dhaka Medical College Hospitals, Dhaka, Bangladeshc Sir Salimullah Medical College Hospitals, Dhaka, Bangladesh

Accepted 17 December 2007

KEYWORDSHaemophilusinfluenzae;Invasive diseases;Children;Bangladesh

* Corresponding author. InternationBangladesh. Tel.: þ880 2 881 1751; fa

E-mail address: mahbubur@icddrb

0163-4453/$30 ª 2008 The British Infedoi:10.1016/j.jinf.2007.12.008

Summary Objective: To prospectively study the epidemiology and antibiotic resistance ofHaemophilus infuenzae isolates from invasive infections in children.Methods: Children (<5 years) with pneumonia, meningitis and septicemia from three hospitalsin Dhaka, Bangladesh were enrolled (1999e2003); clinical and laboratory data, and blood forcultures were collected. Cerebrospinal fluid (CSF) of meningitis cases was analyzed (Gramstain, culture and biochemical tests). Hib antigen was detected by latex agglutination (LA)in culture-negative pyogenic CSF and PCR was done for bexA gene in culture- and LA-negativepyogenic CSF. Antibiotic susceptibility was determined by E-Tests and b-lactamase bynitrocefin stick.Results: Seventy-three cases of H. influenzae infections (46 of 293 meningitis cases, 25 of 1493pneumonia cases, 2 of 48 septicemia cases) were detected; 63%, 34% and 3% of them hadmeningitis, pneumonia and septicemia respectively. H. influenzae type b (Hib) caused infec-tions in 80.8% of cases (60.3% meningitis, 20.5% pneumonia). Most (86%) infections clusteredin 4e12 month infants. The case-fatality in pneumonia was 8% compared to 19% in meningitis.H. influenzae isolates from pneumonia and meningitis children were equally resistant toantibiotics (46% vs 43%). Of 10 drugs tested, isolates were resistant to ampicillin (31%), chlor-amphenicol (42%), trimethoprim-sulfamethoxazole (44%) and azithromycin (1.4%). Multidrug-resistant (MDR) strains were equally prevalent in Hib (31%) and non-b-type (29%) isolates,

al Centre for Diarrhoeal Disease Research, Laboratory Sciences Division, GPO Box 128, Dhaka 1000,x: þ880 2 881 2529..org (M. Rahman).

ction Society. Published by Elsevier Ltd. All rights reserved.

192 M. Rahman et al.

and in pneumonia (31%) and meningitis (34%) cases. None was resistant to amoxicillin-clavula-nate, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin. Of all H. influen-zae infections, 40%, 4.4% and 100% of pneumonia, meningitis and septicemia cases werecaused by other serotypes or non-typeable strains. All ampicillin-resistant-strains producedb-lactamase without detection of b-lactamase-negative-ampicillin-resistant (BLNAR) strains.Conclusion: Hib is a leading cause of invasive bacterial infections in infants. Multidrug-resistant H. influenzae is common and requires amoxicillin-clavulanate, ceftriaxone or azithro-mycin as empirical therapy with specific recommendation for use of ceftriaxone for treatmentof meningitis particularly MDR cases. New fluoroquinolines has potential utility. An effectivenational Hib vaccination programme is essential in Bangladesh although non-Hib infections willremain an issue.ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Haemophilus influenzae is the part of normal flora of therespiratory tract that causes a wide spectrum of life threat-ening invasive diseases predominantly in children in additionto upper respiratory infections with much suffering.1e3 H.influenzae type b (Hib) is the most invasive type of six cap-sular serotypes (aef) and is recognized as a major cause ofmeningitis, community-acquired pneumonia and septice-mia in children <5 years of age resulting in 3 million epi-sodes of serious diseases each year, leading to halfa million deaths predominantly in countries where childrenare not vaccinated against Hib.1e6 In contrast, non-encap-sulated (non-typeable) and non-b-type encapsulated H. in-fluenzae cause invasive infections less frequently.7,8 Hibdisease is thought to be less significant in Asia, becauseof insufficient or poorly characterized data on Hib diseasein many counties of this region. Preliminary data from Ban-gladesh indicate that Hib is the predominant cause of men-ingitis and the second most common cause of pneumonia inchildren of this country.9e11 The epidemiology, serotypepatterns and antimicrobial susceptibility to potentially use-ful new drugs, and the current treatment guidelines of in-vasive H. influenzae diseases in Bangladesh is not wellknown, since optimum laboratory facilities are not rou-tinely available for detecting aetiological agents of menin-gitis, pneumonia and septicemia; the most importantinvasive diseases caused by H. influenzae.9e11 The emer-gence of resistance to antimicrobial agents in H. influenzaestrains significantly increases the treatment cost, durationof hospitalization, risk of mortality and serious complica-tions in children suffering from meningitis and pneumo-nia.3,8 The World Health Organization (WHO) has nowrecommended routine Hib vaccine immunization in youngchildren of all countries, however, surveillance of H. influ-enzae disease be undertaken in developing countries togenerate useful and most-desired data on the burden ofpreventable Hib diseases to ascertain the potential utilityof Hib vaccine as public health priority in those re-gions3,12,13 since Hib diseases have largely disappeared inthe United States, Canada, Europe and Australia after theintroduction of Hib conjugate vaccines in the routine child-hood immunization programs in the 1990s.12e15 Despite thesuccess of the Hib vaccination program, the reemergenceof invasive Hib disease in some countries and the emer-gence of non-encapsulated and non-b-type H. influenzae

strains has been noted in a well-vaccinated population em-phasizing the necessity for continuous surveillance in thepost-vaccination period.16 The purpose of this study wasto conduct prospective active surveillance in multiplehospitals using standard laboratory methods for detectionof H. influenzae to study its epidemiology, proportion ofinvasive disease, serotype distribution, emerging antimi-crobial resistance among children hospitalized with pneu-monia, meningitis and septicemia in three hospitals inDhaka city during April 1999eMay 2003.

Materials and methods

Study population

Surveillance of Haemophilus influenzae invasive diseasessuch as meningitis, pneumonia and septicemia was startedin Dhaka hospital, International Centre for Diarrhoeal Dis-ease Research, Bangladesh (ICDDR,B), Bangladesh (250beds and serves 300 patients of all age groups daily) andDhaka Medical College hospitals (1400 beds and serves3000 patients of all age groups daily) in April 1999. Subse-quently, it was extended to Salimullah Medical Collegehospital (500 beds and serves 1000 patients of all agegroups daily), Dhaka in 2000. Enrolment was completedin May 2003. The research protocol was passed by Re-search Review and Research Ethical committee of ICDDR,Band informed consent from the parents of children wastaken on a consent form. Hospitalized children ages<5 years were assessed for pneumonia by using the WorldHealth Organization criteria: cough or difficult breathingand a respiration rate of 50 breaths per minute or morein children ages 2e11 months or 40 breaths per minuteor more in children ages 12e59 months. A child with symp-toms and signs of meningitis such as lethargy, convulsion,bulged fontanel, neck rigidity etc. is considered as a possi-ble case of meningitis and a spinal tap was performed onthat child. CSF results were considered for confirming theetiology of meningitis. A positive blood culture results re-confirm the diagnosis. All routine investigations were per-formed. Children with suspected septicemia (toxic look,cold and clammy skin/fever, perpura/ecchymosis, irrita-ble/lethargic/unresponsive/comatose state, low bloodpressure, reduced or no urine output, signs with associ-ated diseases) were also included.

H. influenzae invasive diseases in children in Bangladesh 193

Laboratory procedures

Two types of clinical samples were collected for thedetection of H. influenzae. These were blood from patientsof pneumonia, meningitis and septicemia for culture(Bajanca et al., 2004), and CSF from suspected meningitispatients for Gram stain, cytology, biochemical analysis,and culture. Blood samples (1e2 ml, one sample per pa-tient) were obtained from patients by venipuncture and in-oculated directly into 20 ml of Trypticase soy brothcontaining sodium polyanthol sulfonate (Becton Dickinson,Cockeysville, MD) for aerobic cultures at 37 �C. Childrenwith meningitis were subjected to lumber puncture forcollecting CSF. Blood culture broth showing haemolysis, tur-bidity or gases and all CSF were cultured on supplementedchocolate agar (CA, Blood agar base with 2% hemoglobinand 1% isovitalex containing X and V factors) plate incandle-extinction jar (5e10% Co2) at 37 �C for 18e48 h(Bajanca et al., 2004). Suspected bacterial colonies wereidentified by typical Gram stain morphology, catalase, oxi-dase and X and V factors (Difco, Detroit, MI, USA) require-ment tests. H. influenzae isolates were serotyped by slideagglutination using serotype-specific antiserum (Denka-Seiken Co. Ltd, Tokyo, Japan). Antimicrobial susceptibilityof H. influenzae was determined by MICs of the followingantimicrobial agents: ampicillin, chloramphenicol, trimeth-oprim-sulfamethoxazole, azithromycin, ceftriaxone, cipro-floxacin, levofloxacin, moxifloxacin and gatifloxacin byE-tests (AB Biodisk, Sweden) according to CLSI standard.H. influenzae type b ATCC 49247 was used as referencestrain. Blood culture broth showing no hemolysis, turbidityor gas was subcultured on CA on day seven of incubation.

Culture-negative pyogenic CSF (cloudy or turbid CSF orCSF with elevated protein of 100 mg/ml and decreased glu-cose of <40 mg/dl or leucocytosis with WBC >100/mm3

with more than 80 polymorph neutrophil) was tested forHib antigen by Latex agglutination test (Becton Dickinson,Cockeysville, MD, USA) and pyogenic CSF samples, whichwere negative by culture and latex agglutination, were sub-jected to PCR for Hib capsular bex A gene detection asdescribed earlier.17

Results

Of 1834 (pneumonia 1493, meningitis 293 and septicemia 48cases) children studied, 73 (4%) had H. influenzae

Table 1 Detection of Haemophilus influenzae from children (<septicaemia, 1999e2003

Clinical diagnosis No. of cases Clinical

Meningitis 293 CerebroPyogenic meningitis* 122 Cerebro

Pneumonia 1493 BloodBacteremic pneumonia# 177 Blood

Septicemia 48 BloodTotal 1834

*Meningitis cases with cloudy or turbid CSF or CSF with elevated prottosis (WBC >100/mm3 with more than 80 polymorph neutrophil), 122positive for bacterial pathogens in pneumonia cases.

infections (Table 1); 46 (isolation rate Z 15.7%) of themwere detected by testing CSF samples of 293 childrenwith suspected meningitis, 25 were detected by blood cul-tures of 1493 (isolation rate Z 1.7) children with pneumo-nia and 2 by blood culture of 48 (isolation rate Z 4.2%)children with suspected septicemia. Among the 46 con-firmed cases of H. influenzae meningitis, 38 were detectedby culturing CSF samples, seven Hib by LA by examining cul-ture-negative pyogenic CSF samples, and only 1 Hib by PCRby testing culture- and LA-negative pyogenic CSF samples.Eight (18.2%) of 44 Hib meningitis cases were culture-negative and positive by LA (7 cases) and PCR (one case).Blood culture was positive for H. influenzae in 22 (58%) of38 CSF-positive cases. Of 293 suspected meningitis cases,122 (41.64%, H. influenzae) had pyogenic meningitis.When data were confined to cases of pyogenic meningitis,the isolation rate of H. influenzae increased to 37.7% (46of 122 children) and to 14.1% (25) of 177 children withpneumonia who had positive-blood cultures for pathogenicbacteria (bacteremic pneumonia). Overall, 63% (46 of 73) ofconfirmed H. influenzae invasive diseases presented asmeningitis compared to 34% (25 of 73) as pneumonia and3% (2 of 73) as septicemia in our study children (Table 2).Overall 65% of children received antibiotics before comingto hospital.

Fifty-nine (81%) of 73 H. influenzae infections werecaused by Hib being the most frequent serotype with detec-tion of Hib in 95.6% and 60% of all children with meningitisand pneumonia respectively caused by H. influenzae (Table2). The remaining 14 strains were: 3 serotype c, 2 serotypea, 1 serotype d and 8 non-typeable (Table 2). The notablefinding was the isolation of 10 (40%) non-b-type strains of 25H. influenzae isolated in pneumonia cases (6 non-typeable,3 serotype c and 1 serotype d) and 2 (100%) non-typeablestrains in septicemic children and 2 (4.4%) non-b-typestrains in meningitis children. The majority of Hib (91.5%,Fig. 1) as well as non-b-type H. influenzae (91.6%) infec-tions clustered in infant aged between 4 and 12 monthsboth in meningitis and pneumonia.

The antimicrobial resistance rate of 52 Hib strains testedwas: ampicillin 31%, chloramphenicol 42%, TMP-SMX 44%,azithromycin 1.4% respectively. Overall 44% of Hib strainswere drug-resistant and there was no difference in re-sistance rates of isolates from meningitis (43%) and pneu-monia (46%) cases. Interestingly, non-b-type H. influenzaeisolates (n Z 14) had similar resistance to all those drugs(Table 3). Thirty-one percent of Hib isolates were MDR

5 years) with clinical diagnosis of pneumonia, meningitis and

samples tested No. of H. influenzae (isolation rate)

spinal fluid 46 (15.7)spinal fluid 46 (37.7)

25 (1.7)25 (14.1)2 (4.2)

73 (4%)

ein (>100 mg/dl) and decreased glucose (<40 mg/dl) or leukocy-of 293 (41.6%) children had pyogenic meningitis. #Blood culture

Table 2 Number (%) of serotypes of Haemophilus influenzae (73) isolates obtained from invasive diseases in children (<5 year)

Serotypes Invasive diseases

Total (%) Meningitis Pneumonia Septicaemia

H. influenzae type b 59 (81) 44 (95.6) 15 (60) 0 (0)Other serotypes 6 (8) 2 (4.4)* 4 (16)** 0 (0)Non-typeable 8 (11) 0 (0) 6 (24) 2 (100)Total (%) 73 (100) 46 (100) 25 (100) 2 (100)

*2 of 2 serotype a. **Of 4, 3 serotype c and 1 serotype d.

194 M. Rahman et al.

(defined as being resistant to �3 different classes of antimi-crobial agents) compared to 29% of non-b-type. MDR-Hibstrains were equally prevalent in patients with meningitis(34%) and pneumonia (31%). Of six encapsulated non-b-types,oneHic strainwasMDRandoneHid strainwas resistant toTMP-SMX only and the rest four strains (2 Hia and 2 Hic types) weresusceptible to all drugs. Of eight non-typeable strains, 62.5%(5) were drug-resistant compared to 44% of Hib (P < 0.01):three (37.5%) of them were MDR, one was resistant to ampicil-lin and TMP-SMX and another strain was resistant to TMP-SMXonly. All ampicillin-resistant-Hib (31%) and non-b-type H. in-fluenzae (29%) strains produced b-lactamase with no detec-tion of b-lactamase-negative-ampicillin-resistant (BLNAR)and b-lactamase-positive-ampicillin-clavulanate-resistant(BLPACR) strains. All b-lactamase-positive isolates were sus-ceptible to amoxicillin-clavulanate and ceftriaxone. Nonewas resistant to amoxicillin-clavulanate, ceftriaxone, cipro-floxacin, levofloxacin, moxifloxacin and gatifloxacin. All iso-lates were extremely susceptible to ceftriaxone with a MIC90

of <0.016 mg/mL. Ciprofloxacin inhibited 100% of strainswith a MIC50 of �0.006 mg/mL and a MIC90 of �0.012 mg/mLshowing no increase in MIC level exhibiting no reduced suscep-tibility. New fluoroquinolones inhibited 100% of strains witha MIC90 of �0.047 mg/mL to moxifloxacin and MIC90 of�0.008 mg/mL to gatifloxacin. Azithromycin inhibited suscep-tible strains with a MIC90 of�1.0 mg/mL showing good in-vitroactivity.

0

5

10

15

20

25

30

35

40

45

1 2 3 4-6 7-12 13-24 25-36 37-48 49-60

Age (Month)

Num

ber

of c

ases

Non-b type Septicemia

Hib Septicemia

Non-b type Pneumonia

Hib Pneumonia

Non-b type Meningitis

Hib Meningitis

Figure 1 Age distribution of Haemophilus influenzae typeb and non-b-type invasive diseases in hospitalized children un-der 5 years of age.

The case fatality rate in children with pneumonia was 8%compared to 19% in children with meningitis.

Discussion

This is the first prospective largest multicenter study ofinvasive H. influenzae diseases in hospitalized children<5 years of age in Bangladesh. Unlike other reported stud-ies from Bangladesh4,9,10 we studied all important invasivedisease syndromes, included both blood and CSF culture re-sults, used latex agglutination and PCR in a subset of CSFsamples that were pyogenic but culture-negative, and stud-ied serotype distribution and antimicrobial susceptibilitypatterns over the period of 3 years. Our findings suggestedthat H. influenzae was an important cause of life-threateninginfections such as meningitis and pneumonia in young chil-dren of Bangladesh like many other countries. The isolationrate of H. influenzae from blood cultures of children withpneumonia was slightly higher (1.7%) than the reportedstudy (1.2%)10 because of use of multiple diagnostic tests.However, the rates were much higher in children with bac-terimic pneumonia and meningitis. Several lines of evi-dence argue that these Bangladeshi data might representan underestimation of the true H. influenzae disease bur-den because of over the counter availability of antimicro-bial agents and their promiscuous use in Bangladeshresulting in a proportion of the H. influenzae cases beingculture-negative. Ambulatory H. influenzae infectionswere not counted in our hospital-based surveillance. TheHib ‘‘vaccine probe’’ studies in Gambia18 and Chile19

showed that at least 5 cases of blood culture-negative Hibpneumonia occur in the community for each case of bloodculture positive Hib disease detected. In Bangladesh, as inother areas of developing countries,20 many infants andyoung children died at home and never make it to hospitaland H. influenzae is surely responsible for some of thedeaths among infants who die at home because of illnessessimilar to invasive bacterial diseases.

Our study shows that serotype b is the single mostimportant serotype that causes invasive H. influenzae dis-ease in urban children of Bangladesh. The proportion ofbacterial meningitis caused by Hib is similar to reporteddata from Bangladesh,20 India21 and Thailand3 and some-what less than that of United States and Europe beforethe introduction Hib vaccine in their national immunizationprogrammes.12,14,16 We have not observed an increase in in-cidence of Hib diseases over the period of three years,a finding that contrasts with an earlier report, which de-scribed significant increase in the past few years inBangladesh.11

Table 3 Percentage of antimicrobial resistance among invasive Haemophilus influenzae strains isolated from children withmeningitis, pneumonia and septicaemia

Antimicrobial agents H. influenzae type b (N Z 52)* Non-b-type H. influenzae (N Z 14)*

Resistant Intermediate Non-susceptible Resistant Intermediate Non-susceptible

Ampicillin 31 4 35 29 7 36Chloramphenicol 42 2 44 36 7 43Trimethoprim-sulfamethoxazole 44 0 44 43 7 50Azithromycin 1.4 0 1.4 0 0 0

*All are susceptible to amoxicillin-clavulanate, ciprofloxacin, ceftriaxone, levofloxacin, moxifloxacin, and gatifloxacin.

H. influenzae invasive diseases in children in Bangladesh 195

Non-b-type H. influenzae is an uncommon cause of inva-sive disease in children; however, the relative importanceof infections caused by non-encapsulated and non-b-typeencapsulated H. influenzae has increased recently.7,8 A sig-nificant number of non-encapsulated and non-b-type en-capsulated H. influenzae was isolated from blood culturesof children with pneumonia and suspected septicemia rais-ing an important issue of vaccine against non-Hib strains.It’s the first report of isolation of non-encapsulated andnon-b-type encapsulated H. influenzae from invasive dis-ease in children of Bangladesh. A similar finding was re-ported from Pakistan.22 The serotype replacement by Hibvaccine is unlikely to contribute to the occurrence or emer-gence of non-encapsulated and non-b-type encapsulatedH. influenzae since the Hib vaccination is not included inthe Extended Program of Immunization (EPI) program inBangladesh. And its high price limits its wide use in theprivate sector.

Like many other countries, Hib meningitis and pneumo-nia were clustered in young children aged 4e12 months inBangladesh although the age distribution of Hib diseasevaries widely worldwide.1,3 This is an important observationbecause it implies that it should be possible to protect theoverwhelming majority of infants through a well functioningEPI that can immunize infants (at 6, 10 and 14 weeks of age)before they reach the age of peak risk for Hib diseases. Acost-effective Hib vaccination programme targeting thispopulation is likely to reduce Hib invasive diseases in Ban-gladesh. Recently, a combination of Hib vaccine with di-phtheria, pertussis and tetanus (Hib-DPT) was foundeffective in a Hib ‘‘vaccine probe’’ non-randomized case-control study in reducing Hib disease particularly meningitisin children of Bangladesh by passive surveillance of Hib dis-ease.23 It was found less effective for reduction of radiolog-ically confirmed pneumonia possibly because of non-Hib asa cause of pneumonia as shown by our study. It was worthmentioning that Bangladesh recently (2006) applied forfunding from the Global Alliance for Vaccines and Immuniza-tion (GAVI) to introduce a pentavelent combination DTP-HepatisB-Hib vaccines into routine childhood immunizationprogramme in 2008. It should be kept in mind that acellularpertussis vaccine may reduce the efficacy of Hib vaccine.24

High case fatality of H. influenzae diseases indicate theseverity of these infections in Bangladeshi children, whichhas been reported from other countries.2,3 Higher rate ofcase fatality was reported in children infected with drug re-sistant H. influenzae than susceptible strains in a previousreport from Bangladesh.10

Administration of early therapy with proper antimicro-bial agent is the key factor to reduce the mortality,morbidity and complications of invasive H. influenzae dis-ease. We detected a high rate of resistance to TMP-SMX,ampicillin and chloramphenicol among invasive Hib aswell as non-b-type H. influenzae isolates in our study. Ahigh rate of multi-drug resistance to conventional first-line antimicrobial agents was worrying. The limitation ofthe study was that the wide scale use of antibiotic mighthave selected patients with resistant organisms for inclu-sion in the study and the patients might not be represen-tative of community infections. However, an insignificantresistance rate of azithromycin and no resistance toamoxicillin-clavulanate and third generation cephalospo-rin such as ceftriaoxone was observed among H. influen-zae isolates. Thus, these drugs might be useful for thetreatment of invasive H. influenzae infections in Bangla-desh, particularly in case of pneumonia and meningitis.However, it’s a matter of great concern that these drugsare expensive, which are difficult to afford by many pa-tients of developing countries. New respiratory fluoroqui-nolones (levofloxacin, moxifloxacin and gatifloxacin) werefound be effective in the treatment of pneumonia causedby H. influenzae that were resistant to conventional first-line drugs exhibiting the potential therapeutic value ofthese drugs. Recently, MDR strains of Hib were reportedin India.21

Beta-lactamase-mediated ampicillin resistance was com-mon in H. influenzae strains in Bangladesh with no detection ofb-lactamase-negative-ampicillin-resistant (BLNAR) and b-lactamase-positive-ampicillin-clavulanate-resistant (BLPACR)H. influenzae strains. Thus amoxicillin-clavulanate and cef-

triaxone has utility in the treatment of ampicillin-resistantstrains.

In conclusion, Hib was a leading cause of life-threateninginfections, predominantly in infants in Bangladesh. Pneu-monia caused by non-encapsulated and non-b-type encap-sulated H. influenzae was quiet frequent. A high proportionof multidrug-resistant-H. influenzae limited the use offirst-line conventional antibiotics resulting in amoxicillin-clavulanate, ceftriaxone or azithromycin as empirical ther-apy. Specific recommendation for use of ceftriaxone mustbe made for the treatment of meningitis particularly MDRcases. New respiratory fluoroquinolines shows potentialutility for the treatment of H. influenzae infections. A na-tional Hib vaccination programme will prevent a significantproportion of Hib disease in Bangladesh although non-b-type infections will remain an issue.

196 M. Rahman et al.

Acknowledgements

This research protocol was funded by the United StatesAgency for International Development, grant number HRN-A-00-96-90005-00. International Centre for Diarrhoeal Dis-ease Research, Bangladesh, acknowledges with gratitudethe commitment of USAID, Washington, DC, USA to theCentre’s research efforts.

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