820

had spent most of their lives in western Kenya (Bungoma andKitale), neither had an illness suggestive of MVD, andneither showed a rise in titre. The nurse’s contact with

laboratory samples was brief and distant. Maid A had her lastcontact with the first patient 4 days before the onset of hisillness. It seems unlikely that either was a secondary case.Virus persisted in the seminal fluid of patient 2 for 2

months after clinical recovery, and sexual transmission ofMVD is known to occur. The potential for sexualtransmission should be further investigated, since it couldprovide a means of virus spread.Preliminary serological and ecological investigations in the

area of the sugar factory 10 km south-east of Bungomatownship failed to indicate the source or route of infection ofthe primary case. The area (fig. 3), at an altitude of 1450 m, isamong the most densely populated in Kenya. The land, some40% of which is under cultivation with maize and sugar-cane,is open savannah and forms the base of Mount Elgon, whichrises to 4321 m to the north-east astride the Uganda border.Remnant patches of forest with affinities to the West AfricanCongo forest type are found in the area, the flora and fauna ofwhich are unique in Kenya, many species being foundelsewhere only in West and Central Africa. The area is closeto the Lake Kyoga area of Uganda, where monkeysincriminated in the original European outbreaks were

collected.

Attempts to develop a surveillance system for viral

haemorrhagic fevers have emphasised the difficultiesinherent in such programmes. Patients present late in theirillness, when they are unlikely to be viraemic. Necropsy israrely carried out, and indeed in the primary case in thisoutbreak virus particles were not found in liver tissue. Activesurveillance in the area since the outbreak has failed to

provide definite evidence of MVD in the area of residence ofthe primary case, apart from seropositivity in 4 residents.Present evidence, however, suggests the presence of Ebolavirus. 1 surviving patient showed a significant rise in

antibody titre against Ebola virus, and a close friend of anEbola suspect who died was sick and had a significantantibody titre. In addition 4 of 84 individuals in theimmediate area had significant antibody titres against Ebolavirus. Radioimmunoassay may prove a more specificindicator of infection and was strongly positive in 1 of our

suspected cases of Ebola fever (K. M. Johnson, unpublished).Once suspected, isolation of individuals is mandatory until

the diagnostic results are known. The technical, financial,and logistic implications of surveillance and clinical

investigation require close examination to ensure that healthresources are used to maximum advantage, especially incountries with limited health resources. Nosocomialtransmission can probably be prevented with simpleprocedures, and close tissue contact seems necessary to effecttransmission. Simple barrier-nursing techniques andlimitation of laboratory investigation except with adequatecontainment should be established in areas of virus activity.The indirect fluorescent-antibody test appears to be

unsatisfactory especially for epidemiological investigations,and efforts should be increased to develop tests with greaterspecificity.We thank the South African Institute of Medical Research; the National

Institute of Virology, South Africa; and the Special Pathogens Section of theCenters for Disease Control for their support. The South African Institute ofMedical Research and South African Department of Health supplied staff,laboratory facilities, and equipment, especially respirators and plasmapheresis

,

sets. Dr I. M. Spence did the electron microscopy and took the photographs ofthe virus particles in renal tissue. The Special Pathogens Section, C.D.C.,

supplied electron micrographs of the virus isolated from patient 2. We thankthe World Health Organisation, especially Dr Paul Bres, for logistic supportand for protective clothing. We also thank the staff of the Nairobi Hospital fortheir cooperation and the Kenyan Ministry of Health, especially the Divisionof Disease Control and the virology departments, for their support andassistance.

Correspondence should be addressed to D. H. S., Liverpool School ofTropical Medicine, Pembroke Place, Liverpool L3 5QA.

REFERENCES

1. Martini GA. Marburg virus disease, clinical syndrome. In: Martini GA, Siegart R, eds.Marburg virus disease. Berlin: Springer Verlag, 1971: 1-9.

2. Todorovitch K, Mocitch M, Klasnja R.Clinical picture of two patients infected withMarbug vervet virus. In: Martini GA, Siegart R, eds. Marburg virus disease. Berlin.Springer Verlag, 1971: 19-23.

3. Henderson BE, Kissling RE, Williams MC, Kafuko GW, Martin M. Epidemiologicalstudies in Uganda relating to "Marburg’agent". In: Martini GA, Siegart R, eds.Marburg virus disease. Berlin: Springer Verlag, 1971: 166-76.

4. Gear JSS, Cassel GA, Gear AJ, et al. Outbreak of Marburg virus disease in

Johannesburg. Br Med J 1975; iv: 489-93.5. Lyle CJ, Isaacson M, Smith EB, Wulff H, Crees M, Geldenhuys P, Johnston J.

Epidemiological investigation of Marburg virus disease in Southern Africa. Am JTrop Med Hyg 1978; 27: 1210-15.

6. Swoveland PT, Johnson K. Enhancement of fluorescent antibody staining of viralantigens in formalin-fixed tissues by trypsin digestion. J Infect Dis 1979; 140:758-64.

7. Francis DP, Smith DH, Highton RB, et al. Ebola fever in the Sudan, 1976:

Epidemiological aspects of the disease. In: Pattyn SR, ed. Ebola virus haemorrhagicfever. Amsterdam: Elsevier-North Holland, 1976: 129-36.

8. Smith DH, Francis DP, Simpson DIH. African haemorrhagic fever in the SouthernSudan, 1976: the clinical manifestations. In: Pattyn SR, ed. Ebola virus

haemorrhagic fever. Amsterdam: Elsevier-North Holland, 1976: 21-26.9. Piot P, Sureau P, Bremen G, et al. Clinical aspects of Ebola virus infection in Yambuku

area, Zaire, 1976. In: Pattyn SR, ed. Ebola virus haemorrhagic fever. Amsterdam:Elsevier-North Holland, 1976: 7-14.

10. Bremen JG, Piot P, Johnson KM, et al. The epidemiology of Ebola haemorrhagic feverin Zaire, 1976. In: Pattyn SR, ed. Ebola virus haemorrhagic fever. Amsterdam:Elsevier-North Holland, 1976: 103-23.

OUTBREAK OF COXSACKIE A1GASTROENTERITIS: A COMPLICATION OFBONE-MARROW TRANSPLANTATION

TIMOTHY R. TOWNSENDROBERT H. YOLKENCATHERINE A. BISHOPGEORGE W. SANTOS

ELIZABETH A. BOLYARDWILLIAM E. BESCHORNER

WILLIAM H. BURNSREIN SARAL

Epidemiology Unit, Johns Hopkins Hospital; Departments ofPediatrics, Medicine, and Pathology, Johns Hopkins University

School of Medicine; and Johns Hopkins Bone Marrow TransplantUnit at the Johns Hopkins Oncology Center, Baltimore, Maryland,

U.S.A.

Summary In a three-week period 7 of 14 transplantrecipients were infected with coxsackie Al

virus. Diarrhoea and mortality were significantly associatedwith infection (7 of 7 infected compared with 0 of 7 non-infected, and 6 of 7 infected compared with 1 of 7 non-

infected, respectively). Early in the outbreak, the diarrhoeawas presumed to be due to acute graft-versus-host disease(AGVHD). However, the distribution of AGVHD amonginfected and non-infected patients was nearly equal, and atnecropsy 3 of 6 infected patients who had had diarrhoeashowed no evidence of gastrointestinal involvement withAGVHD. Infection with viral enteric pathogens may be animportant factor in the clinical course of transplantrecipients.

Introduction

BONE-MARROW transplantation (BMT) is effective therapyfor aplastic anaemia and lymphoreticular malignancies. 1-3

821

Successful bone-marrow engraftment requires immuno-suppression, and bacterial, fungal, and viral infections duringthis period4 are a major cause of BMT failure. Attention hasfocused on herpesviruses in the aetiology of viral infections inBMT recipients.4 Other viruses, particularly those entericviruses which cause widespread illness in the community,have not been described as causing infections in

immunologically compromised patients.In February and March, 1980, coxsackie A1 virus infected

7 of the 14 patients in the BMT unit of the Johns HopkinsHospital oncology centre. 6 of the 7 infected patients died.

Methods

Laboratory Procedures

Two throat and two stool specimens were obtained with

’Dacron’-tipped swabs. One swab was placed in minimal essentialmedium (containing 0 - 5% gelatin, 50 000 units/ml penicillin G,and 50 pg/ml streptomycin) and stored immediately at - 70°C untiltested for the presence of virus or viral antigen. Specimens wereexamined for the presence of rotavirus, adenoviruses, Escherichiacoli heat-labile toxin, Clostridium difficile toxin, andcoxsackieviruses with previously described enzyme immunoassaymethods. 5-10

Specimens were initially tested for all 23 coxsackie A serotypesusing antisera for each type. Subsequent specimens were tested onlyfor coxsackie Al. Selected specimens positive by enzymeimmunoassay for coxsackievirus were confirmed by inoculation ofstool filtrate into suckling mice following standard procedure.1 t

The other swab was immediately processed according to standardJohns Hopkins Hospital culture methods. Stool specimens weretested for the presence of salmonella, shigella, yersinia, andcampylobacter and throat specimens for respiratory pathogens.

Investigation of the OutbreakThe BMT unit and the clinical management of patients

undergoing transplantation have been described previously.3,4Themedical records of all patients in the BMT unit during the outbreakwere reviewed, and demographic, clinical, and epidemiological datawere abstracted. Nosocomial infections were detected with standardJohns Hopkins Hospital surveillance methods and definitions.Acute graft-versus-host disease (AGVHD) was diagnosed by meansof skin biopsy. Degree-days of fever were defined as the number ofdays a patient’s highest recorded rectal temperature exceeded 38°C,and antimicrobial days were defined as the number of days a patientreceived one or more systemic antimicrobial drugs (excluding oralnon-absorbable drugs). To eliminate possible bias resulting fromthe effects of infection with coxsackie Al, degree-days of fever andantimicrobial-days were tabulated for the average admission-infection interval for infected patients and the same time periodfor uninfected patients. Diarrhoea was defined as 600 ml or more ofliquid stool for three or more consecutive days.

BMT-unit personnel were asked whether they had had diarrhoealillness in the two months preceding the outbreak.

All patients in the BMT unit who died were examined postmortem by W.E.B., who had carried out all necropsies on BMTrecipients since the unit opened in 1977.

Statistical Methods

Mean values and 95% confidence intervals were calculated forcontinuous data. Differences of proportions of discrete data werecompared by means of Fisher’s exact test. A two-tailed test was used,since possible associations between factors were not predictedbeforehand. Levels of statistical significance were set at p<0’05.

Results

Between March 16 and March 22,1980, 4 ofthe 14 patientsin the BMT unit died after an acute diarrhoeal illness. Theunit was closed to new admissions, all remaining patientswere placed on enteric precautions similar to thoserecommended by the Centers for Disease Control, and anepidemiological investigation was begun. I2

Patients’ records revealed that 7 of the 14 patients had haddiarrhoea between Feb. 25, and March 16, 1980. CoxsackieAl antigen was identified from the stools or throats of the 7patients with diarrhoea but not from the 7 patients withoutdiarrhoea. No other enteric pathogens were recovered fromany patient. Infected patients had significantly greater stooloutput volume than non-infected patients during all but oneweek of the epidemic period (fig.l).

6 of the 7 infected patients died. At necropsy these 6patients had both pronounced lymphoid atrophy andabnormalities of the gastrointestinal epithelium. The

epithelium overlying intestinal villi had a foamy vacuolatedappearance, even in patients without AGVHD, who hadadequate numbers of plasma cells in the lamina propria(fig.2). There were patchy areas of severe sloughing in thegastrointestinal mucosa. In addition, acini of the exocrinepancreas contained large pale vacuolated columnar cells.To define risk factors for infection and thereby derive clues

to the source of the outbreak or its mode of transmission, wecompared the characteristics of the infected and non-infectedpatients (see table). Only the presence of diarrhoea and a fataloutcome were significantly associated with coxsackie Alinfection. No temporal association was noted between eitherthe dates of admission or BMT and the date of onset ofinfection. The average BMT-infection interval was 20 days(range 11 to 26 days), and the average admission-infectioninterval was 31 days (range 24 to 38 days). None of thehospital personnel was significantly associated with infectedpatients. Of the 42 hospital personnel working in the unitduring February or March, 1980, 20 were located andinterviewed about antecedent diarrhoeal illness. 5 employeesdescribed diarrhoeal illness occurring in early and midFebruary, characterised by loose stools without significantsystemic symptoms and lasting less than a week. 5 childrenhad been admitted to the paediatric service of the JohnsHopkins Hospital in February, 1980, with diarrhoeal illness,

23 I 1 z

8 z

15 1

22 ’

FEBRUARY MARCH

Pig. 1&mdash;Mean (and 95% confidence intervals) stool output volume (ml)of infected and non-infected patients by week.

BMT unit, February-March, 1980.

822

Fig. 2-Section of jejunum demonstrating villous blunting andvacuolisation of the epithelium.There is loss of polarisation of the nuclei (haematoxylin and eosin, reduced

by a quarter form x 250).

CHARACTERISTICS OF INFECTED AND NON-INFECTED BMT UNIT

PATIENTS, FEBRUARY-MARCH, 1980

NS =not statistically significant.*Mean &plusmn;95% confidence intervals.

and coxsackie A 1 antigen was identified in their stools. Stoolspecimens were not obtained from hospital personnel.

Infected patients who remained in the hospital were kept onenteric precautions until they were no longer excretingdetectable viral antigen. No additional infections occurred,and the BMT unit was reopened to new admissions in earlyApril, 1980.

Discussion

Coxsackie A 1 virus has rarely been recovered from the stoolor throat of an otherwise healthy personY Evidence for aspecific clinical syndrome attributable to coxsackie Al virusis tenuous, but there have been reports of communityoutbreaks of non-specific febrile illness and sporadic cases ofaseptic meningitis or paralytic disease. 13,I4 Diarrhoeal disease

in otherwise healthy people due to coxsackie A 1 virus appearsto be uncommon, although the gastrointestinal tract is thepresumed site of viral replication. Coxsackievirus infectionhas not been reported previously in BMT recipients, so theclinical manifestations, pathophysiology, and pathologicalfindings associated with such infections in this highlycompromised population have not been described.

In February, 1980, a number of children visiting theoutpatients facilities of the Johns Hopkins Hospital haddiarrhoea. 5 of them were admitted to the hospital, andcoxsackie Al antigen was identified in their stools. No otherviral or bacterial pathogens were isolated from these children.The extent of dispersion of coxsackie Al virus through thecommunity during February, 1980, remains unknown, butcircumstantial evidence from this outbreak suggests that thevirus may have caused at least some of the diarrhoea in the

community and it is possible that some hospital personnel orpatients’ visitors may have been infected. Since we wereunable to identify either the source of the virus or its mode oftransmission in this outbreak, we can only speculate thateither visitors or hospital personnel acquired the virus in thecommunity and brought it into the BMT unit. Since viralexcretion in healthy persons rarely lasts more than 30 daysand since no hospital staff interviewed had had an illness inthe 30 days before their interview, we were unable to establishthe role of hospital personnel in the outbreak.

The consistent pathological finding of enlarged palevacuolated columnar cells in the acini of the exocrinepancreas in infected patients was an unexpected finding.While AGVHD may affect the pancreas in animals, this hasnot been described in man 15 The pancreas has been reportedto be a target organ for the coxsackieviruses, and these virusesmay affect the endocrine pancreas in such a way as to induce

juvenile-onset diabetes mellitus.16 However, we did notattempt to recover coxsackie Al virus from the pancreas.

Diarrhoea in BMT recipients has been noted in previousstudies, and in many instances the diarrhoea has beenattributed to AGVHD, since the gastrointestinal tract is amajor target organ for AGVHD; 17 indeed, diarrhoea is one ofthe cardinal clinical symptoms of AGVHD, and stool outputvolume has been proposed as a means of grading the severityof AGVHD.18 However, the role of cultivatable and non-cultivatable enteric pathogens in producing the diarrhoeaobserved among these patients with AGVHD has not beenstudied. The significantly larger liquid stool output volumefrom infected patients than from non-infected patients andthe nearly equal distribution of biopsy-diagnosed AGVHDamong infected and non-infected patients makes AGVHD asthe cause of the diarrhoea in this outbreak less likely.Moreover, at necropsy 3 of the 6 infected patients had no.evidence of gastrointestinal involvement with AGVHD andhad adequate numbers of plasma cells in the lamina propria;yet all had diarrhoea. 19 All 6 infected patients who died hadfoamy vacuolated epithelium overlying their intestinal villi; apathological finding that has been described in associationwith upper gastrointestinal infection with other enteric

viruses, such as rotavirus and Norwalk agent, but not inassociation with AGVHD.2o

Clinical observation is the first step in the epidemiologicalapproach to disease causation, followed by examination ofavailable data to identify statistical associations. The thirdand final step is to design further new studies to demonstratespecific causal inferences. We have documented a cluster of

823

infections due to coxsackie A 1 within a three-week period in 7of 14 BMT recipients. All 7 infected patients had diarrhoea

(>600 ml liquid stool on each of three consecutive days), and 6of these 7 died. Compared with uninfected BMT recipients,diarrhoea and death were statistically significantly associatedwith infection. Post-transplant diarrhoea is a common clinicalfinding and is often attributed to AGVHD. However, wefound no association between AGVHD and either diarrhoeaor infection. Further studies of the relation between entericviral pathogens, AGVHD, and clinical diarrhoea are neededto establish a cause-and-effect relationship. After thisoutbreak we instituted a surveillance programme in ourBMT unit. Gastrointestinal signs and symptoms are nowrecorded daily, clinical and biopsy evidence of AGVHD ismonitored, and stool specimens are tested weekly for entericpathogens. It is hoped that this will help us both to identifycases of enteric infection early to prevent further outbreaksand to define further the relation between enteric pathogens,AGVHD, and clinical disease.This work was supported in part by National Institute of Allergy and

Infectious Disease contract no. 266-792616; National Institutes of Healthgrant no. I-ROIA117604-01; the Thrasher Research Fund, Salt Lake City,Utah; the National Cancer Institute, National Institutes of Health,Department of Health and Human Resources grants nos. CA06973 andCA15396-08; and the Harley W. Howell Foundation.

Correspondence should be addressed to T. R. T., Administration 229, TheJohns Hopkins Hospital, 600 N. Wolfe St., Baltimore, Maryland 21205,U.S.A.

REFERENCES

1. Storb R, Thomas ED, Buckner CD, et al. Allogeneic marrow grafting for treatment ofaplastic anemia: A follow-up on long-term survivors. Blood 1976; 48: 485-90.

2. Thomas ED, Buckner CD, Banaji M, et al. One hundred patients with acute leukemiatreated by chemotherapy, total body irradiation, and allogeneic marrow

transplantation Blood 1977; 49: 522-33.3. Santos GW. Bone marrow transplantation. In: Stollerman GH, ed. Advances in

internal medicine. Chicago: Year Book Medical Publishers, 1979: 157-82.4. Elfenbein GJ, Saral R. Infectious disease during immune recovery after bone marrow

transplantation. In: Allen JC, ed. Infection and the compromised Host. Baltimore:Williams and Wilkins, 1981: 157-96.

5. Yolken RH, Torsch VM. Enzyme-linked immunosorbent assay for detection andidentification of Coxsackieviruses A. Infect Immun 1981; 31: 742-50.

6. Yolken RH, Torsch VM. Enzyme-linked immunosorbent assay for detection andidentification of Coxsackieviruses B antigen in tissue culture and clinical specimens.J Med Virol 1980; 6: 45-52.

7. Yolken RH, Whitcomb LS, Marien G, Bartlett JD. Enzyme immunoassay for thedetection of Clostridium difficile antigen. J Infect Dis 1981; 144: 378.

8. Sarkkinen HK, Tuokko H, Helonen PE. Comparison of enzyme immunoassay andradioimmunoassay for detection of human rotavirus and adenovirus from stoolspecimens. J Virol Methods 1980; 1: 341-51.

9. Merson MH, Yolken RH, Sack RB, Froehlich JL, Greenberg HB, Huq I, Black RW.Detection of Escherichia coli enterotoxins in stools. Infect Immun 1980; 29: 108-13.

10 Yolken RH, Wyatt RG, Zissis GP, et al. Comparative epidemiology of humanRotavirus types I and 2 as studied by enzyme-linked immunosorbent assay. N Engl JMed 1978; 299: 1156-61.

11 Melnick JL, Wenner HA, Philips CA. Enteroviruses. In: Lennette GH, Schmidt NJ,eds. Diagnostic procedures for viral, rickettsial, and chlamydial infections.Washington: American Public Health Association, 1979: 471-534.

12. Centers for Disease Control. Isolation techniques. Washington DC: GovernmentPrinting Office, 1975: 51.

13. Parrott RH. The clinical importance of group A Coxsackie viruses. Ann NY Acad Sci1957; 67: 230-40.

14. Douglas RG. Comparative diagnosis of picornavirus infections. In: Kirstak E, KirstakC, eds Comparative diagnosis of viral diseases, vol 1. New York: Academic Press,1977; 344: 83.

15 Nizze H. Ultrastructural findings in the mouse exocrine pancreas during graft-versus-host reaction. Exp Pathol 1978; 16: 254-66.

16. Yoon J, Austin M, Ondera T, Notkins AL. Virus induced diabetes mellitus. N Engl JMed 1979, 300: 1173-79.

17. Slavin RE, Santos GW. The graft-versus-host reaction in man following bone marrowtransplantation. Pathology, pathogenesis, clinical features and implications. ClinImmunol Immunopathol 1973; 1: 472-98.

18 Thomas ED, Storb R, Clift RA, et al. Bone marrow transplantation. N Engl J Med1975; 292: 832-43, 895-902.

19. Beschorner WE, Yardley JH, Tutschka PJ, Santos GW. Deficiency of local intestinalimmunity with graft-versus-host disease in human bone marrow recipients. J InfectDis 1981; 144: 38-46.

20 Trier JS, Schreiber DS, Blacklow NR. The pathology of acute nonbacterialgastroenteritis. In Yardley JH, Morson BC, Abetts MR, eds. The gastrointestinaltract. International Academy of Pathology monograph. Baltimore: Williams andWilkins, 1977: 36-49.

SUSCEPTIBILITY OF PLASMODIUMFALCIPARUM TO PYRIMETHAMINE ANDSULFADOXINE/PYRIMETHAMINE IN

KISUMU, KENYA

PHUC NGUYEN-DINH HARRISON C. SPENCERSHEMEY CHEMANGEY-MASABAFREDERICK C. CHURCHILL

Parasitic Diseases Division, Center for Infectious Diseases,Centers for Disease Control, Atlanta, Georgia, U.S.A.;

Clinical Research Centre, Kenya Institute of Medical Research;and Division of Vector-Borne Diseases, Ministry of Health, Nairobi,

Kenya

Summary Field studies were conducted in Kisumu,Kenya, to assess the susceptibility of local

strains of Plasmodium falciparum to pyrimethamine alone (bya standard 7-day in-vivo test and a 48 h in-vitro field test) andto sulfadoxine-pyrimethamine (by a 7-day in-vivo test). Bothin-vivo (10/11) and in-vitro (19/21) tests demonstrated thatpyrimethamine resistance was very common. Parasite

susceptibility to sulfadoxine-pyrimethamine was uniformlygreater when 24 isolates were tested in vivo, thus indicatingthat this drug combination remains valuable despite the highfrequency of resistance to pyrimethamine alone.

Introduction

THE continuing spread of chloroquine-resistant strains ofmalaria parasites, particularly their recent emergence in EastAfrica, increases the potential role of pyrimethamine as analternative antimalarial compound, either alone or incombination with other drugs, such as in sulfadoxine/

pyrimethamine (’Fansidar’). This situation underlines theimportance of monitoring closely local strains ofplasmodiumfalciparum for susceptibility not only to chloroquine but alsoto other antimalarial drugs.

Assessments of susceptibility to antimalarial drugs can beconducted in vivo or in vitro. In-vivo tests are technicallysimple to perform but require daily patient follow-up for atleast 7 days, and interpretation of the results may be difficultbecause reinfections may occur during the observation

period.2 These problems can be avoided by use of in-vitrofield tests, such as the Rieckmann macro and micro

methods,3,4 or a more recently developed 48 h test5-7 whichoffers advantages not found in the previously availabletechniques. -

In March-April, 1981, field studies were conducted inKisumu, Kenya, to assess the susceptibility of P. falciparumto pyrimethamine both in vivo and by the 48 h in-vitro fieldtest, and to sulfadoxine/pyrimethamine by in-vivo studies.The results of these studies are reported in this paper.

Patients

Patients

Children infected with P. falciparum were identified throughschool surveys conducted in the municipality of Kisumu, a city(population 150 000) in the malaria-endemic area of Western

Kenya. Thick blood smears obtained by finger prick from pupilsaged 6 to 12 were stained with Giemsa and examined for malariaparasites. Those children found to be infected were tentativelyincluded in the study group if they fulfilled the following criteria: