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HIV-1 viraemia and influenza

SIR,-Cellular activation of CD4 lymphocytes is required forHIV-1 replication in vitro and it is often assumed that immunestimulation due to infection, vaccination, or allogeneic exposure canresult in enhanced HIV-1 replication in vivo. I have examined thispossibility by serial measurement of viral burden in blood fromthree HIV-1 seropositive, symptomless individuals who had acuteinfluenza infection or influenza vaccination. Infectious titres ofHIV-1 in plasma and peripheral blood mononuclear cells (PBMC),measured by end-point-dilution cultures,! increased transiently inpatient 1 who had an acute illness clinically diagnosed as influenza.The levels of infectious HIV-1 in plasma increased by 20-fold in thetwo given influenza A vaccine:

HIV- 7 titrePlasma PBMC

Case Day (TCID/ml) (’7’C/D//0"ce//

TCID=tissue-culture mfective doses.

This rise in viraemia was most prominent in the first week followingimmunisation, and viral titres had returned to or toward baseline by2-3 weeks. An increase in HIV-1 titre in PBMC was seen in onlyone of two cases. The transiently higher levels of viraemia were notassociated with a dramatic decline in the CD4 cell counts.These findings are preliminary and should not be used to make

clinical decisions about influenza vaccinations in HIV-1-infected

persons. However, they should trigger more systematic studies ofthe acute effect of different infections or immunisations on HIV-1 I

replication in vivo. The impact of allogeneic exposures from bloodtransfusions on viral burden should also be determined.

Aaron Diamond AIDS Research Center,NYU School of Medicine,New York, NY 10016, USA DAVID D. Ho

1. Ho DD, Moudgil T, Alam M. Quantitation of human immunodeficiency virus type 1in the blood of infected persons. N Engl J Med 1989; 321: 1621-25.

Insulin autoimmune syndrome and HLA-DR4

SIR,-Dr Uchigata and colleagues (Feb 15, p 393) demonstratethat all of 27 Japanese patients with insulin autoimmune syndromewere positive for HLA-DR4 (DRBI*0406), suggesting that thedevelopment of the syndrome is linked to the presence of specificHLA class II molecules. We have evidence that this may not be sofor other ethnic groups.

In 1986, a 55-year-old Swiss woman was referred to our hospitalfor evaluation of recurrent spontaneous hypoglycaemic attacks thatusually took place 1-3 h after meal ingestion, were improved by foodintake, and were precipitated by exercise. Fasting plasma totalinsulin (1 -22 nmol/1) and proinsulin (0-48 nmol/1) were raised, andhuman insulin specific autoantibodies were detected both byradioimmunoasay (RIA) and by an IgG-specific ELISA. In theRIA, the patient’s serum bound 49-3% of human, but only 3-7% ofadded pork, insulin tracer, and in the ELISA 352% (% of referenceserum binding) human insulin and 2-6% pork insulin were bound.Further analysis revealed a monotypic and monoclonal humaninsulin autoantibody, which showed a restriction to the lambda lightchain. On the basis of these findings insulin autoimmune syndromewas diagnosed.’HLA-typing of this patient by standard microlymphocytic

toxicity tests showed the following HLA haplotype: A30, 32; B27,62; DR 1,2. Thus, apart from the class I antigen B62, which waspresent in 22 of the 27 patients reported by Uchigata, our patient

had HLA antigens that were rarely seen in the Japanese group. Inparticular, the class II antigen DRI was absent in the Japanesepatients with insulin autoimmune syndrome, and HLA-DR2 wasfound in as few as 3 of them. Thus, the high prevalence ofHLA-DR4 (DRB1*0406) in Japanese patients may, at least in part,indicate the high prevalence of that antigen in this ethnic group.Division of Endocrinology and Metabolism,University Hospital,4031 Basel, Switzerland

G. A. SPINASU. KELLER

1. Sklenar I, Wilkin TJ, Diaz JL, Erb P, Keller U. Spontaneous hypoglycemia associatedwith autoimmunity specific to human insulin. Diabetes Care 1987; 10: 152-59.

Mazzotti test for onchocerciasis

SjR,—The Mazzotti test is a dangerous, provocative test that hasbeen used to detect onchocerciasis but is condemned by the WorldHealth Organisation expert committee on onchocerciasis.l It cameinto vogue 40 years ago as an easy way to detect this disease. In

developing areas, one could always give a patient some

diethylcarbamazine (DEC), and if they became sick they had"oncho". In many ways it was easier and cheaper than doing skinsnips.We now know that the use of DEC and provoking a Mazzotti

reaction is not without risk. It can cause severe acute illnesses,irreversible long-term sequelae, and at times even death.l-4 Thesereactions are not manifestations of direct drug toxicity but arerelated to the death of microfilariae. However, the severity of thereaction is not closely linked to the intensity of infection, nor willevery infected person have a reaction. In double-masked, controlledclinical trials, those having a Mazzotti reaction after DEC are clearlydifferent from those who receive placebo therapy, or, for thatmatter, ivermectin.5

I was surprised to read Dr Keystone and Dr Davies’ report(March 14, p 678) in which they recommend this test and describe asingle-blind method for its administration. In patients for whomthey consider the diagnosis of onchocerciasis, they should make adefinitive diagnosis by taking a set of skin snips. If the skin snips arenegative they can be repeated.6 The collection of skin snips is

straightforward. It needs only simple equipment and is defmitivewhen positive. The "trial by fire" approach that the Mazzotti testexemplifies seems hard to justify.

Department of Ophthalmology,University of Melbourne,Melbourne, Victoria, Australia 3002 HUGH R. TAYLOR

1. World Health Organisation Expert Committee on Onchocerdasis: third report.Geneva: WHO. Tech Rep Ser 752, 1987: 55-61.

2. Bird AC, El-Sheikh A, Anderson J, Fuglsang H. Changes in visual function and in theposterior segment of the eye during treatment of onchocerciasis with

diethylcarbamazine citrate. Br J Ophthalmol 1980; 64: 191-200.3. Greene BM, Taylor HR, Humphrey RL. Proteinuria associated with

diethylcarbamazine treatment of onchocerciasis. Lancet 1980; i: 254-55.4. Oomen AP. Fatalities after treatment of onchocerdasis with diethylcarbamazine.

Trans R Soc Trop Med Hyg 1969; 63: 548.5. Greene BM, Taylor HR, Cupp EW, et al. Comparison of ivermectin and

diethylcarbamazine in the treatment of onchocerciasis. N Engl J Med 1985; 313:133-38.

6. Aziz MA, Diallo S, Diop IM, Lariviere M, Porta M. Efficacy and tolerance ofivermectin in human onchocerciasis. Lancet 1982; ii: 171-73.

* ’This letter has been shown to Dr Keystone and Dr Davies, whosereply follows.-ED L.

SIR,-We agree with Dr Taylor that skin snips are the method ofchoice to diagnose onchocerciasis and thank him for emphasisingthe potential dangers of the Mazzotti test, especially in patients withmoderate to heavy infections. However, we do not agree that the testis without merit.The diagnosis of early onchocerciasis is difficult. Onchocerca

volvulus microfilariae can be absent from multiple skin snips in lightor early infections.’ Taylor and his colleagues have confirmed thisby showing that the sensitivity of skin snips declines strikingly whenmicrofilaria density drops below 1 per mg skin.2 Repeat of skin snipsmay not always be possible because of patient intolerance of theprocedure, and may be negative yet again.

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Taylor’s comments on the dangers of the Mazzotti reaction arenot substantiated by Francis and colleagues’3 report showing thatthe severity of the reaction correlates well with intensity ofinfection.3 Their results suggest to us that the risk of a moderate orsevere reaction would be very low in those who are skin-snipnegative.

Taylor points out that the World Health Organisation’scommittee on onchocerciasis condemns the use of the Mazzottitest.4 Yet, in that report the authors write that the Mazzotti test"should be used only in individuals whom a diagnosis ofonchocerciasis is being considered, but in whom parasites cannot bedetected in skin, eyes or blood. In such individuals, the Mazzottitest can be extremely helpful by inducing local pruritis and rash,clinically indicative of the parasite’s presence".’ We agree.

Tropical Disease Unit,Toronto Hospital,Toronto, Ontario M5G 2C4, Canada, UK;Division of Infectious Diseases,Hospital for Sick Children, Toronto

JAY S. KEYSTONEDELE DAVIES

1. Editorial. Diagnosis of early onchocerciasis. Lancet 1991; 337: 1449.2. Taylor HR, Munoz B, Keyvan-Larijani E, Green BM. Reliability of detection of

microfilarariae in skin snips in the diagnosis of onchocerciasis. Am J Trop Med Hyg1989; 41: 467-71.

3. Francis H, Awadzi K, Ottesen EA. The Mazzotti reaction following treatment ofonchocerciasis with diethyl-carbamazine: clinical severity as a function of infectionintensity. Am J Trop Med Hyg 1985; 34: 529-36.

4. WHO Expert Committee on Oncerciasis. Third report. Geneva: World HealthOrganisation. Tech Rep Ser 1987; 752: 60-61.

Factor VIII and factor IX inhibitors inhaemophiliacs

SIR,-Dr Ehrenforth and colleagues report (March 7, p 594) thatof 59 haemophiliac children born since 1970 attending their centre(and treated at least once),52% of those with severe and 5% of thosewith moderate haemophilia A had developed factor VIII inhibitors(antibodies), whereas none of those with haemophilia B had factorIX inhibitors.

By contrast, Dr Ciavarella and Dr Schiavoni (May 23, p 1301)report that in 21 previously untreated patients the incidence ofinhibitor development after treatment with FVIII concentrates ofintermediate or high purity was as low as about 5 %; Dr Verbruggenand colleagues (p 1301) point out that the Bethesda assayEhrenforth used tended to overestimate antibody activity, andtherefore the incidence of FVIII inhibitors. During the period1970-90 (ie, roughly that covered by Ehrenforth’s report), at thethree Swedish centres (which treat all haemophiliacs in the

country), 117 newborn babies with severe or moderate haemophiliawere registered, 81 with haemophilia A (77 severe, 24 moderate),and 16 with haemophilia B (12 severe, 4 moderate), which is muchthe same proportion of severity as Ehrenforth’s population.Inhibitors developed in 21% (16/77) of those with severe

haemophilia A (and at low titre in 1 child with mild disease, whichhave disappeared spontaneously), and in 33% (4/12) of those withsevere haemophilia B; most of these children were less than 2 yearsof age at inhibitor development, and all were less than 7.The inhibitors were of the high response type in 8/20 cases, and of

the low response type in the remaining 12 cases (titre < 10 Bethesdaunits, and no anamnestic response). Of the 6 patients withhigh-response factor VIII antibodies, 2 have been converted to lowresponders and are now on daily high-dose factor VIII treatmentaccording to the Bonn protocol, and 4 underwent the Malimotreatment protocol for the induction of immune tolerance,2.3 theantibodies disappearing in 2 of them. Of the 10 patients withlow-titre factor VIII antibodies, the inhibitors have disappeared in6 (spontaneously in 5, and after immune tolerance induction in 1),enabling them to be put on prophylactic treatment ; no specifictreatment has been prescribed in 2, and the remaining 2 have onlyjust been detected and treatment is not yet decided. The 2 patientswith high-titre factor IX antibodies underwent the Malmotreatment protocol, with disappearance of the antibodies in 1. The 2patients with low-titre factor IX antibodies are siblings, 1 of whomhas had anaphylactoid reactions to two factor IX preparations. 1

patient with high-titre factor IX antibodies has died.

Our findings with respect to age at development of inhibitors andthe amount and duration of replacement therapy are consistent withthose of Ehrenforth and colleagues, though the frequency ofinhibitor development among haemophilia A patients is lower inour series (21% vs 52%). Our patients are not a subgroup in aprospective study of inhibitor development, but represent the entirenational population of haemophiliacs bom in 1970-90, thusexcluding any selection bias that might have been a confoundingfactor. Moreover, in all severe and many moderate cases of

haemophilia, prophylactic treatment is started at an early age inSweden (about age 4 in the early 1970s, and at 1-1-1 now), andinhibitor testing and factor recovery analysis are done routinely 1-3times a year 4 The few patients still untreated do not decisively affectour statistics. The mutation has been characterised in about 60% ofall haemophilia B families in Sweden, and in all six families in whoma family member has developed inhibitors.s Fifteen families withsevere or moderate haemophilia B have a mutation causing grossphysical or functional loss of coding information; in six of thesefamilies, factor IX inhibitor has developed in a family member. Bycontrast, none of the severe or moderate haemophilia B patientswith mutations resulting in single aminoacid substitutions hasdeveloped antibodies. Although the effect of the type of factor VIIImutation on the development of factor VIII inhibitors has yet to beelucidated, data so far suggest that it may be a determinant of

antibody development. In all likelihood, where there is a highprevalence of certain mutations in a specific catchment area therewill be a correspondingly high prevalence of antibody development.This study was supported by grants from The Medical Faculty, University

of Lund, Sweden, and the Swedish Medical Research Council (grant 0087).

Departments of Coagulation Disorders atMalmo General Hospital,

S21401 Sweden;Karolinska Hospital, Stockholm;and Sahlgrenska Hospital, Gothenburg

ROLF LJUNGPIA PETRINIANN-CHRISTINE LINDGRENLILIAN TENGBORNINGA MARIE NILSSON

1. Brackmann HH. Induced immunotolerance in factor VIII inhibitor patients. ProgrClin Biol Res 1984; 150: 181-85.

2. Nilsson IM, Bemtorp E, Zettervall O. Induction of split tolerance and clinical cure inhigh-responding hemophiliacs with factor IX antibodies. Proc Natl Acad Sci USA1986; 83: 9169-73.

3. Nilsson IM, Bemtorp E, Zettervall O. Induction of immune tolerance in patients withhemophilia and antibodies to factor VIII by combined treatment with intravenousIgG, cyclophosphamide, and factor VIII. N Engl J Med 1988; 318: 947-50.

4. Nilsson IM, Bemtorp E, L&ouml;fqvist T, Pettersson H. Twenty-five years’ experience ofprophylactic treatment in severe haemophilia A and B. J Int Med (in press).

5. Green PM, Montandon AJ, Ljung R, et al. Haemophilia B mutations in a completeSwedish population sample: a test of a new strategy for the genetic counselling ofdiseases with high mutational heterogeneity. Br J Haematol 1991; 78: 390-97.

SIR,-Dr Ehrenforth and colleagues report a high incidence ofinhibitors to factor VIII (FVIII) in patients with severe andmoderate haemophilia. 15 of their 46 haemophilia A patientsdeveloped inhibitors. The incidence was 39 per 1000 patient-yearsand the aged-dependent cumulative risk was 33 % by age 6 years. AsEhrenforth et al state, these figures are higher than those reportedpreviously. We agree that earlier studies have probablyunderestimated the risk of acquiring FVIII inhibitors and haveproduced conflicting results because they used prevalence (ratherthan incidence), data have been retrospective, patients with mildhaemophilia have been included, and ages of the study groups havediffered.We have retrospectively reviewed data for 57 patients with

haemophilia A born in 1975 or after. All patients had beenperiodically controlled since their diagnosis and treated at least oncewith commercial FVIII concentrates. Almost all patients weretested for inhibitors at least once a year and whenever their presencewas suspected. In patients in whom an inhibitor was detected,antibody levels were measured on each subsequent visit. Sporadic,low titre, inhibitors ( < 5 Bethesda units) were found in 14 patients,but clinical evidence was noted only once or twice, despite repeatedexposure to FVIII. We did not include these cases in our incidencecalculations, since other similar cases could have been overlooked.

Persistent inhibitors developed in 10 of 52 patients with severe ormoderate haemophilia (FVIIIC < 0’05 IU/ml), resulting in anincidence of 20 per 1000 patient-years. In all cases, inhibitor