- Email: [email protected]
Simplified preparation of malarial blood samples for polymerase chain reaction
303
Locally, high concentrations of this and other cytokines, resulting a trigger for TNP being released when sequestered parasitised erythrocytes undergo schizogony, could be much more relevant than circulating concentrations of these mediators. Indeed, the idea of sequestered schizonts providing a local source of trigger for TNF may provide a much more reasonable link between sequestration and cerebral malaria than does the notion of physical obstruction to blood flow. Although not much is known about the from
effects of TNF on the brain, the evidence suggests that TNF will prove to have widespread and subtle influences. We were surprised that Phillips and Solomon made no mention of TNF with respect to malarial hypoglycaemia. Two groups have shown that serum TNF is the best correlate of malarial hypoglycaemia in African children (’ and Kwiatkowski D, et al, Nov 17, p 1201), and TNF injections cause severe hypoglycaemia in experimental malariaMoreover, TNF has been reported to inhibit gluconeogenesis,8 which is generally agreed to be the most likely mechanism of malarial hypoglycaemia. Phillips and Solomon mention no cytokine other than TNF. The functional significance of TNF cannot be appreciated in isolation:9 it is one of a family of cytokines that duplicate each others’ functions to a remarkable degree. For instance, interleukin-1 (IL-1) and lymphotoxin (LT) duplicate many functions of TNF, and IL-6 has been implicated in TNF-toxicity.11 IL-1 (Kwiatkowski D, et al), LT (Clark IA, et al, unpublished), and Il-62 concentrations are all increased in malaria, and correlate with disease severity. Thus, a trial of anti-TNF antibody, which neutralises only one of these overlapping cytokines, will probably not prove as definitive as Phillips and Solomon assume. A broader array of neutralising antibodies in our view holds more promise. Passive or active immunisation against the malarial components that trigger cytokine release is another useful approach. Division of Biochemistry and Molecular Australian National University, Canberra, ACT 2601, Australia
Biology, I. A. CLARK
Division of Cell Biology, John Curtin School of Medical Research, Australian National University
and TTA GGT TCT CTT AAT AAT GCT
K. A. ROCKETT W. B. COWDEN
1 Grau GE, Taylor TE, Molyneux ME, et al. Tumor necrosis factor and disease severity in children with falciparum malaria. N Engl JMed 1989; 320: 1586-91. 2 Kern P, Hemmer CJ, Van Damme J, et al Elevated tumour necrosis factor alpha and interleukin-6 serum levels as markers for complicated Plasmodium falciparum malaria. Am J Med 1989; 87: 139-43. 3 Jakubowski AA, Casper ES, Gabrilove JL, et al. Phase 1 trial of intramuscularly administered tumor necrosis factor in patients with advanced cancer J Clin Oncol
1989; 7: 298-303. 4 Hesse DG, Tracey KJ, Fong Y, et al. Cytokine appearance in human endotoxemia and primate bacteremia Surg Gynecol Obstet 1988; 166: 147-53. 5. Kwiatkowski D, Cannon JG, Manogue KR, et al. Tumor necrosis factor production in falciparum malaria and its association with schizont rupture. Clin Exp Immunol
1989; 77: 361-66. CAW, Taverne J, Playfair JHL. Malarial parasites induce TNF production by macrophages Immunology 1988; 64: 227-31 7. Clark IA, Cowden WB, Butcher GA, et al. Possible roles of tumor necrosis factor in the pathology of malaria. Am J Pathol 1987; 129: 192-99 8. McCallum RE, Hill MR, Stith RD. Inhibited steroid induction of PEPCK m hepatoma cells treated with hu rIL-1 and hu rTNF In: Powanda MC, Oppenheim JJ, Kluger MJ, et al, eds Monokines and other non-lymphocytic cytokines. New York. Alan R Liss, 1988. 267-72. 9. Clark IA, Chaudhri G, Cowden WB. Roles of tumour necrosis factor in the illness and pathology of malaria Trans R Soc Trop Med Hyg 1989; 83: 436-40. 10. Starnes HF, Pearce MK, Tewarni A, et al Anti-IL-6 monoclonal antibodies protect 6. Bate
against lethal Escherichia coli infection mice. J Immunol 1990, 145: 4185-91.
and
tumor necrosis
factor-&agr; challenge
on samples from control, patient with malaria, and cryopreserved parasitised blood. (1 ) negative control blood, (2) blood from patient on treatment, only one ring seen on thick film (about 0 2 paras!tes/)i! blood); (3) blood from same patient the next day when no parasites were detected, (4), (5), and (6) cryopreserved blood samples containing 16,4 (gametocytes), and 40 parasites per thick film high-power field (7040, 1760, and 17 600 parasites per III blood6)Primers used flanked a 499 nucleotide stretch of the pfmdr 1 gene 1,3 (coded by us as PCR4234) and are GTG GAA AAT CAA CTT TTA TGA
PCR
m
Simplified preparation of malarial blood samples for polymerase chain reaction SIR,-PCR (polymerase chain reaction)l can amplify single copy malaria genes’" but a major constraint on the routine application of PCR to human blood samples is the requirement for purification of parasite DNA. This problem has also been noted in human genetics, where no more than 2 ul whole human blood could be used in 100 III PCR reactions for detection of abundant HLA DQA gene template DNA/ Haemoglobin and other blood proteins inhibit PCR, and even partly purified DNA can contain trace amounts of protein denaturants and inhibitors.
In
the
[3H]-hypoxanthine incorporation procedure for the measuring drug sensitivity of malaria isolatess we use a cell harvester where the infected erythrocyte samples are lysed in situ and washed free of haemoglobin and other proteins on a glass fibre membrane (Flow Laboratories). Nucleic acids remain on the membrane and radioactivity is determined, without quenching by haemoglobin, in a liquid scintillation counter. We found the PCR reactions could be done with high sensitivity using such washed membranes as carriers of template DNA. We have refined and simplified the technique, in part to avoid the possibility of cross-contamination of samples which pass through plastic tubes to the harvester. 20 III fresh (plus EDTA) or cryopreserved blood from malaria patients was applied to the centre of 2cm glass fibre filter discs and allowed to dry. The blood spot was outlined in pencil and lysed on a sintered vacuum filter support with distilled water, followed by saline, and a final distilled water rinse. Very little haemoglobin remained on such filters. When the filter was dry, 1/8th segment of the blood spot circle was cut out with a clean scalpel, and the tip was removed and placed into 95 I.tI PCR buffer (10-5 mmol/1 "tris" HCI, pH83, 52-6mmol/1 KCI, 1-58 mmol/1 MgCl2, z011 % gelatin) containing 2-5 units Taq polymerase, PCR primers, and nucleotides. PCR conditions were: 94’5°C, 45 s (2-5 min, cycle 1); 47°C, 1 min; 72°C, 3 min (10 min, cycle 38) x 38 cycles. PCR products were detected by ultraviolet transillumination in 10 pl samples of the reaction mixture separated by electrophoresis in agarose, and stained with ethidium bromide (figure). Predicted PCR products were readily produced from blood with much fewer than 44 ring stages of Plasmodiumfalciparum per R1, the limit for the sensitivity of routine (100 fields examined) thick blood films,6 but no product was seen with uninfected controls. This simple modification, involving collection of blood spots on glass fibre discs and elution of interfering blood proteins, may greatly facilitate the wider epidemiological and clinical application of PCR to the sensitive detection and analysis of specified malaria genes in fresh and stored materials. It may even prove valuable in diagnostic detection of parasites morphologically affected by drug
304
prophylaxis.7 Our technique should also be applicable to other protozoan parasites found in blood and to human genetic studies.8 Supported by WHO/UNDP/World Bank special programme and by Overseas Development Administration. F.M.A.E.K. is supported by the British Council and the Sir Halley Stewart Trust and is in receipt of an ORS award. We thank John Frean for help in developing the malaria PCR protocol. PHLS Malaria Reference Laboratory and Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, London W1 E 7HT, UK
D. C. WARHURST F. M. AWAD EL KARIEM M. A. MILES
1. Anon. DNA diagnosis and the polymerase chain reaction. Lancet 1988; i: 1372-73. 2. Wilson CM, Serrano AE, Wasley A, et al. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 1989; 244: 1184-86. 3. Foote SJ, Thompson JK, Cowman AF, Kemp DJ. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of Plasmodium falciparum. Cell 1989; 57: 921-30. 4. Mercier B, Gaucher C, Feugeas O, Mazurier C. Direct PCR from whole blood, without DNA extraction. Nucl Acids Res 1990; 18: 5908. 5. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semi-automated microdilution technique. Antimicrob Ag Chemother 1979; 16: 710-18. 6. Dowling MAC, Shute GT. A comparative study of thick and thin blood films in the diagnosis of scanty malaria parasitaemia. Bull WHO 1966; 34: 249-67. 7. Warhurst DC. Diagnosis of malaria. Lancet 1990; 335: 472. 8. Nelson PV, Carey WF, Moms CP. Gene amplification directly from Guthrie blood spots. Lancet 1990; 336: 1451-52.
and adverse reactions to treatment in Onchocerca volvulus-infected British expatriates treated with ivermectin. We report here our experience in American expatriates. Since 1988, the Centers for Disease Control has monitored the release of ivermectin for the treatment of patients with onchocerciasis in the United States. 27 US citizens have been treated for 0 volvulus infections. 14 patients were male, and the median age was 33. Most were in occupations necessitating long stays in onchocerciasis-endemic areas of West and Central Africa; 9 were field scientists, 8 were Peace Corps volunteers, and 8 were missionaries or their family members. Lengths of stay in these areas ranged from 3 months to 8 years, with a median of 2 years. Onchocerciasis was confirmed in 25 by skin snip, nodulectomy, and/or slit-lamp examination of the ocular anterior chamber. The other two patients had compatible clinical symptoms and exposure history. All were treated as outpatients with a standard oral dose of ivermectin 150 Ixg/kg. 10 patients who had recurrent or persistent symptoms after 6 months or more were re-treated with the same dose; all responded favourably. Adverse reactions were noted at a frequency similar to that recorded in British expatriates except that localised oedema was less common in the Americans and none reported fever. All reactions were mild and transient: Reaction Pruritus Rash
Fatigue Localised oedema Headache Chills
BCG, tuberculosis, and leprosy SIR,-Abel and colleagues1 confirm the results of othersz in their case-control study that showed the protective efficacy of BCG against the non-lepromatous form of leprosy in southern Vietnam. These findings may reflect defective cellular immunity against mycobacteria in lepromatous patients. We have reanalysed our data on risk factors for leprosy3.4 to fmd if there was a negative association between tuberculosis and tuberculoid leprosy. 116 patients with lepromatous leprosy (LL) and 73 patients with tuberculoid leprosy (TT) were studied who were seen as outpatients at the Center for Hansen’s disease in Athens. Only cases with either of the two polar types of leprosy were included. 382 patients of low socioeconomic class, admitted to nearby hospitals for other reasons, acted as a control group. All subjects were of caucasian origin, a unique feature of this study. For patients with LL and TT, 10 (8-6%) and 0 subjects had a history of tuberculosis, respectively (=6-64, p=0-01). The frequency of tuberculosis among control patients was 10%. We conclude that there is a negative correlation between tuberculosis and the TT form of leprosy, which is not found in the LL form. These data support the evidence emerging from efficacy studies of BCG vaccination for leprosy prevention, which suggest that cellular immune responses, probably linked to major histocompatibility complex alleles, are important modifiers of the clinical expression of leprosy. 3,4 University of Athens Medical School, 115 27 Goudi, Athens, Greece
EVANGELIA KAKLAMANI YVONNI KOUMANDAKI KLEA KATSOUYANNI
Department of Epidemiology, Harvard School of Public Health, USA
DIMITRIOS TRICHOPOULOS
1. Abel L, Cua VV, Oberti J, et al. Leprosy and BCG in southern Vietnam. Lancet 1990; i: 1536. 2. Fine PEM. BCG vaccination against tuberculosis and leprosy Br Med Bull 1988; 44: 691-703. 3. Papaioannou DJ, Kaklamani EP, Parissis NG, Koumantaki IG, Karalis DT, Trichopoulos DR. Hepatitis B virus (HBV) serum markers in Greek leprosy patients. Int J Lepr 1986; 54: 245-51. 4. Koumantaki IG, Katsouyanni KM, Kaklamani EP, et al. An investigation of family size and birth order as risk factors in leprosy. Int J Lepr 1987; 55: 463-67.
Expatriates treated with ivermectin we are rapidly gaining experience in the of onchocerciasis in residents of Africa and Latin America, little is known about the effects of this disease or its treatment on visitors to onchocerciasis-endemic regions.1,2 Dr Davidson and colleagues (Oct 20, p 1005) describe initial symptoms
SIR,-Although
treatment
No 6 3 3 3 2 2
Reaction Hives Nausea Dizzmess
Myalgia Groin pain
Limbitis/lens opacities
No 1 1 1 1 1 1
This
experience shows that individuals in certain occupational groups who spend more than 3 months in onchocerciasis-endemic areas are at risk for the disease and should be considered for targeted prevention. Adverse reactions to ivermectin are common in 0 volvulus-infected expatriates but in this series the reactions were not severe and are generally well-tolerated by outpatients. Parasitic Diseases Branch, Division of Parasitic Diseases, and Clinical Medicine Branch, Division of Immunologic, Oncologic, and Hematologic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA
RALPH T. BRYAN SUSAN L. STOKES HARRISON C. SPENCER
Pacque M, Munoz B, Greene BM, et al. Safety and compliance with communitybased ivermectin therapy. Lancet 1990; 335: 1377-80 2. Taylor HR, Greene BM. The status of ivermectin in the treatment of human onchocerciasis. Am J Trop Med Hyg 1989; 41: 460-66. 1.
Sudden infant death in Thailand and Alaska S;R,—Dr Wilson (Nov 10, p 1199) questions why sudden infant death syndrome (SIDS) is so uncommon or virtually unknown in south China whereas there is a high frequency in native Indians of Alberta, Canada. The same question could be raised about North Americans of Chinese ancestry, who have a low frequency of SIDS, and Alaska native infants, who have one of the highest rates. In 1990 we visited forensic facilities in Thailand and gathered information about SIDS. In a heavily populated north-west province, a medical examiner who had trained in forensic pathology in the USA could not recall an infant death diagnosed as SIDS during the past several years. In a north-eastern province, a forensic pathologist participating in a government collaborative study on sudden unexplained death in adults (SUDS) could recall only one medical examiner’s case being diagnosed as SIDS. In other provinces the general consensus by medical examiners is that Thai infants are at near zero risk of SIDS. Despite the difficulties of basic sanitation in this developing country, infants may be at less risk for injury in Thailand than those in Western countries. When a Thai mother is occupied with housework, grandparents may attend the infant and provide extra care. In addition, it is culturally unacceptable for Thai (or Chinese) mothers to consume alcohol while nursing or bedsharing with their infants. Fetal alcohol syndrome is not a health problem in Thai or Chinese infants, unlike native North American infants.
Locally, high concentrations of this and other cytokines, resulting a trigger for TNP being released when sequestered parasitised erythrocytes undergo schizogony, could be much more relevant than circulating concentrations of these mediators. Indeed, the idea of sequestered schizonts providing a local source of trigger for TNF may provide a much more reasonable link between sequestration and cerebral malaria than does the notion of physical obstruction to blood flow. Although not much is known about the from
effects of TNF on the brain, the evidence suggests that TNF will prove to have widespread and subtle influences. We were surprised that Phillips and Solomon made no mention of TNF with respect to malarial hypoglycaemia. Two groups have shown that serum TNF is the best correlate of malarial hypoglycaemia in African children (’ and Kwiatkowski D, et al, Nov 17, p 1201), and TNF injections cause severe hypoglycaemia in experimental malariaMoreover, TNF has been reported to inhibit gluconeogenesis,8 which is generally agreed to be the most likely mechanism of malarial hypoglycaemia. Phillips and Solomon mention no cytokine other than TNF. The functional significance of TNF cannot be appreciated in isolation:9 it is one of a family of cytokines that duplicate each others’ functions to a remarkable degree. For instance, interleukin-1 (IL-1) and lymphotoxin (LT) duplicate many functions of TNF, and IL-6 has been implicated in TNF-toxicity.11 IL-1 (Kwiatkowski D, et al), LT (Clark IA, et al, unpublished), and Il-62 concentrations are all increased in malaria, and correlate with disease severity. Thus, a trial of anti-TNF antibody, which neutralises only one of these overlapping cytokines, will probably not prove as definitive as Phillips and Solomon assume. A broader array of neutralising antibodies in our view holds more promise. Passive or active immunisation against the malarial components that trigger cytokine release is another useful approach. Division of Biochemistry and Molecular Australian National University, Canberra, ACT 2601, Australia
Biology, I. A. CLARK
Division of Cell Biology, John Curtin School of Medical Research, Australian National University
and TTA GGT TCT CTT AAT AAT GCT
K. A. ROCKETT W. B. COWDEN
1 Grau GE, Taylor TE, Molyneux ME, et al. Tumor necrosis factor and disease severity in children with falciparum malaria. N Engl JMed 1989; 320: 1586-91. 2 Kern P, Hemmer CJ, Van Damme J, et al Elevated tumour necrosis factor alpha and interleukin-6 serum levels as markers for complicated Plasmodium falciparum malaria. Am J Med 1989; 87: 139-43. 3 Jakubowski AA, Casper ES, Gabrilove JL, et al. Phase 1 trial of intramuscularly administered tumor necrosis factor in patients with advanced cancer J Clin Oncol
1989; 7: 298-303. 4 Hesse DG, Tracey KJ, Fong Y, et al. Cytokine appearance in human endotoxemia and primate bacteremia Surg Gynecol Obstet 1988; 166: 147-53. 5. Kwiatkowski D, Cannon JG, Manogue KR, et al. Tumor necrosis factor production in falciparum malaria and its association with schizont rupture. Clin Exp Immunol
1989; 77: 361-66. CAW, Taverne J, Playfair JHL. Malarial parasites induce TNF production by macrophages Immunology 1988; 64: 227-31 7. Clark IA, Cowden WB, Butcher GA, et al. Possible roles of tumor necrosis factor in the pathology of malaria. Am J Pathol 1987; 129: 192-99 8. McCallum RE, Hill MR, Stith RD. Inhibited steroid induction of PEPCK m hepatoma cells treated with hu rIL-1 and hu rTNF In: Powanda MC, Oppenheim JJ, Kluger MJ, et al, eds Monokines and other non-lymphocytic cytokines. New York. Alan R Liss, 1988. 267-72. 9. Clark IA, Chaudhri G, Cowden WB. Roles of tumour necrosis factor in the illness and pathology of malaria Trans R Soc Trop Med Hyg 1989; 83: 436-40. 10. Starnes HF, Pearce MK, Tewarni A, et al Anti-IL-6 monoclonal antibodies protect 6. Bate
against lethal Escherichia coli infection mice. J Immunol 1990, 145: 4185-91.
and
tumor necrosis
factor-&agr; challenge
on samples from control, patient with malaria, and cryopreserved parasitised blood. (1 ) negative control blood, (2) blood from patient on treatment, only one ring seen on thick film (about 0 2 paras!tes/)i! blood); (3) blood from same patient the next day when no parasites were detected, (4), (5), and (6) cryopreserved blood samples containing 16,4 (gametocytes), and 40 parasites per thick film high-power field (7040, 1760, and 17 600 parasites per III blood6)Primers used flanked a 499 nucleotide stretch of the pfmdr 1 gene 1,3 (coded by us as PCR4234) and are GTG GAA AAT CAA CTT TTA TGA
PCR
m
Simplified preparation of malarial blood samples for polymerase chain reaction SIR,-PCR (polymerase chain reaction)l can amplify single copy malaria genes’" but a major constraint on the routine application of PCR to human blood samples is the requirement for purification of parasite DNA. This problem has also been noted in human genetics, where no more than 2 ul whole human blood could be used in 100 III PCR reactions for detection of abundant HLA DQA gene template DNA/ Haemoglobin and other blood proteins inhibit PCR, and even partly purified DNA can contain trace amounts of protein denaturants and inhibitors.
In
the
[3H]-hypoxanthine incorporation procedure for the measuring drug sensitivity of malaria isolatess we use a cell harvester where the infected erythrocyte samples are lysed in situ and washed free of haemoglobin and other proteins on a glass fibre membrane (Flow Laboratories). Nucleic acids remain on the membrane and radioactivity is determined, without quenching by haemoglobin, in a liquid scintillation counter. We found the PCR reactions could be done with high sensitivity using such washed membranes as carriers of template DNA. We have refined and simplified the technique, in part to avoid the possibility of cross-contamination of samples which pass through plastic tubes to the harvester. 20 III fresh (plus EDTA) or cryopreserved blood from malaria patients was applied to the centre of 2cm glass fibre filter discs and allowed to dry. The blood spot was outlined in pencil and lysed on a sintered vacuum filter support with distilled water, followed by saline, and a final distilled water rinse. Very little haemoglobin remained on such filters. When the filter was dry, 1/8th segment of the blood spot circle was cut out with a clean scalpel, and the tip was removed and placed into 95 I.tI PCR buffer (10-5 mmol/1 "tris" HCI, pH83, 52-6mmol/1 KCI, 1-58 mmol/1 MgCl2, z011 % gelatin) containing 2-5 units Taq polymerase, PCR primers, and nucleotides. PCR conditions were: 94’5°C, 45 s (2-5 min, cycle 1); 47°C, 1 min; 72°C, 3 min (10 min, cycle 38) x 38 cycles. PCR products were detected by ultraviolet transillumination in 10 pl samples of the reaction mixture separated by electrophoresis in agarose, and stained with ethidium bromide (figure). Predicted PCR products were readily produced from blood with much fewer than 44 ring stages of Plasmodiumfalciparum per R1, the limit for the sensitivity of routine (100 fields examined) thick blood films,6 but no product was seen with uninfected controls. This simple modification, involving collection of blood spots on glass fibre discs and elution of interfering blood proteins, may greatly facilitate the wider epidemiological and clinical application of PCR to the sensitive detection and analysis of specified malaria genes in fresh and stored materials. It may even prove valuable in diagnostic detection of parasites morphologically affected by drug
304
prophylaxis.7 Our technique should also be applicable to other protozoan parasites found in blood and to human genetic studies.8 Supported by WHO/UNDP/World Bank special programme and by Overseas Development Administration. F.M.A.E.K. is supported by the British Council and the Sir Halley Stewart Trust and is in receipt of an ORS award. We thank John Frean for help in developing the malaria PCR protocol. PHLS Malaria Reference Laboratory and Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, London W1 E 7HT, UK
D. C. WARHURST F. M. AWAD EL KARIEM M. A. MILES
1. Anon. DNA diagnosis and the polymerase chain reaction. Lancet 1988; i: 1372-73. 2. Wilson CM, Serrano AE, Wasley A, et al. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 1989; 244: 1184-86. 3. Foote SJ, Thompson JK, Cowman AF, Kemp DJ. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of Plasmodium falciparum. Cell 1989; 57: 921-30. 4. Mercier B, Gaucher C, Feugeas O, Mazurier C. Direct PCR from whole blood, without DNA extraction. Nucl Acids Res 1990; 18: 5908. 5. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semi-automated microdilution technique. Antimicrob Ag Chemother 1979; 16: 710-18. 6. Dowling MAC, Shute GT. A comparative study of thick and thin blood films in the diagnosis of scanty malaria parasitaemia. Bull WHO 1966; 34: 249-67. 7. Warhurst DC. Diagnosis of malaria. Lancet 1990; 335: 472. 8. Nelson PV, Carey WF, Moms CP. Gene amplification directly from Guthrie blood spots. Lancet 1990; 336: 1451-52.
and adverse reactions to treatment in Onchocerca volvulus-infected British expatriates treated with ivermectin. We report here our experience in American expatriates. Since 1988, the Centers for Disease Control has monitored the release of ivermectin for the treatment of patients with onchocerciasis in the United States. 27 US citizens have been treated for 0 volvulus infections. 14 patients were male, and the median age was 33. Most were in occupations necessitating long stays in onchocerciasis-endemic areas of West and Central Africa; 9 were field scientists, 8 were Peace Corps volunteers, and 8 were missionaries or their family members. Lengths of stay in these areas ranged from 3 months to 8 years, with a median of 2 years. Onchocerciasis was confirmed in 25 by skin snip, nodulectomy, and/or slit-lamp examination of the ocular anterior chamber. The other two patients had compatible clinical symptoms and exposure history. All were treated as outpatients with a standard oral dose of ivermectin 150 Ixg/kg. 10 patients who had recurrent or persistent symptoms after 6 months or more were re-treated with the same dose; all responded favourably. Adverse reactions were noted at a frequency similar to that recorded in British expatriates except that localised oedema was less common in the Americans and none reported fever. All reactions were mild and transient: Reaction Pruritus Rash
Fatigue Localised oedema Headache Chills
BCG, tuberculosis, and leprosy SIR,-Abel and colleagues1 confirm the results of othersz in their case-control study that showed the protective efficacy of BCG against the non-lepromatous form of leprosy in southern Vietnam. These findings may reflect defective cellular immunity against mycobacteria in lepromatous patients. We have reanalysed our data on risk factors for leprosy3.4 to fmd if there was a negative association between tuberculosis and tuberculoid leprosy. 116 patients with lepromatous leprosy (LL) and 73 patients with tuberculoid leprosy (TT) were studied who were seen as outpatients at the Center for Hansen’s disease in Athens. Only cases with either of the two polar types of leprosy were included. 382 patients of low socioeconomic class, admitted to nearby hospitals for other reasons, acted as a control group. All subjects were of caucasian origin, a unique feature of this study. For patients with LL and TT, 10 (8-6%) and 0 subjects had a history of tuberculosis, respectively (=6-64, p=0-01). The frequency of tuberculosis among control patients was 10%. We conclude that there is a negative correlation between tuberculosis and the TT form of leprosy, which is not found in the LL form. These data support the evidence emerging from efficacy studies of BCG vaccination for leprosy prevention, which suggest that cellular immune responses, probably linked to major histocompatibility complex alleles, are important modifiers of the clinical expression of leprosy. 3,4 University of Athens Medical School, 115 27 Goudi, Athens, Greece
EVANGELIA KAKLAMANI YVONNI KOUMANDAKI KLEA KATSOUYANNI
Department of Epidemiology, Harvard School of Public Health, USA
DIMITRIOS TRICHOPOULOS
1. Abel L, Cua VV, Oberti J, et al. Leprosy and BCG in southern Vietnam. Lancet 1990; i: 1536. 2. Fine PEM. BCG vaccination against tuberculosis and leprosy Br Med Bull 1988; 44: 691-703. 3. Papaioannou DJ, Kaklamani EP, Parissis NG, Koumantaki IG, Karalis DT, Trichopoulos DR. Hepatitis B virus (HBV) serum markers in Greek leprosy patients. Int J Lepr 1986; 54: 245-51. 4. Koumantaki IG, Katsouyanni KM, Kaklamani EP, et al. An investigation of family size and birth order as risk factors in leprosy. Int J Lepr 1987; 55: 463-67.
Expatriates treated with ivermectin we are rapidly gaining experience in the of onchocerciasis in residents of Africa and Latin America, little is known about the effects of this disease or its treatment on visitors to onchocerciasis-endemic regions.1,2 Dr Davidson and colleagues (Oct 20, p 1005) describe initial symptoms
SIR,-Although
treatment
No 6 3 3 3 2 2
Reaction Hives Nausea Dizzmess
Myalgia Groin pain
Limbitis/lens opacities
No 1 1 1 1 1 1
This
experience shows that individuals in certain occupational groups who spend more than 3 months in onchocerciasis-endemic areas are at risk for the disease and should be considered for targeted prevention. Adverse reactions to ivermectin are common in 0 volvulus-infected expatriates but in this series the reactions were not severe and are generally well-tolerated by outpatients. Parasitic Diseases Branch, Division of Parasitic Diseases, and Clinical Medicine Branch, Division of Immunologic, Oncologic, and Hematologic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA
RALPH T. BRYAN SUSAN L. STOKES HARRISON C. SPENCER
Pacque M, Munoz B, Greene BM, et al. Safety and compliance with communitybased ivermectin therapy. Lancet 1990; 335: 1377-80 2. Taylor HR, Greene BM. The status of ivermectin in the treatment of human onchocerciasis. Am J Trop Med Hyg 1989; 41: 460-66. 1.
Sudden infant death in Thailand and Alaska S;R,—Dr Wilson (Nov 10, p 1199) questions why sudden infant death syndrome (SIDS) is so uncommon or virtually unknown in south China whereas there is a high frequency in native Indians of Alberta, Canada. The same question could be raised about North Americans of Chinese ancestry, who have a low frequency of SIDS, and Alaska native infants, who have one of the highest rates. In 1990 we visited forensic facilities in Thailand and gathered information about SIDS. In a heavily populated north-west province, a medical examiner who had trained in forensic pathology in the USA could not recall an infant death diagnosed as SIDS during the past several years. In a north-eastern province, a forensic pathologist participating in a government collaborative study on sudden unexplained death in adults (SUDS) could recall only one medical examiner’s case being diagnosed as SIDS. In other provinces the general consensus by medical examiners is that Thai infants are at near zero risk of SIDS. Despite the difficulties of basic sanitation in this developing country, infants may be at less risk for injury in Thailand than those in Western countries. When a Thai mother is occupied with housework, grandparents may attend the infant and provide extra care. In addition, it is culturally unacceptable for Thai (or Chinese) mothers to consume alcohol while nursing or bedsharing with their infants. Fetal alcohol syndrome is not a health problem in Thai or Chinese infants, unlike native North American infants.