Detection of Y chromosome sequences in Turner's syndrome by Southern blot analysis of amplified DNA

12 13

14

Bagger JP. Coronary sinus blood flow determination: influence of catheter position and respiration. Cardiovasc Res 1985; 19: 27-31. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237: E214-23. Hother-Nielsen O, Schmitz O, Bak J, Beck-Nielsen H. Enhanced

syndrome and normal coronary arteriograms. Am J Cardiol 1973; 32: 375-76. 21 Reaven GM. Role of insulin resistance in human disease. Diabetes

hepatic insulin sensitivity, but peripheral insulin resistance in patients with type 1 (insulin dependent) diabetes. Diabetologia 1987; 30:

24A. 23 Stalder M, Pometta D, Suenram A. Relationship between plasma insulin levels and high density lipoprotein cholesterol levels in healthy man. Diabetologica 1982; 21: 544-48. 24 Fisher BM, Gillen G, Lindop GBM, Dargie HJ, Frier BM. Cardiac function and coronary arteriography in asymptomatic type 1 (insulin dependent) diabetic patients: evidence for a specific heart disease. Diabetologica 1986; 29: 706-12.

834-40. 15 Steele R. Influence of glucose 16

loading and of injected insulin on hepatic glucose output (abstract). Ann N Y Acad Sci 1959; 82: 420. Møller N, Jørgensen JOL, Alberti KGMM, Flyvbjerg A, Schmitz O. Short-term substrate metabolism in normal man. J Clin Endocrinol

Metab 1990; 70: 1179-86. 17 Koivist V, DeFronzo RA. Physical training and insulin sensitivity. Diabetes Metab Rev 1986; 1: 445-81. 18 Ferrannini E, Buzzigoli G, Bonadonna R, et al. Insulin resistance in essential hypertension. N Engl J Med 1987; 317: 350-57. 19 DeFronzo RA. The triumvirate: &bgr;-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 1988; 37: 667-87. 20 Kemp HG. Left ventricular function in patients with the anginal

1988; 37: 1595-607. P, Baily S, Saad R, DeFronzo RA. Insulin resistance and coronary artery disease: the missing link (abstract). Diabetes 1992; 41:

22 Bressler

25

26

Lillioja S, Yuong AA, Culter CL, et al. Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J Clin Invest 1987; 80: 415-23. Baron AD, Laakso M, Brechtel G, Holt B, Watt C, Edelman SY. Reduced postprandial skeletal muscle blood flow contributes to glucose intolerance in human obesity. J Clin Endocrinol Metab 1990; 70: 1525-33.

Detection of Y chromosome sequences in Turner’s syndrome by Southern blot analysis of amplified DNA

Introduction

Summary patients with Turner’s syndrome are monosomy 45,X on karyotyping and there are grounds for supposing that cryptic mosaicism for at least part of the Y chromosome may be present in some patients. If so this would be clinically important because of the risk to patients of gonadal neoplasms and virilisation.

Only

about half of all

We have used

very sensitive method to detect Y chromosomal segments in eighteen patients with Turner’s syndrome, none of whom had evidence of Y chromosomal material by cytogenetic analysis. In DNA from peripheral blood lymphocytes and/or fibroblasts we looked for specific nucleotide sequences from the sex-determining region of the Y chromosome (SRY gene) and repetitive sequences located at the centromeric region (DYZ3). By polymerase chain amplification (PCR) one patient had a definite positive signal and two patients had faintly positive signals for the SRY gene. Southern blot analysis of PCR material with a SRY-specific probe confirmed that these patients were positive for SRY and revealed another three. No patient was positive for DYZ3, suggesting that only a small portion of Y was present. These results suggest that "pure" 45,X monosomy is less frequent than previously supposed. Long-term follow-up of patients with Y sequences is needed to determine their risk for subsequent gonadal neoplasms and virilisation. a

X chromosome was identified as the chromosomal basis of Turner’s syndrome in 1959 but mosaics such as 45,X/46,XX or structural abnormalities of one X chromosome have been recognised. Indeed only 40-60% of karyotypes of phenotypic Turner’s syndrome patients are 45,X1°Z and Y chromosomal material has been detected in individuals with the Turner clinical phenotype and a mosaic karyotype.3 When in association with a 45,X/46,XY karyotype such features are frequently defined as mixed gonadal dysgenesis; however, molecular analysis with marker chromosomes (45,X/46,X, + mar), has revealed Y material in some cases.4,5 Further, when two cases with ring X chromosome were reinvestigated by Southern analysis of genomic DNA and in-situ hybridisation, the ring chromosome was found to be derived from the Y chromosome.** Because of the increased incidence of gonadoblastoma in patients with intra-abdominal testes, such as those with androgen insensitivity syndromes and mixed gonadal dysgenesis, clinicians need to know if Y chromosomal material and dysgenetic testicular tissue are present. Phenotypic females with Turner’s syndrome and Y-specific DNA have been found to have microscopic foci of gonadoblastoma7,8 and virilisation may occur in such individuals.9 A patient with monosomy X by cytogenetic analysis and mild hyperandrogenism has been found to have Y chromosomal material, detected by polymerase chain reaction amplification (PCR) for the SRY gene; no PCR product was detected in 36 other patients with

Monosomy of the

45,X.-" Divisions of Immunogenetics (M Kocova MD PhD, S F Siegel MD, Prof M Trucco MD), Endocrinology (S F Siegel, P A Lee MD PhD), and Medical Genetics (S L Wenger PhD), Children’s Hospital of Pittsburgh, Rangos Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Correspondence to: Dr Massimo Trucco, Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA15213-2583, USA 140

The apparent discrepancy between the 1-2% incidence of Turner’s syndrome in clinically recognised pregnancies and the high prenatal mortality of non-mosaic 45,X fetuses has led to the hypothesis that liveborn 45,X individuals may be cryptic mosaics .12,13 However, detection of mosaicism is influenced by the number of tissues and cells examined. For example, when both peripheral lymphocyte and fibroblast cultures were evaluated only 20 7% of karyotypes of 87 liveborn Turner’s patients were found to be 45,X.14

To look for undetected mosaicism for chromosomal material in another clinical setting (liver transplantation), we developed a PCR protocol followed by Southern blot analysis which increases the sensitivity of detection of Y-positive cells to 1 in 100 000.15 Here we report application of this technique to patients with Turner’s syndrome.

Materials and methods Blood or skin biopsy samples were obtained from eighteen patients with Turner’s syndrome being followed up at the Children’s Hospital of Pittsburgh. Clinical features were noted. The precise cytogenetic analyses from the diagnostic karyotypes were reviewed. This protocol was approved by the hospital’s human rights committee with informed consent from parents and the patient.

Cytogenetic analysis Karyotypes from peripheral blood lymphocytes were analysed by standard techniques. For skin biopsy samples, fibroblasts were cultured and cells were harvested during the log phase growth, 2 days after 1 in 2 subculture with standard cytogenetic techniques. The preparations were G-banded with trypsin G and at least 20 cells per patient were scored for chromosomal abnormalities.

*Short stature= height less than 5% percentile for age. t- =no signal, ± =faint signal, + = def!nite s!gnaL tKarotype in two tissues. §=inv(9)(p11q13) also present. tried at 36 h due to hypoplastic left heart syndrome, karyotype on lung tissue. NA= not available.

Summary of karyotype, clinical features, and molecular genetics

Table :

PCR A portion of the SRY gene located on the distal part of the short arm and a repetitive DNA sequence (DYZ3) at the centromeric region of the Y chromosome were selected. Positive signals for both implied the presence of a larger portion of Y chromosome; a positive signal for SRY alone suggested a smaller portion of the Y chromosome. For the SRY gene, the 270 base-pair sequence representing the DNA binding domain of the SRY gene was amplified with primers SRY-IF and SRY-2R (figure).16 Reaction volume was 100 L and contained 1 ug DNA, 50 mmol/L KCI, 1-5 mmol/L MgCl,, 10 mmol/L "Tris"-HCl at pH 8-4/20 ug gelatin, 0-2 mmol/L each of four different dNTPs, 5 units of Taq polymerase (Perkin Elmer), and 2 umol/L of each primer. Amplification conditions were: denaturing at 95°C for 5 min, 30 cycles of 20 s denaturation at 95°C, 20 s annealing at 60°C, and 20 s extension at 72’C. After the final extension at 72°C for 5 min, 10% of amplified material was loaded on 2%agarose gel which was checked for PCR

products. For DYZ3, primers DYZ3-A and DYZ3-B were used (figure).17 After an initial denaturation at 95°C for 5 min, amplification conditions were 30 s denaturation at 95°C, 30 s annealing at 55°C, and extension at 72°C for 1 min for 30 cycles. Again, the presence of

five such controls were included in each experiment; and patients with karyotypes including cell lines with Y or known Y marker chromosomes were excluded. The dilution experiment was done with healthy male DNA diluted into healthy nulliparous female DNA with ratios of 1 to 10 to 1 to 100 000 before PCR. The signal in the SRY-positive cases was compared with that from different dilutions.

Single-strand conformational polymorphism The primers for the SRY gene were used (0-3 mol) in a reaction volume of 12-5 uL containing 0-12 ug DNA, PCR buffer (Perkin Elmer), 1 25 L lOxdNTP, 007 gL32P-dCTP (370 MBq/mL, Amersham), and 3 units of Taq polymerase. PCR conditions were 94°C for 1 min and 64°C for 4 min for 30 cycles followed by a final extension phase at 72°C for 7 min. 2 uL of PCR product was mixed with 7 J-lL STOP solution (USB Biochem). Samples were heated at 94°C for 3 min and immediately placed on ice. Samples (2-5 uL) were loaded onto Hydro-Link-MDE gel (AT Biochem). After electrophoresis at 8 W for 14 h at room temperature, the gel was transferred to Whatman 3M paper, dried, and autoradiographed at 80°C. -

PCR product was assessed.

Results

Southern blot

DNA from

30 uL samples of amplified material were loaded on 1 % agarose gel. Electrophoresis-separated PCR product from primers was transferred to Hybond N + membrane. 15 The transfer took between 4 h and overnight. The filter was then rinsed in 1 x SSPE for 15 min and air dried. Membranes were prehybrid sodium dodecyl sulphate for 1 h in 50% formamide, 5 x SSPE, 1 x Denhardt’s, 0-1% sodium dodecyl sulphate (SDS), and 0-1 mg/mL herring sperm DNA. The probe to detect the SRY sequence was obtained by amplification of healthy male DNA with SRY-IF and SRY-2R primers and was purified with Gene-Clean (Bio 101). This probe was labelled with 3zP-dCTP. About 200 000 counts per mL hybridisation solution was added to the filter. After the overnight hybridisation at 42°C, the filter was washed in 1 x SSPE for 15 min at room temperature. The stringent wash in 1 x SSPE and 0-1% SDS was at 65°C for 15 min. The filter was exposed to Kodak X-XAR film for 15-60 min. The procedure with DYZ3 probe was the same except that the probe was obtained by amplification from a healthy male. Since false positive bands can be obtained even by the slightest contamination with male DNA all technical aspects of this study (including obtaining the blood samples) were done by women; the negative controls were healthy nulliparous females, and three to

eighteen patients with clinical features of Turner’s syndrome was analysed (table). Patients with Y chromosomal material detected by cytogenetic analysis were excluded. Karyotype analysis was done on fibroblasts and/or blood. Ten patients were 45,X; seven patients were mosaics for 45,X with a structural or numerical anomaly of the X chromosome; one was 45,X/46,XX. One patient had clitoromegaly. patients and the 45,X/46,XX patient yielded positive signal on agarose gel after PCR for SRY. The bands were less intensive than those in healthy males. By Southern blot of the PCR products, positive signals were detected in six individuals (including the three PCR positives). Of these six, four were 45,X, one was 45,X/46,X X, and one was 45,X/47,XXX. One of these patients with X Two of the 45,X a

monosomy had normal stature while another had clito-

romegaly. The height of the patient with the 45,X/46,XX mosaic karyotype was at the fifth percentile for age. The dilution experiment placed the signal in the range of 1 in 100 to 1 in 10 000 male-to-female DNA dilution (figure). With this Southern blot technique, one cell containing SRY could be detected at a ratio of 1 in 100 000 141

Figure:

PCR

amplification with SRY-gene specific primers followed by Southern blot

First lane contains amplified male DNA (3% of PCR reaction). Lanes 1-4 are PCR products of patients corresponding to table. Next lane is amplified female DNA. Lane C contains no DNA (negtive control). Remaining six lanes are male DNA serially diluted into female DNA. Primers used were: SRY-1F=5’-CAGTGTGAAACGGGAGAAAACAGT-3’ SRY-2R=5’-CTTCCGACGAGGTCGATACTTATA-3’ DYZ3-A=5’-TGAAAACTACACAGAAGCTG-3’ DYZ3-B 5’-ACACATCACAAAGAACTATG-3’ =

cells. All patients were negative for DYZ3 by amplification and Southern analysis. Single-strand conformational polymorphism on samples from the six individuals who were positive for SRY on Southern analysis of the PCR products was not different from that in healthy males (data not shown).

Discussion

Using Southern blot analysis of genomic DNA, Muller et al detected Y chromosomal material in a 45,X patient.18 By PCR we have detected the SRY gene in three of eighteen patients. Three more were identified by Southern blot analysis of the amplification product, a technique which increases our ability to distinguish "micromosaicism". Thus, the SRY gene was recognised in six patients with phenotypic Turner’s syndrome but no Y-positive cell line on conventional cytogenetic analysis. Previous analyses have often been limited by the use of a single tissue, by the small number of cells examined, and the detection methods, such as quinacrine staining. Our approach circumvents many of these obstacles. We have thus been able to confirm

that individuals with either 45,X and/or mosaic karyotypes may have some Y chromosome material: we found it in 33 % of our patients and in 40% of those who were 45,X. Our inability to detect DYZ3 implies that only a small portion of the Y chromosome was present in individuals with positive signal for SRY. The need for a sensitive protocol suggests that the ratio of cells with Y sequence is low compared with that in the other cell lines in these patients. The presence of normal female external genitalia in five patients positive for SRY corroborates our impression that the proportion of cells with Y sequence DNA is small. Our findings with this molecular technique support the hypothesis that 45,X monosomy in all tissues may be uncommon in liveborn Turner’s syndrome patients.14 Patients with androgen insensitivity syndromes and mixed gonadal dysgenesis are at increased risk of gonadoblastoma and patients with partial androgen insensitivity syndromes and mixed gonadal dysgenesis assigned to a female sex of rearing may experience excessive virilisation.9 Gonadal tumours have been reported in 45,X patients.19-22 Since gonadectomy is not routine clinical management for patients with Turner’s syndrome, karyotype findings do not exclude micromosaicism in other tissues.23 Long-term follow-up of patients with cells lines containing Y sequence DNA is needed to determine their propensity to develop gonadal tumours. Evaluation of such patients may also provide insight into which portions of the Y chromosome are involved in this neoplastic transformation. Sequences in 142

the fluorescent portion of Yq have been implicated24,25 but reports of gonadoblastoma and dysgerminoma in 45,X and 45,Xj45,X+mar individuals lacking the Yq sequence challenge this supposition.8,19-23 Our results call into question the classification of Turner’s syndrome. Typically, patients with mixed gonadal dysgenesis have genital ambiguity, phenotypic features of Turner’s syndrome, and 45,X/46,XY karoytypes. However, the range extends from phenotypic females with ovarian failure to phenotypic males with dysgeneic testes.26 These patients may be on a spectrum in which the clinical phenotype depends on the specific content and percentage of cells with the SRY gene known to be involved in male sexual differentiation. Importantly, determination of Y sequence positivity may impact on the clinical management of individuals with the phenotypic signs of Turner’s or near

syndrome. References 1

2 3

4

5

6

7

8

9 10

11

12

Ranke MB, Pflüger H, Rosendahl W, et al. Turner’s syndrome: spontaneous growth in 150 cases and review of the literature. Eur J Pediatr 1983; 141: 81-88. Park E, Bailey JD, Cowell CA. Growth and maturation of patients with Turner’s syndrome. Pediatr Res 1983; 17: 1-7. Magenis RE, Breg WR, Clark KA, et al. Distribution of sex chromosome complements in 651 patients with Turner’s syndrome. Am J Hum Genet 1980; 32: 79A. Gemmill RM, Pearce-Birge L, Bixenman H, Hecht BK, Allanson JE. Y chromosome-specific DNA sequences in Turner-syndome mosaic. Am J Hum Genet 1987; 41: 157-67. Lin CC, Meyne J, Sasi R, et al. Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,X/46,X, + mar by use of chromosome-specific DNA probes. Am J Med Genet 1990; 37: 71-78. Jacobs PA, Betts PR, Cockwell AE, et al. A cytogenetic and molecular reappraisal of a series of patients with Turner’s syndrome. An Hum Genet 1990; 54: 209-33. Magenis RE, Tochen ML, Holahan KP, Carey T, Allen L, Brown MG. Turner syndrome resulting from partial deletion ofY chromosome short arm: localization of male determinants. J Pediatr 1984; 105: 916-19. Shah KD, Kaffe S, Gilbert F, Dolgin S, Gertner M. Unilateral microscopic gonadoblastoma in a prepubertal Turner mosaic with Y chromosome material identified by restriction fragment analysis. Am J Clin Pathol 1988; 90: 622-27. Rosen GF, Kaplan B, Lobo RA. Menstrual function and hirsutism in patients with gonadal dysgenesis. Obstet Gynecol 1988; 71: 677-80. Sinclair AH, Berta P, Palmer MS, et al. A gene from the human sex determining region encodes a protein with homology to a conserved DNA binding motif. Nature 1990; 346: 240-44. Medlej R, Lobaccaro JM, Berta P, et al. Screening for Y-derived sex determining gene SRY in 40 patients with Turner syndrome. J Clin Endocrinol Metab 1922; 75: 1289-92. Hook EB, Warburton D. The distribution of chromosomal genotypes associated with Turner’s syndrome: livebirth prevalence rates and evidence for diminished fetal mortality and severity in genotypes associated with structural X abnormalities or mosaicism. Hum Genet

1983; 64: 24-27.

13 Hassold T, Benham F, Leppert M. Cytogenetic and molecuar analysis of sex chromosome monosomy. Am J Hum Genet 1988; 42: 534-41. 14 Held KR, Kerber S, Kaminsky E, et al. Mosaicism in 45,X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes? Hum Genet 1992; 88: 288-94. 15 Starzl TE, Demetris AJ, Trucco M, et al. Systemic chimerism in human female recipients of male livers. Lancet 1992; 340: 876-77. 16 Nakagome Y, Seki S, Fukutani K, Nagafuchi S, Nakahori Y, Tamura T. PCR detection of distal Yp sequences in an XX true hermaphrodite. Am J Med Genet 1991; 41: 112-14. 17 Warburton PE, Greig GM, Haaf T, Willard HF. PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 1991; 11: 325-33. 18 Muller U, Donlon T, Kunkel S, Lalande M, Latt S. Y-190, a DNA probe for the sensitive detection of Y-derived marker chromosomes and mosaicism. Hum Genet 1987; 75: 109-13. 19 Dominguez CJ, Greenblatt RG. Dysgerminoma of the ovary in a patient with Turner’s syndrome. Am J Obstet Gynecol 1962; 83: 674-77.

20 Bonakdar MI, Peisner DB. Gonadoblastoma with a 45,XO karyotype. Obstet Gynecol 1980; 56: 748-50. 21 Lindsay AN, Sills N, MacGillivray MH, Fisher JE, Voorhess ML. Dysgerminoma in a patient with the syndrome of gonadal dysgenesis with a 45,X karyotype. Am J Med Genet 1981; 10: 21-24. 22 Sinisi AA, Perrone L, Quarto C, Barone M, Bellastella A, Faggiano M. Dysgerminoma in 45,X Turner syndrome: report of a case. Clin Endocrinol 1988; 28: 187-93. 23 Bosze P, Magyar E, Tóth A, Lásló J. 45,X streak gonad syndrome associated with bilateral ’burnt out’ gonadoblastoma. Gynecol Obstet Invest 1989; 28: 113-17. 24 Page DC. Hypothesis: a Y-chromosomal gene causes gonadoblastoma in dysgenetic gonads. Development 1987; 101 (suppl): 151-55. 25 De Arce MA, Costigan C, Gosden JR, Lawler M, Humphries P. Further evidence consistent with Yqh as an indicator of risk on gonadal blastoma in Y-bearing mosaic Turner syndrome. Clin Genet

1992; 41: 28-32. 26 Aranoff GS, Morishima A. XO/XY mocaicism in J Adolesc Health Care 1988; 9: 501-04.

delayed puberty.

Spectrum of immunodeficiency in HIV-1-infected patients with pulmonary tuberculosis in Zaire

Summary

Introduction

Tuberculosis (TB) is the most common opportunistic infection in African patients who die from AIDS, yet the stage of immunodeficiency at which TB develops is uncertain. We studied the immune status of HIV-infected outpatients with pulmonary TB in relation to their clinical presentation in a cross-sectional study of 216 HIV-seropositive and 146 HIVseronegative ambulatory incident cases of smear-positive and culture-positive pulmonary TB in Kinshasa, Zaire. HIVseropositive and seronegative patients had median CD4 lymphocyte counts of 316·5/µL and 830·5/µL, respectively. Of the HIV-seropositive patients, 32 9% had less than 200 CD4 lymphocytes/µL, 37% between 200 and 499, and 30·1% 500 or more. Clinical AIDS, as defined by the WHO clinical case-definition or a modified version, was of similar limited use as a predictor of immunodeficiency. Among HIV-seropositive patients, oral candidosis, lymphopenia, a negative tuberculin purified protein derivative test, and cutaneous anergy were strongly associated with CD4 counts of less than 200/µL, and seemed to be better markers of immune dysfunction. We conclude that pulmonary TB develops across a broad spectrum of HIV-induced immunodeficiency and that a diagnosis of pulmonary TB is of limited use as a marker of stage of HIV disease in African HIV-infected outpatients.

Tuberculosis (TB) is the most common opportunistic infection in African patients who die from AIDS.1,2 Reactivation of widespread latent Mycobacterium tuberculosis infection among HIV-infected subjects has resulted in an accelerating TB epidemic that also involved those without HIV.3 An important element in the epidemiology of HIV-associated TB is the stage of immunodeficiency at which TB develops. In the USA, the mean or median CD4 lymphocyte counts of HIV-seropositive patients with TB differed according to the setting in which patients were encountered: 326/L at a county tuberculosis clinic,4 170/L in all patients at a general hospital,5 and 133/tiL among patients in hospital. Limited data are available for Africa on CD4 depletion in HIV-infected patients and their development of TB. We studied the immune status of incident ambulatory cases of pulmonary TB in Kinshasa, Zaire in relation to their clinical presentation.

Lancet 1993; 342: 143-46

Projet SIDA, Kinshasa, Zaire (Y Mukadi MD, J H Perriëns MD, M E St Louis MD, C Brown MD, R W Ryder MD); Institute of Tropical Medicine, Antwerp, Belgium (J H Perriëns, F Portaels PhD, P Piot PhD); Belgian Administration for Development and Cooperation, Brussels, Belgium (J H Perriëns, J-C Willame MD); Division of HIV/AIDS, NCID, CDC, Atlanta, USA (M E St Louis, R W Ryder); NIAID, Bethesda, USA (C Brown); Catholic University of Louvain, Mont Godinne, Belgium (J Prignot PhD, F Pouthier MD); Bureau National de la Tuberculose, Kinshasa (J-C Willame); Centre de Dépistage de la Tuberculose, Kinshasa (M Kaboto MD) Correspondence to: Dr Joseph Perriëns, Clinical Research and Drug Development, Global Programme on AIDS, World Health Organization, CH-1211 Geneva 27, Switzerland

Methods At the TB

in Kinshasa, outpatients with pulscreened for pulmonary TB by sputum microscopy. Between March 1, 1989, and Sept 15, 1991, consecutive patients who had never been treated for TB, but who had sputum smears that were positive for acid-fast bacilli (AFB), were referred to the study team at the screening centre for further investigation and to begin treatment. Consenting consecutive HIV-seropositive patients, diagnosed with pulmonary TB at the centre, whose diagnosis was confirmed by a repeat sputum smear and by the culture of M tuberculosis from sputum, and whose HIV infection was confirmed by western blot, were included in our study. The first 200 patients with a negative HIV-antibody screening test, who were of the same sex and age (within 2 years) as the first 200 HIV-seropositive patients, were also asked to participate, and were included in the study if they satisfied the same criteria, except that their HIV serostatus had to be confirmed by an enzyme-linked immunosorbent assay (ELISA; Vironostica, Organon Teknica, Boxtel, the Netherlands). After written informed consent was obtained, patients were counselled and screened for HIV antibodies with a rapid test (HIVCHEK 1 + 2, Dupont de Nemours, Delaware, USA).

screening

monary symptoms

centre

are

143