HIV infection of undetermined origin during infancy.

The case histories of two children with horizontally acquired HIV infection are described. These children were diagnosed at a paediatric hospital in sub-Saharan Africa. Although the source(s) of infection was not identified, both children had had several contacts with the health service, experienced invasive procedures and ingested expressed milk from their own mothers during hospital admission. Health-care institutions, particularly those located in high HIV prevalence areas, must implement effective infection control measures to ensure that the risk of horizontal infection is minimized. Attention should be given to practices that are unique to each clinical discipline.

X-linked agammaglobulinaemia and the underlying genetics in two kindreds.

OBJECTIVE: Molecular analysis of the Bruton's tyrosine kinase (Btk) gene in two unrelated families, with a combined total of seven boys, affected by X-linked agammaglobulinaemia (XLA). METHODS: Protein electrophoresis and western blotting were used for the examination of Btk protein synthesis in blood leucocytes. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction (RT-PCR) technique. Sequence alterations were screened for by the single-stranded conformation polymorphism (SSCP) method and characterized by standard sequencing protocols. RESULTS: Western blotting revealed Btk protein to be absent in leucocytes of affected males from both families. A novel 3 b.p. deletion in exon 3 of the Btk gene was found to be responsible for the XLA phenotype in the affected proband in one family (kindred I). A diagnostic PCR assay was established to detect this mutation in other affected male siblings and carrier females. For the second family (kindred II), the coding sequence of the Btk gene and the promoter region were found to be normal. CONCLUSIONS: The present study has demonstrated genetic heterogeneity in the Btk gene in South African XLA patients and has identified a novel mutation in this gene in the largest of the affected kindreds. The gene mutation in the second kindred was undetermined and may be indicative of a defect in some other gene associated with Btk function or stability. Western blotting was found to be informative in establishing a deficiency of Btk protein in both probands and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

Disseminated histoplasmosis in an 'immunocompetent' child.

A rare case of severe disseminated histoplasmosis in a 7-year-old boy with apparently normal immune function is described. Current recommendations for diagnostic investigations, monitoring and the treatment of this disease with amphotericin B and itraconazole are reviewed.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

Primary immunodeficiency diseases at Red Cross War Memorial Children's Hospital.

OBJECTIVE: To describe the spectrum of primary immunodeficiency diseases (PIDs) diagnosed at Red Cross War Memorial Children's Hospital. DESIGN: Retrospective, descriptive study. SETTING: Tertiary, referral hospital. PATIENTS: All patients investigated by the immunology service because of suspected PIDs, between January 1983 and December 1996. METHODS: Review of immunology service database and hospital case records. RESULTS: During the 14-year review period, 515 patients were investigated, a mean of 36.8 new patients per annum. Ninety-three patients with PIDs were diagnosed, a mean of 6.6 new patients per annum. The spectrum of PIDs was similar to that reported in developed countries. As in other series, antibody deficiencies predominated, accounting for 56% (52/93) of diagnoses. The male/female ratio was 1.5:1; 73% (62/85) came from the Western Cape, the remaining 27% (23/85) resided in five other provinces. Eighty per cent (70/87) presented with recurrent or atypical infection, with or without failure to thrive. Sinopulmonary infections (80%), diarrhoeal disease (19%) and candidiasis (18%) were the most common preceding infections. By the age of 5 years, only 60% had been diagnosed, compared with about 80% in developed countries. During the study period, 20% (19/93) were known to have died. CONCLUSIONS: The results show a pattern of PIDs incidence similar to that in developed countries. Diagnosis was delayed in many patients, which probably contributed to morbidity. To facilitate earlier diagnosis and to improve outcome, children should be considered for an immunological assessment if they exhibit increased susceptibility to infection.

Immunoglobulin allotypes and genetic susceptibility to invasive Haemophilus influenzae type b and Staphylococcus aureus infections in South African children.

OBJECTIVE: The purpose of this study was to determine whether the G2m(n), G1m(f) and Km(3) immunoglobulin allotypes have any association with susceptibility to invasive Haemophilus influenzae type b (Hib) and Staphylococcus aureus (S. aureus) infections in children. METHODS: Direct enzyme-linked immunosorbent assays with commercially available monoclonal antibodies were established to quantitate G2m(n) and G1m(f) allotypes. A qualitative enzyme-linked immunosorbent assay with polyclonal rabbit anti-Km(3) antibody was established for Km(3) determination. RESULTS: The G2m(n) marker occurred in 34.4% of the mixed ancestry population and 2.9% of the Black population. There was a significantly decreased frequency of the G2m(n) allotype in mixed ancestry children with Hib meningitis (8.5%) and Hib osteomyelitis/septic arthritis and a decreased frequency of Km(3) in black and mixed ancestry children with Hib meningitis. The frequency of G2m(n), G1m(f) and Km(3) allotypes in patients with S. aureus osteomyelitis/septic arthritis were not significantly different from normal population frequency. CONCLUSIONS: This study shows a clear association between the absence of the G2m(n) allotype in mixed ancestry children and susceptibility to invasive infections caused by H. influenzae and an association between the absence of Km(3) and Hib meningitis in both black and mixed ancestry children.

Mutations in the human mannose-binding protein gene: frequencies in several population groups.

Mannose-binding protein (MBP; mannan-binding protein, mannan-binding lectin) is a member of the collectin family of proteins and is thought to be important in innate immunity. We have previously shown high frequencies of two distinct mutations in codon 54 and codon 57 of exon 1 of the MBP gene in non-African and African populations, respectively. These result in low levels of the protein and an opsonic deficiency but the frequencies also suggest some selective advantage for low MBP levels. A third mutation in codon 52 occurs at a much lower frequency. We have now extended our earlier studies to other populations. In the south-west Pacific (Papua New Guinea and Vanuatu) neither the codon 52 nor the codon 57 mutation was detected and the codon 54 mutation was significantly less common (gene frequencies of 0.07 and 0.01, respectively) than in other non-African populations (gene frequencies 0.11-0.16). This could be explained by relatively recent admixture. The ancestral Melanesian population probably diverged some 50,000-60,000 years ago and our data suggest that the codon 54 mutation may have occurred after that even but before the divergence of European-Asian groups (40,000 years ago). Two further sub-Saharan populations were also studied: a group of Xhosa from South Africa were similar to Gambians, with a high gene frequency for the codon 57 mutation (0.27) and no evidence of the codon 52 or 54 mutations. In contrast, San Bushmen from Namibia had low frequencies of both the codon 57 mutation (0.07) and the codon 54 mutation (0.03). Again the codon 52 mutation was not found. This pattern is unique amongst sub-Saharan populations studied to date and suggests that this population may have been subjected to different selective pressures.

Rheumatic fever: autoantibodies against a variety of cardiac, nuclear, and streptococcal antigens.

OBJECTIVE: To measure antibody titres to cardiac, nuclear, and streptococcal antigens in different groups of rheumatic fever (n = 60) and control subjects (n = 80) with the aim of identifying cross reactive antigens of potential laboratory diagnostic value. METHODS: Enzyme linked immunosorbent assays (ELISA), immunocytochemical, and electrophoretic techniques were used to measure titres of antibodies to a variety of cardiac, nuclear, and streptococcal antigens in seven groups comprising patients with rheumatic fever and control subjects. RESULTS: Increased concentrations of antibodies to several streptococcal and cardiac antigens, in addition to increased IgA and IgG levels, were noted in sera from patients with acute rheumatic fever and chronic rheumatic heart disease. Autoantibodies to nuclear antigens were evident in three rheumatic fever sera. CONCLUSION: Although we were unable to identify any unique cross reactivity between cardiac and streptococcal antigens, these results demonstrate that there is an exaggerated humoral response to several cardiac, nuclear and streptococcal antigens in patients with rheumatic fever.

Flow cytometry analysis of OKT4 epitope deficiency in South African black children.

A case of OKT4 epitope deficiency referred for investigation with suspected immunodeficiency is described. Flow cytometry analysis of OKT4 epitope deficiency in a study group of healthy black children showed different manifestations of the lack of OKT4 epitope; a complete lack of OKT4+CD4+ peripheral blood lymphocytes (PBL) with normal numbers of OKT4A+ and Leu3a-CD4+ PBL, decreased percentage OKT4+CD4+ compared with OKT4A+ and Leu-3a+CD4+ PBL, decreased fluorescent staining intensity with OKT4 and a biphasic OKT4 staining pattern associated with a reduced OKT4/Leu-3a ratio. The percentage and fluorescent intensity of OKT4+CD4+ PBL in the study group were significantly lower (P < 0.0001) than Leu-3a+CD4+ and OKT4A+CD4+ PBL. There is thus considerable risk of under-estimating the number of CD4+ cells in black South Africans if the OKT4 MoAb is used.

Identification of cardiac autoantigens in human heart cDNA libraries using acute rheumatic fever sera.

Antigenic mimicry or cross-reactivity between Group A streptococcal antigens and cardiac autoantigens may initiate an autoimmune response resulting in cardiovascular damage in acute rheumatic fever. This study describes a molecular biological approach to the identification of such cross-reactive cardiac antigens. Two human heart cDNA libraries were constructed in the expression vector lambda gt11 and screened with patient sera, monoclonal antibodies and rabbit immune sera cross-reactive with streptococcal and cardiac antigens. Using the serum of a patient with a recurrent acute attack of rheumatic fever containing high titres of antibodies cross-reactive with both sets of antigens, we were able to identify three positive clones with insert sizes of 1.0 kb, 1.4 kb and 0.9 kb in these libraries. Acute rheumatic fever (ARF) sera reacted more strongly with these autoantigen clones than did normal sera. Autoantibodies eluted from the purified plaques of all three clones displayed different patterns of cross-reactivity against immunoblots of streptococcal M5, M6, M19 and M24 protein extracts. The cDNA inserts were sequenced and compared with known sequences in the EMBL and Genbank databases. One clone was 98% homologous with human cytokeratin 8 and showed homologies of 40 to 50% with human cardiac heavy chain myosin, tropomyosin and streptococcal M5 protein--all members of the alpha-helical coiled-coil family of proteins. Another clone was completely homologous to the G-protein alpha-subunit of adenyl cyclase, whilst the sequence of the third clone was not found in any of the data banks.

Chronic granulomatous disease presenting with osteomyelitis: favorable response to treatment with interferon-gamma.

A 3-year-old boy with chronic granulomatous disease presented with abscesses related to underlying osteomyelitis. Treatment with appropriate antibiotics resulted in resistance of the organisms. Treatment with interferon-gamma was encouraging in that it caused the osteomyelitis to heal and prevented its relapse when used as long-term prophylaxis.

IgG subclass values from normal children in Cape Town.

Sensitive and reproducible enzyme-linked immunoabsorbent assays (ELISA) have been developed to quantitate IgG subclass levels using monoclonal antibodies. Normal values for serum IgG subclass levels were determined in 300 healthy children between 6 months and 14 years of age and in 80 adults. High levels of IgG1 and delayed maturational development of IgG2 in children from Cape Town are different to results reported from developed countries. Genetic differences may account for this.

Treatment of chronic granulomatous disease with recombinant gamma interferon.

Chronic granulomatous disease is a rare, primary immunodeficiency associated with serious bacterial and fungal infections caused by phagocytic defects of oxidative metabolism. To date the mainstay of management has been aggressive treatment of infections and the use of prophylactic antibiotics. Two patients, who showed remarkable clinical improvement when treated with recombinant gamma interferon, are reported. Both have been on treatment for at least 18 months and have continued to thrive and remain free of infections.

IgM rheumatoid factor in congenital syphilis: associations with clinical and laboratory findings.

IgM rheumatoid factor (RF), measured by means of an ELISA, was detected in 92% of infants with congenital syphilis. Elevated levels were found to correlate with liver and renal involvement as well as the extent of the disease (P less than 0.05). In addition, levels of circulating immune complexes were closely related to the RF concentration (P less than 0.001). Following treatment of the infants both RF levels and VDRL titres declined at a similar rate (P less than 0.001). These findings indicated a close relationship between the disease process and IgM RF levels. It is postulated that IgM RF may add to immune complex deposition and exacerbate tissue damage in congenital syphilis.

Human monoclonal antibodies: analysis of two antibodies derived from lymphocytes of a patient with acute rheumatic fever.

Human monoclonal antibodies were produced by fusion of peripheral blood lymphocytes from a patient with acute rheumatic fever, with the HGPRT-non-secreting murine (Balb-c) cell line SP2/0Ag14. Heterohybridomas were selected by screening against rheumatic fever-associated group A streptococci using an ELISA, and against paraffin wax-embedded human heart sections using an immunoperoxidase technique. Two human IgM monoclonal antibodies were selected for further analysis by Western blotting and ELISA. Both antibodies demonstrated multispecificity by immunoblotting and ELISA. One of the monoclonals bound to 48 kD and 83 kD bands common to group A streptococcal and heart antigen preparations. Both human monoclonal antibodies bound to a 43 kD constituent band common to human heart and sarcolemma membrane extract. Inhibition studies performed using a competitive solid phase immunoassay confirmed shared epitopes between group A streptococci and human heart. The significance of these monoclonal antibodies to the pathogenesis of rheumatic fever is uncertain.

Hyper IgM with combined immunodeficiency.

This report describes a fourteen year old girl with an unusual immunodeficiency characterized by persistent lymphadenopathy and associated with hypogammaglobulinaemia, excessive IgM production and a severe T cell defect. Total T cell and T helper cell numbers were reduced and T cell proliferative responses to mitogens were poor. Serum IgM levels showed marked fluctuations and peaks correlated with acute tender lymphadenopathy. She was treated with intravenous gammaglobulin and prophylactic antibiotics. Although defective isotype switching of B cells into IgA and IgG producing cells has been accepted as the mechanism of the hyper IgM syndrome, it is becoming increasingly evident that T cell function is not uncommonly involved and may be responsible for impaired isotype switching.