South African Institute for Medical Research

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Y chromosome haplotypes are particularly useful in deciphering human evolutionary history because they accentuate the effects of drift, migration, and range expansion. Significant acceleration of Y biallelic marker discovery and subsequent typing involving heteroduplex detection has been achieved by implementing an innovative and cost-efficient method called denaturing high-performance liquid chromatography (DHPLC). The power of the method resides in its sensitivity and ability to rapidly compare amplified sequences in an automated manner. We have determined the allelic states of 22 Y polymorphisms; 19 of which are unreported, in 718 diverse extant chromosomes; established haplotype frequencies; and deduced a phylogeny. All major geographic regions, including Eurasia, are characterized by mutations reflecting episodes of genetic drift and expansion. Most biallelic markers are localized regionally. However, some show wider dispersal and designate older, core haplotypes. One transversion defines a major haplogroup that distinguishes a previously unknown deep, apparently non-African branch. It provides evidence of an ancient bottleneck event. It is now possible to anticipate the inevitable detailed reconstruction of human Y chromosome genealogy based on several tens to even hundreds of these important polymorphisms.

The frequencies of low-activity alleles of glucose-6-phosphate dehydrogenase in humans are highly correlated with the prevalence of malaria. These "deficiency" alleles are thought to provide reduced risk from infection by the Plasmodium parasite and are maintained at high frequency despite the hemopathologies that they cause. Haplotype analysis of "A-" and "Med" mutations at this locus indicates that they have evolved independently and have increased in frequency at a rate that is too rapid to be explained by random genetic drift. Statistical modeling indicates that the A- allele arose within the past 3840 to 11,760 years and the Med allele arose within the past 1600 to 6640 years. These results support the hypothesis that malaria has had a major impact on humans only since the introduction of agriculture within the past 10,000 years and provide a striking example of the signature of selection on the human genome.

BACKGROUND: Commonly encountered nosocomially acquired gram-negative bacteria, especially Klebsiella pneumoniae, produce extended-spectrum beta-lactamases (ESBLs) as an antibiotic resistance mechanism. OBJECTIVE: To determine whether microbiology laboratories should report the presence of ESBLs and to establish the infection-control implications of ESBL-producing organisms. DESIGN: Prospective observational study. SETTING: 12 hospitals in South Africa, Taiwan, Australia, Argentina, the United States, Belgium, and Turkey. PATIENTS: 440 patients with 455 consecutive episodes of K. pneumoniae bacteremia between 1 January 1996 and 31 December 1997; of these, 253 episodes were nosocomially acquired. MEASUREMENTS: The K. pneumoniae isolates were examined for the presence of ESBLs. Pulsed-field gel electrophoresis was used to analyze the molecular epidemiology of nosocomial bacteremia with ESBL-producing K. pneumoniae. RESULTS: Overall, 30.8% (78 of 253) episodes of nosocomial bacteremia and 43.5% (30 of 69) episodes acquired in intensive care units were due to ESBL-producing organisms. After adjustment for potentially confounding variables, previous administration of beta-lactam antibiotics containing an oxyimino group (cefuroxime, cefotaxime, ceftriaxone, ceftazidime, or aztreonam) was associated with bacteremia due to ESBL-producing strains (risk ratio, 3.9 [95% CI, 1.1 to 13.8]). In 7 of 10 hospitals with more than 1 ESBL-producing isolate, multiple strains with the same genotypic pattern were observed, indicating patient-to-patient spread of the organism. CONCLUSIONS: Production of ESBLs by Klebsiella pneumoniae is a widespread nosocomial problem. Appropriate infection control and antibiotic management strategies are needed to stem the spread of this emerging form of resistance.

Haplotypes consisting of alleles at a short tandem repeat polymorphism (STRP) and an Alu deletion polymorphism at the CD4 locus on chromosome 12 were analyzed in more than 1600 individuals sampled from 42 geographically dispersed populations (13 African, 2 Middle Eastern, 7 European, 9 Asian, 3 Pacific, and 8 Amerindian). Sub-Saharan African populations had more haplotypes and exhibited more variability in frequencies of haplotypes than the Northeast African or non-African populations. The Alu deletion was nearly always associated with a single STRP allele in non-African and Northeast African populations but was associated with a wide range of STRP alleles in the sub-Saharan African populations. This global pattern of haplotype variation and linkage disequilibrium suggests a common and recent African origin for all non-African human populations.

We initiated a worldwide collaborative study, including 455 episodes of bacteremia, to elucidate the clinical patterns of Klebsiella pneumoniae. Historically, community-acquired pneumonia has been consistently associated with K. pneumoniae. Only four cases of community-acquired bacteremic K. pneumoniae pneumonia were seen in the 2-year study period in the United States, Argentina, Europe, or Australia; none were in alcoholics. In contrast, 53 cases of bacteremic K. pneumoniae pneumonia were observed in South Africa and Taiwan, where an association with alcoholism persisted (p=0.007). Twenty-five cases of a distinctive syndrome consisting of K. pneumoniae bacteremia in conjunction with community-acquired liver abscess, meningitis, or endophthalmitis were observed. A distinctive form of K. pneumoniae infection, often causing liver abscess, was identified, almost exclusively in Taiwan.

The prevalence of extended-spectrum beta -lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem (primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Northern Kwazulu/Natal (KZN) Province of South Africa borders on southern Mozambique, between Swaziland and the Indian Ocean. To control malaria vectors in KZN, houses were sprayed annually with residual DDT 2 g/ m2 until 1996 when the treatment changed to deltamethrin 20-25 mg/m2. At Ndumu (27 degrees 02'S, 32 degrees 19'E) the recorded malaria incidence increased more than six-fold between 1995 and 1999. Entomological surveys during late 1999 found mosquitoes of the Anopheles funestus group (Diptera: Culicidae) resting in sprayed houses in some sectors of Ndumu area. This very endophilic-vector of malaria had been eliminated from South Africa by DDT spraying in the 1950s, leaving the less endophilic An. arabiensis Patton as the only vector of known importance in KZN. Deltamethrin-sprayed houses at Ndumu were checked for insecticide efficacy by bioassay using susceptible An. arabiensis (laboratory-reared) that demonstrated 100% mortality. Members of the An. funestus group from Ndumu houses (29 males, 116 females) were identified by the rDNA PCR method and four species were found: 74 An. funestus Giles sensu stricto, 34 An. parensis Gillies, seven An. rivulorum Leeson and one An. leesoni Evans. Among An. funestus s.s. females, 5.4% (4/74) were positive for Plasmodium falciparum by ELISA and PCR tests. To test for pyrethroid resistance, mosquito adults were exposed to permethrin discriminating dosage and mortality scored 24h post-exposure: survival rates of wild-caught healthy males were 5/10 An. funestus, 1/9 An. rivulorum and 0/2 An. parensis; survival rates of laboratory-reared adult progeny from 19 An. funestus females averaged 14% (after 1h exposure to 1% permethrin 25:75cis:trans on papers in WHO test kits) and 27% (after 30 min in a bottle with 25 microg permethrin 40:60cis:trans). Anopheles funestus families showing >20% survival in these two resistance test procedures numbered 5/19 and 12/19, respectively. Progeny from 15 of the families were tested on 4% DDT impregnated papers and gave 100% mortality. Finding these proportions of pyrethroid-resistant An. funestus, associated with a malaria upsurge at Ndumu, has serious implications for malaria vector control operations in southern Africa.

Although extended-spectrum beta-lactamases (ESBLs) hydrolyze cephalosporin antibiotics, some ESBL-producing organisms are not resistant to all cephalosporins when tested in vitro. Some authors have suggested that screening klebsiellae or Escherichia coli for ESBL production is not clinically necessary, and when most recently surveyed the majority of American clinical microbiology laboratories did not make efforts to detect ESBLs. We performed a prospective, multinational study of Klebsiella pneumoniae bacteremia and identified 10 patients who were treated for ESBL-producing K. pneumoniae bacteremia with cephalosporins and whose infecting organisms were not resistant in vitro to the utilized cephalosporin. In addition, we reviewed 26 similar cases of severe infections which had previously been reported. Of these 36 patients, 4 had to be excluded from analysis. Of the remaining 32 patients, 100% (4 of 4) patients experienced clinical failure when MICs of the cephalosporin used for treatment were in the intermediate range and 54% (15 of 28) experienced failure when MICs of the cephalosporin used for treatment were in the susceptible range. Thus, it is clinically important to detect ESBL production by klebsiellae or E. coli even when cephalosporin MICs are in the susceptible range (<or = 8 microg/ml) and to report ESBL-producing organisms as resistant to aztreonam and all cephalosporins (with the exception of cephamycins).

The emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.

The safety, immunogenicity, and impact on carriage of a nonvalent pneumococcal vaccine given at ages 6, 10, and 14 weeks were examined in a double-blind, randomized, placebo-controlled trial in 500 infants in Soweto, South Africa. No serious local or systemic side effects were recorded. Significant antibody responses to all pneumococcal serotypes were observed 4 weeks after the third dose. Haemophilus influenzae type b polyribosylribitol phosphate (geometric mean titer, 11.62 microg/mL) and diphtheria (1.39 IU/mL) antibodies were significantly higher in children receiving pneumococcal conjugate, compared with placebo recipients (4.58 microgram/mL and 0.98 IU/mL, respectively). Nasopharyngeal carriage of vaccine serotypes decreased in vaccinees at age 9 months (18% vs. 36%), whereas carriage of nonvaccine serotypes increased (36% vs. 25%). Carriage of penicillin-resistant pneumococci (21% vs. 41%) and cotrimoxazole-resistant pneumococci (23% vs. 35%) were significantly reduced 9 months after vaccination, compared with controls.

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 x 10(-6) to 1.64 x 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

A prospective study of Klebsiella pneumoniae bacteremia was performed in 12 hospitals in 7 countries. Of 452 episodes of bacteremia, 25 (5.5%) were caused by K. pneumoniae that was resistant in vitro to ciprofloxacin. Extended-spectrum beta-lactamase (ESBL) production was detected in 15 (60%) of 25 ciprofloxacin-resistant isolates, compared with 68 (16%) of 427 ciprofloxacin-susceptible strains (P=.0001). Multivariate analysis revealed that risk factors for ciprofloxacin resistance in K. pneumoniae included prior receipt of a quinolone (P=.0065) and an ESBL-producing strain (P=.012). In all, 18% of ESBL-producing isolates were also ciprofloxacin-resistant. Pulsed-field gel electrophoresis showed that 11 of the 15 ciprofloxacin-resistant ESBL-producing strains belonged to just 4 genotypes, suggesting that patient-to-patient transmission of such strains occurred. The close relationship between ESBL production and ciprofloxacin resistance is particularly worrisome because the first reported instance of plasmid-mediated ciprofloxacin resistance has been in an isolate of K. pneumoniae also possessing an ESBL.

Alu insertion polymorphisms (polymorphisms consisting of the presence/absence of an Alu element at a particular chromosomal location) offer several advantages over other nuclear DNA polymorphisms for human evolution studies. First, they are typed by rapid, simple, PCR-based assays; second, they are stable polymorphisms-newly inserted Alu elements rarely undergo deletion; third, the presence of an Alu element represents identity by descent-the probability that different Alu elements would independently insert into the exact same chromosomal location is negligible; and fourth, the ancestral state is known with certainty to be the absence of an Alu element. We report here a study of 8 loci in 1500 individuals from 34 worldwide populations. African populations exhibit the most between-population differentiation, and the population tree is rooted in Africa; moreover, the estimated effective time of separation of African versus non-African populations is 137,000 +/- 15,000 years ago, in accordance with other genetic data. However, a principal coordinates analysis indicates that populations from Sahul (Australia and New Guinea) are nearly as close to the hypothetical ancestor as are African populations, suggesting that there was an early expansion of tropical populations of our species. An analysis of heterozygosity versus genetic distance suggests that African populations have had a larger effective population size than non-African populations. Overall, these results support the African origin of modern humans in that an earlier expansion of the ancestors of African populations is indicated.

The first recognised outbreak of Marburg virus disease in Africa, and the first since the original epidemic in West Germany and Yugoslavia in 1967, occurred in South Africa in February 1975. The primary case was in a young Australian man , who was admitted to the Johannesburg Hospital after having toured Rhodesia. Two secondary cases occurred, one being in the first patient's travelling companion, and the other in a nurse. Features of the illness included high fever, myalgia, vomiting and diarrhoea, hepatitis, a characteristic maculopapular rash, leucopenia, thrombocytopenia, and a bleeding tendency. The first patient died on the seventh day from haemorrhage resulting from a combination of disseminated intravascular coagulation and hepatic failure. The other two patients were given vigorous supportive treatment and prophylactic heparin and recovered after an acute phase lasting about seven days. During this period on developed pancreatitis, the serum amylase remaining raised until the 32nd day after the onset of the illness. The other developed unilateral uveitis after having been asymptomatic for two months. This persisted for several weeks and Marburg virus was cultured from the anterior chamber of the eye.

SUMMARY The clinical and neurodevelopmental features are presented of four children–two sibling pairs–who were exposed in utero to valproic acid. One of each pair of children presented for diagnosis and assessment of developmental delay; the other sibling was examined at a later date. Three of the children were globally developmentally delayed with marked speech disability, and had dysmorphic features consistent with fetal valproate syndrome. One also had features of infantile autism. The fourth child had some of the dysmorphic features connected with fetal valproate syndrome, but had normal intellect, with his verbal ability being significantly below his non‐verbal ability. He currently attends a school for learning‐disabled children. RÉSUMÉ Le syndrome foetal du valproate: données cliniques et neuro‐développementales dans deux paires de méme fratrie Les données cliniques et neuro‐développementales de quatrc enfants, deux paires de même fratrie, exposés in utero á l'acide valproïque, sont présentées. Dans chaque paire, un enfant fut examiné initialement pour le diagnostic et l'évaluation d'un retard de développement; l'autre membre de la fratrie étant examiné ultérieurement. Chez trois des enfants, il fut noté un trouble net du langage, des caractéres dysmorphiques en rapport avec le syndrome foetal du valproate. Un enfant présentait en outre le tableau d'un autisme infantile. Le quatriéme enfant présentait quelques éléments dysmorphiques caractéristiques du syndrome foetal de valproate mais son intelligence était normale, bien que ses capacités verbales aient été manifestement inférieures á ses capacités non verbales. II fréquente actuellement une école pour enfants en difficultés d'apprentissage. ZUSAMMENFASSUNG Valproai‐Embryofelopathie: klinische und entwieklungsneurologische Merkmale bei zwei Zwillingspaaren Es werden die klinischen und entwicklungsneurologischen Merkmale bei vier Kindern—zwei Zwillingspaaren beschrieben, die in utero eine Exposition zu Valproinsaura hatten. Je ein Zwillingskind wurde wegen einer Entwicklungsverzögerung vorgestellt und untersucht, die beiden anderen Kinder wurden zu einem spateren Zeitpunkt untersucht. Drei der Kinder hatten eine globale Entwicklungsverzogerung mit ausgeprägten Sprachschwierigkeiten und Dysmorphiezeichen, die für die Valproat‐Embryofetopathie typisch sind. Ein Kind hatte außerdem Symptome eines infantilen Autismus. Das vierte Kind hatte einige der typischen Dysmorphiezeichen, aber es hatte einen normalen Intellekt, wobei seine verbalen Fähigkeiten signifikant schlechter waren als seine non‐verbalen. Zur Zeit besucht es eine Schule für lernbehinderte Kinder. RESUMEN Sindrome fetal del valproate: caracteristicas clinicas y neuroevolutivas en dos pares de hermanos Se presentan las caracteristicas clinicas y neuroevolutivas de cuatro niños (dos pares de hermanos) que habian sido expuestos in utero a ácido valproico. Uno de cada pareja de hermanos acudieron a la consulta para diagnóstico y evaluatión de un retraso en el desarrollo; el otro hermano fue examinado un tiempo más tarde. Tres de los niños tenían un retraso global del desarrollo con una marcada dificultad en el lenguaje y tenian caracteristicas dismórficas del tipo del sindrome fetal del valproate. Uno de los niños tenia también signos de autismo infantil. El cuarto niño tenia algunos de los signos dismórficos en relación con el sindrome del valproato pero tenia un intclecto normal y su habilidad verbal era significativamente menor que su habilidad no verbal; acude habitualmente a una escuela especial para niños discapacitados.

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.

A clinicopathological study of 57 unicystic ameloblastomas has been undertaken, which represents 15% of all cases of ameloblastoma accessioned in our department over a 30-yr period. Of the cases where gender was recorded: 30 were male and 23 female. The majority of patients were black (51 cases) and most occurred in the mandible (52). This distribution conforms to that of solid and multicystic ameloblastomas. The mean age at diagnosis was 23.8 years (S.D. 14.9) which is significantly younger than for the conventional counterpart (p less than 0.1%). The lesions were classified histologically into 3 groups: Group 1 (42%) cyst lined by a variable often non-descript epithelium; Group 2 (9%) cyst showing intraluminal plexiform proliferation of epithelium; Group 3 (49%) cyst with invasion of epithelium into the cyst wall in either follicular or plexiform patterns. While Group 1 and 2 lesions may be treated by enucleation, Group 3 lesions should be treated aggressively as for conventional ameloblastomas. The objectives of correct histological diagnosis, subclassification and appropriate therapy are best achieved by enucleation biopsy. There is little evidence to support origin from pre-existing odontogenic cysts.

We studied 455 consecutive episodes of Klebsiella pneumoniae bacteremia occurring in 7 countries. Community-acquired pneumonia and an invasive syndrome of liver abscess, meningitis, or endophthalmitis occurred only in Taiwan and South Africa. Infections by K1 and K2 capsular serotype, the mucoid phenotype, and aerobactin production were important determinants of virulence. The mucoid phenotype was seen in 94% of isolates in patients with community-acquired pneumonia and in 100% of isolates that caused the invasive syndrome in Taiwan and South Africa, compared with only 2% of isolates elsewhere. Mortality of mice injected with mucoid strains (69%) was strikingly higher than that occurring in mice injected with nonmucoid strains (3%, p < 0.001). Differences in clinical features of bacteremic infection with K. pneumoniae are due to the virulence factors expressed by the organism.

Ethionamide (ETA) is an important component of second-line therapy for the treatment of multidrug-resistant tuberculosis. Synthesis of radiolabeled ETA and an examination of drug metabolites formed by whole cells of Mycobacterium tuberculosis (MTb) have allowed us to demonstrate that ETA is activated by S-oxidation before interacting with its cellular target. ETA is metabolized by MTb to a 4-pyridylmethanol product remarkably similar in structure to that formed by the activation of isoniazid by the catalase-peroxidase KatG. We have demonstrated that overproduction of Rv3855 (EtaR), a putative regulatory protein from MTb, confers ETA resistance whereas overproduction of an adjacent, clustered monooxygenase (Rv3854c, EtaA) confers ETA hypersensitivity. Production of EtaA appears to be negatively regulated by EtaR and correlates directly with [(14)C]ETA metabolism, suggesting that EtaA is the activating enzyme responsible for thioamide oxidation and subsequent toxicity. Coding sequence mutations in EtaA were found in 11 of 11 multidrug-resistant MTb patient isolates from Cape Town, South Africa. These isolates showed broad cross-resistance to thiocarbonyl containing drugs including ETA, thiacetazone, and thiocarlide.