Genotyping of human immunodefiency virus isolates in Papua New Guinea.

BACKGROUND: There has been considerable escalation in the incidence of HIV infection in Papua New Guinea since the first cases have been reported in 1987. OBJECTIVES: The study was to identify the genetic subtype in HIV infected patients in Papua New Guinea. It is believed that the result will not only assist in tracing and tracking the sources of the infection, but will also help to evaluate the impact of the genotypes on the natural history of HIV in Papua New Guinea. METHODS: Plasma samples from eighty patients were definitively tested for HIV antibodies at PNG Central Public Health Laboratory using Welcome ELISA, Serodia, Immuno Comb and Hexagon. The samples were also tested for Hepatitis B (HBsAG and HBcAG) and Hepatitis C virus antibodies. The HIV positive samples were reconfirmed by the Western Blot analysis; RNA isolation and reverse transcription. DNA sequencing and phylogenetic analysis and determination of HIV subtypes were determined by using representative sequences A-H, J, N and 0 in the Los Alamos Database. RESULTS: The total number of HIV-1 positive patients' samples was 20 (5 females and 15 males) Out of this, 11 (all males) were successfully subtyped as c (91%) and b (9%) showing the predominant type to be subtype C. Nine isolates were designated not typable. This is attributable to either low viral load or new emerging strains that could not be detected by the database used in phylogenetic analysis. CONCLUSION: Data predicts that there is possible emergence of BC circulating recombinant form (CRF) because we also identified subtype B. We suggest that as subtype C remains a guide for tracking the sources of infection in PNG that both subtypes C and B (and any other subtypes that may be identified in future) be included in the future vaccine for use in Papua New Guinea since some potential vaccines work only against particular subtypes assuming that nearly all subtypes identified so far are responsive to ant-retroviral drugs.

Exchangeability of N termini in the ligand-gated porins of Escherichia coli.

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

Effect of loop deletions on the binding and transport of ferric enterobactin by FepA.

The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.

Biphasic binding kinetics between FepA and its ligands.

The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.

Ataxia in patients infected with Salmonella typhi phage type D2: clinical, biochemical and immunohistochemical studies.

Reversible cerebellar dysfunction has been described in association with typhoid fever by a number of authors. Ataxia and tremors are relatively common manifestations of typhoid fever in Papua New Guinea. A series of 14 patients with typhoid fever and ataxia was compared with non-ataxic typhoid patients. Hyponatraemia and hypoalbuminaemia were common in both groups and more severe in the ataxic group. Hypotension was also more common in the ataxic group. These features may reflect more severe disease. All isolates of Salmonella typhi, from both ataxic and non-ataxic patients, were phage type D2 which is endemic around Port Moresby. Tests for autoantibodies cross-reacting with cerebellar tissue were negative. It is possible that cerebellar dysfunction in typhoid fever is more common in association with phage type D2.

Shigella bacteraemia in adults.

Shigella infections are usually restricted to the intestine. There are few reports of Shigella isolated from the blood and most of these are from children, usually in neonates and the malnourished. In the small number of adult cases of Shigella bacteraemia which have been reported, there appears to be an association with underlying disease and immunosuppression including acquired immunodeficiency syndrome. We report three adult cases in which Shigella were isolated from blood. Two of these patients made an uneventful recovery whilst the third died. An underlying cause of immunosuppression was suspected in this patient but unproven.

Breast filariasis diagnosed by needle aspiration cytology.

A case of nodular lesion of the breast in a 37 year old female caused by filariasis is described. The case is instructive since the diagnosis was made by fine-needle aspiration cytology.

Juvenile laryngeal papillomatosis: report of five cases from Papua New Guinea.

Five cases of juvenile laryngeal papillomatosis encountered in Papua New Guinea during the period from December 1987 to May 1989 are described in detail. They were seen in the age-range of 1 year 7 months-5 years. The common clinical features were change of voice and dyspnoea. They were treated by microlaryngoscopic excision with cupped forceps. One of the cases followed-up for 15 months showed multiple recurrences. This condition of juvenile laryngeal papillomatosis, though not common, is certainly not rare in this country and should be considered as a differential diagnosis in the case of a child presenting with progressive dyspnoea.

The role of percutaneous lung aspiration in the bacteriological diagnosis of pneumonia in adults.

In a prospective study of 175 adults with acute pneumonia, percutaneous lung aspiration (PLA) was performed in 144 in an attempt to obtain a bacteriological diagnosis. Positive cultures were obtained from PLA in 90/144 cases (62.5%). The most common organisms isolated were Streptococcus pneumoniae (60%) gram-negative bacilli including Hemophilus influenzae (26%) and Staphylococcus aureus (11%). In 39% of those patients with positive PLA cultures, the same organism was isolated on blood cultures. Complications of PLA were uncommon, with hemoptysis in 4% and chest wall hematoma in 0.7% of patients. The incidence of post-aspirate pneumothorax was not determined in this study. PLA was found to be a safe and effective method of obtaining a bacteriological diagnosis in adult patients with pneumonia.

Community-acquired acinetobacter pneumonia in adults in Papua New Guinea.

Acinetobacter calcoaceticus, an aerobic gram-negative coccobacillus, is a rare cause of community-acquired pneumonia. It most commonly causes nosocomial infections, particularly in elderly debilitated patients who have undergone surgery, instrumentation, and antibiotic therapy. In a study of acute pneumonia in adults, five cases of community-acquired acinetobacter pneumonia were observed over an 11-month period. Other than chronic pulmonary disease (two patients), no serious underlying diseases existed in these patients. Lobar consolidation was the predominant radiologic pattern. The mortality rate was 40%, and mortality was directly related to therapy with inappropriate antibiotics. The reason for this relatively high prevalence of community-acquired acinetobacter pneumonia in the population studied is not known. Possible explanations include the high prevalence of chronic pulmonary disease, indiscriminate use of penicillin, and an unknown immunodeficiency state.

Mycobacterium ulcerans infections in Papua New Guinea: correlation of clinical, histological, and microbiologic features.

Biopsy material from 46 cases clinically diagnosed as Mycobacterium ulcerans infection was studied. The disease was found to be most frequent in East Sepik Province, and in the first decade of life. In these 46 cases the histological features were characteristic and acid-fast bacilli were seen in tissue sections.

Haemophilus influenzae pneumonia in Melanesian adults: report of 15 cases.

In a prospective study of 170 adult patients with acute pneumonia, Haemophilus influenzae was found to be the aetiological agent in 15 cases (8.8%). The diagnosis in all cases was based on positive cultures of blood or percutaneous lung aspirate, or both. Chronic lung disease was significantly more common in patients with H influenzae pneumonia than in patients with pneumonia due to other organisms but age, sex, and smoking history did not differ significantly. Lobar consolidation was the most common radiological pattern, being present in 10 of the 15 cases. Type b was the commonest serotype isolated, but three cases were due to non-typable (non-capsulate) strains. All patients survived, responding well to treatment with penicillin, ampicillin, or chloramphenicol. Haemophilus influenzae should be considered as a possible cause of pneumonia in adults, particularly those with underlying chronic lung disease.

Penicillin resistant Staphylococcus aureus in Port Moresby.

Since 1971, over 90% of cultures of Staphylococcus aureus isolated from pus specimens obtained in Port Moresby General Hospital have been resistant to penicillin, Since 1979 the percentage has risen to 98% and in addition chloramphenicol resistance has been noted in 26% of isolates since that year. Cases of methicillin or gentamicin resistance have not yet been detected. Even when the possibility of staphylococcal infection is suspected, penicillin or penicillin and chloramphenicol continue to be the initial treatment prescribed in 90.6% of subjects with pyomyositis, 88.5% with acute osteomyelitis, 88.2% with pneumonia and 90% with bacteraemia. Appropriate initial treatment of patients in Port Moresby who are suspected or proven to have serious S. aureus infection should include a beta-lactamase resistant penicillin or suitable alternative bacteriocidal antibiotic.