Killer-cell Immunoglobulin-like Receptor (KIR) gene profiles modify HIV disease course, not HIV acquisition in South African women.

BACKGROUND: Killer-cell Immunoglobulin-like Receptors (KIR) interact with Human Leukocyte Antigen (HLA) to modify natural killer- and T-cell function. KIR are implicated in HIV acquisition by small studies that have not been widely replicated. A role for KIR in HIV disease progression is more widely replicated and supported by functional studies. METHODS: To assess the role of KIR and KIR ligands in HIV acquisition and disease course, we studied at-risk women in South Africa between 2004-2010. Logistic regression was used for nested case-control analysis of 154 women who acquired vs. 155 who did not acquire HIV, despite high exposure. Linear mixed-effects models were used for cohort analysis of 139 women followed prospectively for a median of 54 months (IQR 31-69) until 2014. RESULTS: Neither KIR repertoires nor HLA alleles were associated with HIV acquisition. However, KIR haplotype BB was associated with lower viral loads (-0.44 log10 copies/ml; SE = 0.18; p = 0.03) and higher CD4+ T-cell counts (+80 cells/µl; SE = 42; p = 0.04). This was largely explained by the protective effect of KIR2DL2/KIR2DS2 on the B haplotype and reciprocal detrimental effect of KIR2DL3 on the A haplotype. CONCLUSIONS: Although neither KIR nor HLA appear to have a role in HIV acquisition, our data are consistent with involvement of KIR2DL2 in HIV control. Additional studies to replicate these findings are indicated.

Screening for 'window-period' acute HIV infection among pregnant women in rural South Africa.

OBJECTIVES: The aim of this study was to evaluate the HIV-1 RNA pooled nucleic acid amplification testing (NAAT) strategy to screen pregnant women in the 'window period' of acute HIV infection (AHI) in rural South Africa. METHODS: In 2007 and 2008, 750 consecutive pregnant women on their first antenatal care visit to a primary health care clinic were tested anonymously for HIV infection. HIV-1 RNA pooled NAAT was performed on HIV antibody-negative samples. All positive pools were tested individually and positive samples were classified as incident cases to calculate HIV incidence. RESULTS: The overall HIV prevalence was 37.3% [95% confidence interval (CI) 34.3­41.3]. Of the 467 HIV antibody-negative samples, four (0.9%) were HIV-1 RNA-positive. The mean viral load in the four samples was 386 260 HIV-1 RNA copies/mL (range 64 200­1 228130). The HIV incidence was 11.2%per year (95% CI 0.3­22.1) and all women with AHI were 21 years of age. CONCLUSIONS: Identifying AHI in pregnancy is important for health interventions to reduce perinatal and heterosexual transmission of HIV, and to estimate HIV incidence for epidemiological surveillance.

TB treatment outcomes following directly-observed treatment at an urban outpatient specialist TB facility in South Africa.

The treatment of 450 consecutive new patients with pulmonary TB was evaluated to determine outcome following directly-observed treatment. In all, 176 (39.1%) patients were cured, 23 (5.1%) completed treatment, 80 (17.8%) defaulted treatment, 24 (5.3%) died, 54 (12.0%) were lost to follow-up and 93 (20.7%) were transferred out. Increasing age was significant for death. Males were more likely to default and those with negative pretreatment sputum smears and those who were unemployed were more likely to be lost to follow-up. The overall treatment success rate remains low. Our data suggests that greater emphasis is needed to improve TB treatment success.

Growth and cultural characteristics of Calymmatobacterium granulomatis--the aetiological agent of granuloma inguinale (Donovanosis).

Granuloma inguinale is a chronic destructive granulomatous disease of the genitalia. The clinical diagnosis is often unreliable and the definitive diagnosis is based on the visualisation of 'Donovan bodies' in tissue smears or biopsy specimens. The organism implicated in its aetiology, Calymmatobacterium granulomatis, was reported to have been cultured > 30 years ago, but little is known about the organism because of its fastidious nature and the difficulty in culturing it. Twenty-two biopsy specimens from female patients with clinical and laboratory-confirmed granuloma inguinale were treated with amikacin 10 mg/L and inoculated in a monocyte co-culture system with peripheral blood mononuclear cells (PBMC) from a single donor and autologous sera. The method was subsequently modified by pretreatment of specimens with vancomycin 5 mg/L and metronidazole 10 mg/L in addition to amikacin 10 mg/L for the purpose of decontamination, pooled blood donor PBMC and by the use of heat-inactivated fetal calf serum instead of autologous serum for culture. This modified method was used to culture additional biopsy specimens and genital ulcer scrapings from female and male patients, respectively. All monocyte co-cultures were examined by a rapid Giemsa (RapiDiff) stain and by an indirect immunofluorescence test with immune sera. Representative cultures were examined by transmission electron microscopy. C. granulomatis was successfully isolated in pure culture by the monocyte co-culture system from four biopsy specimens and 14 genital ulcer scrapings. The cultured organisms were visible both intra- and extra-cellularly and were extremely pleomorphic, with characteristic single and biopolar condensation. The numbers of the organisms increased after each passage. All positive cultures showed bright fluorescence when tested with immune sera. Transmission electron microscopy of the cultured bacteria demonstrated a typical gram-negative cell wall consisting of an outer membrane, middle electron opaque layer and an inner plasma membrane. The capsule was thick and electron dense. Numerous electron dense granules were present within the cytoplasm.

Phylogenetic analysis of Calymmatobacterium granulomatis based on 16S rRNA gene sequences.

Calymmatobacterium granulomatis is the aetiological agent of granuloma inguinale - a chronic granulomatous genital infection - and is morphologically similar to members of the genus Klebsiella. This study determined the 16S rRNA gene sequence of C. granulomatis and the taxonomic position of the organism in relation to the genus Klebsiella. Genomic DNA was extracted from C. granulomatis-infected monocytes and from frozen and formalin-fixed paraffin wax-embedded tissue biopsy specimens from patients with histologically proven granuloma inguinale. The 16S rDNA was amplified by PCR with broad range oligonucleotide primers. The amplified DNA fragments were cloned into pMOS vector, digested with Bam HI and Pst1 restriction endonucleases, hybridised with a gram-negative bacterial probe (DL04), sequenced in both directions by the automated ALF DNA sequencer, verified on an ABI Prism 377 automated sequencer and analysed with DNASIS and MEGA software packages. Sequence analysis revealed DNA homology of 99% in C. granulomatis from the different sources, supporting the belief that the bacteria in the culture and the biopsy specimens belonged to the same species, although there was some diversity within the species. Phylogenetically, the strains were closely related to the genera Klebsiella and Enterobacter with similarities of 95% and 94% respectively. C. granulomatis is a unique species, distinct from other related organisms belonging to the gamma subclass of Proteobacteria.

Ultrastructure of Calymmatobacterium granulomatis: comparison of culture with tissue biopsy specimens.

The ultrastructural features of cells of Calymmatobacterium granulomatis from monocyte co-cultures and tissue biopsy specimens were compared. In cultures the bacteria were mainly extracellular, i.e., not within membrane-bound vacuoles. The bacterial body was surrounded by a uniformly extensive homogeneous layer with a relatively high electron density. This layer varied considerably in tissue biopsy specimens, having either homogeneously electron-dense or delicate web-like structures with varying density and thickness. In tissue specimens the bacteria were located predominantly within vacuoles of varying sizes in the cytoplasm of the macrophages and, occasionally, extracellularly within the intercellular spaces of the stroma. The bacterial cytoplasm contained ribosomes scattered throughout with electron-dense granules located peripherally. The trilaminar cell-wall structure was typical of a gram-negative organism, comprising an outer membrane, a middle electron-opaque layer and an inner plasma membrane. Surface structures such as fimbriae, flagella and bacteriophages were not identified in specimens from either source.

Granuloma inguinale in association with pregnancy and HIV infection.

OBJECTIVES: A retrospective study to confirm the clinical impression of an increasing prevalence of granuloma inguinale (GI) in women, and to evaluate its association with pregnancy and HIV infection. METHODS: Clinical records of all patients with a definitive diagnosis of GI attending the gynecology and antenatal clinics at King Edward VIII Hospital, Durban, South Africa, over a period of 36 months (January 1991-December 1993). RESULTS: A total of 123 women were diagnosed with GI. The diagnosis was made by tissue smear alone in 21% (n = 26), histology 43% (n = 53) and by a combination of smear and histology in the rest. Forty-two percent (n = 52) were pregnant. The only difference between pregnant and non-pregnant women were the presence of rectal and pelvic lesions in the latter. Sixty-nine percent (n = 36) delivered vaginally while the remaining (n = 16) were delivered by cesarean section. The indications for cesarean section were obstetric except for a patient in labor with extensive untreated vulval granuloma. In the majority (85%) GI had no influence on pregnancy outcome. There was no evidence of congenital GI in the neonates. Twenty-seven percent (30/113) had positive syphilis serology and 16% (18/110) had antibody to HIV. There were no differences in the clinical features and outcome of HIV positive and negative women. CONCLUSION: This study shows that GI is increasing in pregnancy in Durban, South Africa. Despite the concern that pregnancy promotes dissemination of GI, such an effect could not be established as the clinical response to treatment and outcome were similar in both pregnant and non-pregnant women. Infection with HIV also did not alter the clinical presentation and outcome of the disease in the patients studied.

Serum and tissue concentrations of intravenous cefotaxime during aortic surgery.

Ten patients submitted to prosthetic aortic replacement received repeated intravenous bolus doses of cefotaxime (1 g) during induction of anaesthesia and at 2 hourly intervals during the operation. Hourly blood samples were taken and serum cefotaxime concentrations measured using high performance liquid chromatography. Subcutaneous fat, aortic and peripheral arterial wall samples were taken at various intervals intra-operatively, and tissue antibiotic levels were determined by bio-assay using an agar well diffusion method. Mean serum levels were consistently in excess of 50 mg/l which exceeds the MIC90 of virtually all aerobic and anaerobic pathogens. Tissue levels obtained exceeded the MIC90 of most pathogens. Cefotaxime would appear to provide adequate perioperative prophylactic cover in the dosage regimen used.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Durban, South Africa.

One hundred and forty clinical isolates of Neisseria gonorrhoeae were screened for production of penicillinase by the intralactam strip method and chromogenic cephalosporin test. Minimum inhibitory concentrations (MICs) of penicillin, ampicillin, tetracycline, cefoxitin, cefuroxime, cefotaxime, sulphamethoxazole-trimethoprim (ratio 19/1), and spectinomycin, were measured for 100 strains by the agar dilution method. Seven (5%) of the 140 isolates were identified as penicillinase producing N gonorrhoeae (PPNG). The MICs of penicillin for the seven PPNG strains ranged from 0 X 25 mg/1 to 2 mg/1. Of the 93 non-PPNG strains, 80 (86%) were fully susceptible to penicillin with MICs ranging from 0 X 0037 mg/1 to 0 X 06 mg/1 and 13 (14%) were of intermediate penicillin resistance with MICs greater than or equal to 0 X 125 mg/1. Of the 100 isolates tested, 86% were fully susceptible to tetracycline with MICs of less than 1 mg/1. No spectinomycin resistant strains were encountered in this study. All gonococcal strains were susceptible to the cephalosporins tested as well as to sulphamethoxazole-trimethoprim.

Bacterial vaginosis and lower genital tract infections in women attending out-patient clinics at a tertiary institution serving a developing community.

The prevalence of bacterial vaginosis and other lower genital tract infections were determined in women from a developing community. Patients were recruited from four out-patient clinics of a large urban tertiary referral hospital serving the indigent Black population of KwaZulu/Natal, South Africa. A total of 208 women attending the sexually transmitted diseases (STD) (n = 51), colposcopy (n =50), family planning (n = 52) and antenatal (n = 55) clinics were investigated. Endocervical and vaginal specimens were collected for microbiological investigation of recognised sexually transmitted pathogens. Estimation of vaginal pH, amine test and wet smear microscopy were performed at the bedside. Peripheral venous blood was obtained for serological tests for syphilis, hepatitis B surface antigen and antibody to the human immunodeficiency virus (HIV). Vaginal infections were detected in a total of 50% (104) of women, endocervical infections alone in 9% (18) and concurrent vagino-endocervical infections in 20% (41). Bacterial vaginosis (BV) was diagnosed in 35% (73) and its prevalence amongst different clinic populations ranged from 25% to 41% with no significant differences between any groups. Trichomoniasis was detected significantly more often in women attending the STD and antenatal clinics. Endocervical infections were found mainly in women attending the STD clinic, though the prevalence of Chlamydia trachomatis amongst the other clinic attenders ranged from 13% to 20%. Micro-organisms such as Gardnerella vaginalis, Mycoplasma hominis, anaerobes and curved Gram-negative rods were found in significantly higher number of women with BV. This study confirms the high prevalence of vaginal, endocervical and mixed vagino-endocervical infections in women from developing communities. The high prevalence of bacterial vaginosis as a single infection and its association with other recognised sexually transmitted pathogens in a large proportion of women, is of significance since such infections not only predispose to ascending upper genital tract infections but are also associated with complications in pregnancy such as premature rupture of membranes, preterm labour and endometritis.

Granuloma inguinale (donovanosis) of the oral cavity. A case report.

A case of granuloma inguinale of the oral cavity clinically resembling actinomycosis is reported. This condition should be considered in the differential diagnosis of chronic ulcerative lesions of the mouth, especially in patients with associated genital ulcers.

The microbial aetiology of genital ulcers in black men in Durban, South Africa.

The microbial aetiology of genital ulcers was assessed in 100 black men attending a sexually transmitted disease (STD) clinic in Durban, South Africa. Forty patients harboured Haemophilus ducreyi, one hepes simplex virus, and one Neisseria gonorrhoeae. Syphilis was diagnosed in 44 patients on the basis of dark field microscopy or positive syphilis serology test results, or both. Of these 44 patients, eight also harboured N ducreyi, one herpes simplex virus. Lymphogranuloma venereum was diagnosed in one patient. No cause of ulceration could be found in the remaining 16 patients.

Sudden death caused by myocardial tuberculosis: case report and review of the literature.

A 25-year-old fit man died suddenly while playing social soccer. Autopsy revealed an infiltrative lesion involving the left ventricle with overlying pericarditis. No other significant pathologic changes were observed. Histologic examination showed necrotizing granulomatous inflammation. No acid-fast bacilli were demonstrated in the pericardial fluid or on histologic examination. The presence of Mycobacterium tuberculosis DNA complex was confirmed by use of the ligase chain reaction technique. The differential diagnosis of myocardial tuberculosis includes sarcoidosis, rheumatic fever, rheumatoid arthritis, giant-cell-containing tumors, idiopathic (giant-cell) myocarditis, and bacterial infections such as tularemia and brucellosis. This case illustrates the protean manifestations of tuberculosis and highlights the use of molecular biologic techniques in arriving at a definitive diagnosis in cases of suspected tuberculosis.

Transepithelial elimination of cutaneous vulval granuloma inguinale.

BACKGROUND: Transepithelial elimination (TEE), a distinct and well-known entity, is a process during which the skin eradicates undesirable or irritative dermal substances through intact epidermis or follicular epithelium by passive or active means. Although TEE is being described in an increasing number and range of pathological processes, to date, TEE of granuloma inguinale (GI) remains unrecorded in the English-language literature. The aims of this study were: 1) To appraise the light microscopic and ultrastructural morphological epidermal changes that are associated with TEE of cutaneous vulval GI; and 2) To determine the role of intra-epidermal leucocytes and histiocytes in the pathogenesis of TEE of vulval GI. METHODS: This is a retrospective 9-year histopathological review of all cases diagnosed and coded as vulval granuloma inguinale in the Department of Anatomical Pathology, Nelson R. Mandela School of Medicine, University of Natal, Durban, South Africa. Ultrastructural evaluation was performed on selected cases using a Jeol transmission electron microscope. RESULTS: Of 53 skin biopsies from 47 patients with vulval GI, 43 were suitable for the study. The age range of patients was 15-40 years (mean age=22 years). There were eleven papular, twelve nodular, seven verrucous and thirteen ulcerative lesions. Donovan bodies within macrophages, free-lying Donovan bodies and dense aggregates of neutrophils and plasma cells were seen in the dermis of all biopsies. There was consistent overlying pseudoepitheliomatous hyperplasia. The dermal inflammatory infiltrate hugged the dermo-epidermal junction and appeared entrapped between elongated and acanthotic epidermal rete ridges and pegs. Transepidermal neutrophil microabscesses, histiocytes containing Donovan bodies and neutrophilic and histiocytic fragmentation were present. A variable number of free-lying and intra-histiocytic Donovan bodies and neutrophils were present on the surface of the epidermis. On ultrastructural investigation epidermal spongiosis, intracellular oedema, free-lying, intra-neutrophilic and intra-histiocytic Donovan bodies, and intact and degenerating neutrophils and histiocytes were evident between keratinocytes. The degenerative histiocytes demonstrated marked vacuolation, mitochondrial swelling and bacilli within phagolysosomal vacuoles, bound by intact or disrupted limiting membranes. CONCLUSION: The inflammatory infiltrate at the epitheliomesenchymal interface, pseudoepitheliomatous hyperplasia, intra-epidermal accumulation and disintegration of neutrophils and histiocytes, and the associated release of lytic enzymes, play important contributory roles in TEE of GI. TEE of infectious agents is a poorly recognised mechanism of spread of infectious diseases and represents a public health hazard. In cutaneous vulval GI, TEE is highlighted as a hitherto unrecognised, potential method of spread of Calymmatobacterium granulomatis.

Antimicrobial susceptibilities of Gardnerella vaginalis.

The in vitro susceptibilities of 93 clinical isolates of Gardnerella vaginalis to 25 antimicrobial agents were determined by the agar dilution method. All isolates were susceptible to penicillin, ampicillin, erythromycin, clindamycin, chloramphenicol, and trimethoprim. Activity was poor for vancomycin, LY146032, the cephalosporins, ciprofloxacin, and imipenem. Some resistance was observed with tetracycline and minocycline. The MICs of metronidazole paralleled those of tinidazole, with the hydroxymetabolite of metronidazole being the most active. One strain was resistant to all three agents. Marked resistance to aztreonam, amikacin, and sulfamethoxazole was observed.

A randomized controlled trial of azithromycin versus doxycycline/ciprofloxacin for the syndromic management of sexually transmitted infections in a resource-poor setting.

A randomized controlled trial was carried out to assess the effectiveness of azithromycin versus a standard regimen with doxycycline/ciprofloxacin in the treatment of sexually transmitted infections in a resource-poor environment. Infection with Chlamydia trachomatis was cured in 23/24 (95.8%) of women in the azithromycin arm versus 19/21 (90.5%) in the doxycycline arm (P = 0.6), resulting in three treatment failures. Gonorrhoea was cured in 55/56 (98.2%) women, with one treatment failure in a patient with concomitant C. trachomatis infection. These results indicate that a single oral dose of azithromycin may prove to be a more effective and convenient treatment for sexually transmitted infections in women in a resource-poor environment

The association between sexually transmitted pathogens and cervical intra-epithelial neoplasia in a developing community.

OBJECTIVE--To determine the association of sexually transmitted pathogens in women with cervical intra-epithelial neoplasia (CIN). SETTING--An urban tertiary referral hospital serving a large indigent developing community. PARTICIPANTS--48 women attending a colposcopy clinic and 49 women attending a family planning clinic. METHODS--Vaginal, endocervical, rectal swab specimens and sera were collected for the detection of sexually transmitted pathogens. Cervical cytology was performed on all patients. Women attending the colposcopy clinic had confirmation of abnormal cervical cytology by colposcopic directed biopsy. RESULTS--The mean age of women with CIN (33 years) was significantly greater than that of the women without CIN (28 years) and that of the family planning group (26 years). There was a high prevalence of sexually transmitted pathogens in all women. A significantly higher prevalence of bacterial vaginosis was found in women with CIN compared to those without (50% vs 20%; p = 0.034). The human papilloma virus (HPV) was detected in 46% of women with CIN and 65% of those without CIN. Chlamydia trachomatis (21%) and Trichomonas vaginalis (39%) were detected frequently in women with CIN. C. trachomatis (14%-21%) was detected more frequently than Neisseria gonorrhoeae (3-5%) in all asymptomatic women studied. CONCLUSION--This study demonstrates a high prevalence of sexually transmitted pathogens in women with and without CIN as well as family planning clinic attenders. Bacterial vaginosis was a significant finding in women with CIN. C. trachomatis was detected in a high proportion of all women studied and found more commonly than N. gonorrhoeae. We therefore recommend that all women attending gynaecological services in a developing community be investigated and treated for sexually transmitted diseases.

Susceptibility of penicillinase-producing and non-penicillinase-producing strains of Neisseria gonorrhoeae isolated in Durban, South Africa, to 15 beta-lactam antibiotics.

Using two different inoculum sizes (10(4) and 10(7) organisms), we tested 35 penicillinase-producing (PPNG) and 72 non-PPNG strains of Neisseria gonorrhoeae falling into three categories (penicillin-sensitive, intermediately resistant, and resistant) by the agar-dilution method against 15 beta-lactam antibiotics. With the exception of penicillin, ampicillin, amoxicillin, and cephradine, the isolates were uniformly sensitive to all the other antibiotics tested (including spectinomycin, tetracycline, rosoxacin, sulfamethoxazole-trimethoprim [19:1 ratio], and kanamycin). Ceftriaxone and cefotaxime were the two most active compounds tested; all strains were inhibited at concentrations of 0.007 and 0.015 microgram/ml, respectively. Isolates with intrinsic resistance to penicillin were less susceptible to cefoxitin and cefuroxime than were PPNG strains. The effect of inoculum size varied for the different antibiotics; penicillin and cefaclor showed this effect only with PPNG strains, whereas for ampicillin and amoxicillin it was also seen with non-PPNG strains. Cephamandole showed this effect with both penicillin-sensitive and PPNG strains. In view of the increasing resistance to penicillin, we would recommend that alternative drugs be used as first line therapy for gonorrhea in South Africa.

Tampon sampling for diagnosis of bacterial vaginosis: a potentially useful way to detect genital infections?

Genital tract infections are important causes of ill health in developing countries, but diagnosis is difficult. Bacterial vaginosis (BV) was correctly diagnosed by using a vaginal specimen obtained by tampon sampling in 22 of 24 women (91.6%) for whom BV was diagnosed by Gram staining. The yield for other vaginal infections was higher (28% for Trichomonas vaginalis and 32.7% for Candida albicans) than it was for cervical infections (0% for Neisseria gonorrhoeae and 30% for Chlamydia trachomatis). Tampon sampling was acceptable to patients and may facilitate diagnosis of genital infections in developing countries.

The emergence of penicillinase-producing strains of Neisseria gonorrhoeae in Durban.

Gonorrhoea was diagnosed in 179 (87%) of 206 Black males who presented with urethritis at a sexually transmitted disease clinic at the King Edward VIII Hospital, Durban. Penicillinase-producing strains of Neisseria gonorrhoeae were detected in 7 (5%) of 140 gonococcal isolates, and a further 13 strains were relatively resistant to penicillin. Microscopic examination of Gram-stained smears provided a rapid presumptive diagnosis of gonorrhoea in 162 cases. The modified Thayer-Martin medium proved marginally superior to chocolate agar for the isolation of N. gonorrhoeae from urethral exudates. The causation and laboratory diagnosis of urethritis in males and the antibiotic susceptibility pattern of the gonococcal isolates are discussed.