A continuously monitored colorimetric method for detection of Mycobacterium tuberculosis complex in sputum.

SETTING: Mbeya, Tanzania. OBJECTIVE: To develop a new liquid culture method to detect Mycobacterium tuberculosis complex (MTC) in sputum using 2,3-diphenyl-5-thienyl-(2)-tetrazolium (STC), the nitrate reductase assay (NRA) and p-nitrobenzoic acid (PNB). DESIGN: Ninety-three sputum samples collected from 18 tuberculosis patients were decontaminated with N-acetyl-L-cysteine-sodium hydroxide using MGIT™ 960 and in STC-NRA cultures, both in the presence and in the absence of PNB, an inhibitor of MTC growth. The reduction of STC by colour change indicated mycobacterial growth; NRA was then performed to confirm MTC. RESULTS: STC-NRA culture was positive for acid-fast bacilli in 66/93 (71%) samples, of which 60/93 (64.5%) were identified as MTC-positive and 6/93 (6.5%) as indeterminate mycobacteria. MGIT indicated MTC in 59/93 (63.4%) cultures. Contamination was detected in 12/93 (13%) STC-NRA cultures vs. 29/93 (31.2%) MGIT cultures. The mean time to detection (TTD) of MTC using STC-NRA was 14 days and 7 days using MGIT. CONCLUSION: The STC-NRA method is sensitive for the detection of MTC in sputum. TTD increased with duration of anti-tuberculosis treatment, highlighting the value of this method in monitoring treatment success. The method is simple and inexpensive and, unlike MGIT, does not require technical equipment. The preliminary performance characteristics of the method should be further evaluated in larger studies.

Performance of the Xpert® MTB/RIF assay in an active case-finding strategy: a pilot study from Tanzania.

In this pilot study, we evaluated the Xpert® MTB/RIF assay in an active case-finding strategy, using two spot sputum samples collected within a 1-hour interval from household contacts of smear-positive TB index cases. Tuberculosis (TB) confirmed by culture served as the reference standard. Among 219 enrolled contacts, the yield of active TB was 2.3%. While the sensitivity of smear microscopy was 60% (95%CI 14.7-94.7), Xpert MTB/RIF achieved a sensitivity of 100% (95%CI 47.81-100.0). All culture-confirmed cases tested positive by Xpert MTB/RIF on the first submitted sample, suggesting that the evaluation of only one sample could be sufficient for TB diagnosis in this context.

Evaluation of Diagnos TB AG, a flow-through immunoassay for rapid detection of pulmonary tuberculosis.

We evaluated the diagnostic performance of the Diagnos TB AG immunoassay in 171 Tanzanians with suspected pulmonary tuberculosis (TB). The sensitivity and specificity, and positive and negative predictive values of the rapid test for the detection of pulmonary TB in this population were respectively 60.0%, 33.3%, 40.3% and 52.6%. In its current configuration, this test will not help overcome difficulties in the rapid diagnosis of TB.

Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparumin south-west Tanzania

Background: In Tanzania; drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes. Methods: In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema; Mbeya region; south-western Tanzania; the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps; conferring SP resistance; as well in cytb which is linked to resistance to atovaquone. Reesults: Pfcrt T76 occurs in 50and pfmdr1 Y86 in 51.7. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected.Conclusions: These findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy

Detection of mycobacterial lipoarabinomannan with an antigen-capture ELISA in unprocessed urine of Tanzanian patients with suspected tuberculosis.

A direct antigen-capture ELISA based on the detection of mycobacterial lipoarabinomannan (LAM) in unprocessed urine was evaluated for its usefulness in clinical practice. In Tanzania, 231 patients with suspected pulmonary tuberculosis (TB) and 103 healthy volunteers were screened with standard TB tests and with the new LAM-ELISA. Of 132 patients with confirmed pulmonary mycobacterial disease (positive sputum culture), 106 were positive using the LAM-ELISA (sensitivity 80.3%). In comparison, the sensitivity of acid-fast bacilli (AFB) sputum microscopy was 62.1% (82 of 132 confirmed cases). Of the 231 patients, 17 were both culture- and AFB-negative, but had typical radiographic signs of pulmonary mycobacterial infection and did not respond to antibiotic treatment. Of these 17 patients, 13 (76.5%) had positive LAM-ELISA test results. To define the specificity of the assay, urine samples from 103 healthy volunteers were also screened using LAM-ELISA. All but one had an optical density below the cut-off (specificity 99%). Of interest was a significant correlation between level of microscopic density of mycobacteria in sputum and LAM antigen concentration in urine (chi2=8.44). The LAM-ELISA is a field-adapted tool that can improve screening standards in countries with a high incidence of TB.

In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection.

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.

In-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple PCR primer approach.

Co-infections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of co-infections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.

Methodological lessons from a cohort study of high risk women in Tanzania.

OBJECTIVES: To describe the development, characteristics, and follow up of a high risk cohort of women in Tanzania. Differences in social background and sexual behaviour of women working in traditional and modern alcohol selling workplaces are shown. METHODS: Data from questionnaires four months before the enrollment of the cohort, at enrollment, and at 32 months were compared. Key informant interviews, social mapping exercises, and focus group discussions were held before the start of the cohort. RESULTS: In the absence of organised prostitution, two different groups of women with high risk exposure were identified during the baseline survey: female workers in modern alcohol selling places such as bars, guesthouses, and restaurants (barmaids) and in traditional places (local brew sellers). Overall, the population had a mean age of 27.7 years with barmaids tending to be younger (24.3 years) than local brew sellers (34.2 years). The main duration of stay in the current workplace was 2.1 years (barmaids 0.9 years; local brew sellers 4.1 years). Barmaids were more likely to have paying casual sex partners than local brew sellers and used condoms more regularly. Local brew sellers tend to be more stable with only 10% lost to follow up after 32 months compared with 24.4% of the bar workers. CONCLUSIONS: Preliminary work revealed major differences in characteristics and behaviour between women working in modern and traditional alcohol selling outlets. Thorough preparation of the study, close monitoring of the cohort, and provision of selected benefits resulted in high retention rates over a 32 month project in a highly mobile population.

Baseline survey of sexually transmitted infections in a cohort of female bar workers in Mbeya Region, Tanzania.

OBJECTIVES: To determine baseline prevalence of sexually transmitted infections (STI) and other reproductive tract infections (RTI) and their association with HIV as well as sociodemographic and behavioural characteristics in a newly recruited cohort of female bar workers in Mbeya Region, Tanzania. METHODS: 600 female bar workers were recruited from 17 different communities during September to November 2000 and underwent gynaecological examination, laboratory testing for HIV/STI, and interviews using structured questionnaires. RESULTS: HIV-1 seroprevalence was 68%. Prevalences of STI/RTI were high titre syphilis (TPPA/RPR >/=1/8), 9%; herpes simplex virus 2 antibodies, 87%; chlamydia, 12%; gonorrhoea, 22%; trichomoniasis, 24%; and bacterial vaginosis, 40%. HIV infection was associated with TPPA and HSV-2 seropositivity, bacterial vaginosis and clinically diagnosed genital ulcers, blisters, and warts. Reported high risk sexual behaviour during the past year (having multiple casual partners) was associated with prevalent STI. CONCLUSION: Female bar workers in Mbeya are at high risk of STI and HIV infection. Targeted STI/HIV prevention interventions for these women and their sexual partners need to be reinforced. Methods should be sought to improve healthcare seeking and to provide easily accessible and affordable STI care services.

High proportion of unrelated HIV-1 intersubtype recombinants in the Mbeya region of southwest Tanzania.

BACKGROUND: In Mbeya, a rural region of southwest Tanzania, HIV-1 subtypes A, C and D have been co-circulating since the early 1990s. OBJECTIVE: To define to what extent the co-existence of subtypes has led to recombinant HIV-1 strains and whether there is evidence for epidemic spread of any circulating recombinant form. METHODS: Nine HIV-1-seropositive young adults from Mbeya Town with no evident high-risk behaviour contributed peripheral blood mononuclear cells for this study. Nine virtually full-length-genome-sequences were amplified from this DNA and phylogenetically analysed. RESULTS: Out of the nine samples, two were subtype A (22%), two were subtype C (22%) and five were recombinants (56%): four A/C recombinants and one C/D recombinant. None of the recombinants were related to each other; all of them had different mosaic structures. Most of the genome in the recombinants was subtype C. CONCLUSION: A high proportion of unrelated intersubtype recombinants, none of them apparently spreading in the population, may be present in southwest Tanzania.