Distinguishing between male and female gametocytes.

<p>A) IFAT image of stage V gametocytes showing all fluorophores from the secondary antibodies and DAPI (red, green and blue). B) Rhodamine (red) is visualized, which reacts with anti-α-tubulin II antibodies. C) DAPI (blue) is visualized, reacting with the nuclear material. D) FITC (green) is visualized, reacting with anti-Pfg377 antibodies. The white dotted circle shows gametocytes staining with anti-Pfg377 and anti-α-tubulin II (females), whereas the yellow dotted circle reveals one gametocyte that only reacts with anti-α-tubulin II antibodies (male). It can be seen that gametocytes which reacted with anti-Pfg377 antibodies, also reacted with anti-α-tubulin II antibodies, when comparing B) and D). Parasites were magnified ×1000.</p

Stage III and later gametocytes visualized with anti-Pfg377 and anti-α-tubulin II antibodies.

<p>Fluorescent staining of α-tubuIin (red) generated a characteristic striated pattern, particularly in earlier gametocytes. Expression of Pfg377 (green) was not seen prior to stage III. A) stage III; B) late stage III; C) stage IV; D) late stage IV; E) stage V; F) activated female gametocyte. Nuclear material was stained with DAPI, appearing blue in colour. Parasites were magnified ×1000.</p

RT-PCR of sexual stage and sex specific proteins during gametocytogenesis.

<p>Transcripts of Pfs16, α-tubulin II and Pfg377 were amplified from preparations of stage I–V gametocytes. Gel electrophoresis of amplified products; + and − refer to presence or absence of reverse-transcriptase in the cDNA reaction prior to amplification. PC: positive control; NC: negative control. Cultures were not 100% synchronous. All samples (including positive controls) were run on a single gel at the same time. Lower bands (<100 bp) in each panel, particularly prominent in the absence of cDNA amplification, are primer dimers. Minor contamination of pfg377 DNA is visible in lanes 4, 8 and NC, lower panel.</p

‘Apparent’ sex ratio during gametocytogenesis.

<p>IFAT counts were performed on gametocyte preparations of stage II, stage III, stage IV, stage V, and 30 minutes after induction of activation (AG). Standard error is estimated for the ratio based on error calculated for the mean estimates of the proportion of males across the three independent cultures analysed.</p><p>% Alpha-tub. II+, % Pfg377+: indicate the percentage of gametocytes reacting with the given α-tubulin II and Pfg377 antibodies respectively.</p

Differential staining of gametocytes during gametocytogenesis.

<p>Pfg377 and α-tubulin II double-stained IFAT slides prepared on: A) day 2 (stage II); B) day 5 (stage III); C) day 7 (stage IV); D) day 11 (stage V); E) after gametocyte activation (AG). Parasites were magnified ×1000. Early stage parasites were more likely to be damaged during the purification process, and appear as stained with DAPI alone (panels A, B).</p

Sex ratios established using light microscopy.

<p>Purified stage V gametocytes stained with Giemsa. A.) 3D7 magnet-purified gametocytes magnified ×1000. B.) (Detail) The cytoplasm of male gametocytes (MG) can be seen to stain pink and that of female gametocytes (FG) to stain purple; male gametocytes are smaller than females, the nucleus is bigger in males than in females, the granules of the malaria pigment are centrally located in female gametocytes and more widely scattered in males gametocytes The other 4 discriminatory characters can also be discerned.</p

Trends of Zambia's tuberculosis burden over the past two decades

OBJECTIVES To study trends in Zambia's TB notification rates between 1990 and 2010 and to ascertain progress made towards TB control. METHODS Retrospective review of TB notification returns and TB programme reports for the period from 1990 to 2010. RESULTS Two distinct TB trend periods were identified: a period of rising trends up to a peak between 1990 and 2004 and a period of moderately declining trends between 2004 and 2010. Treatment outcomes improved over the two decades. Data on trends in paediatric TB, TB in prisoners and TB in pregnant women remain scanty and unreliable owing to poor diagnostic capability. There were no data available on trends on drug-resistant TB because of the lack of laboratory services to perform drug sensitivity testing. CONCLUSIONS The period of increasing TB between 1990 and 2000 coincided with an increase in HIV/AIDS. The period of slightly decreasing TB between 2004 and 2010 can be attributed to improved TB care, sustained DOTS implementation and improvement in TB diagnostic services. Newer diagnostics technologies for the rapid diagnosis of active TB cases and for drug-resistant testing, recently endorsed by the WHO, need to be implemented into the national TB programmes to detect more cases and to provide epidemiological and surveillance data from which to obtain an evidence base for guided investments for TB control. Alignment of TB and HIV services is required to achieve improved management outcomes.</p

Tuberculosis and Tuberculosis/HIV/AIDS-Associated Mortality in Africa: The Urgent Need to Expand and Invest in Routine and Research Autopsies

Frequently quoted statistics that tuberculosis and human immunodeficiency virus (HIV)/AIDS are the most important infectious causes of death in high-burden countries are based on clinical records, death certificates, and verbal autopsy studies. Causes of death ascertained through these methods are known to be grossly inaccurate. Most data from Africa on mortality and causes of death currently used by international agencies have come from verbal autopsy studies, which only provide inaccurate estimates of causes of death. Autopsy rates in most sub-Saharan African countries have declined over the years, and actual causes of deaths in the community and in hospitals in most sub-Saharan African countries remain unknown. The quality of cause-specific mortality statistics remains poor. The effect of various interventions to reduce mortality rates can only be evaluated accurately if cause-specific mortality data are available. Autopsy studies could have particular relevance to direct public health interventions, such as vaccination programs or preventive therapy, and could also allow for study of background levels of subclinical tuberculosis disease, Mycobacterium tuberculosis-HIV coinfection, and other infectious and noncommunicable diseases not yet clinically manifest. Autopsies performed soon after death may represent a unique opportunity to understand the pathogenesis of M. tuberculosis and the pathogenesis of early deaths after initiation of antiretroviral therapy. The few autopsies performed so far for research purposes have yielded invaluable information and insights into tuberculosis, HIV/AIDS, and other opportunistic infections. Accurate cause-specific mortality data are essential for prioritization of governmental and donor investments into health services to reduce morbidity and mortality from deadly infectious diseases such as tuberculosis and HIV/AIDS. There is an urgent need for reviving routine and research autopsies in sub-Saharan African countries.</p

Tuberculosis Diagnostics and Biomarkers: Needs, Challenges, Recent Advances, and Opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics.</p