Diagnosis of bubonic plague by PCR in Madagascar under field conditions

The diagnostic value of a PCR assay that amplifies a 501-bp fragment of the Yersinia pestis caf1 gene has been determined in a reference laboratory with 218 bubo aspirates collected from patients with clinically suspected plague managed in a regional hospital in Madagascar. The culture of Y. pestis and the detection of the F1 antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) were used as reference diagnostic methods. The sensitivity of PCR was 89% (57 of 64) for the Y. pestis-positive patients, and 80.7% (63 of 78) for the F1 Ag-positive patients. The specificity of PCR for the culture-, F1 Ag-, and antibody-negative patients (n = 105) was 100%. Because in Madagascar most patients with plague are managed and their clinical samples are collected in remote villages, the usefulness of PCR was evaluated for routine diagnostic use in the operational conditions of the control program. The sensitivity of PCR was 50% (25 of 50) relative to the results of culture and 35.2% (19 of 54) relative to the results of the F1 Ag immunocapture ELISA. The specificity of PCR under these conditions was 96%. In conclusion, the PCR method was found to be very specific but not as sensitive as culture or the F1 Ag detection method. The limitation in sensitivity may have been due to suboptimal field conditions and the small volumes of samples used for DNA extraction. This technique is not recommended as a routine diagnostic test for plague in Madagascar.     

Resurgence of the plague in the Ikongo district of Madagascar in 1998. 2. Reservoirs and vectors implicated

Our survey of mammals and fleas arose as a result of an outbreak of bubonic plague at an usually low altitude in the Ikongo district (Madagascar), while a previous study had found anti-F1 antibodies in an endemic hedgehog. Animals were sampled with live traps in two hamlets (Antanambao-Vohidrotra, 540 m alt. and Ambalagoavy, 265 m alt.) and with pitfall traps in a neighbouring forest (750 m alt.). Rat fleas were collected by brushing the fur and free-living fleas by use of light traps. The introduced shrew Suncus murinus was found only in the village of Ambalagoavy while the black rat (Rattus rattus) was found in all three sites and the only seropositive rat was caught at Antanambao-Vohidrotra. In contrast, among the Tenrecidae (endemic shrews and hedgehogs) found in the forest near the first village, four animals were found seropositive for anti-F1 antibodies. One of them was carrying the endemic flea Paractenopsyllus pauliani, not yet reported as a vector of plague. The endemic vector of plague, Synopsyllus fonquerniei, was found only in the first village of Antanambao-Vohidrotra, and the cosmopolite flea Xenopsylla cheopis only in Ambalagoavy. Although no Yersinia pestis could be isolated and no F1-antigen could be detected in these animals, we found evidence of the recent transmission of plague in Antanambao-Vohidrotra and the nearby forest, but not in Ambalagoavy. These data corroborate with the sylvatic plague cycle hypothesis in Madagascar and its involvement in the outcome of the bubonic plague outbreak in this district.     

Recrudescence and geographic extension of the plague in Madagascar from 1980 to 1999

Plague was introduced to Madagascar in 1898, and it has been characterized by a predominant distribution to the central highlands in the following decades. An increase of plague cases has been observed in the past 20 years, in particular in the capital, Antananarivo, and in the coastal town, Mahajanga, after long periods of silence in 28 and 63 years, respectively. A total of 2,982 confirmed or presumptive cases were reviewed in order to describe the changes in the epidemiological pattern of the disease from 1980 through 1999. The mean annual number of plague cases has increased from 33 during the 1980-1984 period to 298 during the 1995-1999 period. A similar trend of distribution has been observed from the first period to the second by an increase of endemic districts above 800 m altitude from 17 to 37. However, the lethality rate has in the same 20 years observation period decreased from 41.6% to 20.7%, probably due to re-enforcing measures as part of the national control program.     

Variation in Gamma Interferon Responses to Different Infecting Strains of Mycobacterium tuberculosis in Acid-Fast Bacillus Smear-Positive Patients and Household Contacts in Antananarivo,Madagascar

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major cause of global morbidity and mortality throughout the world. It is estimated that there are in excess of new 8 million cases of TB each year, and this represents just the tip of the iceberg. Infection with M. tuberculosis leads to clinically active TB in about 5 to 10% of exposed individuals. A much higher proportion of exposed individuals apparently become latently infected, and these individuals may remain noninfectious and symptom free for years. Approximately one-third of the world population is thought to be latently infected with M. tuberculosis. However, under some circumstances (in about 5% of the latently infected people), the host immune response is perturbed and latent M. tuberculosis infection may develop into clinically active TB (52). This process is most prominent in individuals coinfected with human immunodeficiency virus (HIV), but it can also occur with impairment of the immune system associated with old age, malnutrition, anti-inflammatory drug treatment, etc. Reactivation of latent disease is thought to contribute roughly half of all TB cases, and thus, understanding the factors controlling the development of acute primary TB or latent infection is crucial to TB control (64).Gamma interferon (IFN-γ) production has been widely used to study infection and to examine the human immune response to specific M. tuberculosis antigens. The 6-kDa early secreted antigenic target (ESAT-6) antigen, encoded by genes located within region of difference 1 (RD1) of the M. tuberculosis genome, is much more specific for M. tuberculosis than purified protein derivative (PPD), as these genes were deleted from M. bovis in the development of BCG substrains or are not found in most environmental mycobacteria (29, 53). Some studies showed that the level of IFN-γ release in response to ESAT-6 could identify TB contacts at risk of developing active disease after recent infection (3, 18, 30). CFP7 or TB10.4 is an immunodominant antigen recognized by TB patients and M. bovis BCG-vaccinated subjects, while ESAT-6 is specific to TB patients and induces a strong IFN-γ response (51). Moreover, since CFP7 induces strong protection against infection by M. tuberculosis, it was proposed to be a TB vaccine candidate (1, 19).There is a growing number of observations indicating that TB cases resulting from infection with epidemic strains, such as the W-Beijing strains (22, 35, 39, 44), may display a more severe pathology or more severe symptoms. Beijing strains were also found to induce higher fevers in pulmonary TB patients than other strains (62). In addition, the Beijing genotype, which is responsible for more than 80% of TB cases in China, was associated with virulence and high transmissibility (7, 28). The same has been found more recently with the RD(Rio) strains belonging to the Latin America-Mediterranean (LAM) family (38). Despite the fact that other epidemiological and clinical studies have failed to confirm any association between the mycobacterial genotype and the clinical presentation (8, 41, 43), the immunological aspects of infection with these strains is still of interest and poorly described.Epidemiological studies carried out in Madagascar showed no association between IS6110 patterns and clinical tuberculosis presentation (47), but they did reveal a heterologous population of M. tuberculosis strains, including the existence of a high frequency of unusual genotypes, such as the shared type 109 from the EAI8-MDG family (SpolDB4) (10) or strains with a single copy of IS6110 (24, 46). Since there are limited data on the correlation of the strain genotype with clinical features or the host immune response in patients and their contacts (57, 59), we investigated the IFN-γ response to the ESAT-6, CFP7, and PPD antigens in pulmonary TB patients and their household contacts (as this is commonly used as a biomarker to identify M. tuberculosis infection) and examined the influence of the M. tuberculosis genotype on this response.     

Lutte contre la rage en Afrique: du constat à l’action

As a follow-up to the first AfroREB (Africa Rabies Expert Bureau) meeting, held in Grand-Bassam (Côted’Ivoire) in March 2008, African rabies experts of the Afro-REB network met a second time to complete the evaluation of the rabies situation in Africa and define specific action plans. About forty French speaking rabies specialists from Northern, Western and Central Africa and Madagascar met in Dakar (Senegal), from March 16th to 19th, 2009. With the participation of delegates from Tunisia, who joined the AfroREB network this year, 15 French speaking African countries were represented. Experts from the Institut Pasteur in Paris, the Alliance for Rabies Control, and the Southern and Eastern African Rabies Group (SEARG, a network of rabies experts from 19 English speaking Southern and Eastern African countries) were in attendance, to participate in the discussion and share their experiences. AfroREB members documented 146 known human rabies cases in all represented countries combined for 2008, for a total population of 209.3 million, or an incidence of 0.07 cases per 100,000 people. Even admitting that the experts do not have access to all reported cases, this is far from the WHO estimation of 2 rabies deaths per 100,000 people in urban areas and 3.6 per 100,000 in rural Africa. It was unanimously agreed that the priority is to break the vicious cycle of indifference and lack of information which is the main barrier to human rabies prevention.     

Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and rapid immunogold dipstick

Plague is still prevalent in more than 20 countries. Two F1 antigen diagnostic assays (an immunocapture ELISA and an immunogold chromatography dipstick) were evaluated using bubo aspirates, serum and urine specimens from patients suspected with plague. The specificity of the two F1 assays was found 100%. Using bacteriology as a gold reference diagnostic assay, 52 patients were Yersinia pestis culture positive and 141 negative. The sensitivity of the F1 ELISA test was 100% in bubo, 52% in serum and 58% in urine specimens. In culture negative patients, the F1 antigen could be found in 10% bubo aspirates, 5% serum and 7% urine specimens of culture negative patients for whom a seroconversion for anti-F1 antibodies was also observed. The sensitivity of the dipstick assay was 98% on bubo aspirates specimens. Compared to the ELISA test, the agreement rate was 97.5% and the correlation coefficient tau = 0.90 (p < 10(-3)). In conclusion, the diagnosis of bubonic plague has to be performed on bubo fluid rather than on serum or urine specimens. Both the F1 ELISA and the dipstick assays are valuable tools for an early diagnosis and for the surveillance of plague.     

Mass vaccination campaigns to eradicate poliomyelitis in Madagascar: oral poliovirus vaccine increased immunity of children who missed routine programme

To assess the impact of mass vaccination campaigns using oral poliovirus vaccine (OPV) in Madagascar, serum neutralizing antibodies and geometrical mean titres (GMTs) to poliovirus were measured among 472 children aged up to 59 months, before and after the mass campaign, regardless of their previous history of routine vaccination. In this study, overall coverage with three routine and two mass campaign OPV doses was 69.9 and 93.4%, respectively. Seroprevalences to all poliovirus types were significantly higher after the mass campaign among the children who were not vaccinated through routine programme: 67.5% vs. 90.2% (P < 0.001) for type 1; 66.7% vs. 95.1% (P < 0.001) for type 2; and 55.3% vs. 82.9% (P < 0.001) for type 3. Geometrical mean titres to all poliovirus types also significantly increased after the mass campaign among the same study group: 34.5 vs. 238.9 (P < 0.001) for type 1; 35.1 vs. 402.6 (P < 0.001) for type 2; and 13.3 vs. 92.6 (P < 0.001) for type 3. Post-mass campaign seroprevalences and GMTs for poliovirus, especially types 1 and 3, among children who received up to two routine and two mass campaign OPV doses were significantly higher than pre-mass campaign seroprevalences among children who received three routine OPV doses. Reasons for lack of adherence to the vaccination programme and the mass campaign are discussed. The findings strongly support the WHO strategy of conducting mass campaign in all endemic countries. However, as the mass campaign strategy now has been discontinued, it is crucial to increase the routine coverage and to improve acute flaccid paralysis surveillance in order to fulfil the goal of poliomyelitis eradication.     

液体培养基Bio FM(BIO-RAD)快速分离分枝杆菌的评价

背景:本研究对低收入国家应用简单、快速的分离结核分枝杆菌培养系统进行分析.BioFM(BIO-RAD)是一种浓缩的7H9培养基,适合分枝杆菌生长,并含有显色指示剂.目的:Bio FM系统与本实验室常规使用的罗氏(LJ)培养方法相比较,评价在检出率和检测时间上的差异.材料方法:对270例肺结核病人的样本和178例肺外结核病人的标本分别用Bio FM和LJ方法进行分离培养,比较二者检出率及检测时间.结果:Bio FM和LJ 2种方法在检出率上差别不大(分别为97.9%和93.15%,P＞0.05);但使用Bio FM方法结核分枝杆菌生长速度快于LJ方法(Bio FM平均生长时间为12.42d[3-41 d];LJ平均生长时间为20.7d [10-48],P＜10-6).结论:本研究显示研究,Bio FM液体培养的检测时间快于固体LJ培养基,但检出率并不优于LJ培养基.     

Transmission of tuberculosis in the prison of Antananarivo (Madagascar)

The prevalence of tuberculosis in the Antananarivo prison is 16 times higher than that in the general population of Madagascar. We compared the clustering of Mycobacterium tuberculosis strains within and outside the prison and studied the transmission of strains in the prison. M. tuberculosis strains isolated in 1994 to 1995 from 146 prisoners and from 260 nonprisoner patients from Antananarivo were typed using the genetic markers IS6110 and direct repeat. We compared the strains isolated from prisoners and nonprisoners and found that the clustering rate was higher within (58.9%) than outside the prison (40%) suggesting that the transmission rate was higher in prison. Of the 146 incarcerated patients, 82 were grouped into 22 clusters. We checked for possible tuberculosis transmission between prisoners with identical strains by epidemiological investigation of the various prison clusters. We found that 9.5% of the incarcerated patients could have been sources of infection and that only 15.1% could have been infected in the prison. One hundred and twenty-seven prison patients were new cases. Epidemiological data suggested that 37% of them resulted from a reactivation of an old infection, due to poor living conditions or recent transmission from an index case outside the prison.