A pilot study on the genetic diversity of Mycobacterium tuberculosis complex strains from tuberculosis patients in the Littoral region of Cameroon.

BACKGROUND: The re-emergence of tuberculosis (TB) worldwide, compounded by multi-drug resistance (MDR) of the causative agents constitutes a major challenge to the management of the disease. Rapid diagnosis and accurate strain identification are pivotal to the control of the disease. This pilot study investigated the genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains from TB patients in the Littoral region of Cameroon as well as their resistance to rifampicin (RIF). PATIENTS AND METHODS: This was a cross sectional hospital-based study carried out between January and December 2017 and including 158 isolates from sputum smear positive individuals [105 (66.5%) males and 53 (33.5%) females]. Sputum samples were tested using Xpert MTB/RIF, followed by culture on Lowenstein-Jensen medium. Isolates were further subjected to molecular characterization using IS6110 typing, deletion analysis and spoligotyping. RESULTS: Thirteen (8.8%) of the 147 isolates with susceptibility results available were resistant to RIF. Drug resistance occurred in 5/50 (10%) female compared to 8/97 (8.2%) male (OR, 0.81; 0.25-2.62; p = 0.764), and there was no significant difference across the age ranges (p = 0.448). On the other hand, RIF resistance was associated (OR, 0.18, 95%CI, 0.05-0.69; p = 0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. The 150 identified lineages included among others 54 (36%) Cameroon, 18 (12%) UgandaI, 32 (21.3%) Haarlem, 17 (11.3%) Ghana, 9(6%) West African 1, 7(4.7%) Delhi/CAS, 4 (2.7%) LAM and 3 (2%) UgandaII. Of the 150 isolates, the major cluster was the Cameroon SIT 61, with 43(28.7%) isolates. Six (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74-33.55, p = 0.001). CONCLUSION: The cosmopolitan Littoral region presents with a wide Mycobacterium tuberculosis (MTB) strains diversity and the UgandaI sub-lineage likely associated with RIF resistance. Understanding the spread of this clade through surveillance will enhance TB control in the region.

A public-private partnership to reduce tuberculosis burden in Akwa Ibom State, Nigeria.

BACKGROUND: Tuberculosis (TB) infection and spread are preventable, and TB disease is curable depending on individual and community knowledge of causes of the disease, mode of prevention and cure. An earlier educational intervention carried out in Akwa Ibom State (AKS) of Nigeria in 2006 created awareness of the disease and improved utilization of orthodox medical facilities of residents in 34 communities who had symptoms of TB. OBJECTIVE: The overall aim of this program is to reduce the burden of TB disease in 18 communities of AKS through educational intervention, TB case detection and integration into the State National Tuberculosis and Leprosy Control Programme (NTBLCP), as well as build laboratory capacity to improve TB case detection and control. METHODS: Prior to the educational intervention in each community, standard pretested questionnaires were administered to residents to test their knowledge, attitudes and practices concerning TB. Information about causes, symptoms and prevention of TB was disseminated in community town halls, churches, markets and schools. Individuals who were coughing for three weeks or more were investigated for TB following clinical examination by a physician. Three sputum samples (spot-morning-spot) were obtained from each individual and examined microscopically for the presence of acid-fast bacilli (AFB) using the Ziehl-Neelson staining technique. Those with positive AFB results were integrated into the existing NTBLCP treatment facilities for immediate commencement of Directly-Observed Therapy Short Course (DOTS). Treatment outcome was monitored by retesting patients' sputum after two, five and seven months. Two new laboratories were facilitated while existing laboratory capacity was built by providing higher resolution microscopes, power generating plants, refrigerators, locally-fabricated incinerators and furnishing of staff offices. The program was facilitated by a public-private partnership. Effective Health Care Alliance Research Programme (EHCARP-Nigeria), in collaboration with Nigeria National Petroleum Cooperation and Mobil Producing Nigeria Unlimited (NNPC/MPN) Joint Venture, utilized health personnel from the Akwa Ibom State NTBLCP who conducted laboratory testing and supervised the treatment. RESULTS: The 916 responses to the questionnaires showed that 65.3% (549/841) correctly identified that TB is airborne, and 86% (749/871) were aware that TB could be cured by anti-TB medication. Responses to care-seeking attitudes were provided by 123 respondents. Of this number, fear of stigmatization was the reason for 31% (38) seeking care in unorthodox facilities, while 43.1% (53) did not believe that orthodox medicine could cure their symptoms. Of the 374 detected cases, 9 did not commence treatment. Hence, 365 were placed on DOTS; 36 defaulted, while 11 either died or failed to convert after the seventh month. At the end of month 8, cure was achieved for 87.1% (318). CONCLUSION: Although the previous intervention may have contributed to the good knowledge about TB and care-seeking attitudes displayed by respondents in the communities, sustaining active case finding through public-private partnership can go a long way to reduce TB burden, especially in rural communities where healthcare systems are generally weak or inadequate. Adequate funding of TB control activities is critical in eliminating TB as a public health problem, and the private sector participation such as this is a welcome development.

Utility and diagnostic performance of Mycobacterium tuberculosis complex by two immunochromatographic assays as compared with the molecular Genotype assay in Nigeria.

Among the disadvantages of smear microscopy for detection of tuberculosis cases is its inability to differentiate between Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). This study evaluated two, new immunochromatographic assays - Capilia TB-Neo and SD Bioline - on unheated and heated cultures at 80°C for 30min respectively for their ability to discriminate between MTB complex and NTM as compared with the molecular Genotype assay. Mycobacteria used in the study were obtained from smear-positive specimens collected from patients at four major hospitals in Cross River State, Nigeria. Capilia TB-Neo and SD Bioline showed sensitivities of 98.8% and 93.8% respectively and 100% specificity for both assays. Heating the isolates did not significantly impact the test performance. Both tests are recommended for use in rapid differentiation of strains isolated in Nigeria.

Multidrug resistance and demography of newly diagnosed tuberculosis patients in Cross River State, Nigeria.

SETTING: Nigeria has the world's fourth largest tuberculosis burden, and multidrug resistant tuberculosis (MDR-TB) represents a serious public health problem. OBJECTIVES: To describe the demography of TB patients and determine the susceptibility of Mycobacterium tuberculosis isolates to the major TB drugs. METHODS: One hundred and thirty-seven newly diagnosed TB patients (26 (19%) being HIV positive) from all age groups were recruited into the study. Each specimen was cultured using BACTEC MGIT960, followed by inoculation and growth on Lowenstein-Jensen (LJ) medium. Primary identification was carried out using an immunochromatographic technique (Capilia TB-Neo), and further confirmed by genotyping. Drug susceptibility testing (DST) was carried out by the agar proportion method. RESULTS: Of the 97 pure mycobacterial cultures on LJ medium, 81 (83.5%) isolates were identified as M. tuberculosis complex, while 16 (16.5%) were Capilia negative. DST was carried out on 58 isolates. The drug susceptibility pattern showed that resistance occurred in 16 (27.6%) for streptomycin, 11 (19%) for isoniazid, 9 (16%) for rifampicin, and 10 (17.2%) for ethambutol. Rifampicin monoresistance occurred in 2 (3.4%) cases. MDR (combined resistance to isoniazid and rifampicin), also involving resistance to streptomycin and ethambutol, occurred in 6/58 (10.3%) isolates; although laboratory cross-contamination could not be excluded in 4/6 MDR strains with identical MIRU patterns characterized by consecutive strain numbers. Considering that first out of these 4 isolates was not due to laboratory carryover, the results of this study still report a minimal MDR-TB rate of 3/58 (5.2%) among newly diagnosed TB patients in Cross River State, Nigeria. CONCLUSIONS: An increase in drug resistance was observed in this study as compared with previous studies in the country. Hence, introduction of culture in routine diagnostic mycobacteriology laboratories will prevent the emergence and dissemination of MDR-TB, while improved quality control strategies would in parallel prevent laboratory cross-contamination, thereby reducing mislabeling, unnecessary treatment, and drug toxicity for patients.

Acid-fast bacilli other than mycobacteria in tuberculosis patients receiving directly observed therapy short course in cross river state, Nigeria.

The information on the contribution of non tuberculous mycobacteria (NTM) to mycobacterial infections in Africa is scarce due to limited laboratory culture for its isolation and identification. One hundred and thirty-seven sputum smear positive patients were recruited into a study on the molecular epidemiology of Mycobacterium tuberculosis in Cross River State. Following sputum culture, 97 pure isolates were obtained and identified using Capilia TB-Neo and further confirmed by the GenoType Mycobacterium CM kit. Of the 97 isolates, 81 (83.5%) isolates were Capilia TB-Neo positive while 16 (16.5%) were Capilia TB-Neo negative. Further confirmation with the GenoType Mycobacterium CM kit revealed that 4 (25%) of the 16 isolates belonged to NTM and included M. fortuitum I, M. fortuitum II/M magaritense, M. abscessus, and M. avium ssp. The remaining 12 (75%) Capilia TB-Neo negative isolates were not members of the genus Mycobacterium despite their AFB appearance. Six (33.3%) of the Capilia TB-Neo negative were from HIV positive tuberculosis patients. All subjects in this study were placed on DOTS shortly after the AFB results were obtained. The implication of isolation of 16.5% nontuberculous isolates further emphasizes the need for culture of sputum specimen especially in HIV positive patients prior to administration of antituberculosis therapy.

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State, Nigeria.

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n=64% or 79% of clustered isolates, 2-32 isolates/cluster), with patients in the age group range 25-34 years being significantly associated with shared-type pattern SIT61 (p=0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n=42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n=27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n=52 isolates, 2-9 isolates per cluster), with a resulting recent n-1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p=0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU-VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.

Bacterial isolates from blood cultures of children with suspected septicaemia in Calabar, Nigeria.

BACKGROUND: Septicaemia is a common cause of morbidity and mortality among children in the developing world. This pattern has changed little in the past decade. Physical signs and symptoms, though useful in identifying possible cases have limited specificity. Definitive diagnosis is by bacteriologic culture of blood samples to identify organisms and establish antibiotic susceptibility. These results are usually not available promptly. Therefore a knowledge of epidemiologic and antimicribial susceptibility pattern of common pathogens is useful for prompt treatment of patients. This report highlights the pattern of bacterial isolates in our environment from a retrospective study of our patients' records. METHODS: One thousand, two hundred and one blood samples were analysed from children aged 0-15 years, admitted into the children's wards of the University of Calabar Teaching Hospital, Calabar, Nigeria with features suggesting septicaemia. Samples were collected under aseptic conditions and cultured for aerobic and anaerobic organisms. Isolates were identified using bacteriologic and biochemical methods and antibiotic sensitivity determined by agar diffusion method using standard antibiotic discs. RESULTS: Bacteria was isolated in 552 (48.9%) of samples with highest rates among newborns (271 : 50.8). The most frequent isolates were Staphylococcal aureus (48.7%) and Coliforms (23.4%). Results showed high susceptibilities to the Cephalosporins (Ceftriazone- 100%:83.2%, Cefuroxime-100%:76.5%) and Macrolides (Azithromycin-100%:92.9%) for S. aureus and coliforms respectively. This study underscores the importance of septicaemia as a common cause of febrile illness in children and provides information on common prevalent aetiologic agents and drug susceptibilities of the commonest pathogens. CONCLUSION: Staphylococcus aureus and coliforms were the leading causes of septicaemia in children in this locality, and the third generation cephalosporins and azithromycin were shown to be effective against these pathogens.