Use of Luminex MagPlex magnetic microspheres for high-throughput spoligotyping of Mycobacterium tuberculosis isolates in Port-au-Prince, Haiti.

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct "spacers" separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.

Drug-resistant TB prevalence study in 5 health institutions in Haiti.

OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Multi-drug resistant TB (MDR-TB) is a major public health problem as treatment is long, costly, and associated to poor outcomes. Here, we report epidemiological data on the prevalence of drug-resistant TB in Haiti. METHODS: This cross-sectional prevalence study was conducted in five health centers across Haiti. Adult, microbiologically confirmed pulmonary TB patients were included. Molecular genotyping (rpoB gene sequencing and spoligotyping) and phenotypic drug susceptibility testing were used to characterize rifampin-resistant MTB isolates detected by Xpert MTB/RIF. RESULTS: Between April 2016 and February 2018, 2,777 patients were diagnosed with pulmonary TB by Xpert MTB/RIF screening and positive MTB cultures. A total of 74 (2.7%) patients were infected by a drug-resistant (DR-TB) M. tuberculosis strain. Overall HIV prevalence was 14.1%. Patients with HIV infection were at a significantly higher risk for infection with DR-TB strains compared to pan-susceptible strains (28.4% vs. 13.7%, adjusted odds ratio 2.6, 95% confidence interval 1.5-4.4, P = 0.001). Among the detected DR-TB strains, T1 (29.3%), LAM9 (13.3%), and H3 (10.7%) were the most frequent clades. In comparison with previous spoligotypes studies with data collected in 2000-2002 and in 2008-2009 on both sensitive and resistant strains of TB in Haiti, we observed a significant increase in the prevalence of the drug-resistant MTB Spoligo-International-Types (SIT) 137 (X2 clade: 8.1% vs. 0.3% in 2000-02 and 0.9% in 2008-09, p<0.001), 5 (T1 clade: 6.8% vs 1.9 in 2000-02 and 1.7% in 2008-09, P = 0.034) and 455 (T1 clade: 5.4% vs 1.6% and 1.1%, P = 0.029). Newly detected spoligotypes (SIT 6, 7, 373, 909 and 1624) were also recorded. CONCLUSION: This study describes the genotypic and phenotypic characteristics of DR-TB strains circulating in Haiti from April 2016 to February 2018. Newly detected MTB clades harboring multi-drug resistance patterns among the Haitian population as well as the higher risk of MDR-TB infection in HIV-positive people highlights the epidemiological relevance of these surveillance data. The importance of detecting RIF-resistant patients, as proxy for MDR-TB in peripheral sites via molecular techniques, is particularly important to provide adequate patient case management, prevent the transmission of resistant strains in the community and to contribute to the surveillance of resistant strains.

High prevalence of ESBL-positive bacteria in an obstetrics emergency hospital and neonatal care unit-Haiti, 2016.

A point-prevalence survey of mothers and neonates admitted to an obstetrics emergency hospital in Port-au-Prince, Haiti, revealed that 13 of 127 gram-negative bacteria isolates (10%) from rectal swabs were ESBL-positive in women and 30 of 59 gram-negative bacteria isolates (51%) from rectal swabs were ESBL-positive in neonates. Length of hospital stay and antibiotic consumption were risk factors for ESBL colonization.

Effectiveness of Oral Cholera Vaccine in Haiti: 37-Month Follow-Up.

The first oral cholera vaccine (OCV) campaign, since its prequalification by the World Health Organization, in response to an ongoing cholera epidemic (reactive vaccination) was successfully conducted in a poor urban slum of approximately 70,000 inhabitants in Port-au-Prince, Haiti, in 2012. Vaccine coverage was 75% of the target population. This report documents the impact of OCV in reducing the number of culture-confirmed cases of cholera admitted to the Groupe Haïtien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) cholera treatment center from that community in the 37 months postvaccination (April 2012-April 30, 2015). Of 1,788 patients with culture-confirmed cholera, 1,770 (99%) were either from outside the vaccine area (1,400 cases) or from the vaccinated community who had not received OCV (370 cases). Of the 388 people from the catchment area who developed culture-confirmed cholera, 370 occurred among the 17,643 people who had not been vaccinated (2.1%) and the remaining 18 occurred among the 52,357 people (0.034%) who had been vaccinated (P < 0.001), for an efficacy that approximates 97.5%. Despite not being designed as a randomized control trial, the very high efficacy is a strong evidence for the effectiveness of OCV as part of an integrated package for the control of cholera in outbreak settings.

False-positive rifampin resistant results with Xpert MTB/RIF version 4 assay in clinical samples with a low bacterial load.

We report investigation of 22 TB cases with positive Xpert MTB/RIF result for resistance to Rifampin and "Very Low" MTB detection level. Twelve cases were false positive without rpoB mutations, 2 were false-positives with a silent mutation in rpoB codon T508, and only 10 were true positives.

Whole Genome Sequencing Investigation of a Tuberculosis Outbreak in Port-au-Prince, Haiti Caused by a Strain with a "Low-Level" rpoB Mutation L511P - Insights into a Mechanism of Resistance Escalation.

The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of Mycobacterium tuberculosis rpoB gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDRplus. Such mutations are found in >95% of Mycobacterium tuberculosis strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific "low level" rpoB mutations conferring resistance to RIF below the critical concentration of 1 µg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a "low level" rpoB mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 µg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of embB followed by secondary mutations in rpoB and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing rpoB mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both "gold standard" sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.

Correlation between genotypic and phenotypic testing for resistance to rifampin in Mycobacterium tuberculosis clinical isolates in Haiti: investigation of cases with discrepant susceptibility results.

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95-98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the "gold standard" culture-based drug susceptibility testing (DST). We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay. Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation. Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays.

Treatment outcomes for patients with multidrug-resistant tuberculosis in post-earthquake Port-au-Prince, Haiti.

We report outcomes and 12-month survival for the first cohort of patients to undergo multidrug-resistant tuberculosis (MDR-TB) treatment after the earthquake in Haiti. From March 3, 2010 to March 28, 2013, 110 patients initiated treatment of laboratory-confirmed MDR-TB at the Groupe Haïtien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) Center in Port-au-Prince, Haiti. Twenty-seven patients (25%) were human immunodeficiency virus (HIV)-positive. As of October 31, 2013, 95 (86%) patients were either cured or alive on treatment, 4 (4%) patients defaulted, and 11 (10%) patients died. Culture conversion occurred by 30 days in 14 (13%) patients, 60 days in 49 (45%) patients, and 90 days in 81 (74%) patients. The probabilities of survival to 12 months were 96% (95% confidence interval [95% CI] = 89-99) and 85% (95% CI = 64-94) for HIV-negative and -positive patients, respectively. Despite adverse conditions, outcomes for patients with MDR-TB are highly encouraging. Major efforts are underway to scale up community directly observed therapy and expand care to other regions of Haiti.

Importance of cholera and other etiologies of acute diarrhea in post-earthquake Port-au-Prince, Haiti.

We estimated the proportion of diarrhea attributable to cholera and other pathogens during the rainy and dry seasons in patients seen in two urban health settings: a cholera treatment center (CTC) and oral rehydration points (ORPs). During April 1, 2011-November 30, 2012, stool samples were collected from 1,206 of 10,845 patients who came to the GHESKIO CTC or to the community ORPs with acute diarrhea, cultured for Vibrio cholerae, and tested by multiplex polymerase reaction. Vibrio cholerae was isolated from 409 (41.8%, 95% confidence interval [CI] = 38.7-44.9%) of the 979 specimens from the CTC and in 45 (19.8%, 95% CI = 14.8-25.6%) of the 227 specimens from the ORPs. Frequencies varied from 21.4% (95% CI = 16.6-26.7%) during the dry season to 46.8% (95% CI = 42.9-50.7%) in the rainy season. Shigella, enterotoxigenic Escherichia coli, rotavirus, and Cryptosporidium were frequent causes of diarrhea in children less than five years of age.