Sadia Shakoor

Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED)

BACKGROUND Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specific diarrhoea burdens in the community. METHODS We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defined as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identified through twice-weekly home visits by fieldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1-12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specific burdens of diarrhoea. FINDINGS Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0-24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0-7·1), rotavirus (4·8%, 4·5-5·0), Campylobacter spp (3·5%, 0·4-6·3), astrovirus (2·7%, 2·2-3·1), and Cryptosporidium spp (2·0%, 1·3-2·6) exhibited the highest attributable burdens of diarrhoea in the first year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1-12·1), norovirus GII (5·4%, 2·1-7·8), rotavirus (4·9%, 4·4-5·2), astrovirus (4·2%, 3·5-4·7), and Shigella spp (4·0%, 3·6-4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fifth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. INTERPRETATION There was substantial heterogeneity in pathogen-specific burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These findings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the effect of such interventions on total diarrhoeal incidence at the community level might be limited.

Primary Amebic Meningoencephalitis Caused by Naegleria fowleri, Karachi, Pakistan

We report 13 cases of Naegleria fowleri primary amebic meningoencephalitis in persons in Karachi, Pakistan, who had no history of aquatic activities. Infection likely occurred through ablution with tap water. An increase in primary amebic meningoencephalitis cases may be attributed to rising temperatures, reduced levels of chlorine in potable water, or deteriorating water distribution systems.

Epidemiology and Impact of Campylobacter Infection in Children in 8 Low-Resource Settings: Results From the MAL-ED Study

In a multisite birth cohort study, we document a high burden of Campylobacter infection using enzyme immunoassay, demonstrate an association between Campylobacter and linear growth shortfalls and both increased intestinal permeability and intestinal and systemic inflammation, and identify potential interventions.

Determinants and impact of Giardia infection in the first 2 years of life in the MAL-ED birth cohort

Summary In a multisite birth-cohort study, Giardia spp were detected by enzyme immunoassay at least once in two-thirds of the children. Early persistent infection with Giardia, independent of diarrhea, was associated with deficits in both weight and length at 2 years of age.

Fluoroquinolone-resistant tuberculosis: implications in settings with weak healthcare systems

Fluoroquinolones (FQ) play an essential role in the treatment and control of multidrug-resistant tuberculosis (MDR-TB). They are also being evaluated as part of newer regimens under development for drug-sensitive TB. As newer FQ-based regimens are explored, knowledge of FQ resistance data from high TB burden countries becomes essential. We examine available FQ resistance data from high TB burden countries and demonstrate the need for comprehensive surveys to evaluate FQ resistance in these countries. The factors driving FQ resistance in such conditions and the cost of such resistance to weak healthcare systems are discussed. The need for a comprehensive policy for addressing the issue of FQ resistance is highlighted.

Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins.

ABSTRACT Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S. Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S. Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S. Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally. IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S. Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S. Typhi clone in Sindh, Pakistan. The XDR S. Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S. Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S. Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S. Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S. Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S. Typhi observed here adds urgency to the need for typhoid prevention measures. Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S. Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S. Typhi clone in Sindh, Pakistan. The XDR S. Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S. Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S. Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S. Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S. Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S. Typhi observed here adds urgency to the need for typhoid prevention measures.

Fluoroquinolone-Resistant Mycobacterium tuberculosis, Pakistan, 2005–2009

To the Editor: Pakistan is 1 of 22 countries listed by the World Health Organization (WHO) as having a high incidence of tuberculosis (TB). We recently reported an increase in rates of multidrug-resistant (MDR) TB with emergence of extensively drug-resistant TB (1). Fluoroquinolone resistance is associated with worse outcome in patients with MDR TB (2). Recent evidence suggests emergence and increasing incidence of fluoroquinolone-resistant Mycobacterium tuberculosis from several countries, particularly in MDR strains (3). We present data from a tertiary care referral center laboratory in Pakistan to assess fluoroquinolone resistance in MDR TB strains during 2005–2009. The Aga Khan University Hospital and its clinical laboratory have been accredited by the Joint Commission of International Accreditation and designated as a technical partner of the National TB Program. M. tuberculosis susceptibility testing is also periodically validated by the WHO Supranational Reference Laboratory network. The microbiology laboratory serves different cities across Pakistan with ≈180 peripheral collection units. Specimens for TB cultures are requested by physicians and received through passive collection and thus are not restricted to programmed surveys. All specimens received at each of the collection units are sent to the central laboratory in Karachi for culture and drug susceptibility testing (DST). Specimens reach the main laboratory within 24 hours after receipt. During the past 4 years, the laboratory has received 12,000–15,000 specimens annually for M. tuberculosis culture; positivity rate has been 15%–20%. Culture and DST are performed at the laboratory in accordance with Clinical Laboratory Standards Institute and WHO recommendations, as described (4). During 2005–2008, fluoroquinolone susceptibilities for all MDR and polydrug-resistant isolates were determined by using ciprofloxacin (2 μg/mL). From 2009 onwards, fluoroquinolone susceptibilities were determined by using ofloxacin (2 μg/mL), and second-line DST was performed for all M. tuberculosis isolates. During 2005–2009, a total of 11,263 cultures were reported positive for M. tuberculosis. Of these, 34.4% were MDR, and 50.1% were sensitive to all 4 first-line agents (isoniazid, rifampin, pyrazinamide, ethambutol). Because of inconsistencies in testing criteria for fluoroquinolones (fluoroquinolone testing being conducted primarily for MDR cases during 2005–2008), the overall fluoroquinolone-resistance rate could not be determined. However, for MDR strains, fluoroquinolone susceptibilities were consistently determined, and resistance rates increased from 17.41% in 2005 to 42.92% in 2009 (p<0.001, by χ2 test for trend analysis) (Table). A progressive increase in fluoroquinolone use and its association with increase in resistance against organisms other than M. tuberculosis have been reported from Pakistan (5). We report a progressive increase in fluoroquinolone resistance rate in MDR M. tuberculosis isolates during a 5-year period. This finding is consistent with those of several studies reporting fluoroquinolone resistance from the region. Agrawal et al. have recently reported an exponential increase in fluoroquinolone resistance in India from 3% in 1996 to 35% in 2004 (6). A significant increase in fluoroquinolone resistance from 7.7% to 20% in MDR TB was also reported from Taiwan (7); the authors correlated this finding with the inappropriate use of fluoroquinolones for managing TB rather than with fluoroquinolone misuse in the community. Another study from the United States and Canada reported 4.1% fluoroquinolone resistance in MDR TB strains (8). In addition to detecting increasing fluoroquinolone resistance in MDR isolates, we have also detected fluoroquinolone resistance in non-MDR, polydrug-resistant M. tuberculosis isolates. Moreover, in 2009, a total of 3.1% of isolates susceptible to all first-line agents were fluoroquinolone resistant. Although our dataset includes samples from throughout Pakistan, sampling limitations prevent us from deriving definite conclusions and generalizing results to the entire population of the country. A referral bias attributable to passive sampling exists because cases referred to our laboratory tend to be more complicated. Moreover, treatment history was not available for patients in our dataset; therefore, increased fluoroquinolone resistance could not be correlated with prior fluoroquinolone use. However, our findings have implications for therapy with fluoroquinolones for TB and other infections. Fluoroquinolones are freely available as over-the-counter medications to the general population (9), which creates potential for misuse of fluoroquinolones by the general population for TB and several other infections, such as enteric fever and genitourinary infections. Furthermore, because national guidelines for treating enteric fever and genitourinary infections do not exist, these drugs are also overprescribed by physicians. We propose control of over-the-counter availability of fluoroquinolones and judicious use of this class of drugs by physicians to prevent further escalation in resistance rates in Pakistan. Table Resistance patterns and fluoroquinolone resistance rates of Mycobacterium tuberculosis isolates from the Aga Khan University Hospital laboratory, Karachi, Pakistan, 2005–2009*

Tropical Bacterial Gastrointestinal Infections

The bacterial gastrointestinal infections cholera, salmonellosis, shigellosis, campylobacteriosis, and diarrheagenic Escherichia coli are prevalent in tropical regions. These diseases impose an immense cost and contribute significantly to childhood morbidity and mortality. Management is hampered by limited access to diagnostic facilities and by antimicrobial drug resistance. Rapid point-of-care assays aim to reduce treatment delay and encourage rational use of antimicrobial agents. Control through safe drinking water, good sanitation, and vaccination against typhoid and cholera in high-risk populations is recommended. Vaccines against other Shigella and diarrheagenic E coli infections are under development.

Use of "Parasep filter fecal concentrator tubes" for the detection of intestinal parasites in stool samples under routine conditions

Parasitic gastrointestinal infections are a major cause of morbidity and mortality in the developing world, with stool microscopy being the mainstay of diagnostic practice. Both direct microscopy and concentration techniques can be utilized; direct microscopy may be time consuming and tedious; however clinical laboratories in developing countries lack trained staff who can effectively use concentration methods. In our practice we used the Parasep O and P filter concentrator tubes (manufactured by DiaSys Ltd, Berkshire, England. Product Code 146000) along with direct microscopic techniques and found that Parasep filters enhanced the ability to detect intestinal parasites that would have been missed on routine microscopy. We found the Parasep filter concentration method to be easy, cost-effective and reliable for routine stool examinations.

Vibrio cholerae O1 bacteremia in Pakistan: analysis of eight cases.

Bacteremia caused by Vibrio cholerae O1 has been a rare phenomenon. We report on eight cases of V. cholerae O1 bacteremia from Pakistan which occurred during 1992-2008. Six of the cases were seen in children (two neonates and four infants) and seven of the eight patients were female. Urogenital malignancy, hepatitis B virus-associated end-stage liver disease, concurrent Campylobacter enteritis and prematurity were the underlying conditions in four patients. Two of the eight patients died and one was lost to follow up and this outcome may be due to prior immunity leading to less severe illness.