Lophomonas as a respiratory pathogen-jumping the gun.

Human infections with the protozoan Lophomonas have been increasingly reported in the medical literature over the past three decades. Initial reports were based on microscopic identification of the purported pathogen in respiratory specimens. Later, a polymerase chain reaction (PCR) was developed to detect Lophomonas blattarum, following which there has been a significant increase in reports. In this minireview, we thoroughly examine the published reports of Lophomonas infection to evaluate its potential role as a human pathogen. We examined the published images and videos of purported Lophomonas, compared its morphology and motility characteristics with host bronchial ciliated epithelial cells and true L. blattarum derived from cockroaches, analyzed the published PCR that is being used for its diagnosis, and reviewed the clinical data of patients reported in the English and Chinese literature. From our analysis, we conclude that the images and videos from human specimens do not represent true Lophomonas and are predominantly misidentified ciliated epithelial cells. Additionally, we note that there is insufficient clinical evidence to attribute the cases to Lophomonas infection, as the clinical manifestations are non-specific, possibly caused by other infections and comorbidities, and there is no associated tissue pathology attributable to Lophomonas. Finally, our analysis reveals that the published PCR is not specific to Lophomonas and can amplify DNA from commensal trichomonads. Based on this thorough review, we emphasize the need for rigorous scientific scrutiny before a microorganism is acknowledged as a novel human pathogen and discuss the potential harms of misdiagnoses for patient care and scientific literature.

Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for the detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alpha gene) from standard strains by using six specific primers FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), B3 (backward outer primer), loopF (forward loop primer), and loopB (backward loop primer) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting-curve analysis for specific annealing temperatures (Tm) of 84°C-86°C and 88°C-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Comparative efficacy of natural aquatic predators for biological control of mosquito larvae: A neglected tool for vector control.

Mosquitoes are a dominant fraction of dipteran fauna, occupying a variety of niches. The most common method deployed for their control is the use of insecticides. Throughout their life cycle they are exposed to a wide range of predators in different habitats, thus biological control of mosquitoes by using aquatic predators has been suggested. Therefore, the present study was carried out to explore the type of natural predators coexisting with the mosquito larvae in still water bodies and to determine their efficacy as predators for mosquito larvae. A coexistence of different predators with mosquito larvae was observed in 27 standing water bodies of Chandigarh, India. The predation efficiency of tadpoles of frog was comparable to Gambusia fish, as 97% of the mosquito larvae of all instars of the medically important mosquito genera Anopheles, Aedes, Culex and Armigeres were preyed. The toad tadpoles were found to be least effective and their predation rate was found to be negligible. Further studies on larval source management by frog tadpoles in combination with insecticides or stand-alone would be worthwhile.

Dynamics of Plasmodium berghei NK-65 parasitaemia and CD3+CD4+CD25+Fox-p3+ T-regulatory cells in experimentally induced malaria during early, mid, and late-pregnancy in BALB/c mice.

Introduction: Malaria in pregnancy causes a dual brunt on the mother as well as the foetus. Upregulation of T-regulatory cells (Tregs) during pregnancy allows tolerance towards the growing foetus, their suppression predisposes the mother to infections. This study analyzed the levels of CD3+CD4+CD25+Fox-p3+ Tregs, parasitaemia, maternal and foetal outcomes in BALB/c mice infected with P. berghei NK65 during early-, mid-, and late-pregnancy. Methodology: Total of 114 mice, non-pregnant non-infected (n = 6), non-pregnant infected (n = 12), pregnant non-infected (n = 48) and pregnant infected (n = 48) were included in the study. Infected groups were inoculated intra-peritoneally with 1 × 106 P. berghei infected RBCs during early-, mid-, and late- pregnancy (D6, D10, and D14 respectively). Six mice from each stage were sacrificed on the 5th and 7th day post-infection (DPI) to evaluate parasitaemia (staining) and Tregs from splenocytes (by flow cytometry). Results: The parasitaemia was significantly higher among early pregnancy infected mice (≥ 70%) than mid-pregnancy infected (40-70%), late pregnancy infected (50-65%), and non-pregnant infected mice (≤ 50%) (p < 0.05). The level of Tregs was significantly higher among non-pregnant infected mice as compared to non-pregnant non-infected mice (%Tregs 0.86 vs. 0.44). Among pregnant mice, the levels of Tregs in infected mice were lower than in non-infected mice during all stages of pregnancy. None of the mice infected during early- and mid-pregnancy survived at 6DPI and 7DPI, respectively, and those infected during late-pregnancy delivered premature pups. Conclusion: In contrast to non-pregnant mice, the levels of Tregs among pregnant mice decrease when malaria infection is acquired thereby leading to adverse pregnancy outcomes. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01089-2.

Where Have All the Diagnostic Morphological Parasitologists Gone?

Advances in laboratory techniques have revolutionized parasitology diagnostics over the past several decades. Widespread implementation of rapid antigen detection tests has greatly expanded access to tests for global parasitic threats such as malaria, while next-generation amplification and sequencing methods allow for sensitive and specific detection of human and animal parasites in complex specimen matrices. Recently, the introduction of multiplex panels for human gastrointestinal infections has enhanced the identification of common intestinal protozoa in feces along with bacterial and viral pathogens. Despite the benefits provided by novel diagnostics, increased reliance on nonmicroscopy-based methods has contributed to the progressive, widespread loss of morphology expertise for parasite identification. Loss of microscopy and morphology skills has the potential to negatively impact patient care, public health, and epidemiology. Molecular- and antigen-based diagnostics are not available for all parasites and may not be suitable for all specimen types and clinical settings. Furthermore, inadequate morphology experience may lead to missed and inaccurate diagnoses and erroneous descriptions of new human parasitic diseases. This commentary highlights the need to maintain expert microscopy and morphological parasitology diagnostic skills within the medical and scientific community. We proposed that light microscopy remains an important part of training and practice in the diagnosis of parasitic diseases and that efforts should be made to train the next generation of morphological parasitologists before the requisite knowledge, skills, and capacity for this complex and important mode of diagnosis are lost. In summary, the widespread, progressive loss of morphology expertise for parasite identification negatively impacts patient care, public health, and epidemiology.

Vector and rodent surveillance for Orientia tsutsugamushi in north India.

BACKGROUND & OBJECTIVES: Scrub typhus or chigger borne typhus, caused by Orientia tsutsugamushi is an emerging vector-borne disease as large numbers of cases have been reported in various tropical countries. It is transmitted to humans through bites of infected chiggers (larval mites). The knowledge about the vector, its distribution, density and habitat are important so as to understand the epidemiology of scrub typhus in a given area. To control rickettsial infections, regular rodent-vector surveillance should be planned in areas where the disease transmission is occurring and it will also help to strengthen the existing entomological data related to the vector of scrub typhus in northern India. METHODS: In the present study, rodent-vector surveillance was planned for one whole year, covering both mite active and non-active seasons (October 2019-December 2020) in selected areas of Chandigarh and Punjab in north India. Rodent tissues and mites were also examined for the presence of O. tsutsugamushi by nested PCR for 56 kDa gene and real-time PCR for 47 kDa outer membrane protein gene. 18S gene PCR was performed for molecular identification of mites. RESULTS: In the surveillance, three types of ectoparasite, viz. mites, fleas and ticks were obtained in rodents. All mites found were of Laelapidae family. None of the pooled rodent tissue samples as well as mite samples were found positive for O. tsutsugamushi by nested PCR for rickettsial DNA. INTERPRETATION & CONCLUSION: In the present study, we did not get any evidence of carriage of O. tsutsugamushi in either mites or rodents collected and sampled in selected regions in Chandigarh and Punjab. We need to strengthen the entomological surveillance over a broader region and increase the frequency of trapping rodents to increase clarity on vector-reservoir dynamics in this geographical region.

Circulating filarial antigen detection in brugian filariasis.

Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.

Shigellosis: Epidemiology in India.

Shigellosis is one of the major causes of diarrhoea in India. The accurate estimates of morbidity and mortality due to shigellosis are lacking, though it is endemic in the country and has been reported to cause many outbreaks. The limited information available indicates Shigella to be an important food- borne pathogen in India. S. flexneri is the most common species, S. sonnei and non-agglutinable Shigellae seem to be steadily surfacing, while S. dysenteriae has temporarily disappeared from the northern and eastern regions. Antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world. Especially important is the global emergence of multidrug resistant Shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and also azithromycin. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measures like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such an infrastructure in India is a complex issue and will take time to resolve. Thus, the scientific thrust should be focused towards development of a safe and affordable multivalent vaccine. this review is focused upon the epidemiology, disease burden and the therapeutic challenges of shigellosis in Indian perspective.

Safeguarding health on every plate: Intestinal parasitic infection survey of food handlers in a tertiary care centre.

Food borne infections pose significant public health problem, especially in developing countries of the world. A continuous surveillance to ensure the health of the personnel involved in preparation of the hospital food is important as they can be a source of spreading the infections and possible outbreaks. We analysed the data of the prevalence of intestinal parasitic infections in food handlers in our tertiary care centre from 2018 to 2022 and 6.8% were observed to harbour intestinal parasites during the period. This signifies the importance of routine screening, and the need of awareness and education of the food handlers in hospitals.

Real-time loop-mediated isothermal amplification for the detection of Giardia duodenalis in fecal specimens.

INTRODUCTION: Giardiasis is a leading cause of subacute or chronic diarrhoea and is frequently associated with impaired physical, cognitive and psychosocial development, especially in children. The diagnosis relies mainly on the microscopic evaluation of stool specimens that have a low sensitivity. In contrast, molecular advancements like the polymerase chain reaction and Real-time loop-mediated isothermal amplification (Real-time LAMP) are promising techniques and reportedly have better diagnostic characteristics. METHODS: We have evaluated the performance of Real-time LAMP for detecting Giardia in ninety stool specimens compared to microscopy and nested PCR. RESULTS: A total of 35 fecal samples were detected positive by microscopy, 41 by nested PCR and 43 by real-time LAMP. Microscopy and nested PCR detected 33, microscopy and real-time LAMP detected 35, and nested PCR and real-time LAMP detected 41 positive samples. CONCLUSION: The real-time LAMP assay was found suitable for the rapid and accurate detection of G. duodenalis with a better sensitivity in comparison to nested PCR and microscopy. Furthermore, besides being sensitive and rapid, LAMP had the advantage of an adequate rapid turn-around time of eleven to 15 â€‹min as compared to 5 â€‹h of nested PCR.

Molecular detection of dengue and chikungunya viruses in surveillance of wild-caught Aedes mosquitoes in Punjab, North India.

BACKGROUND: Dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses are transmitted mainly by Aedes mosquitoes and are responsible for a significant global healthcare burden. The current study aimed to detect arboviruses in the Aedes mosquitoes in close proximity of patients during the transmission season. METHODS: Both immature and adult mosquitoes were collected from in and around the patients' houses. Mosquito pools were homogenized and extracted RNA was subjected to reverse transcription polymerase chain reaction for arboviral detection. Transovarian transmission (TOT) was assessed by screening F0 adults. Mosquito positivity was correlated with the aetiological agents identified in patients. RESULTS: Of 46 pools, 19 consisted of wild Aedes, with arboviral positivity in 53% (10/19) of pools. Among wild A. aegypti pools, positivity of DENV mono-infection, CHIKV mono-infection and DENV+CHIKV co-infection was noted in four, two and three pools, respectively. One wild pool of Aedes albopictus was positive for DENV-1. Similarly, A. aegypti F0 (adult Aedes developed from immatures) pools showed 59.2% (16/27) positivity for arboviruses. F0 Aedes showed positivity in three, six and seven pools for DENV-2, CHIKV and DENV+CHIKV, respectively, suggestive of TOT. DENV serotypes and CHIKV from 24 patients' serum samples were matched with strains isolated from Aedes and correlation was observed in four instances. CONCLUSIONS: The study detected DENV and CHIKV from wild-caught Aedes and found evidence of DENV and CHIKV TOT in F0 adults.

Prevalence of multidrug resistance in Mycobacterium tuberculosis isolates from HIV seropositive and seronegative patients with pulmonary tuberculosis in north India.

BACKGROUND: Multidrug resistant (MDR) and extensively-drug resistant (XDR) tuberculosis (TB) are a serious threat to the national TB control programs of developing countries, and the situation is further worsened by the human immunodeficiency virus (HIV) pandemic. The literature regarding MDR/XDR-TB is, however, scanty from most parts of India. We carried out this study to assess the prevalence of MDR/XDR-TB in new and previously treated cases of pulmonary TB and in HIV seropositive and seronegative patients. METHODS: Sputum and blood specimens were obtained from 2100 patients suspected of pulmonary tuberculosis and subjected to sputum microscopy and culture for TB, and HIV serology at our tertiary care centre in north India. The culture positive Mycobacterium tuberculosis isolates were subjected to drug susceptibility testing (DST) for first line anti-tuberculosis drugs, and the MDR isolates were further subjected to second line DST. Various parameters of the patients' were analyzed viz. clinical presentation, radiology, previous treatment history, demographic and socioeconomic data and microbiology results. RESULTS: Of the 2100 patients, sputum specimens of 256 were smear positive for acid-fast bacilli (AFB), 271 (12.9%) grew Mycobacterium spp., and M. tuberculosis was isolated in 219 (10.42%). Of the 219 patients infected with M. tuberculosis, 20.1% (44/219) were found to be seropositive for HIV. Overall, MDR-TB was observed in 17.4% (39/219) isolates. There were 121 newly diagnosed and 98 previously treated patients, of which MDR-TB was found to be associated with 9.9% (12/121) and 27.6% (27/98) cases respectively. There was significantly higher association of MDR-TB (12/44, 27.3%) with HIV seropositive patients as compared to HIV seronegative patients (27/175, 15.4%) after controlling previous treatment status, age, and sex (odd's ratio, 2.3 [95% CI, 1.000-5.350]; p-value, 0.05). No XDR-TB was found among the MDR-TB isolates. CONCLUSION: The present study demonstrated a high prevalence of drug resistance amongst pulmonary TB isolates of M. tuberculosis from north India as compared to the WHO estimates for India in 2010, though this could possibly be attributed to the clustering of more serious or referred cases at our tertiary care centre. The prevalence of MDR-TB in HIV seropositive patients was significantly higher than seronegative individuals. The study emphasizes the need to monitor the trends of drug resistance in TB in various populations in order to timely implement appropriate interventions to curb the menace of MDR-TB.

Evaluation of in-house loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of M. tuberculosis in pulmonary specimens.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost-effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis-specific in-house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR). METHODS: A total of 133 sputum specimens (103 from suspected pulmonary TB cases and 30 from non-TB controls) were subjected to conventional tests, IS6110 PCR and 16s rRNA LAMP assay. RESULTS: Of the 103 patients, the maximum number of cases were found to be positive by LAMP assay, that is, in 87 (84.5%) patients, followed by culture positive in 78 (75.7%), IS6110 PCR in 74 (71.8%), and smear positive in 70 (67.9%) patients. Of the 83 smear positive and/or culture positive cases, LAMP detected 77 (92.77%) cases, and was found to be superior to IS6110 PCR, which could detect 69 (83.1%) cases; a concordance of 0.6 was obtained between the two tests using kappa statistics. CONCLUSION: Overall, LAMP was simple and efficacious for early diagnosis of smear positive, culture positive cases as well as for confirmation of smear negative, culture negative cases, and was found to be superior to IS6110 PCR.

Extra-intestinal complications of Ascaris lumbricoides infections in India: A systematic review and meta-analysis.

The aberrant migration of Ascaris lumbricoides may cause extra-intestinal ascariasis (EIA) involving hepato-biliary-pancreatic (HBP) or other extra-gastro-intestinal (EGI) organs. We performed a systematic review and meta-analysis to study the risk factors and clinical presentations of EIA, and differences in HBP and EGI ascariasis. Medline, Web of Science and Embase were searched for cases of EIA in the English language from India. From 1204 articles, 86 studies (105 cases) were included. The majority of the cases involved the HBP system (78%). Among HBP ascariasis, the most commonly involved site was the bile duct (53.6%). Females had 11.3 times higher odds (95% CI 2.852 to 44.856; p=0.001) of HBP ascariasis, while the pediatric population had lower odds (OR=0.323). Previous gallbladder disease was significantly associated with HBP ascariasis in adults (p=0.046), while a significantly higher number of cases of EGI ascariasis were observed among pediatric patients (p=0.003). Ocular symptoms occurred exclusively in the pediatric population (p=0.017). Overall, death was reported in 3.8% of patients (n=4). This review emphasizes the importance of the complications of EIA. It encourages future research into issues such as the reasons of higher gall bladder ascariasis in females and the implications of Ascaris-related complications following biliary tract interventions. It also suggests considering Ascaris as a differential diagnosis for airway obstuction in intubated critically ill patients.

Primate malaria of human importance.

Nonhuman primate (NHP) malaria poses a major threat to the malaria control programs. The last two decades have witnessed a paradigm shift in our understanding of the malaria caused by species other than the traditionally known human Plasmodium species - Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The emergence of the malaria parasite of long-tailed macaque monkeys, Plasmodium knowlesi, as the fifth malaria species of humans has made the scientific community consider the risk of other zoonotic malaria, such as Plasmodium cynomolgi, Plasmodium simium, Plasmodium inui, and others, to humans. The development of knowledge about P. knowlesi as a pathogen which was earlier only known to experimentally cause malaria in humans and rarely cause natural infection, toward its acknowledgment as a significant cause of human malaria and a threat of malaria control programs has been made possible by the use of advanced molecular techniques such as polymerase chain reaction and gene sequencing. This review explores the various aspects of NHP malaria, and the association of various factors with their emergence and potential to cause human malaria which are important to understand to be able to control these emerging infections.

Seroprevalence of human cystic echinococcosis: A study from a tertiary care center of North India.

Present study retrospectively analysed the serological data of patients suspected of cystic echinococcosis (CE) attending the outpatient clinics or admitted in our hospital. An enzyme-linked immunoassay was used to analyse anti-CE antibodies in serum samples of 3680 patients. Microscopy of aspirated cystic fluid was performed on 170 cases only. CE seropositive cases were 595 (16.2%), of which 293 (49.2%) were males and 302 (50.8%) were females. A higher percentage of seropositivity was found in adults within age range of 21-40 years of age. There has been a decrease in seropositivity in the study years (2016-2021) in comparison to previous years (1999-2015).