Hematological indicators for neglected tropical diseases

Neglected tropical diseases (NTDs) flourish in areas of poor socio-economic status in developing countries. However, increased international travels, armed conflicts, and effects of environmental changes on vector ecology have influenced the emergence of such diseases also in developed countries. As many of the parasitic infections are periodically asymptomatic, there is a risk of spread of pathogens by unaware carriers. Tests for accurate and rapid diagnosing and monitoring are crucial in the fight against NTDs.

Background

NTDs are a group of conditions prevalent to impoverished regions in tropical areas (1). They are neglected in the sense of having limited resources and being almost absent from the global health agenda (1). They are also neglected by research, with a lack of comprehensive studies and with knowledge on their management mostly shared in case studies. The causative pathogens, such as viruses, bacteria, fungi, protozoans, and parasitic worms, thrive in hard-to-reach areas with limited access to clean water, sanitization, and healthcare (1). Many of the pathogens are vector borne, with animal reservoirs and associated with complex life cycles, making their control challenging. Co-infections due to different NTDs occurring in the same populations add to the complexity (2).

Available diagnostics for NTDs

Rapid and accurate diagnosis is crucial in the fight against NTDs, yet clinical laboratories in low-income communities are constantly facing financial and human resource constraints (3). Microscopic examination of biological samples is the reference method in parasitology and a relatively cheap method for morphology-based diagnosis, however, requires a high level of parasitological knowledge (3). Pathogen-specific tests such as PCR or immunoassays can provide rapid and accurate test results, but their relatively higher cost per test might omit their broad use in affected communities (3).

Impaired host resistance, co-infections, and pathogen-mediated suppression of inflammatory mediators make diagnosis challenging. As many NTDs cause anemia—either directly through blood loss or indirectly through bone marrow suppression, inflammation, hypersplenism, hemolysis, or anorexia—the prevalence of anemia has been suggested to be added to the choice of indicators (4). Anemia is particularly common in individuals infected with soil-transmitted helminths or schistosoma (4) and can easily be diagnosed with a simple blood cell count.

Anemia as an indicator for NTDs

A complete blood count (CBC) is a simple and low-cost test, and therefore widely available in laboratories worldwide. Compact, benchtop analyzers with a robust performance enables their use also in remote areas. In addition to hemoglobin concentration for diagnosing anemia, a CBC test can give enhanced insights into anemia type (Table 1).

 

Table 1. Anemia classified based on cell morphology (6, 7)
RBC morphology Cause Etiological factors Typical RBC parameter values
Normochromic and macrocytic anemias Nuclear maturation defects: deficient chromatin condensation and extrusion from the cell, with low cell division resulting in large ovalocytes or megaloblasts. Vitamin B12 deficiency Vitamin B9 deficiency Low RBC/HCT Normal HGB/MCHC High MCV/MCH/RDW
Normochromic and normocytic anemias Hemolytic: increased RBC destruction due to intra- or extra-cellular defects. Hemorrhagic: increased blood loss, acute or chronic. Hereditary spherocytosis Liver disease Renal diseases Sickle cell disease Hemolytic anemia Acute hemorrhage Low RBC/HGB/HCT Normal MCV/RDW/MCH/MCHC
Hypochromic and microcytic anemias Cytoplasmic maturation defects: deficient hemoglobin synthesis in the cytoplasm Iron deficiency anemia Thalassemia Low HGB/MCV/MCH/MCHC Normal RBC/HCT High RDW

Both iron deficiency anemia (IDA) and thalassemia are manifested as microcytosis and hypochromia, with low hemoglobin concentration as a result. However, while IDA is a nutrient disorder that can be treated with iron supplementation, thalassemia is an inherited disorder that lacks management protocol and for which blood transfusion is a mainstay of treatment (9, 10). The ability to differentiate IDA from thalassemia is important, as blood hemoglobin will not be improved by iron supplementation in thalassemia patients (11). Together with determination of, for example, serum iron levels, RBC indices can be of good help (8, 11). Many formulas that include the RBC indices have been suggested to discriminate thalassemia from IDA (Table 2), of which RDW index (RDWI) followed by Mentzer index were found to provide the highest rate of correctly diagnosed patients (8, 11).

Table 2. Suggested formulas to distinguish beta-thalassemia trait (β-TT) from iron deficiency anemia (IDA) (adopted from Naizi et al. [8] and Jameel et al. [11])
Index Formula β-TT IDA
Red cell distribution width RDW < 14 < 14
RDWI MCV × RDW / RBC < 220 > 220
Mentzer MCV / RBC < 13 > 13
England & Fraser MCV – (5 × HGB) - RBC < 0 (neg) > 0 (pos)
Srivastava MCH / RBC < 3.8 > 3.8
Shine & Lal MCV × MCV × MCH / 100 < 1530 > 1530
Green & King MCV × MCV × RDW / (HGB × 100) < 72 > 72
Ricerca RDW / RBC < 3.3 > 3.3

Complete blood count in diagnosis of NTDs

In addition to parameters useful in anemia investigations, a cell count analyzer reports a range of other parameters related to inflammation and infections. For example, dengue fever is associated with thrombocytopenia, neutrophilia is associated with acute infections caused by bacteria or certain viruses (e.g., rabies), and eosinophilia suggests nematodes or cestodes, typically acute schistosomiasis (serology and parasitology are often negative at this stage), or filariasis (5, 12). A summary of hematological findings based on the literature on NTDs are given in Table 2.

Table 3. Hematological implications for neglected tropical diseases caused by infections (1, 13–25)
Disease Pathogen Transmission Manifestation Hematology implications
Buruli ulcer Bacteria (Mycobacterium ulcerans) Not known Begins with a painless nodule or papule in the skin Normal
Chagas disease Protozoan parasite (Trypanosoma cruzi) Vector-borne (triatomine bug) Food-borne Congenital (pregnancy, birth Through blood/blood products Organ transplantation Laboratory accidents Often asymptomatic Leukocytosis Lymphocytosis Anemia
Chromoblastomycosis Fungi (e.g., Fonsecaea pedrosoi, Fonsecaea monophora, Cladophialophora carrionii) Infected skin injury Wart-like lesions Normal
Dengue Virus (genus Flavivirus) Mosquito bites Hemorrhage Neutropenia Thrombocytopenia
Chikungunya Virus Mosquito bites Rash, joint pain Lymphopenia
Dracunculiasis Parasitic worm (Dracunculus medinensis) Contaminated drinking water Ulcer Anemia
Echinococcosis Parasitic worms (tapeworms, genus Echinococcus) Contaminated food Cysts, often in liver and lungs, containing watery fluid Eosinophilia Anemia
Foodborne trematodiases Parasitic worms (flatworms, e.g., Clonorchis sinensis, Opisthorchis viverrini, O. felineus, Fasciola hepatica, F. gigantica, Paragonimus spp) Raw fish, aquatic vegetables Initially, often asymptomatic Eosinophilia
Human African trypanosomiasis (HAT) Protozoan parasite (genus Trypanosoma) Tsetse fly bites Local reaction (trypanosomal chancre) Leukocyte count in cerebrospinal fluid (CSF) Anemia
Leishmaniasis Protozoan parasite (genus Leishmania) Sandfly bites Ulcer Lymphocytosis
Leprosy Bacteria (Mycobacterium leprae) Droplets from the nose and mouth Akin lesions Lymphocytosis
Lymphatic filariasis Parasitic worms (Wuchereria bancrofti, Brugia malayi, Brugia timori) Mosquito bites Tissue swelling Eosinophilia
Mycetoma Different species of fungi (eumycetoma) or bacteria (actinomycete) Infected skin injury Hard swelling, discharging sinuses and grains Eumycetoma: Normal Actinomycosis: Leukocytosis Neutrophilia
Noma Non-specific polymicrobial organisms Gum injury Initial soft tissue lesion (a sore) of the gums Leukocytosis Neutropenia Anemia
Onchocerciasis Parasitic worm (nematode, Onchocerca volvulus) Black fly bites Decrease in visual acuity, narrowing of the visual field Eosinophilia
Rabies Virus (genus Lyssavirus) Zoonotic (e.g., dog bite) Early symptoms, e.g., itching, pain around site of exposure Neutrophilia
Scabies Parasitic mite (Sarcoptes scabiei hominis) Skin-to-skin contact Itching, skin lesions Eosinophilia
Schistosomiasis Parasitic worms (trematodes, e.g., Schistosoma haematobium, Schistosoma mansoni, Schistosoma japonicum) Skin contact with infested water Early symptoms, e.g., itching, allergic, gastrointestinal Eosinophilia Anemia
Soil-transmitted helminthiases Parasitic worms, e.g., roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura), hookworms (Necator americanus and Ancylostoma duodenale) Contaminated food and drinking water Diarrhea, abdominal pain Anemia
Taeniasis/cysticercosis Parasitic worm (tapeworm, Taenia solium) Contaminated food Often asymptomatic Eosinophilia
Trachoma Bacteria (Chlamydia trachomatis) Contaminated hands, cloths, and flies Respiratory infection Eosinophilia
Yaws Bacteria (Treponema pallidum) From person to person through minor injuries Hard swelling, ulcer Anemia Thrombocytopenia Either leukopenia or leukocytosis. Monocytosis is common.

Enhancing the outreach of diagnostic testing

NTDs are mainly endemic in rural areas, in conflict zones, and hard-to-reach regions (1). At the same time, early diagnosis is essential but largely dependent of availability of testing facilities (3). Initiatives to enhance outreach is therefore of importance.

Science House Medicals, Boule’s distribution partner based in Bhopal, India, is a provider of diagnostic services to government healthcare facilities. As part of a public-private partnership, utilizing government’s will for social good and private sector’s ability to support infrastructure and enhance service standards, Science House Medicals was awarded a hub-and-spoke type of project by the government of Madhya Pradesh (26).

Under this project, they operate 324 hub laboratories and 1690 spoke centers in both urban and rural areas, allowing a presence at about every 7.5 km. This way, patients will neither need to travel long distances nor wait for long periods to get timely medical care. Blood collected at the regional centers is transported by motorcycle or drone for testing at the hub laboratory (Fig 1). Results are reported back to the regions through LIS/HIS connection, with a turnaround time of about 1 to 1.5 hour.

Science House Medicals

Fig 1. Blood collected at spoke centers is getting tested on a Swelab™ Alfa Plus hematology analyzer at one of the hub laboratories.

Q-Line Biotech, Boule’s distribution partner based in Lucknow, India, has received many honorary awards for contributions to improving public health in India (27). One of the later initiatives is mobile vans for coronavirus virus testing with 24-hour report turnaround time, to support RT-PCR investigation of SARS CoV 2 virus spread out in the villages (Fig 2).

Mobile vans for coronavirus virus testing

Fig 2. Mobile vans for coronavirus virus testing.

Reliable equipment performance a prerequisite

Hematology systems intended for CBC testing in remote areas pose special challenges to the engineers (28). The systems need to be robust to minimize maintenance and service needs, as such support can be at a far distance. These systems also need to be cost-efficient and simple to use, while providing laboratory-quality results.

Fluidic system – the heart of automated hematology analysis

Although simple to use, automated hematology analysis is technically complex. To allow reporting of CBC results, multiple methods are run in parallel to obtain results for the full range of parameters. The fluidic system is therefore the heart of the analyzer, and it plays a pivotal role in analyzer performance (29). Commercially available analyzers employ different design solutions for their fluidic systems, each with its advantages and disadvantages. A thorough understanding of flow dynamics and control of pressurized fluids is critical when designing the fluidic system.

Piston-driven flow control versus flow control using air and vacuum pumps

One common approach is piston-driven fluidic control. This method offers the benefit of lower liquid circuit volume and reduced reagent consumption, however, comes with the trade-off of typically requiring daily cleanings to prevent clogging by protein buildup within the system.

In contrast, flow control using air and vacuum pumps stands out for its robustness. This method is less prone to leakage and reduces the need for moving mechanical parts and other components that increases maintenance requirements, ultimately enhancing instrument uptime.

The importance of precise sample aspiration

The aspiration method employed is a critical component of hematology analysis that significantly impacts measurement quality. Precise sample aspiration is fundamental to obtaining accurate and reliable results. Two primary aspiration methods are commonly used— micro-pipette aspiration and shear valve-aided aspiration—each with its own advantages and considerations.

A micro-pipette aspiration method has the advantage of a small sample volume. However, even a slight decrease in the sample volume can have a substantial impact on the accuracy of the results. Additionally, the method requires that the micro-dilutor is completely sealed to avoid leakage from the syringe. The method also requires an aspiration module that moves the sample to an open mixing cup through an X-Y moving assembly, a solution that might increase the maintenance needs.

Although the aspirated volume is greater with a shear valve, the method excels in robustness. Shear valve aspiration is controlled by a blood sensor and a precise volume is cut and brought to a closed mixing chamber using diluent reagent. Cutting the same precise volume for each measurement, the analyzer is not required to be as frequently calibrated. Additionally, this method allows the use of a closed mixing cup, reducing the maintenance needs by being less susceptible to entry of dust and other impurities.

Boule hematology solutions designed for decentralized testing

The difference between hematology analyzers may not be apparent simply by comparing technical or performance specifications, nor may it be revealed during installation, operation, and performance qualification (IQ/OQ/PQ). It is therefore advisable to get basic knowledge about the strengths and weaknesses of different design solution before selecting an automated hematology system for clinical use.

Boule hematology systems are tailored for use in laboratories at small hospitals and primary care units (Fig 3). With a closed fluidic system and automated cleaning procedures, the analyzers are designed for a reliable performance to minimize maintenance and service needs, maximizing uptime and system availability to provide patient results.

“A blood count is a reliable, low-cost, and immediate reading test that helps us in the screening of people suspected of being infected by SARS-COV-2, and it is one of the first filters we use in decision-making”, says Dr. Fernando Bonilla, Clinical Microbiologist at Permanent Contingency Commission, Triage Center Honduras (30). “With Swelab Alfa Plus, we have a highly reliable instrument, easy to use, with an autoloader for the automated handling of a significant number of samples, and with the capillary blood functionality for urgent cases that require an immediate count and where a tube of venous blood sample is not necessarily available. In such cases, we can use a drop of blood from the patient’s finger to obtain a hemogram with 22 parameters in one minute.”

Dr. Bonilla uses a Swelab Alfa Plus analyzer

Fig 3. Dr. Bonilla uses a Swelab Alfa Plus analyzer equipped with space-saving automation wheels to cope with high workloads, and with the MPA inlet that reports a full CBC from a finger-stick sample in about one minute.

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